Pituitary Adenylate Cyclase-activating Polypeptide Receptor Expression Research Articles

Reference gene recommendations and PACAP receptor expression in murine sympathetic ganglia of the autonomic nervous system that innervate adipose tissues after chronic cold exposure.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important regulator of the stress response in mammals, influencing both the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). PACAP has been reported to influence energy homeostasis, including adaptive thermogenesis, an energy burning process in adipose tissue regulated by the SNS in response to cold stress and overfeeding. While research suggests PACAP acts centrally at the level of the hypothalamus, knowledge of PACAP's role within the sympathetic nerves innervating adipose tissues in response to metabolic stressors is limited. This work shows, for the first time, gene expression of PACAP receptors in stellate ganglia and highlights some differential expression with housing temperature. Additionally, we present our dissection protocol, analysis of tyrosine hydroxylase gene expression as a molecular biomarker for catecholamine producing tissue and recommend three stable reference genes for the normalization of quantitative real time-polymerase chain reaction (qRT-PCR) data when working with this tissue. This study adds to information about neuropeptide receptor expression in peripheral ganglia of the sympathetic nervous system innervating adipose tissue and provides insight into PACAP's role in the regulation of energy metabolism.

PMON182 PACAP Receptor Expression in Sympathetic Ganglia of the Autonomic Nervous System

Abstract In obesity, energy intake exceeds energy expenditure and concomitantly increases risk of chronic diseases, including metabolic diseases such as diabetes. Therapeutics to correct dysregulations in energy balance are needed and one notable neuropeptide being studied is pituitary adenylate cyclase-activating polypeptide (PACAP), an overarching regulator of the stress response1,2. In the context of metabolism, PACAP has been shown to regulate adaptive thermogenesis, an energy burning process regulated by the sympathetic nervous system (SNS) in response to cold stress and overfeeding. While research suggests PACAP acts centrally at the level of the hypothalamus to regulate energy balance3-5, PACAP is also known to be expressed in the SNS. In preganglionic neurons innervating the adrenal medulla6, PACAP is co-localized with acetylcholine and regulates catecholamine synthesis and release. Recent neuronal tracing studies have shown that postganglionic fibres of the stellate ganglia innervate intrascapular brown adipose tissue (BAT), the main thermogenic tissue in mammals7. However, PACAP's role within the sympathetic nerves innervating and regulating energy metabolism in adipose tissues is not known. As such, our group is interested in characterizing PACAP and PACAP receptor expression in sympathetic nerves innervating and regulating energy metabolism in BAT (via the stellate ganglia) in response to metabolic stressors, such as cold stress. We hypothesize that PACAP is released from preganglionic nerves which binds PACAP receptors (PAC1, VPAC1, VPAC2) expressed in postganglionic nerves of the stellate ganglia. Here we report, for the first time, PAC1 and VPAC1 expression in the stellate ganglia with sex-specific differential gene expression based on housing temperature. Analysis of PAC1 splice variant expression identified at least two variants in tissues of the SNS (adrenal gland, superior cervical ganglia and the stellate ganglia). These results will support future functional studies characterizing PACAP regulation of catecholamine production in peripheral ganglia which will contribute to assessing G-protein coupled receptors (GPCRs) as potential therapeutic targets for obesity and metabolic disease.

Pituitary Adenylate Cyclase Activating Polypeptide Has Inhibitory Effects on Melanoma Cell Proliferation and Migration In Vitro.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide which is distributed throughout the body. PACAP influences development of various tissues and exerts protective function during cellular stress and in some tumour formation. No evidence is available on its role in neural crest derived melanocytes and its malignant transformation into melanoma. Expression of PACAP receptors was examined in human skin samples, melanoma lesions and in a primary melanocyte cell culture. A2058 and WM35 melanoma cell lines, representing two different stages of melanoma progression, were used to investigate the effects of PACAP. PAC1 receptor was identified in melanocytes in vivo and in vitro and in melanoma cell lines as well as in melanoma lesions. PACAP administration did not alter viability but decreased proliferation of melanoma cells. With live imaging random motility, average speed, vectorial distance and maximum distance of migration of cells were reduced upon PACAP treatment. PACAP administration did not alter viability but decreased proliferation capacity of melanoma cells. On the other hand, PACAP administration decreased the migration of melanoma cell lines towards fibronectin chemoattractant in the Boyden chamber. Furthermore, the presence of the neuropeptide inhibited the invasion capability of melanoma cell lines in Matrigel chambers. In summary, we provide evidence that PACAP receptors are expressed in melanocytes and in melanoma cells. Our results also prove that various aspects of the cellular motility were inhibited by this neuropeptide. On the basis of these results, we propose PACAP signalling as a possible target in melanoma progression.

