Mary Ann Liebert, Inc.

Cannabis and Cannabinoid Research

Oleoylethanolamide and palmitoylethanolamide protect

cultured cortical neurons against hypoxia through TRPV4
receptors

Journal: Cannabis and Cannabinoid Research

Manuscript ID Draft

Manuscript Type: Original Research

Fo
Date Submitted by the Author: n/a

Complete List of Authors: Portavella, Manuel; Universidad de Sevilla, Psicología Experimental

Rodriguez-Espinosa, Nieves; Universidad de Sevilla, Fisiologia Medica
rP

Galeano, Pablo; Instituto de Investigaciones Bioquímicas de Buenos Aires

(IIBBA-CONICET)
Blanco, Eduardo; Universitat de Lleida
Romero, Juan; Instituto de Investigaciones Bioquímicas de Buenos Aires
ee

(IIBBA-CONICET)
Holubiec, Mariana; Instituto de Investigaciones Bioquímicas de Buenos
Aires (IIBBA-CONICET)
rR

Rodriguez de Fonseca, Fernando; Instituto de Biomedicina de Málaga

(IBIMA), Unidad de Gestión Clínica de Salud Mental, Hospital Regional
Universitario
Fernandez-Espejo, Emilio; Universidad de Sevilla, Fisiologia Medica
ev

Keywords: Endocannabinoid system, Psychopharmacology

Manuscript Keywords (Search Hypoxia-ischemia, Oleoylethanolamide, Palmitoylethanolamide, PPARalpha,

iew

Terms): TRPV1, TRPV4

Introduction: Perinatal hypoxia-ischemia encefalopathy is defined as a

neurological syndrome where the newborn suffers from acute ischemia and
hypoxia during the perinatal period. New therapies are needed. The
acylethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide
(PEA) possess neuroprotective properties, and they could be effective
against perinatal hypoxia-ischemia. These lipid mediators act through
peroxisome proliferator-activated receptors subtype α (PPARα), or
transient vanilloid receptors (TRPV) such as TRPV subtype 1 and 4.
Material and methods: The objectives of this study were to discern: a) the
neuroprotective role of OEA and PEA in parieto-temporal cortical neurons of
Abstract: newborns rats and mice subjected to hypoxia, and b) the role of the
receptors PPARα, TRPV1 and TRPV4 in neuroprotective effects. Cell culture
of cortical neurons, and the lactate-dehydrogenase assay were carried out.
The role of receptors was discerned by using selective antagonist and
agonist ligands, as well as knock-out mice.
Results: The findings indicate that OEA and PEA exert neuroprotective
effects on culture cortical neurons subjected to a hypoxic episode. These
effects are not mediated by PPARα or TRPV1. However TRPV4 seems to be
involved in neuroprotective effects of acylethanolamides, because blocking
or stimulating TRPV4 facilitates or inhibit the neuroprotective effects of
OEA and PEA, respectively.

ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com

Page 1 of 24 Mary Ann Liebert, Inc.

1
2
3
Conclusion: The lipid mediators OEA and PEA exert neuroprotective effects
4
on culture cortical neurons subjected to hypoxia through TRPV4 receptors.
5 These in vitro results could be of utility for developing new therapeutic
6 tools against perinatal hypoxia-ischemia.
7
8
9
10
11
12
13
14
15
16
17
18
19
Fo
20
21
22
23
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 2 of 24

1
2
3 Oleoylethanolamide and palmitoylethanolamide protect
4
5 cultured cortical neurons against hypoxia through
6 TRPV4 receptors
7
8
9
Manuel Portavella 1 *, Nieves Rodriguez-Espinosa 2 *, Pablo Galeano3, Eduardo
10
11 Blanco4, Juan I. Romero3, Mariana I. Holubiec3, Fernando Rodriguez de
12 Fonseca 5, Emilio Fernández-Espejo 2
13
14 1 Laboratorio de Conducta Animal y Neurociencia. Departamento de Psicología
15 Experimental, Facultad de Psicología, Universidad de Sevilla, Sevilla, Spain
16
17 2 Laboratorio de Neurofisiología y Neurología Molecular, Departamento de
18 Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla,
19 Sevilla, Spain.
Fo
20
21 3 Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET),
22
Avda. Patricias Argentinas 435, C1405BWE, Ciudad Autónoma de Buenos
23
rP

24
Aires, Argentina.
25
26 4 Universitat de Lleida, Instituto de Investigación Médica de Lleida, Dr. Pifarré
ee

27 Foundation (IRBLleida), Lleida, Spain.

28
29 5 Unidad de Gestión Clínica de Salud Mental, Hospital Regional Universitario,
rR

30 Instituto IBIMA, Málaga, Spain

31
32
ev

33
34
35 *Both authors contributed equally to this work
36
iew

37 Running head: acylethanolamides and hypoxia

38
39
Number of words: 4377
40
41
42 _______________________________________________________________
43
44 Address correspondence to:
45
46
47 Emilio Fernández-Espejo, Departamento de Fisiologia Medica y Biofisica,
48 Universidad de Sevilla, Av. Sanchez Pizjuan 4, E-41009 Sevilla, SPAIN. Tel:
49
50 34-954556584. E-mail: efespejo@us.es
51
52 Fernando Rodríguez de Fonseca, Unidad de Gestión Clínica de Salud Mental,
53
54 Hospital Regional Universitario, Instituto IBIMA, Málaga, Spain. E-mail:
55
56
fernando.rodriguez@ibima.eu
57
58 1
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 3 of 24 Mary Ann Liebert, Inc.

