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.     

Possible association between the pituitary adenylate cyclase-activating polypeptide (PACAP) gene and major depressive disorder

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1: adenylate cyclase-activating polypeptide 1) is a neuropeptide with neurotransmission modulating activity. The associations of the PACAP gene with schizophrenia and hippocampal volume have been reported. We recently reported depression-like behavior in the forced swimming test in PACAP deficient mice. Here we examined a possible association between the PACAP gene and major depressive disorder (MDD) in 637 patients and 967 controls and found that a genetic variant in the gene was associated with MDD. The present results suggest that PACAP signaling might contribute to the pathogenesis of MDD.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the brain

Recent progress in research on pituitary adenylate-activating polypeptide (PACAP) with a special emphasis on the brain is reviewed. PACAP is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide family. PACAP functions as a hypothalamic hormone, neurotransmitter, neuromodulator, and neurotrophic factor. Studies on the gene encoding the PACAP precursor and the specific PACAP receptor (PAC1-R) and its subtypes have provided information on the control of gene expression for PACAP, and the relationship between the receptor subtypes and the signal transduction pathways. The PAC1-R is a G protein-coupled receptor with seven transmembrane domains and belongs to the VIP receptor family. At least eight subtypes of PAC1-R result from alternate splicing. Each subtype is coupled to specific signaling pathways, and its expression is tissue or cell specific. PACAP stimulates the release of arginine vasopressin and increases cytosolic Ca2+ ([Ca2+]i). PACAP serves as a neurotransmitter and/or neuromodulator and the activation of the PAC1-R stimulates a cAMP-protein kinase A signal transduction pathway which in turn evokes the [Ca2+]i signaling system. More importantly, PACAP is a neurotrophic factor that may play an important role during the development of the brain. The PAC1-R is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development. In the adult brain, PACAP appears to function as a neuroprotective factor that attenuates the neuronal damage resulting from various insults.     

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.     

Role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the action of ginsenoside Rh2 against beta-amyloid-induced inhibition of rat brain astrocytes

Ginsenoside, the active principles in Panax ginseng root, has been demonstrated to show neurotrophic and neuroprotective actions for prevention of neuron degeneration. Deposition of β-amyloid peptide (Aβ) causes neurotoxicity through the formation of plaques in brains with Alzheimer's disease. Pituitary adenylate cyclase-activating polypeptide (PACAP) is introduced as a neurotrophic factor to promote cell survival. However, effect of Rh2, one of ginsenosides, on PACAP expression induced by Aβ remains unclear. In the present study, we found that Rh2 stimulates PACAP gene expression and cell proliferation in type I rat brain astrocytes (RBA1) cells and both effects were not modified by the estrogen antagonists (MPP or ICI 182780). Also, Rh2 ameliorates the RBA1 growth inhibition of Aβ. Moreover, blockade of PACAP receptor PAC1 using PACAP (6-38) inhibits all the actions of Rh2. These results suggest that Rh2 can induce an increase of PACAP to activate PAC1, but not estrogen receptor, and thereby leads to attenuate Aβ-induced toxicity. Thus, ginseng seems useful in the prevention of dementia.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive neuronal elements in rat hypothalamus and median eminence with special reference to morphological background of its effect on anterior pituitary — light and electron microscopic immunocytochemistry

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellular portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.     

Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic AMP formation in the duck and goose brain

Two molecular forms of pituitary adenylate cyclase-activating polypeptide (PACAP), i.e., PACAP27 and PACAP38 (0.0001-1 microM), as well as vasoactive intestinal polypeptide (VIP; 0.1-3 microM), have been studied for their effects on cyclic AMP formation in the hypothalamus and cerebral cortex of duck and goose. All three peptides concentration-dependently stimulated cyclic AMP production in the tested brain regions of 2-3-weeks-old (young) ducks, with VIP showing at least one order of magnitude weaker activity than PACAP. This characteristics suggests the existence in the duck's brain of adenylyl cyclase-linked PAC1 receptors. Both forms of PACAP also stimulated the nucleotide formation in the cerebral cortex and hypothalamus of 5-6-months-old (adult) ducks or geese grown under natural environment. The peptides-evoked effects in adult and young ducks were comparable, and clearly greater than those found in adult geese. The present data extend our recent observations made on chicks, and suggest PACAP to be a potent stimulator of the cyclic AMP generation in the avian central nervous system.     

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.     

