Reviews of Physiology - Biochemistry and Pharmacology Vol. 169

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Reviews of Physiology, Biochemistry
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Reviews of Physiology,
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Reviews of Physiology, Biochemistry and Pharmacology
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Contents
Hyperforin: To Be or Not to Be an Activator of TRPC(6) . . . . . . . . . . . . . . . . . 1

Kristina Friedland and Christian Harteneck
The Piezo Mechanosensitive Ion Channels: May the Force

Be with You! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Eric Honore, Joana Raquel Martins, David Penton,
Amanda Patel, and Sophie Demolombe
Chronobiology and Pharmacologic Modulation of the

ReninAngiotensinAldosterone System in Dogs:
What Have We Learned? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Jonathan P. Mochel and Meindert Danhof
v
Rev Physiol Biochem Pharmacol (2015) 169: 124
DOI: 10.1007/112_2015_25
Springer International Publishing Switzerland 2015
Published online: 19 September 2015
Hyperforin: To Be or Not to Be an Activator

of TRPC(6)
Kristina Friedland and Christian Harteneck
Abstract Meantime, it is well accepted that hyperforin, the chemical instable

phloroglucinol derivative of Hypericum perforatum, St. Johns wort, is the
pharmacophore of St. Johns wort extracts. With the decline of this scientific
discussion, another controversial aspect has been arisen, the question regarding the
underlying mechanism leading to the pharmacological profile of the plant extract
used in therapy of depression. We will summarize the different concepts described
for hyperforins antidepressive activity. Starting with unspecific protein-independent
mechanisms due to changes in pH, we will summarize data of protein-based
concepts beginning with concepts based on involvement of a variety of proteins
and will finally present concepts based on the modulation of a single protein.
Keywords Calcium homeostasis Depression Hyperforin Ion channel

Protonophore St. Johns wort TRP channel
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Chemical Stability of Hyperforin and Stable Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Hyperforin as Protonophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4 Hyperforin as Inducer of Cytochrome P450 Enzymes, Hyperforin as Pregnane X
Receptor Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5 Hyperforin as Activator of Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
K. Friedland (*)
Department of Molecular and Clinical Pharmacy, Friedrich-Alexander University Erlangen/
Nuremberg, Cauerstr. 4, 91058 Erlangen, Germany
e-mail: kristina.leuner@fau.de
C. Harteneck (*)
Institute of Pharmacology and Toxicology and Interfaculty Centre for Pharmacogenomics
and Drug Research, Eberhard Karls Universitat, Wilhelmstr. 56, 72074 Tubingen, Germany
e-mail: christian.harteneck@uni-tuebingen.de
2 K. Friedland and C. Harteneck
6 The Role of TRPC6 Channels for Neuronal Effects of Hyperforin: Depression,

Epilepsy, Autism, Ischemia, Cognition, and Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . 14
7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1 Introduction
St. Johns wort, Hypericum perforatum, is used for centuries to treat mild and
moderate depression (Gastpar 2013). Depression is one of the most frequent disease
worldwide and the most common psychiatric diseases (Duman and Aghajanian
2012; Penninx et al. 2013). Approximately 1 of 5 women and 1 of 8 men suffer from
a depressive episode during their lifetime (Mu~noz et al. 2010; Murray et al. 2012).
Depression refers to a set of mental symptoms such as loss of interest and pleasure,
decreased cognition and memory, and disrupted sleeping, eating, ambulation, and
sexual activity (Duman and Aghajanian 2012). A large number of clinical trials
(Lecrubier et al. 2002; Gastpar et al. 2006; Kasper et al. 2010; Singer et al. 2011) as
well as a recent Cochrane meta-analysis (Linde et al. 2008) confirm the antidepres-
sant activity in patients of the plant and its extracts. St. Johns wort extracts contain
several constituents like hyperforin (Fig. 1a), a phloroglucinol derivative, the
naphtodianthrone hypericin (Fig. 1b), and some flavonoids such as isoquercitrin,
biapigenine, or rutin, which were discussed to represent the antidepressive principle
(Noldner and Sch otz 2002; Muller 2003; Butterweck and Schmidt 2007; Paulke
et al. 2008). The first molecule in focus of representing the antidepressive principle
was hypericin mediating its effect via the inhibition of the monoamine oxidase
(Suzuki et al. 1984). These results could not be validated by other groups arguing
for a minor role of hypericin in the antidepressant effects of St. Johns wort extract
(Bladt and Wagner 1994; Thiede and Walper 1994; Cott 1997). Other mechanisms
were investigated such as interaction of hypericin with monoamine receptors
(Butterweck et al. 2002; Caccia and Gobbi 2009). However, the concentration
gap of hypericin affinities in the micromolar range for binding these receptors
and nanomolar plasma concentrations measured in humans after intake of thera-
peutic doses made it unlikely to account for the antidepressive principle (Staffeldt
et al. 1994; Kerb et al. 1996; Brockmoller et al. 1997). Phototoxicity is a feature
attracting attention to hypericin (Boiy et al. 2008; Davids et al. 2008), being
currently under investigation regarding its antimetastatic and antiangiogenic prop-
erties in the treatment of glioblastomas or melanoma (Davids et al. 2008; Barliya
et al. 2011; Dror et al. 2013). The flavonoids, biapigenine, hyperoside, and
isoquercitrin, showed moderate antidepressive activity in the forced swim test, a
behavioral animal model for depression (Noldner and Schotz 2002; Paulke
et al. 2008) with yet unknown mechanism.
Hyperforin (Fig. 1a) was long neglected as the putative antidepressant active
constituent of H. perforatum due to its chemical instability in response to light and
air. Under appropriate storage conditions, 15% hyperforin can be found in the
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 3
A H3 C CH 3 B OH O OH
H 3C
CH 3
OH
O
CH 3 HO CH 3
O
HO CH 3
H3 C H3 C O CH 3
CH 3
H3C OH O OH
CH 3
hypericin
hyperforin
H3 C CH 3
C D
H 3C
CH 3
O O
O O HO O
HO CH 3
O
H3 C CH 3
H 3C H 3C O CH 3 OH
HO
CH 3
Hyp9
H 3C
CH 3
aristoforin
E
O
O CH 3
O
CH 3
OH
O
1-stearoyl-2-arachidonoyl-sn-glycerol
Fig. 1 Structure of hyperforin (A), hypericin (B), aristoforin (C), Hyp9 (D), and 1-stearoyl-2-
arachidonoyl-sn-glycerol (E)
St. Johns wort extracts (Chatterjee et al. 1998). The phloroglucinol derivative
hyperforin showed pronounced effects in behavioral models for depression includ-
ing the forced swim test (15 mg/kg BW), the learned helplessness test (15 mg kg/
BW), the elevated plus maze test, or the light/dark test (Chatterjee et al. 1998;
Zanoli et al. 2002). Importantly, one clinical trial showed loss of antidepressant
properties of a St. Johns wort extract containing 0.5% hyperforin instead of 5%
hyperforin (Laakmann et al. 1998). On the biochemical level, several groups
showed hyperforin-mediated inhibition of neurotransmitter uptake such as seroto-
nin (IC50 205 nM in whole brain rat synaptosomes), norepinephrine (IC50 102 nM in
rat synaptosomes from the occipital cortex), dopamine (IC50 80 nM in synapto-
somes isolated from rat striatum), GABA (IC50 184 nM in whole brain rat
synaptosomes), and L-glutamate (IC50 143 nM in rat synaptosomes isolated from rat
forebrain) (Chatterjee et al. 1998; Singer et al. 1999; Wonnemann et al. 2000;
Marsh and Davies 2002). All transmitters are known for their potential role in the
pathogenesis of depressive disorders (Duman and Aghajanian 2012; Duric and
Duman 2013). Increases in extracellular neurotransmitter levels in response to
hyperforin were shown in rat synaptosomes (Chatterjee et al. 2001) and in different
brain areas using microdialysis (Philippu 2001; Buchholzer et al. 2002; Coskun
et al. 2004; Kiewert et al. 2004; Yoshitake et al. 2004) or pushpull analysis
(Kaehler et al. 1999). In rat hippocampus, hyperforin (10 mg/kg BW i.p. or
10 M via the dialysis probe) resulted in increased acetylcholine levels which
was completely reverted by local perfusion with calcium-free buffer or in the
presence of tetrodotoxin, an inhibitor of voltage-dependent sodium-channels
(Buchholzer et al. 2002; Kiewert et al. 2004). Hence, hyperforin-mediated neuro-
transmitter release is a calcium-dependent mechanism requiring intact neuronal
communication and cell firing (Buchholzer et al. 2002; Kiewert et al. 2004). The
impact of hyperforin in increased extracellular accumulation of neurotransmitters
like dopamine, norepinephrine, serotonin, and glutamate was measured in the rat
locus coeruleus upon application of hyperforin (10 mg/kg BW, i.p. application)
(Kaehler et al. 1999). In rat synaptosomes, hyperforin (5 M) also increased the
release of several neurotransmitters such as glutamate or GABA (Marsh and Davies
2002). These observations argued for the modulation of synaptosomal neurotrans-
mitter transport by hyperforin.
Pharmacological relevant concentrations regarding inhibition of neurotransmit-
ter uptake are achieved in human plasma. The intake of a single dose of 300 mg
Hypericum extract containing 14.8 mg hyperforin by healthy volunteers resulted in
human plasma levels of 150 ng/mL hyperforin (280 nM) (Biber et al. 1998). The
application of the clinical dosage (3 300 mg St. Johns wort extract per day) given
for one day resulted in hyperforin concentration of 100 ng/mL or 180 nM in plasma
of healthy volunteers (Biber et al. 1998). Comparable plasma level as well concen-
trations in brain tissues can be achieved by the application of hyperforin sodium
salt. In rats, the application of 300 mg/kg BW Hypericum extract (WS5572, 5%
hyperforin) results in plasma concentrations of 370 ng/mL hyperforin (690 nM).
The maximum concentration was detected after 3 h and an estimated half-life time
of 6 h could be calculated (Biber et al. 1998). The effective dosage used in
behavioral test like the forced swim test given as hyperforin sodium salt (15 mg/
kg BW) or as St. Johns wort extract (containing 5% hyperforin), resulted in organ
concentrations of 28.8 and 15.8 ng/g (hyperforin per brain tissue) respectively
(Keller et al. 2003). Based on its lipophilic structure, hyperforin might accumulate
in the brain reaching higher nanomolar concentrations in the brain than found in the
plasma.
The diversity of neurotransmitter being extracellularly increased in response to
hyperforin makes it unlikely that hyperforin directly modulates neurotransmitter
transport comparable to amitriptyline, a tricyclic antidepressant, or fluoxetine, a
selective serotonin reuptake inhibitor (Chatterjee et al. 1998; Singer et al. 1999).
This is underscored by the fact that the neurotransmitter being modulated by
hyperforin is mediated by neurotransmitter transporters of different classes. The
serotonin, norepinephrine, dopamine, and GABA transporter belong to the SLC6

gene family; the L-glutamate transporter belongs to the class of SLC1 plasma
membrane transporters (Scimemi 2014; Jensen et al. 2015; Lin et al. 2015).
Therefore alternative mechanisms involved in the antidepressant activity of
hyperforin have been described like modulation of intracellular pH (Chatterjee
et al. 2001; Roz and Rehavi 2003, 2004; Sell et al. 2014), upregulation of metab-
olizing enzymes of the cytochrome P450 family (Moore et al. 2000; Obach 2000;
Cantoni et al. 2003; Lee et al. 2006; Whitten et al. 2006), and modulation of ion
channels (Chatterjee et al. 1999; Marsh and Davies 2002; Treiber et al. 2005;
Muller et al. 2008; Gibon et al. 2010). Below, we will summarize the different
concepts described for hyperforins antidepressive activity. Starting with unspecific
protein-independent mechanisms due to changes in intracellular pH, we will sum-
marize data of protein-based concepts beginning with concepts based on involve-
ment of a variety of proteins and will finally present concepts based on the
modulation of a single protein. Prior to the summary of the different concepts, we
will shed light on the chemical stability of hyperforin an important issue preventing
the acceptance of hyperforin as the pharmacophore of St. Johns wort in the past.
2 Chemical Stability of Hyperforin and Stable Analogues
Hyperforin, the prenylated acylphloroglucinol, is unstable in water and aprotic

solvents such as n-hexane and possesses a high sensitivity toward oxidative atten-
uation and degradation (Maisenbacher and Kovar 1992; Verotta et al. 1999;
Wolfender et al. 2003; Isacchi et al. 2007). The main degradation products are
furohydrohyperforin and oxidized forms of hyperforin in which a hydroxy-
dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric
form) and the prenyl chain at C-8 of the core nucleus of hyperforin (Wolfender
et al. 2003). Verotta et al. studied the biological activity of the oxidized hyperforin
derivatives highlighting the role of the enolized cyclohexanedione moiety for
activity on neurotransmitter reuptake (Verotta et al. 2004). The solvent in which
the hyperforin salt is dissolved or stored as well as the period in which hyperforin is
stable in an aqueous solution should be chosen very carefully and time lags based
on experimental approach should be taken into account. Stable hyperforin deriva-
tives such as aristoforin (Fig. 1c) (Gartner et al. 2005; Rothley et al. 2009) or
tetrahydrohyperforin (Inestrosa et al. 2011; Abbott et al. 2013) may be the alterna-
tives under certain circumstances. The activity of aristoforin was tested regarding
antitumor (Gartner et al. 2005; Semelakova et al. 2012) and antiangiogenic prop-
erties (Rothley et al. 2009). Tetrahydrohyperforin was mainly investigated in the
context of Alzheimers disease models and was shown to decrease amyloid beta
levels and improves synaptic plasticity including neurogenesis as well as cognitive
impairment (Inestrosa et al. 2011; Abbott et al. 2013). In contrast to aristoforin and
tetrahydrohyperforin, the chemically simplified phloroglucinol derivatives (Hyps,
e.g., Hyp9; Fig. 1d) represent hyperforin analogues with respect to their
antidepressant effects comprising neurotransmitter uptake and neuronal differenti-

ation (Leuner et al. 2010).
3 Hyperforin as Protonophore
In 2002, the group around Rehavi suggested that hyperforin might be a