1
2
3 Abstract
4
5
6
Introduction: Perinatal hypoxia-ischemia encefalopathy is defined as a
7
8 neurological syndrome where the newborn suffers from acute ischemia and
9
10 hypoxia during the perinatal period. New therapies are needed. The
11 acylethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide (PEA)
12
13 possess neuroprotective properties, and they could be effective against
14
15 perinatal hypoxia-ischemia. These lipid mediators act through peroxisome
16 proliferator-activated receptors subtype α (PPARα), or transient vanilloid
17
18 receptors (TRPV) such as TRPV subtype 1 and 4.
19
Material and methods: The objectives of this study were to discern: a) the
Fo
20
21 neuroprotective role of OEA and PEA in parieto-temporal cortical neurons of
22
23 newborns rats and mice subjected to hypoxia, and b) the role of the receptors
rP

24 PPARα, TRPV1 and TRPV4 in neuroprotective effects. Cell culture of cortical

25
26 neurons, and the lactate-dehydrogenase assay were carried out. The role of
ee

27
28 receptors was discerned by using selective antagonist and agonist ligands, as
29 well as knock-out mice.
rR

30
31 Results: The findings indicate that OEA and PEA exert neuroprotective effects
32
on culture cortical neurons subjected to a hypoxic episode. These effects are
ev

33
34 not mediated by PPARα or TRPV1. However TRPV4 seems to be involved in
35
36 neuroprotective effects of acylethanolamides, because blocking or stimulating
iew

37 TRPV4 facilitates or inhibit the neuroprotective effects of OEA and PEA,

38
39 respectively.
40
41
Conclusion: The lipid mediators OEA and PEA exert neuroprotective effects
42 on culture cortical neurons subjected to hypoxia through TRPV4 receptors.
43
44 These in vitro results could be of utility for developing new therapeutic tools
45
against perinatal hypoxia-ischemia.
46
47
48
49
50 Key words: hypoxia-ischemia; neuroprotection; oleoylethanolamide;
51
52 palmitoylethanolamide; PPARα; TRPV1, TRPV4.
53
54
55
56
57
58 2
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 4 of 24

1
2
3 Introduction
4
5
6 Perinatal hypoxia-ischemia (HI) encefalopathy is defined as a neurological
7
8 syndrome where the newborn suffers from acute ischemia and hypoxia during
9 the perinatal period. Brain damage after HI insults is caused by a deleterious
10
11 combination of glial activation, excitotoxicity, inflammation, and oxidative stress
12
13 with overproduction of oxidative radicals such as nitric oxide (NO) and reactive
14 oxygen species (ROS) 1-4
The clinical relevance of this syndrome requires
15
16 further research in search for protective therapies.
17
18
19 Peroxisome proliferator-activated receptor α (PPARα) is a potential target for
Fo
20
21 neuroprotection against acute ischemia and hypoxia because this receptor
22
plays a prominent role in the modulation of inflammatory and oxidant stress
23
rP

24 responses. 5-10
Thus PPARα activation is known to activate astrocytes and glial
25
26 cells, to reduce the transcription of inflammatory response genes, and to
ee

27 promote neurological recovery by exerting anti-inflammatory effects. 11-18

28
29 Neuroprotective effects of activating PPARα are also associated with a
rR

30
31 decrease in cerebral oxidative stress. 19 In this context, there is strong evidence
32
that normal PPARα function is necessary to protect cells from inflammation and
ev

33
20
34 oxidative damage in several tissues such as liver, heart, and spleen.
35
36 Furthermore, PPARα agonists have potentially therapeutic efficacy in several
iew

37
neurological diseases such as Parkinsonism, multiple sclerosis, or autoimmune
38
39 encephalomyieltis. 21-23
PPARα can be modulated by lipid mediators, such as
40
41 the acylethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide
42 24-26
(PEA). These fatty acids act as endogenous ligands for PPARα, and
43
44 several authors have reported that OEA could exert neuroprotective effects
45 22, 27-29
46 through PPARα. Although OEA is an analogue of the endocannabinoid
47 anandamide, and PEA enhances anandamide activity, both acylethanolamides
48
49 do not act directly through cannabinoid CB receptors. 24-26, 30
50
51
52 Another family of receptors that are involved in the response to hypoxia is the
53
54 transient receptor potential vanilloid (TRPV) family. These vanilloid receptors
55 are widely distributed in the central nervous system. 31
It seems that blocking
56
57
58 3
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 5 of 24 Mary Ann Liebert, Inc.

1
2
3 the transient receptor potential vanilloid subtype 1 or TRPV1 could mediate
4
neuroprotective effects, and OEA-induced neuroprotection could be explained,
5
6 in part, by its blocking effects on TRPV1. 32-35
There is also evidence showing
7
8 that the transient receptor potential vanilloid subtype 4 or TRPV4 is involved in
9 cerebral ischemic reperfusion injury, and recovery of brain edema. 36-40
The
10
11 TRPV4 channel induces an increase in intracellular calcium concentration and
12
13 plays an important role under physiological and pathological conditions.
14 Blocking TRPV4 may inhibit brain edema in cerebral ischemia. 37-38
TRPV4
15
16 channels participate in the pathogenic mechanisms of astroglial reactivity
17
following ischemic insult. 41
18
19
Fo
20
21 The neuroprotectant role of OEA and PEA in brain damage after acute hypoxia
22 remains to be fully determined. It is worth noting that these compounds might
23
rP

24 play a physiological role against deleterious effects of hypoxia, because it is

25
26
known that their brain levels are increased after a traumatic or hypoxia-ischemia
ee

27 insult. 42-44
The objectives of this study were to discern: a) the neuroprotective
28
29 role of OEA and PEA in culture cortical neurons subjected to hypoxia, and b)
rR

30
the role of PPARα, TRPV1 and TRPV4 on possible OEA and PEA-mediated
31
32 neuroprotective effects by using selective receptor antagonist and agonist
ev

33
34 ligands as well as knock-out PPARα mice.
35
36
iew

37 Material and Methods

38
39
40 Animals
41
42 Pregnant Wistar rats (250-350 g) from the breeding colony of the University of
43 Seville were used. Pups at P0 were used for in vitro experiments. Laboratory
44
45 temperature was kept at 22 ± 1°C, and a 12-h light-dark cycle (lights on at 08:00
46
47 hours) was maintained throughout the experiment. Food (lab chow) and water
48 were available ad libitum.
49
50
51
For further studying the role of PPARα, one-week-old male PPARα +/+ wild-
52
53 type (WT) and PPARα -/- knockout (KO) mice were also used. WT and KO C57
54
55 BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA).
56 KO mice on a C57 BL/6J genetic background were bred in accordance with
57
58 4
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 6 of 24

