Bilateral irradiation of head and neck induces an enhanced expression of substance P in the parasympathetic innervation of the submandibular gland.

Substance P and calcitonin gene-related peptide (CGRP) are present in nerve fibers innervating the submandibular gland. Radiotherapy of tumors in the head and neck region usually embraces the salivary glands in the irradiated field and consequently a dramatic decrease in salivary function is seen. In this study, the submandibular glands and ganglia of rats subjected to fractionated irradiation were examined by use of immunohistochemical techniques for demonstration of substance P and CGRP. Irradiation was given on five consecutive days (daily doses of 6-9 Gray) with unilateral or bilateral irradiation techniques. Specimens of control and experimental animals were processed in parallel. A marked increase in the expression of substance P in the ganglionic cells--presumably parasympathetic--and in the number of fibers showing substance P-like immunoreactivity in association with acini and small ducts was seen in response to bilateral irradiation. (Surprisingly, unilateral irradiation of the parotid area had no effect on peptide distribution in the irradiated gland and ganglion). No changes in the pattern of CGRP immunoreactivity occurred. In the trigeminal ganglion, which supplies the submandibular gland with the majority of the sensory substance P- and CGRP-containing nerve fibers, no changes in the expression of substance P or CGRP immunoreactivity were seen. The results suggest that bilateral irradiation leads to an increase in the synthesis of substance P-like substance in the parasympathetic ganglionic cells supplying the submandibular gland with secretory nerves, and can thus be an additional factor in explaining the altered secretory capacity of salivary glands.     

Vasoactive intestinal peptide evoked secretion of fluid and protein from rat salivary glands and the development of supersensitivity

Vasoactive intestinal peptide (VIP) injected intravenously was found to induce a flow of saliva from both the parotid and the submaxillary gland in the rat. The secretion was slow in onset. The amount of saliva secreted from the parotid gland was less than that from the submaxillary gland. Parotid saliva was very viscous. VIP-evoked parotid saliva was more protein rich than both submaxillary saliva and saliva secreted in response to other sialagogue drugs including the beta-adrenergic receptor agonist isoprenaline. The effect of VIP was direct; it occurred after removal of the adrenals, after degeneration of intraglandular nerves and in the presence of autonomic blockers. A supersensitivity to VIP was demonstrable. In the parotid and the submaxillary glands the secretory response to VIP was enlarged following parasympathetic denervation and decentralization, respectively, while after sympathetic denervation supersensitivity was only found to develop in the submaxillary gland.     

An electrophysiological in-vivo study on the effects of nerve stimulation, drugs and denervation in the parotid gland of the rat

Electrical stimulation of the sympathetic and parasympathetic nerves to the parotid gland of the rat gave rise to membrane potential changes. Responses were in several cases elicited by single stimuli which did not cause secretion. They were very similar to the ones seen after the administration of noradrenaline or acetylcholine. The peptides substance P (SP) and vasoactive intestinal polypeptide (VIP), in some cells, evoked membrane potential changes. The response was clearly enhanced during concurrent stimulation of the auriculotemporal nerve at a frequency below the secretory threshold. The membrane potential changes always occurred at stimulation frequencies or agonist doses that were lower or the same as those required for secretion. After atropine the cells became less sensitive to both substance P and VIP. Sympathetic or parasympathetic denervation about 2 weeks in advance, to induce supersensitivity, lowered the thresholds for most agonists just like in vitro. Also as in vitro, a greater number of cells responded to SP and VIP in the denervated than in the innervated gland.     

Motor nerves of salivary myoepithelial cells in dogs

1. The pressures in the ducts of the parotid and submaxillary glands were recorded in dogs under chloralose-urethane anaesthesia. A single stimulus applied to the parasympathetic nerve regularly caused a pressure rise of short duration. Usually it did not evoke secretion.2. In both glands a single stimulus applied to the vagosympathetic trunk usually caused a pressure response of short duration. It had no secretory effect.3. In the parotid gland, repetitive vagosympathetic stimulation caused a sustained increase of pressure in the duct, usually without inducing secretion.4. In the submaxillary gland, repetitive vagosympathetic stimulation caused a sustained pressure rise, even when the secretory response had been abolished by propranolol.5. It is concluded that the salivary myoepithelial cells of dogs are supplied with fibres from both sympathetic and parasympathetic divisions of the autonomic nervous system and both can cause the cells to contract.     

