Atypical antipsychotics - effects of amisulpride on salivary secretion and on clozapine-induced sialorrhea

Oral Diseases (2012) 18, 680-691 Objective: Amisulpride is suggested for treatment of clozapine-induced sialorrhea. However, objective measurements of its effectiveness are lacking and, preclinically, amisulpride has no effect. We currently hypothesise that amisulpride acts by reducing the nervous- rather than the clozapine-driven salivary secretion. Material and Methods: Effects of intravenous amisulpride (as well as of clozapine and raclopride, a dopamine D2/D3 antagonist) were investigated in rats, including those subjected to chronic preganglionic parasympathetic denervation (submandibular glands) or combined postganglionic parasympathetic and sympathetic denervation (parotid glands). In duct-cannulated glands, secretion was evoked reflexly, at low and maximum flow rates, and by electrical stimulation of the parasympathetic and sympathetic innervations, and administration of autonomimetics (including substance P). Results: Unlike clozapine, amisulpride had no effect on the reflexly evoked secretion at maximum rate. With respect to reflex secretion at low rate and to the secretion evoked by muscarinic, α-adrenergic, β-adrenergic and substance P receptors, amisulpride (in contrast to raclopride) dose dependently potentiated the responses. Amisulpride had no effect on gland blood flow. Conclusions: No support for any inhibitory influence of amisulpride was found. Conversely, amisulpride universally enhanced secretion, suggesting that amisulpride is a potential drug for dry-mouth treatment. The mechanism behind the potentiation is currently unknown.     

Secretion of amylase from the rat parotid salivary gland after degeneration of the auriculotemporal nerve

The output of amylase into saliva secreted after injection of methacholine or substance P was increased after parasympathetic denervation, but the salivary concentration of amylase was unchanged. The increased output corresponded to the increased flow. Isoprenaline injected during the methacholine-induced secretion raised the output, more being secreted from the denervated than from the contralateral gland. Vasoactive intestinal peptide, given while substance P caused salivation, also increased the amylase output, but equally from the two glands.     

N-Desmethylclozapine exerts dual and opposite effects on salivary secretion in the rat

Ekström J, Godoy T, Riva A. N -Desmethylclozapine exerts dual and opposite effects on salivary secretion in the rat. Eur J Oral Sci 2010; 118: 1–8. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci
N -Desmethylclozapine is a major metabolite of the atypical antipsychotic drug clozapine, used in the treatment of therapy-resistant schizophrenia. Patients under clozapine treatment report a troublesome sialorrhea. Recent experiments show clozapine to exert mixed agonist/antagonist actions on salivary secretion in rats. The present study was performed to define the secretory role of N -desmethylclozapine and to compare it with that of its parent compound. N -Desmethylclozapine evoked secretion by acting directly on the muscarinic acetylcholine M1-receptors of 'silent' duct-cannulated parotid and submandibular glands of the anaesthetized rat. In chronic surgically denervated glands, the secretory response was enlarged. The methacholine-evoked secretion, as well as the parasympathetic nerve-evoked secretion, were reduced by N -desmethylclozapine and involved blockade of M3-receptors, while the sympathetic nerve-evoked response was reduced, involving blockade of α₍₁₎-adrenergic receptors. Synergistic interactions between N -desmethylclozapine and the β-adrenergic receptor agonist, isoprenaline, occurred. Compared with clozapine, the excitatory efficacy of N -desmethylclozapine was higher and the inhibitory efficacy was lower (parasympathetic activity) or about the same (sympathetic activity). Theoretically, in humans treated with clozapine, an increase in the N -desmethylclozapine : clozapine ratio would contribute to salivation during the night and at rest, and, furthermore, the magnitude of the reduction in the reflexly elicited secretion is likely to diminish.     

Amylase in parotid saliva of rats after sympathetic nervous decentralization

The marked increase in the amylase secretion that occurs in parasympathetic saliva some days after sympathetic ganglionectomy occurred after decentralization also; hence, it results from lack of impulses from the central nervous system. It occurred whether salivation was evoked by activating muscarinic receptors with methacholine, peptidergic receptors with substance P or physalaemin, or alpha-adrenoceptors with phenylephrine. Unilateral operation affected to some degree the contralateral gland also.     