protonophore (Fig. 2a) and thereby inhibit neurotransmitter reuptake (Roz
et al. 2002; Roz and Rehavi 2003). They used synaptic vesicles isolated from rat
brain and showed that hyperforin inhibited the uptake of serotonin, norepinephrine,
as well as dopamine. They compared the IC50 values needed for the inhibition of
synaptosomal and vesicular uptake. Half-maximal inhibition of serotonin uptake in
synaptosomes was achieved by IC50 values of 1.1 M and an IC50 values of
0.32 M for synaptic vesicles (Roz et al. 2002). The noncompetitive inhibition of
the synaptosomal uptake as well as the vesicular uptake found in their experiments
provided the basis for the concept that hyperforin might interfere with the driving
force of the vesicular uptake, the pH gradient across the vesicular membrane
generated by the H+-ATPase (Fig. 2b). In a second publication, they studied the
impact of hyperforin on the acidification of rat brain synaptic vesicles analyzing
ATP-dependent proton uptake in synaptic vesicles using the fluorescence indicator
acridine orange (Roz and Rehavi 2003). They demonstrated that the protonophore
FCCP (1 M) and hyperforin (0.43 M) both inhibited the proton transport into
synaptic vesicles. In addition, both compounds blocked serotonin uptake in synap-
tosomes in similar concentrations (1 M). The authors concluded that FCCP and
hyperforin dissipate the pH gradient and thereby inhibit the vesicular uptake. They
suggest that the reduced vesicular storage leads to enhanced cytoplasmic mono-
amine concentrations which in turn decrease the neurotransmitter transmembrane
gradient and consequently an apparent inhibition of neurotransmitter uptake is
observed. However, protonophores such as the mitochondrial uncoupler FCCP
show no antidepressant effects.
The impact of hyperforin on intracellular pH regulation was further studied by
Froestl et al. (2003) in the context of Alzheimers disease and amyloid precursor
protein processing (APP). Several compounds affecting intracellular pH reduce
APP processing such as ammonium chloride, chloroquine, monensine,
bafilomycine, and FCCP. The group compared pH changes and APP processing
in the presence of bafilomycin A1, FCCP, and hyperforin. Bafilomycin A1 is a
specific inhibitor of vacuolar ATPases and thus inhibits the acidification of a
number of intracellular organelles. FCCP as a mitochondrial uncoupler of oxidative
phosphorylation also shows effects on the level of the plasma membrane. Using the
intracellular pH-sensitive fluorescence indicator BCECF, Froestl and colleagues
found similar effects on intracellular pH in the neuronal-like cell line PC12 induced
either by hyperforin or FCCP at concentrations of 5 or 10 M. The effects on APP
processing by FCCP were reduced compared to hyperforin, whereas bafilomycin
A1 only moderately affected APP processing and intracellular pH. These results
H3C CH 3
H 3C
A
CH 3
H+
OH
O
CH 3
O
H3C H3C O CH 3
CH 3
H 3C
CH 3
outside H 3C CH 3
H+
H 3C
CH 3
H3C CH 3 H3C CH 3
+ H3C CH 3 OH + H3C CH 3
N H +
H H3C CH N H +
H
H N H 3 O H 3C C H3 H N H
CH 3 N
+
N
+ CH 3 CH 3 N
+
N
+
CH 3 CH 3
H 3C O
H H H H
O O CH H 3C O O
O O 3
CH O O
P O CH
3 P
O O O P O O H 3C OH H 3C 3
OH O O O P O O
O P O P O P O P
O O O O
O O CH 3 CH O O
O O O 3 H 3C C H3 C H3 O O
O
HH33CC CH 3
O H3C 3C O
O
O O H3C OH CH 3 C H3
C H3
O CH
O
O O
H 3C H 3C H 3C 3
O O O O O O O O O O O O
O O CH CH 3
OH O O
3
O O O O CH 3 O O O O
OH O
H 3C CH 3 CH 3
H 3C O O H 3C CH 3
CH 3 CH H 3C
O 3 CH 3
H 3C
CH C
CH 3 H 3C H 3C HO3 C H3 3 CH 3 CH 3
H3C OH H3C O CH 3 OH
CH 3 H 3C
O
CH 3 CH 3 CH 3 O
C H3
O H 3C OH
CH 3
O
H C O
H 3C H 3C O C H3 3 CH
CH
3
O H 3C H 3 CCH 3 O CH 3
H 3C 3
CH 3
H3C CH 3
H 3C H3C O CH
H+
3
CH 3 H 3C
OH C H3 CH 3
H 3C
CH 3
O H3C CH
C H3 H 3C
3
O H 3C CH 3 CH 3
H 3C
C H3
H 3C H 3C H 3C O CH 3
CH 3 OH
H3C CH 3 OH O H3C
H3C H 3C CH 3 CH3 CH H3C
H 3 CO 3
H 3C H 3C O H 3C H 3C
CH 3
CH CH H 3C CH CH
3
H3C 3 H 3C H 3HC CH 3 HO 3C H 3C CH 3 3
H3C 3
H3C
C 3 CH H3C H3C O CH
3 3
CH 3 CH 3 CH 3 CH 3
H 3C OH H 3C O CH
OH H 3C
H 3C H 3C H 3C 3
CH H 3C
CH 3 CH 3 CH 3 3 CH 3 CH 3
O O
C H3 CH H
OCH
CH 3 CH 3 3 3 CH 3 CH 3
O H 3C CH 3 O H 3C
O C H3
CH 3
O CH H 3C
H 3C H 3C H 3C
3 H 3C H 3 C CH 3 O O CH 3 H 3C CH 3
CH 3
CH 3 OH CH
H 33C H 3C H 3C O C H3
CH 3
O
H 3C CH 3 CHH 3C OH
C H3 3
O CH 3 O
H+
CH 3
H 3C
H 3C H 3C O CH 3 C H3 O
C HC3H H 3C H 3C O CH 3
H 3C 3
H 3C C H3
H 3C CH3
H 3C CH 3 H 3C C H3
CH 3 H 3C
CH 3 H 3C
OH H 3C CH
OH 3
CH 3
O
C H3 O OH
O C H3
C C H3
OH 3 O
CH 3
H 3C H 3C O CH 3 H 3C O CH 3
C
O O O O H 3C H 3 C
H 3CH 3
O C H3
H 3C C H3 O O O O
O O O CH 3 O O HH3C
3C H 3C O CH 3
O O O O
O O O O CH3 CH O O O O
3
O H 3C
O O O O C H3 OH CH O O O O
H 3 CH 3 C CH3 3
CH 3
C H3 O H 3C OH
CH 3
O
O O H 3C H 3C CH 3 O O O
O O O O
CH 3 H 3C H 3C O CH 3O CH 3 C H3
O P O P O O P O P
O
O O O
O O OH O
O O O
O O
P P H 3C H 3C O CH P P
O CH 3 3

O O H O O H CH 3 H 3C H 3C O CH
O O 3 H O O H
+ + O CH3 + +
H3C N H3C N H 3C H3C N H3C N
H H CH 3 H 3C C H3 3
CH H H
+ H + H + H + H
N N H 3C H 3C O CH 3 H 3C N N
H+
H3C CH H3C CH CH CH CH
3 3 H 3C 3 H 3HC3 C 3 H3C 3
CH 3
C H3 C H3
OH
H 3C O
C H3 CH 3
O
inside
H 3C H 3C O CH 3
CH 3
H 3C
CH 3
H+
B
physiological processes pharmacological interference
of serotonin re-uptake by protonophoric activity
& vesicle loading of hyperforin
+
ATP H ADP
H -ATPase P
5-HT H+ H+ [H +]
+ +
H H +
+ H
H + HYP H
+
HYP
VMAT H
+
H +
H
5-HT H
+
SERT
SLC6A4 + H +
+ [H +] Na
+
Na + H
H [Na+] NHE
5-HT
+
H
+ +
Na Na
Fig. 2 Model of hyperforin as protonophore. (a) Adapted to the models known for the action
mechanisms of other ionophores, the association of hyperforin molecules is shown forming a
proton-permeable tube of the plasma membrane. (b) The model of physiological processes of
serotonin reuptake and vesicular loading as well as pharmacological interference by the
protonophoric activity of hyperforin is adapted from the scheme developed by Sell et al. (2014).
The reuptake of neurotransmitter across the presynaptic plasma membrane is mediated by various
monoaminesodium symporter. Here, we show the reuptake of serotonin (5-HT) by the serontonin
transporter (SERT/SLC6A4). Cytosolic neurotransmitter is transported into vesicles against a
concentration gradient by the activity of the vesicular monoamine transporter (VMAT), a
monoamineproton antiporter. The proton gradient across the vesicular membrane is established
by the activity of the vacuolar proton ATPase (H+-ATPase) transporting protons across the
suggest that hyperforin might mediate its effects on APP processing via a different
mechanism.
Recently, Sell et al. (2014) published that hyperforin acts as a protonophore.
Analyzing whole-cell currents in HEK293 cells stably expressing TRPC6, they
observed differences in currentvoltage relationship depending on the activating
ligand they use (Sell et al. 2014). While OAG and flufenamic acid resulted in the
typical double-rectifying currentvoltage relationship known for currents mediated
by TRPC6, hyperforin activated a current with a divergent currentvoltage rela-
tionship which could also be found in response to hyperforin in untransfected
HEK293 cells as well as in microglia. Although TRPC6 expression is absent in
microglia, they tested microglia isolated from TRPC6/TRPC3-deficient mice where
they found identical hyperforin-induced currents as they recorded in their initial
experiments. BCECF imaging used for monitoring intracellular pH showed that
extracellular solutions of different pH induced changes in intracellular pH in a
hyperforin-dependent manner. Based on these data, they conclude that hyperforin
acts as a protonophore in microglia and artificial lipid bilayers. In order to confirm
this hypothesis, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a well-
documented protonophore, was used in side-by-side experiments. In microglia,
CCCP-induced current resulted in currentvoltage relationship which could be
nevertheless distinguished from hyperforin-induced currents. Surprisingly, CCCP
was inefficient to induce currents in the lipid bilayer experiments using an
undefined commercial lipid mix. Based on their data, the authors provide a concept
for the antidepressive effect of hyperforin by dually interfering with intracellular
proton and ion homeostasis (Fig. 2b) (Sell et al. 2014). At the plasma membrane,
hyperforin induces an increased proton transport being compensated by the
sodiumproton exchanger resulting in increased intracellular sodium concentra-
tions and thereby indirectly inhibiting the monoamine transporter, a monoamine
sodium symporter protein. At the membrane of intracellular monoamine storage
vesicles, hyperforin-mediated proton flux disturbs the proton gradient across the
vesicular membrane and thereby inhibits the vesicular monoamine transporter, a
monoamineproton antiporter. In summary, the authors presume that the antide-
pressant activity is mediated by hyperforin-dependent indirect inhibition of trans-
mitter reuptake and vesicular transmitter loading based on the protonophoric
activity of hyperforin. These results dont exclude other effects of hyperforin
such as TRPC6 activation (see below).
Ionophores forming ion-permeable pores in lipid bilayers (Fig. 2a) like
natamycin, nystatin, amphotericin B as potassium ionophores, or nigericin or
monensin as protophores are therapeutically used as antimicrobiotics. Based on
Fig. 2 (continued) vesicular membrane. The protonophoric activity of hyperforin (HYP) results in
acidification of the cytosol by increased proton concentration resulting in the activity of the
sodiumproton exchanger (NHE). With the activity of the sodiumproton exchanger, a sodium
proton antiporter, the sodium gradient across the plasma membrane is reduced diminishing the
activity of the serotoninsodium symporter
their unselective mechanism of action, the antimicrobiotic activity is associated

with serious side effects limiting the use of these drugs. In the case of protonophoric
properties, hyperforin would have pronounced cytotoxic effects not only in the
brain but in all cells exposed to hyperforin during absorption and distribution
processes. This is in contrast to the fact that Hypericum extracts have been used
for decades as a safe remedy.
4 Hyperforin as Inducer of Cytochrome P450 Enzymes,

Hyperforin as Pregnane X Receptor Ligand
Cost-intensive safety pharmacology and clinical studies necessary for the approval
of modern drugs are substituted in the marketing of plant-derived remedies of the
traditional medicine by an evolutionary selection process over centuries. Under
these evolutionary conditions, not all aspects of modern safety pharmacology can
be identified and therefore the discovery of drug interference by St. Johns wort
extracts was a big surprise. Several case reports provided evidence for interference
reactions reporting reduced cyclosporine and theophylline levels during simulta-
neous intake of St. Johns wort extract (Ernst 1999; Breidenbach et al. 2000a, b;
Novelli et al. 2014). The initial suspicion was confirmed by subsequent clinical
studies showing that St. Johns wort extract induces a decline in the concurrent
therapy with immunosuppressants cyclosporine A, tacrolimus, and mycophenolic
acid (Bauer et al. 2003; Mai et al. 2003). The mechanism leading to increase
expression levels of cytochrome P450 enzymes (CYP) was soon deciphered by
experiments showing that constituents of St. Johns wort extracts in particular
hyperforin induce hepatic drug metabolism through activation of the pregnane X
receptor (PXR, Fig. 3) (Moore et al. 2000; Wentworth et al. 2000; Watkins
et al. 2003). Hyperforin binds to PXR with an EC50 value of 27 nM (Moore
et al. 2000). The binding of hyperforin to PXR results in transcriptional activation
and increased expression of proteins involved in drug metabolism. CYP enzymes,
mainly CYP3A4, as well as other proteins like the multidrug resistance-associated
protein (MDR1), are upregulated [for review see (Zhou et al. 2004)] and represent
the cause for the unwanted effects of St. Johns wort in therapy. While the
upregulation of multidrug resistance-associated protein (MDR1) is critical in can-
cer therapy by triggering the efflux of cytotoxic compounds, the upregulation of
CYP3A4 affects 50% of our therapeutic drug armamentarium.
Several hyperforin-derived compounds have been developed and analyzed.
Derivatives resulting from chemically oxidation processes of the instable
phloroglucinol, hyperforin, lost their antidepressive effects. In symmetric
phloroglucinol structures, the antidepressive effect of hyperforin was preserved
(Leuner et al. 2010). 2,4-Diacylphloroglucinol compounds (Hyp9; see Fig. 1d)
blocked serotonin uptake in murine synaptosomes and were able to mimic nearly
all features shown for hyperforin, e.g., induction of neurite growth and TRPC6
HYP
MDR1
DRUG
ATP ATP
DRUG
HYP DRUG DRUG
DRUG
DRUG
HYP CYP DRUG
PXR
RUG
RUG
RUG
PXR HYP
PXR HYP
MDR1 DRUG CYP

mRNA CYP3A4
POL POL mRNA
other CYP
mRNA mRNAs
Fig. 3 Illustration of hyperforin-triggered transcriptional regulation of MDR-1 and CYP450
enzymes. Hyperforin binds to the pregnane X receptor, thereby modulating the transcriptional
activity of CYP and MDR genes. The hyperforin-induced activation of pregnane X receptor results
in increases in mRNA species coding for CYP enzymes and transporters like MDR1. PXR
pregnane X receptor, POL DNA-dependent RNA polymerase, MDR1 multidrug resistance trans-
porter 1, CYP cytochrome P450 enzymes
activation [(Leuner et al. 2010) for further aspects see below]. However, the
absence of PXR binding is the most impressive feature of the
2,4-diacylphloroglucinol compounds (Kandel et al. 2014). Docking studies, model-
ing the compounds into the crystal structure of PXR provided evidence for the
inefficiency of the compounds to bind to PXR (Kandel et al. 2014). The modeling
results could be validated by biochemical experiments testing the induction of the
expression of drug metabolizing proteins. While hyperforin and rifampicin effi-
ciently elevated CYP3A4 mRNA transcription, this effect was absent in side-by-
side experiment using 2,4-diacylphloroglucinol compounds (Kandel et al. 2014).
The 2,4-diacylphloroglucinol structure provide an interesting lead in the develop-
ment of a new generation of antidepressants acting via a quite different mechanism
(see below) compared to the known synthetic antidepressants of today.
5 Hyperforin as Activator of Ion Channels
The impact of hyperforin on the modulation of ion channels became obvious by the
work of Chatterjee et al. (1999). In acutely dissociated hippocampal neurons, they
found a complex modulation of ion channels by the application of hyperforin
(Chatterjee et al. 1999). Hyperforin induced a current, which they pharmacologi-
cally characterized by the determination of an EC50 value of 9.1 M, the hyperforin
concentration needed for half-maximal activation (Chatterjee et al. 1999). On the
other hand, steady-state conductances of a variety of ion channels were blocked by
the application of hyperforin. Ligand-induced conductances like AMPA-, NMDA-,
and GABA-currents were blocked by hyperforin as well as voltage-dependent K+,
Na+, and Ca2+ channels (Chatterjee et al. 1999; Fisunov et al. 2000). While the
inhibition of K+ or Na+ channels was left without further characterization, the
authors showed inhibition of N-type and P-type voltage-dependent Ca2+ channels
at low micromolar concentrations (Chatterjee et al. 1999; Fisunov et al. 2000;
Krishtal et al. 2001).
The activation of a sodium-permeable ion channel leading to intracellular
sodium increase by hyperforin was shown by Singer et al. reporting an EC50 of
2 M for this process and an inhibition of serotonin uptake in human platelets as a
model system for serotonin transport in neurons (Singer et al. 1999). Based on the
sodium entry measured and the inhibition of the uptake of GABA and L-glutamate
in synaptosomes by monensin and ouabain, an inhibitor of the Na+/K+-ATPase, the
modulation of the Na+/K+-ATPase by hyperforin has been discussed (Wonnemann
et al. 2000). However the inability to substantiate the interaction of hyperforin with
the Na+/K+-ATPase resulted in the falsification of this hypothesis (Wonnemann
et al. 2000).
Different other features of hyperforin-induced currents were stepwise
complemented by imaging and pharmacological approaches. The imaging
approaches using SBFI-AM demonstrated hyperforin-mediated sodium influx
(EC50 0.72 M), while the use of fura-2/AM allowed the recording of increases
in intracellular Ca2+ concentrations (EC50 1.16 M) (Treiber et al. 2005). The
permeability of the hyperforin target in PC12 cells for sodium and calcium as
well as other divalent cations argued for a nonselective cation channel to be the
target of hyperforin. Several inhibitors of nonselective cation channels such as the
organic inhibitors SK&F96365 and LOE 908 as well as the anorganic inhibitors
lanthanum and gadolinium ions validated this hypothesis and pointed to the partic-
ipation of a TRP channel in mediating hyperforin-induced currents (Treiber
et al. 2005; Leuner et al. 2007).
TRP channels comprise a superfamily of 28 mammalian members (27 in
humans) subdivided in 6 subfamilies the TRPC, TRPV, TRPM, TRPP, TRPML,
and TRPA subfamilies (Harteneck et al. 2000; Clapham et al. 2001; Montell
et al. 2002; Flockerzi 2007; Wu et al. 2010; Nilius and Szallasi 2014). Several
TRP channel inhibitors such as 2-APB, ACA (Harteneck et al. 2007; Harteneck and
Gollasch 2011), and ruthenium red allowed to narrow down the spectrum of
putative candidates to members of the TRPC family and finally to TRPC6 (Leuner
et al. 2007). The relevance for TRPC6 in hyperforin-induced cation entry was
validated in analyses of recombinantly expressed TRPC6 by imaging and electro-
physiological methods (whole-cell, inside-out, and outside-out configurations)
showing that TRPC6 is a target of hyperforin (Leuner et al. 2007, 2010; Muller
et al. 2008). Approaches using siRNA knockdown and the expression of a selective
dominant-negative TRPC6 mutant enabled to unravel the physiological role of
TRPC6 as hyperforin target in the neuronal-like PC12 cell line. The knockdown
of TRPC6 resulted in reduced growth of neurites and thereby argued for a role of
TRPC6 in neuronal plasticity and differentiation processes (Leuner et al. 2007).
The relevance of hyperforin-triggered TRPC6 activation in differentiation pro-
cesses was validated in the quite different cell model of keratinocytes, which
express TRPC6. During differentiation of keratinocytes, the expression pattern of
keratins changes. The change as measure of keratinocytes differentiation was
modulated by hyperforin in a TRPC6-dependent manner in these experiments
(Muller et al. 2008).
The current concept for TRPC6-dependent antidepressive activity of hyperforin
is visualized in Fig. 4. TRPC6 localized in the presynaptic membranes mediates
sodium and calcium entry upon activation by hyperforin. The sodium entry medi-
ated by TRPC6 impairs the sodium gradient across the plasma membrane and
thereby indirectly reduces the driving force for the serotoninsodium symporter,
while calcium entry via calcium-dependent kinase IV as well as the activation of the
MAPK and PI3K pathways is involved in differentiation processes modulating
neuronal plasticity (Heiser et al. 2013).
TRPC1TRPC7 channels (TRPC2 is a pseudogene in humans) are permeable for
mono- as well as divalent cations such as Ca2+, Ba2+, Sr2+, Zn2+, or Mn2+ and are
integrated in G protein-coupled receptor-mediated signaling cascades leading to
phospholipase C activation. Phospholipase C isoforms mediate the breakdown of
phosphatidyl-3,4-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate and
diacylglycerol (DAG), intracellular signaling molecules activating downstream
processes. Inositol-1,4,5-trisphosphate acts as a second messenger on inositol-
1,4,5-trisphosphate receptors mediating calcium release from the intracellular
endoplasmic storage compartment. Diacylglycerols, 1,2-fatty acid esters of glyc-
erol, stimulate protein kinase C on one hand and on the other directly activate
TRPC3, TRPC6, and TRPC7 channels (Hofmann et al. 1999). Stearoyl-
arachidonoyl-sn-glycerol (SAG, Fig. 1e) represents a naturally occurring
diacylglycerol, whereas in most of the in vitro experiments, the oleoyl-acetyl-sn-
glycerol (OAG) is used as diacylglycerol analogue due to better solubility.
The expression of TRPC channels in the developing as well as in the adult brain
makes TRPC channels to interesting targets in the control of growth cone guidance,
neurotransmitter release, regulation of synaptic plasticity, and brain development
(Strubing et al. 2001; Fusco et al. 2004; von Bohlen Und Halbach et al. 2005;
Chung et al. 2006; Faber et al. 2006; McGurk et al. 2011). With respect to the
pharmacological use of hyperforin as antidepressant, the activation of TRPC3 and
TRPC6 channels by brain-derived neurotrophic factor via tyrosine kinase receptors
P P
CREB
CREB
POL POL
CREB
CaMKII
2+
Ca 2+
5-HT 2+ Ca
Ca
2+ +
Na +Ca , Na
+
5-HT Na
+
Na
5-HT TRPC6
2+ +
SERT Ca , Na
SLC6A4
5-HT
+
Na Hyperforin
synthetic
antidepressants
Fig. 4 Illustration of hyperforin-triggered, TRPC6-dependent indirect inhibition of neurotrans-
mitter reuptake. TRPC6 localized in the presynaptic membranes mediates sodium and calcium
entry upon activation by hyperforin. The sodium entry mediated by TRPC6 impairs the sodium
gradient across the plasma membrane and thereby indirectly reduces the driving force for the
serotoninsodium symporter (SERT/SLC6A4: solute carrier family 6A4 transporter), while cal-
cium entry via calcium/calmodulin-dependent kinase II (CaMKII) is involved in differentiation
processes modulating neuronal plasticity via phosphorylation of cAMP response element-binding
protein (CREB). 5-HT serotonin, CREB P phosphorylated form of the cAMP response element-
binding protein, POL DNA-dependent RNA polymerase
(TrK), e.g., TrKB and phospholipase C, is of particular interest (Li et al. 2005,
2012; Amaral and Pozzo-Miller 2007; Sossin and Barker 2007). Reduced BDNF
levels have been observed in the hippocampus of patients with major depression,
and reduced levels of BDNF are considered to be an important trigger in the
pathophysiology of major depression. The antidepressant treatment results in
increased BDNF levels (Duman and Aghajanian 2012; Duric and Duman 2013;
Duman and Duman 2015).
Interested in neuronal zinc homeostasis, Gibon et al. studied TRPC6-mediated
currents in HEK293 cells and in cortical neurons (Gibon et al. 2011b; Bouron and
Oberwinkler 2014). Analyses of recombinantly expressed TRPC6 in HEK293 cells
elicited a pronounced uptake of Zn2+ in the presence of OAG or hyperforin. In
primary cortical neurons, hyperforin resulted in an increase of intracellular Ca2+

