Abstract

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Autonomic Neuroscience: Basic and Clinical 133 (2007) 3182. Reflex salivary secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43. Salivary glands and their innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1. The development of salivary glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2. The development of nerves in salivary glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3. Innervation of adult salivary glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. The short term effects of autonomic nerves on salivary function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85. The coupling of autonomic nerve stimulation to secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116. Trophic effects of nerves and salivary gland regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6.1. Effects of denervations on salivary secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13M3 and (to a lesser extent) M1 muscarinic receptors, evoking the secretion of saliva by acinar cells in the endpieces of the salivary gland ductaltree. Most salivary glands also receive a variable innervation from sympathetic nerves which released noradrenaline from which tends to evokegreater release of stored proteins, mostly from acinar cells but also ductal cells. There is some cross-talk between the calcium and cyclic AMPintracellular 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 parasympatheticallyderived vasointestinal peptide, acting through endothelial cell derived nitric oxide, plays a role in the reflex vasodilatation that accompaniessecretion. Neuronal type, calcium-activated, soluble nitric oxide within salivary cells appears to play a role in mediating salivary proteinsecretion in response to autonomimetics. Fluid secretion by salivary glands involves aquaporin 5 and the extent to which the expression ofaquaporin 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 thatglands can regenerate provided that the autonomic innervation remains intact. The mechanisms by which nerves integrate with salivary cellsduring 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

Contentscontrolled by autonomic nerves. All salivary glands are supplied by chOral homeostasis is dependent upon saliva and its content of proteins. Reflex salivary flow occurs at a low resting rate and for short periodsof the day more intense taste or chewing stimuli evoke up to ten fold increases in salivation. The secretion of salivary fluid and proteins is

olinergic parasympathetic nerves which release acetylcholine that binds toReview

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

www.elsevier.com/locate/autneu6.2. The effects of denervation on salivary gland structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.3. The involvement of nerves in salivary gland regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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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modified, principally with the removal of sodium chlorideduring passage through the ductal system to themouth (Melvin

tions compared to during stimulation. In contrast thesubmandibular/sublingual glands secrete relatively more

euroset al., 2005; Turner and Sugiya, 2002). Saliva is thereforeconverted from an isotonic to a hypotonic solutionwhichaids the detection of salt in the diet. This is an energy richprocess so that the most active ducts have large numbers ofmitochondria located in the basolateral part of the cells leading

saliva under resting conditions (Shannon et al., 1969). Ithas been shown that different afferent stimuli can change thecomposition of saliva secreted by a single gland. A relativelygreater amount of IgA was present in chewing stimulatedhuman parotid saliva compared to citric acid evoked saliva7. Conclusions and clinical perspective . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Saliva in the mouth is the mixed product of 3 pairs ofmajor salivary glands, the parotid, submandibular and sub-lingual glands as well as numerous minor salivary glandsfound in the submucosa under most soft tissue surfaces in themouth. Whole mouth saliva also contains small amounts ofother fluids and products of the mucosal surface. Like othermucosal fluids saliva forms a mobile layer on the mucosalsurface and contains an array of components that fulfilimportant functions. In fact protection of the oral mucosalsurface is provided by components which are also present inother mucosal fluids, such as mucins (MUC5B and MUC7)and secretory IgA (sIgA) as well as components whichappear to be less widely detected on other surfaces or may bepeculiar to saliva (e.g. histatins, agglutinin). Unlike othermucosal fluids saliva contains a range of components thatinteract with and protect teeth (e.g. proline rich proteins,statherins). Saliva is crucial in maintaining the integrity ofthe oral mucosal surface and in preserving an ecologicalbalance (Hay and Bowen, 1999). The roles played bydifferent salivary components in protecting the soft and hardtissues of the mouth has been reviewed by others (Ameron-gen and Veerman, 2002).

Most of the components of saliva in the mouth water,ions, proteins are actively secreted by salivary glands. Thesecretory endpiece of salivary glands consists of acinarsecretory units made up of acinar cells which are responsiblefor synthesising and secreting most of the functionallyimportant protein components of saliva (Segawa andYamashina, 1998; Proctor, 1998). The acini are specialisedto each gland and are classified to reflect the proteins se-creted by each type of acinithe parotid glands have mainlyserous, the sublingual mainly mucous and the submandibulara mixture of the two. In the same way that diets vary fromone species to another so too are the salivary glands whichare specialised for the diet (Tandler and Phillips, 1998) andtherefore this simple classification of acinar cells does not fitall species. Water and electrolytes are actively transported byacinar cells and then the electrolyte content of saliva is

4 G.B. Proctor, G.H. Carpenter / Autonomic Nto their description as striated ducts. In addition to removingions they also add potassium and bicarbonatethe latter forms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

an important component of the buffering system of saliva thatprevents dissolution of teeth by acid producing bacteria. Mostductal cells secrete only small amounts of protein. However,granular ductal cells, which are peculiar to submandibularglands of some rodents including mouse and rat, are packedfull of tissue kallikreins. In addition to the parenchymalcomponent of salivary glands there are myoepithelial cells (seeFig. 1) which support the acini in some but not all salivaryglands and may help expel secretions from the ductal system.Plasma cells present in the gland interstitium, secreteimmunoglobulins which are transported by salivary cellsinto saliva and a dense network of blood vessels supply thefluid component of saliva (via the interstitial space).Controlling and influencing all these cells are parasympatheticand sympathetic autonomic nerves which work togetherharmoniously to evoke secretion.

2. Reflex salivary secretion

Salivary gland secretion is a nerve mediated reflex andonce the autonomic nerve supply, particularly the parasym-pathetic nerve, has been interrupted then secretion from mostglands ceases almost entirely. There are a few salivary glandsthat maintain a spontaneous secretion in the absence ofnerve mediated stimuli but even in these glands a normal rateof secretion requires an intact autonomic nerve supply(Emmelin, 1972). Salivary glands provide a resting flow ofsaliva into the mouth that fulfils the protective role referredto above. Then for short periods during overt reflexstimulation provided by the taste and chewing of food, anenormously increased activity is superimposed upon theresting flow (Emmelin, 1972). Hector and Linden (1999)have reviewed in detail how taste, mastication and otherstimuli evoke reflex salivary secretion through variousreceptors including gustatory receptors, mechanoreceptors,olfactory receptors and nociceptors. Thus not only thevolume but also the composition of mixed saliva in themouth can vary depending upon the contribution of differentglands during reflex stimulation. The parotid gland has avery low secretory rate under resting (unstimulated) condi-

cience: Basic and Clinical 133 (2007) 318(Proctor and Carpenter, 2002). Others have shown that sweetstimulated human parotid saliva has a higher protein

eurosG.B. Proctor, G.H. Carpenter / Autonomic Nconcentration compared to acid stimulated saliva (Mackieand Pangborn, 1990). There have been a few studies inwhich the effects of different reflex stimuli have been studiedin animal models. Gjorstrup (1980) and later Ikawa et al.(1991) showed that higher concentrations of salivaryamylase and other proteins were secreted into rabbit parotidsaliva evoked by carrots compared to standard pelletedchow.