Physical Activity Protects the Pathological Alterations of Alzheimer's Disease Kidneys via the Activation of PACAP and BMP Signaling Pathways.

Alzheimer’s disease (AD) is a neurodegenerative disorder with typical amyloid beta (Aβ) aggregations. Elimination of the Aβ precursors via the kidneys makes the organ a potential factor in the systemic degeneration leading to AD. Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neuroprotective effects in AD and plays a protective role in kidney pathologies. Increased physical activity is preventive of the formation of AD, but its detailed mechanism and possible connections with PACAP have not been clarified. In the kidneys of AD mice, the effects of physical activity were investigated by comparing wild-type and AD organs. Aβ plaque formation was reduced in AD kidneys after increased training (TAD). Mechanotransduction elevated PACAP receptor expression in TAD mice and normalized the protein kinase A (PKA)-mediated pathways. BMP4/BMPR1 elevation activated Smad1 expression and normalized collagen type IV in TAD animals. In conclusion, our data suggest that elevated physical activity can prevent the AD-induced pathological changes in the kidneys via, at least in part, the activation of PACAP–BMP signaling crosstalk.

Alzheimer’s Disease Mouse as a Model of Testis Degeneration

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with protective functions in the central nervous system and various peripheral organs. PACAP has the highest expression level in the testes, among the peripheral organs, and has a positive regulative role in spermatogenesis and in sperm motility. In the present study, we explored testicular degenerative alterations in a mouse model of Alzheimer’s disease (AD) (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) and demonstrated changes in PACAP-regulated signaling pathways. In addition, the effects of increased physical activity of AD (trained AD (TAD)) mice on testis were also followed. Reduced cell number and decreased thickness of basement membrane were detected in AD samples. These changes were compensated by physical activity. Expression of PACAP receptors and canonical signaling elements such as PKA, P-PKA, PP2A significantly decreased in AD mice, and altered Sox transcription factor expression was also detected. Via this signaling mechanism, physical activity compensated the negative effects of AD on the expression of type IV collagen. Our findings suggest that the testes of AD mice can be a good model of testis degeneration. Moreover, it can be an appropriate organ to follow the effects of various interventions such as physical activity on tissue regeneration and signaling alterations.

SAT-601 Development of a Protocol for Stellate and Celiac Ganglia Dissection for Characterization of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Protein and Receptor Expression in Male and Female Mice Following Cold Acclimation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is being studied to understand the endocrine regulation of energy balance and has been shown to be important in the regulation of the stress response (1,2). Specifically, PACAP has been shown to regulate thermogenesis, an energy burning process regulated by the sympathetic nervous system that contributes to achieving energy homeostasis in response to cold stress and overfeeding. PACAP is expressed in the sympathetic nervous system and is required at the adrenomedullary synapse to maintain epinephrine secretion from the adrenal medulla in response to physiological stress (3). Across the branches of the sympathetic nervous system, PACAP receptor expression is most well characterized in the superior cervical ganglia (SCG) (4). However, a detailed characterization of PACAP and its receptors has not been performed in ganglia whose postganglionic fibres innervate adipose tissues (stellate and celiac ganglia) in response to thermogenic stress. We hypothesized that PACAP is produced by preganglionic neurons innervating the stellate and celiac ganglia, and act on PACAP receptors expressed on the post-ganglionic neurons, and this expression will be upregulated in response to chronic cold stress. Due to their small and amorphous shape, we have developed a protocol to reliably isolate the stellate and celiac ganglia and validate their identity through the presence of tyrosine hydroxylase mRNA, using adrenal and SCG samples as positive controls. PACAP receptor expression (VPAC1, VPAC2, PAC1) was examined in the ganglia utilizing real-time PCR, and PACAP protein was visualized in the ganglia of transgenic mice that express eGFP under the control of the PACAP promoter (PACAP-eGFP mice) (5). This research demonstrates the expression of PACAP receptors in ganglia whose postganglionic fibres innervate adipose tissue, enhancing our understanding of PACAP’s role in the SNS, and its contribution to the regulation of adaptive thermogenesis.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Hippocampal Synaptic Transmission and Plasticity: New Therapeutic Suggestions for Fragile X Syndrome.

