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)

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.
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PMID:Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture. 348 83

Pancreastatin is a 49 amino acid peptide first isolated, purified and characterized from porcine pancreas. Its biological activity in different tissues can be assigned to the C-terminal part of the molecule. Pancreastatin has a prohormonal precursor, chromogranin A, which is a glycoprotein present in neuroendocrine cells, including the endocrine pancreas. We have been interested in pancreastatin action in the liver. We found that pancreastatin has a glycogenolytic effect in the hepatocyte both in vivo and in vitro. We then studied and characterized the specific pancreastatin receptor in the rat liver plasma membrane, as well as the specific signal transduction. This receptor appears to be coupled to two different G proteins. A pertussis toxin-insensitive G proteins leads to the activation of phospholipase C, and therefore mediates the glycogenolytic effect in the liver by increasing cytoplasmic free calcium and stimulating protein kinase C. The role of cyclic GMP in the action of pancreastatin is not known yet, although it seems to regulate negatively the activation of phospholipase C. The precise mechanism by which pancreastatin stimulates guanylate cyclase activity remains to be studied.
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PMID:Pancreastatin action in the liver: dual coupling to different G proteins. 877 44

Pancreastatin (PST), a recently discovered regulatory peptide derived from chromogranin A, has been shown to have a glycogenolytic effect in the hepatocyte that is mediated by increasing intracellular calcium. Our previous studies on pancreastatin signaling suggested that PST receptor is coupled to some G proteins in the plasma membrane of the hepatocyte. The nature of this interaction was investigated using antisera against G(q/11)alpha by different approaches. Indirect evidence of a pertussis toxin (PT)-insensitive G protein of the family of G(q/11)alpha was obtained by measuring high-affinity guanosine triphosphatase (GTPase) activity in soluble rat liver membranes. PST increased GTPase activity in a dose-dependent manner. This effect was only slightly inhibited by PT pretreatment of the membranes, whereas anti-G(q/11)alpha antisera blocked most of the PST-stimulated GTPase activity. The selective association of the PST receptor with this G protein was further studied by the coelution in wheat germ agglutinin semipurification of the receptor and by immunoprecipitation of the G protein-PST receptor complexes using G-protein-specific antisera. A G protein of the family of G(q/11)alpha was found to be associated with the semipurified PST receptor. Moreover, anti-G(q/11)alpha antisera immunoprecipitated most PST-binding activity (95%), bringing down most of the specific G protein, whereas anti-G(il,2)alpha and -G(o,i3)alpha failed to immunoprecipitate the PST-binding activity. Finally, the coupling of the PST receptor with the effector phospholipase C was disrupted by blocking with G(q/11)alpha antisera, suggesting that a G protein of the family of G(q/11)alpha is a signal mediator from PST receptors to phospholipase C activation in rat liver membranes.
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PMID:Pancreastatin receptor is coupled to a guanosine triphosphate-binding protein of the G(q/11)alpha family in rat liver membranes. 946 64

Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a "neuronal" phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5'-deletions narrowed the nerve growth factor response element to a region from - 77 to - 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter approximately 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C-gamma or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Rafl, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor --> TrkA --> Shc/Grb2/Sos --> Ras --> Raf --> MEK --> mitogen-activated protein kinase --> ribosomal protein S6 serine kinase II --> CREB cyclic AMP response element.
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PMID:Neurotrophin activation of catecholamine storage vesicle protein gene expression: signaling to chromogranin a biosynthesis. 1019 63

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.
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PMID:Characterization of pancreastatin receptors and signaling in adipocyte membranes. 1044 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

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).
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PMID:Characterization of pancreastatin receptor and signaling in rat HTC hepatoma cells. 1084 19

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor Gq/11 protein-phospholipase C (PLC)-beta pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulin-stimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis.
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PMID:Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk. 1092 27

Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Galpha(q/11) pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K(d) of 0.5 nM and a B(max) of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCbeta(2)>PLCbeta(1)>PLCbeta(3) by activating Galpha(16) in rat heart membranes. These data suggest that PST may modulate the cardiac function.
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PMID:Pancreastatin, a chromogranin A-derived peptide, activates Galpha(16) and phospholipase C-beta(2) by interacting with specific receptors in rat heart membranes. 1125 46

Secretin evokes catecholamine secretion from PC12 pheochromocytoma cells. We tested whether secretin activates transcription of the major vesicular core protein chromogranin A (CgA). Secretin stimulated both endogenous CgA gene transcription (approximately 4-6-fold) as well as transfected CgA promoter activity (approximately 8-10-fold; EC50, approximately 7 nm) in PC12 cells. Studies on CgA promoter 5'-deletion mutant/luciferase reporter constructs, point mutations of the CgA cAMP response element (CRE), and their transfer to a heterologous promoter implicated CRE in cis as both necessary and sufficient for secretin-stimulated CgA gene transcription. Secretin-induced CgA gene transcription was inhibited/abolished by cytosolic Ca2+ chelation, chemical blockade of phospholipase C, protein kinase A (PKA), or mitogen-activated protein (MAP) kinase extracellular signal regulated kinase (ERK) 1/2 and the expression of dominant negative mutants of ERK1/2, CRE binding protein (CREB) kinase RSK2, or CREB. Secretin also augmented (approximately 4-fold) phosphorylation of ERK1/2. Trans-activation (approximately 21-fold) of GAL4-CREB fusion protein by secretin indicates involvement of CREB in secretin signaling to gene transcription. Electrophoretic mobility shift assays also identified CREB as the mediator of secretin-induced CgA gene transcription, and pCREB supershifts indicated Ser-133 as the active CREB moiety in vitro. This conclusion was reinforced in vivo by results of chromatin pCREB immunoprecipitation assays. We conclude that secretin signals to CgA gene transcription through the CRE domain in cis and through cAMP, Ca2+, PKA, MAP kinase, and the transcription factor CREB in trans. Thus, multiple signal transduction pathways seem to subserve the function of stimulus-transcription coupling after this peptidergic stimulus to chromaffin cells.
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PMID:Secretin activation of chromogranin A gene transcription. Identification of the signaling pathways in cis and in trans. 1264 81


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