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)

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.
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PMID:Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. 199 39

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.
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PMID:Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization. 199 9

Renomedullary interstitial cells cultured from the Dahl salt-resistant rat have higher levels of basal cytosolic calcium and prostaglandin E2 and are more responsive to vasopressin than interstitial cells from the Dahl salt-sensitive rat. We examined the potential role of inositol phospholipid hydrolysis in mediating these differences. Vasopressin-induced increases in labeled inositol phosphates were enhanced in renomedullary interstitial cells from Dahl salt-resistant compared with those from salt-sensitive rats. Addition of neomycin reduced basal production of labeled inositol phosphates and abolished the increase in inositol phosphates induced by vasopressin. Neomycin also prevented the peak decline pattern in cytosolic Ca2+ seen with vasopressin but did not influence basal cytosolic Ca2+. In the presence of neomycin, vasopressin induced a modest but prolonged increase in cytosolic calcium. In contrast to its marked effects on inositol phosphate production, neomycin was without effect on basal or vasopressin-responsive prostaglandin E2 production. Moreover, basal and vasopressin-induced increases in cytosolic Ca2+ remained higher in renomedullary interstitial cells from Dahl salt-resistant versus those from salt-sensitive rats exposed to neomycin. The results do not support a requirement for phospholipase C-induced inositol phospholipid hydrolysis in the mediation of vasopressin actions on prostaglandin E2 production by renomedullary interstitial cells and imply that the differences in cytosolic Ca2+ and prostaglandin E2 seen in these two cell lines are not related to differences in inositol phospholipid metabolism.
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PMID:Calcium and prostaglandin E2 in renomedullary interstitial cells. 199 61

The effects of angiotensin II (AII), acetylcholine and vasopressin on the intracellular concentration of Ca2+ have been little studied in adrenocortical cells from the zona fasciculata/reticularis (ZFR). Primary cultures of bovine ZFR cells maintained in suspension cultured for 72 h produce cortisol in response to AII (0.1 microM), acetylcholine (0.1 mM) and vasopressin (1 microM). This response is accompanied by a breakdown of membrane phosphoinositides from [3H]inositol-prelabelled cells. Using cells loaded with the Ca2+ indicator fura-2, the intracellular concentration of Ca2+ was measured in response to increasing doses of all three agonists and found to increase in a graded fashion in each case. The basal intracellular concentration of Ca2+ was 75 +/- 3 nM (mean +/- S.E.M., n = 52), rising to a maximum 1.82 +/- 0.14-fold (n = 6) for AII (0.1 microM), 1.35 +/- 0.05-fold (n = 7) for acetylcholine (0.1 mM) and 1.27 +/- 0.10-fold (n = 6) for vasopressin (1 microM). In the case of AII and acetylcholine, agonists were added sequentially in medium of normal extracellular Ca2+ concentration (1.2 mM) or in medium in which the Ca2+ concentration was buffered to approximate to the intracellular concentration of Ca2+ (75-100 nM). Evidence was thereby obtained that both AII and acetylcholine mobilize a common intracellular pool of Ca2+. Our findings suggest that these three agonists, all of which stimulate phospholipase C, increase intracellular Ca2+ through a mechanism which depends, at least in part, on the release of Ca2+ from a common intracellular pool.
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PMID:Dose-dependent effects of angiotensin II, acetylcholine and vasopressin on the cytosolic concentration of Ca2+ in suspension primary cultures of zona fasciculata/reticularis cells from bovine adrenal cortex. 204 45

Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 mM to 8 mM. If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]i spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2(+)-stimulation of phospholipase C production of Ins(1,4,5)P3.
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PMID:Imaging and manipulation of cytosolic ions and messengers during cell activation. 208 12