and Zn2+ concentrations which were comparable to the effects of OAG. In whole
patch-clamp recordings, hyperforin-induced inward current was recorded. In the
recombinant system, the current density measured at 60 mV was around 8 pA/pF,
whereas in cortical neurons the values were around fourfold reduced (2 pA/pF),
arguing for a fourth less channel density in cortical neurons. With respect to the
discussion whether or not TRPC6 represents the hyperforin target, it is important to
emphasize that currents were undetectable in the absence of extracellular Ca2+ and
Zn2+ ions. The hyperforin- and OAG-mediated Ca2+ increases were blocked by
SK&F96365 and Gd3+ (Tu et al. 2009).
Further evidence for the activation of TRPC6 by hyperforin comes from a study
on the impact of platelet-activating factor on lung endothelial cells and endothelial
permeability (Samapati et al. 2012). Besides showing increased recruitment of
TRPC6 channels to caveolae upon activation of the acidic sphingomyelinase by
platelet-activating factor, the authors measured hyperforin-induced, TRPC6-like
currents in pulmonary microvascular endothelial cells. The currentvoltage rela-
tionship of the currents recorded showed an inwardly rectifying current that
reversed nearly +20 mV (Samapati et al. 2012).
Two recent publications validated the impact of hyperforin-mediated activation
of TRPC6 by their analyses of hyperforin-induced effects in TRPC6 knockout mice
(Ding et al. 2011; Chen et al. 2013). Ding et al. focused on reactive oxygen species-
mediated TRPC6 protein activation in vascular myocytes (Ding et al. 2011), a
mechanism controlling vasoconstrictor-regulated vascular tone. In freshly isolated
and endothelium-denuded thoracic aortas, hyperforin induced a dose- and time-
dependent constriction in wild-type mice, which was absent in the presence of
vehicle and by the use of preparations obtained from TRPC6 knockout mice.
Consistent with the ex vivo study, hyperforin stimulated a robust Ca2+ entry in
the aortic vascular smooth muscle cells isolated from wild-type mice, but not in
cells isolated from TRPC6 knockout mice. Chen et al. tested the involvement of
TRPC6 channels in histamine-mediated microvascular endothelial leakage. In
wild-type mice, hyperforin strongly increases vascular micro leakage being mark-
edly attenuated in TRPC6 knockout mice (Chen et al. 2013).
6 The Role of TRPC6 Channels for Neuronal Effects

of Hyperforin: Depression, Epilepsy, Autism, Ischemia,
Cognition, and Alzheimers Disease
The participation of TRPC6 channels is discussed for several neuronal disorders

such as epilepsy, ischemia, autism, or depression (Nilius 2012, 2015; Vennekens
et al. 2012; Harteneck and Leuner 2014). In the pilocarpine-induced status
epilepticus in rats, TRPC6 channel expression was reduced in CA1 and CA3
pyramidal neurons as well as dentate granule cells. The activation of TRPC6
channels by hyperforin protected against these neuronal damages following the

status epilepticus (Kim et al. 2013) and prevented the reduction of TRPC6 protein
expression in these areas. Several lines of evidence point to an involvement of
reduced expression and activation of TRPC6 channels in ischemia (Du et al. 2010;
Lin 2013) and a protective effect of TRPC6 overexpression or activation using
hyperforin (Du et al. 2010; Lin et al. 2013). When hyperforin was applied directly
after middle cerebral artery occlusion, it reduced infarct volumes, improved neu-
rological scores after 24 h, and enhanced the expression of TRPC6 and the
phosphorylation of CREB in the ipsilateral cortex via MAPK pathway as well as
CAMKIV phosphorylation (Lin et al. 2013).
A de novo balanced translocation disruption of TRPC6 has been reported in a
non-syndromic autistic patient (Griesi-Oliveira et al. 2014). TRPC6 reduction or
haploinsufficiency leads to altered neuronal development, morphology, and func-
tion. Importantly, the phenotype could be rescued by hyperforin (0.3 M). The role
of TRPC6 in a syndromic autism spectrum disorder, the Rett syndrome, is also
discussed (Li et al. 2012).
While the molecular understanding of the role of TRPC6 and hyperforin is less
understood in epilepsy, ischemia, and autism, several reports allow the assembly of
the molecular mechanism providing the basis for the antidepressive therapy.
Changes in neurotrophic factor levels such as the brain-derived neurotrophic factor
(BDNF) are considered to be critically involved in the pathogenesis of depression
(Duman and Aghajanian 2012; Duric and Duman 2013; Duman and Duman 2015).
Disruption of hippocampal function contributes to several aspects and symptoms of
depression, such as deficits in concentration. Indeed, brain imaging studies in
depressed patients revealed decreased hippocampal volume, and postmortem
brain studies showed moderate apoptosis and atrophy in the CA1 region and the
dentate gyrus (McKinnon et al. 2009; Travis et al. 2014). Classical antidepressants,
such as selective serotonin reuptake inhibitors (SSRI), may counteract these
changes by increasing neuronal plasticity through enhanced serotonin and norepi-
nephrine levels paralleled by increased expression of the neurotrophic factor
BDNF. Importantly, neuroplastic changes mediated by neurotrophic factors rang-
ing from neurogenesis to synaptogenesis and associated morphological changes of
dendritic spines are considered as the final biological action of antidepressants on
the reversal of clinical symptoms (Duman and Aghajanian 2012; Duric and Duman
2013). TRPC channels especially TRPC3 and TRPC6 channels are downstream
effectors of neurotrophin signaling in CNS neurons, which regulate BDNF-induced
increase in quantal neuronal transmitter release, enhance synapse density, and
improve spine morphology as well as dendritic lengths (Li et al. 2005; Amaral
and Pozzo-Miller 2007; Sossin and Barker 2007; Fortin et al. 2012).
In hippocampal preparations, hyperforin acts as a BDNF or NGF mimetic and
modulates neuronal plasticity via the activation of TRPC6 channels (Leuner
et al. 2007, 2013; Heiser et al. 2013). Hyperforin (0.3 M) induces neurite out-
growth via TRPC6 channels and modulates spine morphology in CA1 as well as
CA3 pyramidal neurons in organotypic hippocampal slices. These effects were
blocked by TRPC6-specific shRNA-mediated knockdown or an expression of a
pore-dead mutant of TRPC6. The effect is mediated by a strong hyperforin-

dependent phosphorylation of the transcription factor CREB, one of the most
important targets of the neurotrophic factors BDNF and NGF in different neuronal
cells such as hippocampal neurons as well as PC12 cells (Leuner et al. 2007; Heiser
et al. 2013). Hyperforin increased the quantal neurotransmitter release in CA1
neurons and thereby elicited enhanced frequency of miniature excitatory potentials
in CA3 neurons. These results were emphasized by studies of Gibon et al. treating
mice for 4 weeks with 4 mg/kg BW hyperforin and analyzing CREB phosphoryla-
tion and TrkB expression in postmortem tissues (Gibon et al. 2012). Hyperforin
elicits an increase in CREB protein levels as well as the phosphorylation of CREB
in the cortex. The physiological effects are paralleled by hyperforin-dependent
increases in TRPC6 as well as of TrkB expression in the cortex. In the hippocam-
pus, comparable features were detectable without showing hyperforin-dependent
neurogenesis (Gibon et al. 2012).
With respect to the discussion of 2,4-diacylphloroglucinol structures as leads in
the development of new antidepressants, it is interesting to note that the Hyp
derivative Hyp 2 mimicked BDNF function in CA1 and CA3 pyramidal neurons
of the rat hippocampus (Leuner et al. 2013). It increased the quantal neurotrans-
mitter release in CA1 neurons and thereby elicited enhanced frequency of miniature
excitatory potentials in CA3 neurons. Similar effects were observed after applica-
tion of St. Johns wort extract in the nucleus of the solitary tract which is located in
the dorsal medulla (Vance et al. 2014). In voltage-clamp recordings of fura-2-
loaded CA3 pyramidal neurons in acute hippocampal slices, the application of Hyp
derivatives induced a slowly developing inward current that was accompanied by a
similar slow calcium elevation. The effects were significantly reduced by the
unselective TRP channel blocker La3+ (Leuner et al. 2013).
Several recent publications point to an improvement of cognition in different
animals models for Alzheimers disease and a reduction of amyloid beta plaques,
one of the hallmarks of Alzheimers disease by St. Johns wort extract, hyperforin,
or tetrahyperforin (Dinamarca et al. 2006; Cerpa et al. 2010; Griffith et al. 2010;
Carvajal and Inestrosa 2011; Inestrosa et al. 2011; Abbott et al. 2013; Carvajal
et al. 2013; Brenn et al. 2014; Montecinos-Oliva et al. 2014). Tetrahyperforin was
also shown to improve adult neurogenesis in wild-type as well as a transgenic
Azheimers animal model (Abbott et al. 2013) as well as a reduction of
synaptotoxicity (Montecinos-Oliva et al. 2015). Recently, Montecinos-Oliva dem-
onstrated that TRPC3/6/7 channels might also be the molecular target of tetrahydro-
hyperforin (Montecinos-Oliva et al. 2014, 2015). They used an electrophysiological
as well as a behavioral approach. The effect of tetrahydrohyperforin was first
investigated on A-induced reduction of field excitatory postsynaptic potential
under basal conditions and after long-term potentiation. Coadministration of
tetrahydrohyperforin significantly recovered these defects. In addition, under the
co-application of SK&F9365, the protective effect of tetrahydrohyperforin was lost.
Next, cognition, e.g., performance in the Morris water maze test, was investigated.
Ten-week treatment with tetrahydrohyperforin (6 mg/kg BW) led to low escape
latencies. However, the treatment with SK&F96365 itself resulted in higher escape
latencies in the group also treated with tetrahydrohyperforin. The role of TRPC6
channels for Alzheimers disease pathology is also supported by Lessard
et al. (2005) demonstrating that Alzheimers disease-linked presenilin 2 variants
reduce agonist-induced TRPC6 activation when co-expressed in HEK293 cells.
7 Summary
The data analyzing the molecular mechanism of St. Johns wort antidepressive
action are divers and controversial. Despite older data, there is growing evidence
that hyperforin represents the pharmacological principle of St. Johns wort and its
extracts. The transcriptional activation via pregnane X receptor is mainly seen as
the major drawback in therapy reducing steady-state levels of simultaneously
applied remedies. The increase in CYP enzyme might contribute to the normaliza-
tion of cortisol levels, reported to be increased in depression; however the relevance
has not been studied so far. An obvious discrepancy in the studies analyzing the
activation mechanism of hyperforin results from electrophysiological data provid-
ing different currentvoltage relationships and current characteristics reported in
the absence of divalent cations (Leuner et al. 2007; Muller et al. 2008; Gibon
et al. 2010, 2011a, b; Tu et al. 2010; Sell et al. 2014). The discrepancy is linked to
the question whether or not the activity of hyperforin is dependent on TRPC6. A
recent report revived data on the protonophoric character of hyperforin, in contrast
to many showing the dependence on TRPC6 of hyperforin-stimulated currents.
Currently, the basis for the discrepancies in the shape of the currentvoltage
relationships is mysterious as well as the dependence of hyperforin-triggered
currents on divalent cation. It is obvious that the report claiming protonophoric
activity of hyperforin is able to show hyperforin-induced currents in the absence of
divalent cations in particular calcium, whereas the report providing data on cal-
cium- and zinc-based currents is unable to record currents in the absence of the
divalent cation. It will be interesting to understand the origin of these discrepancies
in the future.
With respect to drug developments, the view of hyperforin as protonophore-
mediating currents in a protein-independent manner is problematic. The side effects
reported for protonophoric and ionophoric compounds are serious. The problematic
is becoming much more evident in the light of the concentrations needed. TRPC6-
dependent modulation of synaptic plasticity by hyperforin is induced at concentra-
tions of 100300 nM, whereas the effect on the intracellular pH changes was
induced in the presence of much higher hyperforin levels. The view of TRPC6-
dependent, hyperforin-induced antidepressive activity provides a more optimistic
view as already lead structures have been described integrating the antidepressant
activity in animal models with simultaneously eliminating the therapy-limiting
pregnane X receptor-dependent side effects.
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DOI: 10.1007/112_2015_26
Published online: 16 September 2015
The Piezo Mechanosensitive Ion Channels:

May the Force Be with You!
Eric Honore, Joana Raquel Martins, David Penton, Amanda Patel,

and Sophie Demolombe
Abstract Piezo1 and Piezo2 are critically required for nonselective cationic
mechanosensitive channels in mammalian cells. Within the last 5 years, tremen-
dous progress has been made in understanding the function of Piezo1/2 in
embryonic development, physiology, and associated disease states. A recent break-
through was the discovery of a chemical opener for Piezo1, indicating that
mechanosensitive ion channels can be opened independently of mechanical stress.
We will review these new exciting findings, which might pave the road for the
identification of novel therapeutic strategies.
Keywords Electrophysiology Mechanosensation Mechanotransduction Piezo1

Piezo2 Xerocytosis
Contents
1 Piezo1/2 Are Essential Components of Distinct Mechanically Activated Cationic
Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2 Piezo1/2 Are Pore-Forming Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3 Mapping the Ionic Pore in Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4 The Small Synthetic Molecule Yoda1 Is an Opener of Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5 Upregulation of Piezo1 by Phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6 Dehydrated Stomatocytosis (Xerocytosis) Is Caused by Gain-of-Function Mutations
in Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
7 A Possible Link Between Piezo1 and Sickle Cell Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
E. Honore, J.R. Martins, D. Penton, A. Patel, and S. Demolombe (*)

Institut de Pharmacologie Moleculaire et Cellulaire, LabEx ICST, UMR 7275 CNRS,
Universite de Nice Sophia Antipolis, Valbonne, France
e-mail: demolombe@ipmc.cnrs.fr
26 E. Honore et al.
8 Role of Endothelial Piezo1 in the Vascular Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

9 Role of Piezo1 in Bladder Mechanosensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
10 Gain-of-Function Mutations in Piezo2 Cause Distal Arthrogryposis . . . . . . . . . . . . . . . . . . . . . 33
11 Piezo2 and the Sense of Touch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
12 Tuning Piezo2 Influences Touch Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
13 Drosophila Piezo Is Involved in Nociception, Although Not in Light Touch
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
14 Regulation of Piezo1 in Renal Tubular Epithelial Cells by TRPP2 . . . . . . . . . . . . . . . . . . . . . . . 36
15 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Mechanotransduction concerns the molecular and cellular mechanisms responsible

for the conversion of mechanical stimuli into an electrochemical response. Special-
ized cells, such as cutaneous mechanoreceptors that are exquisitely sensitive to a
variety of tactile stimuli, are responsible for mechanosensory transduction. How-
ever, even nonspecialized cells, including endothelial cells that can be activated by
very subtle changes in blood flow, are also intrinsically mechanosensitive.
Opening of nonselective cationic stretch-activated channels (SACs) mediates cell
depolarization and promotes an increase in intracellular calcium. In a recent tour de
force, Patapoutian and colleagues demonstrated that Piezo1 and Piezo2 are criti-
cally required for SAC activity in mammalian cells. Within the space of 5 years,
tremendous progress has been made in understanding the function of Piezo/SACs in
embryonic development, physiology, and associated disease states. Another recent
breakthrough was the discovery of a chemical opener for Piezo1, indicating that
SACs can be opened independently of mechanical stress. We will review these
recent exciting findings about Piezo1 and Piezo2 which will pave the road for the
identification of novel therapeutic strategies for the treatment of associated patho-
logies, including inherited xerocytosis and possibly sickle cell disease.
How cells respond to a variety of mechanical stimuli has been the topic of
intensive research over the years (Gillespie and Walker 2001; Sukharev and Corey
2004; Kung 2005; Chalfie 2009; Delmas et al. 2011; Haswell et al. 2011; Nilius and
Honore 2012; Wood and Eijkelkamp 2012; Booth 2014). Although important
progress was made with the early identification of the MscL and MscS channels
in bacteria, DEG/ENaC channels in the nematode, and TREK/TRAAK K2P chan-
nels in human cells, the molecular basis for mammalian nonselective cationic SACs
was only discovered 5 years ago (Sukharev et al. 1994; Patel et al. 1998; Miller
et al. 2003; OHagan et al. 2005; Coste et al. 2010). Piezo1 and Piezo2 play a major
role in sensing mechanical inputs, both in specialized mechanosensory cells and
nonspecialized cells. We will summarize the major discoveries concerning this new
class of mammalian mechanosensitive ion channels, with a particular emphasis on
their role in physiology, as well as in various disease states.
The Piezo Mechanosensitive Ion Channels: May the Force Be with You! 27
1 Piezo1/2 Are Essential Components of Distinct

Mechanically Activated Cationic Channels
Piezo1 was identified using a siRNA knockdown screening in the N2A cell line that
is characterized by a particularly high endogenous SAC activity (Coste et al. 2010).
A second protein Piezo2 was subsequently discovered by sequence homology
(Coste et al. 2010). Piezos are already present in paramecium, protozoan parasites,
chordates, plants, and invertebrates, although no Piezo homologue is found in
bacteria or yeast (Coste et al. 2010). The predicted Piezo proteins contain between
2,100 and 4,700 amino acids, depending on the species. In the adult mouse, the
strongest expression of Piezo1/2 was found in the lung and bladder, while expres-
sion is low in the heart and absent in the brain (Coste et al. 2010). Of note, Piezo2 is
also abundantly expressed in DRG neurons, unlike Piezo1 (Coste et al. 2010;
Ranade et al. 2014b). Interestingly, Piezo1 is also expressed in brain-derived
human neural stem/progenitor cells, and its activation was proposed to be involved
in neurogenesis and enhanced astrogenesis (Pathak et al. 2014).
Plasma membrane expression of tagged Piezo1 was reported in various cell
types (Coste et al. 2010; Peyronnet et al. 2013). Moreover, nonselective cationic
SAC activity (with a reversal near 0 mV) was observed upon heterologous expres-
sion of Piezo1 or Piezo2, with a single-channel conductance of about 2535 pS and
activation in response to mechanical stress, including cell poking, membrane
stretch, substrate deflexion, or fluid flow (Coste et al. 2010; Poole et al. 2014;
Ranade et al. 2014a, b). Sodium, potassium, calcium, and magnesium all permeate
Piezo1 with a slight preference for calcium (Coste et al. 2010; Gnanasambandam
et al. 2015). Piezo1 is inhibited by ruthenium red and Gd3+ (despite the low
specificity of these agents for SACs), as well as the spider peptide GsTMx4, a
more specific inhibitor of mechanosensitive cationic channels (Suchyna et al. 2000;
Bae et al. 2011). GsMTx4 produces a 30 mmHg rightward shift in the pressure-
gating curve of Piezo1, thus acting as a gating modifier (Bae et al. 2011). Both Piezo
isoforms show pronounced inactivation, with a relatively faster kinetics of inacti-
vation for Piezo2. Interestingly, protonation of human Piezo1 stabilizes inacti-
vation, thus inhibiting channel activity (Bae et al. 2015). Of note, Drosophila Piezo
(dPiezo) isoform shows a much smaller single-channel conductance of about 3 pS
and is in addition resistant to inhibition by ruthenium red (Coste et al. 2012).
2 Piezo1/2 Are Pore-Forming Subunits
Using bilayer reconstitution experiments, it was demonstrated that purified Piezo

proteins are pore-forming subunits (Coste et al. 2012). Of note, Piezo1 channels
were constitutively active upon reconstitution in asymmetrical bilayers (droplet
interface lipid bilayers as well as in proteoliposomes), suggesting that residual
tension in the bilayer might be sufficient to gate open Piezo1 (Coste et al. 2012).
28 E. Honore et al.
Using total internal reflection fluorescence (TIRF) microscopy to measure discrete

photobleaching steps of green fluorescent protein (GFP)-mPiezo1 fusion proteins, it
was estimated that mouse Piezo1 (mPiezo1) proteins would assemble preferentially
as tetramers (Coste et al. 2012). In line with this finding, purified glutathione
S-transferase (GST)-mPiezo1 fusion protein complex has a molecular weight of
about 1.2 million daltons, four times the molecular weight of a single GST-mPiezo1
polypeptide (318 kDa) (Coste et al. 2012). Thus, Piezos are thought to assemble as
tetramers (120160 predicted transmembrane segments in total) and represent one
of the largest plasma membrane ion channel complexes identified so far (Coste
et al. 2012). A functional synergy between native Piezo1 and Piezo2 was reported
in articular chondrocytes and upon exogenous expression in N2A cells (Lee
et al. 2014). However, whether heteromultimerization between Piezo1 and Piezo2
subunits may occur has not yet been demonstrated.
Exposing cells to the actin-disrupting reagent cytochalasin D increased the
frequency of Piezo1 openings in cell-attached patches, probably by reducing
mechanoprotection (Gnanasambandam et al. 2015). Thus, Piezo1 is a pore-forming
channel subunit that is mechanosensitive but can be influenced by cytoskeletal
elements (Sharif Naeini et al. 2009; Peyronnet et al. 2013; Gnanasambandam
et al. 2015).
3 Mapping the Ionic Pore in Piezo1
Through the combination of bioinformatical analysis, immunostaining of

Myc-labeled putative loop regions, and determination of intracellular phosphory-
lation sites by mass spectrometry, a model predicting 38 transmembrane domains
was recently proposed for mPiezo1 (Fig. 1A) (Coste et al. 2015). Moreover,
chimeras between mPiezo1 and dPiezo suggest that the C-terminal region (starting
at amino acid 1974), comprising the last two transmembrane (TM) domains and the
neighboring hydrophobic region, is responsible for the pore properties of Piezos
(Fig. 1A, B) (Coste et al. 2015). Moreover, alignment of Piezo sequences from
various protozoan parasites identified a conserved sequence in this region that
might be involved in channel pore properties (Prole and Taylor 2013). This short
stretch of residues includes the PFEW motif which is conserved among species
(Fig. 1B) (Prole and Taylor 2013; Coste et al. 2015). In line with this prediction, it
was shown that the E2133A mutation reduces Piezo1 single-channel conductance
by about half (Coste et al. 2015). Similarly, the E2133K mutation dramatically
decreased single-channel conductance, altered ionic selectivity, and reduced block
by extracellular ruthenium red (Coste et al. 2015). Remarkably, introducing a
positive charge at position E2133 decreased mPiezo1 preference for cations over
anions, while the E2133D mutant had the opposite effect. Since the mutation
E2133A did not prevent ruthenium red block (unlike E2133K), it is unlikely that
it comprises the ruthenium red-binding site (Coste et al. 2015). Nevertheless, the
E2133K mutation is predicted to affect the charge in the vicinity of a putative
A mPiezo1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
LNLFLFQGFRLVPFLVELRAVMDWVWTDTTLSLSNWMCVEDIYANIFIIKC
2133
C
M 2225R R2456H
Hydrophobic T2127M
5 hPiezo1
R1358P A2020T E2496ELE
Hydropathicity scale
Xerocytosis mutations
2.5
2.5
5
Hydrophilic
0 250 500 750 1000 1250 1500 1750 2000 2250 2500
Fig. 1 Hypothetical transmembrane topology of mPiezo1. (A) Cartoon showing the proposed
topological structure of mPiezo1, as predicted by bioinformatical analysis. The conserved PFEW
motif is located within a hydrophobic domain at the C-terminal region. (B) Putative structural
models of the C-terminal domain of mPiezo1 comprising the PFEW motif, with green segments
representing hydrophobic stretches. The E2133 residue involved in permeation is indicated by a
red sphere. (C) Hydropathicity plot of hPiezo1 with gain-of-function mutations associated with
xerocytosis (indicated in red). The mutant R2456H (squared in red) produces the most dramatic
impairment of inactivation. Peaks with scores greater than 1.8 on the hydrophobicity scale indicate
possible transmembrane segments. Adapted with permission from Coste et al. (2015)
ruthenium red-binding site that remains to be identified. Importantly, E2133

mutants did not affect pressure sensitivity, indicating that E2133 is involved in
permeation but not mechanosensitivity (Coste et al. 2015). A similar role for E2416
was found in Piezo2, demonstrating that this conserved glutamate residue clearly
plays a major role in the pore properties of Piezos (Coste et al. 2015). Notably,
mutation of the P and W conserved residues in the PFEW motif killed channel
30 E. Honore et al.
activity, further indicating that this region plays a key role in pore properties (Coste
et al. 2015). Altogether these findings indicate that E2133 is either within or at least
in close proximity to the Piezo1 pore domain. However, since this conserved
residue is also present in dPiezo (E2091), the major difference in pore properties
between mPiezo1 and dPiezo has to be explained by other differences present in the
C-terminal domain that remain to be identified.
4 The Small Synthetic Molecule Yoda1 Is an Opener

of Piezo1
In another remarkable effort, Patapoutian and colleagues screened over three

million synthetic small molecules using a fluorescence cellular assay to monitor
calcium influx through Piezo1 and identified a single compound called Yoda1 that
selectively opens Piezo1 but not Piezo2 (Fig. 2) (Syeda et al. 2015). Yoda1, at the
micromolar range, shifted the pressure effect curve of Piezo1 toward lower pressure
values and markedly slowed down Piezo1 inactivation (Syeda et al. 2015).
Yoda1
Xerocytosis
Piezo1 knock-out
Force Sickle cell
disease
Piezo1
H2O H2O
Ca2+
K+
K+
H2O
KCa3.1 KCa3.1
open closed
RBC shrinkage RBC overhydrated
Fig. 2 The opening of Piezo1 shrinks red blood cells. The opening of Piezo1 by the agonist Yoda1
or by mechanical stress promotes calcium influx that stimulates the opening of the Gardos channel
KCa3.1 in RBCs (left panel). The resulting K+ efflux leads to water loss and RBC shrinkage. Gain-
of-function mutations of Piezo1 slowing down inactivation similarly result in dehydration of
RBCs, causing xerocytosis. Inhibition of Piezo1 by antagonists might become valuable for the
treatment of the sickle cell disease, when Piezo1 activity in RBCs appears to be enhanced. By
contrast, red blood cells from Piezo1 knockout mice (right panel) are overhydrated since water is
retained because of low intracellular calcium and closing of the Gardos channels
Importantly, even in the absence of mechanical stimulation, the addition of Yoda1

increased Piezo1 channel activity. Yoda1 comprises two chlorines and a thioether
group, which are both critically required for Piezo1 opening (Fig. 2) (Syeda
et al. 2015). Remarkably, Yoda1 was also able to open Piezo1 after reconstitution
into artificial bilayers. Of note, in a symmetrical bilayer, Piezo1 channels were in
the closed state, unlike the ones initially shown in asymmetrical bilayers (Coste
et al. 2012; Syeda et al. 2015). Kinetic analysis revealed that Yoda1 mainly
stabilizes the open state of Piezo1 (Syeda et al. 2015). These findings suggest that
Yoda1 does not require additional proteins and may act either directly on Piezo1
and/or on the lipid bilayer. However, since Piezo2 is resistant to Yoda1, a mem-
brane effect appears to be unlikely (Syeda et al. 2015). These findings are very
significant since they suggest that a natural agonist might exist that could open
Piezo1 in the absence of any mechanical stimulation.
5 Upregulation of Piezo1 by Phosphoinositides
Unexpectedly, activation of TRPV1 channels with capsaicin either in dorsal root

ganglion neurons or in a heterologous expression system inhibited Piezo1 and
Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]
and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma mem-
brane through Ca2+-induced phospholipase C (PLC) activation (Borbiro
et al. 2015). Moreover, PI(4,5)P2 and PI(4)P applied to excised inside-out patches
prevented the rundown of Piezo1 activity. These data also suggest that inhibition of
Piezo2 channels by TRPV1 activation might contribute to the analgesic effect of
capsaicin (Borbiro et al. 2015). The upregulation of Piezos by membrane phosphoino-
sitides also indicates that endogenous lipids might act as natural Piezo openers
(as discussed above for Yoda1), as previously demonstrated for the stretch-sensitive
K2P channels TREK/TRAAK (Honore 2007).
6 Dehydrated Stomatocytosis (Xerocytosis) Is Caused

by Gain-of-Function Mutations in Piezo1
Gain-of-function (GOF) mutations in Piezo1 are associated with xerocytosis, an

autosomal dominant hemolytic anemia characterized by dehydration of erythro-
cytes (Zarychanski et al. 2012; Bae et al. 2013a, b; IAndolfo et al. 2013). Most of
the mutations found are located in the C-terminal 1/5th of the Piezo1 protein and
result in a significant slowing of the inactivation kinetics (Fig. 1C). In particular, the
R2456H mutant produced the strongest effect on the inactivation kinetics
(Albuisson et al. 2013). Thus, an increase in Piezo1 channel activity (GOF) due
32 E. Honore et al.
to a slower inactivation process is associated with red blood cell (RBC)