Supersensitivity to substance P and physalaemin in rat salivary glands after denervation or decentralization

Substance P, a putative neurotransmitter in mammals, and physalaemin, present in the skin of an amphibian, are both undecapeptides and belong to the family of tachykinins. The secretory effect of these tachykinins on parotid and submaxillary glands of the rat was examined. Dose-response curves showed that in the unoperated glands maximal secretory responses were obtained to an intravenous dose of 5–10μg/kg of the tachykinins, that the amount of saliva secreted from the submaxillary gland was twice that from the parotid gland, and that physalaemin was more potent than substance P. Parasympathetic denervation of the parotid gland and decentralization of the submaxillary gland caused a marked sensitization to the tachykinins, as judged by lowered threshold doses for secretion and increased secretory responses to a series of submaximal doses 3 weeks postoperatively. Sensitization was less marked after sympathetic denervation and decentralization; in the parotid gland decentralization caused, in fact, no sensitization while in the submaxillary gland the degree of sensitization was about the same after the two types of operation. The tachykinins acted directly on the gland cells and the effect was not exerted via cholinergic, α-adrenergic or β-adrenergic receptors. The pattern of sensitization to the tachykinins, found in the present study, after the different types of operation is similar to that previously found to cholinergic and α-adrenergic agonists and different from that to a β-adrenergic agonist. Studies by others have shown that in the rat parotid gland peptidergic receptors share a common intracellular pathway with cholinergic and α-adrenergic receptors, whereas β-adrenergic receptors use another pathway. In the present study it is suggested that this intracellular arrangement is of importance for the development of supersensitivity.     

Nitric oxide-dependent in vitro secretion of amylase from innervated or chronically denervated parotid glands of the rat in response to isoprenaline and vasoactive intestinal peptide

The basal in vitro release of amylase was similar from rat parotid lobules of innervated and chronically denervated glands and was unaffected by the inhibitors used in this study. The secretion of amylase induced by isoprenaline or vasoactive intestinal peptide (VIP) was reduced by one-third to one-half from the lobules of the innervated glands and even more so from the lobules of the denervated glands by ODQ, an inhibitor of soluble guanyl cyclase which is activated by nitric oxide (NO) and catalyses the cGMP production. The use of N (omega)-propyl-L-arginine (N-PLA) revealed that the evoked secretion of amylase in the denervated glands depended on the activity of neuronal type NO synthase to synthesize NO. Since the denervated gland is virtually devoid of NO synthase-containing nerve fibres, the neuronal type NO synthase was most probably of a non-neuronal source. NO-dependent amylase secretion was agonist related, since amylase secretion evoked by bethanechol and neuropeptide Y was not reduced by ODQ or N-PLA. Hence, under physiological conditions, activation of beta-adrenoceptors (sympathetic activity) and VIP receptors (parasympathetic activity) is likely to cause secretion of parotid amylase partly through a NO/cGMP-dependent intracellular pathway involving the activity of neuronal type NO synthase, possibly of acinar origin.     

Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenaline, neuropeptide Y, vasoactive intestinal peptide, peptide histidine-isoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenaline- and noradrenaline/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin gene-related peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenaline-, noradrenaline/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C2-C5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradrenaline/neuropeptide Y and noradrenaline cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C2-C5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)     

The otic ganglion in rats and its parotid connection: cholinergic pathways, reflex secretion and a secretory role for the facial nerve

Otic ganglionectomy in rats was found to have affected the parotid gland more profoundly than section of the auriculotemporal nerve as assesssed by reduction in gland weight (by 33 versus 20%) and total acetylcholine synthesizing capacity (by 88 versus 76%) 1 week postoperatively and, when assessed on the day of surgery under adrenoceptor blockade, by reflex secretion (by 99 versus 88%). The facial nerve contributed to the acetylcholine synthesizing capacity of the gland. Section of the nerve only, at the level of the stylomastoid foramen, reduced the acetylcholine synthesis by 15% and, combined with otic ganglionectomy, by 98% or, combined with section of the auriculotemporal nerve, by 82%. The facial nerve was secretory to the gland, and the response was of a cholinergic nature. The nerve conveyed reflex secretion of saliva and caused secretion of saliva upon stimulation. Most of the facial secretory nerve fibres originated from the otic ganglion, since after otic ganglionectomy (and allowing for nerve degeneration) the secretory response to facial nerve stimulation was markedly reduced (from 23 to 4 μl (5 min)⁻¹). The persisting secetory response after otic ganglionectomy, exaggerated due to sensitization, and the residual acetylcholine synthesizing capacity (mainly depending on the facial nerve) showed that a minor proportion of pre- and postganglionic nerve fibres relay outside the otic ganglion. The great auricular nerve, which like the facial nerve penetrates the gland, caused no secretion of saliva upon stimulation. Avulsion of the auriculotemporal nerve was more effective than otic ganglionectomy in reducing the acetylcholine synthesizing capacity (by 94 versus 88%) and as effective as otic ganglionectomy in abolishing reflex secretion (by 99%). When aiming at parasympathetic denervation, avulsion may be the preferable choice, since it is technically easier to perform than otic ganglionectomy.     

