Parasympathetic reflex vasodilatation in rat submandibular gland

The present study was designed to investigate 1) whether parasympathetic reflex vasodilatation occurs in the submandibular gland (SMG) in deeply urethan-anesthetized, cervically vagotomized, and sympathectomized rats when the central cut end of the lingual nerve (LN) is electrically stimulated and 2) to what extent the neural mechanisms underlying such responses are the same as those involved in the response to direct stimulation of the chorda-LN (CLN). Stimulation of each nerve separately elicited a marked blood flow increase in SMG. Section of the chorda tympani abolished the SMG blood flow response but had no effect on the lip blood flow increase evoked by LN stimulation. Section of the CLN abolished the SMG blood flow increases evoked by stimulation of either nerve. The SMG blood flow increases (regardless of whether they were evoked by LN or CLN stimulation) were markedly reduced by the autonomic cholinergic ganglion blocker hexamethonium. The present study demonstrates that a parasympathetic reflex vasodilator mechanism is present in the rat SMG and that it can express its effects under deep general anesthesia.     

Submandibular responses to stimulation of the parasympathetic innervation in anesthetized sheep.

Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation and the output of vasoactive intestinal peptide (VIP) were investigated in anaesthetized sheep in the presence and absence of atropine (>/=0.5 mg/kg). In the absence of atropine, parasympathetic stimulation caused an increase in the flow of saliva and a decrease in submandibular vascular resistance; the latter response persisted after the administration of atropine and was then significantly reduced at the lowest but not at the higher frequencies tested. The output of VIP from the gland was frequency dependent over the range of 10-20 Hz (continuously) and significantly increased after atropine (P < 0.02). Furthermore, the fall in vascular resistance was linearly related to log VIP output after total muscarinic blockade. Intracarotid infusions of synthetic VIP produced dose-dependent falls in submandibular vascular resistance, together with a corresponding increase in submandibular blood flow. It is concluded that the atropine-resistant vasodilatation that occurs in this gland during parasympathetic stimulation is likely to be due largely, if not entirely, to the release of VIP.     

Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation

In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) +/- atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion.     

Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation in anesthetized cats.

Submandibular secretory responses to stimulation of the parasympathetic chorda-lingual nerve in anaesthetized cats have been investigated before, during, and after intracarotid infusion of endothelin-1 (ET-1), which reduced blood flow through the gland by 64+/-7%. Stimulation at different frequencies (2, 4, 8, and 16 Hz) evoked a frequency-dependent increase in the flow of submandibular saliva, sodium concentration and output, and output of both potassium and protein. The reduction in submandibular blood flow, which occurred in response to the infusion of ET-1, was associated with a decreased flow of saliva and a diminished output of both sodium and protein. The flow of saliva was linearly related to submandibular blood flow both in the presence and absence of ET-1. It is concluded that submandibular secretory responses to electrical stimulation of the parasympathetic innervation can be significantly attenuated by reducing the blood flow through the gland by ET-1 infusion, just as it is when the blood flow is reduced by hypotension.     

Effects of prolonged reduction in blood flow on submandibular secretory function in anesthetized sheep.

Submandibular vascular and secretory responses to parasympathetic chorda-lingual (C-L) stimulation were investigated in anesthetized sheep before, during, and after an intracarotid (ic) infusion of endothelin-1 (ET-1). Stimulation of the peripheral end of the C-L nerve at 4 and 8 Hz produced a frequency-dependent reduction in submandibular vascular resistance (SVR) associated with a frequency-dependent increase in submandibular blood flow, salivary flow, and Na+, K+, and protein output from the gland. During stimulation at 4 Hz, ic ET-1 significantly increased SVR (P < 0.01), without significantly affecting either the aortic blood pressure or heart rate. Submandibular blood flow (SBF) was reduced by 48 +/- 4% and the flow of saliva by 50 +/- 1%. The effect on blood and salivary flow persisted for at least 30 min after the infusion of ET-1. The reduction in SBF was associated with a diminution in the output of Na+,K+, and protein in the saliva (P < 0.01). These effects persisted for 30 min after the infusion of ET-1 had been discontinued and were linearly related to the flow of plasma throughout.     

VIP-containing nerve fibres in the submandibular gland of the dog and protein secretion in vitro in response to VIP

Previously, administration of VIP has been shown to elicit no flow of saliva from the submandibular gland of the dog. However, in the present study we found VIP to cause release of protein in vitro from canine submandibular gland tissue. Furthermore, VIP-containing nerve fibres were demonstrated in large numbers in association with acini. Thus, VIP may be involved in the nervous regulation of salivary protein secretion in the dog.     