dehydration.
Using a Piezo1 fluorescent reporter mouse line, it was shown that Piezo1 is
expressed in both peripheral mature RBCs and developing bone marrow pro-RBCs.
A specific deletion in the hematopoietic system (Vav1-iCre) was used to decipher
the functional role of Piezo1 in RBCs. Piezo1-deficient RBCs were overhydrated,
with an increased osmotic fragility (i.e., they lyse at a higher relative tonicity),
showed an enlarged size, and tended to be retained in the spleen (Fig. 2). These
findings indicate that Piezo1 is important for RBC integrity and recirculation.
Mechanical stress induced a calcium influx which was absent in RBCs lacking
Piezo1 (Fig. 2) (Cahalan et al. 2015). Along the same line, Piezo1 is involved in
erythrocyte volume homeostasis in zebrafish, where disruption of the channel
resulted in RBC swelling/lysis (Faucherre et al. 2014).
Taking advantage of the Yoda1 agonist, it was shown that Piezo1 opening
resulted in RBC shrinkage through the downstream activation of the Gardos
channel KCa3.1 (Fig. 2) (Cahalan et al. 2015). It is interesting to note that RBCs
lacking KCa3.1 have also been shown to be overhydrated (Grgic et al. 2009).
Remarkably, Yoda1 decreased the osmotic fragility of WT but not Piezo1-deficient
RBCs. This effect could be prevented by inhibition of KCa3.1 with the antagonist
TRAM-34 or by deletion of Piezo1 in RBCs (Cahalan et al. 2015). Thus, Piezo1
opening leads to RBC dehydration through activation of KCa3.1, efflux of K+, and
consequent water loss (Fig. 2).
7 A Possible Link Between Piezo1 and Sickle Cell Disease
Previous work had suggested that activation of stretch-activated ion channels

contributed to the cationic conductance (Psickle) occurring in sickle cell disease
(drepanocytosis), where RBCs display an abnormal rigid sickle-like shape under
certain circumstances such as hypoxia (Fig. 2) (Vandorpe et al. 2010; Ma
et al. 2012). Notably, Psickle induced by deoxygenation was inhibited by the
peptide GsMTx-4, an inhibitor of Piezo1 (Vandorpe et al. 2010). This inherited
disease is associated with frequent attacks of severe pain due to the accumulation of
abnormal sickled RBCs in capillaries. It is tempting at this stage to propose that the
opening of Piezo1 might contribute to Psickle (Fig. 2). The future generation of
mice with conditional deletion of Piezo1 together with hemoglobin carrying the
sickle cell mutations will allow further exploration of this hypothesis. If this is
indeed verified, the use of Piezo1 antagonists might become highly valuable for the
treatment of this severe genetic disease affecting millions of people, predominantly
in sub-Saharan Africa (Patel et al. 2015).
8 Role of Endothelial Piezo1 in the Vascular Development
Global knockout of Piezo1 is embryonically lethal, thus indicating an important

functional role for this mechanosensitive ion channel in early development
(Li et al. 2014; Ranade et al. 2014a). Piezo1 is expressed in the endothelium of
developing blood vessels and its genetic deletion profoundly alters vascular archi-
tecture (Li et al. 2014; Ranade et al. 2014a). Haploinsufficiency is viable, but
endothelial abnormality occurs in adult vessels (Li et al. 2014). Notably, Piezo1
confers sensitivity to shear stress and results in the downstream activation of
proteases by intracellular calcium (Li et al. 2014). Loss of Piezo1 in endothelial
cells leads to a major change in stress fiber organization and cellular orientation in
response to the polarity of the applied force, linking Piezo1 opening to the regu-
lation of vascular architecture (Li et al. 2014; Ranade et al. 2014a).
9 Role of Piezo1 in Bladder Mechanosensation
Piezo1 expression is particularly strong in the bladder and in the urothelium, both in
mice and humans (Coste et al. 2010; Miyamoto et al. 2014). The urothelium is
centrally involved in bladder mechanosensation. A Piezo1-dependent and
GsMTx4-sensitive increase in cytosolic Ca2+ concentrations in response to stretch
was demonstrated in urothelial cells (Miyamoto et al. 2014). Furthermore, this
effect was associated with the release of ATP. These findings suggest that urothelial
Piezo1 is involved in bladder mechanotransduction and might represent a putative
pharmacological target for the treatment of bladder dysfunction (Miyamoto
et al. 2014).
10 Gain-of-Function Mutations in Piezo2 Cause Distal

Arthrogryposis
Distal arthrogryposis is a rare autosomal dominant disorder characterized by cleft

palate and congenital contractures of the hands and feet (Coste et al. 2013;
McMillin et al. 2014). Two distinct Piezo2 mutations were identified in patients
with a subtype of distal arthrogryposis type 5 (DA5) characterized by generalized
autosomal dominant contractures with limited eye movements, restrictive lung
disease, and variable absence of cruciate knee ligaments (Coste et al. 2013). Both
E2727del and I802F mutations speed up the rate of recovery from inactivation,
while E2727del also slows down inactivation. Mutations in Piezo2 also cause distal
arthrogryposis type 3 (Gordon syndrome; DA3), another previously reported auto-
somal dominant disorder (McMillin et al. 2014). Although DA3 and DA5 had
traditionally been considered separate disorders, these novel findings indicate that
34 E. Honore et al.
they are etiologically related (Coste et al. 2013; McMillin et al. 2014). Thus, both
Piezo1 and Piezo2 GOF mutations are associated with inherited diseases
(Zarychanski et al. 2012; Bae et al. 2013a, b; Coste et al. 2013; IAndolfo
et al. 2013; McMillin et al. 2014).
11 Piezo2 and the Sense of Touch
Piezo2 is localized to the peripheral nerve endings of low-threshold mechano-

receptors innervating hairy as well as glabrous skin (Ranade et al. 2014b) (Fig. 3).
Remarkably, specific deletion of Piezo2 in both sensory dorsal root ganglion (DRG)
neurons and Merkel cells, using the Advil-creERT2 driver, dramatically impaired
touch sensation, although without influencing nociception (Ranade et al. 2014b). In
line with these findings, most rapidly inactivating mechanically activated currents
were lost in cultured DRG neurons lacking Piezo2. Moreover, mechanosensitivity
of an isolated nerve-skin preparation was blunted upon Piezo2 deletion (Ranade
et al. 2014b). Thus, the opening of the Piezo2 mechanosensitive ion channel in
sensory DRG afferents plays a central role in light touch sensitivity (Ranade
et al. 2014b).
Epidermal Merkel cells make synapse-like contacts with slowly adapting type
I (SAI) low-threshold afferents (Ikeda et al. 2014; Maksimovic et al. 2014; Ranade
et al. 2014b; Woo et al. 2014) (Fig. 3). The opening of Piezo2 in Merkel cells was
demonstrated to shape the mechanotransduction current of SAI neurons (Ikeda
et al. 2014; Maksimovic et al. 2014; Ranade et al. 2014b; Woo et al. 2014). Indeed,
Touch dome
Moving stimulus
Keratinocytes
Static displacement
SAI response
Merkel cells
Piezo2 IA firing
(Transduction in afferents)
Afferents
SA firing
(Transduction in Merkel cells)
Fig. 3 The Merkel-cell neurite complexes in touch domes of the skin. Tactile stimuli evoke
responses from sensory afferents innervating touch domes. A two-receptor-site model has been
proposed for type I slowly adapting firing responses (Ranade et al. 2014b; Woo et al. 2015). The
opening of Piezo2 in Merkel cells mediates sustained firing during static displacement, while the
opening of Piezo2 in neurite afferents mediate fast-adapting firing in response to moving mecha-
nical stimuli. Adapted with permission from Woo et al. (2015)
Merkel cells are essential to induce sustained neuronal activity in tactile afferents
(Fig. 3) (Ikeda et al. 2014; Maksimovic et al. 2014; Woo et al. 2014).
Thus, the opening of Piezo2 in neuronal afferents underlies the transient (fast-
adapting) response to moving mechanical stimuli, while the opening of Piezo2 in
Merkel cells mediates the sustained discharge in response to static displacement
(Fig. 3) (Maksimovic et al. 2014; Ranade et al. 2014b; Woo et al. 2014). Although
Piezo2 is a fast-inactivating SAC, the small plateau current of Piezo2 might be
sufficient to mediate the sustained activation of Merkel cells by static mechanical
stimuli, because of a high resistance of the membrane (Coste et al. 2010; Woo
et al. 2014). In the same line, Piezo2 was shown to be required for
mechanotransduction in human stem cell-derived touch receptors (Schrenk-
Siemens et al. 2015).
12 Tuning Piezo2 Influences Touch Sensitivity
Molecular-scale displacements are sufficient to gate mechanosensitive channels in

mouse touch receptors (Poole et al. 2014). Remarkably, stomatin-like protein
3 (STOML) brought down the activation threshold for Piezo1/2 currents from
~100 nm down to ~10 nm (Poole et al. 2014). Thus, STOML3 tunes Piezo channels,
allowing the detection of molecular-scale stimuli responsible for fine touch (Wetzel
et al. 2007; Poole et al. 2014).
Piezo2 current amplitude was also increased and inactivation slowed down by
stimulation of the bradykinin receptor beta 2 (BDKRB2), an effect mediated by
protein kinase A and C activation (Dubin et al. 2012). Moreover, Piezo2 was
involved in EPAC1-dependent mechanical allodynia (Eijkelkamp et al. 2013).
Thus, Piezo2 sensitization by inflammatory mediators is likely to contribute to
mechanical hyperalgesia (Dubin et al. 2012; Eijkelkamp et al. 2013).
13 Drosophila Piezo Is Involved in Nociception, Although

Not in Light Touch Sensitivity
dPiezo is widely expressed in every sensory neuron of Drosophila larvae (Kim

et al. 2012). dPiezo knockout (KO) flies were viable and fertile and did not lack
motor coordination (Kim et al. 2012). No defect in bristle mechanoreceptor poten-
tial and sensitivity to gentle innocuous touch mediated by ciliated sensory neurons
was observed upon dPiezo deletion (Kim et al. 2012). Notably, dPiezo KO larvae
showed impaired escape responses to painful mechanical stimuli. However,
responses to high-noxious-temperature stimuli which also elicit an escape behavior
were not altered in the absence of dPiezo (Kim et al. 2012). Thus, mechanical
36 E. Honore et al.
nociception in Drosophila larvae is specifically mediated by dPiezo, unlike light

touch detection.
The TRPA channel Painless was previously associated with Drosophila mechan-
ical nociception (Tracey et al. 2003). Interestingly, the double dPiezo:Painless
mutant had a defect in mechanical nociception comparable to the individual
mutants (Kim et al. 2012). Therefore, both dPiezo and Painless are likely to operate
in the same pain pathway (Kim et al. 2012). Since Painless activity is modulated by
intracellular calcium, it might be downstream of dPiezo (Nilius and Honore 2012).
According to this possible scheme, Painless would amplify the nociceptive
mechanical stimuli but would not act as a primary sensory channel activated by
mechanical stress. While light touch, proprioception, and hearing in Drosophila are
associated with TRP channels (Walker et al. 2000; Gong et al. 2004), dPiezo in
multiple dendritic neurons mediates mechanical nociception (harsh touch). Thus,
during evolution, the role of Piezo has evolved from nociception (dPiezo) in
Drosophila to light touch (mPiezo2) in the mouse (Kim et al. 2012; Maksimovic
et al. 2014; Ranade et al. 2014b; Woo et al. 2014).
14 Regulation of Piezo1 in Renal Tubular Epithelial Cells

by TRPP2
Kidney epithelial cells respond to both changes in intraluminal pressure and fluid
flow (Patel and Honore 2010; Weinbaum et al. 2010). Peristaltic pressure generated
by rhythmic papillary contractions varies between 15 and 45 mmHg, resulting in
the stretching of both apical and basolateral membranes (Jensen et al. 2007).
Intraluminal pressure can also be abnormally elevated in various kidney diseases
(Patel and Honore 2010). For instance, obstructive uropathy leads to an increase in
intratubular pressure, in excess of 60 mmHg (Wyker et al. 1981; Cachat et al. 2003;
Power et al. 2004; Quinlan et al. 2008; Rohatgi and Flores 2010). Stretching, as well
as compression, of renal epithelial cells is also common in PKD patients (Patel and
Honore 2010). Abnormal fluid accumulation in renal cysts causes the cyst wall to
stretch (Derezic and Cecuk 1982; Tanner et al. 1995; Praetorius et al. 2009).
Moreover, compression of healthy tubules by neighboring cysts results in the
upstream accumulation of urine and consequent tubular distension. Thus, the
stretch of tubular epithelial cells is relevant to both physiological and pathological
renal conditions (Patel and Honore 2010). Moreover, the apical side of tubular
epithelial cells is also continuously subjected to urine flow stimulation (i.e., shear
stress).
Nonselective SACs were recorded at the basolateral side of renal tubular epi-
thelial cells and were characterized by a lack of inactivation and a very slow
deactivation when recorded in the cell-attached patch clamp configuration
(Peyronnet et al. 2013). Piezo1 is critically required for SAC activity in mouse
renal tubular cells (Peyronnet et al. 2013). The lack of Piezo1 inactivation in renal
epithelial cells remains unexplained.
Unexpectedly, overexpression of TRPP2 (polycystin-2; PC2), or to a greater
extent its pathogenic mutant PC2-740X (which lacks interaction with polycystin-1;
PC1), impaired native SACs in renal tubular epithelial cells, as well as in arterial
myocytes (Sharif Naeini et al. 2009; Peyronnet et al. 2013). The inhibitory effect of
PC2 on native SACs could be reversed by overexpressing PC1, while it was
mimicked by Pkd1 deletion (Sharif Naeini et al. 2009; Peyronnet et al. 2013).
Moreover, PC2 inhibited exogenous Piezo1/SAC activity expressed in a variety of
cell types (Peyronnet et al. 2013). PC2 co-immunoprecipitated with Piezo1 and
deletion of its N-terminal domain prevented both this interaction and inhibition of
SAC activity. Altogether, these findings indicate that renal SACs depend on Piezo1
but are critically conditioned by the PC1/PC2 ratio. Interestingly, Piezo1 opening is
thought to control cell extrusion by tissue overcrowding in developing epithelia of
the zebrafish (Eisenhoffer et al. 2012). Thus, inhibition of Piezo1, as observed upon
Pkd1 deletion or expression of the PC2-740X mutant, might contribute to
cystogenesis by impairing cell extrusion from developing cysts (Peyronnet
et al. 2013).
These findings, together with the previously reported downstream activation of
Painless by dPiezo, indicate a possible functional interaction between TRP chan-
nels and Piezos (Kim et al. 2012; Peyronnet et al. 2013).
15 Conclusions and Perspectives
In conclusion, recent findings regarding Piezo1 and Piezo2 represent a major

breakthrough for a better understanding of molecular mechanotransduction in
both specialized and nonspecialized mammalian cells. Piezos do not appear to
need a sophisticated machinery of associated proteins to be active, as seen in
heterologous expression systems or upon reconstitution in artificial bilayers
(Coste et al. 2010). However, regulation by additional elements including
STOML3, cytoskeletal elements, and lipids, possible heteromultimerization,
as well as functional interaction with TRP channels might tune the Piezos activity
and influence their function in specific cell types (Kim et al. 2012; Peyronnet
et al. 2013; Lee et al. 2014; Poole et al. 2014; Borbiro et al. 2015). The recent
identification of the Piezo1 opener Yoda1 suggests that endogenous molecules may
similarly open Piezos, independently of mechanical stress. Among the important
questions that need to be answered, we might consider: Does tension directly open
Piezos? Where is the mechanosensor in Piezos? What mediates Piezo inactivation?
What is the 3D structure of Piezos? Are Piezos expressed in the inner ear and what
is their function? Are Piezos involved in baroreceptor function? Are Piezos impli-
cated in lung mechanotransduction, where its expression is the highest? What is the
role of Piezo1 in the myogenic control of smooth muscle cells? Is Piezo1 a putative
drug target for the treatment of sickle cell disease?
38 E. Honore et al.
The discovery of Piezos is definitely an amazing finding in the ion channel field
and more surprises are certainly to be expected in the near future.
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DOI: 10.1007/112_2015_27
Published online: 2 October 2015
Chronobiology and Pharmacologic

Modulation of the ReninAngiotensin
Aldosterone System in Dogs: What Have
We Learned?
Jonathan P. Mochel and Meindert Danhof
Abstract Congestive heart failure (CHF) is a primary cause of morbidity and

mortality with an increasing prevalence in human and canine populations. Recog-
nition of the role of reninangiotensinaldosterone system (RAAS) overactivation
in the pathophysiology of CHF has led to significant medical advances. By decreas-
ing systemic vascular resistance and angiotensin II (AII) production, angiotensin-
converting enzyme (ACE) inhibitors such as benazepril improve cardiac hemody-
namics and reduce mortality in human and dog CHF patients. Although several
experiments have pointed out that efficacy of ACE inhibitors depends on the time of
administration, little attention is paid to the optimum time of dosing of these
medications. A thorough characterization of the chronobiology of the renin cascade
has the potential to streamline the therapeutic management of RAAS-related
diseases and to help determining the optimal time of drug administration that
maximizes efficacy of ACE inhibitors, while minimizing the occurrence of adverse
effects. We have developed an integrated pharmacokineticpharmacodynamic
model that adequately captures the disposition kinetics of the paradigm drug
benazeprilat, as well as the time-varying changes of systemic reninangiotensin
aldosterone biomarkers, without and with ACE inhibition therapy. Based on these
chronobiological investigations, the optimal efficacy of ACE inhibitors is expected
with bedtime dosing. The data further show that benazepril influences the dynamics
of the reninangiotensinaldosterone cascade, resulting in a profound decrease in
J.P. Mochel (*)

