Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.
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PMID:Induction of the fibrinogen receptor on human platelets by intracellular mediators. 310 May 33

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.
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PMID:Differential effects of peptide histidine isoleucine (PHI) and related peptides on stimulation and suppression of neuroblastoma cell proliferation. A novel VIP-independent action of PHI via MAP kinase. 967 97

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. We previously reported that PACAP coupled to the PAC1 receptor to evoke dihydropyridine-sensitive early (15 to 20 minutes) catecholamine secretion and cAMP response element binding protein-mediated trans-activation of the secretory protein chromogranin A promoter in PC12 pheochromocytoma cells. In this report, we studied whether the secretory and transcriptional responses elicited by PACAP were subject to desensitization. We found that PACAP evoked distinct immediate (initial, 0 to 20 minutes) and long-lasting (20 to 180 minutes) effects on catecholamine secretion. Initial secretory and chromogranin A trans-activation responses induced by PACAP were desensitized in a dose-dependent fashion after preexposure of cells to PACAP, and the IC(50) doses of PACAP for desensitization were approximately 18- to approximately 32-fold lower than the EC(50) activating doses for secretion or transcription. Desensitization of the initial secretion response was associated with decreased Ca(2+) influx through L-type voltage-operated Ca(2+) channels. Acute exposure to PACAP also triggered long-lasting (up to 3 hours), extracellular Ca(2+)-dependent, pertussis toxin-insensitive catecholamine secretion; indeed, even after short-term (20 minutes) exposure to PACAP and removal of the secretagogue, PC12 cells continued to secrete norepinephrine up to 76.9+/-0.22% of cellular norepinephrine content after 3 hours. A phospholipase C-beta inhibitor (U-73122) blocked this extended secretory response, which was dependent on low-magnitude Ca(2+) influx resistant to several L-, N-, P/Q-, or T-type Ca(2+) channel antagonists, but sensitive to Zn(2+), Ni(2+), Cd(2+), or to the store-operated Ca(2+) channel blocker SKF96365. A less than additive effect of the sarco-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin plus PACAP on this sustained secretion also supported a contribution of store-operated Ca(2+) entry to the sustained secretory response. We propose that PACAP-evoked secretion and transcription are subject to homologous desensitization in PC12 cells; however, PACAP also induces long-lasting secretion, even under dose and time circumstances in which acute, dihydropyridine-sensitive secretion has been desensitized. Although initial secretion is mediated by an L-type voltage-operated Ca(2+) channel, extended secretion may involve a store-operated Ca(2+) channel that is activated through a G(q/11)/phospholipase C-beta/phosphoinositide signaling pathway.
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PMID:Time-dependent effects of the neuropeptide PACAP on catecholamine secretion : stimulation and desensitization. 1056 98

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.
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PMID:Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate. 1061 84

Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin-induced PACAP mRNA expression. Down-regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.
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PMID:Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells. 1064

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G protein-coupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. (125)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G protein coupling did not account for differences in internalization. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.
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PMID:Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization. 1090 67

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel member of the secretin-glucagon peptide family. In mammals, this peptide has been located in a wide range of tissues and is involved in a variety of biological functions. In lower vertebrates, especially fish, increasing evidence suggests that PACAP may function as a hypophysiotropic factor regulating pituitary hormone secretion. PACAP has been identified in the brain-pituitary axis of representative fish species. The molecular structure of fish PACAP is highly homologous to mammalian PACAP. The prepro-PACAP in fish, however, is distinct from that of mammals as it also contains the sequence of fish GHRH. In teleosts, the anterior pituitary is under direct innervation of the hypothalamus and PACAP nerve fibers have been identified in the pars distalis. Using the goldfish as a fish model, mRNA transcripts of PACAP receptors, namely the PAC1 and VPACI receptors, have been identified in the pituitary as well as in various brain areas. Consistent with the pituitary expression of PACAP receptors, PACAP analogs are effective in stimulating growth hormone (GH) and gonadotropin (GTH)-II secretion in the goldfish both in vivo and in vitro. The GH-releasing action of PACAP is mediated via pituitary PAC1 receptors coupled to the adenylate cyclase-cAMP-protein kinase A and phospholipase C-IP3-protein kinase C pathways. Subsequent stimulation of Ca2+ entry through voltage-sensitive Ca2+ channels followed by activation of Ca2+-calmodulin protein kinase II is likely the downstream mechanism mediating PACAP-stimulated GH release in goldfish. Although the PACAP receptor subtype(s) and the associated post-receptor signaling events responsible for PACAP-stimulated GTH-II release have not been characterized in goldfish, these findings support the hypothesis that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to regulate GH and GTH-II release in fish.
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PMID:Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish. 1094 84

