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)

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.
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PMID:5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells. 1037 10

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments (86)Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased (86)Rb efflux rate from 0.015 min(-1) to a maximal of 0. 049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba(2+) (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased (86)Rb efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin.
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PMID:Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes. 1041 26

In a previous publication we provided evidence of a novel neuronal pathway for the control of GnRH secretion by bradykinin. The action of bradykinin appeared to be exerted through the bradykinin B2 receptor. In this study we demonstrated that the bradykinin B2 receptor is densely localized in the arcuate nucleus, median eminence, organum vasculosum of the lamina terminalis, and preoptic area, regions known to be critical for the control of GnRH secretion. To determine the mechanism of action of bradykinin in stimulating GnRH release, we used immortalized GnRH (GT1-7) cells in vitro. Bradykinin stimulation of GnRH secretion from GT1-7 cells appears to involve activation of the phospholipase C signaling pathway and mobilization of extracellular and intracellular calcium stores. Evidence to support this contention was derived from the observations that incubation of the phospholipase C inhibitor, U-73122 with bradykinin, blocked the ability of bradykinin to stimulate release from GT1-7 cells. This effect was specific, as a nitric oxide synthase inhibitor and a cyclooxygenase inhibitor were found to have no effect on bradykinin-induced GnRH secretion, suggesting that nitric oxide and PGs do not mediate bradykinin effects. Pertussis toxin also had no effect on bradykinin action. This suggests that the bradykinin B2 receptor may be coupled to a pertussis toxin-insensitive G protein in GT1-7 cells. With respect to calcium involvement in bradykinin action, fura-2 calcium indicator studies revealed that bradykinin can rapidly increase intracellular Ca2+ levels in GT1-7 cells. A role for intracellular Ca2+ in bradykinin action was further suggested by the finding that an intracellular calcium chelator, 1,2-bis(O-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, significantly attenuated the effects of bradykinin on GnRH release. The elevation of intracellular calcium by bradykinin appears to be due to mobilization of calcium from the endoplasmic reticulum, as incubation of the Ca2+-adenosine triphosphatase inhibitor thapsigarin, which depletes endoplasmic reticulum Ca2+ stores, significantly attenuated bradykinin action on GnRH release. Extracellular calcium may also be involved in bradykinin action, as the L-type Ca2+ channel blockers verapamil and nifedipine had no effect on bradykinin-induced GnRH release, whereas the nonselective Ca2+ channel blocker, nickel chloride, attenuated bradykinin-induced GnRH release. Taken as a whole, these studies demonstrate that the bradykinin B2 receptor is densely localized in key hypothalamic nuclei responsible for regulation of GnRH release, and that the mechanism of bradykinin stimulation of GnRH secretion involves activation of the phospholipase C signaling pathway, with a critical role implicated for calcium in bradykinin action in GT1-7 cells.
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PMID:Bradykinin receptor localization and cell signaling pathways used by bradykinin in the regulation of gonadotropin-releasing hormone secretion. 1049 24

Exocrine secretions proceed in two phases which can be studied individually in submandibular glands. We have investigated the response to neuropeptides and purinergic agonists of rat submandibular glands. Pituitary Adenylate Cyclase Activating Peptide (PACAP), an analog of VIP increased the intracellular concentration of cyclic AMP in acinar cells. PACAP also stimulated the activity of the Na(+)-K(+)-2Cl(-)-cotransporter. Extracellular ATP increased the [Ca2+]i in ductal cells. Two distinct receptors were involved in this response. A metabotropic purinergic receptor of the P2Y1 type raised the cellular concentration of IP3 after activating a phospholipase C. The second component of the purinergic response involved an ionotropic P2X7 receptor. After binding an agonist, this receptor formed a non-specific cation channel permeant to calcium and manganese, highly sensitive to inhibition by nickel. Two phospholipases A2 were activated following the occupancy of this receptor. The calcium-independent enzyme triggered kallikrein secretion in response to extracellular ATP. In conclusion, neuropeptides and purinergic agonists activate the acinar and ductal phases of the salivary secretion and are therefore promising candidates for the development of new sialagogues for therapeutic use.
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PMID:[Value of new agonists of the acinar and ductal phases of exocrine secretions]. 1099 84