Department of Pharmacology, Leiden-Academic Centre for Drug Research, 2300 Leiden,
The Netherlands
Department of Integrated Quantitative Sciences, Novartis Campus, St. Johann, 4002 Basel,
Switzerland
e-mail: jonathan.mochel@hotmail.com
M. Danhof
Department of Pharmacology, Leiden-Academic Centre for Drug Research, 2300 Leiden,
The Netherlands
44 J.P. Mochel and M. Danhof
AII and aldosterone (ALD), while increasing renin activity for about 24 h. From the
results of recent investigations in human, it is hypothesized that reduction of AII
and ALD is one of the drivers of increased survival and improved quality of life in
dogs receiving ACE inhibitors. To support and consolidate this hypothesis, addi-
tional efforts should be directed toward the collection of circulating RAAS peptides
in spontaneous cases of canine CHF. If such a link could be established, profiling of
these biomarkers could support determination of the severity of heart failure,
complement clinical and echocardiographic findings, and be used for therapeutic
drug monitoring purposes.
Keywords Chronobiology Heart failure PKPD modeling RAAS
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2 An Overview of the ReninAngiotensinAldosterone System: Past and Present . . . . . . . . . 46
2.1 A Complex and Highly Regulated Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2 ReninAngiotensinAldosterone Activation in Vascular Inflammation,
Remodeling, and Congestive Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3 Modeling and Simulation: A Basis for Optimizing the Dosing Schedule of RAAS
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4 Components of the Renin Cascade, Blood Pressure, and Urinary Electrolytes Fluctuate
with Clear Circadian Changes in Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5 The Chronobiology of Renin Activity, Blood Pressure, and Urinary Electrolytes Is
Synchronized to the Dogs Feeding Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
6 Benazepril Markedly Influences the Dynamics of the Circulating RAAS . . . . . . . . . . . . . . . . . 58
7 Conclusions and Research Perspectives in Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Abbreviations
ACE Angiotensin-converting enzyme

AI Angiotensin I
AII Angiotensin II
ALD Aldosterone
ARA Aldosterone receptor antagonists
ARB Angiotensin receptor blocker
ARC Arcuate nucleus
BP Blood pressure
CHF Congestive heart failure
CKD Chronic kidney disease
CVHD Chronic valvular heart disease
HT Hypertension
LH Lateral hypothalamus
LSD Low-sodium diet
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 45
MMVD Myxomatous mitral valve disease

NLME Nonlinear mixed effects
PB Physiologically based
PD Pharmacodynamics
PK Pharmacokinetics
RA Renin activity
RAAS Reninangiotensinaldosterone system
SCN Suprachiasmatic nucleus
UA:C Urinary aldosterone to creatinine ratio
UK,fe Potassium fractional excretion
UNa,fe Sodium fractional excretion
1 Introduction
Congestive heart failure (CHF) is a major cause of morbidity and mortality with an
increasing prevalence in human and canine populations (Guglielmini 2003; George
et al. 2014). In dogs, CHF most often develops consequent to chronic valvular heart
defect, also known as myxomatous mitral valve disease (MMVD) or chronic left
valvular heart disease (CVHD) (Borgarelli and Buchanan 2012). MMVD is char-
acterized by thickening and shortening of the atrioventricular valves and affects
about 75% of dogs over the age of 16 (Guglielmini 2003). While MMVD has been
recognized in dogs for over a century, histopathological and clinical studies have
not been able to reveal its cause or why it occurs ten times more frequently in dogs
than in humans (Borgarelli and Buchanan 2012). In humans, the majority of heart
diseases are caused by atherosclerosis, a condition which is not spontaneously
observed in dogs even under a high-cholesterol regime (Mahley et al. 1974). The
first large animals used to study heart failure were dogs, in which models of
myocardial infarction and serial microembolization of the coronary artery were
developed (Zaragoza et al. 2011). Similar to humans, the -myosin heavy chain
isoforms predominate in the dog myocardium (Hasenfuss 1998), such that the
excitation/contraction coupling in the myocardium of dogs appears to be similar
to that in the human myocardium. More importantly, the pathophysiological
scheme of renin activation, as observed in the course of CHF, is similar between
dogs and humans, which motivated the choice of this animal species in the
experimental work on the reninangiotensinaldosterone system (RAAS) and
blood pressure (BP) pioneered by Guyton, Hall, and co-workers (Cowley and
Guyton 1972; Guyton et al. 1972; McCaa et al. 1975; Young and Guyton 1977;
DeClue et al. 1978; Lohmeier et al. 1978; Hall et al. 1980, 1984; Wilczynski and
Osmond 1983). Renin release from the juxtaglomerular apparatus is a common
compensatory mechanism to the reduced cardiac output observed in symptomatic
stages of canine and human heart failure (Watkins et al. 1976; Hall 1991). Recog-
nition of the dysregulation of the RAAS in the pathophysiology of CHF has led to
significant medical advances (McMurray et al. 2012). Reduction of angiotensin II
(AII) and aldosterone (ALD) levels is paramount to prevent life-threatening com-

plications associated with myocardial fibrosis and systemic hypertension.
In recent years, the measurement of neurohormonal rhythms and the subsequent
discovery of molecular clocks have renewed scientific interest in the importance of
chronobiological concepts (Sole and Martino 2009). Cardiac and vascular tissues
display remarkable circadian variations in gene expression, metabolism, growth,
and remodeling (Martino et al. 2007). In addition, a rich body of literature has
demonstrated that components of the circulating RAAS oscillate with a circadian
periodicity in rodents (Hilfenhaus 1976) and humans (Cugini et al. 1981; Kawasaki
et al. 1990). In contrast, little is known about the periodicity of the systemic RAAS
in dogs.
One approach to increase the efficiency of pharmacotherapy lies in the admin-
istration of drugs at a time where they are most likely to be effective and/or best
tolerated. Accumulating knowledge on the 24-h biological rhythm of the renin
angiotensin cascade has the potential to optimize the therapeutic management of
RAAS-related diseases by determining the time of drug administration that would
maximize efficacy while minimizing the occurrence of adverse effects. This con-
cept, referred to as chronotherapy, has been shown to be effective in the treatment
of various chronic diseases, such as rheumatoid arthritis (Staessen et al. 1992), lung
cancer (Mazzoccoli et al. 2012), or cardiovascular diseases (Nicholls et al. 1993).
Studies in human patients have documented differences in the efficacy and duration
of action of RAAS inhibitors depending on the time of drug administration (Tata
et al. 2005; Hermida and Ayala 2009). Taken together, these findings indicate that
tuning in to bodys rhythms could have a substantial impact on the effectiveness of
drugs targeting the reninangiotensin cascade in dogs suffering from CHF.
2 An Overview of the ReninAngiotensinAldosterone

System: Past and Present
2.1 A Complex and Highly Regulated Machinery
Various authors have amply reviewed the role of the RAAS in the regulation of BP
and volume homeostasis (Ferrario and Strawn 2006; Moon 2013; Sayer and Bhat
2014). The expression of certain RAAS components even in simple organisms like
crustaceans, insects, and leeches underscores the importance of the renin cascade in
the control of cell volume and water homeostasis throughout evolution (De Mello
2014). The history of the RAAS and its discovery has recently been retraced with
great accuracy in a review paper by Tsukamoto and Kitakaze (2013).
A common description of the functioning of the systemic RAAS cascade begins
with the release of renin from granular cells of the juxtaglomerular apparatus, in
response to changes in sodium chloride concentrations, decreased renal blood flow,
and sympathetic stimulation. Many studies have established that renin secretion is
Angiotensinogen Renin
Angiotensin I
Bradykinin
Compensatory
Chymase ACE
feedback loop
Inactive
fragments
Angiotensin II
AT1 Receptor
End-Organ Damage
(heart, vasculature, kidneys)
Aldosterone
Fig. 1 Schematic view of the systemic reninangiotensin cascade. Angiotensinogen is converted

into angiotensin I (AI) by the activated form of renin. Angiotensin II (AII) is derived from AI by
enzymatic cleavage catalyzed by ACE although alternative, ACE-independent pathways can also
lead to the production of AII. AII stimulatory effect on aldosterone (ALD) release is mediated by
type 1 AII receptors (AT1R). Compensatory mechanisms (e.g., AIIrenin negative feedback loop)
contribute to the regulation of renin release. Long-term increases in AII and ALD lead to
end-organ damage and progression of heart failure. ACE inhibitors, like benazepril, act by
preventing the formation of AII and the degradation of bradykinin, which acts as a potent
vasodilator. Source: Mochel et al. (2013b). Reprinted by permission of Wiley
inversely related to renal perfusion pressure (Hackenthal et al. 1990; Bock

et al. 1992), while -adrenergic activation has been shown to stimulate renin release
in several species, including the dog (Lew and Summers 1987). Renin catalyzes the
conversion of the precursor angiotensinogen to angiotensin I (AI), which in turn is
converted to the octapeptide AII by the angiotensin-converting enzyme (ACE) as it
passes through the pulmonary capillaries (Fig. 1). Enzymes other than ACE may
contribute to the conversion of AI to AII. Chymase, cathepsin G, tonin, and other
proteases have been described as alternative pathways of AII production (Weber
et al. 1995; Roig et al. 2000). AII is a potent vasoconstrictor with additional endocrine
(e.g., ALD and arginine vasopressin secretion), neuronal (e.g., sympathetic noradren-
aline release), and renal (e.g., glomerular filtration rate modulation) actions
(Tsukamoto and Kitakaze 2013). The majority of these effects are mediated through
selective binding of AII to AT1 receptors. In most cases AT2 receptor binding elicits
vasodilation, but cardiomyocyte hypertrophy and cell death have also been reported
with stimulation of AT2 receptors (Henrion et al. 2001). Aldosterone secretion from
adrenocortical cells of the zona glomerulosa contributes to body fluid and acidobasic
Fig. 2 Classic view of reninangiotensin system cascade (blue) and recent view of renin
angiotensin system cascade (green). AP aminopeptidase, APA aminopeptidase A, APN
aminopeptidase N, CP carboxypeptidase, EP endopeptidase, ACE angiotensin-converting
enzyme, ACE2 angiotensin-converting enzyme 2, CPP carboxypeptidase P, PRCP prolyl car-
boxypeptidase, NEP neprilysin, PO prolyl oligopeptidase, Mas Ang-(1-7) Mas receptor, Mrg
Mas-related G-protein-coupled receptor, AT4 angiotensin type 4 receptor. Letters in green repre-
sent amino acids using the one-letter code. Source: Ferr~ao et al. (2014). Reprinted with permission
from Baishideng Publishing Group
homeostasis via sodium, potassium, and hydrogen ion exchanges in the distal renal
tubules and collecting ducts of Bellini (Quinn and Williams 1988). Note that the effect
of ALD on the regulation of natriuresis and BP would be quantitatively less important
than the action of AII on proximal tubular sodium reabsorption. This direct intrarenal
effect of AII further results in reduced urinary flow in the tubular segments of the
medulla, thereby increasing medullary osmolality and fluid reabsorption in the
descending loop of Henle and the collecting ducts of Bellini (Hall, 1991).
Next to the systemic (circulatory) renin cascade, several RAAS components are
also produced at the tissue level, in the heart, the vascular endothelium, or the
kidneys (Danser 1996; Danser et al. 1997). This local RAAS functions as an
autocrine or paracrine system and regulates tissue growth and repair processes.
It is now recognized that the conventional renin/ACE/AII/AT1 cascade is no
longer the sole signaling pathway of the RAAS. At least three new axes have
recently been identified in the kidneys and other tissues (Zhuo et al. 2013)
(Fig. 2). These include (i) the ACE2/ANG(1-7)/Mas receptor pathway that may
play an opposing role to the renin/ACE/AII/AT1 axis (Esteban et al. 2009), (ii) the
prorenin/PRR/MAP kinases ERK1/2 axis, which appears to be pivotal in the
development of diabetic nephropathy in rodents (Ichihara et al. 2004, 2006), and
(iii) the ANGIV/AT4/IRAP cascade, whose implication in the regulation of BP and
renal modulation remains controversial. With the discovery of these additional
pathways, the action of the RAAS has been extended beyond the regulation of
BP, sodium, and fluid homeostasis by the AT1 receptor.
2.2 ReninAngiotensinAldosterone Activation in Vascular

Inflammation, Remodeling, and Congestive Heart
Failure
Excessive activation of the RAAS plays an essential role in vascular inflammation

and remodeling (Pacurari et al. 2014). Animal and human studies have shown that
AII possesses pro-inflammatory actions by regulating the expression of cytokines
and chemokines in the kidneys, vessels, and the heart (Hahn et al. 1994; Tummala
et al. 1999). Consequently, chronic infusion of AII has been associated with
increased BP, myocardial infiltration of inflammatory cells, and cardiac fibrosis
(Qi et al. 2011). Many of these pathophysiological changes can be attributed to
mechanical injury from elevated BP and AII-induced oxidative stress (Weir 2006)
and will eventually result in end-organ damage manifested by myocardial infarc-
tion, CHF, and chronic kidney disease (CKD) (Chobanian et al. 2003). The
pro-inflammatory and pro-fibrotic effects of the RAAS are also mediated by
ALD, which further promotes insulin resistance and vascular remodeling (Martinez
2010; Cascella et al. 2010). While the relation of systemic hypertension (HT) to the
development of CKD has not been extensively documented in small animals, there
is reasonable evidence to justify extrapolation of these considerations from human
to dog patients (Lefebvre et al. 2007). Elevations of AII and ALD systemic levels
have been associated with poorer prognosis and increased mortality in human
patients with CHF. In a study by Roig et al. (2000), increased AII levels in patients
with left ventricular dysfunction was found to be an independent predictor of
increased mortality and morbidity risk. Likewise, results from Guder et al. (2007)
provide evidence that high ALD is a predictor of increased mortality risk in heart
failure patients of any cause and severity, consistent with earlier findings from
Latini et al. (2004). In dog and human species, the time of systemic RAAS
activation approximates the development of symptoms (Oyama 2009).
3 Modeling and Simulation: A Basis for Optimizing

the Dosing Schedule of RAAS Inhibitors
In veterinary drug development, dose selection is commonly based on dose ranging/

titration studies that bear a series of limitations: (i) the dose selected as the most
effective is not necessary the optimal dose, as it heavily depends on the power of the
design (Toutain 2002), while (ii) trials in which the sample size is small frequently
lead to the selection of high doses, which could be detrimental in patients with CHF.
As opposed to concentrationeffect time data, doses per se do not contain any
pharmacological information.
The value of modeling and simulation lies in its ability to use in silico models for
better integration and understanding of quantitative pharmacology. Modeling and
simulation approaches are becoming increasingly adopted in cardiovascular
research to evaluate dosing schedules and to streamline overall drug development
(Hong et al. 2008). Such a paradigm is based on the construction of mathematical
models that quantitatively describe our current understanding of physiological
systems and their response to drug exposure under (patho-)physiological condi-
tions. In this context, pharmacokineticpharmacodynamic (PKPD) models are
good candidates for integrating the large body of available information on RAAS
activation, regulation, and modulation, to accurately select dose ranges of
cardioactive agents at an early stage of drug development. Additionally, in an
attempt to capture and estimate variability in drug exposure and response, nonlinear
mixed-effects (NLME) models allow separating the between- and within-subject
variabilities from the measurement error (noise).
For model building, already existing data derived from several sources, such as
human, animal, in vitro, or in silico studies, can be integrated. Yet, only a few
models of the RAAS have been established to date. Takahashi et al. (2003) have
developed a qualitative model for describing the effect of ACE expression on BP in
mice. In a more recent publication from Hong et al. (2008), a semi-mechanistic
PKPD model was developed to evaluate the effects of aliskiren on the time course
of plasma renin activity (RA), AI, and AII in humans. Using a physiologically
based (PB) modeling approach, Guillaud and Hannaert (2010) and Hallow
et al. (2014) have extended the model of BP regulation pioneered by Guyton
(1990) to incorporate a more detailed representation of the renin cascade. Finally,
Ramusovic and Laeer (2012) have built a comprehensive model for the interaction
of AI and AII following single administrations of various RAAS blockers (i.e.,
enalapril, benazepril, aliskiren, and losartan).
Results from Hong et al. (2008) underscore the importance of characterizing the
time-varying changes in RAAS peptides to properly quantify the modulatory effect
of drugs on the circulating RAAS. In this experiment, the authors reported substan-
tial variations in renin, AI, and AII during placebo treatment, which they described
using a periodic function (sum of 2 harmonics) of renin production as the driving
force for the episodic profiling of downstream biomarkers AI and AII. In the case of
benazepril, the use of a straight line approximation of the mean (instead of a cosine)
for modeling of the placebo data would result in overestimating the effect of ACE
inhibition on AII and ALD, while underestimating its effect on RA (Mochel
et al. 2015). Circadian changes in the functioning of the circulating RAAS provide
a strong scientific rationale for tailored administration of cardioactive drugs at a
time that would optimize efficacy. However, additional influences to the underlying
biological rhythm may further contribute to temporal changes in the effectiveness
of drug therapies. Several physiological factors such as gastrointestinal pH and
motility, cardiac blood flow, or liver enzyme activity are prone to diurnal variations
(Reinberg and Smolensky 1982; Bruguerolle 1998; Ohdo 2007) and could impact
the disposition kinetics (i.e., absorption, distribution, metabolism, and elimination)
and the related efficacy of RAAS inhibitors. These fluctuations could be taken into
account using ad hoc PB PKPD modeling techniques.
4 Components of the Renin Cascade, Blood Pressure,