Differential calcium responses to the pituitary adenylate cyclase-activating polypeptide (PACAP) in the five main cell types of rat anterior pituitary

We have compared the effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on the five main cell types of rat anterior pituitary in primary culture by monitoring changes in cytosolic Ca2+ concentration ([Ca2+]i) in single fura-2-loaded cells. Cells were typed by multiple sequential primary immunocytochemistry at the end of the Ca2+ measurements. PACAP-27 increased [Ca2+]i by three different mechanisms, each one dominant in a given cell type. These involved Ca2+ entry or release from the stores and mediation through different second messenger pathways: (1) stimulation of Ca2+ entry mediated by cAMP was the main mechanism in somatotrophs; (2) Ca2+ release from the intracellular Ca2+ stores mediated by phospholipase C (PLC) was the dominant modality in gonadotrophs; (3) stimulation of Ca2+ entry not mediated by cAMP was the main mechanism in lactotrophs. A minor fraction of somatotrophs (11%) may also use mechanism 3. Corticotrophs and thyrotrophs exhibited weak responses to PACAP (<10% of the cells responded), which in all cases were mediated by mechanism 1. Mechanism 3 represents a novel effect of PACAP which cannot be explained by interaction with the conventional PACAP receptor families.     

The functional recombinant first extracellular (EC1) domain of PACAP receptor PAC1 normal form (PAC1-EC1(N)) recognizes selective ligands and stimulates the proliferation of PAC1-CHO cells

PAC1 is a pituitary adenylate cyclase-activating polypeptide (PACAP) preferring receptor, which is abundant in the central and peripheral nervous systems. PAC1 belongs to the class B family of G protein-coupled receptors (GPCRs). The N-terminal first extracellular (EC1) domain of PAC1 is responsible for ligand recognition and binding. In this study, the recombinant EC1 domain of the PAC1 normal (N) form (amino acids 21–155) with 6His tag at the C-terminus (named PAC1-EC1(N)) was first expressed in an Escherichia coli strain and purified by an Ni-NTA affinity column. About 6–8 mg of recombinant PAC1-EC1(N) protein with purity above 95% was produced from 1 L of bacterial culture. Mass spectrum and western blot were used to identify the recombinant PAC1-EC1(N). Intrinsic tryptophan fluorescence (ITF) assays showed that the purified PAC1-EC1(N) protein was able to recognize and bind to the PAC1 selective agonist maxadilan, the antagonist M65 and vasoactive intestinal polypeptide (VIP). Maxadilan and M65 had higher affinities for PAC1-EC1(N) than VIP. The results of MTT assays showed that PAC1-EC1(N) stimulated the viability of PAC-CHO cells but blocked the effects of maxadilan on the proliferation of CHO cells expressing PAC1 (PAC1-CHO), indicating that the functional soluble PAC1-EC1(N) may act as a regulator for the activation of PAC1.     

人脑星形细胞瘤纤溶酶原激活抑制因子1基因表达研究

目的研究人脑星形细胞瘤纤溶酶原激活抑制因子１（ＰＡＩ１）基因表达及其临床意义。方法采用Ｎｏｒｔｈｅｒｎ杂交和免疫组化ＡＢＣ方法检测３６例人脑星形细胞瘤ＰＡＩ１ｍＲＮＡ和蛋白表达，分析其与临床病理因素之间的关系。结果所有星形细胞瘤组织均可表达３．０ｋｂ和２．２ｋｂ的ＰＡＩ１ｍＲＮＡ转录物；高分级星形细胞瘤ＰＡＩ１ｍＲＮＡ表达水平显著高于低分级星形细胞瘤（Ｐ＜００１）；正常脑组织未检测出ＰＡＩ１ｍＲＮＡ表达。ＰＡＩ１ｍＲＮＡ表达水平与星形细胞瘤的坏死（ｒ＝０．５１，Ｐ＜０．０１）、微血管数（ｒ＝０．３３，Ｐ＜０．０１）及脑水肿（ｒ＝０．２７，Ｐ＜０．０１）呈显著正相关，与患者性别、年龄及瘤体大小无显著相关性。免疫组化染色显示，ＰＡＩ１蛋白主要分布在高分级星形细胞瘤的瘤细胞和内皮细胞，尤以血管增殖部位和坏死灶周围较为显著，低分级星形细胞瘤呈低水平表达。结论ＰＡＩ１基因表达与人脑星形细胞瘤的分级、坏死、血管生成及脑水肿密切相关，可作为星形细胞瘤恶性程度的分子标记。