Pituitary Adenylate Cyclase-activating Polypeptide Stimulation Research Articles

PACAP and acetylcholine cause distinct Ca2+ signals and secretory responses in chromaffin cells.

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.

PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization

Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) display psychomotor abnormalities, most of which are ameliorated by atypical antipsychotics with serotonin (5-HT) 2A receptor (5-HT2A) antagonism. Heterozygous Pacap mutant mice show a significantly higher hallucinogenic response than wild-type mice to a 5-HT2A agonist. Endogenous PACAP may, therefore, affect 5-HT2A signaling; however, the underlying neurobiological mechanism for this remains unclear. Here, we examined whether PACAP modulates 5-HT2A signaling by addressing cellular protein localization. PACAP induced an increase in internalization of 5-HT2A but not 5-HT1A, 5-HT2C, dopamine D2 receptors or metabotropic glutamate receptor 2 in HEK293T cells. This PACAP action was inhibited by protein kinase C inhibitors, β-arrestin2 silencing, the PACAP receptor PAC1 antagonist PACAP6-38, and PAC1 silencing. In addition, the levels of endogenous 5-HT2A were decreased on the cell surface of primary cultured cortical neurons after PACAP stimulation and were increased in frontal cortex cell membranes of Pacap−/− mice. Finally, intracerebroventricular PACAP administration suppressed 5-HT2A agonist-induced head twitch responses in mice. These results suggest that PACAP–PAC1 signaling increases 5-HT2A internalization resulting in attenuation of 5-HT2A-mediated signaling, although further study is necessary to determine the relationship between behavioral abnormalities in Pacap−/− mice and PACAP-induced 5-HT2A internalization.

SUN-484 Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Regulation of Hypothalamic Kisspeptin Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor are broadly distributed in the brain, and PACAP is known to work as a multifunctional peptide. However, it is still largely unknown how PACAP affects the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we examined the effect of PACAP on hypothalamic kisspeptin expression, a known regulator of gonadotropin-releasing hormone. We used two hypothalamic cell models, mHypoA-50 and mHypoA-55, which were originated from kisspeptin-expressing neuron in anterioventral periventricular nucleus and arcuate nucleus regions in the hypothalamus, respectively. Expression of Kiss-1 gene, which encodes kisspeptin, was significantly increased by PACAP stimulation in both mHypoA-50 and mHypoA-55 cells, by up to 2.69 ± 0.93-fold and 4.89 ± 1.13-fold, respectively. PACAP6-38, a PACAP receptor antagonist did not antagonize the action of PACAP on Kiss-1 gene expression but increased Kiss-1 gene by itself in these cells. PACAP-induced Kiss-1 gene expression in both mHypoA-50 and mHypoA-55 cells was almost completely prevented in the presence of H89, a protein kinase A inhibitor. PACAP was expressed in both these hypothalamic cell models and its expression was up-regulated by estradiol in mHypoA-50 cells but not in mHypoA-55 cells. Stimulation of mHypoA-50 and mHypoA-55 cells with PACAP increased the expression levels of corticotropin-releasing hormone and neurotensin, both of which could modulate HPG axis. Our present observations suggest that hypothalamic PACAP might modulate the HPG axis by directly or indirectly modulating Kiss-1 gene expression.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of hypothalamic kisspeptin expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor are broadly distributed in the brain, and PACAP is known to work as a multifunctional peptide. However, it is still largely unknown how PACAP affects the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we examined the effect of PACAP on hypothalamic kisspeptin expression, a known regulator of gonadotropin-releasing hormone. We used two hypothalamic cell models, mHypoA-50 and mHypoA-55, which were originated from kisspeptin-expressing neuron in anterioventral periventricular nucleus and arcuate nucleus regions in the hypothalamus, respectively. Expression of Kiss-1 gene, which encodes kisspeptin, was significantly increased by PACAP stimulation in both mHypoA-50 and mHypoA-55 cells, by up to 2.69 ± 0.93-fold and 4.89 ± 1.13-fold, respectively. PACAP6-38, a PACAP receptor antagonist did not antagonize the action of PACAP on Kiss-1 gene expression but increased Kiss-1 gene by itself in these cells. PACAP-induced Kiss-1 gene expression in both mHypoA-50 and mHypoA-55 cells was almost completely prevented in the presence of H89, a protein kinase A inhibitor. PACAP was expressed in both these hypothalamic cell models and its expression was up-regulated by estradiol in mHypoA-50 cells but not in mHypoA-55 cells. Stimulation of mHypoA-50 and mHypoA-55 cells with PACAP increased the expression levels of corticotropin-releasing hormone and neurotensin, both of which could modulate HPG axis. Our present observations suggest that hypothalamic PACAP might modulate the HPG axis by directly or indirectly modulating Kiss-1 gene expression.

