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Autonomic Neuroscience: Basic and Clinical 133 (2007) 3 18

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Review

Regulation of salivary gland function by autonomic nerves


Gordon B. Proctor , Guy H. Carpenter
Salivary Research Unit, King's College London Dental Institute, Floor 17 Guy's Tower, London SE1 9RT, UK
Received 16 August 2006; received in revised form 6 October 2006; accepted 20 October 2006

Abstract
Oral homeostasis is dependent upon saliva and its content of proteins. Reflex salivary flow occurs at a low resting rate and for short periods
of the day more intense taste or chewing stimuli evoke up to ten fold increases in salivation. The secretion of salivary fluid and proteins is
controlled by autonomic nerves. All salivary glands are supplied by cholinergic parasympathetic nerves which release acetylcholine that binds to
M3 and (to a lesser extent) M1 muscarinic receptors, evoking the secretion of saliva by acinar cells in the endpieces of the salivary gland ductal
tree. Most salivary glands also receive a variable innervation from sympathetic nerves which released noradrenaline from which tends to evoke
greater release of stored proteins, mostly from acinar cells but also ductal cells. There is some cross-talk between the calcium and cyclic AMP
intracellular pathways coupling autonomic stimulation to secretion and salivary protein secretion is augmented during combined stimulation.
Other non-adrenergic, non-cholinergic neuropeptides released from autonomic nerves evoke salivary gland secretion and parasympathetically
derived vasointestinal peptide, acting through endothelial cell derived nitric oxide, plays a role in the reflex vasodilatation that accompanies
secretion. Neuronal type, calcium-activated, soluble nitric oxide within salivary cells appears to play a role in mediating salivary protein
secretion in response to autonomimetics. Fluid secretion by salivary glands involves aquaporin 5 and the extent to which the expression of
aquaporin 5 on apical acinar cell membranes is upregulated by cholinomimetics remains uncertain. Extended periods of autonomic denervation,
liquid diet feeding (reduced reflex stimulation) or duct ligation cause salivary gland atrophy. The latter two are reversible, demonstrating that
glands can regenerate provided that the autonomic innervation remains intact. The mechanisms by which nerves integrate with salivary cells
during regeneration or during salivary gland development remain to be elucidated.
2006 Elsevier B.V. All rights reserved.
Keywords: Saliva; Salivary glands; Autonomic nerves; Sympathetic; Parasympathetic; Secretion

Contents
1.
2.
3.

4.
5.
6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reflex salivary secretion . . . . . . . . . . . . . . . . . . . . .
Salivary glands and their innervation . . . . . . . . . . . . . . .
3.1. The development of salivary glands . . . . . . . . . . . .
3.2. The development of nerves in salivary glands . . . . . .
3.3. Innervation of adult salivary glands . . . . . . . . . . . .
The short term effects of autonomic nerves on salivary function.
The coupling of autonomic nerve stimulation to secretion . . . . .
Trophic effects of nerves and salivary gland regeneration . . . .
6.1. Effects of denervations on salivary secretion . . . . . . .
6.2. The effects of denervation on salivary gland structure . .
6.3. The involvement of nerves in salivary gland regeneration .

Corresponding author.
E-mail address: gordon.proctor@kcl.ac.uk (G.B. Proctor).
1566-0702/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.autneu.2006.10.006

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G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

7. Conclusions and clinical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Saliva in the mouth is the mixed product of 3 pairs of
major salivary glands, the parotid, submandibular and sublingual glands as well as numerous minor salivary glands
found in the submucosa under most soft tissue surfaces in the
mouth. Whole mouth saliva also contains small amounts of
other fluids and products of the mucosal surface. Like other
mucosal fluids saliva forms a mobile layer on the mucosal
surface and contains an array of components that fulfil
important functions. In fact protection of the oral mucosal
surface is provided by components which are also present in
other mucosal fluids, such as mucins (MUC5B and MUC7)
and secretory IgA (sIgA) as well as components which
appear to be less widely detected on other surfaces or may be
peculiar to saliva (e.g. histatins, agglutinin). Unlike other
mucosal fluids saliva contains a range of components that
interact with and protect teeth (e.g. proline rich proteins,
statherins). Saliva is crucial in maintaining the integrity of
the oral mucosal surface and in preserving an ecological
balance (Hay and Bowen, 1999). The roles played by
different salivary components in protecting the soft and hard
tissues of the mouth has been reviewed by others (Amerongen and Veerman, 2002).
Most of the components of saliva in the mouth water,
ions, proteins are actively secreted by salivary glands. The
secretory endpiece of salivary glands consists of acinar
secretory units made up of acinar cells which are responsible
for synthesising and secreting most of the functionally
important protein components of saliva (Segawa and
Yamashina, 1998; Proctor, 1998). The acini are specialised
to each gland and are classified to reflect the proteins secreted by each type of acinithe parotid glands have mainly
serous, the sublingual mainly mucous and the submandibular
a mixture of the two. In the same way that diets vary from
one species to another so too are the salivary glands which
are specialised for the diet (Tandler and Phillips, 1998) and
therefore this simple classification of acinar cells does not fit
all species. Water and electrolytes are actively transported by
acinar cells and then the electrolyte content of saliva is
modified, principally with the removal of sodium chloride
during passage through the ductal system to the mouth (Melvin
et al., 2005; Turner and Sugiya, 2002). Saliva is therefore
converted from an isotonic to a hypotonic solutionwhich
aids the detection of salt in the diet. This is an energy rich
process so that the most active ducts have large numbers of
mitochondria located in the basolateral part of the cells leading
to their description as striated ducts. In addition to removing
ions they also add potassium and bicarbonatethe latter forms

14
15
15

an important component of the buffering system of saliva that


prevents dissolution of teeth by acid producing bacteria. Most
ductal cells secrete only small amounts of protein. However,
granular ductal cells, which are peculiar to submandibular
glands of some rodents including mouse and rat, are packed
full of tissue kallikreins. In addition to the parenchymal
component of salivary glands there are myoepithelial cells (see
Fig. 1) which support the acini in some but not all salivary
glands and may help expel secretions from the ductal system.
Plasma cells present in the gland interstitium, secrete
immunoglobulins which are transported by salivary cells
into saliva and a dense network of blood vessels supply the
fluid component of saliva (via the interstitial space).
Controlling and influencing all these cells are parasympathetic
and sympathetic autonomic nerves which work together
harmoniously to evoke secretion.
2. Reflex salivary secretion
Salivary gland secretion is a nerve mediated reflex and
once the autonomic nerve supply, particularly the parasympathetic nerve, has been interrupted then secretion from most
glands ceases almost entirely. There are a few salivary glands
that maintain a spontaneous secretion in the absence of
nerve mediated stimuli but even in these glands a normal rate
of secretion requires an intact autonomic nerve supply
(Emmelin, 1972). Salivary glands provide a resting flow of
saliva into the mouth that fulfils the protective role referred
to above. Then for short periods during overt reflex
stimulation provided by the taste and chewing of food, an
enormously increased activity is superimposed upon the
resting flow (Emmelin, 1972). Hector and Linden (1999)
have reviewed in detail how taste, mastication and other
stimuli evoke reflex salivary secretion through various
receptors including gustatory receptors, mechanoreceptors,
olfactory receptors and nociceptors. Thus not only the
volume but also the composition of mixed saliva in the
mouth can vary depending upon the contribution of different
glands during reflex stimulation. The parotid gland has a
very low secretory rate under resting (unstimulated) conditions compared to during stimulation. In contrast the
submandibular/sublingual glands secrete relatively more
saliva under resting conditions (Shannon et al., 1969). It
has been shown that different afferent stimuli can change the
composition of saliva secreted by a single gland. A relatively
greater amount of IgA was present in chewing stimulated
human parotid saliva compared to citric acid evoked saliva
(Proctor and Carpenter, 2002). Others have shown that sweet
stimulated human parotid saliva has a higher protein

