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)

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores--melanocyte stimulating hormone, light, (-) norepinephrine, 5-hydroxytrptamine, and the beta2-adrenergic receptor--were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP3 in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide beta responses were blocked only by H89, predicting coupling to adenylyl cyclase.
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PMID:Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C. 869 26

Neurotransmitters and hormones, by binding to receptors linked to adenylate cyclase or phospholipase C (PLC), increase cytosolic free Ca2+ and potentiate glucose-induced insulin release from beta-cells. Interactions between both signaling pathways may occur and be of relevance to the regulation of insulin secretion. We demonstrate here that in single insulin-secreting HIT cells, forskolin and 8-bromo-cAMP, which stimulate Ca2+ influx through voltage-dependent Ca2+ channels (VDCC), cause a marked increase in the frequency, amplitude, and duration of Ca2+ transients evoked by hormones linked to PLC, such as arginine vasopressin (AVP) or bombesin. Forskolin also potentiates AVP- or bombesin-induced insulin secretion from populations of HIT cells in the presence of elevated glucose (10 mM). BAY K 8644, an activator of VDCC, mimicked the effects of elevated cAMP on both AVP- and bombesin-induced Ca2+ transients and insulin release, which suggests that enhanced Ca2+ influx through VDCC activated by cAMP-dependent mechanisms underlies the positive interactions of both signaling pathways on Ca2+ signaling and insulin secretion. Physiologically, synergistic cross-signaling between the cAMP- and Ca2+ -phosphoinositide signaling pathway could be important for the regulation of insulin release under conditions where extracellular glucose is high and beta-cells are exposed to multiple stimuli activating adenylate cyclase or PLC at the same time.
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PMID:CYCLIC adenosine 3',5'-monophosphate potentiates Ca2+ signaling and insulin secretion by phospholipase C-linked hormones in HIT cells. 877 Sep 28

Bombesin stimulation of inositol 1,4,5-trisphosphate (Ins P3) formation in rat sonicated pancreatic acinar cells was inhibited by an antibody directed against the pertussis toxin (PTX)-sensitive GTP-binding G alpha i3 protein but not by an anti-G alpha q-11 antibody. After solubilization and gel filtration, [125I-Tyr4]bombesin binding sites were recovered in a peak of protein of 67 approximately 90 kDa with a maximal enrichment corresponding to a molecular mass of 83-kDa. Results obtained from the non-hydrolysable GTP analog guanosine-5'-[gamma-thio]triphosphate (GTP gamma S) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the G alpha i3 protein but not the G alpha q-11 protein. Furthermore, GTP gamma S increased the bombesin binding dissociation constant (KD) from 0.32 to 0.60 nM, while the anti-G alpha i3 antibody decreased the maximal binding capacity (Bmax) from 50 to 25 fmol/mg protein without affecting the KD. Mixing solubilized bombesin binding sites with a phospholipase C (PLC) preparation from rat pancreas reconstituted a bombesin-stimulated PLC activity which was markedly inhibited by the anti-G alpha i3 antibody but unaffected by the anti-G alpha q-11 antibody. In addition, this stimulation was inhibited by an anti-PLC beta 1 antibody. This result supports the involvement of the PLC beta 1 isoform in bombesin receptor activation.
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PMID:Bombesin activation of phospholipase C beta 1 in rat acinar pancreatic cells involves the pertussis toxin-sensitive G alpha i3 protein. 879 79

Imaging [Ca2+]i at high temporal resolution and measuring the properties of Ca2+ signaling in streptolysin O (SLO)-permeabilized cells were used to study the spacial organization of signaling complexes. Sequential stimulation of single cells within pancreatic acini with several Ca2+-mobilizing agonists revealed an agonist-specific pattern and propagation rate of Ca2+ waves in the same cells, with CCK8 stimulating the fastest and bombesin the slowest waves. More importantly, each agonist initiated the wave in a different region of the same cell. On the other hand, repetitive stimulation with the same agonist induced Ca2+ waves of the same pattern that were initiated from the same region of the cell. The agonist-specific Ca2+ signaling does not appear to be the result of coupling to different G proteins as infusion of an anti-Galphaq antibody into the cells through a patch pipette equally inhibited Ca2+ signaling by all agonists. Further evidence for compartmentalization of signaling complexes was developed in permeabilized cells. The time-dependent loss of Ca2+ signaling due to SLO permeabilization occurred in an agonist-specific manner in the sequence cabachol > bombesin > cholecystokinin. Signaling by all agonists could be completely restored with as low as 2 micro guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). At this low concentration GTPgammaS recoupled inositol 1,4,5-trisphosphate production and Ca2+ release, rather than enhancing phospholipase C activity. Priming of Ca2+ signaling by GTPgammaS was agonist-specific. Guanosine 5'-O-(thio)diphosphate (GDPbetaS) uncoupled the ability of signaling complexes to release Ca2+ much better than stimulating inositol 1,4,5-trisphosphate production. The uncoupling of Ca2+ signaling by GDPbetaS was also agonist-specific. The combined findings of agonist-specific initiation sites of the Ca2+ wave and differential access of guanine nucleotides to signaling complexes suggest spacial compartmentalization of Ca2+ signaling complexes. Each complex must include a receptor, G protein, and phospholipase C that are coupled to a specific portion of the Ca2+ pool.
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PMID:Spacial compartmentalization of Ca2+ signaling complexes in pancreatic acini. 879 36

Mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory neuropeptides involved in numerous physiologic processes, and have been implicated as autocrine and/or paracrine growth factors in human lung carcinoma. Three structurally and pharmacologically distinct bombesin receptor subtypes have been isolated and characterized: the gastrin releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The three receptors are structurally related, sharing about 50% amino acid identity. They are members of the G-protein coupled receptor superfamily with a seven predicted transmembrane segment topology characteristic of receptors in this family. The signal transduction pathway for GRP-R and NMB-R involves coupling to a pertussis-toxin insensitive G-protein, activation of phospholipase C (PLC), generation of inositol trisphosphate (IP3), release of intracellular calcium, and activation of protein kinase C. While all three bombesin receptors are activated by bombesin agonists, GRP-R, NMB-R, and BRS-3 have very different affinities for the mammalian bombesin-like peptides GRP and NMB, as well as bombesin receptor antagonists. The three bombesin receptor subtypes are expressed in an overlapping subset of human lung carcinoma cell lines. Any therapeutic strategy based on modulation of bombesin growth responses in human lung carcinoma would be well served to take into account the pharmacologic heterogeneity of the relevant receptors.
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PMID:Bombesin receptor structure and expression in human lung carcinoma cell lines. 880 6

The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+]i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 microM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+]i fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hyperpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca(2+)-ATPase of endoplasmic reticulum and by affecting Ca(2+)-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-cresol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i. It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin.
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PMID:Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells. 884 21

Gastrin-releasing peptide and other bombesin-like peptides stimulate secretion, cell proliferation, and smooth muscle contraction via a family of G protein-coupled receptors that activate phospholipase C. Second messenger formation by one of these receptors, called BR1, is rapidly desensitized after treatment of cells with either agonists or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether receptor phosphorylation was involved in BR1 desensitization, we generated antibodies to a peptide corresponding to a unique sequence within the COOH terminus of this receptor. One antibody (BR1-517) immunoprecipitated 60% of the solubilized [125I-Tyr4]bombesin/receptor complex prepared from either Swiss 3T3 fibroblasts or CHO-K1 cells transfected to express high levels of mouse BR1 (CHO-mBR1). Furthermore, immunoprecipitation of photoaffinity-labeled receptors yielded the expected 87-kDa radiolabeled band on gel electrophoresis. Phosphorylation of this immunoprecipitated receptor protein was markedly stimulated when [32P]orthophosphate-labeled Swiss 3T3 cells or CHO-mBR1 cells were treated with 100 nM bombesin for 5 min. 32PO4 incorporation into immunoprecipitated receptor was detectable after 2 min and maximal after 15 min of bombesin treatment. Phosphoamino acid analysis showed 32P labeling of serine and theonine but not tyrosine residues. Pretreatment of CHO-mBR1 cells with 100 nM TPA for 30 min also desensitized bombesin stimulation of inositol-1,4,5-trisphosphate formation. However, TPA did not increase 32PO4 incorporation into the immunoprecipitated receptor, although protein kinase C inhibition potentiated bombesin-induced receptor phosphorylation. Subsequent studies showed that TPA did stimulate receptor phosphorylation, but the antibody did not recognize this phosphorylated state of the receptor. Thus, TPA decreased the efficiency of receptor immunoprecipitation, and subsequent incubation of receptor with alkaline phosphatase reversed this TPA inhibition. The differential specificity of the antibody for various phosphorylated forms of BR1 demonstrates that agonist-induced and TPA-induced phosphorylations of the receptor occur at distinct sites.
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PMID:Agonist binding and protein kinase C activation stimulate phosphorylation of the gastrin-releasing peptide receptor at distinct sites. 886 15

The carboxyl terminus of the G protein alpha subunit is a key determinant of the fidelity of receptor activation. We have previously shown that the Gq alpha subunit (alpha q) can be made to respond to alpha i-coupled receptors by replacing its carboxyl terminus with the corresponding alpha i2, alpha o, alpha z residues. We now extend these findings in three ways: 1) carboxyl-terminal mutations of alpha q/alpha i chimeras show that the critical amino acids are in the -3 and -4 positions, 2) exchange of carboxyl termini between alpha q and alpha z allows activation by receptors appropriate to the carboxyl-terminal residues, and 3) we identify receptors that either do or do not activate the expected carboxyl-terminal chimeras (alpha q/alpha i, alpha q/alpha s, alpha s/alpha q). Replacement of the five carboxyl-terminal amino acids of alpha q with the alpha s sequence permitted an alpha s-coupled receptor (the V2 vasopressin receptor but not the beta 2-adrenergic receptor) to stimulate phospholipase C. Replacement of the five carboxyl-terminal amino acids of alpha z with residues of alpha q permitted certain alpha q-coupled receptors (bombesin and V1a vasopressin receptors but not the oxytocin receptor) to stimulate adenylyl cyclase. Thus, the relative importance of the G alpha carboxyl terminus in permitting coupling to a new receptor depends on the receptor with which it is paired. These studies refine our understanding and provide new tools with which to study the fidelity of receptor/G alpha activation.
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PMID:Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. 886 34

Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.
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PMID:CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes. 903 86

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.
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PMID:RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. 911 54


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