Pituitary Adenylate Cyclase-activating Polypeptide Stimulation Research Articles

Pacap stimulation of gonadotropin-II secretion in goldfish pituitary cells: mechanisms of action and interaction with gonadotropin releasing hormone signalling.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.

Autocrine Expression and Ontogenetic Functions of the PACAP Ligand/Receptor System during Sympathetic Development

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC1) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC1 receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC1 receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca2+, the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis.

Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells.

Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology.

Hypophysiotropic Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Goldfish: Immunohistochemical Demonstration of PACAP in the Pituitary, PACAP Stimulation of Growth Hormone Release from Pituitary Cells, and Molecular Cloning of Pituitary Type I PACAP Receptor

Hypophysiotropic Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Goldfish: Immunohistochemical Demonstration of PACAP in the Pituitary, PACAP Stimulation of Growth Hormone Release from Pituitary Cells, and Molecular Cloning of Pituitary Type I PACAP Receptor

Hypophysiotropic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: immunohistochemical demonstration of PACAP in the pituitary, PACAP stimulation of growth hormone release from pituitary cells, and molecular cloning of pituitary type I PACAP receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family, and its molecular structure is highly conserved in vertebrates. In this study, the functional role of PACAP in regulating GH release in the goldfish was investigated. Using immunohistochemical staining, nerve fibers with PACAP immunoreactivity were identified in the vicinity of goldfish somatotrophs, suggesting that this neuropeptide may influence GH release in the goldfish. The direct regulatory action of PACAP on GH secretion was demonstrated in vitro in perifused goldfish pituitary cells. PACAPs (0.01 nM to 1 microM) from different species, including ovine PACAP27, ovine PACAP38, frog PACAP38, zebra fish PACAP27, and zebra fish PACAP38, were all effective in stimulating GH release with ED50 values of 8.9 +/- 3.5, 3.3 +/- 1.6, 14.4 +/- 3.5, 15.4 +/- 4.1, and 1.4 +/- 0.2 nM, respectively. Similar concentrations of vasoactive intestinal polypeptide (VIP), a peptide related to PACAP, was not effective in this respect. In addition, the GH-releasing action of ovine PACAP38 (10 nM) was inhibited by the PACAP antagonist PACAP(6-38) (10 microM), but not by the VIP antagonist [4-Cl-D-Phe6,Leu17]VIP (10 microM). The pharmacology of these GH responses is consistent with the mammalian type I PACAP receptors, suggesting that a similar receptor subtype is present in the goldfish pituitary and mediates the GH-releasing action of PACAP. To establish the structural identity of this goldfish PACAP receptor, a complementary DNA (cDNA) clone sharing a high degree of sequence homology with mammalian type I PACAP receptors was isolated from a goldfish pituitary cDNA library. This cDNA was 5.2 kb in size with a 1.4-kb open reading frame and encoded a 465-amino acid protein with the typical structure of a 7-transmembrane domain-containing, G protein-coupled receptor. Functional expression of this cDNA in COS-7 cells revealed that this fish type I PACAP receptor could be activated by ovine PACAP27 and PACAP38 to increase cAMP synthesis with ED50 values of 2.4 +/- 0.8 and 4.2 +/- 1.2 nM, respectively. Other structurally related peptides, including VIP (100 nM), GH-releasing hormone (100 nM), glucagon (100 nM), secretin (100 nM), gastric inhibitory polypeptide (100 nM), and PTH (100 nM), were not effective in altering cAMP production. Using Northern blot and RT-PCR, messenger RNA transcripts of this PACAP receptor were identified in the brain, heart, and pituitary of the goldfish. These results, taken together, support the hypothesis that PACAP functions as a novel GH-releasing factor in the goldfish through activation of type I PACAP receptors.

Transcriptional and Posttranscriptional Control of Tyrosine Hydroxylase Gene Expression During Persistent Stimulation of Pituitary Adenylate Cyclase‐Activating Polypeptide Receptors on PC12 Cells: Regulation by Protein Kinase A‐Dependent and Protein Kinase A‐Independent Pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).

Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone alpha-subunit transcription in alphaT3-1 gonadotropes.