Nerve impulses in the afferent limbs of the salivary reflexpass to the salivary nuclei within the medulla oblongata andfrom these centres efferent parasympathetic secretomotornerves emerge to supply the parenchyma of salivary glands(see Fig. 2). The integration of impulses from primarysalivary centres to glands depends on central modulation.The central neural connections between primary salivarycentres and other nuclei are not well understood and continueto be investigated. Retrograde labelling of neurons has dem-onstrated that the primary parasympathetic salivary centresform connections with the lateral hypothalamus where theregulation of feeding, drinking and body temperature occurs.

Fig. 1. Structural elements within rat submandibular glands digested with collagenasbe separate or joined. B, myoepithelial cells surrounding acinar groups demonstrattaken by confocal microscopy, colour relates to depth of field. C, tight junctions blarger than the adjacent acinar lumena. D, Cholinesterase staining of parasympath(arrow) terminating at a cell.5cience: Basic and Clinical 133 (2007) 318It appears that both excitatory (gamma aminobutyric acidcontaining) and inhibitory (glycine containing) nervessynapse with the salivary centres (Matsuo, 1999). Furtherevidence of the involvement of central mechanisms in mod-ulating salivary secretion was reported following intracer-ebroventricular injection of pilocarpine or atropine whichwere found to respectively stimulate and inhibit salivation(Renzi et al., 2002; Takakura et al., 2003). However, a morerecent study questioned the high doses used in the earlierwork (Sato et al., 2006). Alpha(2) adrenoceptor agonists(e.g. clonidine) and antagonists (e.g. yohimbine) have beendemonstrated to act centrally in studies of reflex secretion inhuman subjects and cholinergically evoked secretion inanimal models. Alpha(2) adrenoceptor blockade increasessalivary secretion whilst stimulation by alpha(2) adrenocep-tor agonists inhibits secretion (Moreira et al., 2002; Phillipset al., 2000). The primary sympathetic salivary centres arelocated in the upper thoracic segments of the spinal cordalthough it remains unclear precisely where in this region(Bradley et al., 2005; Matsuo, 1999). It has been understood

e. A, Unstained phase contrast image. Distinct acini (Ac) and ducts (Dc) mayed by actin staining. Images B and C are composites of a Z-series of imagesetween cells demonstrated by occludin staining. The ductal lumen is muchetic nerves surrounding the acinar units. Thinner fibres are sometimes seen

ary saar thehere srasymretion

eurosfor some time that central inhibition as a result ofconnections between the primary salivary centres and the

Fig. 2. Salivary reflex secretion. Afferent stimuli are integrated in the primconduct signals to salivary glands via parasympathetic ganglia stituated nesympathetic centre in the upper thoracic segments of the spinal cord and fromcervical ganglion. Nerves project (upper broken line) from the cortex to the paeffect on salivary secretion. Efferent autonomic nerves stimulate salivary sec

6 G.B. Proctor, G.H. Carpenter / Autonomic Nhigher centres of the brain are responsible for the dry mouthassociated with anxiety. It is worth repeating at this point thatthere is no peripheral inhibition of salivary secretion undernormal conditions. The concept of peripheral sympatheticinhibition of salivary secretion which later became widelyaccepted, was appreciated as an experimental artefact over acentury ago (see Garrett, 1987). Experimental electricalstimulation of the sympathetic nerve supply to salivaryglands or use of alpha(1) adrenoceptor agonists in anaes-thetized animals leads to a vasoconstriction of glandularblood vessels in addition to activation of parenchymal cells.In contrast under reflex conditions only sympathetic secreto-motor nerve fibres and not vasoactive nerve fibres to salivaryglands are activated. Thus vasoconstriction is not part of thesalivary reflex.

3. Salivary glands and their innervation

3.1. The development of salivary glands

During embryogenesis the major salivary glands developfrom the ectoderm whereas the minor glands originate fromthe mesoderm. In the genetic syndrome salivary glandagenesis there is an absence of ectodermal-derived structuressuch as sweat glands and the major salivary glands but minorsalivary glands are present (Nordgarden et al., 2001). Thefirst visible sign of salivary glands in the embryo (e13 in rats,e8 wks in humans) involves interactions between theepithelial layer and the mesenchyme (derived from theneural crest) resulting in a local thickening of the epithelium

livary centres of the medulla. Autonomic parasympathetic efferent nervestarget gland. Nerves project (lower broken line) from the medulla to theympathetic efferent nerves conduct signals to salivary glands via the superiorpathetic centres in the medulla and these can have an excitatory or inhibitoryand there is no peripheral inhibition of secretion via sympathetic nerves.

cience: Basic and Clinical 133 (2007) 318to form a placode. Some of the signalling occurring at thisstage involves Sonic Hedgehog (Shh) (Jaskoll et al., 2004),Epithelial Growth factor (EGF) and Transforming growthfactor (TGF) (Jaskoll and Melnick, 1999) and Ectodys-plasin (Eda) and its receptor (EdaR) (Jaskoll et al., 2003).Each of these signalling molecules only has a partial rolesince mice with one of these genes knocked out have defectsin, but not a complete absence of, salivary glands. Followingthe formation of the salivary placode salivary glanddevelopment has been well characterised as in Fig. 3.

EGF receptor and its two ligands, EGF and TGF appearto be important in co-ordinating the regulated cell prolifer-ation during salivary gland development. EGF is not presentin the initial bud stage and so signalling occurs via TGFlocalised on the cell surface and opposing mesenchymelayer. By early canalicular stage EGF is first detected in theepithelia of the terminal end buds. By late canalicular stageTGF is newly located on the ductal epithelia facing thelumen, i.e. where most proliferation and apoptosis isoccurring (Jaskoll and Melnick, 1999). If glands are removedat the canalicular stage and stimulated in vitro by auto-nomimetics a specific group of proteins are secreted re-flecting the two main cell typesType 1 terminal tubulecells and Type 3 proacinar cells (Fig. 3D). Interestingly thereare differences in which agonists cause the secretion of theseproteins (Ball et al., 1988) suggesting that the differentmechanisms of protein secretion (and the control thereof)seen in the adult are also present in the neonatal salivary

when catecholamine-containing nerves start to appear(Bottaro and Cutler, 1984). The development of sympatheticnerves occurs at a similar time to the development of pro-acinar and terminal tubule Types 1 and 3 cells into matureacinar and ductal cells and a neonatal sympatheticdenervation reduced parotid acinar development (Henriks-son et al., 1985) which implies a role for the innervation inthe final differentiation of Types 1 and 3 cells into functionalacinar and ductal structures (Danielsson et al., 1988; Bottaroand Cutler, 1984). A dichotomy of sympathetic nerve devel-opment in the rat submandibular and sublingual glands existssince despite developing in the same capsule surrounded bythe same mesenchymal cap the sublingual gland containsvery few sympathetic nerves whereas the submandibular