Protein secretion in salivary glands of cats in vivo and in vitro in response to vasoactive intestinal peptide

In anaesthetized cats exogenous vasoactive intestinal peptide failed to elicit any secretion of saliva from the submandibular and parotid glands. However, protein release from both glands occurred in response to VIP in the presence of alpha- and beta-adrenoceptor blocking agents and was dose-dependent. This response was revealed by means of a subsequent washout flow of saliva evoked by intravenous injections of methacholine or stimulations of the parasympathetic innervation. The submandibular glands responded to vasoactive intestinal peptide at a lower dose than the parotid glands. In the presence of atropine (but in the absence of adrenoceptor blockers), stimulation of the parasympathetic chorda-lingual nerve, which of itself elicited no secretion of saliva, contributed to the release of protein within the submandibular gland, since the output of protein in response to a subsequent stimulation of the sympathetic innervation was increased. Vasoactive intestinal peptide administered in combination with methacholine or during ongoing parasympathetic nerve-induced salivary secretion revealed positive interactions, particularly with respect to protein release. In-vitro protein release in response to vasoactive intestinal peptide was also demonstrated by perfusing small pieces of the two glands in the presence of muscarinic and adrenoceptor blockers. As in vivo, submandibular tissue responded at a lower concentration of vasoactive intestinal peptide than the parotid tissue. One to two weeks after combined parasympathetic and sympathetic denervation of the parotid glands, the glands were sensitized to vasoactive intestinal peptide when tested in vitro. It is concluded that vasoactive intestinal peptide or a structurally related peptide is a potential transmitter in the parasympathetic control of protein secretion in salivary glands of cats.     

Motor and Secretory Effects of Nerves on the Parotid Gland of the Rat

The influence of parasympathetic and sympathetic nerves on the parotid gland of the rat was investigated. It was found that both divisions of the autonomic nerves evoke secretion and probably also motor effects in this gland. Secretion elicited on sympathetic stimulation was mediated both via α- and β-adrenoceptors, while motor effects were mediated via α-adrenoceptors. On stimulation of the autonomic nerves a lower duct pressure was reached in the parotid than in the submaxillary gland, and on sympathetic nerve stimulation the flow of saliva always started later from the parotid than from the submaxillary gland. These findings are discussed in the view of the different arrangement of the myoepithelial cells in the 2 glands.     

Secretion of kallikrein and its role in vasodilatation in the submaxillary gland

1. The effects of parasympathetic (chorda) and sympathetic nerve stimulation on the concentration and output of kallikrein secreted in saliva from the cat's submaxillary gland were compared. Sympathetic stimulation always produced a much higher concentration (up to 500 times) and output (up to 390 times) of kallikrein than parasympathetic stimulation. In the dog, in which sympathetic nerve stimulation produces little or no secretion from the submaxillary gland, there was also a marked increase in the secretion of kallikrein when sympathetic was superimposed on parasympathetic secretion. This effect did not occur, however, in the rabbit's submaxillary gland.2. It was possible to deplete the cat's submaxillary gland of kallikrein, either by ligation of the duct for several days or by duct ligation and sympathetic nerve stimulation, so that it was undetectable either in the gland or in saliva after stimulation of the chorda. Such glands, nevertheless, responded to chorda stimulation with a normal atropine-resistant vasodilatation.3. There is a close parallelism between the rate of secretion of salvia and vasodilatation over a range of frequencies of chorda stimulation, but the output (and concentration) of kallikrein in saliva is distinctly different for the same frequencies of nerve stimulation.4. Our results are consistent with the view that vasodilator nerves exist in the parasympathetic nerves to the submaxillary gland. We suggest that they are cholinergic in nature despite the fact that chorda vasodilatation is resistant to atropine. It is further suggested that neither the kallikrein-kinin system nor adrenergic vasodilator nerve fibres play a significant role in chorda vasodilatation.     