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Fig. 1. Structural elements within rat submandibular glands digested with collagenase. A, Unstained phase contrast image. Distinct acini (Ac) and ducts (Dc) may
be separate or joined. B, myoepithelial cells surrounding acinar groups demonstrated by actin staining. Images B and C are composites of a Z-series of images
taken by confocal microscopy, colour relates to depth of field. C, tight junctions between cells demonstrated by occludin staining. The ductal lumen is much
larger than the adjacent acinar lumena. D, Cholinesterase staining of parasympathetic nerves surrounding the acinar units. Thinner fibres are sometimes seen
(arrow) terminating at a cell.

concentration compared to acid stimulated saliva (Mackie


and Pangborn, 1990). There have been a few studies in
which the effects of different reflex stimuli have been studied
in animal models. Gjorstrup (1980) and later Ikawa et al.
(1991) showed that higher concentrations of salivary
amylase and other proteins were secreted into rabbit parotid
saliva evoked by carrots compared to standard pelleted
chow.
Nerve impulses in the afferent limbs of the salivary reflex
pass to the salivary nuclei within the medulla oblongata and
from these centres efferent parasympathetic secretomotor
nerves emerge to supply the parenchyma of salivary glands
(see Fig. 2). The integration of impulses from primary
salivary centres to glands depends on central modulation.
The central neural connections between primary salivary
centres and other nuclei are not well understood and continue
to be investigated. Retrograde labelling of neurons has demonstrated that the primary parasympathetic salivary centres
form connections with the lateral hypothalamus where the
regulation of feeding, drinking and body temperature occurs.

It appears that both excitatory (gamma aminobutyric acid


containing) and inhibitory (glycine containing) nerves
synapse with the salivary centres (Matsuo, 1999). Further
evidence of the involvement of central mechanisms in modulating salivary secretion was reported following intracerebroventricular injection of pilocarpine or atropine which
were found to respectively stimulate and inhibit salivation
(Renzi et al., 2002; Takakura et al., 2003). However, a more
recent study questioned the high doses used in the earlier
work (Sato et al., 2006). Alpha(2) adrenoceptor agonists
(e.g. clonidine) and antagonists (e.g. yohimbine) have been
demonstrated to act centrally in studies of reflex secretion in
human subjects and cholinergically evoked secretion in
animal models. Alpha(2) adrenoceptor blockade increases
salivary secretion whilst stimulation by alpha(2) adrenoceptor agonists inhibits secretion (Moreira et al., 2002; Phillips
et al., 2000). The primary sympathetic salivary centres are
located in the upper thoracic segments of the spinal cord
although it remains unclear precisely where in this region
(Bradley et al., 2005; Matsuo, 1999). It has been understood

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Fig. 2. Salivary reflex secretion. Afferent stimuli are integrated in the primary salivary centres of the medulla. Autonomic parasympathetic efferent nerves
conduct signals to salivary glands via parasympathetic ganglia stituated near the target gland. Nerves project (lower broken line) from the medulla to the
sympathetic centre in the upper thoracic segments of the spinal cord and from here sympathetic efferent nerves conduct signals to salivary glands via the superior
cervical ganglion. Nerves project (upper broken line) from the cortex to the parasympathetic centres in the medulla and these can have an excitatory or inhibitory
effect on salivary secretion. Efferent autonomic nerves stimulate salivary secretion and there is no peripheral inhibition of secretion via sympathetic nerves.

for some time that central inhibition as a result of


connections between the primary salivary centres and the
higher centres of the brain are responsible for the dry mouth
associated with anxiety. It is worth repeating at this point that
there is no peripheral inhibition of salivary secretion under
normal conditions. The concept of peripheral sympathetic
inhibition of salivary secretion which later became widely
accepted, was appreciated as an experimental artefact over a
century ago (see Garrett, 1987). Experimental electrical
stimulation of the sympathetic nerve supply to salivary
glands or use of alpha(1) adrenoceptor agonists in anaesthetized animals leads to a vasoconstriction of glandular
blood vessels in addition to activation of parenchymal cells.
In contrast under reflex conditions only sympathetic secretomotor nerve fibres and not vasoactive nerve fibres to salivary
glands are activated. Thus vasoconstriction is not part of the
salivary reflex.
3. Salivary glands and their innervation
3.1. The development of salivary glands
During embryogenesis the major salivary glands develop
from the ectoderm whereas the minor glands originate from
the mesoderm. In the genetic syndrome salivary gland
agenesis there is an absence of ectodermal-derived structures
such as sweat glands and the major salivary glands but minor
salivary glands are present (Nordgarden et al., 2001). The
first visible sign of salivary glands in the embryo (e13 in rats,
e8 wks in humans) involves interactions between the

epithelial layer and the mesenchyme (derived from the


neural crest) resulting in a local thickening of the epithelium
to form a placode. Some of the signalling occurring at this
stage involves Sonic Hedgehog (Shh) (Jaskoll et al., 2004),
Epithelial Growth factor (EGF) and Transforming growth
factor (TGF) (Jaskoll and Melnick, 1999) and Ectodysplasin (Eda) and its receptor (EdaR) (Jaskoll et al., 2003).
Each of these signalling molecules only has a partial role
since mice with one of these genes knocked out have defects
in, but not a complete absence of, salivary glands. Following
the formation of the salivary placode salivary gland
development has been well characterised as in Fig. 3.
EGF receptor and its two ligands, EGF and TGF appear
to be important in co-ordinating the regulated cell proliferation during salivary gland development. EGF is not present
in the initial bud stage and so signalling occurs via TGF
localised on the cell surface and opposing mesenchyme
layer. By early canalicular stage EGF is first detected in the
epithelia of the terminal end buds. By late canalicular stage
TGF is newly located on the ductal epithelia facing the
lumen, i.e. where most proliferation and apoptosis is
occurring (Jaskoll and Melnick, 1999). If glands are removed
at the canalicular stage and stimulated in vitro by autonomimetics a specific group of proteins are secreted reflecting the two main cell typesType 1 terminal tubule
cells and Type 3 proacinar cells (Fig. 3D). Interestingly there
are differences in which agonists cause the secretion of these
proteins (Ball et al., 1988) suggesting that the different
mechanisms of protein secretion (and the control thereof)
seen in the adult are also present in the neonatal salivary