7G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318gland. Between birth and adult (Fig. 3E) Type 3 proacinarcells develop into acini (Ac) and eventually sero-mucous

Fig. 3. The development of rat submandibular salivary glands. Starting atembryonic day e12/13 the initial bud stage (A) there is a proliferation andinfolding of the epithelial layer (E) of the oral cavity (OC) into the adjoiningcondensed mesenchyme (CM) to form the pseudoglandular stage (B) inwhich an elongated cord of epithelial (E) cells extends into the surroundingmesenchyme (M). The appearance of a ductal cell lumen (L) characterises theonset of the canalicular stage (C) and the terminal bud stage is reached oncelumena extend into the epithelial buds (D). At this stage two cell types areapparent-type 3 pro-acinar cells and type 1 terminal tubules cells. From birthto day 30 salivary glands develop their mature phenotype (E) of acinar cellsand intercalated, granular and striated ductal cells.cells whilst Type 1 cells maintain a central location possiblyforming intercalated cells (Hand et al., 1996; Jaskoll andMelnick, 1999); striated ducts (SD) appear to develop post-natally (Redman et al., 2002).

3.2. The development of nerves in salivary glands

Saliva can be produced from rat salivary glands at birth bystimulating parasympathetic nerves implying a functionalneuro-epithelial junction although sympathetic nerves andsecretory granule movement in response to sympatheticnerve stimulation does not occur until 35 days after birth

Fig. 4. Sympathetic nerves in rat salivary glands. Catecholamine fluorescence of cellBoth parotid and submandibular glands have a dense sympathetic innervation partiwhereas the sublingual gland has a sparse sympathetic innervation except around tgland has a rich innervation (see Fig. 4). This presumablyreflects the lack of expression of the neurotrophin NGF insublingual glands as the expression of this molecule cor-relates closely with sympathetic innervation in other tissues(Glebova and Ginty, 2004).

Parasympathetic nerves develop in parallel with the sali-vary parenchyma and require an interaction with the paren-chyma in order to develop (Coughlin, 1975). By separatingthe submandibular ganglion from the salivary epithelium atan early stage (e 11 in the mouse) the development ofcholinesterase containing nerves was inhibited although thesalivary parenchyma developed normally in the absence ofthe submandibular ganglion (note the opposite relationshipoccurs later at pro-acinar to mature cell conversion stagewhich requires parasympathetic input). Which signallingmolecules interact between nerves and salivary cells is notclear although, as indicated above, EGF signalling is active.Several recent studies using knockout mice have indicated asignificant role for glial cell derived neurotrophins (GDNFs)affecting peripheral nerve innervation. Glial cells (alsoknown as Schwann cells) support, guide and interact withnerve axons right up to the target cell. Although para-sympathetic and sympathetic nerves enter glands fromseparate sources they quickly align themselves and com-bined parasympathetic/sympathetic/Schwann cell bundlesare common throughout most salivary glands (Garrett and

s from digested rat parotid (A), sublingual (B) and submandibular (C) glands.

cularly surrounding the acini (Ac) but also present on ductal structures (Dc),he occasional duct.

eurosKidd, 1993). For parasympathetic nerves the main GDNFappears to be neurturin acting on GDNF family receptoralpha 2 (GFR2) molecules expressed by nerve axons whichappears to be important for the development of peripheralfibres. Knocking out GFR2 greatly reduced the parasym-pathetic innervation of acini in the sublingual gland (Rossi etal., 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. NGFacts through a high (TrkA) and a low affinity receptor (p75)-TrkA promotes cell survival and axon elongation and p75activation invokes cell death (Dechant and Barde, 2002).Knocking out p75 actually enhanced sympathetic innerva-tion of mouse submandibular glands (Jahed and Kawaja,2005) whereas knocked out NGF inhibited sympatheticinnervation of salivary glands (Ghasemlou et al., 2004).

The guidance molecules for nerves in salivary glands areunknown but by comparison with other models (Zou, 2004)Wnt binding to frizzled receptors could be important to axonguidance by acting as both attractants and repellents whilstectodysplasin A (Eda) and its receptor (EdaR) also haveforward and reverse signalling capabilities (Hinck, 2004).Secreted frizzled (1 and 4) are strongly expressed by salivarycells at e15.5, (Leimeister et al., 1998) and WNT4 mRNAexpression has been shown to be increasingly expressed inmouse from e14 to adultin fact, with NGF it is one of thefew proteins to be actively increased from post-partum day 5to adult (Hoffman et al., 2002).

3.3. Innervation of adult salivary glands

From the two separate nerve supplies to the salivary glandsympathetic and parasympathetic nerves run together withSchwann cells as one bundle up to the target cell (Garrett andKidd, 1993). From there some nerves may supply severalmore target cells as unmyelinated axons. There appears to beno specialised neuro-effector sites, neurotransmitter releasemay occur anywhere along the nerve, although nerveterminals may have a bulbous ending and varicositiesalong the length of the nerve (Ohtani et al., 1983) theseshould not be thought of as the only place whereneurotransmitter release may occur. Furthermore electronmicroscopical studies have indicated that two types of nerve-epithelial cell relationship exist-epilemmal and hypolemmal(Garrett and Kidd, 1993). Epilemmal nerve arrangements siton top of the epithelial cell, surrounded by a Schwann cell,and are considered less efficient, in terms of neurotransmis-sion, as the neurotransmitter must pass through twobasement membranes (Schwann cell and epithelial cell) toreach the receptor. Hypolemmal nerves sit between epithelialcells and are not surrounded by Schwann cell. However, noevidence exists for any difference between their relativeefficiencies of each nerve arrangement. The relative

8 G.B. Proctor, G.H. Carpenter / Autonomic Nproportion of epi- and hypolemmal nerves varies accordingto the gland being studied for instance the rat parotid hasepi- and hypolemmal nerves whereas the rat submandibularhas only epilemmal nerves (Garrett and Kidd, 1993)although interestingly, hypolemmal nerves exist in thedeveloping rat submandibular gland (Yohro, 1971).