β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking.

A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.

Expression and Regulation of Pituitary Adenylate Cyclase-Activating Polypeptide in Rat Placental Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified as a hypophysiotropic factor that regulates pituitary cell functions and has been subsequently shown to be widely distributed and have multiple functions. The PACAP is known to be expressed in placental tissues and is suggested to have a critical role in physiological function of the placenta. In addition to PACAP, the hypothalamic peptides kisspeptin and gonadotropin-releasing hormone (GnRH) are also expressed in placental cells. In this study, we used primary cultures of placental tissues from rats of 16 to 18 days gestation and examined the regulation and function of PACAP. The PACAP messenger RNA (mRNA) expression and PACAP-immunoreactive cells were detected in primary cultures of rat placental cells. The PACAP mRNA expression in placental cells was upregulated in the presence of the sex steroids estradiol and progesterone; however, their combined treatment failed to enhance their individual effects. When the cells were stimulated with kisspeptin, PACAP mRNA expression was increased. Similarly, GnRH had a stimulatory effect on PACAP expression. Conversely, kisspeptin expression in placental cells was increased by PACAP stimulation, whereas PACAP failed to stimulate GnRH mRNA expression in these cells. Finally, we found that PACAP had a stimulatory effect on human chorionic gonadotropin expression in placental cells. Our current observations suggest that the hypothalamic peptides PACAP, kisspeptin, and GnRH are interrelated and maintain placental functions.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases expression of the gonadotropin-releasing hormone (GnRH) receptor in GnRH-producing GT1-7 cells overexpressing PACAP type I receptor

The present study demonstrates the action of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone (GnRH)-producing neuronal cells, GT1-7. Because we found the expression levels of PACAP type 1 receptor (PAC1R) to be low in these cells, we transfected them with PAC1R expression vector and observed the outcome. PACAP increased the activity of the serum response element (Sre) promoter, a target of extracellular signal-regulated kinase (ERK), as well as the cAMP response element (Cre) promoter in GT1-7 cells overexpressing PAC1R. We also observed ERK phosphorylation and cAMP accumulation upon PACAP stimulation. PACAP stimulated the promoter activity of GnRH receptor (GnRHR) with increasing levels of GnRHR proteins. Notably, the increase in GnRHR promoter activity from kisspeptin was potentiated in the presence of PACAP. A similar increasing effect of PACAP on the action of kisspeptin was observed for Cre promoter activity. On the other hand, the Sre promoter activated by kisspeptin was inhibited by co-treatment with kisspeptin and PACAP. Likewise, kisspeptin-increased GnRHR promoter activity and Cre promoter activity were both potentiated in the presence of cAMP, whereas the Sre promoter activated by kisspeptin was inhibited in the presence of cAMP. Our observations show that PACAP increases GnRHR expression and stimulates kisspeptin’s effect on GnRHR expression in association with the cAMP/PKA signaling pathway in GT1-7 cells overexpressing PAC1R. In addition, PACAP was shown to have an inhibitory effect on ERK-mediated kisspeptin action.

PACAP-Evoked Adrenal Excitation is Due to Membrane Depolarization and Facilitation of an LVA Calcium Channel

Pituitary adenylate cyclase activating polypeptide (PACAP) is a peptide transmitter released from the sympathetic splanchnic nerve to stimulate neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that PACAP is preferentially released under heightened splanchnic firing of the sympathetic stress response. PACAP-dependent stimulation results in an immediate and robust Ca2+-dependent catecholamine secretion from chromaffin cells. Yet, PACAP stimulation does not evoke action potential firing in the chromaffin cell.