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone alpha-subunit synthesis and release from pituitary cells. We have used alphaT3-1 clonal gonadotropes to investigate the intracellular mechanisms involved in PACAP regulation of alpha-subunit gene transcription; and using deletion, mutation, and heterologous constructs of the alpha-promoter linked to a luciferase reporter gene, we have defined DNA sequences responsive to PACAP. Stimulation of alphaT3-1 cells for 24 h with PACAP, GnRH, or vasoactive intestinal peptide (VIP) resulted in a time- and concentration-dependent increase in alpha-promoter transcription at 100 nM for GnRH (17.5-fold, P < 0.001), PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold, P < 0.05). Incubation of alphaT3-1 cells in calcium-depleted medium suggested that the transcriptional response to PACAP was less dependent on changes in intracellular calcium concentration, in contrast to the results seen with GnRH or VIP, where alpha-subunit transcription was significantly reduced. Transfection of an alpha-promoter construct containing a mutant cAMP response element (CRE) suggested that the CRE region is involved in PACAP and VIP responsiveness, with stimulatory effects on the mutant construct by PACAP (11.1-fold) and VIP (7.6-fold) being significantly (P < 0.001) reduced, compared with their stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native alpha-promoter. In the same experiment, the transcriptional response of the mutant CRE construct and the native CRE construct to GnRH was not significantly different. Both PACAP and VIP enhanced GnRH-stimulated alpha-subunit gene transcription, but this additive effect was lost when their combined effects on the mutant CRE were examined. Deletion analysis indicated that sequences between -244 and -195 bp were involved in mediating the response to PACAP, with a dramatic reduction in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct, compared with the -244-bp construct (15.8-fold). Constructs containing only upstream alpha-promoter sequences from -517 bp to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited a similar loss of responsiveness to PACAP below -298 bp. Thus, our studies show that, unlike GnRH, PACAP stimulation of alpha-subunit gene transcription in alphaT3-1 cells is less dependent on changes in intracellular calcium concentration; and full transcriptional activation of the alpha-subunit by PACAP requires an intact CRE. PACAP responsiveness involves sequences between -244 and -195 bp of the alpha-promoter. These sequences have been implicated also in GnRH-responsiveness and may thus provide a mechanism for coordinated regulation of the alpha-subunit gene by PACAP and GnRH in alphaT3-1 cells.

Evidence that pituitary adenylate cyclase activating polypeptide suppresses follicle-stimulating hormone-beta messenger ribonucleic acid levels by stimulating follistatin gene transcription.

There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator ofgonadotrope function. One of the actions of PACAP identified previously is to decrease FSHbeta messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHbeta mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived alphaT3-1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in alphaT3-1 cells PACAP activation of the follistatin promoter appears to be via the cAMP-dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHbeta mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.

Comparative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide on Aldosterone Secretion in Normal Bovine and Human Tumorous Adrenal Cells*

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.

Stimulation of guanosine 5'-O-(3-[35S] thiotriphosphate) binding by cholinergic muscarinic receptors in membranes of rat olfactory bulb.

In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 mM NaCl. Exposure to N-ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [35S]GTP gamma S binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [35S]GTP gamma S binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [35S]GTP gamma S binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [35S]GTP gamma S binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G1/G0, but not to Gs, and support the possibility that activation of G1/G0 mediates the stimulatory effect on adenylyl cyclase activity.

Presence of pituitary adenylate cyclase-activating polypeptide receptors in Y-79 human retinoblastoma cells.