Parasympathetic and sympathetic nerves appear to be incontact with most cell types in salivary glands. Most nervestaining is associated with the acinar cells and theirassociated myoepithelial cells which both receive dualinnervation (see Figs. 1 and 4). Although the rat sublingualhas a paucity of adrenergic innervation some sympatheticnerves were seen associated with striated ducts and bloodvessels (Garrett et al., 1991). Blood vessels also have dualinnervation-parasympathetic stimulation causing vasodila-taion and sympathetic stimulation vasoconstriction, althoughit should be remembered that such vasoconstriction is notpart of the salivary reflex (Bernard, 1858 cited in (Garrett,1999)). Vasodilatation in the rat submandibular caused byacetylcholine and VIP release from parasympathetic nerves(Anderson and Garrett, 1998) is mediated by nitric oxide andother endothelium-derived hyperpolarizing factors (Ander-son et al., 2006) nitric oxide appears to be less importantas the size of the blood vessel decreases.

As well as the main neurotranmitters acetylcholine andadrenaline 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 adenylatecyclase activating peptide (PACAP), neuronal nitric oxidesynthase (nNOS) and calcitonin gene-related peptide(CGRP) have all been detected within either parasympathet-ic, sympathetic or sometimes both nerves (reviewed in(Ekstrom, 1999)). These neuropeptides can have effects onthe blood vessels and on the salivary cells enhancing proteinand/or fluid secretory responses (see later). There is somespecific localisation of peptide containing nerves as VIPnerves are more numerous around the mucous acinar cells inthe human submandibular gland (Kusakabe et al., 1998),their expression may change with salivary gland develop-ment (Virta et al., 1992) and focal infiltrates of inflammatorycells may downregulate nerve expression of neuropeptides(Pedersen et al., 2000). Some of these NANC transmittersare also found in sensory nerve fibres within the salivaryglands. By using the sensory neurotoxin capsaicin to destroysensory nerve fibres only nerves containing both CGRP andSP, usually located around ducts and blood vessels, werediminished (Dunerengstrom et al., 1985).

4. The short term effects of autonomic nerves on salivaryfunction

The efferent autonomic secretomotor nerves supplyingsalivary glands stimulate secretion and there is no antago-nism between the two branches of the autonomic nervoussystem (Emmelin, 1987). Garrett (1987) produced a useful

cience: Basic and Clinical 133 (2007) 318summary of the effects of parasympathetic and sympatheticimpulses on salivary glands (Table 1).

sympathetic nerve supplies to the rat parotid (Asking andGjorstrup, 1987) or submandibular glands (Anderson et al.,1995; Carpenter et al., 2000) is superimposed upon abackground of parasympathetic nerve stimulation, then largeincreases in protein secretion are observed (Figs. 5 and 6a).Such dual stimulation experiments are thought to better

Table 1Effects of autonomic nerves on salivary gland function (modified fromGarrett, 1987)

Parasympathetic stimulation

1) Is mediated mainly by acetylcholine in combination with NANCpeptides (e.g. VIP)2) Evokes most of the salivary fluid secreted. Mainly acts through M3 andto a lesser extent M1 muscarinic cholinergic receptors3) Causes variable degrees of exocytosis from salivary cells but isresponsible for most mucin secretion by mucous glands4) Induces contraction of myoepithelial cells5) Increases glandular blood flow as part of the salivary reflex

Sympathetic stimulation

1) Is mediated mainly by noradrenaline and acts essentially on cellsreceiving parasympathetic impulses, which tends to produce synergisticeffects, but exerts little effect on mucous gland secretion2) Often does not cause much mobilization of fluid but DOES NOTinhibit salivary secretion3) Tends to modulate the composition of saliva by increasing exocytosisfrom salivary cells4) Induces contraction of myoepithelial cells

9G.B. Proctor, G.H. Carpenter / Autonomic Neuroscience: Basic and Clinical 133 (2007) 318It can be seen from Table 1 that parasympathetic impulsesusually evoke most of the fluid secretion into saliva whilstsympathetic nerves have less of a fluid evoking role. Therehas been a tendency to dichotomize the respective roles ofthe nerves further by attributing salivary protein secretionalmost entirely to sympathetic nerve impulses. The latter isan oversimplification since various studies have demonstrat-ed that parasympathetically mediated impulses can give riseto substantial protein secretion (e.g. Asking and Gjorstrup,1987). Nevertheless, the importance of sympathetically

5) Exerts control on glandular blood flow but NOT as part of the salivaryreflexmediated impulses in evoking protein secretion is demon-strated by experiments in which electrical stimulation of the

Fig. 5. Augmented or synergistic secretion of amylase during dual nervestimulation of the rat parotid gland. Parasympathetic (PS) and sympathetic(S) stimulation frequencies have been adjust to evoke similar outputs ofamylase into saliva.When these stimulations are combined amylase secretionis 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 continuoussympathetic nerve stimulation or high frequency (10, 20 Hz) discontinuous

stimulation onto a background of parasympathetic nerve stimulation inanaesthetised rats. Both patterns of stimulation greatly increase the amount ofperoxidase secreted in a frequency dependent manner (Anderson et al.,1995). Statistically different (pb0.05) from c, parasympathetic alone and d,sympathetic alone. (B) The effects of acute sympathectomy on reflex secre-tion in the conscious rat with indwelling submandibular cannulae. Peroxidasesecretion in grooming (g), rejection (r) and feeding (f) reflexes is partlydependent upon sympathetic impulses since it is greatly reduced by

sympathectomy (Sx). In contrast heat (h) reflex secretion is almost entirelyparasympathetically mediated (Matsuo et al., 2000).

sympathetic nerve stimulation in vivo and adrenergicstimulation in vitro evoked little mucin secretion. Rather,mucin secretion from these mucous glands is dependentupon parasympathetic stimulation and peptidergic stimula-tion (Culp et al., 1991). Thus, although parasympatheticnerve mediated stimuli appear to universally cause fluidsecretion from salivary glands, the role of the sympatheticinnervation in evoking protein secretion is variable.

The functional significance of vesicular transport withregard to salivary protein secretion remains uncertain. How-ever, vesicular transport is crucial for the delivery of membraneion transport proteins to cell membranes. Such proteins allowthe movement of sodium, chloride and bicarbonate into thelumen of the secretory endpiece and the osmotic gradientcreated 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 andmany of the significant membrane transport proteins have beenidentified. The route by which water crosses epithelial cells,whether transcellular or paracellular, has been debated forsome time (Young et al., 1987). The discovery of water chan-