1
2
3 European Union guidelines for animal care. PPARα +/+ and PPARα -/- mice
4
were housed five per cage in temperature- (21 ± 1ºC) and humidity (55 ± 10%)-
5
6 controlled rooms with a 12-h light/12-h dark cycle (light between 8:00 AM and
7
8 8:00 PM). Food and water were available ad libitum during the whole
9 experiment.
10
11
12
13 Genotyping protocol for PPARα
α
14 Mice homozygous for the Pparatm1Gonz targeted mutation (129S4/SvJae-
15
16 Pparatm1Gonz) were obtained from Jackson Laboratories (Bar Harbor, ME).
17 45
18
Genotyping protocol has been described by the authors elsewhere. The
19 following primers were used:
Fo
20
21 IMR0013 (50-CTTGGGTGGAGAGGCTATTC-3; Tm = 59 ºC),
22
IMR0014 (50-AGGTGAGATGACAGGAGATC-30; Tm=54 ºC),
23
rP

24 IMR11999 (50-CCATCCAGATGACACCTTCC-30; Tm=60 ºC),

25
26 IMR1200 (50-TCTCTTGCAACAGTGGGTGC -30; Tm=62 ºC).
ee

27
28
29 Compounds and protocol
rR

30
Oleoylethanolamide (OEA) and palmithoylethanolamide (PEA) were purchased
31
32 from Tocris. OEA and PEA were dissolved in ethanol until use, and they were
ev

33
34 used for cultures after further dilution at 10% ethanol in Neurobasal, at doses of
35 0, 5, 10, 20 and 40 µM. The selective PPARα antagonist GW6471 or [(2S)-
36
iew

37 2-[[(1Z)-1-Methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl] amino]-3-[4-[2-
38
39 (5-methyl-2-phenyl-4-oxazolyl)ethoxyphenylpropyl-carbamic acid ethyl ester
40 was purchased from Tocris, and it was dissolved in 10% DMSO until use (IC50
41
42 value of GW6471 is 0.24 µM). The selective antagonist of vanilloid TRPV1 SB
43
452533 or N-(2-Bromophenyl)-N'-[2-[ethyl(3-methylphenyl)amino]ethyl]-urea
44
45 was purchased from Tocris, and it was and it was dissolved in 10% DMSO until
46
47 use (pIC50 = 7.0). The selective antagonist of vanilloid TRPV4, RN1734 or 2,4-
48 Dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzene sulfonamide, was
49
50 purchased from Tocris (IC50 value of RN1734 is 3.2µM for rTRPV4). Finally,
51
52 GSK1016790A, selective agonist of vanilloid TRPV4, was purchased to Sigma-
53 Aldrich. GSK1016790A or (N-((1S)-1-{[4-((2S)-2-{[(2,4-
54
55 Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-
56
methylbutyl)-1-benzothiophene-2-carboxamide) is known to evoke a dose-
57
58 5
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 7 of 24 Mary Ann Liebert, Inc.

1
2
3 dependent activation of TRPV4 whole-cell currents at concentrations above
4
1nM.
5
6
7
8 Cell culture and lactate-dehydrogenase (LDH) assay
9 Primary cultures of parieto-temporal cortical neurons were established as
10
46 47, 48
11 previously described, with some modifications suggested by others.
12
13 Postnatal rat pups (P0) or 1-month old PPAR-α +/+ and PPAR-α -/- mice were
14 killed by decapitation, and brains were removed. All animals were humanely
15
16 sacrificed.
17
18
19 Exposure to pharmacological compounds and hypoxia was initiated after 4 days
Fo
20 48
21 of in vitro conditions. Treatments were carried out either before or after
22 hypoxia exposure. Regarding experiments before hypoxia, OEA and PEA were
23
rP

24 added to the culture medium for 30min, at different concentrations (0, 5, 10, 20
25
26
and 40 µM, all compounds). If selective receptor ligands were used (GW6471,
ee

27 SB 452533, RN1734), they were added to the medium 15 min before OEA and
28
29 PEA. SB 452533 and RN1734 were used at doses of 0.1, 0.1, 1, 5 and 10 µM.
rR

30
GSK1016790A was used at doses of 1 and 5 nM. In these cases,
31
32 acylethanolamides were used at the most effective dose, or not added for
ev

33
34 studying per se effects of antagonists. Thereafter neurons were exposed during
35 40 min to hypoxia by using hypoxic medium, consisting on Neurobasal without
36
iew

37 B27 which had been exposed to 95% N2/5% CO2 air bubbling for 30 min. After
38
this 40-min hypoxia period, the medium was replaced with fresh incubation
39
40 solution equilibrated with 95% O2/5% CO2. As regards experiments after
41
42 hypoxia, the protocol was similar but Neurobasal without B27 was used as the
43 incubation solution equilibrated with 95% O2/5% CO2, and OEA and PEA were
44
45 added to the medium for 30 min, just after the 40-min hypoxia exposure
46
47 (antagonists were not used).
48
49
50 After all treatments, the medium was removed, and the cultures were further
51 22
incubated for 24 hours, in order to carry out the LDH assay. Citotoxicity was
52
53 evaluated by release of the cytosolic enzyme lactate dehydrogenase (LDH) into
54
55 the culture medium by dying cells (Cytotoxicity Detection kit, Roche,
56 Indianapolis, USA). Total LDH release was calculated by incubating untreated
57
58 6
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 8 of 24

1
2
3 cells with 0.5% Triton X-100 for 1 hour to induce maximal cell lysis. Basal death
4
was calculated from untreated wells without B27. Treatment values were then
5
6 expressed as percent of the maximum LDH release. Background LDH release
7
8 (media alone) was subtracted from the experimental values.
9
10
11 Statistics and ethics
12
13 For statistics, when the effects of lipid ligand compounds on culture dopamine
14 neurons were compared, one-way ANOVA was used, followed by Newman-
15
16 Keuls test. When a receptor blocker was added, two-way ANOVA was used for
17
statistics (lipid ligand dose and receptor blocker dose as factors), followed by
18
19 Newman-Keuls test. Experiments were performed according the animal care
Fo
20
21 guidelines of the European Communities Council (86/609/ECC, 90/679/ECC,
22 98/81/CEE, 2003/65/EC, Commission Recommendation 2007/526/EC),
23
rP