Cytochemical analysis demonstrated that a high percentage of human Y-79 retinoblastoma cells displayed a specific labeling by the biotinyl derivative of pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide of the secretin-vasoactive intestinal peptide (VIP) family of peptides. In cell membranes, the two molecular forms of PACAP, the one with 38 (PACAP 38) and the other with 27 (PACAP 27) amino acids, displaced the binding of 125I-PACAP 27 with IC50 values in the picomolar range and increased adenylyl cyclase activity by 100-fold with EC50 values of 27 and 180 pM, respectively. VIP, human peptide histidine-isoleucine, glucagon, and secretin were much less effective and potent in both receptor assays. The PACAP receptor antagonists PACAP 6-27 and PACAP 6-38 and an antiserum directed against the stimulatory G protein Gs inhibited the PACAP stimulation of adenylyl cyclase. In intact cells, both PACAPs and VIP failed to stimulate the phosphoinositide hydrolysis, whereas in cell membranes PACAP 38, but not the other peptides, produced a modest increase (40%) of inositol phosphate formation with an EC50 value of 22 nM. However, this effect was not antagonized by either PACAP 6-38 or PACAP 6-27. These data demonstrate the presence in human Y-79 retinoblastoma cells of specific PACAP receptors and provide further evidence that PACAP may act as a neurotransmitter/neuromodulator in mammalian retina.

Tissue‐specific and Developmental Expression of Pituitary Adenylate Cyclase‐activating Polypeptide (PACAP) Receptors in Rat Brain

The two forms of pituitary adenylate cyclase-activating polypeptide, PACAP27,and PACAP38, are novel members of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylate cyclase and phospholipase C have been recently identified. We examined the expression of PACAP receptors in the rat cortex, hippocampus, cerebellum and hypothalamus during postnatal development. Functional studies revealed PACAP stimulation of cAMP formation in all the brain areas examined and [3H]inositol monophosphate ([3H]InsP) accumulation only in the cerebellum and hypothalamus. Throughout development, the efficacy or PACAP in stimulating cAMP formation slightly increased in the cortex and hypothalamus and decreased in the hippocampus and cerebellum; PACAP stimulation of [3H]InsP formation decreased in the cerebellum and remained steady in the hypothalamus. The effects of PACAP27 and PACAP38 on cAMP levels and inositol phospholipid hydrolysis were dose-dependent between 1 and 100 nM. In the same brain areas, treatment with VIP increased cAMP formation at doses greater than 100nM and failed to affect [3H]InsP content, thus suggesting the existence of type-1 PACAP receptors. The reverse transcription polymerase chain reaction (RT-PCR) was used to analyse the mRNA expression of type-1 PACAP receptor splice variants. PACAP receptor gene expression in the central nervous system was regulated in a developmental- and tissue-specific manner. The PACAP-R transcript was detected in all the brain areas examined whereas PACAP-R-hop mRNA ocurred only in the cerebellum and hypothalamus. The different expression profiles and functional properties of PACAP receptors in the developing rat brain suggest an involvement of PACAP in histogenesis, maturation and neurotransmission.

Pituitary adenylate cyclase-activating polypeptide induces the voltage- independent activation of inward membrane currents and elevation of intracellular calcium in HIT-T15 insulinoma cells

The secretion of insulin by pancreatic beta-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockade by La3+, and which exhibits a reversal potential of about -28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at -70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic beta-cells during the transition period from fasting to feeding.

Pituitary adenylate cyclase-activating polypeptide induces the voltage-independent activation of inward membrane currents and elevation of intracellular calcium in HIT-T15 insulinoma cells.

The secretion of insulin by pancreatic beta-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockade by La3+, and which exhibits a reversal potential of about -28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at -70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic beta-cells during the transition period from fasting to feeding.

Expression of pituitary adenylate cyclase activating polypeptide and receptors in human brain tumors.

The capacity of brain tumor samples to synthesize pituitary adenylate cyclase activating polypeptide (PACAP) was evaluated by the reverse transcriptase-polymerase chain reaction technique (RT-PCR). The expression of PACAP receptors was assessed by a combination of RT-PCR techniques, conventional binding techniques, and also by the ability of PACAP to stimulate adenylate cyclase activity. A weak PACAP mRNA and PACAP receptor mRNA expression was detected in only 3 of 16 meningiomas. A weak PACAP-stimulated adenylate cyclase activity (+20%) was detected in 10 of the 16 samples but binding of labeled PACAP was never observed. In the 16 gliomas studied (including two oligodendrogliomas and two ependymomas), PACAP mRNA was identified in 13 samples and PACAP receptor mRNA in 15 samples. PACAP receptors were identified in all the samples by binding studies and/or by PACAP stimulation of the adenylate cyclase activity. PACAP mRNA was never detected in pituitary adenomas (three prolactinomas, two mixed PRL-GH-producing tumors, three GH-secreting tumors, three gonadotrophinomas, one ACTH-producing tumor, two nonsecreting tumors) whereas PACAP receptor mRNA was highly expressed in all the tumors except prolactinomas, where it was at the limit of detection, confirming the binding and adenylate cyclase activation results. Thus, it is unlikely that the neuropeptide PACAP could influence meningioma's cell growth; PACAP secreted from extratumoral areas may influence pituitary tumors and PACAP could participate to gliomas development.