PACAP‐regulated phenylethanolamine N‐methyltransferase gene expression

Pituitary adenylate cyclase activating polypeptide (PACAP) induces the proximal -893 bp of rat phenylethanolamine N-methyltransferase (PNMT) gene promoter in PC12 cells via PACAP type I receptors. Deletion mutation analysis suggested that the initial -392 bp of promoter, containing early growth response protein (Egr-1), specificity protein 1 (Sp1) and activator protein 2 (AP-2) binding sites (-165, -168 and -103 bp, respectively), was sufficient for PACAP activation. Egr-1 and AP-2 involvement was supported by PACAP induction of their mRNA and protein. Mutation of the Egr-1, Sp1 and AP-2 elements showed that the Egr-1 site was essential for PACAP stimulation. Mutation of the -103 bp AP-2 site partially reduced PACAP activation of the promoter. Mutation of two upstream AP-2 sites at -573 and -650 bp, separately or in tandem, also prevented promoter induction by PACAP. siRNA knock-down of Egr-1 and AP-2 suppressed promoter activation for the -893 bp construct. Egr-1 siRNA knock-down also eliminated the residual activation observed for the -103 bp AP-2 mutant construct, suggesting that Egr-1 and AP-2 through respective -165 and -650/-573/-103 bp sites cooperatively stimulate the promoter. PACAP responses appear orchestrated through cAMP-protein kinase A and phospholipase C signaling as MDL12,330A, H89 and U73122, respectively, inhibited promoter induction by PACAP and reduced PACAP-stimulation of Egr-1, AP-2 and PNMT mRNA and protein and Egr-1 and AP-2 protein/DNA complex formation. Findings are the first to show that PACAP stimulates PNMT promoter-driven gene expression via PACAP type I receptors and cAMP-protein kinase A and phospholipase C signaling, recruiting Egr-1 and AP-2 as cooperative regulators, and the first to associate the transcription factor AP-2 to PACAP-mediated gene induction.

GnRH-induced PACAP and PAC1 receptor expression in pituitary gonadotrophs: A possible role in the regulation of gonadotropin subunit gene expression

We examined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP type 1 receptor (PAC1-R) mRNA following gonadotropin-releasing hormone (GnRH) stimulation using the gonadotroph cell line LβT2. GnRH stimulation increased PACAP and PAC1-R mRNA expression in a static culture. Increase in the cell surface density of the PAC1-R following transfection with PAC1-R expression vectors significantly increased gonadotropin LHβ and FSHβ subunit promoter activities following 100 nM PACAP stimulation. In addition, increasing concentrations of PACAP stimulation augmented the promoter activities for both LHβ and FSHβ in PAC-1R overexpressing cells. In the cells with PAC1-R, the effect of GnRH was further potentiated in the presence of PACAP from 5.31 ± 0.93 to 9.89 ± 0.38-fold for LHβ and for FSHβ subunit, respectively; from 2.58 ± 0.31-fold by GnRH alone to 10.90 ± 2.79-fold with PACAP. The combination treatment with GnRH and PACAP did not augment the ERK phosphorylation induced by GnRH alone. PACAP expectedly increased cAMP accumulation and this effect was significantly attenuated in the presence of GnRH. PACAP gene expression was more prominent following lower frequency GnRH pulses (every 120 min) in a perifused culture. Our results suggest that PACAP and PAC1-R are produced locally within the gonadotrophs following GnRH stimulation. They subsequently affect the gonadotrophs in an autocrine manner and modulate the GnRH pulse-dependent specific regulation of gonadotropin subunits.

Src-dependent TrkA transactivation is required for pituitary adenylate cyclase-activating polypeptide 38-mediated Rit activation and neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Galpha/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both G(i)alpha and G(s)alpha/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y(499)) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.

Increased Stathmin1 Expression in the Dentate Gyrus of Mice Causes Abnormal Axonal Arborizations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.

Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.