24 European Directive 2010/63/EU and the Spanish Royal Decree 53/2013 on the
25
26
protection of animals used for research and other scientific purposes. Animal
ee

27 experiments were approved by the local ethical committee (CEEA; University of

28
29 Seville, BIO127).
rR

30
31
32 Results
ev

33
34
35 OEA exerts neuroprotection if given either before or after hypoxia
36
iew

37 Two-way ANOVA revealed a dose effect after OEA treatment (F4, 70=11.7,
38
p<0.001), without interaction. Thus OEA given either before or after hypoxia
39
40 exerted similar effects. One-way ANOVA revealed a significant dose effect
41
42 after OEA treatment prior to hypoxia exposure (F4, 44=5.6, p<0.01), 20 and 40
43 µM OEA reliably enhancing cell survival relative to 0 dose-treated cells (20µM
44
45 OEA p<0.05; 40µM OEA, p<0.01; Newman-Keuls). If OEA was given after
46
47 hypoxia, one-way ANOVA revealed a significant dose effect (F4, 39=8.9,
48 p<0.01), and post-hoc analysis indicated that 40 µM OEA exerted a
49
50 neuroprotective effect (p<0.01, Newman-Keuls), as shown in Figure 1.
51
52
53
54
55 ==== Figure 1 about here ====
56
57
58 7
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 9 of 24 Mary Ann Liebert, Inc.

1
2
3 PEA exerts neuroprotection if given either before or after hypoxia
4
Two-way ANOVA revealed a dose effect after PEA treatment (F4, 70=12.6,
5
6 p<0.001), without interaction. Thus PEA given either before or after hypoxia
7
8 exerted similar effects. One-way ANOVA revealed a significant dose effect after
9 PEA treatment prior to hypoxia exposure (F4, 44=17.3, p<0.001). Thus 10, 20
10
11 and 40 µM PEA reliably enhanced cell survival relative to 0 dose-treated cells
12
13 (10µM PEA p<0.05; 20µM and 40µM PEA, p<0.01; Newman-Keuls). If PEA was
14 given after hypoxia, similar changes were found (dose effect, F4, 39=3.2;
15
16 p<0.02; 10, 20 and 40µM PEA, p<0.01; Newman-Keuls), as shown in Figure 2.
17
18
19 ==== Figure 2 about here ====
Fo
20
21
22 Neuroprotective properties of OEA and PEA are not mediated by PPARα
α
23
rP

24 receptors
25
26 As explained, in order to block PPARα, the selective antagonist GW6471 was
ee

27 added to the culture medium. Following GW6471 administration, the survival

28
29 effects of 40µM OEA or PEA before hypoxia episode were not significantly
rR

30
modified, as shown in Table 1.
31
32
ev

33
34 ==== Table 1 about here ====
35
36
iew

37 Furthermore, after using PPARα -/- mice, the neuroprotective effects of OEA
38
39 and PEA remain, without significant differences between both groups, as shown
40 in Fig. 3. Two-way ANOVA revealed dose effects without interaction for every
41
42 compound (OEA, F4, 50=155, p<0.001; PEA, F4, 50=69, p<0.001). Regarding
43
OEA, one-way ANOVA revealed a significant dose effect after OEA treatment
44
45 prior to hypoxia exposure (PPARα -/- mice, F4, 29=72, p<0.001; WT mice, F4,
46
47 29=83.3, p<0.001), and 10, 20 and 40µM OEA were observed to significantly
48 enhance cell survival relative to 0 dose-treated cells in both type of cells (10µM
49
50 OEA, p<0.05; 20µM OEA p<0.01; 40µM OEA, p<0.01; Newman-Keuls). As
51
52 regards PEA, one-way ANOVA revealed a significant dose effect after PEA
53 treatment prior to hypoxia exposure (PPARα -/- mice, F4, 29=33.9, p<0.001; WT
54
55 mice, F4, 29=35, p<0.001). Thus 10, 20 and 40 µM PEA reliably enhanced cell
56
57
58 8
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 10 of 24

1
2
3 survival relative to 0 dose-treated cells in both type of cells (10µM PEA p<0.05;
4
20µM and 40µM PEA, p<0.01; Newman-Keuls).
5
6
7
8 ==== Figure 3 about here ====
9
10
11 Neuroprotective properties of OEA and PEA are not mediated by TRPV1
12
13 The selective antagonist SB 452533 was given for blocking TRPV1. Following
14 SB 452533 at different doses and 40µM OEA or PEA, two-way ANOVA did not
15
16 reveal significant effects, as shown in Table 2.
17
18
19 ==== Table 2 about here ====
Fo
20
21
22 Neuroprotective properties of OEA and PEA are facilitated or inhibited
23
rP

24 after blocking or stimulating TRPV4, respectively

25
26
The selective antagonist RN1734 was added to the medium for blocking
ee

27 TRPV4. Following RN1734 at different doses and 40µM OEA, one-way ANOVA
28
29 revealed a significant dose effect, as shown in Table 3 (F4, 24= 24.5, p<0.005).
rR

30
The highest RN1734 dose (10 µM) significantly enhanced neuroprotective
31
32 effects of 40µM OEA (p<0.05 versus the remainder doses, Newman-Keuls). As
ev

33
34 regards PEA, after RN1734 at different doses and 40µM PEA, one-way ANOVA
35 revealed a significant dose effect (F4, 24= 25.2, p<0.05). Thus, the highest
36
iew

37 RN1734 doses (5 and 10µM) significantly enhanced neuroprotective effects of

38
40µM PEA, as shown in Table 3 (p<0.05 versus the remainder doses, Newman-
39
40 Keuls). In fact, complete protection was afforded by TRPV antagonists at high
41
42 doses. Thus neuroprotective properties of OEA and PEA are further enhanced
43 after blocking TRPV4 (Table 3). RN1734 was neuroprotectant per se, as
44
45 revealed by ANOVA (F4, 24= 19.1, p<0.05). Thus, the highest RN1734 doses (5
46
47 and 10µM) significantly reduced cell death, as shown in Table 3 (p<0.05 versus
48 the remainder doses, Newman-Keuls).
49
50
51
GSK1016790A was found to induce full cell death at doses higher than 10 nM
52
53 (data not shown). Since lower doses induced less cell death, as shown in Table
54
55 3, 0, 1 and 5 nM GSK1016790A was used. Following GSK1016790A at 1 and
56 5nM, one-way ANOVA revealed a significant dose effect on cell death (F2,
57
58 9
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 11 of 24 Mary Ann Liebert, Inc.

