PACAP-specific Receptor Research Articles

Pituitary Adenylate Cyclase Activating Polypeptide Inhibits A10 Dopamine Neurons and Suppresses the Binge-like Consumption of Palatable Food

Pituitary adenylate cyclase-activating polypeptide (PACAP) binds to PACAP-specific (PAC1) receptors in multiple hypothalamic areas, especially those regulating energy balance. PACAP neurons in the ventromedial nucleus (VMN) exert anorexigenic effects within the homeostatic energy balance circuitry. Since PACAP can also reduce the consumption of palatable food, we tested the hypothesis that VMN PACAP neurons project to the ventral tegmental area (VTA) to inhibit A10 dopamine neurons via PAC1 receptors and KATP channels, and thereby suppress binge-like consumption. We performed electrophysiological recordings in mesencephalic slices from male PACAP-Cre and tyrosine hydroxylase (TH)-Cre mice. Initially, we injected PACAP (30 pmol) into the VTA, where it suppressed binge intake in wildtype male but not female mice. Subsequent tract tracing studies uncovered projections of VMN PACAP neurons to the VTA. Optogenetic stimulation of VMN PACAP neurons in voltage clamp induced an outward current and increase in conductance in VTA neurons, and a hyperpolarization and decrease in firing in current clamp. These effects were markedly attenuated by the KATP channel blocker tolbutamide (100 μM) and PAC1 receptor antagonist PACAP6–38 (200 nM). In recordings from A10 dopamine neurons in TH-Cre mice, we replicated the outward current by perfusing PACAP1–38 (100 nM). This response was again completely blocked by tolbutamide and PACAP6–38, and associated with a hyperpolarization and decrease in firing. These findings demonstrate that PACAP activates PAC1 receptors and KATP channels to inhibit A10 dopamine neurons and sex-dependently suppress binge-like consumption. Accordingly, they advance our understanding of how PACAP regulates energy homeostasis via the hedonic energy balance circuitry.

Adipose Tissue Expression of PACAP, VIP, and Their Receptors in Response to Cold Stress

Obesity arises from disrupted energy balance and is caused by chronically higher energy intake compared to expenditure via basal metabolic rate, exercise, and thermogenesis. The brown adipose tissue (BAT), the primary thermogenic organ, has received considerable attention as a potential therapeutic target due to its ability to burn lipids in the production of heat. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as a key regulator of the physiological stress response both centrally and peripherally. While PACAP has been shown to increase thermogenesis by acting at the hypothalamus to increase sympathetic output to BAT, a peripheral role for PACAP-activated thermogenesis has not been studied. We identified PACAP receptor (PAC1, VPAC1/2) expression for the first time in murine BAT and confirmed their expression in white adipose tissues. PAC1 receptor expression was significantly altered in all three adipose tissues studied in response to 3.5-week cold acclimation, with expression patterns differing by depot type. In primary cell culture, VPAC1 was increased in differentiated compared to non-differentiated brown adipocytes, and the same trend was observed for the PACAP-specific receptor PAC1 in gonadal white fat primary cultures. The primary PAC1R mRNA splice variant in interscapular BAT was determined as isoform 2 by RNA-Seq. These results show that PACAP receptors are present in adipose tissues and may have important functional roles in adipocyte differentiation, lipid metabolism, or adipose sensitization to sympathetic signaling in response to thermogenic stimuli.

Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. To investigate the effect of PACAP on eccrine sweat secretion. Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders.

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.

Noradrenaline-mediated protection against TNF-alpha-induced neuronal atrophy

injury (SCI) in rodent. However, it is still unclear that the PACAP induced by hMSCs contributes to the cellar protection. Therefore, we examined effect of hMSCs on SCI of PACAP gene deficient mice. We also determined that hMSCs partially suppressed neural inflammation through PACAP with host tissues. Under inhalation of anesthesia, mice either PACAP+/+ (wild) or +/− (KO) were subjected to spinal cord transection by a razor at level of Th9–10 intervertebral spinal cord. One day later, the mice were injected hMSCs (5 × 10 cells/0.5 μL) or vehicle one caudal the intervertebral spinal cord. The mice were scored locomotor activity detected by Basso Mouse Scale (BMS) and evaluated injury area with glial fibrillary acidic protein (GFAP) surrounded area at 7 days post operation. We also examined mouse PACAP and PACAP specific receptor (PAC1R) and human or mice proand anti-inflammatory cytokine gene expressions. WTmice implanted hMSCs into spinal cord ameliorated significantly locomotor activity and injury volume to compare with vehicle-treated one. The protections were canceled by inviable hMSCs implanted into WT mice and by viable hMSCs implanted into KO mice. Spinal cord implanted hMSCs expressed an increase of mouse PACAP gene, but not PAC1R. The hMSCs implanted WT animals were decreased mouse IL-1, TNF-alpha, IL-10 and TGF-beta, and increased IL-4 compared with vehicle treatedWT one. However, given hMSCs into KOmice, IL-1, TGFbeta and IL-4 expressed similar to vehicle-treatedWT one. These results suggest that hMSCs suppress neural damage partially mediated by PACAP of mice and decrease of IL-1 and TGF-beta and increase of IL-4 were regulated by the PACAP expression.

