Pituitary adenylate cyclase‐activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice

Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9‐week‐old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293–1299, 2016. © 2016 Wiley Periodicals, Inc.     

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunoreactivity distribution in the small intestine of the adult New Hampshire chicken

We conducted a study in which we demonstrated by means of immunoperoxidase and immunofluorescence methods the presence of pituitary adenylate cyclase-activating peptide 38 (PACAP-38) immunoreactivity in the small intestine of adult New Hampshire chickens and its co-localization with VIP. In particular we describe for the first time the presence of PACAP-positive cells in the epithelium of crypts and villi. Using double immunostaining, we observed that these two peptides were widely co-localized in the nerve structures of duodenum and jejunum with the exception of the ileum, where we noticed a faint co-localization regarding the nerve fibers of the lamina propria of the villi. Furthermore, the two peptides were occasionally co-stored in the epithelial cells of the mucosa. Our findings suggest that in the chicken small intestine, PACAP can be considered, not only as a neuromodulator released by nerve elements, but also as a gut hormone secreted by endocrine cells, and it appears likely to have a role in the regulation of important intestinal physiological functions.     

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.     

Masticatory-salivary reflexes mobilize noradrenergic,non-cholinergic secretory mechanisms in parotid glands of conscious rats

All rats were maintained on liquid diet, prepared from their ordinary hard pelleted diet, for a week, then fasted 32–33 h and subsequently either fed liquid or pelleted diet for 60–90 min or serving as non-fed controls. Both the fed and the non-fed animals had received atropine and α- and β-adrenoceptor antagonists 15 min before the test. The rats given the liquefied chow consumed about twice as much as those given the hard chow. In the parotid glands of the rats given hard chow the number of acinar granules and the total amylase activity were reduced, by 50 and 70%, respectively, as compared with the glands of control rats. In the parotid glands of the rats given liquefied chow the number of acinar granules and the total activity of amylase remained unchanged. The results suggest that non-adrenergic, non-cholinergic secretory mechanisms of the rat parotid gland participate in masticatory-salivary reflexes.     

Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands

The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10–CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin‐1 (ANXA1), which has immunomodulatory and anti‐inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26‐negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)‐positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS‐positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10‐CC26–ANXA1‐positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a defensive and protective role. Protein expression heterogeneity in the different portions of the glands could be an important clue in further investigations of their role.     

Neurokinin A in the parotid and submandibular glands of the rat: immunohistochemical localization and effect on protein and peroxidase secretion

An indirect immunofluorescence technique was used to study the distribution of neurokinin A immunoreactive (NKA-IR) nerve fibres in submandibular and parotid glands of the rat. The functional role of neurokinin A on protein and peroxidase secretion in these glands was evaluated by using in vitro methods. In the parotid gland neurokinin A immunoreactive fibres were mainly distributed around the secretory acini, but some were also in evidence around the stromal blood vessels and ducts. The number of the neurokinin A immunoreactive nerve fibres was lower in the submandibular gland than in the parotid gland. They were mainly distributed around the secretory acini and stromal blood vessels and ducts. In vitro, neurokinin A significantly stimulated the release of total amount of released proteins and peroxidase from parotid gland fragments, while in the submandibular gland only the release of peroxidase was increased. By using SDS polyacrylamide gel electrophoresis (SDS-PAGE) specific changes were found in the release of proteins after neurokinin A stimulation. The results of the present study demonstrate that neurokinin A immunoreactive nerve fibres are present in the rat parotid and submandibular glands. Their localization around the secretory elements of the glands and the effect of neurokinin A in vitro experiments indicates that neurokinin A might have a significant role in the regulation of salivary secretion.     

Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain

Distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites was investigated in the rat brain and pituitary gland by means of in vitro autoradiography. High densities of specific [125I]PACAP binding were observed in the anterior pituitary, hippocampus (CA1-4 and dentate gyrus) and in the superior colliculus. Moderate to high labeling was observed in the periaqueductal gray matter, substantia nigra pars compacta, and in the habenula. The hypothalamus, thalamus, ventral tegmental area (VTA), mammillary body and medial geniculate body were moderately labeled. The present results support possible actions of PACAP on the pituitary functions, and further suggest that PACAP is a neurotransmitter/neuromodulator in the central nervous system.     

Phenotypes in canalicular adenoma of human minor salivary glands reflect the interplay of altered secretory product, absent neuro-effector relationships and the diversity of the microenvironment