1
2
3 19=12.9, p<0.01), as shown in Table 3. GSK1016790A induced cell death per
4
se. 0µM OEA did not induce significant changes in cell death after cotreatment
5
6 with GSK1016790A. As regards GSK1016790A plus 40µM PEA, one-way
7
8 ANOVA did not reveal significant dose effect. Thus neuroprotective properties
9 of OEA and PEA were not observed after stimulating TRPV4 (Table 3).
10
11
12
13 ==== Table 3 about here ====
14
15
16 Discussion
17
18
19 Brain damage after hypoxia-ischemia (HI) insults is caused by the deleterious
Fo
20
21 combination of glial activation, excitotoxicity, inflammation, and oxidative stress
22 with over-production of reactive oxygen species. 1-4
We have tested in a
23
rP

24 culturing cell model of neuronal hypoxia some lipid mediators that are known to
25
26
act through receptors which are otherwise involved in post-HI reactions. These
ee

27 receptors encompass PPARα, and the vanilloid receptors TRPV1 and TRPV4.
28
29 These lipid mediators belong to the family of acylethanolamides, that are
rR

30 24-
cannabinoid derivatives which do not act through cannabinoid CB receptors.
31
32 26, 30
ev

33
34
35 The findings of the present study indicate that the fatty acid acylethanolamides
36
iew

37 oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), exert

38
39 neuroprotective effects on culture cortical neurons subjected to a hypoxic
40 episode. These effects are dose-dependent in a linear fashion. The 40µM
41
42 OEA/PEA dose exerted the maximum effects. Contrary to expectation, these
43
44
effects are not mediated by PPARα or TRPV1. In this context, these fatty acids
45 exert neuroprotective effects in other cellular and animal models through these
46
24-26, 32, 33
47 receptors, and OEA and PEA act as endogenous ligands for PPARα,
48 22-27, 29
exerting neuroprotective effects . OEA is also known to act as
49
50 neuroprotectant through the transient receptor potential vanilloid subtype 1 in
51 34, 35
52 other situations. To sum up, neuroprotective effects on cortical neurons
53 against acute hypoxia are mediated by mechanisms other than PPARα or
54
55 TRPV1.
56
57
58 10
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 12 of 24

1
2
3 It is of note that TRPV4 is the receptor which is involved in neuroprotection
4
against hypoxia following OEA and PEA rather than PPARα or TRPV1. The
5
6 findings indicate that TRPV4 blocking further enhances the neuroprotective
7
8 effects of OEA and PEA, and its activation fully inhibits the neuroprotective
9 effects of the acylethanolamides. Simulation of TRPV4 was observed to be
10
11 cytotoxic per se for culture cortical neurons, as expected since the activation of
12 37-40
13 TRPV4 is known to induce cytotoxicity in many types of cells. A complete
14 protection against hypoxia of cortical neurons was afforded by high doses of the
15
16 TRPV4 antagonist RN1734. It seems that acylethanolamides could somehow
17
modulate TRPV4 activity, and blocking TRPV4 can enhance their
18
19 neuroprotective effects against hypoxia.
Fo
20
21
22 These results point to a new therapeutic approach for fighting cortical neuron
23
rP

24 death after a hypoxia episode, because co-treatment with acylethanolamides

25
26
and TRPV4 antagonists might be a pharmacological tool with protective effects
ee

27 in vivo as does it in vitro. Interestingly TRPV4 channels participate in important

28
29 mechanisms that are involved in deleterious effects of hypoxia such as changes
rR

30 37, 38, 41
in intracellular calcium concentration and astroglial reactivity. TRPV4 is
31
32 known to be sensitive to cell swelling and arachidonic acid and its metabolites,
ev

33 38
34 which are associated with cerebral ischemia. These hypoxia-induced
35 deleterious effects might be attenuated after blocking TRPV4. The
36
iew

37 neuroprotective role of these vanilloid receptors in ischemia episodes has been

38
highlighted recently. TRPV4 is involved in cerebral ischemic reperfusion injury
39
40 and recovery of brain edema. 36-37
Blocking TRPV4 may inhibit brain edema in
41 37, 40
42 cerebral ischemia, and reactive astrocytosis after stroke. TRPV4 is
43 upregulated and involved in neuronal injury during cerebral ischemia. 39
44
45
46
47 Funders
48 Supported by grants to EFE and FRF from Consejeria de Economia,
49
50 Innovacion, Ciencia y Empleo, Junta de Andalucia, Spain (EFE, group BIO127;
51
FRF, group BIO-339), Red de trastornos adictivos (RD16/0001/0017, Instituto
52
53 Carlos III), and from Fundació “La Marató de TV3” (386/C/2011). Pablo
54
55 Galeano and Juan I. Romero are research members of the CONICET
56 (Argentina). Eduardo Blanco is an associate professor of the Serra-Hunter
57
58 11
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 13 of 24 Mary Ann Liebert, Inc.