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic synaptic transmission and plasticity in the hippocampus, a brain area with a key role in learning and memory. In agreement, several studies have demonstrated that PACAP modulates learning in physiological conditions. Recent publications show reduced PACAP levels and/or alterations in PACAP receptor expression in different conditions associated with cognitive disability. It is noteworthy that PACAP administration rescued impaired synaptic plasticity and learning in animal models of aging, Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea. In this context, results from our laboratory demonstrate that PACAP rescued metabotropic glutamate receptor-mediated synaptic plasticity in the hippocampus of a mouse model of fragile X syndrome (FXS), a genetic form of intellectual disability. PACAP is actively transported through the blood–brain barrier and reaches the brain following intranasal or intravenous administration. Besides, new studies have identified synthetic PACAP analog peptides with improved selectivity and pharmacokinetic properties with respect to the native peptide. Our review supports the shared idea that pharmacological activation of PACAP receptors might be beneficial for brain pathologies with cognitive disability. In addition, we suggest that the effects of PACAP treatment might be further studied as a possible therapy in FXS.

Comparison of expression and proliferative effect of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors on human astrocytoma cell lines.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10(-11)M) for 3days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.

Interaction of Gonadal Steroids and Gonadotropin-Releasing Hormone on Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP Receptor Expression in Cultured Rat Anterior Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine-paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH.

Development-Related Splicing Regulates Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Receptors in the Retina

The ubiquitous pituitary adenylate cyclase-activating peptide (PACAP) has a disparate array of functions in development (e.g., proliferation and apoptosis). Among three types of PACAP receptor (VPAC1, VPAC2, and PAC1), PAC1 is subject to alternative splicing that generates isoforms. Although the literature documenting the presence of PACAP receptors in the central nervous system is vast, their expression during development has not been established yet. Here, we performed quantitative analyses on the expression of PACAP receptors during the postnatal development of the rat retina. Retinas were harvested from postnatal days 0 to 20 (P0-P20). Using a comprehensive primer system, expression changes were followed employing quantitative real-time PCR. Changes at the protein level were detected by immunoblotting using anti-VPAC1, -VPAC2, and -PAC1 receptor antibodies. The expression of VPAC1 showed increases at P10 and P15. Peaks in VPAC2 expression were observed at P5 and P15. Using splicing variant-specific primers for PAC1 receptor, splicing regulation of Null, Hip, Hop1, and Hiphop1 variants was revealed in correlation with postnatal development. Transcript levels of the Null and Hip variants showed a decline, while Hop1 became the major PACAP receptor by P20. Hiphop1 transcript levels did not display remarkable changes except for a transient increase at P10. Immunoblotting confirmed the presence and expression level changes of the receptors. We conclude that both VPAC1 and VPAC2 could have roles at all stages of retinal development, that PACAP acts through a specific set of PAC1 isoforms, and that Hip and Hop1 are predominantly involved in the postnatal development of rat retina.