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Fig. 3. The development of rat submandibular salivary glands. Starting at


embryonic day e12/13 the initial bud stage (A) there is a proliferation and
infolding of the epithelial layer (E) of the oral cavity (OC) into the adjoining
condensed mesenchyme (CM) to form the pseudoglandular stage (B) in
which an elongated cord of epithelial (E) cells extends into the surrounding
mesenchyme (M). The appearance of a ductal cell lumen (L) characterises the
onset of the canalicular stage (C) and the terminal bud stage is reached once
lumena extend into the epithelial buds (D). At this stage two cell types are
apparent-type 3 pro-acinar cells and type 1 terminal tubules cells. From birth
to day 30 salivary glands develop their mature phenotype (E) of acinar cells
and intercalated, granular and striated ductal cells.

gland. Between birth and adult (Fig. 3E) Type 3 proacinar


cells develop into acini (Ac) and eventually sero-mucous
cells whilst Type 1 cells maintain a central location possibly
forming intercalated cells (Hand et al., 1996; Jaskoll and
Melnick, 1999); striated ducts (SD) appear to develop postnatally (Redman et al., 2002).
3.2. The development of nerves in salivary glands
Saliva can be produced from rat salivary glands at birth by
stimulating parasympathetic nerves implying a functional
neuro-epithelial junction although sympathetic nerves and
secretory granule movement in response to sympathetic
nerve stimulation does not occur until 35 days after birth

when catecholamine-containing nerves start to appear


(Bottaro and Cutler, 1984). The development of sympathetic
nerves occurs at a similar time to the development of proacinar and terminal tubule Types 1 and 3 cells into mature
acinar and ductal cells and a neonatal sympathetic
denervation reduced parotid acinar development (Henriksson et al., 1985) which implies a role for the innervation in
the final differentiation of Types 1 and 3 cells into functional
acinar and ductal structures (Danielsson et al., 1988; Bottaro
and Cutler, 1984). A dichotomy of sympathetic nerve development in the rat submandibular and sublingual glands exists
since despite developing in the same capsule surrounded by
the same mesenchymal cap the sublingual gland contains
very few sympathetic nerves whereas the submandibular
gland has a rich innervation (see Fig. 4). This presumably
reflects the lack of expression of the neurotrophin NGF in
sublingual glands as the expression of this molecule correlates closely with sympathetic innervation in other tissues
(Glebova and Ginty, 2004).
Parasympathetic nerves develop in parallel with the salivary parenchyma and require an interaction with the parenchyma in order to develop (Coughlin, 1975). By separating
the submandibular ganglion from the salivary epithelium at
an early stage (e 11 in the mouse) the development of
cholinesterase containing nerves was inhibited although the
salivary parenchyma developed normally in the absence of
the submandibular ganglion (note the opposite relationship
occurs later at pro-acinar to mature cell conversion stage
which requires parasympathetic input). Which signalling
molecules interact between nerves and salivary cells is not
clear although, as indicated above, EGF signalling is active.
Several recent studies using knockout mice have indicated a
significant role for glial cell derived neurotrophins (GDNFs)
affecting peripheral nerve innervation. Glial cells (also
known as Schwann cells) support, guide and interact with
nerve axons right up to the target cell. Although parasympathetic and sympathetic nerves enter glands from
separate sources they quickly align themselves and combined parasympathetic/sympathetic/Schwann cell bundles
are common throughout most salivary glands (Garrett and

Fig. 4. Sympathetic nerves in rat salivary glands. Catecholamine fluorescence of cells from digested rat parotid (A), sublingual (B) and submandibular (C) glands.
Both parotid and submandibular glands have a dense sympathetic innervation particularly surrounding the acini (Ac) but also present on ductal structures (Dc),
whereas the sublingual gland has a sparse sympathetic innervation except around the occasional duct.

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Kidd, 1993). For parasympathetic nerves the main GDNF


appears to be neurturin acting on GDNF family receptor
alpha 2 (GFR2) molecules expressed by nerve axons which
appears to be important for the development of peripheral
fibres. Knocking out GFR2 greatly reduced the parasympathetic innervation of acini in the sublingual gland (Rossi et
al., 1999). For sympathetic nerves, Nerve growth factor
(NGF) has been known for a long time to be important.
Indeed NGF was first isolated from rodent salivary glands
(Cohen, 2004) as they have such a high concentration. NGF
acts through a high (TrkA) and a low affinity receptor (p75)TrkA promotes cell survival and axon elongation and p75
activation invokes cell death (Dechant and Barde, 2002).
Knocking out p75 actually enhanced sympathetic innervation of mouse submandibular glands (Jahed and Kawaja,
2005) whereas knocked out NGF inhibited sympathetic
innervation of salivary glands (Ghasemlou et al., 2004).
The guidance molecules for nerves in salivary glands are
unknown but by comparison with other models (Zou, 2004)
Wnt binding to frizzled receptors could be important to axon
guidance by acting as both attractants and repellents whilst
ectodysplasin A (Eda) and its receptor (EdaR) also have
forward and reverse signalling capabilities (Hinck, 2004).
Secreted frizzled (1 and 4) are strongly expressed by salivary
cells at e15.5, (Leimeister et al., 1998) and WNT4 mRNA
expression has been shown to be increasingly expressed in
mouse from e14 to adultin fact, with NGF it is one of the
few proteins to be actively increased from post-partum day 5
to adult (Hoffman et al., 2002).
3.3. Innervation of adult salivary glands
From the two separate nerve supplies to the salivary gland
sympathetic and parasympathetic nerves run together with
Schwann cells as one bundle up to the target cell (Garrett and
Kidd, 1993). From there some nerves may supply several
more target cells as unmyelinated axons. There appears to be
no specialised neuro-effector sites, neurotransmitter release
may occur anywhere along the nerve, although nerve
terminals may have a bulbous ending and varicosities
along the length of the nerve (Ohtani et al., 1983) these
should not be thought of as the only place where
neurotransmitter release may occur. Furthermore electron
microscopical studies have indicated that two types of nerveepithelial cell relationship exist-epilemmal and hypolemmal
(Garrett and Kidd, 1993). Epilemmal nerve arrangements sit
on top of the epithelial cell, surrounded by a Schwann cell,
and are considered less efficient, in terms of neurotransmission, as the neurotransmitter must pass through two
basement membranes (Schwann cell and epithelial cell) to
reach the receptor. Hypolemmal nerves sit between epithelial
cells and are not surrounded by Schwann cell. However, no
evidence exists for any difference between their relative
efficiencies of each nerve arrangement. The relative
proportion of epi- and hypolemmal nerves varies according
to the gland being studied for instance the rat parotid has