Phosphatidylinositol 4,5-bisphosphate (PIP2) affects profoundly several cardiac ion channels and transporters, and studies of PIP2-sensitive currents in excised patches suggest that PIP2 can be synthesized and broken down within 30 s. To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and PIP(2) levels actually change in intact heart, we used a new, nonradioactive HPLC method to quantify anionic phospholipids. Total PIP and PIP2 levels (10-30 micromol/kg wet weight) do not change, or even increase, with activation of Galpha(q)/phospholipase C (PLC)-dependent pathways by carbachol (50 microM), phenylephrine (50 microM), and endothelin-1 (0.3 microM). Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1microM) both cause 30% reduction of PIP2 in ventricles, suggesting that diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac PIP2. PIP2, but not PIP, increases reversibly by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig left atria; the increase is blocked by nickel (2 mM). Both PIP and PIP2 increase within 3 min in hypertonic solutions, roughly in proportion to osmolarity, and similar effects occur in multiple cell lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism might be sensitive to membrane tension, per se.
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PMID:Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions. 1205 91

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca(2+) and did not involve intracellular Ca(2+) mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A(2) (PLA(2)) inhibitor ONO-RS-082 and partially attenuated by the selective Ca(2+)-dependent cytosolic PLA(2) inhibitor, arachidonyl trifluoromethylketone (AACOCF(3)), or by bromoenol lactone, an inhibitor of Ca(2+)-independent cytosolic PLA(2). Magnesium, which decreases the concentration of ATP(4-), and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X(7) purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X(7) receptor antagonist. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca(2+) influx, cytosolic PLA(2), and PKC. Also, the data suggest an involvement of P2X(7) receptors in PLD-2 stimulation by ATP.
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PMID:Activation of phospholipase D-2 by P2X(7) agonists in rat submandibular gland acini. 1217 68

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP(3)). The PLC beta isoforms of PLCs are activated by G proteins after hormone or neurotransmitter stimulation of G protein-coupled receptors (GPCR). PLC epsilon is a recently identified PLC isoform that is activated by Ras and G beta gamma subunit although the physiological role of this enzyme is not well understood. Methods for purification of PLC beta and PLC epsilon from Sf9 cells are described. In the case of hexahistidine (6-His)-tagged PLC beta the purification involves two steps, affinity chromatography with Ni-NTA agarose followed by heparin Sepharose chromatography. 6-His-tagged PLC epsilon can be purified in a single step with nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity chromatography.
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PMID:Purification of phospholipase C beta and phospholipase C epsilon from Sf9 cells. 1450 Oct 38

Inhibition of Ca2+ uptake by the sarcoplasmic reticulum decreases cytosolic Ca2+ clearance and also triggers Ca2+ influx in response to Ca2+ store depletion. The role of extracellular Ca2+ in the contractures evoked by cyclo-piazonic acid (CPA) and thapsigargin (TG), Ca2+ pump inhibitors, was assessed in mouse diaphragm. At 3-100 microM, CPA elicited a rapid-onset contracture followed by a large elevation of muscle tone, which corresponded temporally to the monophasic slow contracture evoked by TG (1-30 microM). Irrespective of the differences in profiles, contractures were prevented and inhibited by the removal of extracellular Ca2+, but not by nicardipine and SK&F96365, blockers of voltage-gated (L-type) and receptor-operated Ca2+ channels. Mn2+ and Ni2+ preferentially depressed the fast-phase contracture, whereas long-term pretreatment with LY294002, U73122, and 2-aminoethoxydiphenylborance, inhibitors of phosphatidylinositol kinase, phospholipase C, and inositol trisphosphate receptors, suppressed the slow-phase contracture. When contracture was inhibited, the twitch response remained augmented and prolonged by CPA and TG, indicating that the inhibition was not due to malfunction of the contractile apparatus. For preparations incubated in Ca2+-free medium containing CPA, a monophasic fast upstroke of muscle tone developed as extracellular Ca2+ was restored. The results suggest that the bimodal contracture induced by CPA is mediated by the recruitment of distinct Mn2+- and U73122-sensitive Ca2+ entries. The ongoing two-component Ca2+ entries might merge if the muscle preparation was preconditioned with CPA in Ca2+-free medium to deplete cellular Ca2+ stores.
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PMID:Dependence of cyclopiazonic-acid-induced muscle contractures on extracellular Ca2+. 1471 28