1
2
3 Programme from the Catalan Government. Mariana I. Holubiec is a fellowship
4
holder from ANPCyT (Argentina).
5
6
7
8 Acknowledgements
9 The authors thank Silvia Castellano and M.V. López for their help with cell
10
11 cultures, Mara Guerra for genotyping, and Itziar Benito for animal care
12
13 (Universidad de Sevilla).
14
15
16 Conflict of interest
17
The authors declare that there are no conflicts of interest
18
19
Fo
20
21
22
23
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 12
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 14 of 24

1
2
3 References
4
5
6 1.Ferriero DM. Neonatal brain injury. N Engl J Med. 2004; 351: 1985-1995.
7
8
9 2. Fernandez-Lopez D, Martinez-Orgado J, Casanova I, et al. Immature rat
10
11 brain slices exposed to oxygen–glucose deprivation as an in vitro model of
12
13 neonatal hypoxic–ischemic encephalopathy. J Neurosci Methods 2005;145:
14 205–212.
15
16
17
3. Fernandez-Lopez D, Martinez-Orgado J, Nunez E, et al. Characterization of
18
19 the neuroprotective effect of the cannabinoidagonist WIN-55212 in an in vitro
Fo
20
21 model of hypoxic–ischemic brain damage in newborn rats. Pediatr Res. 2006;
22 60: 169–173.
23
rP

24
25
26
4. Martinez-Orgado J, Fernandez-Lopez D, Moro MA, Lizasoain I. Nitric oxide
ee

27 synthase as a target for the prevention of hypoxic–ischemic newborn brain

28
29 damage. Curr Enzym Inhib. 2006; 2: 219–229.
rR

30
31
32 5. Peters JM, Hennuyer N, Staels B, et al. Alterations in lipoprotein metabolism
ev

33
34 in peroxisome proliferator-activated receptor alpha-deficient mice. J Biol Chem.
35 1997; 272(43): 27307-27312.
36
iew

37
38
6. Aoyama T, Peters JM, Iritani N, et al. Altered constitutive expression of fatty
39
40 acid-metabolizing enzymes in micelacking the peroxisome proliferator-activated
41
42 receptor alpha (PPARalpha). J Biol Chem. 1998; 273(10): 5678-5684.
43
44
45 7. Akiyama TE, Nicol CJ, Fievet C, et al. Peroxisome proliferator-activated
46
47 receptor-alpha regulates lipid homeostasis, but is not associated with obesity:
48 studies with congenic mouse lines. J Biol Chem. 2001; 276: 39088-39093.
49
50
51
8. Bełtowski J, Wójcicka G, Mydlarczyk M, Jamroz A. The effect of peroxisome
52
53 proliferator-activated receptors alpha (PPARalpha) agonist, fenofibrate, on lipid
54
55 peroxidation, total antioxidant capacity, and plasma paraoxonase 1 (PON 1)
56 activity. J Physiol Pharmacol. 2002 ; 53(3): 463-475.
57
58 13
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 15 of 24 Mary Ann Liebert, Inc.

1
2
3
4
9. Cuzzocrea S, Mazzon E, Di Paola R, et al.The role of the peroxisome
5
6 proliferator-activated receptor-alpha (PPAR-alpha) in the regulation of acute
7
8 inflammation. J LeukBiol. 2006; 79(5): 999-1010.
9
10
11 10. Kreisler A, Gelé P, Wiart JF, et al. Lipid-lowering drugs in the MPTP mouse
12
13 model of Parkinson's disease: fenofibrate has a neuroprotective effect,
14 whereas bezafibrate and HMG-CoA reductase inhibitors do not. Brain Res.
15
16 2007; 1135(1): 77-84.
17
18
19 11. Schmidt A, Vogel R, Holloway MK, et al. Transcription control and neuronal
Fo
20
21 differentiation by agents that activate the LXR nuclear receptor family. Mol Cell
22 End. 1999; 155: 51-60.
23
rP

24
25
26
12. Delerive P, Fruchart JC, Staels B. Peroxisome proliferator-activated
ee

27 receptors in inflammation control. J Endocrinol. 2001 ; 169: 453-459.

28
29
rR

30
13. Lleo A, Galea E, Sastre M. Molecular targets of non-steroidal anti-
31
32 inflammatory drugs in neurodegenerative diseases. Cell Mol Life Sci. 2007; 64:
ev

33
34 403-418.
35
36
iew

37 14. Kono K, Kamijo Y, Hora K, et al. PPAR{alpha} attenuates the

38
proinflammatory response in activated mesangial cells. Am J Physiol Renal
39
40 Physiol. 2009; 296(2): F328-36.
41
42
43 15. Xu J, Chavis JA, Racke MK, Drew PD. Peroxisome proliferator-activated
44
45 receptor-alpha and retinoid X receptor agonists inhibit inflammatory responses
46
47 of astrocytes. J Neuroimmunol. 2006; 176(1-2): 95-105.
48
49
50 16. Drew PD, Xu J, Storer PD, et al. Peroxisome proliferator-activated receptor
51
agonist regulation of glial activation: relevance to CNS inflammatory disorders.
52
53 Neurochem Internat. 2006; 49(2): 183-189.
54
55
56
57
58 14
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 16 of 24

1
2
3 17. Chen XR, Besson VC, Palmier B, et al. Neurological Recovery-Promoting,
4
Anti-Inflammatory, and Anti-Oxidative Effects Afforded by Fenofibrate, a PPAR
5
6 Alpha Agonist, in traumatic Brain Injury. J Neurotrauma 2007;24(7): 1119-1131.
7
8
9 18. Xu J, Racke MK, Drew PD. Peroxisome proliferator-activated receptor-alpha
10
11 agonist fenofibrate regulates IL-12 family cytokine expression in the CNS:
12
13 relevance to multiple sclerosis. J Neurochem. 2007; 103(5): 1801-1810.
14
15
16 19. Deplanque D, Gelé P, Pétrault O, et al. Peroxisome proliferator-activated
17
receptor-alpha activation as a mechanism of preventive neuroprotection
18
19 induced by chronic fenofibrate treatment. J Neurosci. 2003 ; 23(15): 6264-
Fo
20
21 6271.
22
23
rP

24 20. Poynter ME, Daynes RA. Peroxisome proliferator-activated receptor alpha

25
26
activation modulates cellular redox status, represses nuclear factor-kappaB
ee

27 signaling, and reduces inflammatory cytokine production in aging. J Biol Chem.