Luteinizing Hormone-stimulated Pituitary Adenylate Cyclase-activating Polypeptide System and its Role in Progesterone Production in Human Luteinized Granulosa Cells

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

Differential involvement of nitric oxide signaling in dopamine and PACAP stimulation of growth hormone release in goldfish

Previous studies in goldfish pituitary cells have shown that nitric oxide synthase (NOS)/nitric oxide (NO) signaling is involved in mediating the growth hormone (GH) release response to gonadotropin-releasing hormones. In this study, the involvement of this signaling pathway in mediating the action of two cAMP-mobilizing neuroendocrine stimulators of GH release, pituitary adenylate cyclase-activating polypeptide (PACAP) and dopamine (DA), was investigated in cell column perifusion experiments with primary cultures of dispersed pituitary cells. GH responses to PACAP were unaffected by three NOS inhibitors, aminoguanidine hemisulfate, 1400W and 7-nitroindazole (7-Ni). PACAP-stimulated GH release was also not reduced by two NO scavengers, rutin hydrate and PTIO, but NO-donor sodium nitroprusside (SNP)-elicited GH release was additive to the GH response to PACAP. In contrast, DA-induced GH secretion was reduced by 7-Ni, rutin hydrate and PTIO while not being additive to the GH response induced by SNP. These results indicate that although both PACAP and DA stimulation of acute GH release involve activation of adenylate cyclase/cAMP, DA- but not PACAP-signaling also utilizes the NOS/NO second messenger system.

Signaling mechanisms for α2-adrenergic inhibition of PACAP-induced growth hormone secretion and gene expression grass carp pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent growth hormone (GH)-releasing factor in lower vertebrates. However, its functional interactions with other GH regulators have not been fully characterized. In fish models, norepinephrine (NE) inhibits GH release at the pituitary cell level, but its effects on GH synthesis have yet to be determined. We examined adrenergic inhibition of PACAP-induced GH secretion and GH gene expression using grass carp pituitary cells as a cell model. Through activation of pituitary alpha2-adrenoreceptors, NE or the alpha2-agonist clonidine reduced both basal and PACAP-induced GH release and GH mRNA expression. In carp pituitary cells, clonidine also suppressed cAMP production and intracellular Ca2+ levels and blocked PACAP induction of these two second messenger signals. In GH3 cells transfected with a reporter carrying the grass carp GH promoter, PACAP stimulation increased GH promoter activity, and this stimulatory effect could be abolished by NE treatment. In parallel experiments, clonidine reduced GH primary transcript and GH promoter activity without affecting GH mRNA stability, and these inhibitory actions were mimicked by inhibiting adenylate cyclase (AC), blocking protein kinase A (PKA), removing extracellular Ca2+ in the culture medium, or inactivating L-type voltage-sensitive Ca2+ channels (VSCC). Since our recent studies have shown that PACAP can induce GH secretion in carp pituitary cells through cAMP/PKA- and Ca2+/calmodulin-dependent mechanisms, these results, taken together, suggest that alpha2-adrenergic stimulation in the carp pituitary may inhibit PACAP-induced GH release and GH gene transcription by blocking the AC/cAMP/PKA pathway and Ca2+ entry through L-type VSCC.

A novel DISC1-interacting partner DISC1-Binding Zinc-finger protein: implication in the modulation of DISC1-dependent neurite outgrowth

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a (1;11) (q42.1;q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. To investigate how DISC1 confers susceptibility to psychiatric disorders, we previously identified fasciculation and elongation protein zeta-1 and Kendrin as DISC1-interacting molecules in a yeast two-hybrid screen of a human brain complementary DNA library. Here, we have further identified a novel DISC1-interacting protein, termed DISC1-Binding Zinc-finger protein (DBZ), which has a predicted C(2)H(2)-type zinc-finger motif and coiled-coil domains. DBZ was co-immunoprecipitated with DISC1 in lysates of PC12 cells and rat brain tissue. The domain of DISC1 interacting with DBZ was close to the translocation breakpoint in the DISC1 gene. DBZ messenger RNA (mRNA) was expressed in human brains, but not in peripheral tissues. In situ hybridization revealed high expression of DBZ mRNA in the hippocampus, olfactory tubercle, cerebral cortex and striatum in rats. Because this pattern of localization was similar to that of the pituitary adenylate cyclase (PAC(1)) receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which has recently been implicated in neuropsychological functions, we examined whether DISC1/DBZ interaction was involved in the PACAP signaling pathway. PACAP upregulated DISC1 expression and markedly reduced the association between DISC1 and DBZ in PC12 cells. A DISC1-binding domain of DBZ reduced the neurite length in PC12 cells after PACAP stimulation and in primary cultured hippocampal neurons. The present results provide some new molecular insights into the mechanisms of neuronal development and neuropsychiatric disorders.