epi- and hypolemmal nerves whereas the rat submandibular


has only epilemmal nerves (Garrett and Kidd, 1993)
although interestingly, hypolemmal nerves exist in the
developing rat submandibular gland (Yohro, 1971).
Parasympathetic and sympathetic nerves appear to be in
contact with most cell types in salivary glands. Most nerve
staining is associated with the acinar cells and their
associated myoepithelial cells which both receive dual
innervation (see Figs. 1 and 4). Although the rat sublingual
has a paucity of adrenergic innervation some sympathetic
nerves were seen associated with striated ducts and blood
vessels (Garrett et al., 1991). Blood vessels also have dual
innervation-parasympathetic stimulation causing vasodilataion and sympathetic stimulation vasoconstriction, although
it should be remembered that such vasoconstriction is not
part of the salivary reflex (Bernard, 1858 cited in (Garrett,
1999)). Vasodilatation in the rat submandibular caused by
acetylcholine and VIP release from parasympathetic nerves
(Anderson and Garrett, 1998) is mediated by nitric oxide and
other endothelium-derived hyperpolarizing factors (Anderson et al., 2006) nitric oxide appears to be less important
as the size of the blood vessel decreases.
As well as the main neurotranmitters acetylcholine and
adrenaline there are other non-adrenergic, non-cholinergic
(NANC) transmitters within nerves in salivary glands.
Neuropeptide Y (NPY), neurokinin A (NKA), substance P
(SP), vasoactive intestinal peptide (VIP), pituitary adenylate
cyclase activating peptide (PACAP), neuronal nitric oxide
synthase (nNOS) and calcitonin gene-related peptide
(CGRP) have all been detected within either parasympathetic, sympathetic or sometimes both nerves (reviewed in
(Ekstrom, 1999)). These neuropeptides can have effects on
the blood vessels and on the salivary cells enhancing protein
and/or fluid secretory responses (see later). There is some
specific localisation of peptide containing nerves as VIP
nerves are more numerous around the mucous acinar cells in
the human submandibular gland (Kusakabe et al., 1998),
their expression may change with salivary gland development (Virta et al., 1992) and focal infiltrates of inflammatory
cells may downregulate nerve expression of neuropeptides
(Pedersen et al., 2000). Some of these NANC transmitters
are also found in sensory nerve fibres within the salivary
glands. By using the sensory neurotoxin capsaicin to destroy
sensory nerve fibres only nerves containing both CGRP and
SP, usually located around ducts and blood vessels, were
diminished (Dunerengstrom et al., 1985).
4. The short term effects of autonomic nerves on salivary
function
The efferent autonomic secretomotor nerves supplying
salivary glands stimulate secretion and there is no antagonism between the two branches of the autonomic nervous
system (Emmelin, 1987). Garrett (1987) produced a useful
summary of the effects of parasympathetic and sympathetic
impulses on salivary glands (Table 1).

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318
Table 1
Effects of autonomic nerves on salivary gland function (modified from
Garrett, 1987)
Parasympathetic stimulation
1) Is mediated mainly by acetylcholine in combination with NANC
peptides (e.g. VIP)
2) Evokes most of the salivary fluid secreted. Mainly acts through M3 and
to a lesser extent M1 muscarinic cholinergic receptors
3) Causes variable degrees of exocytosis from salivary cells but is
responsible for most mucin secretion by mucous glands
4) Induces contraction of myoepithelial cells
5) Increases glandular blood flow as part of the salivary reflex

sympathetic nerve supplies to the rat parotid (Asking and


Gjorstrup, 1987) or submandibular glands (Anderson et al.,
1995; Carpenter et al., 2000) is superimposed upon a
background of parasympathetic nerve stimulation, then large
increases in protein secretion are observed (Figs. 5 and 6a).
Such dual stimulation experiments are thought to better

Sympathetic stimulation
1) Is mediated mainly by noradrenaline and acts essentially on cells
receiving parasympathetic impulses, which tends to produce synergistic
effects, but exerts little effect on mucous gland secretion
2) Often does not cause much mobilization of fluid but DOES NOT
inhibit salivary secretion
3) Tends to modulate the composition of saliva by increasing exocytosis
from salivary cells
4) Induces contraction of myoepithelial cells
5) Exerts control on glandular blood flow but NOT as part of the salivary
reflex

It can be seen from Table 1 that parasympathetic impulses


usually evoke most of the fluid secretion into saliva whilst
sympathetic nerves have less of a fluid evoking role. There
has been a tendency to dichotomize the respective roles of
the nerves further by attributing salivary protein secretion
almost entirely to sympathetic nerve impulses. The latter is
an oversimplification since various studies have demonstrated that parasympathetically mediated impulses can give rise
to substantial protein secretion (e.g. Asking and Gjorstrup,
1987). Nevertheless, the importance of sympathetically
mediated impulses in evoking protein secretion is demonstrated by experiments in which electrical stimulation of the

Fig. 5. Augmented or synergistic secretion of amylase during dual nerve


stimulation of the rat parotid gland. Parasympathetic (PS) and sympathetic
(S) stimulation frequencies have been adjust to evoke similar outputs of
amylase into saliva. When these stimulations are combined amylase secretion
is greater than the sum of the individual stimulations (Asking, 1985).

Fig. 6. Peroxidase secretion from acinar cells of rat submandibular gland. (A)
The effects of adding increasing low frequencies (0.12.0 Hz) of continuous
sympathetic nerve stimulation or high frequency (10, 20 Hz) discontinuous
stimulation onto a background of parasympathetic nerve stimulation in
anaesthetised rats. Both patterns of stimulation greatly increase the amount of
peroxidase secreted in a frequency dependent manner (Anderson et al.,
1995). Statistically different (p b 0.05) from c, parasympathetic alone and d,
sympathetic alone. (B) The effects of acute sympathectomy on reflex secretion in the conscious rat with indwelling submandibular cannulae. Peroxidase
secretion in grooming (g), rejection (r) and feeding (f) reflexes is partly
dependent upon sympathetic impulses since it is greatly reduced by
sympathectomy (Sx). In contrast heat (h) reflex secretion is almost entirely
parasympathetically mediated (Matsuo et al., 2000).