28
29 1998; 273: 32833-32841.
rR

30
31
32 21. Racke MK, Gocke AR, Muir M, et al. Nuclear receptors and autoimmune
ev

33
34 disease: the potential of PPAR agonists to treat multiple sclerosis. J Nutr. 2006;
35 136(3): 700-703.
36
iew

37
38
22. Galan-Rodriguez B, Suárez J, Gonzalez-Aparicio R, et al.
39
40 Oleoylethanolamide exerts partial and dose-dependent neuroprotection of
41
42 substantia nigra dopamine neurons. Neuropharmacology 2009; 56: 653-664.
43
44
45 23. Diab A, Hussain RZ, Lovett-Racke AE, et al. Ligands for the peroxisome
46
47 proliferator-activated receptor-gamma and the retinoid X receptor exert additive
48 anti-inflammatory effects on experimental autoimmune encephalomyelitis. J
49
50 Neuroimmunol. 2004; 148: 116-126.
51
52
53 24. Rodriguez de Fonseca F, Navarro M, Gomez R, et al. Anorexic lipid
54
55 mediator regulated by feeding. Nature 2001; 414(6860): 209-212.
56
57
58 15
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 17 of 24 Mary Ann Liebert, Inc.

1
2
3 25. Fu J, Gaetani S, Oveisi F, Lo Verme J, et al. Oleoylethanolamide regulates
4
feeding and body weight through activation of the nuclear receptor PPAR-alpha.
5
6 Nature 2003; 425(6953): 90-93.
7
8
9 26. Lo Verme J, Fu J, Astarita G, et al. The nuclear receptor peroxisome
10
11 proliferator-activated receptor-alpha mediates the anti-inflammatory actions of
12
13 palmitoylethanolamide. Mol Pharmacol. 2005; 67(1): 15-19.
14
15
16 27. Lombardi G, Miglio G, Varsaldi F, et al. Oxyhomologation of the amide bond
17
potentiates neuroprotective effects of the endolipid N-palmitoylethanolamine. J
18
19 Pharmacol Exp Ther. 2007; 320: 599-606.
Fo
20
21
22 28. Bisogno T, Martire A, Petrosino S, et al. Symptom-related changes of
23
rP

24 endocannabinoid and palmitoylethanolamide levels in brain areas of R6/2 mice,

25
26
a transgenic model of Huntington’s disease. Neurochem Internat 2008; 52:
ee

27 307-313.
28
29
rR

30
29. Sun Y, Alexander SP, Garle MJ, et al. Cannabinoid activation of PPAR
31
32 alpha, a novel neuroprotective mechanism. Brit J Pharmacol. 2007; 152: 734-
ev

33
34 743.
35
36
iew

37 30. Ho WS, Barrett DA, Randall MD. 'Entourage' effects of N-

38
palmitoylethanolamide and N-oleoylethanolamide on vasorelaxation to
39
40 anandamide occur through TRPV1 receptors. Br J Pharmacol. 2008;
41
42 155(6):837-46.
43
44
45 31. Mezey E, Tóth ZE, Cortright DN, et al. Distribution of mRNA for vanilloid
46
47 receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous
48 system of the rat and human. Proc Natl Acad Sci USA 2000; 97(7):3655-3660.
49
50
51
32. Wang X, Miyares RL, Ahern GP. Oleoylethanolamide excites vagal sensory
52
53 neurones, induces visceral pain and reduces short-term food intake in mice via
54
55 capsaicin receptor TRPV1. J Physiol. 2005; 564: 541-547.
56
57
58 16
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 18 of 24

1
2
3 33. Thabuis C, Tissot-Favre D, Bezelgues JB, et al. Biological functions and
4
metabolism of oleoylethanolamide. Lipids 2008; 43: 887-894.
5
6
7
8 34. Kim SR, Lee DY, Chung ES, et al. Transient receptor potential vanilloid
9
10 subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo
11 and in vitro. J Neurosci. 2005; 25(3): 662-671.
12
13
14
15 35. Marinelli S, Di Marzo V, Florenzano F, et al. N-Arachidonoyl-Dopamine Tunes
16 Synaptic Transmission onto Dopaminergic Neurons by Activating both
17
18 Cannabinoid and Vanilloid Receptors. Neuropsychopharmacology 2006; 32,
19
298-308.
Fo
20
21
22
23 36. Ding H, Lin YX, Shen QW, et al. Research progress of TRPV4 and cerebral
rP

24 ischemic reperfusion injury. Sheng Li Xue Bao 2015; 67(5):527-32.

25
26
ee

27
28 37. Jie P, Tian Y, Hong Z, et al. Blockage of transient receptor potential
29 vanilloid 4 inhibits brain edema in middle cerebral artery occlusion mice. Front
rR

30
31 Cell Neurosci. 2015; 9:141.
32
ev

33
34 38. Jie P, Hong Z, Tian Y, et al. Activation of transient receptor potential
35
36 vanilloid 4 induces apoptosis in hippocampus through downregulating PI3K/Akt
iew

37 and upregulating p38 MAPK signaling pathways. Cell Death Dis. 2015;
38
39 6:e1775.
40
41
42 39. Jie P, Lu Z, Hong Z, et al. Activation of Transient Receptor Potential
43
44 Vanilloid 4 is Involved in Neuronal Injury in Middle Cerebral Artery Occlusion in
45
Mice. Mol Neurobiol. 2016; 53(1):8-17.
46
47
48
49 40. Rakers C, Schmid M, Petzold GC . TRPV4 channels contribute to calcium
50 transients in astrocytes and neurons during peri-infarct depolarizations in a
51
52 stroke model. Glia 2017; 65(9):1550-1561.
53
54
55
56
57
58 17
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 19 of 24 Mary Ann Liebert, Inc.

