Forms Of Pituitary Adenylate Cyclase-activating Polypeptide Research Articles

Kynurenic Acid Inhibits the Electrical Stimulation Induced Elevated Pituitary Adenylate Cyclase-Activating Polypeptide Expression in the TNC.

Migraine is a primary headache of imprecisely known mechanism, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Intensive research has recently focused on pituitary adenylate cyclase-activating polypeptide (PACAP) and the kynurenine systems as potential pathogenic factors. We investigated the link between these important mediators and the effects of kynurenic acid (KYNA) and its synthetic analog (KYNA-a) on PACAP expression in the rat trigeminal nucleus caudalis (TNC) in a TS stimulation model related to migraine mechanisms. Adult male Sprague-Dawley rats were pretreated with KYNA, KYNA-a, the NMDA receptor antagonist MK-801, or saline (vehicle). Next, the trigeminal ganglion (TRG) was electrically stimulated, the animals were transcardially perfused following 180 min, and the TNC was removed. In the TNC samples, 38 amino acid form of PACAP (PACAP1-38)-like radioimmunoactivity was measured by radioimmunoassay, the relative optical density of preproPACAP was assessed by Western blot analysis, and PACAP1-38 mRNA was detected by real-time PCR. Electrical TRG stimulation resulted in significant increases of PACAP1-38-LI, preproPACAP, and PACAP1-38 mRNA in the TNC. These increases were prevented by the pretreatments with KYNA, KYNA-a, and MK-801. This is the first study to provide evidence for a direct link between PACAP and the kynurenine system during TS activation.

Intranasal administration of PACAP: Uptake by brain and regional brain targeting with cyclodextrins

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood–brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2–4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood–brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, β-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-β-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Its Receptors in the Zebrafish Ovary: Evidence for Potentially Dual Roles of PACAP in Controlling Final Oocyte Maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia.PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an ∼14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.

PACAP-27 radioimmunoassay: Description and application of a novel method

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed neuropeptide that has two molecular forms with 38 and 27 amino acid residues. The aim of the present study was to develop a new, highly specific PACAP-27 assay to investigate the quantitative distribution of PACAP-27 in the central nervous system of various vertebrate species applying the same technical and experimental conditions. Our results show that the antiserum used turned to be PACAP-27 specific. The average ID50 value was 51.5±3.6 fmol/ml and the detection limit was 2 fmol/ml. PACAP-27 immunoreactivity was present in the examined brain areas, with highest concentration in the rat diencephalon and telencephalon. Swine and pigeon brain also contained significant amount of PACAP-27. Our results confirm the previously described data showing that PACAP-38 is the dominant form of PACAP in vertebrates, since PACAP-38 levels exceeded those of PACAP-27 in all examined brain areas. Furthermore, our study describes for the first time, the comparative quantitative distribution of PACAP-27 and-38 in the swine and pigeon brain.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp: II. Solution Structure of a Brain-Specific PACAP by Nuclear Magnetic Resonance Spectroscopy and Functional Studies on GH Release and Gene Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been proposed to be the ancestral GHRH. Recently, using grass carp as a model for modern-day bony fish, we demonstrated that PACAP nerve fibers are present in close proximity to carp somatotrophs, and mammalian PACAPs can induce GH secretion in carp pituitary cells. To further examine the role of PACAP as a GH-releasing factor in fish, the structural identity of grass carp PACAP was established by molecular cloning. The newly cloned PACAP was found to be a single-copy gene and expressed in the brain but not other tissues. The mature peptides of PACAP, namely PACAP(27) and PACAP(38), were synthesized. As revealed by nuclear magnetic resonance spectroscopies, carp PACAP(38) is composed of a flexible N terminal from His(1) to Ile(5), an extended central helix from Phe(6) to Val(26), and a short helical tail in the C terminal from Arg(29) to Arg(34). The C-terminal helix is located after a hinge region at Leu(27) to Gly(28) and is absent in the solution structures of PACAP(27). The two forms of PACAPs were effective in elevating GH release and GH transcript expression in grass carp pituitary cells. These stimulatory effects occurred with parallel rises in cAMP and Ca(2+) entry via voltage-sensitive Ca(2+) channels in carp somatotrophs. The present study represents the first report for solution structures of nonmammalian PACAPs and provides evidence that a brain-specific isoform of PACAP in fish can stimulate GH synthesis and release at the pituitary level, presumably by activating the appropriate postreceptor signaling mechanisms.