Ca2+ transfer across the syncytiotrophoblast (ST) of the human placenta is essential for normal fetal development. However, the nature of Ca2+ conductance in the ST and the mechanisms by which it is regulated are poorly understood. With the major signal transduction pathway of endothelin-1 (ET1) acting via phospholipase C (PLC) and Ca2+, we used ET1 to analyse the nature of Ca2+ channels on cultured trophoblastic cells by means of cytofluorimetric analysis using the ratiometric Ca2+ indicator Indo-1. Results indicate that ET1 (10(-7) M) stimulates a biphasic (transient and sustained) increase in [Ca2+]i in trophoblastic cells. This response is mediated by the endothelin receptor B (ETB) coupled to PLC, since treatment with BQ788 (10(-6) M) or U73122 (2 microM) totally abolished the response. Persistence of the rapid transient rise in [Ca2+]i in Ca2+-free extracellular medium confirms the release of Ca2+ from intracellular stores in response to ET1 stimulation. Furthermore, abolition of the sustained increase in [Ca2+]i in Ca2+-free extracellular medium argues in favour of the entry of Ca2+ during the plateau phase. Abolition of this plateau phase by Ni2+ (1 mM) in the presence of extracellular Ca2+ confirmed the existence of an ET1-induced Ca2+ entry. No evidence for the presence of voltage-operated channels was demonstrated during ET1 action since nifedipine (10(-6) M) did not reduce the Ca2+ response and depolarization with a hyper-potassium solution had no effect. Pharmacological studies using the imidazole derivatives SK&F96365 (30 microM) and LOE 908 (10 microM) partially inhibited the ET1-evoked Ca2+ response, thus providing evidence for the presence of both store-operated Ca2+ channels and non-selective cationic channels in the human ST.
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PMID:Calcium channels activated by endothelin-1 in human trophoblast. 1535 10

Lysophosphatidic acid (LPA) is a bioactive lipid, which is structurally similar to sphingosine 1-phosphate (S1P) and which can mobilize Ca2+ in multiple cell types. We recently showed that S1P induces Ca2+ entry directly through store-operated Ca2+ entry (SOCE) channels in human polymorphonuclear neutrophils (PMN). We therefore examined the mechanisms by which LPA induces intracellular Ca2+ mobilization in PMN. External application of low micromolar LPA caused dose-dependent Ca2+ influx without releasing Ca2+ stores, whereas G-protein-coupled (GPC) LPA receptors respond to nanomolar LPA. Additive Ca2+ influx by LPA compared with 100 nM ionomycin-induced Ca2+ influx suggests that LPA-induced Ca2+ influx does not pass through SOCE channels. Ca2+ influx was resistant to inhibition of Gi/o by pertussis toxin, of phospholipase C by U73122, and of G12/13/Rho by Y27632, all demonstrating GPC receptor independence. This Ca2+ influx was inhibited by Gd3+, La3+, Zn2+, or MRS1845 but not by Ni2+ or the sphingosine kinase inhibitor dimethylsphingosine. In addition, we found that LPA has no effect on neutrophil chemotaxis; however, it has stimulatory effects on neutrophil respiratory burst in a dose-response manner. These findings suggest that LPA-induced Ca2+ influx in PMN occurs through a mechanism other than SOCE channels, independent of Ca2+ store-depletion and S1P synthesis, and that the characteristics of LPA-induced Ca2+ influx are similar to those of S1P-induced influx in terms of sensitivity to inorganic inhibitors. Unlike S1P, LPA has stimulatory effects on neutrophil respiratory burst.
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PMID:Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils. 1552 18


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