Reinnervated nerves contribute to the secretion function and regeneration of denervated submandibular glands in rabbits

This study aimed to investigate the contribution of redistributed nerves in the secretory function and regeneration of a denervated submandibular gland (SMG). The postganglionic parasympathetic and sympathetic denervated SMGs of rabbits were wrapped in polyester or acellular dermal matrices to block nerve regeneration either partially or completely. Submandibular glands were removed 4, 8, 16, and 24 wk after the operation and examined histologically. Furthermore, the aquaporin‐5 (AQP5), muscarinic‐3 (M3), and β1‐adrenergic receptors were evaluated by immunofluorescence and western blot analysis. After denervation, salivary flow was decreased and acinar cells were atrophic, and the expression levels of the M3, β1‐adrenergic, and AQP5 receptors were decreased. However, both impaired secretion function and atrophic parenchyma were gradually ameliorated with the growing redistribution of parasympathetic and sympathetic nerves. Apoptosis was markedly inhibited and expression of the M3, β1‐adrenergic, and AQP5 receptors was increased after reinnervation. In contrast, SMGs without reinnervated nerves maintained hyposecretion and atrophic parenchyma. In conclusion, reinnervated nerves in a rabbit's denervated SMG played an important role in the secretion function and regeneration of SMGs via up‐regulation of the expression of neurotransmitter receptors and AQP5.     

Regulation of hemodynamics in major salivary glands by parasympathetic vasodilation

Background

Since salivary fluid is created from blood plasma, hemodynamics in the salivary glands play an important role in the production of saliva. Trigeminal sensory input induces both salivary secretion and reflex parasympathetic vasodilation in salivary glands. This glandular vasodilation is thought to be important for the regulation of glandular hemodynamics due to the rapidity with which blood flow is increased. This review article summarizes recent research on the involvement of parasympathetic vasodilation in regulating hemodynamics in the salivary gland.

Acetylcholine synthesis, muscarinic receptor subtypes, neuropeptides and secretion of ferret salivary glands with special reference to the zygomatic gland

Studies on salivary secretion are usually focused on parotid and submandibular glands. However, the film of mucin, that protects the oral structures and is responsible for the feeling of oral comfort, is produced by the submucosal glands. The submucosal zygomatic and molar glands are particularly large in carnivores such as the ferret. Comparisons between the mucous sublingual, zygomatic and molar glands, serous parotid and sero-mucous submandibular glands showed the acetylcholine synthesis, in terms of concentration, to be three to four times higher in the mucous glands than in the parotid and submandibular glands. Bromoacetylcholine inhibited 95-99% of the synthesis of acetylcholine in the incubates of the five types of glands, showing the acetylcholine synthesis to depend on the activity of choline acetyltransferase. The high acetylcholine synthesis in the zygomatic gland was of nervous origin, since cutting the buccal nerve, aiming at parasympathetic denervation, and allowing time for nerve degeneration, reduced the acetylcholine synthesising capacity of the gland by 95%. A similar reduction (96%) in the parotid gland followed upon the avulsion of the parasympathetic auriculo-temporal nerve. Zygomatic saliva was very viscous. The salivary flow rate in response to electrical stimulation (20 Hz) of the buccal nerve (zygomatic gland), expressed per gland weight, was one-third of that to stimulation of the auriculo-temporal nerve (parotid gland) or the chorda-lingual nerve (submandibular gland). As previously shown for the parotid and submandibular gland, a certain fraction (25%) of the parasympathetic secretory response of the zygomatic gland depended on non-adrenergic, non-cholinergic transmission mechanisms, probably involving substance P and vasoactive intestinal peptide and possibly calcitonin gene-related peptide. Particularly, high concentrations of vasoactive intestinal peptide were found in the sublingual and molar glands, and of substance P in the submandibular, zygomatic and molar glands; notably, the concentration of calcitonin gene-related peptide of the sublingual gland was not detectable. All five muscarinic receptor subtypes were detected in the five glands. The receptor protein profile, as judged by immunoblotting and semi-quantitative estimations, was about the same in the glands: high level of M3, low level of M2 and levels roughly in the same range of M1, M4 and M5. Compared to the parotid and submandibular glands, the M5 receptor level was particularly low in the mucin-secreting glands. The present study points out both similarities and dissimilarities between the five types of glands investigated. The zygomatic gland, in particular, appears to be a suitable model for future studies aiming at causing relief of dry mouth by local pharmacological treatment.     