10

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

reflect the events leading to reflex secretion of saliva, since it


is expected that both parasympathetic and sympathetic impulses are acting on secretory cells simultaneously. Studies
of reflex salivary secretion have shown that the protein
content of saliva is decreased in the presence of betaadrenoceptor blockade or acute sympathetic denervation
compared to glands without such blockade (e.g. Ikawa et al.,
1991) or denervation (Fig. 6B; (Matsuo et al., 2000).
Most of the protein secreted by salivary glands is derived
from protein storage granules in acinar cells by a process of
exocytosis (Castle et al., 1975; Segawa and Yamashina,
1998). Sympathetic stimulation causes a morphologically
obvious depletion of storage granules from acinar cells in
most salivary glands studied. However, in studies of the rat
parotid and submandibular glands the morphological effects
of parasympathetic stimulation were barely discernable,
compared to obvious degranulation with sympathetic
stimulation, even when the conditions of parasympathetic
and sympathetic stimulation were adjusted to evoke similar
amounts of protein secretion (Asking and Gjorstrup, 1987;
Garrett et al., 1991). These observations fuelled the some
times furious debate concerning the role of acinar cell nonstorage granule vesicular secretory routes in salivary protein
secretion. More recent in vivo studies have shown that
salivary proteins accumulate in the ductal system of salivary
glands in the absence of stimulation (Garrett et al., 1996;
Proctor et al., 2003) whilst in vitro radiolabelling studies
have demonstrated the rapid secretion of newly synthesised
secretory proteins via a non-storage granule route (Castle
and Castle, 1996). The latter protein secretion pathway was
referred to as the minor regulated pathway in view of its upregulation by low doses of autonomimetics. The composition of proteins secreted by storage granules and vesicles
differs and the mechanisms enabling selective sequestration
of different proteins are still being studied in a variety of
exocrine cells including salivary acinar cells (Gorr et al.,
2005). So the existence of autonomically regulated vesicular
protein transport pathways is now more widely accepted. A
recent series of studies by the present authors have established that vesicular transcytosis of IgA across glandular
epithelial cells is also subject to autonomic regulation (see
Proctor and Carpenter, 2002). Both parasympathetic and
sympathetic stimuli cause increased salivary secretion of
sIgA (Fig. 7) and appear to regulate such secretion through
the transcytosis of the epithelial polymeric immunoglobulin
receptor containing vesicles.
It must be emphasized that the effects of parasympathetic
and sympathetic nerve impulses on protein secretion from
salivary glands can differ between glands in the same species
and between the same gland in different species. The rat
parotid and submandibular glands and more recently the
mouse submandibular gland are the most frequently
employed models for salivary gland studies. Consequently
these glands have become our reference points and the many
earlier studies on glands in other species are largely ignored.
Morphologically the cat submandibular gland more closely

Fig. 7. Effects of parasympathetic (ps) and sympathetic (sy) nerve


stimulation on secretion of immunoglobulin A from the rat salivary glands.
Although IgA is not a product of salivary epithelial cells, its secretion into
saliva is increased by stimulation from autonomic nerves. Other results
suggest that this effect is exerted through increased transcytosis via the
epithelial cell polymeric immunoglobulin receptor. a and c statistically
significantly (P b 0.05) from unstimulated (un); b statistically significantly
(P b 0.05) different from ps.

resembles the human gland than does the rat submandibular


gland and studies of the effects of nerve stimulation on the
main parenchymal cells proved revealing (see Garrett, 1987).
Both divisions of the autonomic nervous system exerted an
effect on protein secretion from central acinar, demilunar and
striated duct cells. The amounts of protein secreted from each
cell type differed depending upon the nerve being stimulated.
The sparse adrenergic (sympathetic) innervation of
mucous secreting glands such as the rat and human sublingual and the human minor salivary glands appears to be
directed to the vasculature rather than the parenchyma
(Garrett and Anderson, 1991; Rossoni et al., 1979). Thus
sympathetic nerve stimulation in vivo and adrenergic
stimulation in vitro evoked little mucin secretion. Rather,
mucin secretion from these mucous glands is dependent
upon parasympathetic stimulation and peptidergic stimulation (Culp et al., 1991). Thus, although parasympathetic
nerve mediated stimuli appear to universally cause fluid
secretion from salivary glands, the role of the sympathetic
innervation in evoking protein secretion is variable.
The functional significance of vesicular transport with
regard to salivary protein secretion remains uncertain. However, vesicular transport is crucial for the delivery of membrane
ion transport proteins to cell membranes. Such proteins allow
the movement of sodium, chloride and bicarbonate into the
lumen of the secretory endpiece and the osmotic gradient
created leads to water movement and hence saliva is formed
(Melvin et al., 2005). This anion (chloride and bicarbonate)
dependent model of salivary secretion is now accepted and
many of the significant membrane transport proteins have been
identified. The route by which water crosses epithelial cells,
whether transcellular or paracellular, has been debated for
some time (Young et al., 1987). The discovery of water channels or aquaporins (see Verkman, 2005) led to studies of the
role played by these channels in fluid secretion. Active fluid

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

transport in some tissues, e.g. sweat glands and intestinal cells,


is aquaporin independent but in tissues such as kidney and
salivary gland, where fluid transport per unit surface area is
much higher, aquaporins play a role (Verkman, 2005). Aquaporins 1, 3, 5, 6 and 8 have been detected in salivary cells and
salivary glands (Gresz et al., 2001; Hoque et al., 2002;
Ishikawa et al., 2000). Of these, aquaporin 5 is expressed in
apical membranes and aquaporin 3 in basolateral membranes
of acinar cells and therefore have been implicated in mediating
water transport into saliva (Gresz et al., 2001). The importance
of aquaporin 5 has been confirmed by the observation that
salivary fluid secretion is decreased by 50% in aquaporin 5
knockout mice but is unaffected in aquaporin 3 knockout mice
(Melvin et al., 2005). A series of studies on rat parotid gland
slices in vitro utilized membrane fractionation in order to
quantify aquaporin 5 in the apical plasma membrane. The
results of these experiments indicated that aquaporin 5
expression in the apical plasma membrane is subject to rapid
increases following stimulation with autonomimetics (Ishikawa et al., 2000). Subsequent in vivo studies utilizing autonomimetics failed to show changes in aquaporin 5 expression
on apical plasma membranes as determined immunocytochemically by light and electron microscopy (Gresz et al.,
2004). Given the great increase in vesicular and storage granule
trafficking and membrane turnover during stimulated salivary
secretion it would appear that aquaporin 5 is somehow anchored to the apical plasma membrane in order to be retained
there for the duration of stimulated secretion.
In addition to the effects on parenchymal cells autonomic
nerves exercise short term regulation of salivary gland blood
flow. Parasympathetic vasodilation is an integral part of the
salivary reflex as demonstrated recently in the rat submandibular gland (Mizuta et al., 2000; Anderson et al., 2006).
Sympathetically mediated vasoconstriction is under separate
vasomotor control and independent of the salivary reflex
(Emmelin and Engstrom, 1960). Parasympathetic and sympathetic nerves also cause contraction of myoepithelial cells
which embrace the secretory endpieces of salivary glands
like an octopus sitting on a rock (Tamarin, 1966). In fact the
extent of the embrace does vary between glands, for example
in the rat parotid gland myoepithelial cells are mainly associated with intercalated duct cells. Since myoepithelial cells
respond to lower doses of autonomimetics and low electrical
nerve stimulation frequencies than parenchymal cells it
seems that contraction may slightly precede secretion and
possibly serve to enhance the expulsion of saliva, particularly the more viscous secretions of mucous glands. Such
contraction may help to propel saliva from the acinus and to
prevent extravasation of saliva into the gland interstitium
(Garrett, 1998).
5. The coupling of autonomic nerve stimulation to secretion
The principal neurotransmitters activating salivary cell
secretion are acetylcholine, secreted by parasympathetic
nerves and noradrenaline, secreted by sympathetic nerves.

