Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway

Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca(2+) channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6-38, an antagonist at type-I PACAP receptor. 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2',5'-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca(2+) channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.     

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC₁ receptor, but not by VPAC₁ and VPAC₂ receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC₁ receptor, adenylyl cyclase, or protein kinase A. Activation of Ca²⁺ channels by PACAP38 was also observed, which could be antagonized by the PAC₁ receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca²⁺ concentration, which was decreased by PAC₁ receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC₁ receptor, inhibiting voltage-dependent potassium channels, activating Ca²⁺ channels and increasing intracellular Ca²⁺ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

In vivo effect of pituitary adenylate cyclase activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats.

In view of the recent demonstrations that pituitary adenylate cyclase activating polypeptide (PACAP) 38 stimulates the release of LH from superfused pituitary cells and that the hypothalamus and anterior pituitary have highly selective binding sites for the peptide, we have surveyed the effect of intraatrial injections of PACAP 38 and vasoactive intestinal peptide (VIP), which has 68% homology with PACAP 38, in intact adult male rats. Furthermore the effect of intracerebroventricular (icv) injection of PACAP 38 was investigated. Intraatrial (10, 30, 100 micrograms) and icv (8, 32 micrograms) administration of PACAP 38 stimulated LH release significantly (P less than 0.01) in a dose-related fashion. Icv injection at a dose of 0.8 microgram was ineffective. The time course pattern of LH release by intraatrial injection and that by icv injection was similar, but the LH levels increased by intraatrial injection were much higher than that by icv injection. Intraatrial administration of VIP had almost no effect on LH release. These findings suggested that PACAP 38 stimulates LH release in vivo.     

Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1–299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The β-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC₁-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.     

The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1(null), at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC(1) receptor isoform activation of specific intracellular signaling pathways.

Pituitary adenylate cyclase-activating polypeptides (PACAP) have potent regulatory and neurotrophic activities on superior cervical ganglion (SCG) sympathetic neurons with pharmacological profiles consistent for the PACAP-selective PAC(1) receptor. Multiple PAC(1) receptor isoforms are suggested to determine differential peptide potency and receptor coupling to multiple intracellular signaling pathways. The current studies examined rat SCG PAC(1) receptor splice variant expression and coupling to intracellular signaling pathways mediating PACAP-stimulated peptide release. PAC(1) receptor mRNA was localized in over 90% of SCG neurons, which correlated with the cells expressing receptor protein. The neurons expressed the PAC(1)(short)HOP1 receptor but not VIP/PACAP-nonselective VPAC(1) receptors; low VPAC(2) receptor mRNA levels were restricted to ganglionic nonneuronal cells. PACAP27 and PACAP38 potently and efficaciously stimulated both cAMP and inositol phosphate production; inhibition of phospholipase C augmented PACAP-stimulated cAMP production, but inhibition of adenylyl cyclase did not alter stimulated inositol phosphate production. Phospholipase C inhibition blunted neuron peptide release, suggesting that the phosphatidylinositol pathway was a prominent component of the secretory response. These studies demonstrate preferential sympathetic neuron expression of PACAP-selective receptor variants contributing to regulation of autonomic function.     

Presence of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors in Y-79 Human Retinoblastoma Cells

Abstract: 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 p M , 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 G_s 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 n M . 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.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

The neuropeptide PACAP attenuates beta-amyloid (1-42)-induced toxicity in PC12 cells

Pituitary adenylate cyclase activating polypeptide (PACAP) modulates neurotransmission in the central and peripheral nervous systems. In vitro and in vivo studies have shown the protective effects of PACAP against neuronal damage induced by ischemia and agonists of NMDA-type glutamate receptors. Here, we demonstrated that PACAP also protected against neuronal toxicity induced by beta-amyloid (Abeta) peptide, aggregation of which is a causative factor for Alzheimer's disease. PACAP (10(-9)M) rescued 80% of decreased cell viability and 50% of elevated caspase-3 activity that resulted from exposure of PC12 cells to Abeta. PACAP was at least 10(4)-fold more effective than other neuropeptides including vasoactive intestinal peptide (VIP) and humanin, which correlated with the level of cAMP accumulation. Thus, our results suggested that PACAP attenuates Abeta-induced cell death in PC12 cells through an increase in cAMP and that caspase-3 deactivation by PACAP is involved in the signaling pathway for this neuroprotection.     