Differential Mechanisms for PACAP and GnRH cAMP Induction Contribute to Cross‐talk between both Hormones in the Gonadotrope LβT2 Cell Line

The effects and respective influence of pituitary adenylate cyclase-activating polypeptide (PACAP) and gonadotropin-releasing hormone (GnRH) on cyclic AMP (cAMP) production in pituitary gonadotropes were analyzed using the LbetaT2 cell line. Both hormones induced cAMP with, however, different intensity and time course. In addition, the GnRH effect was markedly reduced by PKC inhibitors. Despite its positive coupling to cAMP pathway, GnRH counteracted PACAP induction of cAMP and this effect was mimicked by the PKC activator phorbol 12-myristate 13-acetate (PMA). The data reveal major differences in the mechanisms by which PACAP and GnRH activate cAMP/PKA pathway in LbetaT2 cells and suggest that PKC activation serves GnRH not only to increase cAMP but also to counteract the PACAP stimulation of this signaling pathway.

Dual effect of pituitary adenylate cyclase activating polypeptide on prostate tumor LNCaP cells: short- and long-term exposure affect proliferation and neuroendocrine differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that elicits the increase of intracellular cAMP levels and protein kinase A activity in various cell systems. Here we show that the pattern of cAMP elevation triggered by PACAP is critical for the fate of LNCaP prostate cancer cells. We demonstrate that these cells express PACAP and its type 1 receptor. A short-term stimulation with PACAP, which generates a transient cAMP rise, induces proliferation of LNCaP cells through a protein kinase A-dependent activation of the MAPK cascade. On the contrary, we observed that chronic PACAP stimulation, giving rise to a sustained cAMP accumulation, leads to proliferation arrest and neuroendocrine differentiation. Moreover, PACAP stimulates phosphory-lation and activation of the cAMP response element binding transcription factor (CREB), and MAPK activation is necessary for its full transcriptional activity, indicating a direct involvement of cAMP response element in PACAP action. These findings demonstrate that a crucial event determining the outcome of prostatic cancer cells progression is the sustained vs. transient intracellular cAMP increase.

Stimulation of extracellular signal-regulated kinase by pituitary adenylate cyclase-activating polypeptide in alpha T3-1 gonadotrophs.

The putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates glycoprotein hormone alpha-subunit (alpha GSU) gene transcription and secretion in the clonal gonadotroph alpha T3-1 cell line. The specific signalling pathways regulating these actions of PACAP have not been clearly defined. We have examined the possibility that mitogen activated protein kinases (MAPKs) may play a role in mediating the effects of PACAP on alpha T3-1 gonadotrophs. Treatment of alpha T3-1 cells with PACAP (100 nM) or epidermal growth factor (EGF, 10 nM) for 5 min significantly stimulated extracellular signal-regulated kinase activity (ERK, a component of the MAPK pathway) as determined by an immunocomplex assay. Pre-treatment of alpha T3-1 cells with the specific MAPK kinase (MEK) inhibitor, U0126, blocked PACAP and EGF-induced activation of ERK. Transcriptional stimulation of a human alpha GSU-luciferase reporter construct by PACAP was unaffected by U0126 treatment. However, pre-treatment with U0126 significantly inhibited PACAP stimulation of [(3)H]-thymidine incorporation in alpha T3-1 cells. Thus our results suggest that PACAP stimulates ERK activation in alpha T3-1 cells, and that the functional effect of this ERK activation is increased DNA synthesis and cell proliferation rather then transcriptional activation of the alpha GSU gene.