Analysis of PAC1 receptor gene variants in Caucasian and African American infants dying of sudden infant death syndrome

Stress peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has been implicated in sudden infant death syndrome (SIDS). The aim of this exploratory study was to determine whether variants in the gene encoding the PACAP-specific receptor, PAC1, are associated with SIDS in Caucasian and African American infants. Polymerase chain reaction and Sanger DNA sequencing was used to compare variants in the 5'-untranslated region, exons and intron-exon boundaries of the PAC1 gene in 96 SIDS cases and 96 race- and gender-matched controls. The intron 3 variant, A/G: rs758995 (variant 'h'), and the intron 6 variant, C/T: rs10081254 (variant 'n'), were significantly associated with SIDS in Caucasians and African Americans, respectively (p<0.05). Also associated with SIDS were interactions between the variants rs2302475 (variant 'i') in PAC1 and rs8192597 and rs2856966 in PACAP among Caucasians (p<0.02) and rs2267734 (variant 'q') in PAC1 and rs1893154 in PACAP among African Americans (p<0.01). However, none of these differences survived post hoc analysis. Overall, this study does not support a strong association between variants in the PAC1 gene and SIDS; however, a number of potential associations between race-specific variants and SIDS were identified that warrant targeted investigations in future studies.

Expression of pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 in the periodontal ligament after tooth luxation.

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.

Evidence for pituitary adenylate cyclase-activating peptide as a direct immunoregulator in teleost head kidney

In mammals, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent anti-inflammatory factor, showing that it inhibits the expression and release of proinflammatory cytokines and enhances the production of anti-inflammatory factors. However, whether fish PACAP plays similar regulatory roles as seen in mammals remains unclear. In the present study, expression of PACAP-specific receptor PAC1-R was shown in grass carp head kidney and spleen, supporting that PACAP may have a direct effect on fish immune cells. To test this hypothesis, the immunoregulatory role of grass carp PACAP (gcPACAP) was examined in head kidney leucocytes (HKLs). Results showed that gcPACAP inhibited basal and further attenuated lipopolysaccharide (LPS)-stimulated cell viability of HKLs, indicating that gcPACAP may possess similar inhibitory property at cellular level as seen in mammals. Curiously, in vitro and in vivo studies revealed that gcPACAP stimulated proinflammatory factors (IL-1β and TNF-α) but not IL-10 mRNA expression in HKLs and head kidney. Moreover, bacterial infection and LPS enhanced IL-1β, TNF-α and IL-10 mRNA expression in grass carp head kidney and HKLs, respectively, and these stimulatory effects were not influenced by gcPACAP. These findings suggest that PACAP plays distinct roles, at least does not function as an anti-inflammatory factor, in fish compared with that in mammals.

Cellular Localization of PACAP and Its Receptors in the Ovary of the Spotted Ray Torpedo marmorata Risso 1880 (Elasmobranchii: Torpediniformes)

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family and occurs in two amidated forms, PACAP38 and PACAP27, with 38 and 27 amino acids, respectively. PACAP acts by binding to three different receptors, that are classified by their binding affinity for PACAP and VIP (vasoactive intestinal polypeptide): PAC(1)R (PACAP-specific receptor) exclusively binds PACAP, while VPAC(1)R (VIP/PACAP receptor, subtype 1) and VPAC(2)R (VIP/PACAP receptor, subtype 2) bind both PACAP and VIP. PACAP, first discovered in the brain, was then localized in several peripheral tissues of mammals, including the ovary. Besides mammals, PACAP and its receptors have been reported in fish too; however, less is known about the presence of PACAP in the fish ovary and the studies are limited to teleosts. The aim of our work was to study the distribution of the PACAP/PACAP-Rs system in the ovary of the cartilaginous fish Torpedo marmorata. Using in situ hybridization (ISH) and immunohistochemistry techniques, we demonstrated that PACAP and its receptors are widely represented in the Torpedo ovary in a stage-dependent manner. Moreover, our findings suggest an involvement of this peptide in the whole follicologenesis, probably influencing steroidogenesis, follicle development, and oocyte growth.