eurosreflect the events leading to reflex secretion of saliva, since itis expected that both parasympathetic and sympathetic im-pulses are acting on secretory cells simultaneously. Studiesof reflex salivary secretion have shown that the proteincontent of saliva is decreased in the presence of beta-adrenoceptor blockade or acute sympathetic denervationcompared 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 derivedfrom protein storage granules in acinar cells by a process ofexocytosis (Castle et al., 1975; Segawa and Yamashina,1998). Sympathetic stimulation causes a morphologicallyobvious depletion of storage granules from acinar cells inmost salivary glands studied. However, in studies of the ratparotid and submandibular glands the morphological effectsof parasympathetic stimulation were barely discernable,compared to obvious degranulation with sympatheticstimulation, even when the conditions of parasympatheticand sympathetic stimulation were adjusted to evoke similaramounts of protein secretion (Asking and Gjorstrup, 1987;Garrett et al., 1991). These observations fuelled the sometimes furious debate concerning the role of acinar cell non-storage granule vesicular secretory routes in salivary proteinsecretion. More recent in vivo studies have shown thatsalivary proteins accumulate in the ductal system of salivaryglands in the absence of stimulation (Garrett et al., 1996;Proctor et al., 2003) whilst in vitro radiolabelling studieshave demonstrated the rapid secretion of newly synthesisedsecretory proteins via a non-storage granule route (Castleand Castle, 1996). The latter protein secretion pathway wasreferred to as the minor regulated pathway in view of its up-regulation by low doses of autonomimetics. The composi-tion of proteins secreted by storage granules and vesiclesdiffers and the mechanisms enabling selective sequestrationof different proteins are still being studied in a variety ofexocrine cells including salivary acinar cells (Gorr et al.,2005). So the existence of autonomically regulated vesicularprotein transport pathways is now more widely accepted. Arecent series of studies by the present authors have esta-blished that vesicular transcytosis of IgA across glandularepithelial cells is also subject to autonomic regulation (seeProctor and Carpenter, 2002). Both parasympathetic andsympathetic stimuli cause increased salivary secretion ofsIgA (Fig. 7) and appear to regulate such secretion throughthe transcytosis of the epithelial polymeric immunoglobulinreceptor containing vesicles.

It must be emphasized that the effects of parasympatheticand sympathetic nerve impulses on protein secretion fromsalivary glands can differ between glands in the same speciesand between the same gland in different species. The ratparotid and submandibular glands and more recently themouse submandibular gland are the most frequentlyemployed models for salivary gland studies. Consequentlythese glands have become our reference points and the many

10 G.B. Proctor, G.H. Carpenter / Autonomic Nearlier studies on glands in other species are largely ignored.Morphologically the cat submandibular gland more closelyresembles the human gland than does the rat submandibulargland and studies of the effects of nerve stimulation on themain parenchymal cells proved revealing (see Garrett, 1987).Both divisions of the autonomic nervous system exerted aneffect on protein secretion from central acinar, demilunar andstriated duct cells. The amounts of protein secreted from eachcell type differed depending upon the nerve being stimulated.

The sparse adrenergic (sympathetic) innervation ofmucous secreting glands such as the rat and human sub-lingual and the human minor salivary glands appears to bedirected to the vasculature rather than the parenchyma(Garrett and Anderson, 1991; Rossoni et al., 1979). Thus

Fig. 7. Effects of parasympathetic (ps) and sympathetic (sy) nervestimulation on secretion of immunoglobulin A from the rat salivary glands.Although IgA is not a product of salivary epithelial cells, its secretion intosaliva is increased by stimulation from autonomic nerves. Other resultssuggest that this effect is exerted through increased transcytosis via theepithelial cell polymeric immunoglobulin receptor. a and c statisticallysignificantly (Pb0.05) from unstimulated (un); b statistically significantly(Pb0.05) different from ps.

cience: Basic and Clinical 133 (2007) 318nels or aquaporins (see Verkman, 2005) led to studies of therole played by these channels in fluid secretion. Active fluid

eurostransport in some tissues, e.g. sweat glands and intestinal cells,is aquaporin independent but in tissues such as kidney andsalivary gland, where fluid transport per unit surface area ismuch higher, aquaporins play a role (Verkman, 2005). Aqua-porins 1, 3, 5, 6 and 8 have been detected in salivary cells andsalivary glands (Gresz et al., 2001; Hoque et al., 2002;Ishikawa et al., 2000). Of these, aquaporin 5 is expressed inapical membranes and aquaporin 3 in basolateral membranesof acinar cells and therefore have been implicated in mediatingwater transport into saliva (Gresz et al., 2001). The importanceof aquaporin 5 has been confirmed by the observation thatsalivary fluid secretion is decreased by 50% in aquaporin 5knockout mice but is unaffected in aquaporin 3 knockout mice(Melvin et al., 2005). A series of studies on rat parotid glandslices in vitro utilized membrane fractionation in order toquantify aquaporin 5 in the apical plasma membrane. Theresults of these experiments indicated that aquaporin 5expression in the apical plasma membrane is subject to rapidincreases following stimulation with autonomimetics (Ishi-kawa et al., 2000). Subsequent in vivo studies utilizing auto-nomimetics failed to show changes in aquaporin 5 expressionon apical plasma membranes as determined immunocyto-chemically by light and electron microscopy (Gresz et al.,2004). Given the great increase in vesicular and storage granuletrafficking and membrane turnover during stimulated salivarysecretion it would appear that aquaporin 5 is somehow an-chored to the apical plasma membrane in order to be retainedthere for the duration of stimulated secretion.

In addition to the effects on parenchymal cells autonomicnerves exercise short term regulation of salivary gland bloodflow. Parasympathetic vasodilation is an integral part of thesalivary reflex as demonstrated recently in the rat subman-dibular gland (Mizuta et al., 2000; Anderson et al., 2006).Sympathetically mediated vasoconstriction is under separatevasomotor control and independent of the salivary reflex(Emmelin and Engstrom, 1960). Parasympathetic and sym-pathetic nerves also cause contraction of myoepithelial cellswhich embrace the secretory endpieces of salivary glandslike an octopus sitting on a rock (Tamarin, 1966). In fact theextent of the embrace does vary between glands, for examplein the rat parotid gland myoepithelial cells are mainly asso-ciated with intercalated duct cells. Since myoepithelial cellsrespond to lower doses of autonomimetics and low electricalnerve stimulation frequencies than parenchymal cells itseems that contraction may slightly precede secretion andpossibly serve to enhance the expulsion of saliva, particu-larly the more viscous secretions of mucous glands. Suchcontraction may help to propel saliva from the acinus and toprevent extravasation of saliva into the gland interstitium(Garrett, 1998).