Parasympathectomy increases resting salivary secretion in normal and irradiated submandibular glands of rats

Fluid and ion secretion from the submandibular gland (SMG) is mainly regulated by parasympathetic nerves. This study evaluated the effect of parasympathectomy on salivary secretion from normal and irradiated rat SMGs from 1 to 24 wk after denervation. Although stimulated salivary secretion was significantly lower in denervated SMGs compared with contralateral self‐controls, the resting salivary flow rates were markedly higher in the denervated SMGs at 1, 12, and 24 wk after denervation. The levels of muscarinic acetylcholine M1 and M3 receptors, as well as of aquaporin 5, were up‐regulated. Notably, although irradiated SMGs showed significantly lower resting and stimulated salivary secretion rates than non‐irradiated SMGs, the resting salivary secretion rates of the irradiated and denervated SMGs were markedly higher than seen in the irradiated self‐control SMGs at 1, 12, and 24 wk after parasympathectomy, and were even higher than seen in the non‐irradiated sham‐operated rats. The expression of M1 and M3 receptors was similarly elevated. Taken together, our results suggest that parasympathetic denervation increases resting salivary secretion of both normal and irradiated SMGs. This approach might provide a potential modality for relieving radiation‐induced xerostomia, which is a common complication following treatment of head and neck cancer.     

Oral Dryness and Thirst: —The Central Effect of Acetylcholine on Drinking Behavior—

Saliva is secreted from the salivary glands, and its production is controlled by the autonomic nervous system. Acetylcholine released by the parasympathetic nervous system stimulates muscarinic receptors and triggers the secretion of serous saliva. We found that intraperitoneally injected pilocarpine, which is known as a cholinergic muscarinic receptor agonist and a sialogue, increases not only saliva secretion but also drinking behavior in rats. This indicates that while pilocarpine increases wetness in the oral cavity, it also evokes the sensation of thirst and elicits water intake. Therefore, the thirst sensation can be evoked independently of oral wetness. Further, we found that nicotine, which is a stimulant of cholinergic nicotinic receptor, evokes the thirst sensation. We demonstrated that thirst was induced both by nicotine acting on nicotinic receptors in the brain and by pilocarpine acting on muscarinic receptors in the brain using behavioral, electrophysiological and molecular biological experiments. In this review, we discuss the relationship between oral dryness and thirst, and the different mechanisms underlying these two phenomena. We propose that while the symptom of xerostomia includes both oral dryness and thirst, it is important to understand that oral dryness and the sensation of thirst are intrinsically different.     

Sympathectomy-induced increases in calcitonin gene-related peptide (CGRP)-, substance P- and vasoactive intestinal peptide (VIP)-levels in parotid and submandibular glands of the rat

The neuropeptide contents of rat salivary glands were increased four weeks after sympathetic postganglionic denervation (but not after preganglionic denervation): calcitonin gene-related peptide (CGRP) by 400 and 65% in the parotid and submandibular glands, respectively; substance P by 30% in the submandibular gland; and vasoactive intestinal peptide (VIP) by 30% in the parotid gland. The sensory neurotoxin capsaicin prevented the expected increases of CGRP and substance P in the submandibular glands and of VIP in the parotid glands. The CGRP-increase in the parotid gland was, however, only reduced (by 65%). Parasympathetic otic ganglionectomy reduced the peptide levels in the parotid glands (CGRP--50%, VIP--98% and substance P--99%). From these residual levels, CGRP increased almost 8-fold and substance P 3-fold in response to the sympathetic denervation, while VIP was unaffected. In the parasympathetically denervated glands, the capsaicin-sensitive contribution to the CGRP-response to sympathetic denervation was roughly estimated to be more than 25% but less than 40%, while the corresponding contribution to the substance P-response was roughly estimated to be more than 6% but less than 58%. Most likely not only CGRP/substance P-containing sensory C-fibres (submandibular and parotid glands) but also parasympathetic VIP-containing secretomotor and vasomotor fibres (parotid glands) contributed to the capsaicin-sensitive response to sympathetic denervation.     