Pituitary adenylate cyclase activating peptide (PACAP) in salivary glands of the rat: origin,and secretory and vascular effects

Pituitary adenylate cyclase activating peptide (PACAP)-38. injected Lv. to the anaesthetized rat. evoked secretion of saliva from the three major salivary glands. the submandibular glands responding with the greatest and the sublingual glands with the smallest volumes. The parotid saliva was rich in amylase and protein. In vitro. pieces of parotid and submandibular gland tissues released K⁺ and protein in response to PACAP-38. with atropine and adrenoceptor antagonists present. The blood flow in the submandibular gland increased in response to PACAP-38. despite a marked fall in mean aortic blood pressure. PACAP is a vasoactive intestinal peptide (VIP)-like neuropeptide. A comparison between the two peptides showed PACAP-38 to be more effective than VIP with respect to vascular responses and less or equi-effective with VIP with respect to the secretory responses. thus suggesting the involvement of PACAP type I and type II receptors. respectively PACAP-38 and -27 were present in the parotid gland as judged by radioimmunoassay. the concentration of the former being about twice that of the latter. Parasympathetic denervation. by cutting the auricula-temporal nerve. reduced the total parotid gland contents of PACAP-38 and -27 by 23 and 44%. respectively (compared with a previously demonstrated 95% reduction of VIP). Sympathetic de nervation. section of the facial nerve or treatment with the sensory neurotoxin capsaicin did not affect the content of PACAP. The difference in efficacy between PACAP and VIP in the vascular and secretory responses as well as the difference in localization suggest that the two peptides play different physiological roles in the salivary glands.     

Substance P: Indirect and direct effects on parotid acinar cell membrane potential

Direct effects of exogenously applied substance P on salivary acinar cells have been previously reported. This electro-physiological study confirms these direct effects in rat but not mouse parotid gland. We demonstrate that in the absence of autonomic blockade the peptide evokes marked responses which are blocked by atropine (10^–6 M). These effects cannot be attributed to direct activation of acinar cells and are presumably due to release of acetylcholine from parasympathetic nerve endings. We consider that substance P, in addition to direct effects, could act to modulate neuronal activity in salivary glands; a role already assigned to the peptide in the central nervous system.     

Acinar degranulation in the rat parotid gland induced by neuropeptides

Vasoactive intestinal peptide (VIP, 4 microg kg(-1) min(-1)), substance P (3 microg kg(-1) min(-1)) and neurokinin A (2.5 microg kg(-1) min(-1)) were infused intravenously for 30 min in anaesthetized rats and the effects of these peptides on the parotid gland were examined. VIP reduced the numerical density of parotid acinar secretory granules, storing proteins, by 29 % and the glandular amylase activity by 33 %. Substance P reduced the number of secretory granules by 18 % but the amylase activity was unchanged. These results make VIP and substance P likely contributors to the parasympathetic non-adrenergic, non-cholinergic (NANC)-evoked parotid acinar degranulation. Neurokinin A, on the other hand, caused no reduction in granular number but reduced the glandular amylase activity by 19 %, indicating vesicular protein secretion.     

Amylase secretion in response to activation of different autonomic receptors in the rabbit parotid gland

The contribution by different autonomic receptors to the amylase secretion from the parotid gland of the anaesthetized rabbit was studied as the response to various parasympathomimetic and sympathomimetic drugs. Amylase secretion by infusions of pilocarpine or parasympathetic nerve stimulation was low, but regularly higher in response to pilocarpine than to parasympathetic nerve stimulation. These effects were reduced to the same level by β-adrenoceptor block indicating the presence and for pilocarpine also the release of catecholamines, probably from the adrenals, Isoprenaline injections produced a high amylase secretion, that was blocked by atenolol, indicating that predominantly β₁-adrenoceptors were activated. Phenylephrine was without amylase secretory effects. By accepting isoprenaline maximum as maximum for sympathetically produced amylase secretion, a theoretical frequency-response relationship for amylase secretion by sympathetic nerve stimulation could be calculated; ED₅₀ was 0.9 Hz. The results indicate that under experimental conditions in vivo there are certain differences between the rat and the rabbit parotid glands in the autonomic control not only of fluid, but particularly of amylase secretion.     