11

The release of acetylcholine from parasympathetic nerves and


its interaction with muscarinic cholinergic receptors
(mAChRs) regulates many fundamental functions in the
periphery (smooth muscle contraction, glandular secretion,
modulation of cardiac output) and CNS (motor control,
thermoregulation, memory). Five subtypes of mAChR, M1
M5, have been designated on the basis of cDNA cloning and
the interactions of agonists and antagonists with these
receptors along with their intracellular coupling through
different G proteins, continues to be characterized (Caulfield
and Birdsall, 1998). It is clear that release of acetylcholine
from parasympathetic nerves acting via mAChRs plays the
main role in evoking fluid secretion since secretion is almost
completely abolished by atropine. There has been a widespread acceptance based on studies of the rat parotid gland, that
salivary secretion is mediated entirely by M3 receptors (Baum
and Wellner, 1999). Pharmacological studies of rabbit and rat
submandibular and rat sublingual salivary glands indicate that
fluid secretion is also partially mediated by M1 and other nonM3 receptors (Culp et al., 1996; Tobin et al., 2002; Tobin,
1995). Recent studies of knockout mice has enabled a greater
understanding of the involvement of different mAChR
subtypes in salivary secretion (Gautam et al., 2004; Nakamura
et al., 2004). These studies indicate that both M1 and M3
receptors make a contribution to the secretion of whole mouth
saliva (the combined secretion of all of the salivary glands)
evoked by pilocarpine and other muscarinic agonists. The M1
receptor appears to make more of a contribution at higher
doses of pilocarpine whilst the M3 receptor mediates secretion
in response to lower doses of agonist. Stimulation via both M1
and M3 receptors is coupled to secretion through a G-protein/
phospholipase C generation of inositol triphosphate (IP3) and
diacylglycerol. The interaction of IP3 with IP3 receptors
(IP3R's) on the endoplasmic reticulum causes release of stored
calcium (Baum and Wellner, 1999; Gallacher and Smith,
1999). Rises in intracellular calcium open apical membrane
chloride channels and basolateral membrane potassium channels in acinar cells leading to electrolyte and water secretion
(Melvin et al., 2005). Studies of carbachol induced calcium
signalling in mAChR knock-out mice suggest that, unlike M3
receptors, M1 receptors are not ubiquitously expressed on
submandibular acinar cells (Nakamura et al., 2004).
Noradrenaline released from sympathetic nerves stimulates salivary secretion through alpha1- and beta1-adrenoceptors. Alpha1-adrenoceptor mediated signals follow a
similar intracellular calcium pathway as described for M1
and M3 (above) leading to fluid secretion (Baum and
Wellner, 1999). Beta1-adrenoceptor signalling mainly occurs
through G-protein/adenylate cyclase generation of intracellular cAMP followed by activation of protein kinase A and
phosphorylation of endogenous proteins leading to exocytosis of protein storage granules and salivary protein
secretion (Baum and Wellner, 1999).
Signalling from parasympathetic nerves can give rise to
substantial salivary protein secretion. Such protein secretion
may be due in part to release of the neuropeptide co-

12

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

transmitter vasointestinal polypeptide (VIP) (Ekstrom,


1999). VIP intracellular signalling occurs through cAMP
as described for noradrenaline signalling. However, cholinergic stimuli can give rise to the release of protein by a
coupling mechanism independent of cAMP, involving
elevated intracellular calcium and activation of protein
kinase C (Moller et al., 1996).
Experiments have been undertaken in which salivary
glands are stimulated simultaneously through the sympathetic
nerve supply on a background of parasympathetic nerve stimulation or through combined use of sympatho- and
parasympathomimetics, since such combined stimulation
occurs during reflex secretion. Under these experimental conditions an augmented secretion of salivary fluid is observed
in cats and dogs (Emmelin, 1987) and an augmented secretion
of amylase occurs in rabbit and rat parotid glands (see Fig. 5).
The latter effect appears to be dependent upon beta adrenergically mediated stimuli since it is produced by
isoprenaline and abolished by beta blockers (Asking, 1985).
A similar augmented secretion has been observed in vitro in rat
acinar cells (Tanimura et al., 1999) and appears to reflect a
cross-talk between the intracellular calcium and cAMP
secretory signalling pathways. It has also been observed with
VIP, a cAMP mobilising agonist, in combination with either
phentolamine, acetylcholine or substance P, all of which
mobilize intracellular calcium (Bobyock and Chernick, 1989).
The mechanism of cross-talk may involve a potentiation of the
release of calcium due to phosphorylation of inositol triphosphate receptors by cAMP dependent protein kinase A
(Straub et al., 2002).
Parasympathetically evoked vasodilation persists in the
presence of atropine, an observation originally made at the end
of the 19th century. Thulin (1976) observed that a small
secretion of parasympathetically evoked rat submandibular
saliva also persisted in the presence of atropine. Further studies
undertaken by Ekstrom and other researchers has demonstrated that in addition to the main neurotransmitters there are a
variety of NANC neuropeptide transmitters (Ekstrom, 1999).
The study of peptidergic NANC stimulated salivary secretion
mainly concerns the neuropeptides released by parasympathetic nerves in response to relatively high nerve stimulation
frequencies. Such overt atropine resistant secretion fades with
continued stimulation, reflecting the exhaustable stores of
peptide in dense cored vesicles in nerve endings (Ekstrom
et al., 1989). It is likely that neuropeptides are released from
parasympathetic nerves with acetylcholine even at lower
stimulation frequencies and that they modulate the protein and
fluid secretory responses of salivary glands. The coupling of
peptide stimulation to secretion differs according to the peptide. For example, VIP has been shown to evoke rises in
adenosine 3,5-(cyclic) monophosphate (cAMP) whilst
substance P causes increases in intracellular calcium. Studies
of atropine resistant vasodilation demonstrated that VIP acted
largely through activation of endothelial cell nitric oxide
synthase (NOS) and the generation of nitric oxide (Ekstrom,
1999; Edwards and Garrett, 1993) leading to the activation