Rat hepatocytes were maintained in primary monolayer culture for 24 h in the presence of serum. Treatment of hepatocytes with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for 5-15 min increased membrane-associated protein kinase C activity and concomitantly decreased soluble activity. Membrane protein kinase C activity returned to basal values within 1 h then decreased by more than 50% within 2 h. Prolonged (2-18 h) incubation with PMA did not further decrease protein kinase C activity. Pretreatment of hepatocytes with PMA for 5-15 min had little effect on the subsequent actions of 100 nM vasopressin but abolished the stimulation of inositol phosphate accumulation by 3 nM vasopressin and 20 microM norepinephrine. Long-term exposure (2-18 h) of hepatocytes to 1 microM PMA actually enhanced the effects of vasopressin and 20 microM norepinephrine. The stimulation by norepinephrine (20 microM) of inositol phosphate accumulation was abolished by the alpha 1-adrenergic antagonist prazosin (1 microM), whereas the beta-adrenergic antagonist propranolol (30 microM) had little effect. Addition of 8Br-cAMP (100 microM) or glucagon (10 nM) for 5 min or 8 h had no significant effect alone, but enhanced the subsequent vasopressin stimulation of inositol phosphate accumulation. There was no effect of 8Br-cAMP or glucagon on norepinephrine stimulation of phosphoinositide breakdown. These data indicate that the stimulation of phospholipase C activity in rat hepatocytes by 3 nM vasopressin is enhanced by cyclic AMP-dependent kinase but inhibited by protein kinase C. In contrast, down regulation of protein kinase C markedly enhanced the maximal phosphoinositide response due to both vasopressin and norepinephrine.
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PMID:Vasopressin and norepinephrine stimulation of inositol phosphate accumulation in rat hepatocytes are modified differently by protein f1nase C and protein kinase A. 210 81

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.
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PMID:Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells. 211 56

The hormonal control of glycogen synthase and phosphorylase interconversion was investigated in hepatocytes isolated from lean and genetically obese (fa/fa) rats. In cells from obese animals, the inactivation of synthase by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), phospholipase C, vasopressin and the alpha 1-adrenergic agonist phenylephrine was markedly impaired, and the property of PMA to counteract phosphorylase activation by phenylephrine was attenuated. The maximal response of phosphorylase activation to phenylephrine and vasopressin was increased in obese-rat hepatocytes, but the sensitivity to these hormones was similar to that in lean-rat hepatocytes. These observations indicate that the defect in protein kinase C that we reported previously in heart of insulin-resistant fa/fa rats [van de Werve, Zaninetti, Lang, Vallotton & Jeanrenaud (1987) Diabetes 36, 310-319] is probably also expressed in liver.
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PMID:Altered regulation of glycogen metabolism by vasopressin and phenylephrine in hepatocytes from insulin-resistant obese (fa/fa) rats. Role of protein kinase C. 211 21

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.
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PMID:Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor. 214 82

In order to clarify the mechanism(s) by which cyclic GMP inhibits the generation of inositol phosphates in rat aorta segments and cultured bovine aortic smooth muscle cells, we studied phosphoinositide hydrolysis and GTPase activity in homogenates and membrane preparations of cultured bovine aortic smooth muscle cells. Pretreatment of homogenate preparations with cyclic GMP plus ATP did not inhibit [8-arginine, 3H] vasopressin (AVP) binding, but resulted in a total suppression of the AVP-induced GTPase activation. The pretreatment with cyclic GMP and ATP also inhibited the formation of inositol phosphates induced by AVP in the presence of low concentrations of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), or by high concentrations of GTP gamma S alone. However, the formation of inositol phosphates by high concentrations of Ca2+ alone was not blocked. These results suggest that the ability of cyclic GMP to inhibit phosphoinositide hydrolysis results from an inhibition of a guanine nucleotide regulatory protein activation, and the interaction between guanine nucleotide regulatory protein and phospholipase C. While the precise site of this inhibition is not presently known, the inhibition by cyclic GMP is dependent upon the addition of ATP and probably entails a phosphorylation event since adenylylimidodiphosphate can not substitute for the ATP requirement.
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PMID:Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells. 215 23


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