The objectives of the study reported here were to identify amino acid residues of the C-terminus that are critical for intracellular signaling. A total of nine amino acid substitution and truncation mutants were constructed by PCR and confirmed by sequencing. Mutant and wildtype receptors were stably transfected into NIH/3T3 fibroblasts and studied for their ability to bind PACAP-27 and activate phospholipase C (PLC) and adenylyl cyclase (AC). Receptor affinity of 125I-PACAP-27 for the wildtype and mutants were similar (Kd = 0.6-1.5 nM). However, truncation of the entire 63 amino acids of the hPAC1 resulted in no signaling to either AC or IP. Addition of the proximal 10 amino acids of the C-terminus failed to restore AC or IP signaling, whereas addition of the proximal 27 amino acids of the C-terminus resulted in reconstitution of complete AC and IP responses, identical to the WT. Point mutations within this 17 amino acid region identified specific amino acids involved in PAC1 signaling. These results indicate that a structural motif within the proximal region of the carboxyl terminus is critical for G protein coupling.
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PMID:Essential structural motif in the C-terminus of the PACAP type I receptor for signal transduction and internalization. 1119 23

Morphological studies identified PACAP-immunoreactive nerve fibers in dense pericellular arrangements around virtually every cholinergic parasympathetic neuron of guinea pig cardiac ganglia; all postganglionic cardiac neurons expressed membrane-associated PAC1 receptor protein. Characterization of the alternative splice variants established predominant expression of the PAC1(very short) receptor transcript containing neither HIP nor HOP exons. PACAP depolarized cardiac neurons and increased membrane excitability; the excitability resulted from neither altered action potential properties nor inhibition of IM. Treatment of cardiac ganglia explants with PACAP significantly reduced the numbers of cholinergic neurons coexpressing somatostatin immunoreactivity, which did not appear to be correlated with prosomatostatin mRNA expression. The PACAP-mediated decrease in somatostatin immunoreactive neurons required calcium influx through L-type calcium channels and activation of adenylyl cyclase, whereas activation of phospholipase C or protein kinase A was not required. These observations indicate that PACAP through the PAC1 receptors elicits complex actions on guinea pig parasympathetic cardiac ganglia neurons, including modulation of membrane ion conductances and modulation of neuropeptide expression.
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PMID:PACAP peptides modulate guinea pig cardiac neuron membrane excitability and neuropeptide expression. 1119 24

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the nervous system across phyla. PACAP has an amino acid sequence homology of 68% with that of vasoactive intestinal polypeptide (VIP) and of 37% with that of secretin, indicating that PACAP is a member of the VIP/glucagon/secretin superfamily. PACAP exerts its actions via three heptahelical G-protein-linked receptors: one PACAP-specific (PAC1) receptor and two receptors (VPAC1 and VPAC2) shared with VIP. PACAP stimulates several different signaling cascades in neurons, leading to the activation of adenylate cyclase, phospholipase C, and mitogen-activated protein kinase and mobilization of calcium. Although PACAP and VIP have no apparent homology with calcitonin and parathyroid hormone (PTH), PAC1, VPAC, secretin, glucagon, glucagon-like peptide 1, growth hormone-releasing hormone, calcitonin, and PTH/PTH-related peptide receptors are related to each other and constitute a subfamily of the G-protein-coupled receptors. Distribution analysis of PACAP and its receptors and pharmacological studies have elucidated its pleiotropic effects in the central and peripheral nervous systems. However, the relevance of the pharmacological PACAP effects to the actual physiological activities of endogenous PACAP has not been addressed, because potent and selective low-molecular-weight PACAP antagonists have not yet been developed. To assess the function of PACAP in vivo, we have recently generated PAC1 receptor- and PACAP-targeted mice, and provided evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors. In this review, we focus on the physiological and or pathophysiological roles mediated by PACAP in the nervous system.
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PMID:[Physiological significance of pituitary adenylate cyclase-activating polypeptide (PACAP) in the nervous system]. 1251 Mar 88


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