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

---

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

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
...
PMID:Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. 131 10

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.
...
PMID:Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. 131 81

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate. 131 12

Fructose-1,6-diphosphate (FDP) is a physiological product which exhibits pharmacological properties. This study shows that FDP (1-3 mM) inhibits platelet aggregation induced by the agonists thrombin, vasopressin, platelet activating factor, ADP, adrenaline, arachidonate and the stable thromboxane analogue U 44069. Thrombin-promoted ATP secretion and cytosolic Ca2+ rise are also drastically inhibited by FDP, which decreases, although to a lesser extent, the protein kinase C-dependent phosphorylation of the 47 kDa protein. The inhibition on thrombin-induced aggregation is shared, albeit less efficiently, by glucose-1,6-diphosphate and fructose-2,6-diphosphate but not by other phosphorylated monosaccharides (fructose-1:2 cyclic,6-diphosphate, glucose-1- and glucose-6-phosphate, fructose-1- and fructose-6-phosphate, mannose-6-phosphate and 5-phosphoryl ribose-1-pyrophosphate). FDP does not affect platelet activation induced by the protein kinase C activators dioctanoylglycerol or phorbol 12-myristate 13-acetate. No increase of cAMP concentration is observed in FDP-treated platelets. Altogether, these results indicate that FDP inhibits platelet activation at a level preceding phospholipase C. The data are consistent with a general inhibitory action of FDP on signal transmission.
...
PMID:Fructose-1,6-diphosphate inhibits platelet activation. 131 5

Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation.
...
PMID:Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets. 132 53

We have studied the cellular mechanism responsible for induction of preproendothelin (preproET)-1 mRNA and release of ET-1 by thrombin in cultured bovine endothelial cells (ECs). Thrombin induced an immediate and dose-dependent formation of inositol-1,4,5-trisphosphate (IP3) with a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). The thrombin-induced ET-1 release was abolished either by a phospholipase C inhibitor, a protein kinase C (PKC) inhibitor, or an intracellular Ca(2+)-chelator, whereas a Ca(2+)-channel antagonist was ineffective. A selective thrombin inhibitor (argatroban) decreased IP3 formation and the increase in [Ca2+]i and ET-1 release stimulated by thrombin. Northern blot analysis revealed that thrombin-induced expression of preproET-1 mRNA was inhibited completely by a PKC inhibitor and partially by argatroban. These data suggest that thrombin is involved in the mechanism of preproET-1 mRNA expression and subsequent ET-1 release, possibly through activation PKC and mobilization of intracellular Ca2+ resulting from the receptor-mediated phosphoinositide breakdown in ECs.
...
PMID:Cellular mechanism of thrombin on endothelin-1 biosynthesis and release in bovine endothelial cell. 147 6

Agonists-induced platelet shape change, inositol metabolism, and Ca mobilization were investigated in patients with various platelet dysfunctions. The platelet shape change determined by our method revealed that arachidonate-induced platelet shape change was completely defective in patients with cyclo-oxygenase (CO) deficiency (A). STA2-induced platelet shape change was also defective in one of five patients with impaired aggregation to STA2 (B). Thrombin-induced platelet shape change was weak in patients with Bernard-Soulier syndrome. In patient with Hermansky-Pudlak syndrome, the platelets did not respond normally to STA2, arachidonate or PMA. These findings suggested that the determinations of platelet shape change by our method was useful in diagnosing platelet dysfunctions. Inositol metabolism and Ca mobilization in response to thrombin, STA2, or NaF were also investigated in patient A,B, and impaired aggregation to A23187 in patient C. The responses were normal in patient A, suggested that CO activity did not affect them. Inositol metabolism was also normal in patient C, although Ca mobilization in response to A23187 was delayed, and that in response to thrombin was defective in the absence of extracellular Ca2+. This suggests that the patient's platelets have a defective IP3-induced Ca mobilization pathway. STA2 selectively failed to induce IP3 formation and Ca mobilization in patient B, although 3H-labelled thromboxane ligand (3H-U46619) bound to the patient's platelets, normally. These findings suggested that the patient's platelets have a defect in postreceptor signal transduction, especially thromboxane receptor-mediated phospholipase C activation pathway.
...
PMID:[Analysis of platelet shape change, inositol metabolism, and Ca mobilization in patients with platelet dysfunction]. 151 80

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.
...
PMID:Thrombin signal transduction mechanisms in human glomerular epithelial cells. 153 79

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.
...
PMID:Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells. 154 34

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.
...
PMID:Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells. 169 2


1 2 3 4 5 6 7 8 9 10 Next >>

---