Pituitary adenylate cyclase-activating polypeptide stimulates prolactin gene expression in a rat pituitary cell line

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to activate adenylate cyclase and stimulate PRL secretion in dispersed pituitary cells. We have employed the GH3 rat pituitary cell line to investigate whether PACAP can regulate expression of the PRL gene. PACAP increased cellular levels of cAMP in a concentration-dependent fashion (EC50, approximately 6 x 10(-9) M). PACAP also increased PRL mRNA levels in GH3 cells, implying that this peptide stimulates a step in expression of the PRL gene. In addition, PACAP strongly stimulated chloramphenicol acetyltransferase (CAT) activity in GH3 cells transiently transfected with a plasmid containing the first 187 basepairs of the rat PRL promoter cloned up-stream of the CAT gene, implying that PACAP stimulates transcription directed by the PRL promoter. The PACAP stimulation of CAT activity was observed at concentrations as low as 10(-11) M. We examined the action of PACAP on expression of a 5'-deletion series of PRL-CAT constructs. The PACAP response is completely lost when PRL promoter sequences between positions -187 and -113 are removed, implying that neither a previously described sequence resembling a cAMP response element nor the most proximal pit-1-binding site 1P plays a major role in the actions of PACAP on PRL gene transcription. This observation together with the ability of low concentrations of PACAP to stimulate PRL promoter activity without detectably increasing cellular cAMP levels suggest that the action of PACAP on PRL gene transcription might involve a cAMP-independent pathway.

Pituitary adenylate cyclase-activating polypeptide stimulates prolactin gene expression in a rat pituitary cell line.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to activate adenylate cyclase and stimulate PRL secretion in dispersed pituitary cells. We have employed the GH3 rat pituitary cell line to investigate whether PACAP can regulate expression of the PRL gene. PACAP increased cellular levels of cAMP in a concentration-dependent fashion (EC50, approximately 6 x 10(-9) M). PACAP also increased PRL mRNA levels in GH3 cells, implying that this peptide stimulates a step in expression of the PRL gene. In addition, PACAP strongly stimulated chloramphenicol acetyltransferase (CAT) activity in GH3 cells transiently transfected with a plasmid containing the first 187 basepairs of the rat PRL promoter cloned up-stream of the CAT gene, implying that PACAP stimulates transcription directed by the PRL promoter. The PACAP stimulation of CAT activity was observed at concentrations as low as 10(-11) M. We examined the action of PACAP on expression of a 5'-deletion series of PRL-CAT constructs. The PACAP response is completely lost when PRL promoter sequences between positions -187 and -113 are removed, implying that neither a previously described sequence resembling a cAMP response element nor the most proximal pit-1-binding site 1P plays a major role in the actions of PACAP on PRL gene transcription. This observation together with the ability of low concentrations of PACAP to stimulate PRL promoter activity without detectably increasing cellular cAMP levels suggest that the action of PACAP on PRL gene transcription might involve a cAMP-independent pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin synthesis from rat pineal gland

The effect of pituitary adenylate cyclase polypeptide (PACAP) on rat pineal was examined. PACAP stimulated melatonin release from cultured dissociated pinealocytes with a 10 4 higher potency than isoproterenol (EC 50 were 30 pM and 250 nM, respectively). The 10 −9 M PACAP stimulation was not inhibited by 5 × 10 −6 M VIP antagonist whereas that of 10 −9 M VIP was reduced by 54%. Kinetic analysis of melatonin release indicated that PACAP acts postsynaptically via receptor activation.