5. The coupling of autonomic nerve stimulation to secretion

The principal neurotransmitters activating salivary cell

G.B. Proctor, G.H. Carpenter / Autonomic Nsecretion are acetylcholine, secreted by parasympatheticnerves and noradrenaline, secreted by sympathetic nerves.The release of acetylcholine from parasympathetic nerves andits interaction with muscarinic cholinergic receptors(mAChRs) regulates many fundamental functions in theperiphery (smooth muscle contraction, glandular secretion,modulation of cardiac output) and CNS (motor control,thermoregulation, memory). Five subtypes of mAChR, M1M5, have been designated on the basis of cDNA cloning andthe interactions of agonists and antagonists with thesereceptors along with their intracellular coupling throughdifferent G proteins, continues to be characterized (Caulfieldand Birdsall, 1998). It is clear that release of acetylcholinefrom parasympathetic nerves acting via mAChRs plays themain role in evoking fluid secretion since secretion is almostcompletely abolished by atropine. There has been a wide-spread acceptance based on studies of the rat parotid gland, thatsalivary secretion is mediated entirely byM3 receptors (Baumand Wellner, 1999). Pharmacological studies of rabbit and ratsubmandibular and rat sublingual salivary glands indicate thatfluid secretion is also partially mediated byM1 and other non-M3 receptors (Culp et al., 1996; Tobin et al., 2002; Tobin,1995). Recent studies of knockout mice has enabled a greaterunderstanding of the involvement of different mAChRsubtypes in salivary secretion (Gautam et al., 2004; Nakamuraet al., 2004). These studies indicate that both M1 and M3receptors make a contribution to the secretion of whole mouthsaliva (the combined secretion of all of the salivary glands)evoked by pilocarpine and other muscarinic agonists. The M1receptor appears to make more of a contribution at higherdoses of pilocarpine whilst theM3 receptor mediates secretionin response to lower doses of agonist. Stimulation via bothM1and M3 receptors is coupled to secretion through a G-protein/phospholipase C generation of inositol triphosphate (IP3) anddiacylglycerol. The interaction of IP3 with IP3 receptors(IP3R's) on the endoplasmic reticulum causes release of storedcalcium (Baum and Wellner, 1999; Gallacher and Smith,1999). Rises in intracellular calcium open apical membranechloride channels and basolateral membrane potassium chan-nels in acinar cells leading to electrolyte and water secretion(Melvin et al., 2005). Studies of carbachol induced calciumsignalling in mAChR knock-out mice suggest that, unlike M3receptors, M1 receptors are not ubiquitously expressed onsubmandibular acinar cells (Nakamura et al., 2004).

Noradrenaline released from sympathetic nerves stimu-lates salivary secretion through alpha1- and beta1-adreno-ceptors. Alpha1-adrenoceptor mediated signals follow asimilar intracellular calcium pathway as described for M1and M3 (above) leading to fluid secretion (Baum andWellner, 1999). Beta1-adrenoceptor signalling mainly occursthrough G-protein/adenylate cyclase generation of intracel-lular cAMP followed by activation of protein kinase A andphosphorylation of endogenous proteins leading to exocy-tosis of protein storage granules and salivary proteinsecretion (Baum and Wellner, 1999).

Signalling from parasympathetic nerves can give rise to

11cience: Basic and Clinical 133 (2007) 318substantial salivary protein secretion. Such protein secretionmay be due in part to release of the neuropeptide co-

eurostransmitter vasointestinal polypeptide (VIP) (Ekstrom,1999). VIP intracellular signalling occurs through cAMPas described for noradrenaline signalling. However, cholin-ergic stimuli can give rise to the release of protein by acoupling mechanism independent of cAMP, involvingelevated intracellular calcium and activation of proteinkinase C (Moller et al., 1996).

Experiments have been undertaken in which salivaryglands are stimulated simultaneously through the sympatheticnerve supply on a background of parasympathetic nerve sti-mulation or through combined use of sympatho- andparasympathomimetics, since such combined stimulationoccurs during reflex secretion. Under these experimental con-ditions an augmented secretion of salivary fluid is observedin cats and dogs (Emmelin, 1987) and an augmented secretionof amylase occurs in rabbit and rat parotid glands (see Fig. 5).The latter effect appears to be dependent upon beta ad-renergically mediated stimuli since it is produced byisoprenaline and abolished by beta blockers (Asking, 1985).A similar augmented secretion has been observed in vitro in ratacinar cells (Tanimura et al., 1999) and appears to reflect across-talk between the intracellular calcium and cAMPsecretory signalling pathways. It has also been observed withVIP, a cAMP mobilising agonist, in combination with eitherphentolamine, acetylcholine or substance P, all of whichmobilize intracellular calcium (Bobyock and Chernick, 1989).The mechanism of cross-talk may involve a potentiation of therelease of calcium due to phosphorylation of inositol tri-phosphate receptors by cAMP dependent protein kinase A(Straub et al., 2002).

Parasympathetically evoked vasodilation persists in thepresence of atropine, an observation originally made at the endof the 19th century. Thulin (1976) observed that a smallsecretion of parasympathetically evoked rat submandibularsaliva also persisted in the presence of atropine. Further studiesundertaken by Ekstrom and other researchers has demonstrat-ed that in addition to the main neurotransmitters there are avariety of NANC neuropeptide transmitters (Ekstrom, 1999).The study of peptidergic NANC stimulated salivary secretionmainly concerns the neuropeptides released by parasympa-thetic nerves in response to relatively high nerve stimulationfrequencies. Such overt atropine resistant secretion fades withcontinued stimulation, reflecting the exhaustable stores ofpeptide in dense cored vesicles in nerve endings (Ekstromet al., 1989). It is likely that neuropeptides are released fromparasympathetic nerves with acetylcholine even at lowerstimulation frequencies and that they modulate the protein andfluid secretory responses of salivary glands. The coupling ofpeptide stimulation to secretion differs according to the pep-tide. For example, VIP has been shown to evoke rises inadenosine 3,5-(cyclic) monophosphate (cAMP) whilstsubstance P causes increases in intracellular calcium. Studiesof atropine resistant vasodilation demonstrated that VIP actedlargely through activation of endothelial cell nitric oxide