Effects of vasoactive intestinal polypeptide on acetylcholine stimulation of rat submandibular gland

Studies were carried out on the role of vasoactive intestinal polypeptide (VIP) in the regulation of secretion and blood flow in the rat salivary gland. The first experiments to investigate the spontaneous secretory pattern revealed a clear diurnal fluctuation with a significant increase at night, so that the subsequent experiments were performed during the daytime where the secretion was consistently low. Intravenous administration of VIP at a dose smaller than 40 pmole caused a dose-dependent vasodilatory response, but at a high dose such a local effect was hampered by a decrease in systemic blood pressure. VIP potentiated the acetylcholine chloride (AcCho)--evoked salivary secretion, but VIP (0-100 pmole/kg) alone did not cause salivary secretion. Atropine reduced the salivary secretion evoked by AcCho and VIP, and the blood flow change evoked by AcCho. However, the blood flow change evoked by VIP was not affected by atropine. Hexamethonium exerted no significant effect on the response to administration of AcCho or VIP. The results indicate that VIP has a significant vasodilatory action and cooperates with AcCho in the regulation of salivary secretion in the rat, and VIP effects are atropine resistant, as in other species of animals.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE1 or PGE2, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva; PGF2 alpha was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 micrograms/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE1, PGE2, PGF2 alpha or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF2 alpha, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion, Endogenous prostaglandins themselves may not play a role in secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, and inhibitor of prostaglandin biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion, The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Secretory responses in granular ducts and acini of submandibular glands in vivo to parasympathetic or sympathetic nerve stimulation in rats

Summary The roles of sympathetic and parasympathetic nerves in the secretion of saliva from submandibular glands of rats have been tested by electrical stimulation of either nerve for 1 h unilaterally in separate animals. The flows of saliva thereby induced and their protein content were monitored. Structural changes in each gland were assessed by light- and electron microscopy and compared with the unstimulated contralateral control gland, and the extent of the changes was determined morphometrically. Sympathetic nerve stimulation induced a relatively low flow of saliva that was rich in protein and was accompanied by extensive degranulation from both acinar and granular duct cells. In contrast parasympathetic nerve stimulation induced a considerable flow of saliva that had a low protein content and no detectable degranulation occurred from the secretory cells. It is possible, therefore, that some protein in parasympathetic saliva may have arisen from a non-granular pathway.     

Stimulation of lacrimal secretion by sympathetic nerve impulses in the rabbit

Direct electrical stimulation of the preganglionic nerve trunk of the ipsilateral superior cervical sympathetic ganglion induced fluid secretion from the cannulated main excretory duct of the non-stimulated rabbit lacrimal gland. The optimum strength and frequency of stimulation were 4 volts and 25 Hz. At this stimulus parameter, the rate of secretion was 2.1 +/- 0.2 microliter/10 min. The sympathetic-induced lacrimal secretion was markedly depressed after intravenous administration of hexamethonium (1 mg/kg) and by post-ganglionic neurectomy. The results suggest that, in addition to parasympathetic nerve activity, sympathetic nerve impulses, which pass through the superior cervical sympathetic ganglion, also play a role in initiating fluid secretion in the rabbit lacrimal gland.     

Expression and characteristics of vanilloid receptor 1 in the rabbit submandibular gland

Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE₁ or PGE₂, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva: PGF_2α was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 μg/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE₁, PGE₂, PGF_2α or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF_2α, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion. Endogenous prostaglandins themselves may not play a role in a secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, an inhibitor of prostaglandins biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion. The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

A study of vasoactive intestinal peptide and acetylcholine as possible mediators of vasodilatation in the cat submandibular gland

Vasoactive intestinal polypeptide (VIP) is a powerful vasodilator agent in the submandibular gland of the cat, and its effect can be reduced by avian pancreatic polypeptide (APP), or by desensitization of the gland's blood vessels to VIP. However, the vasodilatation caused by parasympathetic nerve stimulation is not reduced by either of these means. We conclude, therefore, that VIP is unlikely to be a major mediator of this atropine-resistant vasodilatation. Experiments with a potentiator of acetylcholine, eserine, and with a depleter, suggest that acetylcholine plays some role in this vasodilatation, but it too does not appear to be the major physiological mechanism. Substance P and ATP were neither potent nor consistent vasodilators and are unlikely mediators.     

Effects of chronic clonidine administration on parasympathetic-evoked rat saliva.

Effects of chronic administration of clonidine on parasympathetic-evoked saliva from both parotid and submandibular glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Ion concentrations (Na, K and Ca) of parasympathetically nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study.     

Peptidergic innervation of the major salivary glands of the ferret

In parotid, sublingual and submandibular glands of the ferret, morphological correlates were looked for, using immunocytochemistry, to previous physiological findings showing parasympathetic "atropine-resistant" salivary secretion and neuropeptide-evoked salivation in this species. Nerve fibers storing VIP were numerous in association with acini, ducts and blood vessels, while the number of fibers storing substance P was moderate and those containing CGRP and galanin few; also the number of NPY-containing fibers was low around acini and ducts but relatively high around vessels. Sympathectomy eliminated all NPY- and almost all dopamine beta-hydroxylase-containing fibers. Parasympathectomy of the parotid gland resulted in a total loss of the VIP-containing fibers, and a profound reduction in the number of substance P- and CGRP-containing fibers.     