soluble guanylate cyclase and generation of guanosine 3,5(cyclic) monophosphate or cGMP. In earlier studies it was
apparent that the cGMP second messenger pathway could
mediate amylase secretion in response to parasympathetic
stimulation, at least in some glands (Watson et al., 1982).
However, cAMP was recognised as being the most potent
mediator of amylase secretion, being up-regulated in response
to sympathetic stimulation and cGMP was concluded to be of
secondary importance (Butcher and Putney, 1980). More
recently, interest in cGMP as a mediator in salivary secretion
has been re-awakened, due mainly to the studies of atropine
resistant vasodilation when it became apparent that L-NAME
(N-nitro-L-arginine methyl ester), an inhibitor of NOS, also
partially inhibited salivary protein secretion. Further studies
confirmed a role for nitric oxide in mediating protein secretion
in response to parasympathetic and sympathetic stimulation
(e.g. Buckle et al., 1995). It is now apparent from studies of rat
and mouse salivary glands that nitric oxide and generation of
cGMP can be linked to the release of calcium from ryanodinesensitive intracellular stores via production of the calcium
mobilising nucleotide cADP-ribose (Harmer et al., 2001;
Looms et al., 2001). The source of nitric oxide in the above
signalling cascade appears to be from neuronal type calcium
dependent NOS of non-neuronal origin as shown by
fluorescence microscopy of acinar cells loaded with a nitric
oxide indicator (Looms et al., 2001) and in vivo denervation
studies (Sayardoust and Ekstrom, 2003). Studies in the rabbit
parotid gland suggest that nitric oxide production and subsequent downstream signalling through cGMP are calcium
dependent (Sugiya et al., 1998). These data suggest that
initially elevated intracellular calcium leads to further increases in intracellular calcium via nitric oxide, cGMP and
cADPribose as shown in Fig. 8. It may also be that diffusion of
nitric oxide into adjacent cells plays a role in co-ordinating the
secretory response in acini (Harmer et al., 2001). Nitric oxide
has therefore been demonstrated to make a significant
contribution to salivary protein secretion in response to
autonomic stimulation. However, there remain some unexplained observations. For example, rat parotid acinar cells
appear to show a degree of dependency upon nitric oxide
production during isoprenaline evoked amylase secretion and
it is not clear how NOS activation takes place under these
circumstances. Cholinergic stimulation, which leads to rises in
intracellular calcium, does not appear to lead to significant
nitric oxide evoked amylase secretion in some studies
(Sayardoust and Ekstrom, 2003). Nevertheless, rat parotid
acinar cells contain calcium dependent neuronal type NOS and
soluble guanylate cyclase activity (Shimomura et al., 2004). It
is apparent that the extent of nitric oxide induced protein
secretion differs in different salivary glands.
6. Trophic effects of nerves and salivary gland
regeneration
In the same way that acute electrical stimulation of the
nerves supplying salivary glands has helped to define their

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Fig. 8. Signalling pathways for nitric oxide (NO) in salivary cells. Salivary
cells contain neural type nitric oxide synthase (NOS) which can generate NO
and activate guanylate cyclase with the formation of cyclic guanosine
monophosphate (cGMP). The activation of NOS (NOS) may occur through
inositol triphosphate (IP3) signalling from muscarinic receptor or alphaadrenoceptor occupation. cGMP can cause further release of calcium from
ryanodine sensitive stores via protein kinase G, and cADPribose which may
lead to further NO generation. NO may also diffuse to neighbouring acinar
cells and act through similar intracellular signalling. NO may also be
generated as a result of raised intracellular cyclic adenosine monophosphate
(cAMP) from beta-adrenoceptor or vasointestinal peptide (VIP) receptor
occupation and activation of guanylate cylase (GC).

roles so cutting of the nerves, either singly or together, has


proved useful to evaluate their longer term effects. However,
interpretation of the effects of simply cutting the nerves
requires caution due to some short term paradoxical effects.
Post-ganglionic denervations can cause degeneration secretion to occur-caused by the release of neurotransmitters
from degenerating nerve fibres (Emmelin, 1968). Preganglionic denervation stops nerve impulses reaching the
gland whilst leaving axons within the salivary gland intact.
In this way it is possible to show the effect of neural traffic on
salivary cells but leaving intact any trophic effects from
nerve-epithelial cell interactions. To study trophic effects,
post-ganglionic denervation would be most useful. Postganglionic sympathetic denervation and post-ganglionic
parasympathetic denervation can be achieved in the rat
parotid gland since the parasympathetic ganglion is distant
from the gland. However, the latter is difficult to achieve in
the submandibular gland since the ganglia lie within the
gland itself.
6.1. Effects of denervations on salivary secretion
Secretion from denervated glands is complex and varies
according to the different routes of protein secretion, the
form of stimulation given and the gland studied. For
instance, amylase secretion (released from protein storage
granules) from parotid glands following 1 week of sympathectomy increased in response to parasympathetic stimulation (Asking and Emmelin, 1989) but decreased in

13

response to isoprenaline (a beta-adrenoceptor agonist) from


isolated parotid cells (Melvin et al., 1988). Whereas 1 week
of sympathectomy reduced parasympathetically mediated
secretion of secretory IgA, a non-storage product transported
by polymeric immunoglobulin receptor (Proctor et al.,
2000). The decreased IgA secretion appeared to be related
to decreased routing of pIgR to the basolateral membranes of
epithelial cells and others have shown an effect of
parasympathetic stimulation on localisation of proteins to
membranes (Ishikawa et al., 2000). A further difference between stored proteins was seen in the rat submandibular gland
following 1 week of pre-ganglionic sympathectomy where
parasympathetic nerve stimulation increased stored protein
secretion but not IgA secretion into saliva (Proctor et al.,
2000). So, sympathectomy can increase cholinergicallymediated protein secretion and interestingly the opposite may
also be true, i.e. 1 week parasympathectomy can increase
sympathetically-mediated fluid secretion (Carpenter et al.,
2005; Proctor et al., 1990). These cross-over effects, whereby
removing the influence of one nerve can heighten the functions of the opposite nerve may reflect the cross-talk between
the parasympathetic and sympathetic inputs to individual
(and mostly acinar) cells that has been suggested by dual
stimulation experiments on intact glands (Carpenter et al.,
2000; Anderson et al., 1995). Interestingly the changes in
cellular sensitivity following denervation are not reflected by
changes in receptor density nor binding constants (or to
changes in the cellular content of the secretory protein, see
(Ekstrom, 1999) for a review) and further work is required
therefore to untangle these complexities.
6.2. The effects of denervation on salivary gland structure
Acute sympathectomy (1 week) caused an increased
gland weight due to the accumulation of secretory material
within glands whereas longer sympathetic denervation
caused the gland to reduce in weight (Proctor and Asking,
1989). Following 1 week of pre-ganglionic parasympathectomy (Carpenter et al., 2005; Chaparro et al., 1998;
Katsukawa et al., 1990) the size of submandibular acini
was reduced (although DNA levels stayed the same) whereas
granular tubules which appear to be more dependant on
hormonal influences remained unchanged (Anderson et al.,
1994). In contrast, knocking out both the M1 and M3 had no
apparent effects on parenchymal structure (Nakamura et al.,
2004) though a detailed analysis was not published. This
may suggest that the presence of parasympathetic nerves (in
the knock out mouse) was enough to prevent atrophy seen in
the rat suggesting a role for other non-cholinergic neurotransmitters such as substance P and VIP (see Ekstrom,
1999).
6.3. The involvement of nerves in salivary gland regeneration
Switching the feed for a rat from solid chow to a liquid
diet causes a severe atrophy of the salivary glands (Hand and

