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)

Endothelin (ET) exerts various biological actions in mesangial cells, including stimulation of proliferation, contraction and phospholipase C activation. We investigated the presence of specific ET receptors on cultured rat mesangial cells, incubating the cells in the presence of [125I]ET-1 both at 22 and 4 degrees C. ET binding was time- and temperature-dependent and achieved equilibrium at 2 hr at 22 degrees C and at 5 hr at 4 degrees C. Scatchard analyses of equilibrium saturation curves with [125I]ET-1 and homologous competition curves revealed the presence of a single class of high-affinity binding sites (Kd = 31.4 +/- 7.08 pM). Heterologous competition experiments with ET-3 and sarafotoxin, however, indicated the presence of two binding sites for ET-related peptides in mesangial cells with a Kd for ET-3 of 41.5 +/- 19.2 and of 374 +/- 38.5 nM. Nifedipine and arginine-vasopressin failed to compete for ET binding sites. Preincubation of the cells with 1 nM ET-1 caused a dramatic decrease in ET binding capacity (from 0.5-0.02 fmol/100,000 cells) without affecting the Kd for the receptors (38 pM). ET receptor down regulation was not prevented by protein kinase C inhibition with H-7 and sangiovamycin, or after down regulation of protein kinase C induced by 24-hr preincubation with phorbol myristate acetate. ET receptor down regulation also exerts functional effects, as we found a decrease in intracellular-free calcium response to ET-1 after long-term preincubation with the same agonist. Our results are consistent with the presence of two binding sites for ET in rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endothelin binding and receptor down regulation in rat glomerular mesangial cells. 184 3

Previous autoradiographic studies have delineated the renal medullas the predominant site of renal endothelin (ET) receptors. Accordingly, cultured rat renal medullary interstitial cells (RMICs) were studied as a target tissue for ET action. Scatchard analysis revealed presence of a single class of high-affinity receptor sites (Kd, 57 +/- 10 pM; receptor density, 749 +/- 124 fmol/mg protein). Relative potency order for displacing 125I-ET-1 was ET-1 greater than ET-2 greater than sarafotoxin greater than big endothelin (human) = big endothelin (porcine). ET-3, unrelated pressor substances, vasodilators, Ca2+ channel antagonists, atrial natriuretic factor, GTP, and GppNHp did not inhibit binding. Challenge of monolayers with ET-1 evoked a biphasic elevation in cytosolic free Ca2+ concentration [Ca2+]i). Initial transient rise in [Ca2+]i observed in absence of extracellular Ca2+ and accumulation of inositol trisphosphate (IP3) was consistent with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Half-maximal activation concentration of ET-1 for the process was 0.5 and 1 nM for [Ca2+]i and IP3, respectively. The late sustained phase in [Ca2+]i elevation was completely blocked by Ni2+, unperturbed by nimodipine, and accompanied by influx of Mn2+, indicating presence of receptor-operated Ca2+ channels. Ca2+ channel opening was detected at 10(-16) MET-1, whereas greater than 10(-12) M agonist was required to mobilize Ca2+ from intracellular stores and/or stimulate phosphoinositol hydrolysis, indicating that ET activation of PI-PLC and Ca2+ channel opening were independent events. ET-1 markedly stimulated prostaglandin E2 synthesis in a concentration-dependent manner that paralleled PI-PLC activation and mobilization of [Ca2+]i. In summary, cultured rat RMICs possess ET receptors that are linked to PI-PLC, Ca2+ channels, and perhaps phospholipase A2.
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PMID:Characterization of endothelin 1 receptor and signal transduction mechanisms in rat medullary interstitial cells. 184 65

Endothelin (ET)-1 is a powerful vasoconstrictor known to be produced and secreted by endothelial cells lining large vessels. Because ET-1 stimulates glomerular mesangial cell contraction, glomerular capillary endothelial cells (GEN), normally situated in close apposition to mesangial cells, were examined for potential ET expression and secretion. Cultured bovine GEN released ET in a time-dependent fashion. ET secretion was significantly stimulated by bradykinin, an agonist known to activate phospholipase C in these cells. Preproendothelin 1 (preproET-1) mRNA levels in GEN rose in a biphasic manner on stimulation with bradykinin. The early increments (at 30 min) were not dependent on new protein synthesis, whereas the late rise (6 h after addition of bradykinin) appeared to be protein synthesis dependent. Neither early or late bradykinin-stimulated preproET-1 mRNA expression in glomerular endothelial cells was due to inhibition of mRNA breakdown. Both phases of preproET-1 mRNA expression were observed with other glomerular endothelial cell calcium-mobilizing agonists, namely thrombin, and were mimicked by the calcium ionophore ionomycin. By contrast, the protein kinase C activator phorbol myristate acetate only enhanced preproET-1 mRNA expression at 30 min and suppressed expression thereafter. It is concluded that GEN have the potential to express and secrete ET-1 in a phospholipase C-regulated fashion. Furthermore, because glomerular mesangial cells respond to this peptide, the findings raise the possibility of paracrine regulation of mesangial cell tone by glomerular endothelial cell-derived ET-1.
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PMID:Regulated expression of endothelin 1 in glomerular capillary endothelial cells. 185 92