Vagal vasodilatory mechanisms in the pig bronchial circulation preferentially involves sensory nerves

The present study shows that in contrast to the upper trachea, where the parasympathetic vasodilatory components of both cholinergic and non-cholinergic nature are dominating, the vagal blood flow regulation in the peripheral airways of the pig supplied by the bronchial artery is entirely carried out by local release of vasodilatory mediators from capsaicin-sensitive sensory nerves. Also inhalation of the vapour phase from the major airway irritant cigarette smoke was associated with a marked increase in bronchial blood flow possibly via local axon reflexes. Capsaicin, substance P (SP) and calcitonin gene-related peptide (CGRP) caused vasodilatation in both the trachea and bronchi while vasoactive intestinal polypeptide (VIP) was most active in the trachea. These functional data were supported by immunohistochemical studies showing the presence of SP- and CGRP-containing nerves of presumably sensory origin around bronchial blood vessels while VIP-positive perivascular fibres of local parasympathetic origin were found mainly in the trachea.     

Degeneration secretion from parotid glands after section of the auriculotemporal nerves at different levels

1. In cats under ether or hexobarbitone anaesthesia the auriculotemporal nerve was cut near the parotid gland on one side and 12-20 mm more proximally on the other. After 22-64(1/2) hr the cats were anaesthetized with chloralose and the parotid ducts cannulated. Degeneration secretion of saliva which appears after post-ganglionic parasympathetic denervation was found to start 2-5(1/2) hr later in the gland denervated proximally than in that denervated distally. It ceased, on the other hand, later in the former than in the latter gland.2. Before degeneration secretion had started spontaneously it could be provoked by intravenous injection of acetylcholine, methacholine, carbachol or eserine and the effect was more pronounced on the gland denervated distally. When it had ceased spontaneously it could also be provoked, and the effect on the other gland was now more marked.3. Earlier it has been assumed that while a nerve is degenerating there is a period when the nerve endings are unable to retain in a normal way acetylcholine still being synthesized. It is now suggested that this period starts later after proximal than after distal denervation because more of the material required for the normal function of the endings is available in a long piece than in a short piece of nerve.     

Depletion by capsaicin of substance P-immunoreactivity and acetylcholinesterase activity from nerve fibres in the guinea-pig heart

The sensitivity to capsaicin of substance P-immunoreactive nerve fibres in the hearts of guinea-pigs was examined. Capsaicin decreased considerably the substance P-immunoreactive material in nerve fibres of the parietal pericardium, atria, bicuspid and tricuspid valves. Pericardial and valvar nerve fibres localized by acetylcholinesterase (AChE) histochemistry had a distribution and density pattern similar to that of the substance P immunoreactive fibres. Capsaicin treatment also decreased the number of visible AChE-positive nerve fibres. It is known that capsaicin has a selective action on those substance P-immunoreactive fibres that are of sensory origin; thus, these results imply that substance P-immunoreactive fibres in the heart are sensory. Moreover, the results suggest that some of the nerve fibres localized in the heart by AChE histochemistry are substance P-containing sensory fibres.     

Sprouting of non-sympathetic myelinated and unmyelinated fibres in response to chronic sympathetic denervation in the pineal gland of the Chinese hamster,Cricetulus griseus

Summary We have examined the effects of chronic sympathetic denervation on non-sympathetic myelinated and unmyelinated fibres in the superficial pineal gland of the Chinese hamster (Cricetulus griseus), using LM, EM and immunohistochemistry. The results suggest that non-sympathetic, myelinated and unmyelinated fibres enter the superficial pineal gland at its distal portion by way of the nervi conarii, and that these fibres are immunoreactive for calcitonin gene-related peptide or substance P. Non-sympathetic, myelinated and unmyelinated fibres in the superficial pineal gland increased in number following chronic superior cervical ganglionectomy. The number of unmyelinated fibres in the nervi conarii also increased in ganglionectomized animals. Thus, the numerical increase of calcitonin gene-related peptide or substance P fibres found in the superficial pineal gland after long-term sympathectomy may be due to sprouting of these fibres. It is speculated that the growth of non-sympathetic, myelinated and unmyelinated fibres and myelination of the former fibres occurring after sympathectomy are caused by nerve growth factor-related mechanisms.     

Salivary Secretion in the Rat in a Hot Environment

Conscious rats were found to secrete saliva from the submaxillary gland when exposed to heat. In rats with sympathetically denervated glands the secretory rate was a little lower and in parasympathetically denervated glands the secretion was almost abolished. The salivary flow appeared to increase with increasing ambient temperature and the secretion started earlier at higher temperatures. The secretion elicited by heat stress was compared to the secretion evoked by electrical stimulation of the parasympathetic nerve to the gland. It is concluded that the salivary secretion evoked by heat stress is mainly mediated by the parasympathetic secretory nerves, although the sympathetic fibres also take part.