and Urinary Electrolytes Fluctuate with Clear Circadian
Changes in Dogs
The circadian system is organized in a hierarchical scheme, in which a master

pacemaker in the suprachiasmatic nucleus (SCN) regulates downstream oscillators
in peripheral tissues and organs (Ko and Takahashi 2006). This complex machinery
ensures that biological activities, from gene expression to cellular and physiological
manifestations, occur in the right sequence and at the right time of day (Reppert and
Weaver 2002). Core clock components are defined as genes whose protein products
are essential to the genesis and regulation of circadian rhythms within individual
cells throughout the organism. Within the SCN, clock genes, such as Clock
(Circadian Locomotor Output Cycles Kaput) and Arntl (Aryl hydrocarbon receptor
nuclear translocator-like, also known as Bmal1), are responsible for the genesis and
persistence of circadian rhythms (Dunlap 1999). Clock is expressed constitutively
in the SCN, while BMAL1 expression varies with a 24-h periodic rhythmicity.
These genes encode basic helix-loop-helix transcription factors that heterodimerize
and initiate the transcription of target genes containing E-box cis-regulatory
enhancer sequences, including Period (Per1, Per2, and Per3) and Cryptochrome
(Cry1 and Cry2) (Van der Zee et al. 2008; Storch and Weitz 2009) (Fig. 3).
Circadian rhythms are further coordinated by the transcription of another set of
genes, referred to as clock-controlled genes, which are expressed in phase with
Per and Cry (Dardente et al. 2004).
In mammals, the circadian clock influences many physiological and behavioral
variables including locomotor activity, body temperature, and heart rate (Reppert
and Weaver 2002; Van Esseveldt et al. 2000). Peptides of the reninangiotensin
cascade oscillate with a circadian periodicity in humans (Cugini et al. 1981, 1984,
1985, 1986, 1987; Kawasaki et al. 1990), and data from telemetry monitoring have
consistently demonstrated that BP had a reproducible diurnal rhythm, with highest
Fig. 3 The core oscillator of the mammalian circadian clock. (a) The transcriptionaltranslational
feedback loop that makes up the core oscillator of the mammalian circadian clock. CLOCK and
BMAL1 bind to E-box DNA sequences to activate clock-controlled genes (cogs), including those
that code for the oscillator proteins PER and CRY. PER and CRY are translated in the cytoplasm
and then cycle back into the nucleus to directly repress CLOCK:BMAL1. As PER and CRY level
drop, CLOCK:BMAL1 reactivates another round of transcription. (b) The CLOCK (green) and
BMAL1 (blue) proteins heterodimerize to bind DNA through their N-terminal bHLH domains.
The structure reported by Huang et al. (2011) shows that the PAS A domains dimerize symmet-
rically; in contrast, the PAS B domains form a head-to-tail interaction, mediated by a conserved
Trp residue on BMAL1 that binds into CLOCK PAS B. The analogous Trp on CLOCK projects
into solvent for putative interactions with CRY. To inhibit transcription, the PER tandem PAS
domains may interact with those of CLOCK:BMAL1. Unstructured regions of CLOCK and
BMAL1 (dotted lines) are important for transcriptional activation and histone acetyltransferase
(HAT) activity. Gln glutamine, ccgs clock-controlled gene sequences. Source: Crane (2012).
Reprinted with permission from the American Association for the Advancement of Science
(AAAS)
levels measured in the morning and lowest values around midnight (Staessen
et al. 1992; Smolensky and Haus 2001). Several factors have been shown to
influence daynight variations of the systemic RAAS, including alterations in
posture (Muller et al. 1958), sleep cycles (Brandenberger et al. 1985, 1994), and
age (Cugini et al. 1985). However, investigations on the effect of feeding time on
the periodicity of the RAAS have led to conflicting results (Kunita et al. 1976;
Ikonomov et al. 1981).
Although few observations of time variations of RAAS peptides have been
reported in dogs (Corea et al. 1996; Gordon and Lavie 1985; Reinhardt
et al. 1996), no systematic characterization of the chronobiology of these variables
is presently available in the literature. In addition, the question of whether BP
oscillates over the 24-h span in dogs is still a matter of debate (Miyazaki et al. 2002;
Piccione et al. 2005; Soloviev et al. 2006; Mochel et al. 2013a, 2014a). Results from
our research (Mochel et al. 2013a) showed that plasma renin (Fig. 4), urinary
Potassium fractional excretion (% change from baseline)

Sodium fractional excretion (% change from baseline)
200
400
160

Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 07:00 h

Renin activity (% change from baseline)
Dogs fed at 13:00 h Dogs fed at 13:00 h Dogs fed at 13:00 h
140

150
300

120

200

100
100

80

100
50
60

Night Night
Night
40
0
0
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
Time (hours) Time (hours) Time (hours)
Potassium fractional excretion (% change from baseline)

Sodium fractional excretion (% change from baseline)
200
400
160

Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Renin activity (% change from baseline)
Dogs fed at 19:00 h Dogs fed at 19:00 h Dogs fed at 19:00 h
140

150
300
120

100
200
100

80

100
50
60

Night Night Night
40
0
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
Time (hours) Time (hours) Time (hours)
Fig. 4 Circadian changes in renin activity, urinary sodium, and potassium fractional excretion
under various feeding schedules. Top. Average plasma renin activity (left pane), urinary sodium
(middle pane), and potassium (right pane) fractional excretion (% change from baseline) in dogs
fed a normal-sodium diet (0.5% sodium) at 07:00 h (continuous line) and 13:00 h (dashed line).
Bottom. Average plasma renin activity (left pane), urinary sodium (middle pane), and potassium
(right pane) fractional excretion (% change from baseline) in dogs fed a normal-sodium diet (0.5%
sodium) at 07:00 h (continuous line) and 19:00 h (dashed line). Source: Mochel et al. (2014a).
Reprinted by permission of Taylor & Francis LLC
aldosterone (UA:C), BP (Fig. 5), and urinary electrolytes (UK,fe and UNa,fe for
potassium and sodium fractional excretion, respectively) oscillate with a circadian
periodicity in trained and relaxed healthy dogs fed a regular diet at 07:00 h. An
approximately twofold (1.63.2-fold) difference between day and night measure-
ments was found for RA ( p < 0.01), UA:C ( p: 0.01), UK,fe ( p: 0.01), and UNa,fe
( p: 0.007).
Our results are consistent with previous investigations in dogs (Corea
et al. 1996), horses (Clarke et al. 1978, 1988), and humans (Cugini et al. 1981,
1985), which underlines the similarity of body fluid homeostasis in mammals. A
cosine model with a fixed 24-h period was found to fit the periodic variations of RA,
BP, urinary aldosterone, and potassium excretion well, as suggested by the quality
of the model diagnostics. In contrast, circadian changes in urinary sodium were best
characterized by means of a surge model, reflecting an afternoon peak sodium
excretion followed by a monotonous decay (Fig. 6). Here, NLME modeling
allowed borrowing information from the densely sampled plasma variables (i.e.,
RA) to improve parameter estimation of the (more sparse) urinary endpoints.
Similar to fluctuations in RA and UA:C, renal excretion of potassium was low in
the morning, increased in the afternoon, and peaked in the early evening. Systolic
140
140

Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 19:00 h Dogs fed at 19:00 h
Diastolic blood pressure (% change from baseline)

Systolic blood pressure (% change from baseline)
120
120
100

100
Night 80 Night
80
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
Time (hours) Time (hours)
Fig. 5 Circadian changes in systolic and diastolic blood pressure under various feeding schedules.
Average systolic (left pane) and diastolic (right pane) blood pressure vs. time profile (% change
from baseline) in dogs fed a normal-sodium diet (0.5% sodium) at 07:00 h (continuous line) and
19:00 h (dashed line). Source: Mochel et al. (2014a). Reprinted by permission of Taylor &
Francis LLC
and diastolic BP rose during the first half of the night, before returning to baseline
values in the morning.
From a physiological standpoint, the morning decrease in RA would be related
to body fluid expansion consecutive to sodium and water intake, while the simili-
tude between RA, urinary aldosterone, and potassium fluctuations would reflect
aldosterone-stimulated secretion by the reninangiotensin pathway and
aldosterone-mediated excretion of potassium in the kidney distal tubules. Likewise,
the main contribution of the reninangiotensin cascade to the chronobiology of BP
would be related to the sodium-retaining effects of AII and ALD, associated with
the strong vasomotor effect of AII. Compared with urinary potassium, the peak
renal elimination of sodium occurred much earlier during the day (around 15:00 h).
This phenomenon is known as the impulse-response pattern of sodium excretion
and is characterized by a peak natriuresis 48 h after feeding (Boemke et al. 1995).
Finally, the increase in sodium excretion during daytime was not associated to an
elevated BP, which is consistent with earlier publications in dogs (Andersen
et al. 2000; Bie and Sandgaard 2000; Sandgaard et al. 2000), and suggests that
pressure natriuresis is not a prime determinant of sodium homeostasis in
intact dogs.
140
Observed variable (unit)
: period

120

A: amplitude
M : mesor
100
: acrophase

80
7 am 1 pm 7 pm 1 am 7 am 1 pm 7 pm 1 am 7 am
Time (hours)
140
Observed variable (unit)
: period

120

A: amplitude B : baseline
w : width
100
: acrophase
80
7 am 1 pm 7 pm 1 am 7 am 1 pm 7 pm 1 am 7 am
Time (hours)
Fig. 6 Model parameters of a cosine and a surge function. Top: The shape of a cosine model is
determined by a set of parameters: (M, A, , and ), where M is the mesor (daily average of
rhythm), A is the amplitude of the cosine, is the acrophase (or time of peak), and is the period
(herein fixed to a value of 24 h). Bottom: The structure of a surge function is similar to that of a
cosine, with the substitution of the mesor by the baseline (initial value of rhythm, B), and the
addition of another parameter: the width of the surge (w). Source: Mochel et al. (2013a). Reprinted
by permission of Taylor & Francis LLC
5 The Chronobiology of Renin Activity, Blood Pressure,

and Urinary Electrolytes Is Synchronized to the Dogs
Feeding Schedule
In order to synchronize the internal clock to external circadian changes, biological

clocks are constantly reset by environmental cues (i.e., zeitgebers) in a process
known as entrainment. Lightdark cycles are among the most influential external
stimuli for synchronizing circadian oscillators (Hankins et al. 2008). Other,
nonphotic synchronizers exist, some of which act through the SCN (e.g., arousal,
Werner et al. 2010), while others, such as the timing of food intake, are more
directly affecting the peripheral circadian machinery (Huang et al. 2011; Patton and
Mistlberger 2013). The manipulation of feeding schedules has been shown to
modify the rhythmicity of liver glycogen, serum glucose, blood cells counts
(Nelson et al. 1975; Pauly et al. 1975; Philippens et al. 1977), BP, and heart rate
(Van den Buuse and Malpas 1997) in laboratory animals. In humans, plasma
glucocorticoid levels exhibit an anticipatory increase prior to the time of feeding
(Saito et al. 1989). As opposed to ad libitum feeding, restriction of food to
predefined time intervals elicits well-characterized physiological responses (i.e.,
food seeking behavior) to maintain metabolic homeostasis. Feeding schedules have
the ability to entrain the rhythmicity of clock-gene expression in various peripheral

organs and brain regions, dissociating these from the SCN zeitgeber, which remains
synchronized to lightdark cycles (Patton and Mistlberger 2013). This has been
shown in many studies where restriction to food access to a 26-h-time period
shifted the periodicity of clock-gene expression in most peripheral organs to align
with the expected meal time (Boulos and Terman 1980; Dibner et al. 2010).
The effect of food intake on the periodicity of the systemic RAAS has already
received wide attention across species. Studies have been performed under
(i) sodium restriction (Cugini et al. 1981, 1985), (ii) episodic vs. continuous feeding
(Blair-West and Brook 1969; Clarke et al. 1978, 1988), and (iii) fasting conditions
(Cugini et al. 1987). In contrast, the impact of timed feeding on the chronobiology
of the renin cascade remains controversial. In a study by Ikonomov et al. (1981),
diurnal changes in food intake did not affect the rhythmicity of renin and sodium
excretion. These results deviate from earlier findings by Kunita et al. (1976) in
healthy volunteers where circadian changes in RA and ALD disappeared when
meals were taken at night instead of the usual times of the day. In addition, previous
research on the influence of meal timing on the chronobiology of BP revealed that
reapportionment of food intake was accompanied by a peak shift in rabbits (Van
den Buuse and Malpas 1997) or the suppression of BP rhythmicity in rats (Van den
Buuse 1999). Few telemetry studies in dogs have suggested that food intake was
followed by a rapid drop in BP and heart rate (Mishina et al. 1999; Miyazaki
et al. 2002).
Our data have shown that feeding schedules exert a substantial effect on the
periodicity of RA, urinary electrolytes, and BP in dogs (Mochel et al. 2014a).
Precisely, introducing a 6- or 12-h delay in the dogs meal time caused a shift of
similar magnitude in the rhythm of these biomarkers (Figs. 4 and 5), as confirmed
by the model-based estimates of the phase shift parameter. Taken together, these
results suggest that food intake provides cues that are able to act as synchronizers
for the chronobiology of the reninangiotensin system, BP, and renal sodium and
potassium exchanges. Postprandial changes in RA are likely to be related to
sodium- and water-induced body fluid expansion, while variations in urinary
potassium would be the consequence of RAAS-mediated exchanges of electrolytes
in the kidney distal tubules. As discussed above, the increased natriuresis observed
after food intake is reflective of the impulse-response pattern of sodium excre-
tion. The marked post-meal drop in systolic and diastolic BP would be the result of
reduced RA levels combined with the secretion of vasodilatory gut peptides, such
as neurotensin and insulin (Shibao et al. 2007). Note that the decrease in RA and BP
could also reflect the predominance of parasympathetic activity during the post-
prandial state, as described by Kobayashi and Kamiya (1997). Finally, although BP
does not drop at night in healthy dogs fed a normal-sodium diet at 07:00 h (Mochel
et al. 2013a), it certainly does in dogs fed at 19:00 h (Mochel et al. 2014a),
indicating that similar to humans, lower BP levels are to be expected at nights
when dogs are given an evening meal.
The mechanisms by which peripheral oscillators can be entrained by food
remain unclear. Feeding-related signals that are capable of entraining peripheral
Stomach Melatonin
LH
Ghrelin Raphe
SCN
Leptin
ARC
GCs
Adiponectin Glucose
White adipose Adrenal
tissue
Liver & muscle Insulin
Pancreas
Fig. 7 Endocrine feedback to the circadian clock. Various hormones can directly or indirectly
feedback on central and peripheral clock function. In the brain endocrine targets with connections
to the SCN include the orexinergic neurons of the lateral hypothalamus (LH), the arcuate nucleus
(ARC), and the raphe nuclei of the brainstem. Other endocrine effects may be mediated via
peripheral tissues and clocks such as the liver and muscle. Source: Tsang et al. (2013). Reprinted
with permission from Bioscientifica Ltd
oscillators include autonomic outputs from the central nervous system, dietary
sodium, and feeding-dependent hormones (Mistlberger and Antle 2011; Tsang
et al. 2013) (Fig. 7). Although they are important to metabolic homeostasis,
glucocorticoids would not play a role. In contrast, ghrelin levels do exhibit a
clear circadian rhythm which appears to be aligned with the timing of food intake.
It has been hypothesized that ghrelin-secreting cells are themselves entrained by
feeding and that their endocrine signal serves as a messenger to other cells, both in
the brain and in peripheral tissues (LeSauter et al. 2009). Importantly, ghrelin can
also modify the phase of the SCN and its response to light (Brown and Azzi 2013).
Timed feeding has the ability to synchronize the activity of central oscillators, as
shown by Kurumiya and Kawamura (1991) in a rodent experiment where the peak
activity of neurons located in the hypothalamus was driven by the time of food
intake. Entrainment signals may also be provided by the intracellular ratio of
reduced to oxidized nicotinamide adenine dinucleotide cofactors (Rutter
et al. 2001). Next to the effect of feeding time on the chronobiology of the
RAAS, food composition per se (i.e., dietary sodium) also influences the periodicity
of renin. This was shown (i) from the results of the covariate analysis performed
under regular diet conditions (0.5% sodium) (Mochel et al. 2013a, 2014a) and
(ii) from the comparison of the mesor and amplitude estimates of RA in dogs fed a
normal (0.5% sodium) vs. a low-sodium (0.05% sodium) regime (Mochel