14

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

Ho, 1981) and the hyperplastic response of switching back to


a solid diet or moving to a bulk diet was inhibited by a
double denervation (Schneyer et al., 1992). Chronic beta
adrenoceptor stimulation with isoprenaline can also induce
salivary gland hyperplasia with the upregulation of a specific
set of secretory proteins (Johnson, 1984). Interestingly both
isoprenaline-induced and reflexly-induced increases in gland
size were mediated in part by NGF (Purushotham et al.,
1993). Conversely chronic pilocarpine administration to
mimick increased parasympathetic impulses did not cause a
change in gland size (Muller et al., 1985) although just
30 min of parasympathetic nerve stimulation is enough to
cause significant mitogenic activity (Schneyer et al., 1993)
again suggesting a greater trophic role for neuropeptides
rather than the classical neurotransmitters.
Nerves also play a role in salivary gland regeneration
following duct ligation induced atrophy. Our own experiments have used the ligation and deligation of the rat submandibular duct to examine the influence of nerves on the
regeneration of salivary glands from an atrophic state. Initial
studies indicate that parasympathetic, but not sympathetic,

Fig. 9. H and E staining of formol sucrose fixed rat submandibular glands


following ligation (1 week) and deligation (4 weeks). In addition the gland
(panel B) was parasympathectomised at the point of deligation. The added
effect of a parasympathectomy has reduced acinar (Ac) size and increased
the length of the intercalated ducts (ID) making them more obvious. Other
ducts (Dc) appear similar in both glands.

Fig. 10. Comparison of salivary flow from affected and contra-lateral control
submandibular glands in a group of ten patients with sialolithiasis. Saliva
was collected using a novel suction technique pre-operatively and at
6 months post-operative follow-up. Mean salivary flow was reduced to 16%
in the affected glands and recovered to approximately 47% by 6 months
(Osailan, 2004).

nerves are important in regenerating salivary glands.


Following 1 week of ligation, a pre-ganglionic parasympathectomy reduced gland recovery by 50% after 4 weeks of
deligation compared with control glands that had been
deligated alone. Secretory capacity, as evaluated by whole
body methacholine stimulation, revealed that denervated and
deligated glands only secreted 60% of the salivary flow
compared to normally innervated deligated glands. Morphologically (see Fig. 9) acini in denervated and deligated glands
did not regenerate as completely since they were smaller and
the intercalated ducts longer than those of regenerating
glands with intact parasympathetic innervation. These
findings are interesting because the intercalated ducts have
some features of the immature pro-acinar phenotype possibly
suggesting they are the source of developing new acinar cells
(Denny et al., 1997). The lack of parasympathetic input
therefore may prevent the conversion of intercalated cells
into acinar units. A similar role for parasympathetic nerves
has also been shown in the developing gland where a parasympathectomy at birth limited subsequent salivary gland
weight to 40% and greatly reduced the spreading and development of the myoepithelial cells surrounding acini
(Murakami et al., 1991). Likewise sympathetic denervation
at birth (when pro-acinar and terminal tubule cells are
maturing) caused a significant reduction in gland size
(9 weeks later) with a reduction in secretory granule content
(Henriksson et al., 1985). In the same series of experiments
chronic beta blockade of adrenoceptors did not cause the
same changes, again suggesting a role for NANC transmitters in the development of salivary glands although this has
not been followed-up.
7. Conclusions and clinical perspective
It is clear that autonomic nerves have a considerable
influence on salivary glands. In addition to controlling immediate secretion from salivary cells nerves are also involved

G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318

in maintaining salivary gland size in order to deliver the


volume and composition of secretion that meets functional
demands. The latter must involve influencing mitosis to
generate new cells, developing cells into their mature
functional state and controlling responsiveness to parasympathetic and sympathetic inputs. The effects of denervations
are complex and underline the complex roles that nerves play
in maintaining salivary glands. Our knowledge of the
different intracellular signalling cascades involved in mediating nerve derived stimuli has greatly increased but it
remains unclear how these different signalling mechanisms
are integrated and the extent to which cross-talk occurs. The
role of muscarinic receptor sub-types in mediating salivary
secretion is of particular clinical interest at present since a
range of muscarinic receptor agonists and antagonists have
been developed for the treatment of Alzheimer's disease,
nociceptive pain, schizophrenia, Parkinson's disease, urinary
incontinence, irritable bowel syndrome, gastric ulceration
and chronic obstructive pulmonary disease (Eglen et al.,
1999). A frequent side effect of the use of muscarinic
antagonists is dry mouth (xerostomia). In order to develop
more selective muscarinic antagonists without the xerostomic
side effect we need to gain an understanding of how different
muscarinic receptor subtypes are involved in the physiological control of salivary secretion (Proctor, 2006). In addition
to action at peripheral autonomic receptors many prescribed
drugs appear to exert side effects on salivary secretion via a
central action (Scully, 2003). The list of drugs producing
xerostomia as a side effect is extensive (see Sreebny and
Schwartz, 1997; http://www.drymouth.info).
The mechanisms by which nerves exert trophic influences
on salivary glands requires further study and may lead to
novel approaches for therapeutic intervention in salivary
gland disease. The latter may involve the use of exogenous
stem cells to regenerate salivary glands following irreversible
damage from irradiation or chronic inflammation. Clearly the
success of such approaches will depend upon the presence of
autonomic nerves and the integration of newly formed secretory structures with nerves. Recent studies have demonstrated that human salivary glands like animal models, have
the capacity to regenerate. Following removal of salivary
ductal stones by minimally invasive surgical techniques
inflamed, atrophic salivary glands regain secretory function
progressively over the ensuing 612 months (Fig. 10;
(Osailan, 2004). The latter indicates that human glands are
able to regenerate and re-integrate secretory structures with
the autonomic innervation.
Acknowledgements
The authors thank colleagues who appear as co-authors on
publications. Particular thanks goes to Katherine Paterson who
provided technical assistance. The support of the Wellcome
Trust and GlaxoSmithKline is gratefully acknowledged. The
authors would like to dedicate this chapter to Professor John R.
Garrett, who followed in the footsteps of the great pioneers in

15

the subject of salivary glandular innervation, and thank him


for those lively discussions concerning salivary phenomena.
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