Structural and functional characterization of pituitary adenylyl cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) and its receptor in olive flounder (Paralichthys olivaceus)

We identified full-length cDNAs encoding pituitary adenylyl cyclase-activating polypeptide (PACAP), PACAP-related peptide (PRP), and PACAP-specific receptor (PAC1R) from olive flounder, Paralichthys olivaceus. Two variant mRNA forms were created by alternative splicing. Comparison of genomic and cDNA sequences of the PRP-PACAP precursor revealed that skipping of exon 4 within PRP resulted in two variant transcripts: a long form encoding both PRP and PACAP and a short form encoding PACAP only. Both transcripts were constitutively observed only in the brain, whereas the short form appeared in gut tissues, such as the intestine and pyloric cecum in fish challenged with a pathogen, but not in healthy fish. Furthermore, expression of the long PRP/PACAP transcript gradually increased in the intestine of flounder challenged with bacteria, suggesting that PRP and/or PACAP may serve as a regulator(s) of the immune system, especially in the gastrointestinal tract of olive flounder. The biological functions of PACAP and PRP were investigated by exogenous treatment of flounder embryogenic cells (hirame natural embryonic cells, HINAE cells) with synthetic peptides of fPACAP-38 and/or fPRP-45. Intracellular cyclic adenosine monophosphate (cAMP) production in PAC1R-overexpressing HINAE cells was regulated by fPACAP-38 in a concentration-dependent manner, but was not regulated by fPRP-45. Results from real-time quantitative polymerase chain reaction revealed that PAC1R mRNA was specifically induced by fPACAP-38 but not by fPRP-45; PACAP significantly increased TNF-α mRNA but not growth hormone (GH) mRNA in HINAE cells; however, PRP affected GH but not TNF-α mRNA expression. These results suggest that the expression ratio of PRP and PACAP is regulated at the transcriptional level depending on the tissues and conditions, and that the unique biological roles of PRP and PACAP differ from that of mammalian PRP.

The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood–brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood–brain barrier (BBB), blood–air barrier (BAB) and blood–testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.

Effects of cyclopeptide C*HSDGIC* from the cyclization of PACAP (1–5) on the proliferation and UVB-induced apoptosis of the retinal ganglion cell line RGC-5

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that confers potent neurotrophic and neuroprotective effects. Cyclopeptide C*HSDGIC* (CHC), which results from the cyclization of PACAP (1–5) with disulfide, has been demonstrated to represent a potent agonist for the PACAP-specific receptor PAC1 which mediates the majority of PACAP's effects. In this study, the expression of PAC1 in a rat retinal ganglion cell line (RGC-5) was confirmed using a western blot analysis, and it was determined that CHC promoted the proliferation of RGC-5 cells using the cell counting kit-8 (CCK8) assay and flow cytometry. Furthermore, the treatment of CHC attenuated the decrease of cell viability in cells exposed to UVB irradiation. Flow cytometry and a JC-1 assay revealed that the CHC treatment protected the RGC-5 cells against UVB-induced apoptosis. In addition, similar to PACAP, the anti-apoptotic effect of CHC was related to the down-regulation of caspase-3. In summary, these results demonstrate for the first time that PAC1 is present in RGC-5 cells and that CHC, a cyclopeptide from PACAP, promotes RGC-5 cell proliferation and attenuates UVB-induced apoptosis.

Role of Mitochondrial Activation in PACAP Dependent Neurite Outgrowth

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases neurite outgrowth, although signaling via its receptor PACAP-specific receptor (PAC1R) has not been fully characterized. Because mitochondria also play an important role in neurite outgrowth, we examined whether mitochondria contribute to PACAP-mediated neurite outgrowth. When mouse primary hippocampal neurons and Neuro2a cells were exposed to PACAP, neurite outgrowth and the mitochondrial membrane potential increased in both cell types. These results were reproduced using the PAC1R-specific agonist maxadilan and the adenylate cyclase activator forskolin, whereas the protein kinase A inhibitor H89 and mitochondrial uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) inhibited these effects. Expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1α), a master regulator of mitochondrial activation, and its downstream effectors, such as cytochrome C and cytochrome C oxidase subunit 4, increased in response to PACAP. Knocking down Pgc1α expression using small interfering RNA or treatment with CCCP significantly attenuated neurite outgrowth and reduced the mitochondrial membrane potential in PACAP-treated cells. These data suggest that mitochondrial activation plays a key role in PACAP-induced neurite outgrowth via a signaling pathway that includes PAC1R, PKA, and Pgc1α.

Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

Cloning and differential expression pattern of pituitary adenylyl cyclase-activating polypeptide and the PACAP-specific receptor in darkbarbel catfish Pelteobagrus vachelli

Pituitary adenylyl cyclase-activating polypeptide (PACAP) can mediate growth hormone and gonadotropin release in teleost pituitary via PACAP receptors. In this study, the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) and the PACAP-specific receptor (PAC1-R) were cloned from the brain of darkbarbel catfish Pelteobagrus vachelli. The PRP-PACAP cDNA had two variants expressed by alternative splicing: a long form encoding both PRP and PACAP and a short form encoding PACAP only. Our data showed that the exon skipping on the PACAP transcripts was a possible mechanism regulating the expression ratio of PACAP to PRP in non-mammalian vertebrates. Based on multi-sequence alignments and phylogenetic analysis, the catfish PACAP and PAC1-R were highly conserved during evolution. Real-time quantitative PCR revealed that the PACAP-short and PAC1-R tanscripts were mainly expressed in the brain and gonad of darkbarbel catfish, though a small amount of mRNAs was also found in other tissues. Immunofluorescent staining studies showed wide distribution and high levels of PAC1-R in the catfish brain, suggesting that the PAC1-R form may play a central role in growth hormone release. The expressions of PACAP and PAC1-R in gonads and the occurrence of PACAP-immunoreactive material in testis suggest that PACAP may act as a paracrine/autocrine factor for gonad development.

PACAP-mediated sperm–cumulus cell interaction promotes fertilization

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus-oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus-oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

Regulation of Spinal Dynorphin 1-17 Release by Endogenous Pituitary Adenylyl Cyclase-Activating Polypeptide in the Male Rat: Relevance of Excitation via Disinhibition

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC(1)R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC(1)R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213-1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

PAC1 Deficiency in a Murine Model Induces Gastric Mucosa Hypertrophy and Higher Basal Gastric Acid Output

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to increase the histamine release from gastric enterochromaffin-like (ECL) cells and promote gastric acid secretion in rats. In contrast, in mice, PACAP has been demonstrated to induce a decrease of gastric acid secretion, an effect presumably due to somatostatin release. To more clearly define the role of PACAP in the regulation of gastric acid output, a knockout mouse model for the PACAP-specific receptor PAC1 was applied in this study. Measurements of the basal and stimulated gastric acid secretion and morphological studies on the gastric mucosa were performed in both wild-type and PAC1-deficient mice. Compared with the wild-type mice, the PAC1-deficient mice showed a nearly threefold higher basal gastric acid output, increased gastric mucosa thickness and glands height, and proportional increases in parietal and total cell counts in the gastric mucosa. The PAC1-deficient mice also showed a trend of increased plasma gastrin levels and gastrin gene expression in the gastric mucosa. This study indicates that the expression of PAC1 is clearly important for maintaining the homeostasis of gastric acid secretion. Loss of PACAP receptor during development may lead to a compensatory mechanism regulating gastric acid secretion.

Pituitary adenylyl cyclase‐activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase-activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP-cAMP-dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP-specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [(3)H]thymidine as well as the number of 5-bromo-2'-deoxyuridine-positive and cyclin D1-positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP-PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell-extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.

Alternative Splicing of the Pituitary Adenylate Cyclase-activating Polypetide (PACAP) Receptor Contributes to Function of PACAP-27

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides performing a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA region encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. To characterize the molecular forms of alternative splicing variants of PAC1, we examined the binding affinity and activation of two major second messenger pathways (cAMP production and changes in [Ca(2+)]( i )) by PACAP-27. Activation of cAMP was influenced by the variant in both of the EC1 domain and IC3 loops. In the N form in the EC1 domain, the suppressive effect of the HOP1 form in the IC3 loop was enhanced. Regarding the intracellular calcium mobilization assay, the rank order of the potency of PACAP-27 for the different PAC1 isoforms was S/HOP1>>N/R~S/R>>N/HOP1. In particular, PACAP-27 exhibited remarkable potency of calcium mobilization in the S/HOP1-expressing cells at sub-picomolar concentrations even though the affinities of PACAP-27 to the four PAC1 isoforms were not significantly different. This suggests the specific functions of PACAP-27 due to PACAP-27 preferring PAC1 activation, and leads in clarification of the pleiotoropic function of PACAP.