Effects of heterologous cross-suture between the postganglionic sympathetic and the preganglionic parasympathetic nerve trunks of submandibular glands in cats

Summary After sectioning the postganglionic adrenergic sympathetic nerve trunk for the submandibular gland, as close to the submandibular artery as practicable, its central end was sutured to the peripheral end of the preganglionic cholinergic parasympathetic nerve trunk for the gland, the chorda, which had been sectioned where it left the lingual nerve. The effects of this heterologous cross-sature were studied at different times, up to 1 year afterwards, by assessing the physiological and pharmacological responses of the glands and the neuro-histochemical changes in the nerve trunks and in the nerves within the glands.In all cases adrenergic sympathetic nerves grew across the site of suture and down the erstwhile cholinergic parasympathetic trunk, eventually to develop connections in the gland. In some cases the functional adrenergic reinnervation of the submandibular gland appeared to result exclusively or predominantly from the direct downgrowth of adrenergic axons to the gland, via the crossed nerves. In other cases however, in addition to a direct glandular reinnervation, there was some physiological and morphological evidence which suggested that possible heterogenous synaptic contacts may have been created between postganglionic sympathetic axons and cholinergic ganglion cells in the chorda nerve.This work was supported by a grant from the Joint Research Committee, King's College Hospital.     

Protein secretion from rat submandibular acini and granular ducts: effects of exogenous VIP and substance P during parasympathetic nerve stimulation

The influences of exogenous vasoactive intestinal peptide (VIP) and substance P on the release of peroxidase from acini and true tissue kallikrein (rK1) from granular ducts of the rat submandibular gland were studied during continuous parasympathetic stimulation. Parasympathetic nerve impulses caused a moderate flow of saliva (mean +/- SD, 108+/-26 microl/g tissue/min) that had a low protein concentration (174+/-88 microg/ml). The outputs of peroxidase and rK1 were minimal (14.3+/-11.8 pmol DCF/g tissue/min and 6.5+/-3.4 nmol AFC/g tissue/min, respectively). When administered intravenously, VIP had no apparent effect on the overall flow rate, but caused a significant increase in the output of peroxidase; 450% at 1 microg/kg and a further 10-fold increase at 10 microg/kg. In contrast, substance P (1 microg/kg) evoked a marked increase in flow rate (68%), and peroxidase secretion increased only 3-fold. The output of rK1 was unaffected by either VIP or substance P. Our results support the hypothesis that acinar, but not granular duct, protein secretion is evoked by non-adrenergic, non-cholinergic peptides released from parasympathetic nerve terminals.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.     

Nervous control of mammalian salivary glands

In the production and flow of saliva, sympathetic and parasympathetic nerves generally cooperate, although variations between the different salivary glands are considerable, particularly in the sympathetic innervation. In the submandibular gland of the dog, sympathetic impulses cause secretion via beta-adrenoceptors, and since sympathetic motor effects are elicited via alpha-adrenoceptors it is possible to study separately motor and secretory effects in this gland. Such experiments indicate that myoepithelial contractions serve to accelerate the salivary flow and to support the secreting acinar cells and prevent back-flow of fluid from the luminal system into the glandular tissues. The contractions are elicited reflexly from the oral mucosa together with secretion. A potentiation interaction between sympathetic and parasympathetic nerves occurs in the formation of the primary saliva. In parotid glands of rabbits and rats such an interaction has been demonstrated in the secretion of amylase.     

Na+, K(+)-ATPase in cat salivary glands and changes induced by nerve stimulation: an immunohistochemical study

The enzyme Na+, K(+)-ATPase was localized immunohistochemically in major salivary glands of the cat before and after autonomic nerve stimulation. Immunostaining was limited to basolateral plasma membranes. Cells lining striated and excretory ducts contained abundant Na+, K(+)-ATPase and showed no changes with neural stimulation. Serous-type cells in resting glands varied in reactivity, showing weak to moderate staining intensity in the parotid gland and more uniform staining of greater intensity in the sublingual gland. In contrast, demilune cells in the resting submandibular gland showed little if any staining. Mucous-type cells were negative in all glands. Parasympathetic stimulation promoted a gradual increase in immunostaining of submandibular demilune cells, which became marked with time. Sympathetic stimulation produced no detectable changes in Na+, K(+)-ATPase immunoreactivity in any site. These results support the concept that basolateral Na+, K(+)-ATPase is essential to the formation of a near-isotonic primary saliva by serous-type cells. The mechanism whereby parasympathetic stimulation evokes a marked flow of submandibular saliva remains unexplained, but has now been shown to involve a marked increase in the immunoreactivity of Na+, K(+)-ATPase at the base of the gland's demilune cells.