12 G.B. Proctor, G.H. Carpenter / Autonomic Nsynthase (NOS) and the generation of nitric oxide (Ekstrom,1999; Edwards and Garrett, 1993) leading to the activationsoluble guanylate cyclase and generation of guanosine 3,5-(cyclic) monophosphate or cGMP. In earlier studies it wasapparent that the cGMP second messenger pathway couldmediate amylase secretion in response to parasympatheticstimulation, at least in some glands (Watson et al., 1982).However, cAMP was recognised as being the most potentmediator of amylase secretion, being up-regulated in responseto sympathetic stimulation and cGMP was concluded to be ofsecondary importance (Butcher and Putney, 1980). Morerecently, interest in cGMP as a mediator in salivary secretionhas been re-awakened, due mainly to the studies of atropineresistant vasodilation when it became apparent that L-NAME(N-nitro-L-arginine methyl ester), an inhibitor of NOS, alsopartially inhibited salivary protein secretion. Further studiesconfirmed a role for nitric oxide in mediating protein secretionin response to parasympathetic and sympathetic stimulation(e.g. Buckle et al., 1995). It is now apparent from studies of ratand mouse salivary glands that nitric oxide and generation ofcGMP can be linked to the release of calcium from ryanodine-sensitive intracellular stores via production of the calciummobilising nucleotide cADP-ribose (Harmer et al., 2001;Looms et al., 2001). The source of nitric oxide in the abovesignalling cascade appears to be from neuronal type calciumdependent NOS of non-neuronal origin as shown byfluorescence microscopy of acinar cells loaded with a nitricoxide indicator (Looms et al., 2001) and in vivo denervationstudies (Sayardoust and Ekstrom, 2003). Studies in the rabbitparotid gland suggest that nitric oxide production and sub-sequent downstream signalling through cGMP are calciumdependent (Sugiya et al., 1998). These data suggest thatinitially elevated intracellular calcium leads to further in-creases in intracellular calcium via nitric oxide, cGMP andcADPribose as shown in Fig. 8. It may also be that diffusion ofnitric oxide into adjacent cells plays a role in co-ordinating thesecretory response in acini (Harmer et al., 2001). Nitric oxidehas therefore been demonstrated to make a significantcontribution to salivary protein secretion in response toautonomic stimulation. However, there remain some un-explained observations. For example, rat parotid acinar cellsappear to show a degree of dependency upon nitric oxideproduction during isoprenaline evoked amylase secretion andit is not clear how NOS activation takes place under thesecircumstances. Cholinergic stimulation, which leads to rises inintracellular calcium, does not appear to lead to significantnitric oxide evoked amylase secretion in some studies(Sayardoust and Ekstrom, 2003). Nevertheless, rat parotidacinar cells contain calcium dependent neuronal typeNOS andsoluble guanylate cyclase activity (Shimomura et al., 2004). Itis apparent that the extent of nitric oxide induced proteinsecretion differs in different salivary glands.

6. Trophic effects of nerves and salivary glandregeneration

cience: Basic and Clinical 133 (2007) 318In the same way that acute electrical stimulation of thenerves supplying salivary glands has helped to define their

eurosFig. 8. Signalling pathways for nitric oxide (NO) in salivary cells. Salivarycells contain neural type nitric oxide synthase (NOS) which can generate NOand activate guanylate cyclase with the formation of cyclic guanosinemonophosphate (cGMP). The activation of NOS (NOS) may occur throughinositol triphosphate (IP3) signalling from muscarinic receptor or alpha-adrenoceptor occupation. cGMP can cause further release of calcium fromryanodine sensitive stores via protein kinase G, and cADPribose which maylead to further NO generation. NO may also diffuse to neighbouring acinarcells and act through similar intracellular signalling. NO may also begenerated as a result of raised intracellular cyclic adenosine monophosphate(cAMP) from beta-adrenoceptor or vasointestinal peptide (VIP) receptoroccupation and activation of guanylate cylase (GC).

G.B. Proctor, G.H. Carpenter / Autonomic Nroles so cutting of the nerves, either singly or together, hasproved useful to evaluate their longer term effects. However,interpretation of the effects of simply cutting the nervesrequires caution due to some short term paradoxical effects.Post-ganglionic denervations can cause degeneration secre-tion to occur-caused by the release of neurotransmittersfrom degenerating nerve fibres (Emmelin, 1968). Pre-ganglionic denervation stops nerve impulses reaching thegland whilst leaving axons within the salivary gland intact.In this way it is possible to show the effect of neural traffic onsalivary cells but leaving intact any trophic effects fromnerve-epithelial cell interactions. To study trophic effects,post-ganglionic denervation would be most useful. Post-ganglionic sympathetic denervation and post-ganglionicparasympathetic denervation can be achieved in the ratparotid gland since the parasympathetic ganglion is distantfrom the gland. However, the latter is difficult to achieve inthe submandibular gland since the ganglia lie within thegland itself.

6.1. Effects of denervations on salivary secretion

Secretion from denervated glands is complex and variesaccording to the different routes of protein secretion, theform of stimulation given and the gland studied. Forinstance, amylase secretion (released from protein storagegranules) from parotid glands following 1 week of sym-pathectomy increased in response to parasympathetic sti-mulation (Asking and Emmelin, 1989) but decreased inresponse to isoprenaline (a beta-adrenoceptor agonist) fromisolated parotid cells (Melvin et al., 1988). Whereas 1 weekof sympathectomy reduced parasympathetically mediatedsecretion of secretory IgA, a non-storage product transportedby polymeric immunoglobulin receptor (Proctor et al.,2000). The decreased IgA secretion appeared to be relatedto decreased routing of pIgR to the basolateral membranes ofepithelial cells and others have shown an effect ofparasympathetic stimulation on localisation of proteins tomembranes (Ishikawa et al., 2000). A further difference be-tween stored proteins was seen in the rat submandibular glandfollowing 1 week of pre-ganglionic sympathectomy whereparasympathetic nerve stimulation increased stored proteinsecretion but not IgA secretion into saliva (Proctor et al.,2000). So, sympathectomy can increase cholinergically-mediated protein secretion and interestingly the opposite mayalso be true, i.e. 1 week parasympathectomy can increasesympathetically-mediated fluid secretion (Carpenter et al.,2005; Proctor et al., 1990). These cross-over effects, wherebyremoving the influence of one nerve can heighten the func-tions of the opposite nerve may reflect the cross-talk betweenthe parasympathetic and sympathetic inputs to individual(and mostly acinar) cells that has been suggested by dualstimulation experiments on intact glands (Carpenter et al.,2000; Anderson et al., 1995). Interestingly the changes incellular sensitivity following denervation are not reflected bychanges in receptor density nor binding constants (or tochanges in the cellular content of the secretory protein, see(Ekstrom, 1999) for a review) and further work is requiredtherefore to untangle these complexities.

6.2. The effects of denervation on salivary gland structure

Acute sympathectomy (1 week) caused an increasedgland weight due to the accumulation of secretory materialwithin glands whereas longer sympathetic denervationcaused the gland to reduce in weight (Proctor and Asking,1989). Following 1 week of pre-ganglionic parasympathect-omy (Carpenter et al., 2005; Chaparro et al., 1998;Katsukawa et al., 1990) the size of submandibular aciniwas reduced (although DNA levels stayed the same) whereasgranular tubules which appear to be more dependant onhormonal influences remained unchanged (Anderson et al.,1994). In contrast, knocking out both the M1 and M3 had noapparent effects on parenchymal structure (Nakamura et al.,2004) though a detailed analysis was not published. Thismay suggest that the presence of parasympathetic nerves (inthe knock out mouse) was enough to prevent atrophy seen inthe rat suggesting a role for other non-cholinergic neuro-transmitters such as substance P and VIP (see Ekstrom,1999).