Role of Two Genes Encoding PACAP in Early Brain Development in Zebrafish

To study the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in early brain development, we examined PACAP and its receptors for first expression and then separately knocked down the two forms of PACAP in zebrafish where development is rapid and observable. We injected morpholinos (antisense oligonucleotides) into fertilized eggs to block PACAP. Morphological changes in the brain were observed in embryos at 27 h post fertilization (hpf). Using in situ hybridization of early brain marker genes, we found that the most striking effects were an increase in pax2.1 expression in eye stalks associated with absence of either form of PACAP or an increase in eng2 and fgf8 in the midbrain-hindbrain boundary after loss of PACAP2. These marker genes are among the earliest factors in the formation of the midbrain-hindbrain boundary, an early organizing center. We suggest that PACAP is a target gene with feedback inhibition on pax2.1, eng2, or fgf8 in specific brain areas. In the hindbrain, the absence of either form of PACAP had little effect, as shown by expression of ephA4 and meis1.1. During midbrain development, our evidence suggests that PACAP1 can activate mbx. In both the diencephalon and/or forebrain, lack of PACAP1 or PACAP2 led to an increase in fgf8, again suggesting a suppressive effect of PACAP during development on these important genes that help to define cells in the forebrain. The early expression of transcripts for PACAP and its receptors by 0.5-6 hpf make both PACAP1 and PACAP2 candidates for factors that influence brain development.

Cyclic AMP Formation in C6 Glioma Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert their actions via common receptors, VPAC1 and VPAC2, which are equally sensitive to both peptides, while PACAP stimulates its specific PAC1-type receptors. Both peptides potently stimulate cAMP production in different biological systems. In the present article, we examined the effects of PACAP and VIP on cAMP formation in C6 rat glioma cells used between passages 12-28 (early) and 120-136 (late). In the presence of the PDE inhibitor IBMX (0.1 mM), PACAP (0.1 microM) and VIP (1 microM) strongly stimulated cAMP synthesis in C6 cells in early passages, but not in C6 cells in late passages. In contrast, forskolin (10 microM), a direct activator of adenylyl cyclase, and isoprenaline (10 microM), a beta-adrenergic receptor agonist, strongly stimulated cAMP production in both early and late C6 cell passages. Concentration-dependent studies carried out in early passages with PACAP-38, PACAP-27, mammalian and chicken VIPs, and PHI/PHM peptides (1-5 microM) revealed that both forms of PACAP produced strong cAMP accumulation, VIP peptides were less effective than PACAP, while the cAMP effects of PHI/PHM peptides were noticeable only at the highest doses tested. These results suggest that C6 glioma cells in early passages possess functional PAC1 and possibly VPAC-type receptors, but either the density of PACAP/VIP receptors progressively declines or the PACAP/VIP receptor-Gs protein coupling becomes less effective through culture passages.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.

Possible Role of a Neuropeptide PACAP (Pituitary Adenylate Cyclase‐Activating Polypeptide) on Stimulus‐Secretion Coupling in Catecholamine Neuron

AbstractFor Abstract see ChemInform Abstract in Full Text.

A newly developed enzyme-immunoassay for measuring the tissue contents of PACAP in fish

We have developed a novel and easy enzyme-immunoassay (EIA) for pituitary adenylate cyclase-activating polypeptide (PACAP). We used it to determine immunoreactive PACAP levels in the central nervous system (CNS) and peripheral tissues of two fishes, a teleost (the stargazer) and an elasmobranch (a stingray). An antiserum was raised in a white rabbit immunized with a conjugate of synthetic stargazer PACAP27 plus keyhole limpet hemocyanin. The EIA system used an antiserum/biotin-labeled PACAP/avidin/biotin-conjugated enzyme complex, and a double antibody method was used to precipitate the immune complexes. We call the system the avidin–biotin complex detectable EIA (ABCDEIA) for PACAP. ABCDEIA with biotin-labeled PACAP27 detected only PACAP27, recognizing neither the longer forms of PACAP nor any other peptides. PACAPs with 27, 38, and 44 residues cross-reacted in another ABCDEIA with biotin-labeled PACAP38 or PACAP44. Whole brains of both fishes contained much higher levels of PACAP, 6–30 times as high as the levels in the mammalian brain, but unexpectedly, no immunoreactive PACAP27 was found in any CNS or peripheral tissue in either fish. The gastrointestinal tracts of fish also contained lower, but significant amounts of PACAP.

Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Activation of Trk Neurotrophin Receptor Signaling by Pituitary Adenylate Cyclase-activating Polypeptides

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation.

Comparative distribution of pituitary adenylate cyclase‐activating polypeptide (PACAP) binding sites and PACAP receptor mRNAs in the rat brain during development

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites as well as PACAP-specific receptor 1 (PAC1-R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1-R), and VPAC2-R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27-amino-acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3- to 5-fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [35S] uridine triphosphate-specific riboprobes. The evolution of the distribution of PAC1-R and VPAC1-R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1-R mRNA being much lower than that of PAC1-R mRNA. In contrast, intense expression of VPAC2-R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1-R and VPAC1-R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1-R and/or VPAC1-R, may play a crucial role in the regulation of neurogenesis in the rat brain. J. Comp. Neurol. 425:495–509, 2000. © 2000 Wiley-Liss, Inc.