The effect of porcine endothelin (endothelin 1) on rings of isolated rabbit pulmonary vein was studied using tissue bath techniques. Endothelin was found to be a potent constrictor of these vessels, producing concentration-dependent contractions with an EC50 value of 3.2 +/- 0.6 x 10(-9) M. Contractions were not significantly affected by the Ca2+ channel antagonists verapamil, nifedipine, or nicardipine. Contractions were greatly attenuated by 3 mM LaCl3 (85.8 +/- 8.0% relaxation) and were diminished in Ca(2+)-free media (51 +/- 9% of control). The protein kinase C (PKC) inhibitor H7 (20 microM) potently and reversibly inhibited endothelin-induced contractions by 82 +/- 6% when used as post-treatment. Incubation of tissues with 20 microM H7 did not significantly affect either the strength of contractions induced with endothelin or the time required for contraction to reach plateau, but these contractions were poorly sustained compared to controls. The phospholipase C (PLC) inhibitor neomycin (10 mM) inhibited endothelin-induced contractions and it also affected the rate of force development. The phospholipase A2 (PLA2) inhibitor quinacrine had no significant effect on endothelin-induced contractions. Extracellular Ca2+ appears to enter the cell via non-potential dependent channels that can be blocked by La3+. Moreover, the potent vasoconstrictor properties of endothelin on rabbit pulmonary veins involves activation of both PLC and PKC, but not PLA2. PKC is intimately associated with maintaining the tonic phase of smooth muscle contraction.
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PMID:Signal transduction in endothelin-induced contraction of rabbit pulmonary vein. 213 7

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.
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PMID:Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells. 215 94

Endothelin (ET-1) receptors were studied in the C-6 glia cell line. ET-1 binds to C-6 cells in a temperature- and time-dependent manner, with an apparent Kd of 1.16 +/- 0.07 10(-10) M and a Bmax of 96,500 +/- 6000 sites/cell (mean +/- SEM, n = 27). Stimulation of protein kinase C (PKC) with the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA) resulted in a decrease in the number of receptors in a dose-dependent manner. Inhibition of PKC with H-7 eliminated the effect of PMA on the reduction of binding sites. Treatment with exogenous 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), release of endogenous DAG with phospholipase C, and inhibition of the metabolism of DAG with the diacylglycerol kinase inhibitor R 59022 also resulted in a decrease in the number of receptors. The effect of these agents was inhibited by H-7. ET-1-mediated down-regulation of receptors was also demonstrated, but the down-regulation was not affected by H-7 or by depletion of cellular PKC with chronic, high dose of PMA. Internalization constants of ET-1-receptor complex was also measured according to the model of Wiley and Cunningham (Cell 25 (1981) 433). PMA- and ET-1-mediated down-regulation of receptors was associated with an increase in the endocytosis constant for the hormone-receptor complex and a decrease in the rate of insertion of receptor into the plasma membrane. PMA, but not ET-1, increased the rate of endocytosis of unoccupied receptors. Radioiodinated ET-1 was crosslinked to the receptor after binding, extracted and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A band at 66 kDa was obtained. These studies show that ET-1 and PKC activation produce down-regulation of ET-1 membrane receptors and that ET-1-mediated down-regulation probably does not involve the activation of PKC.
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PMID:ET-1 receptors in C-6 cells: homologous down-regulation and modulation by protein kinase C. 216 63

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.
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PMID:Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. 217 94

The activities of three isoforms of the endothelin (ET) family peptides, ET-1, ET-2 and ET-3, were studied in cultured osteoblastic cells from neonatal rat calvariae. All three isoforms induce stimulation of DNA synthesis and reductions in cellular alkaline phosphatase activity in a dose-dependent manner with the rank order of potency: ET-1 congruent to ET-2 greater than ET-3. The 125I-labeled ET binding and affinity-cross linking experiments show the presence of a single class of the ET binding sites with a more than 10-fold higher affinity for ET-1 and ET-2 as compared to ET-3. The endothelins dose-dependently stimulate the production of inositol phosphates and induce mobilization of Ca2+ with the similar relative potency to that for the receptor binding. These results indicate that osteoblastic cells possess the endothelin receptor with a high affinity for ET-1 and ET-2 that is coupled to phospholipase C, and that the endothelins modulate cellular functions via this receptor.
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PMID:The effects of the endothelin family peptides on cultured osteoblastic cells from rat calvariae. 220 4

We have shown previously that in liver, endothelin (ET) binding to plasma membranes causes a rise in cytosolic calcium and activation of glycogenolysis. Here we show that the calcium extrusion pump in liver plasma membranes is inhibited by ET peptides, with ET-1 > or = ET-3 = sarafotoxin S6C-inhibition of the system being potentiated by GTP gamma S. Also, ET-1 stimulates PIP2 hydrolysis in liver plasma membranes in a guanine nucleotide-dependent manner, with ET-1 > or = ET-3 = sarafotoxin S6C. In order to determine the nature of G protein(s) coupling of the ETB receptor to both effectors, antibodies against the C-terminus of different G-protein alpha-subunits were used. Antibodies reactive with Gs alpha blocked ET-1 inhibition of the calcium pump, but they had no effect on ET-1 stimulation of PIP2 hydrolysis. Antibodies reactive with Gq alpha dose-dependently antagonized stimulation of PIP2 breakdown by ET-1 without affecting ET-1 inhibition of the calcium pump. Antibodies reactive with Gi1 alpha/Gi2 alpha had no effect on both systems. We conclude that the calcium signal induced by endothelins in hepatocytes may be consequent to both an activation of phospholipase C and inhibition of the calcium pump, both effectors being coupled to the ETB receptor by different G proteins, Gq and Gs, respectively.
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PMID:Endothelin inhibits the calcium pump and stimulates phosphoinositide phospholipase C in liver plasma membranes via two different G proteins, Gs and Gq. 750 31

Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10(-8) M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10 microM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10(-4) M) induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10 microM) treatment of cells did not affect either basal or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 microM ethylene glycol bis(beta-aminoethyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 microM) or Ca(2+)-channel agonist Bay K8644 (1 microM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTP gamma S (30 microM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 microM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTP gamma S, is likely be a Ca2+ dependent process.
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PMID:Calcium and the endothelin-1 and alpha 1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes. 752 83


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