6.3. The involvement of nerves in salivary gland regeneration

13cience: Basic and Clinical 133 (2007) 318Switching the feed for a rat from solid chow to a liquiddiet causes a severe atrophy of the salivary glands (Hand and

Ho, 1981) and the hyperplastic response of switching back toa solid diet or moving to a bulk diet was inhibited by adouble denervation (Schneyer et al., 1992). Chronic betaadrenoceptor stimulation with isoprenaline can also inducesalivary gland hyperplasia with the upregulation of a specificset of secretory proteins (Johnson, 1984). Interestingly bothisoprenaline-induced and reflexly-induced increases in glandsize were mediated in part by NGF (Purushotham et al.,1993). Conversely chronic pilocarpine administration tomimick increased parasympathetic impulses did not cause achange in gland size (Muller et al., 1985) although just30 min of parasympathetic nerve stimulation is enough tocause significant mitogenic activity (Schneyer et al., 1993)again suggesting a greater trophic role for neuropeptidesrather than the classical neurotransmitters.

Nerves also play a role in salivary gland regenerationfollowing duct ligation induced atrophy. Our own experi-ments have used the ligation and deligation of the rat sub-mandibular duct to examine the influence of nerves on theregeneration of salivary glands from an atrophic state. Initialstudies indicate that parasympathetic, but not sympathetic,

therefore may prevent the conversion of intercalated cellsinto acinar units. A similar role for parasympathetic nerveshas also been shown in the developing gland where a para-sympathectomy at birth limited subsequent salivary glandweight to 40% and greatly reduced the spreading and devel-opment of the myoepithelial cells surrounding acini(Murakami et al., 1991). Likewise sympathetic denervationat birth (when pro-acinar and terminal tubule cells arematuring) 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 experimentschronic beta blockade of adrenoceptors did not cause thesame changes, again suggesting a role for NANC transmit-ters in the development of salivary glands although this hasnot been followed-up.

7. Conclusions and clinical perspective

It is clear that autonomic nerves have a considerable

14 G.B. Proctor, G.H. Carpenter / Autonomic NeurosFig. 9. H and E staining of formol sucrose fixed rat submandibular glandsfollowing ligation (1 week) and deligation (4 weeks). In addition the gland(panel B) was parasympathectomised at the point of deligation. The addedeffect of a parasympathectomy has reduced acinar (Ac) size and increased

the length of the intercalated ducts (ID) making them more obvious. Otherducts (Dc) appear similar in both glands.nerves are important in regenerating salivary glands.Following 1 week of ligation, a pre-ganglionic parasym-pathectomy reduced gland recovery by 50% after 4 weeks ofdeligation compared with control glands that had beendeligated alone. Secretory capacity, as evaluated by wholebody methacholine stimulation, revealed that denervated anddeligated glands only secreted 60% of the salivary flowcompared to normally innervated deligated glands. Morpho-logically (see Fig. 9) acini in denervated and deligated glandsdid not regenerate as completely since they were smaller andthe intercalated ducts longer than those of regeneratingglands with intact parasympathetic innervation. Thesefindings are interesting because the intercalated ducts havesome features of the immature pro-acinar phenotype possiblysuggesting they are the source of developing new acinar cells(Denny et al., 1997). The lack of parasympathetic input

Fig. 10. Comparison of salivary flow from affected and contra-lateral controlsubmandibular glands in a group of ten patients with sialolithiasis. Salivawas collected using a novel suction technique pre-operatively and at6 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).

cience: Basic and Clinical 133 (2007) 318influence on salivary glands. In addition to controlling im-mediate secretion from salivary cells nerves are also involved

eurosin maintaining salivary gland size in order to deliver thevolume and composition of secretion that meets functionaldemands. The latter must involve influencing mitosis togenerate new cells, developing cells into their maturefunctional state and controlling responsiveness to parasym-pathetic and sympathetic inputs. The effects of denervationsare complex and underline the complex roles that nerves playin maintaining salivary glands. Our knowledge of thedifferent intracellular signalling cascades involved in medi-ating nerve derived stimuli has greatly increased but itremains unclear how these different signalling mechanismsare integrated and the extent to which cross-talk occurs. Therole of muscarinic receptor sub-types in mediating salivarysecretion is of particular clinical interest at present since arange of muscarinic receptor agonists and antagonists havebeen developed for the treatment of Alzheimer's disease,nociceptive pain, schizophrenia, Parkinson's disease, urinaryincontinence, irritable bowel syndrome, gastric ulcerationand chronic obstructive pulmonary disease (Eglen et al.,1999). A frequent side effect of the use of muscarinicantagonists is dry mouth (xerostomia). In order to developmore selective muscarinic antagonists without the xerostomicside effect we need to gain an understanding of how differentmuscarinic receptor subtypes are involved in the physiolog-ical control of salivary secretion (Proctor, 2006). In additionto action at peripheral autonomic receptors many prescribeddrugs appear to exert side effects on salivary secretion via acentral action (Scully, 2003). The list of drugs producingxerostomia as a side effect is extensive (see Sreebny andSchwartz, 1997; http://www.drymouth.info).

The mechanisms by which nerves exert trophic influenceson salivary glands requires further study and may lead tonovel approaches for therapeutic intervention in salivarygland disease. The latter may involve the use of exogenousstem cells to regenerate salivary glands following irreversibledamage from irradiation or chronic inflammation. Clearly thesuccess of such approaches will depend upon the presence ofautonomic nerves and the integration of newly formed se-cretory structures with nerves. Recent studies have demon-strated that human salivary glands like animal models, havethe capacity to regenerate. Following removal of salivaryductal stones by minimally invasive surgical techniquesinflamed, atrophic salivary glands regain secretory functionprogressively over the ensuing 612 months (Fig. 10;(Osailan, 2004). The latter indicates that human glands areable to regenerate and re-integrate secretory structures withthe autonomic innervation.

Acknowledgements

The authors thank colleagues who appear as co-authors onpublications. Particular thanks goes to Katherine Patersonwhoprovided technical assistance. The support of the WellcomeTrust and GlaxoSmithKline is gratefully acknowledged. The

G.B. Proctor, G.H. Carpenter / Autonomic Nauthors would like to dedicate this chapter to Professor JohnR.Garrett, who followed in the footsteps of the great pioneers inthe subject of salivary glandular innervation, and thank himfor those lively discussions concerning salivary phenomena.

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Regulation of salivary gland function by autonomic nervesIntroductionReflex salivary secretionSalivary glands and their innervationThe development of salivary glandsThe development of nerves in salivary glandsInnervation of adult salivary glands

The short term effects of autonomic nerves on salivary functionThe coupling of autonomic nerve stimulation to secretionTrophic effects of nerves and salivary gland regenerationEffects of denervations on salivary secretionThe effects of denervation on salivary gland structureThe involvement of nerves in salivary gland regeneration

Conclusions and clinical perspectiveAcknowledgementsReferences