1
2
3 41. Butenko O, Dzamba D, Benesova J, et al. The increased activity of TRPV4
4
channel in the astrocytes of the adult rat hippocampus after cerebral
5
6 hypoxia/ischemia. PLoS One 2012; 7(6):e39959.
7
8
9 42. Hansen HS, Moesgaard B, Petersen G, Hansen HH. Putative
10
11 neuroprotective actions of N-acyl-ethanolamines. Pharmacol Ther. 2002;
12
13 95(2):119-126.
14
15
16 43. Hansen HS, Moesgaard B, Hansen HH, Petersen G. N-Acylethanolamines
17
and precursor phospholipids - relation to cell injury. Chem Phys Lipids 2000;
18
19 108(1-2):135-150.
Fo
20
21
22 44. Franklin A, Parmentier-Batteur S, Walter L, et al. Palmitoylethanolamide
23
rP

24 increases after focal cerebral ischemia and potentiates microglial cell motility. J
25
26
Neurosci. 2003; 23:7767-7775.
ee

27
28
29 45. Gonzalez-Aparicio R, Flores JA, Tasset I, et al. Mice lacking the peroxisome
rR

30
proliferator-activated receptor alpha gene present reduced number of dopamine
31
32 neurons in the substantia nigra without altering motor behavior or dopamine
ev

33
34 neuron decline over life. Neuroscience 2011; 186:161-169.
35
36
iew

37 46. Cardozo DL. Midbrain dopaminergic neurons from postnatal rat in long-term
38
primary culture. Neuroscience 1993; 56(2): 409-421.
39
40
41
42 47. Mena MA, Davila V, Sulzer D. Neurotrophic effects of L-DOPA in postnatal
43 midbrain dopamine neuron/cortical astrocyte cocultures. J Neurochem. 1997; 69:
44
45 1398-1408.
46
47
48 48. Molina-Holgado F, Pinteaux E, Heenan L, et al. Neuroprotective effects of the
49
50 synthetic cannabinoid HU-210 in primary cortical neurons are mediated by
51
phosphatidylinositol 3-kinase/AKT signaling. Mol Cell Neurosci. 2005; 28(1):
52
53 189-194.
54
55
56
57
58 18
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 20 of 24

1
2
3 Figure legends
4
5
6 Figure 1. Percent cell survival of cortical neurons after OEA given before and
7
8 after hypoxia episode. Mean ± SEM. * p<0.05, ** p<0.01 versus 0 dose
9 (Newman-Keuls).
10
11
12
13
14
15
16
17
18
19
Fo
20
21
22
23
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 19
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 21 of 24 Mary Ann Liebert, Inc.

1
2
3 Figure 2. Percent cell survival of cortical neurons after PEA given before and
4
after hypoxia episode. Mean ± SEM. * p<0.05, ** p<0.01 versus 0 dose
5
6 (Newman-Keuls).
7
8
9
10
11
12
13
14
15
16
17
18
19
Fo
20
21
22
23
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 20
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 22 of 24

1
2
3 Figure 3. Percent cell survival of cortical neurons after OEA and PEA in
4
PPARα +/+ wild-type (WT) and PPARα -/- knockout (KO) mice. All compounds
5
6 were given before hypoxia episode. Mean ± SEM. * p <0.05, ** p<0.01 versus 0
7
8 dose (Newman-Keuls).
9
10
11
12
13
14
15
16
17
18
19
Fo
20
21
22
23
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 21
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 23 of 24 Mary Ann Liebert, Inc.

1
2
3 Table 1. Percent cell survival effects of 40µM OEA and PEA after adding
4
5
GW6471, selective PPARα antagonist.
6
7
8 40 µM OEA 40 µM PEA GW6471 GW6471
9
alone dose (µM)
10
11 94.1±3 94.5±3 34.3±3 0
12
13 93.3±4 93.6±2 36.1±2 0.1
14 91.4±2 95.4±4 37.2±4 1
15
16 95.2±3 95.3±2 38.3±3 5
17
18 93.4±2 96.2±1 38.4±4 10
19
Fo
20
21 Mean ± SEM. Compounds were added to culture media before hypoxia
22
23
episode. Abbrev.: OEA, oleoylethanolamide; PEA, palmitoylethanolamide.
rP

24
25
26
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 22
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
 Mary Ann Liebert, Inc. Page 24 of 24

1
2
3 Table 2. Percent cell survival effects of 40µM OEA and PEA after blocking
4
TRPV1 with SB 452533.
5
6
7
8 40 µM OEA 40 µM PEA SB 45253 SB 452533
9 alone dose (µM)
10
11 95.2±5 95±3 52.8±4 0
12
13 94.4±3 98.1±5 52.6±5 0.1
14
15 98.7±4 97.8±2 51.8±5 1
16
17 97±5 96.3±2 52±5 5
18
19 93±4 95.6±2 54.3±4 10
Fo
20
21
22
23 Mean ± SEM. All compounds were given before hypoxia. Abbrev.: OEA,
rP

24
oleoylethanolamide; PEA, palmitoylethanolamide; TRPV1, transient receptor
25
26 potential vanilloid type 1.
ee

27
28
29
rR

30
31
32
ev

33
34
35
36
iew

37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 23
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com
Page 25 of 24 Mary Ann Liebert, Inc.

1
2
3 Table 3. Percent cell survival effects of 40µM OEA and PEA after blocking
4
or stimulating TRPV4 with RN1734 or GSK1016790A, respectively.
5
6
7
8 Blocking TRPV4
9
40 µM OEA 40 µM PEA RN1734 alone RN1734 dose
10
11 (µM)
12
13 92.1±3 93.8±4 47.4±5 0
14
15 91.2±4 97.1±3 48.3±5 0.1
16
17 94.3±4 97±3 55.4±4 1
18
19 98±1 100* 64.4±3 # 5
Fo
20
21 100 * 100* 65.5±5 # 10
22
23 Stimulating TRPV4
rP

24
25
40 µM OEA 40 µM PEA GSK1016790A GSK1016790A
26 alone (nM)
ee

27
28 92.8±4 92.2±3 45.4±7 0
29
rR

30 91.2±4 94.1±3 33.4±5 ^ 1

31
32 93.3±3 92±3 31.5±5 ^ 5
ev

33
34
35
36
Mean ± SEM. * p<0.01 versus corresponding 0 and 0.01 dose of RN1734 +
iew

37 OEA/PEA (Newman-Keuls). # p<0.01 versus corresponding 0 and 0.01 dose of

38
39 RN1734 alone (Newman-Keuls). ^ p<0.01 versus corresponding 0 dose of
40
GSK1016790A alone (Newman-Keuls). All compounds were given before the
41
42 hypoxia episode. Abbrev.: OEA, oleoylethanolamide; PEA,
43
44 palmitoylethanolamide; TRPV4, transient receptor potential vanilloid type 4.
45
46
47
48
49
50
51
52
53
54
55
56
57
58 24
59
60 ScholarOne Support phone: 434-964-4100 email: ts.mcsupport@thomson.com