et al. 2015). In all studies, decreasing the amount of dietary sodium was found to
increase the mesor and amplitude of RA oscillations, suggesting that sodium not
only influences the tonic (i.e., mesor), but also the phasic (i.e., amplitude) secretion
of renin. Sodium intake was also found to affect the mesor, but not the amplitude of
urinary aldosterone oscillations in dogs (Mochel et al. 2013a).
6 Benazepril Markedly Influences the Dynamics

of the Circulating RAAS
Inhibition of the RAAS, as part of a global therapeutic scheme to decrease AII and
ALD exposure, and lower BP for preventing or delaying end-organ damage has
proven to be effective in human and canine CHF (Chobanian et al. 2003; Lefebvre
et al. 2007). Among RAAS inhibitors, two classes of drug directly target AII
through complementary mode of actions: (i) ACE inhibitors prevent the formation
of AII and the degradation of bradykinin, which increases the stimulation of nitric
oxide and has positive effects on endothelial function, while (ii) angiotensin recep-
tor blockers (ARBs) selectively antagonize AII at AT1 receptors. A theoretical
advantage of ARBs lies in their ability to increase activation of the AT2 receptor
and modulate the effects of AII breakdown products (Liu 1997), while reducing the
risk of ALD escape. Nevertheless, the escape phenomenon has also been reported
during long-term use of ARBs (Naruse et al. 2002), and the use of non-peptide
ARBs in small animal patients was shown to be ineffective (Adams 2009).
More recently, ALD receptor antagonists (ARAs) have also been registered for
use in canine patients suffering from CHF. In a study from Bernay et al. (2010),
spironolactone reduced by a factor of 2 the risk of cardiac-related death, euthanasia,
or severe worsening when used in addition to conventional therapy (ACE inhibi-
tion, plus furosemide and digoxin if required) in dogs with CVHD. These results
were however disputed by Kittleson and Bonagura (2010) on the grounds of several
methodological flaws (e.g., patient categorization, definition of CHF). In addition,
Schuller et al. (2011) could not find any significant effect of low-dose
spironolactone on survival when used as adjunct treatment to conventional conges-
tive heart failure treatment in dogs. Interestingly enough, ARAs have shown a
significant reduction in mortality in human CHF patients when combined with ACE
inhibitors, whereas ARBs have not (Werner et al. 2010). Lately, results of the
PARADIGM-HF clinical trial comparing the angiotensin receptorneprilysin
inhibitor LCZ696 with enalapril in patients with reduced ejection fraction CHF
were disclosed in the New England Journal of Medicine (McMurray et al. 2014).
LCZ696 was found to be superior by ca. 20% to enalapril in reducing the risks of
death and of hospitalization for heart failure ( p < 0.001). In a preliminary dog
study, valsartan, LCZ696 at 15 and 45 mg/kg decreased ALD levels to a significant
extent ( 23%, 45%, and 43%, respectively, p < 0.05). The greatest reductions
were observed in the LCZ696 groups, where LCZ696 15 mg/kg at 2 h reduced ALD
twofold lower than valsartan ( p < 0.05) (Mochel et al. 2014b).
By decreasing systemic vascular resistance, ACE inhibitors are known to
improve cardiac hemodynamics and exercise capacity in human and dog patients
(Levine et al. 1984; Uretsky et al. 1988; Lefebvre et al. 2007). Benazepril, enalapril,
imidapril, and ramipril are currently approved for use in dogs with CHF. Benazepril
hydrochloride (Fortekor; Novartis Animal Health, Basel, Switzerland) is a
nonsulfhydryl prodrug which is converted in vivo by esterases into its active
metabolite, benazeprilat, a highly potent and selective inhibitor of ACE (Webb
1990) with well-documented effectiveness in symptomatic canine CHF (King
et al. 1995; Lefebvre et al. 2007). In the BENCH (BENazepril in Canine Heart
Disease) Study Group (1999), the mean survival time of benazepril-treated dogs
with mild to moderate CHF was improved by a factor of 2.7, as compared with the
placebo group (428 vs. 158 days, p < 0.05). Although most of the preclinical
investigations for dose selection of benazepril have used ACE activity as a surro-
gate marker of efficacy in dogs, recent literature suggests that this may not be a
sensitive approach to properly assess the modulatory effect of ACE inhibitors on
the RAAS (Van de Wal et al. 2006). Using a low-sodium diet (LSD) model of
RAAS activation, our research shows that benazeprilat markedly influences the
dynamics of the systemic RAAS following single and repeated oral administrations
of benazepril at its recommended dose (0.251.0 mg/kg q24 h) in dogs (Mochel
et al. 2013b). In this study, treatment with benazepril triggered an apparent decrease
in AII and ALD, together with a sustained elevation of RA, as a consequence of
benazeprilat-induced interruption of the AIIrenin negative feedback loop (Bussien
et al. 1986; Steele et al. 2002). As expected, changes in ALD were followed by a
significant reduction of potassium renal excretion. The modulatory action of
benazeprilat on the functioning of the circulating RAAS was further characterized
using an integrated mechanism-based PKPD modeling approach (Fig. 8) (Mochel
et al. 2015). The final model, which also described the natural time course of the
biomarkers in the absence of the ACE inhibitor (i.e., under placebo treatment),
predicted no time delay between the dynamics of benazeprilat, RA, AII, and ALD,
which is an indication of a rapid turnover of RAAS biomarkers in plasma. As
previously mentioned, the use of a straight line approximation of the mean (in lieu
of a time-varying function) for modeling of the placebo data would have resulted in
overestimating the effect of ACE inhibition on AII and ALD, while
underestimating its effect on RA. Simulations from the PKPD model allowed
quantifying the extent and duration of effect of benazepril (0.34 mg/kg PO,
q24 h) on the RAAS. Results showed a two- to threefold change in systemic
RAAS levels at steady-state benazeprilat peak concentrations, and a more
prolonged effect on RA (at least 16 h) compared with AII and ALD (between
5 and 10 h). Such discrepancies could be related to the production of AII by
upregulation of ACE-independent pathways in response to renin and AI accumu-
lation during acute and long-term use of ACE inhibitors (Geary et al. 1992;
Fyhrquist and Saijonmaa 2008). Alternatively, the relatively short-lasting effect
of benazeprilat on AII and ALD might have been the result of the opposite
Depot Tinf
Benazepril
compartment Renin
M(RA), A(RA), (RA)
Angiotensinogen
ka
Angiotensin I
Imax (AII), (AII)
k1
k10 Free ACE-bound IC50 (AII)
Benazeprilat Benazeprilat
Emax (RA), (RA)
k2
Angiotensin II EC50 (RA)
Total benazeprilat M(AII), A(AII), (AII)
[Angiotensin II]
Imax (ALD), (ALD)

IC50 (ALD)
Aldosterone
M(ALD), A(ALD), (ALD)
Fig. 8 Integrated pharmacokinetic/pharmacodynamic model of benazeprilat disposition and

effect on the dynamics of the reninangiotensin cascade. A stepwise integrated PKPD model
was used, which includes the chronobiology of renin activity (RA), angiotensin II (AII), and
aldosterone (ALD) during placebo treatment, and the subsequent changes in dynamics following
inhibition of ACE. Benazeprilat data were analyzed using the class of pharmacokinetic models
developed by Lees et al. (1989) for ACE inhibitors. A compartmental approach was used where the
total amount of benazeprilat, as measured by the bioanalytical assay, is the sum of (i) benazeprilat
specifically and reversibly bound to circulating ACE (termed Abound) and (ii) benazeprilat free of
binding (referred to as Afree). A sequential zero and first-order absorption model was found to best
fit the data, where Tinf is the duration of the hypothetical infusion into the depot compartment (not
measured, i.e., shaded in gray), and ka is a first-order rate constant representing the absorption of
benazepril into the central compartment and its in vivo conversion to benazeprilat. k1 is the second-
order rate constant of association of the benazeprilatACE complex, and k2 is the first-order rate
constant of dissociation of the benazeprilatACE complex. The free fraction represents the amount
of benazeprilat that is systemically cleared from the central compartment, according to the first-
order rate constant k10. The modulatory effect of benazeprilat on the RAAS was described using a
combination of immediate response models, where benazeprilat concentrations vs. time data
served as the driving force for prediction of AII, while RA and ALD levels were derived from
the predicted difference in AII during placebo and benazepril treatment. Source: Mochel
et al. (2015). Reprinted by permission of Springer
stimulatory effect of sodium depletion on the RAAS. The reduction of AII and ALD
systemic levels certainly explains part of the clinical efficacy observed in
benazepril-treated CHF dogs, but additional effects on bradykinin degradation
and BP are likely to also come into play.
7 Conclusions and Research Perspectives in Dogs
Similar to humans, peptides of the systemic RAAS and BP oscillate with a clear
circadian periodicity in dogs. In both species, the timing of food intake appears to
be pivotal to the circadian organization of these biomarkers. Our data further show
that benazepril influences the dynamics of the reninangiotensinaldosterone cas-
cade, resulting in a profound but temporary decrease in AII and ALD, while
increasing RA for about 24 h.
Results from our model-based approach provide new insights into the relation of
dietary sodium to the chronobiology of the renin cascade, which would have been
impossible using standard statistics. Specifically:
1. The amount of sodium intake was shown to influence the tonic (i.e., mesor) and
the phasic (i.e., amplitude) secretion of renin; the greater the intake of sodium,
the smaller the mesor and amplitude of RA.
2. The time of food (i.e., sodium) intake appeared to exert a synchronizing effect on
the acrophase of RA and BP oscillations, which consolidates preliminary data
from the literature (Itoh et al. 1996).
Based on our findings on the dynamics of the circulating RAAS under physio-
logical (Mochel et al. 2013a, 2014a) and RAAS-activated conditions (Mochel
et al. 2013b, 2015), various strategies could in theory improve the therapeutic
management of cardiovascular diseases in dogs. Essentially, one could think of:
1. Adjusting the time of dosing. In dogs, cardioactive medications are commonly
given with morning food for the sake of convenience. However, results from our
chronobiological investigations with morning feeding indicate that the peak RA
and BP occurs in the evening and at night. Assuming that drug efficacy is
maximum when the peak effect time of the drug is synchronized with the peak
of the underlying biological rhythm, one would expect optimized efficacy with
bedtime dosing.
2. Adjusting dietary sodium intake. Because high dietary sodium is thought to play
a role in the development of HT and cardiovascular and renal diseases in
humans, a common practice in veterinary cardiology was to restrict sodium
intake in the diet of CHF dogs. There is however no substantial evidence that
elevated sodium intake increases the risk of HT in dogs (see results from
Anderson et al. 1986 and Greco et al. 1994 showing that fluctuations in sodium
intake has no apparent effect on BP and heart rate), and the current recommen-
dation is to avoid highly elevated dietary salt intake, without making a specific
effort to restrict it (Chandler 2008). Furthermore, because the mesor and ampli-
tude value of RA oscillations was found to be much greater in dogs fed a
low-sodium regime (Mochel et al. 2015), we could assume that CHF dogs
would rather benefit from a normal-sodium diet.
The research summarized herein is not a static and completed piece of work but
is, instead, a starting point for further data integration and hypothesis testing. As the
mathematical model of RAAS dynamics builds on new preclinical data, it should

also be refined by including additional measures from the target dog population
(i.e., symptomatic cases of CHF). Accumulating knowledge from diseased animals
will not only help assessing the reliability of the LSD model (comparing estimates
of RAAS oscillations under LSD vs. CHF conditions), but it will also determine
whether AII and ALD levels are predictors of increased morbidity and mortality
risk in canine CHF patients. In this case, profiling of RAAS peptides could
complement clinical and echocardiographic findings and serve as supportive infor-
mation for diagnostic, prognostic, and therapeutic monitoring purposes. Adapting
the drug dosing schedule of ACE inhibitors to intrinsic biological rhythms has been
shown to improve therapeutic management of RAAS-related diseases (i.e., CHF
and HT) in human patients. Another perspective of this work therefore consists in
evaluating whether the efficacy and safety profile of these RAAS inhibitors could
be optimized by selecting the appropriate time of treatment in dogs. A number of
publications in laboratory animals have already provided solid evidence that car-
diovascular growth and remodeling is dynamic and does not occur uniformly over
the 24-h span (Bray and Young 2008; Sole and Martino 2009). In a study by
Martino et al. (2011) in mice, captopril significantly improved cardiovascular
functioning and reduced adverse remodeling with bedtime dosing, while no effect
was reported when the drug was administered during active hours. Likewise,
several trials (Kuroda et al. 2004) have shown that dosing of ACE inhibitors during
sleeping hours was more effective and safe in human patients. These findings,
combined with those of our research on the chronobiology of the reninangiotensin
cascade in dogs, encourage future investigations on the chronotherapy of RAAS
inhibitors in prospective clinical trials with CHF dogs.
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ISSN 0303-4240 ISSN 1617-5786 (electronic)

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Springer International Publishing AG Switzerland is part of Springer Science+Business Media

Hyperforin: To Be or Not to Be an Activator of TRPC(6) . . . . . . . . . . . . . . . . . 1

The Piezo Mechanosensitive Ion Channels: May the Force

Chronobiology and Pharmacologic Modulation of the

Hyperforin: To Be or Not to Be an Activator

Kristina Friedland and Christian Harteneck

Abstract Meantime, it is well accepted that hyperforin, the chemical instable

Keywords Calcium homeostasis Depression Hyperforin Ion channel

6 The Role of TRPC6 Channels for Neuronal Effects of Hyperforin: Depression,

serotonin, norepinephrine, dopamine, and GABA transporter belong to the SLC6

2 Chemical Stability of Hyperforin and Stable Analogues

Hyperforin, the prenylated acylphloroglucinol, is unstable in water and aprotic

antidepressant effects comprising neurotransmitter uptake and neuronal differenti-

In 2002, the group around Rehavi suggested that hyperforin might be a

their unselective mechanism of action, the antimicrobiotic activity is associated

4 Hyperforin as Inducer of Cytochrome P450 Enzymes,

MDR1 DRUG CYP

5 Hyperforin as Activator of Ion Channels

primary cortical neurons, hyperforin resulted in an increase of intracellular Ca2+

6 The Role of TRPC6 Channels for Neuronal Effects

The participation of TRPC6 channels is discussed for several neuronal disorders

channels by hyperforin protected against these neuronal damages following the

pore-dead mutant of TRPC6. The effect is mediated by a strong hyperforin-

Fortin DA, Srivastava T, Dwarakanath D, Pierre P, Nygaard S, Derkach VA et al (2012) Brain-

Tu P, Kunert-Keil C, Lucke S, Brinkmeier H, Bouron A (2009) Diacylglycerol analogues activate

The Piezo Mechanosensitive Ion Channels:

Eric Honore, Joana Raquel Martins, David Penton, Amanda Patel,

Keywords Electrophysiology Mechanosensation Mechanotransduction Piezo1

E. Honore, J.R. Martins, D. Penton, A. Patel, and S. Demolombe (*)

8 Role of Endothelial Piezo1 in the Vascular Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Mechanotransduction concerns the molecular and cellular mechanisms responsible

1 Piezo1/2 Are Essential Components of Distinct

2 Piezo1/2 Are Pore-Forming Subunits

Using bilayer reconstitution experiments, it was demonstrated that purified Piezo

Using total internal reflection fluorescence (TIRF) microscopy to measure discrete

3 Mapping the Ionic Pore in Piezo1

Through the combination of bioinformatical analysis, immunostaining of

ruthenium red-binding site that remains to be identified. Importantly, E2133

4 The Small Synthetic Molecule Yoda1 Is an Opener

In another remarkable effort, Patapoutian and colleagues screened over three

RBC shrinkage RBC overhydrated

Importantly, even in the absence of mechanical stimulation, the addition of Yoda1

5 Upregulation of Piezo1 by Phosphoinositides

Unexpectedly, activation of TRPV1 channels with capsaicin either in dorsal root

6 Dehydrated Stomatocytosis (Xerocytosis) Is Caused

Gain-of-function (GOF) mutations in Piezo1 are associated with xerocytosis, an

to a slower inactivation process is associated with red blood cell (RBC)

7 A Possible Link Between Piezo1 and Sickle Cell Disease

Previous work had suggested that activation of stretch-activated ion channels

8 Role of Endothelial Piezo1 in the Vascular Development

Global knockout of Piezo1 is embryonically lethal, thus indicating an important

9 Role of Piezo1 in Bladder Mechanosensation

10 Gain-of-Function Mutations in Piezo2 Cause Distal

Distal arthrogryposis is a rare autosomal dominant disorder characterized by cleft

11 Piezo2 and the Sense of Touch

Piezo2 is localized to the peripheral nerve endings of low-threshold mechano-

12 Tuning Piezo2 Influences Touch Sensitivity

Molecular-scale displacements are sufficient to gate mechanosensitive channels in

13 Drosophila Piezo Is Involved in Nociception, Although

dPiezo is widely expressed in every sensory neuron of Drosophila larvae (Kim