Clozapine-induced salivation: interaction with N-desmethylclozapine and amisulpride in an experimental rat model

Many drugs (e.g. amisulpride) have been used to treat troublesome clozapine-induced salivation; however, varying success has been achieved in this respect, probably because, until recently, the salivatory action of clozapine has been largely unexplained. In the rat, clozapine and its main metabolite, N-desmethylclozapine, were found to exert mixed secretory actions: excitatory, through muscarinic acetylcholine M1-receptors giving rise to a long-lasting, low-level flow of saliva; and inhibitory, through muscarinic M3-receptors and α(1) -adrenoceptors reducing the parasympathetically and sympathetically nerve-evoked flow of saliva. The aim of the present study was to define the interactions between clozapine and N-desmethylclozapine, and clozapine and amisulpride, with respect to the excitatory response. Submandibular glands, sensitized by chronic parasympathetic preganglionic denervation, were studied in pentobarbitone-anaesthetized rats. To prevent clozapine from being metabolized to N-desmethylclozapine by hepatic enzymes, the liver was, under terminal anaesthesia, excluded from the circulation. The weak receptor-stimulating clozapine prevented the strong receptor-stimulating N-desmethylclozapine, at specific ratios in humans and in rats, from exerting its full agonistic action. In conclusion, the contribution of N-desmethylclozapine to the clozapine-induced sialorrhoea was, at most, only partly additive. Furthermore, the present experimental set-up failed to demonstrate any anti-salivatory action of amisulpride on the clozapine-induced flow of saliva.     

调控涎腺细胞分泌的受体及其信号转导的研究进展

涎腺细胞膜表面的受体及其所介导的信号转导通路是调控涎腺分泌的重要途径。本文概要介绍了毒蕈碱型胆碱受体、α1-肾上腺素受体、β-肾上腺素受体及辣椒素受体在正常颌下腺的表达及其对唾液分泌的调控作用,重点介绍了作者所进行的涎腺细胞受体对失神经支配的移植颌下腺分泌调控作用的研究结果。     

Autonomic control of salivary secretion

The view is generally held that the secretory elements of the major salivary glands are supplied with parasympathetic (cholinergic) nerve fibres, while the extent of the sympathetic (adrenergic) innervation varies considerably not only between different species but also between the glands of the same species. Parasympathetic stimulation evokes a copious flow of saliva. In contrast, sympathetic stimulation produces either a small flow, which is rich in protein, or no flow at all. In recent years it has become evident that secretion of saliva may not be elicited only by cholinergic or adrenergic agonists but also by peptides, injected into the bloodstream, and further, that the salivary secretion in response to stimulation of the parasympathetic innervation is not always completely abolished by the muscarinic receptor blocker atropine (and adrenoceptor antagonists). The present communication focuses on this "atropine-resistant" parasympathetic influence on the salivary glands and on a possible role for neuropeptides in mediating "atropine-resistant" parasympathetic responses in the glands. Immediate effects as well as long-term trophic effects are considered.     

Involvement of vasoactive intestinal polypeptide in the parasympathetic vasodilatation of the rat masseter muscle

The parasympathetic vasodilatory fibres are known to innervate vessels in a rat masseter muscle via both cholinergic and non-cholinergic mechanisms. However, the non-cholinergic mechanisms are still unclear. Recently, vasoactive intestinal polypeptide (VIP) was convincingly shown to be involved in the parasympathetic vasodilatation in orofacial areas, such as submandibular glands and lower lip. However, very little is known about the rat masseter muscle. The present study was designed in the rat masseter muscle to assess (1) whether the parasympathetic nerve innervating vessels have VIP immunoreactivities, (2) whether intravenous administration of VIP induces the vasodilatation, and (3) effects of selective VIP receptor antagonist ([4Cl-d-Phe⁶, Leu¹⁷] VIP) in the presence or absence of atropine on the parasympathetic vasodilatation. The VIP immunoreactivities were found at two sites of the parasympathetic otic ganglion and nerve fibres located around vessels. The intravenous administration of VIP induced the vasodilatation, and [4Cl-d-Phe⁶, Leu¹⁷] VIP markedly decreased the vasodilatation evoked by VIP administration. The parasympathetic vasodilatation was not inhibited by [4Cl-d-Phe⁶, Leu¹⁷] VIP. However, treatment with [4Cl-d-Phe⁶, Leu¹⁷] VIP markedly decreased the parasympathetic vasodilatation when [4Cl-d-Phe⁶, Leu¹⁷] VIP was administered together with atropine. These results suggest that (1) VIP exists in the postganglionic parasympathetic nerve innervating the vessels in the masseter muscle, (2) the intravenous administration of VIP induces the vasodilatation in the masseter muscle, and (3) VIP may be involved in the parasympathetic vasodilatation in the masseter muscle when muscarinic cholinergic receptors are deactivated by either atropine or the suppression of the ACh release.     