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 μM), as well as vasoactive intestinal polypeptide (VIP; 0.1-3 μM), 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.

Comparative distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites and PACAP receptor mRNAs in the rat brain during development

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites as well as PACAP-specific receptor 1 (PAC1-R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1-R), and VPAC2-R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27-amino-acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3- to 5-fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [(35)S] uridine triphosphate-specific riboprobes. The evolution of the distribution of PAC1-R and VPAC1-R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1-R mRNA being much lower than that of PAC1-R mRNA. In contrast, intense expression of VPAC2-R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1-R and VPAC1-R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1-R and/or VPAC1-R, may play a crucial role in the regulation of neurogenesis in the rat brain.

Reduction of lipopolysaccharide-induced neurotoxicity in mixed cortical neuron/glia cultures by femtomolar concentrations of pituitary adenylate cyclase-activating polypeptide

Stimulation of murine primary mixed cortical neuron/glia cultures with lipopolysaccharide, an endotoxin, was used as a model for inflammatory disorders of the central nervous system. Lipopolysaccharide (20 μg/ml) increased the secretion of lactate dehydrogenase, a marker for cell injury, and nitric oxide into the culture medium. The lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium was reduced by pituitary adenylate cyclase-activating polypeptide (PACAP) at 10 −14–10 −12 M. The 27- and 38-amino-acid forms of PACAP were equipotent and their dose–response curves were U-shaped. PACAP6-38, a specific type I PACAP receptor antagonist, blocked the reduction by PACAP38 of the lipopolysaccharide-induced release of lactate dehydrogenase. The lipopolysaccharide-induced secretion of nitric oxide into the culture medium was reduced by PACAP at 10 −14–10 −12 M and 10 −8–10 −6 M. The 27- and 38-amino-acid forms of PACAP were equipotent. PACAP6-38 blocked the reduction of the lipopolysaccharide-induced secretion of nitric oxide by PACAP38 at 10 −12 M, but not at 10 −8 M. Vasoactive intestinal polypeptide reduced the lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium at 10 −14–10 −12 M, but these concentrations of vasoactive intestinal polypeptide had no effect on the lipopolysaccharide-induced secretion of nitric oxide. PACAP6-38 did not effect the reduction of the lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium by 10 −12 M vasoactive intestinal polypeptide. These results indicate that stimulation of type I PACAP receptors by femtomolar concentrations of PACAP can prevent neuron death in a model for inflammatory disorders of the CNS. These results suggest that PACAP is also an extraordinarily potent inhibitor of some microglial functions.

PACAP and PACAP receptors in insulin producing tissues: localization and effects

We have studied the localization, receptor occupancy and potency of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) in insulin-producing tissues. Immunocytochemistry showed that PACAP-like immunoreactivity (PACAP-IR) was localized to pancreatic nerves with accumulation in intrapancreatic ganglia in both mouse and rat. In contrast, PACAP-IR could not be demonstrated in endocrine cells. Furthermore, in situ hybridization, using oligodeoxyribonucleotide probes recognizing mRNA for PACAP receptors, demonstrated that mouse and rat pancreas, and the insulinoma cell lines HIT-T15 and RINm5F, expressed both the PACAP type 1 and the VIP2/PACAP receptors. Moreover, both PACAP27 and PACAP38 dose-dependently (0.1 nM to 100 nM) and equipotently stimulated insulin secretion in isolated mouse and rat islets and in HIT-T15 and RINm5F cells. Furthermore, in mouse islets, vasoactive intestinal polypeptide (VIP) was of equal potency as PACAP at stimulating insulin secretion. In mouse, PACAP also stimulated insulin secretion in a subfraction of the isolated islets also at the low dose of 1 fM. Thus, (1) PACAP is exclusively a neuropeptide in the pancreas, (2) insulin-producing cells express PACAP type 1 and VIP2/PACAP receptors and (3) the two forms of PACAP equipotently stimulate insulin secretion. Based on these results, we suggest that PACAP is involved in the neural regulation of insulin secretion.

Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca 2+ concentration in porcine somatotropes through common and distinct mechanisms

Ca 2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca 2+ concentration ([Ca 2+] j) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca 2+ sources and of CAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca 2+] i rises that could be followed by a refractory plateau (type I response), or by a decrease in [Ca 2+] i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca 2+ profiles in response to both peptides were similar, PACAP38-induced [Ca 2+] i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca 2+, blocking Ca 2+ entry through L-type voltage sensitive Ca 2+ channels (VSCC), or inhibiting PKA. Conversely, Ca 2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca 2+] j induced by PACAP38. We conclude that both peptides stimulate extracellular Ca 2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca 2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.