IL-6 and PACAP Receptor Expression and Localization after Global Brain Ischemia in Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a neuroprotective action against ischemic damage. This action is mediated by the interleukin-6 (IL-6) pathway. However, as the expression patterns of PACAP receptors and IL-6 following ischemia are not understood, we evaluated them in the mouse hippocampus in response to ischemia induced by bilateral common carotid artery occlusion. Real-time PCR determination of PAC1R mRNA expression in the hippocampus was significantly elevated on day 7 after ischemia. VPAC1R mRNA expression was significantly decreased 3 days after the ischemic episode, while VPAC2R mRNA expression showed a nonsignificant tendency to increase on day 7. IL-6 mRNA expression was significantly increased on day 3 and peaked on day 7 after ischemia. The mRNA expression of activity-dependent neuroprotective protein, which is a neuroprotective factor stimulated by PACAP, remained virtually unchanged in response to ischemia. IL-6 immunoreactivity was detected in the CA1 pyramidal cell layer and colocalized with the neuronal marker NeuN on day 1 after ischemia. On day 3, irregularly shaped IL-6-immunopositive cells colocalized with the astrocytic marker glial fibrillary acidic protein but not with the microglial marker Iba1. PAC1R immunoreactivity co-labeled with IL-6 immunoreactivity. These results suggest that PACAP could stimulate IL-6 secretion by neurons during the acute phase after an ischemic episode and thereafter by astrocytes during the subacute phase.

Pituitary adenylate cyclase‐activating polypeptide counteracts the impaired adult neural stem cell viability induced by palmitate

Diabetes and obesity are characterized by hyperlipidemia and represent risk factors for premature neurological disorders. Diabetic/obese animals have impaired adult neurogenesis. We hypothesize that lipotoxicity leading to neurogenesis impairment plays a role in the development of neurological complications. If so, normalizing neurogenesis in diabetes/obesity could be therapeutically useful in counteracting neurological dysfunction. The goal of this study was to determine the potential of pituitary adenylate cyclase-activating polypeptide (PACAP) to protect adult neural stem cells (NSCs) from lipotoxicity and to study the expression of PACAP receptors in NSCs under lipotoxic conditions in vitro and in the subventricular zone in vivo. The viability of NSCs isolated from the adult mouse brain subventricular zone was assessed in the presence of a high-fat milieu, as mimicked by palmitate, which characterizes diabetic lipotoxicity. Regulation studies of PACAP receptors were performed by quantitative PCR on NSCs in vitro or on subventricular tissues isolated from obese ob/ob mice and their lean littermates. We show that palmitate impairs NSC viability by promoting lipoapoptosis. We also show that PACAP counteracts lipotoxicity via PAC-1 receptor activation. Studies on PACAP receptor expression revealed that PAC-1 and VPAC-2 are expressed by NSC in vitro and are upregulated by palmitate treatment and that PAC-1, VPAC-1, and VPAC-2 are expressed in the subventricular zone/striatum in vivo and are upregulated in ob/ob mice. The present study reveals a previously uncharacterized role of PACAP to protect NSC from lipotoxicity and suggests a potential therapeutic role for PACAP receptor agonists in the treatment of neurological complications in obesity and diabetes.

Differential expression of PACAP receptors in postnatal rat brain

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a multi-functional neuropeptide that acts through activation of three common G-protein coupled receptors (VPAC1, VPAC2 and PAC1). In this study, we have investigated the gene expression profile of PAC1 isoforms (Hop1, Hip, Hip-Hop) and VPAC1, VPAC2 receptors in distinct brain regions during different stages of rat postnatal development. Using quantitative real time PCR approach we found that PAC1 isoforms were highly expressed in the cortex of newborns with marked decrease in expression during later stages of development. In contrast, mRNA levels of VPAC1, VPAC2 receptors were markedly lower in newborns in comparison to later developmental stages. Expression of PAC1 isoforms predominated in the hippocampus, while expression of VPAC1 was more prominent in the cortex and VPAC2 in the striatum and hippocampus. In addition we found that during early stages of postnatal development the expression of PAC1 receptor in the hippocampus was significantly higher in females than in males. No sex dependent differences in expression were observed for the VPAC1 and VPAC2 receptors. In summary, differential expression of PAC1, VPAC1 and VPAC2 receptors during postnatal development as well as gender dependent differences of PAC1 receptor expression in the hippocampus, will contribute to our understanding of the role of PACAP/VIP signaling system in normal brain development and function.