AIMS: Uncertainty about the factors influencing phenotypes in salivary canalicular adenoma prompted the present investigation. METHODS AND RESULTS: Specimens of canalicular adenoma from 15 patients were examined with the use of histology, histochemistry for protein, mucosubstances and pigments, nerve staining and immunocytochemistry for cytoskeleton components. The tumours consisted largely of simple cells lining tubules that were occasionally cystic or branching and budding, and were set in loose, vascular and often haemorrhagic stroma. Other phenotypes recognized were mucous cells, apocrine-like cells, pigmented cells, microliths and stromal macrophages, detected in 26.6%, 20%, 33.3%, 20% and 53. 3% of the patients, respectively. Simple cells showed moderate levels of -SH groups and strong immunoreactivity for 'simple' epithelial phenotype cytokeratin. The simple cells lining cystic tubules showed additional immunoreactivity for 'stratified' epithelial phenotype cytokeratin, possibly an adaptation to mechanical pressure. Lumina showed variable levels of neutral and carboxylated glycoproteins, and chondroitin sulphate. Stroma showed high levels of chondroitin sulphate and hyaluronic acid. Mucous cells showed high levels of -SS- groups and nonsulphated glycoproteins. Apocrine-like cells contained lipofuscin. Pigmented cells contained haemosiderin, possibly a consequence of localized iron overload. Microliths contained mucosubstances. Macrophages often contained lipofuscin. No nerves were found in relation to the tumours. CONCLUSIONS: The results suggest that, contrary to popular belief, phenotypes in canalicular adenoma do not reflect histogenetic concepts but rather may derive from the interplay between an altered secretory product, consisting of glycosaminoglycan and an immature form of glycoprotein, the lack of neuro-effector relationships and the different microenvironments throughout the tumour.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an upstream regulator of prodynorphin mRNA expression in neurons

Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA.     

Developmental studies on vasoactive intestinal peptide,substance P and calcitonin gene-related peptide in salivary glands of postnatal rats

The development of vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in parotid, submandibular and sublingual glands of the male rat was followed by immunochemistry and immunocytochemistry. The total amounts of these peptides increased in surges during the first 8 weeks of the animal's life; one within 2–4 weeks and the other beginning 1–2 weeks later. Nerve fibres containing these peptides were present at birth showing a pattern of distribution similar to that in adults. During the first 4 weeks the nerve fibres increased in number.     

The ultrastructure of the gastric glands and its relation to induced secretory activity of cod,Gadus morhua (Day)

Light and electron microscopic studies have been performed on different parts of cod stomach. The studies have been concentrated on the gastric glands of the gastric mucosa. The glands consist of only one cell type. This cell contains an apically located, comprehensive tubular system as well as zymogen granules and therefore probably secretes both acid and zymogen. The high degree of development of certain organelles and their mutual localization shows a structural organization which appears suitable for an effective secretion. What appears to be intermediate organelle forms are common. When acid secretion is increased by continuous intramuscular infusion of histamine or of carbachol the most marked structural changes are increases in the size of the gland lumen and in the cell membrane facing the lumen. Histamine in particular causes the appearance of numerous long and slender cell processes in an enlarged gland lumen. When the acid secretion, increased by histamine, had reached a plateau, metiamide was infused. This histamine H₂-receptor antagonist caused a decrease in acid secretion as well as in the size of the gland cell surface to values characteristic of untreated cod.     

Fluid and protein secretion from ferret submandibular and parotid glands in response to sympathetic nerve stimulation or administration of sympathomimetics.

Electrical stimulation of the sympathetic innervation evoked secretion of submandibular and parotid saliva. By changing the mode of stimulation from a continuous to an intermittent one the fluid response increased and glandular blood flow improved. The volumes from the submandibular glands were larger than those from the parotid glands and further, the protein concentration of submandibular saliva was higher than that of parotid saliva. Adrenaline, isoprenaline and phenylephrine evoked larger fluid responses from submandibular than from parotid glands. However, the fluid response was small compared to the parasympathetic one. Substance P-evoked saliva was used as carrier for protein released by sympathetic nerve stimulation or administration of adrenaline and isoprenaline. In vitro tissues of submandibular and parotid glands responded to adrenaline with a dose-dependent release of protein. Taken together, the analytical pharmacology performed in vivo and in vitro, and including the antagonists phentolamine, dihydroergotamine, propranolol and metoprolol, showed that in submandibular glands, α(α₁)adrenoceptors were predominantly involved in fluid secretion and β(β₁)-adrenoceptors predominantly involved in protein secretion. In parotid glands, fluid secretion seemed solely to depend on α(α₁)-adrenoceptors, while β(β₁)-adrenoceptors seemed almost solely involved in protein secretion.     

Existence and distribution of melanocytes and HMB-45-positive cells in the human minor salivary glands

The existence and distribution of melanocytes in the human minor salivary glands were investigated in a series of autopsy and biopsy materials. The cells with the following characteristics were regarded as melanocytes; spindle-shaped or dendritic cells with fine granular pigments: (i) stained brownish-black by hematoxylin-eosin stain, and black by Masson-Fontana's silver impregnation method; and (ii) disappeared after treatment with peroxide and potassium permanganate solution. In addition, the expression of antigen identified by anti-HMB-45 antibody in serial sections with melanocytes was examined. Melanocytes were found in eight (1.8%) of 445 cases, and there was no relationship between the existence of melanocytes and significant diseases of the subjects. Various numbers of melanocytes were distributed in fibrous tissue around the interlobular ducts, intralobular ducts and acini, but were not in direct contact with the epithelia. Neither melanocytes nor melanin granules were found in the salivary gland epithelia. HMB-45-positive cells without intracytoplasmic fine granules were found solitarily or in small groups in periductal and periacinar fibrous tissues with or without slight infiltration of small mononuclear cells.