Carbachol improves secretion in the early phase after rabbit submandibular gland transplantation

Oral Diseases (2010) 16 , 351–359 Objectives: To investigate the changes in the muscarinic receptor signaling pathway with submandibular gland (SMG) transplantation and whether carbachol improves secretion in transplanted SMGs. Materials and methods: SMG autotransplantation was performed in a rabbit model. Carbachol (1 μM) was infused into the transplanted glands from postoperative day 1–7. The expression of the M1 and M3 muscarinic receptors, aquaporin‐5 (AQP5), and phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK1/2) was measured by RT‐PCR, immunoblotting or immunofluorescence. The content of inositol 1, 4, 5‐trisphosphate (IP₃) was measured by radioimmunoassay. Results: Salivary flow of the transplanted SMGs was decreased after transplantation. As well, the expressions of M1 and M3 receptors and their downstream signaling molecules, IP₃, p‐ERK1/2 and AQP5, were all reduced. Atrophy of acinar cells was shown in transplanted glands. However, all these alterations were reversed after carbachol treatment for 7 days. Furthermore, carbachol directly increased the mRNA expression of AQP5 and phosphorylation of ERK1/2 in cultured neonatal rabbit SMG cells. Conclusion: A lack of acetylcholine and downregulation of the muscarinic receptor signaling pathway is involved in the early hypofunction of transplanted SMGs. Carbachol treatment could be a new therapeutic strategy to improve secretion and prevent the obstruction of Wharton’s duct in the early phase after SMG transplantation.     

大鼠腮腺M受体亚型分布的实验研究

目的：检测大鼠腮腺组织中Ｍ受体亚型的分布。方法：取ＳＤ大鼠腮腺制备膜蛋白,应用３Ｈ－ＱＮＢ测定特异性饱和曲线;选择Ｍ１、Ｍ２、Ｍ３的选择性拮抗剂Ｐｉｒｅｎｚｅｐｉｎｅ,Ｇａｌａｍｉｎｅ,４－ＤＡＭＰ进行竟争性结合实验。结果：饱和曲线示平衡解离常数Ｋｄ为０．８４±０．０８ｎＭ,最大结合量Ｂｍａｘ为１００．２±７．７ｆｍｏｌ／ｍｇ蛋白,Ｈｉｌ系数为０．８８,这说明３Ｈ－ＱＮＢ与Ｍ受体为单一结合位点;竟争性结合实验示三种拮抗剂的半数最大抑制浓度ＩＣ５０分别为１．９８×１０－９（Ｍ１）,７．８×１０－８（Ｍ２）,４．７６×１０－１０ｍｏｌ／Ｌ。结论：以上结果表明腮腺Ｍ受体以Ｍ３最多,Ｍ１次之,Ｍ２最少,这对阐明唾液腺的分泌机制、Ｍ受体与疾病状态均有重要的意义。     

Soluble guanylyl cyclase is localised in the acinar cells and participates in amylase secretion in rat parotid gland

It is well known that the muscarinic cholinergic agonists, carbachol and methacholine, enhance nitric oxide synthase (NOS) activity, and also stimulate salivary secretion. In the present study, we investigated whether salivary secretion by muscarinic cholinergic stimulation is mediated through the NO/cGMP signaling pathway in rat salivary glands. Since NO activates soluble guanylyl cyclase (sGC) and cGMP may function as a mediator, the localisation of sGC was investigated in the salivary glands. sGC was localized in both the acinar and duct cells of the rat parotid and sublingual glands, and localized only in the acinar cells of the submandibular glands. S-Nitroso-glutathione (NO generator; GSNO) and YC-1 (NO-independent sGC activator) stimulated sGC in the cytosol to synthesise cGMP. The combination of GSNO and YC-1 stimulated sGC synergistically. Carbachol, GSNO and YC-1 enhanced amylase release from the rat parotid glands. Amylase release stimulated by carbachol and GSNO was inhibited by addition of the sGC inhibitor, ODQ, and cGMP-dependent protein kinase inhibitor, KT-5823. These results indicate that amylase release may be mediated through the NO/cGMP signaling pathway.     

Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man

In rat parotid, submandibular and sublingual glands and in ovine parotid and in human labial glands, the expression of muscarinic receptor subtypes was examined by immunoblotting and immunohistochemistry. Functional correlates were searched for in rat salivary glands. In the rat submandibular and sublingual glandular tissues clear signals of muscarinic M1 and M5 receptors could be detected in the immunoblotting and vague bands for muscarinic M3 and, in particular for, M4 receptors. The rat parotid gland differed. In this gland, the signal was less obvious for the muscarinic M1 receptor, and further, muscarinic M4 receptors appeared more strongly marked than in the submandibular glands. The results from the immunohistochemistry could be interpreted as the muscarinic M4 receptors are located on nerve fibres, since the outer layer of lobuli were densely stained. Intraglandular vessels in the rat submandibular and parotid glands showed expression of M3 receptors. In contrast to the parotid gland, the submandibular vessels also expressed M1 and M2 receptors. Occasionally M5 receptors appeared in the arteries and veins also. The functional studies in the rat confirmed muscarinic M1 receptor mediated secretion in the submandibular gland. Since the M1 receptor blockade did not affect submandibular blood flow, indirect vascular effects could not in total explain the secretory inhibition. Also in the human labial glands, muscarinic M1, M3 and M5 receptors occurred. No or low amounts of muscarinic M2 and M4 receptors could be detected. In patients with Sj?gren-like symptoms an up-regulation of M3, M4 and M5 receptors was apparent in the labial glands. In ovine parotid glands all receptors could be detected, but constantly with vague bands for muscarinic M2 receptors. In conclusion, muscarinic M1 receptors seem to be expressed in seromucous/mucous glands. A secretory effect by muscarinic M5 receptors is not to be excluded, since they were expressed in all the glands examined. However, other functions, such as promotion of inflammation, cell growth and proliferation are possible as well.     

Effect of chronic constant light on sensitivity of rat submandibular gland

A consistent difference in the secretory response between submandibular (SM) glands of rats maintained under constant light (CL) during 50 days and those of rats under a photoperiod (14 h light: 10 h dark) was found. We have used alpha 1-adrenergic, muscarinic, peptidergic and beta-adrenergic secretagogue agents to study the secretory response of rat SM glands "in vivo". The response to phenylephrine, methacholine and substance P, was increased by exposure CL, while that to isoproterenol was diminished. The changes in the sensitivity of the secretory response from SM gland of rats under CL might be related to changes in the normal interplay of various receptors as well as to possible alteration in the intracellular signal transduction. It may represent and adaptive process of the nervous control of saliva secretion by environmental light and be of physiological and clinical interest.     

Cholinergic nerves of unknown origin in the parotid glands of rats.

The effectiveness of the parasympathetic denervation of the gland caused by avulsion of the auriculo-temporal nerve was investigated. In the first type of experiment, the cholinesterase inhibitor, eserine, was injected through the salivary duct. The substance is thought to preserve acetylcholine leaking from nerve endings, thereby causing secretion of saliva; a secretory response was thus taken as evidence for the presence of cholinergic nerves. In the second type of experiment, the acetylcholinesterase activity of the gland was examined histochemically. From the denervated glands, eserine caused secretion and acetylcholinesterase positive nerves were found in the glands. Both the amount of secretion and the number of nerves were greatly diminished when compared with the intact glands. The results indicate that the denervation procedure was incomplete, leaving some cholinergic nerves unsevered. That cholinergic nerves remain offers an explanation to the residual choline acetyltransferase activity, which has earlier been shown in the gland after avulsion of the auriculo-temporal nerve. To extend the denervation procedure, either avulsion of the facial nerve or avulsion of the superior cervical ganglion was included. Neither procedure abolished the response to eserine or the acetyl-cholinesterase activity, indicating that the routes for the remaining nerves are to be found elsewhere.