Partial rescue of functional interactions of a nonpalmitoylated mutant of the G-protein G alpha s by fusion to the beta-adrenergic receptor

Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha(s) palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (betaAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc(-) cells, which lack endogenous Galpha(s), showed that wild-type Galpha(s) was predominantly localized at the plasma membrane, but the mutant C3A-Galpha(s), which does not incorporate [(3)H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha(s) showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high-affinity agonist binding, all of which were present in the wild-type Galpha(s) expressing cells. Fusion of C3A-Galpha(s) with the betaAR [betaAR-(C3A)Galpha(s)] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha(s) and betaAR (betaAR-Galpha(s)) fusion protein, the betaAR-(C3A)Galpha(s) fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing betaAR-Galpha(s) reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in betaAR-(C3A)Galpha(s) expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha(s) and facilitated bimolecular interactions of Galpha(s) with the betaAR and adenylyl cyclase.     

Preferential inhibition of the oncogenic form of RasH by mutations in the GAP binding/"effector" domain

The double mutation, D33H/P34S, reduced the transforming activity of oncogenic RasH proteins, G12V and Q61L, 400- and 20-fold, respectively. Remarkably, this same mutation did not reduce the transforming activity of normal RasH, nor did it impair the ability of the protein to restore a functional Ras pathway in cells whose endogenous Ras proteins were inhibited. Another mutation in this region, D38N, had similar effects. The mutations reduced downstream coupling efficiency of normal Ras as assessed by yeast adenylyl cyclase stimulation. However, this was offset by decreased GTPase activating protein (GAP) binding, since the latter resulted in elevated GTP-bound mutant Ras in cells. The mutations produced a similar decrease in downstream coupling efficiency of oncogenic Ras, but decreased GAP binding did not compensate because the GTPase activity of oncogenic Ras is not stimulated by GAP. These results imply that preferential inactivation of oncogenic Ras in human tumors may be achieved by reagents designed to inhibit the GAP-binding/"effector" domain of Ras proteins.     

PACAP protects neuronal differentiated PC12 cells against the neurotoxicity induced by a mitochondrial complex I inhibitor, rotenone

In vivo and in vitro studies have suggested a neuroprotective role for Pituitary adenylate cyclase activating polypeptide (PACAP) against neuronal insults. Here, we showed that PACAP27 protects against neurotoxicity induced by rotenone, a mitochondrial complex I inhibitor that has been implicated in the pathogenesis of Parkinson's disease (PD). The neuroprotective effect of PACAP27 was dose-dependent and blocked by its specific receptor antagonist, PACAP6-27. The effects of PACAP27 on rotenone-induced cell death were mimicked by dibutyryl-cAMP (db-cAMP), forskolin and prevented by the PKA inhibitor H89, the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PACAP27 administration blocked rotenone-induced increases in the level of caspase-3-like activity, whereas could not restore mitochondrial activity damaged by rotenone. Thus, our results demonstrate that PACAP27 has a neuroprotective role against rotenone-induced neurotoxicity in neuronal differentiated PC12 cells and the neuroprotective effects of PACAP are associated with activation of MAP kinase pathways by PKA and with inhibition of caspase-3 activity; the signaling mechanism appears to be mediated through mitochondrial-independent pathways.     

垂体腺苷酸环化酶激活肽的发现及研究进展

垂体腺苷酸环化酶激活肽是最初在绵羊下丘脑发现的一种新的具有多种生物活性的多肽。它广泛分布于中枢神经系统、周围神经系统以及非神经组织内。此外,它在某些类型细胞的旁分泌和自分泌主财节中也发挥作用。     

Pituitary adenylyl cyclase-activating polypeptide 38 reduces astroglial proliferation by inhibiting the GTPase RhoA

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G(1) phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G(1) phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.