PACAP Induces Neurite Outgrowth in Cultured Trigeminal Ganglion Cells and Recovery of Corneal Sensitivity After Flap Surgery in Rabbits

To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery. Animal study. The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase. The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity. PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.

Expression of PACAP Receptors in the Frog Brain during Development

This study describes the expression of PAC1 and VPAC1 receptor (PAC1-R and VPAC1-R) mRNAs in the brain of frog (Rana esculenta) during development. PAC1-R mRNA was detected in the periventricular and subependymal layers of the thalamus and epithalamus and in the ependymal layer of the mesencephalon and rhombencephalon (stage 20), in the amygdala, in the habenular complex, in the periventricular nucleus of the hypothalamus, and in the ventral cerebellum (stage 30). VPAC1-R mRNA expression was observed only at stage 20, in the floor of the hypothalamus. These results suggest that, in amphibians, pituitary adenylate cyclase-activating polypeptide (PACAP) may play a role in brain development.

Pituitary adenylate cyclase-activating polypeptide and PACAP receptor expression and function in the rat adrenal gland

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a basic 38-amino acid peptide, which acts through three main G protein-coupled VIP/PACAP receptor subtypes, called PAC1, VPAC1 and VPAC2. We have investigated the expression and function of PACAP and its receptors in the rat adrenal gland. Reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmune assay (RIA) allowed the detection of PACAP expression as mRNA and protein exclusively in adrenal medulla (AM). RT-PCR and quantitative autoradiography, using [(125)I]PACAP and selective VIP/PACAP receptor ligands, demonstrated the expression of PAC1 only in AM, and VPAC1 and VPAC2 in both AM and zona glomerulosa (ZG), PACAP receptor expression being absent in zona fasciculata/reticularis (ZF/R). PACAP38 concentration-dependently increased aldosterone secretion from dispersed ZG cells and catecholamine secretion from AM tissue, the maximal effective concentration being 10(-7) M. ZF/R cells did not display any secretory response to PACAP38. Aldosterone response of ZG cells to 10(-7) M PACAP38 was unaffected by the PAC1-antagonist (A) PACAP(6-38), and significantly decreased by the VPAC1-A [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP(3-7) GRF(8-27)-NH(2). Catecholamine response of AM tissue to PACAP38 was reduced, but not abolished, by both PAC1-A and VPAC1-A. The VPAC2 agonist (ago) Ro25-1553 elicited sizeable secretory responses from both ZG cells and AM tissue. PACAP38 (10(-7) M) evoked a marked rise in cyclic-AMP (cAMP) and inositol-1,4,5-triphosphate (IP3) production by ZG cells and AM tissue. cAMP response of ZG cells was lowered by VPAC1-A, and that of AM tissue by both PAC1-A and VPAC1-A. IP3 response of ZG cells and AM tissue was unaffected by PAC1-A and decreased by VPAC1-A. VPAC2-ago did not affect cAMP release, but raised IP3 production by both ZG cells and AM tissue. Aldosterone response of ZG cells and catecholamine response of AM tissue to PACAP38 (10(-7) M) were reduced by the adenylate cyclase (AC) and phospholipase-C (PLC) inhibitors (I) SQ-22536 and U-73122, as well as by the protein kinase (PK)A-I H-89 and PKC-I calphostin-C. Conversely, the secretory responses of both ZG and AM preparations to VPAC2-ago were annulled by PLC-I, lowered by PKC-I, and unaffected by either AC-I or PKA-I. Collectively, our findings allow us to conclude that in the rat adrenals: i) PACAP biosynthesis exclusively occurs in the AM; ii) ZG cells are provided with functional VPAC1 and VPAC2 receptors, whose activation by PACAP evokes a moderate aldosterone response; iii) AM cells possess all the subtypes of VIP/PACAP receptors, whose activation by PACAP elicits a marked catecholamine response; and iv) PAC1 receptors are coupled to the AC-dependent cascade, VPAC1 receptors to both the AC- and PLC-dependent cascades, and VPAC2 receptors exclusively to the PLC-dependent cascade.

Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel member of the secretin-glucagon peptide family. In mammals, this peptide has been located in a wide range of tissues and is involved in a variety of biological functions. In lower vertebrates, especially fish, increasing evidence suggests that PACAP may function as a hypophysiotropic factor regulating pituitary hormone secretion. PACAP has been identified in the brain-pituitary axis of representative fish species. The molecular structure of fish PACAP is highly homologous to mammalian PACAP. The prepro-PACAP in fish, however, is distinct from that of mammals as it also contains the sequence of fish GHRH. In teleosts, the anterior pituitary is under direct innervation of the hypothalamus and PACAP nerve fibers have been identified in the pars distalis. Using the goldfish as a fish model, mRNA transcripts of PACAP receptors, namely the PAC1 and VPAC1 receptors, have been identified in the pituitary as well as in various brain areas. Consistent with the pituitary expression of PACAP receptors, PACAP analogs are effective in stimulating growth hormone (GH) and gonadotropin (GTH)-II secretion in the goldfish both in vivo and in vitro. The GH-releasing action of PACAP is mediated via pituitary PAC1 receptors coupled to the adenylate cyclase-cAMP-protein kinase A and phospholipase C-IP3-protein kinase C pathways. Subsequent stimulation of Ca2+ entry through voltage-sensitive Ca2+ channels followed by activation of Ca2+-calmodulin protein kinase II is likely the downstream mechanism mediating PACAP-stimulated GH release in goldfish. Although the PACAP receptor subtype(s) and the associated post-receptor signaling events responsible for PACAP-stimulated GTH-II release have not been characterized in goldfish, these findings support the hypothesis that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to regulate GH and GTH-II release in fish.Key words: PACAP, VIP, PAC1 receptor, VPAC1 receptor, VPAC2 receptor, growth hormone, gonadotropin-II, cAMP, protein kinase A, protein kinase C, calcium, pituitary cells, goldfish, and teleost.

Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel member of the secretin-glucagon peptide family. In mammals, this peptide has been located in a wide range of tissues and is involved in a variety of biological functions. In lower vertebrates, especially fish, increasing evidence suggests that PACAP may function as a hypophysiotropic factor regulating pituitary hormone secretion. PACAP has been identified in the brain-pituitary axis of representative fish species. The molecular structure of fish PACAP is highly homologous to mammalian PACAP. The prepro-PACAP in fish, however, is distinct from that of mammals as it also contains the sequence of fish GHRH. In teleosts, the anterior pituitary is under direct innervation of the hypothalamus and PACAP nerve fibers have been identified in the pars distalis. Using the goldfish as a fish model, mRNA transcripts of PACAP receptors, namely the PAC1 and VPACI receptors, have been identified in the pituitary as well as in various brain areas. Consistent with the pituitary expression of PACAP receptors, PACAP analogs are effective in stimulating growth hormone (GH) and gonadotropin (GTH)-II secretion in the goldfish both in vivo and in vitro. The GH-releasing action of PACAP is mediated via pituitary PAC1 receptors coupled to the adenylate cyclase-cAMP-protein kinase A and phospholipase C-IP3-protein kinase C pathways. Subsequent stimulation of Ca2+ entry through voltage-sensitive Ca2+ channels followed by activation of Ca2+-calmodulin protein kinase II is likely the downstream mechanism mediating PACAP-stimulated GH release in goldfish. Although the PACAP receptor subtype(s) and the associated post-receptor signaling events responsible for PACAP-stimulated GTH-II release have not been characterized in goldfish, these findings support the hypothesis that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to regulate GH and GTH-II release in fish.

Pituitary adenylate cyclase-activating polypeptide causes Ca2+ release from ryanodine/caffeine stores through a novel pathway independent of both inositol trisphosphates and cyclic AMP in bovine adrenal medullary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues using in situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptor-related proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.