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)

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.
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PMID:A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts. 149 Nov 4

Through kinetic analysis, the relationships between the glucose-6-phosphatase system and constituent phospholipids were studied in rat liver microsomes. When phosphoglycerides such as phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane were hydrolyzed by phospholipase C of C. perfringens, the activities of glucose-6-P phosphohydrolase and glucose-6-P:glucose phosphotransferase both decreased with or without subsequent exposure to taurocholate. In these cases, the Michaelis constants (Km) for glucose-6-P were increased, concomitant with the decrease in the maximal velocities (Vmax) for glucose-6-P hydrolysis. On exposure to taurocholate, the apparent Km for glucose of phosphotransferase was decreased. When phosphatidylinositol was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the activities of phosphohydrolase and phosphotransferase were both decreased on exposure to taurocholate. In this case, the value of Vmax of phosphohydrolase was decreased and that of Km for glucose-6-P was slightly decreased, while the apparent Km for glucose of phosphotransferase was increased. Without exposure to detergent, the activities of phosphohydrolase and phosphotransferase both decreased at glucose-6-P concentrations higher than 10 mM. However, at a concentration lower than 1 mM, the activity of phosphohydrolase became higher than that of the control, and Vmax and Km for glucose-6-P were decreased. A similar tendency was also observed in microsomes where membranous phosphatidylinositol was hydrolyzed, when they were treated with DIDS (an anion-transport inhibitor). From these results, it is concluded that the activity of glucose-6-phosphatase is greatly influenced by changes of the phospholipids on the microsomal membrane, and the activity of glucose-6-P translocase is stimulated by the breakdown of phosphatidylinositol.
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PMID:Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on kinetic properties of the glucose-6-phosphatase system in rat liver microsomes. 630 99

In C6 glioma cells, extracellular ATP and other nucleotide analogs stimulated phosphoinositide (PI) breakdown and inhibited isoproterenol-induced cyclic AMP (cAMP) accumulation. The rank orders of potencies of 15 nucleotide analogs for both responses were clearly different. ATP and adenosine 5'-O-(3-thiotriphosphate) are the most potent agonists for stimulating PI hydrolysis; 2-methylthio-ATP (2-MeSATP) is the most potent agonist for inhibiting cAMP accumulation. P1-mediated responses of PI turnover and cAMP formation are not present in C6 glioma cells. Pertussis toxin (PTX) blocked the nucleotide-induced inhibition of cAMP accumulation but exerted no effect on inositol phosphate formation. Short-term treatment with phorbol 12-myristate 13-acetate inhibited both signal transduction pathways. The effects of three P2 purinergic antagonists, suramin, reactive blue and 4,4'-diisothiocyanatostilbene sulfonic acid (DIDS), on ATP- and 2-MeSATP-induced stimulation of PI turnover and inhibition of cAMP formation, respectively, were compared. For stimulating PI turnover, suramin is a competitive antagonist (pA2, 4.4); reactive blue and DIDS are noncompetitive antagonists at 30 microM and 100 microM, respectively. For the inhibition of cAMP formation, reactive blue and DIDS competitively antagonized the response of 2-MeSATP (pA2 values, 6.3 for reactive blue and 5.7 for DIDS); suramin was only slightly effective at 100 microM. It was concluded that the nucleotide receptor is linked to phospholipase C by a PTX-insensitive Gp protein and the P2Y receptor is linked to adenylyl cyclase by a PTX-sensitive Gi protein. Suramin is a competitive antagonist for the nucleotide receptor; reactive blue and DIDS are more selective antagonists for the P2Y receptor.
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PMID:Different signal transduction pathways are coupled to the nucleotide receptor and the P2Y receptor in C6 glioma cells. 801 79

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a significant role in transepithelial salt absorption as well as secretion by a number of epithelial tissues including sweat glands, airways and intestine. Early studies suggested that in absorption significant cross talk occurs between CFTR Cl(-) channels and epithelial Na(+) channels (ENaC). Studies based primarily on cultured cells of the airways and on ex vivo expression systems suggested that activating CFTR inhibits ENaC channels so that activation of CFTR and deactivation of ENaC seem reciprocal. Lack of CFTR Cl(-) conductance (g(CFTR)) in the plasma membranes was seen to enhance ENaC conductance (g(ENaC)) and Na(+) absorption from the airway surface liquid causing airway pathology in cystic fibrosis (CF). To determine if these events hold true for a purely absorptive epithelium, we investigated the role of CFTR in regulating g(ENaC) in native human sweat gland ducts. After permeabilizing the basilateral membrane of the duct with alpha-toxin, the relative activities of ENaC and CFTR in the apical membrane were characterized by correlating the effect of activating CFTR with ENaC function. We found that in contrast to reciprocal activities, activating g(CFTR) by either cAMP, cGMP or the G-proteins plus 5 mM ATP was accompanied by a concomitant activation, not inhibition, of g(ENaC). The activation of g(ENaC) appeared to be critically dependent on CFTR Cl(-) channel function because removal of Cl(-) from the medium, blockage of CFTR with inhibitor DIDS or the absence of CFTR in the DeltaF508 CF ducts prevented activation of g(ENaC) by cAMP, GMP or G-proteins. Most significantly, g(ENaC) was dramatically reduced, not increased, in CF as compared to non-CF sweat ducts. These results showed that lack of CFTR in the plasma membranes is not characteristically coupled to elevated ENaC activity or to increased Na(+) absorption in CF epithelial cells. Not only are CFTR and ENaC activated together in duct salt absorption, but ENaC activation depends on functioning CFTR. NaCl is poorly absorbed in the CF duct because CFTR activity appears to impose a loss of ENaC activity as well.
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PMID:Functional interaction of CFTR and ENaC in sweat glands. 1254 96

We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl- conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl(-) -channel function is required for activation. We used basilaterally alpha-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl(-) -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl- channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 microM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl- current (I(Cl-)) through CFTR was blocked by: 1) Cl- removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 microM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous DeltaF508 CF ducts). However, Cl- gradients in the direction of absorption supported, while Cl- gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated I(Cl-) through CFTR in the direction of absorption (Cl- gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing I(Cl-) through CFTR in the direction of secretion (Cl- gradient from cell to lumen) prevents ENaC activation even in the presence of I(Cl-) through CFTR.
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PMID:ENaC activity requires CFTR channel function independently of phosphorylation in sweat duct. 1646 40

The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.
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PMID:Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes. 1729 97

Apical and/or basolateral membranes of polarized epithelia express P2Y receptors, which regulate the transport of fluid and electrolytes. In the airway, P2Y receptors modulate Cl(-) secretion through the phospholipase C and calcium signaling pathways. Recent evidence suggests that P2Y(6) receptors are expressed in bronchial epithelium and coupled to the cAMP/protein kinase A (PKA) pathways. We examined P2Y receptor subtype expression, including P2Y(6,) and the effect of extracellular nucleotides on basal short-circuit current (I(SC)) and intracellular calcium concentration ([Ca(2+)](i)) in a human bronchial epithelial cell line (16HBE14o-). Real-time PCR demonstrated P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor expression and confirmed that transcript levels were not altered when cells were grown under varied conditions. It was determined that P2Y agonists (ATP, UTP, UDP) stimulated a concomitant increase in I(SC) and [Ca(2+)](i). Apical nucleotides stimulated an increase in [Ca(2+)](i) more efficiently than basolateral nucleotides; however, P2Y agonistic effects on I(SC) were greater when applied basolaterally. Since the P2Y(6) receptors differentially regulate apical and basolateral UDP-induced I(SC) and [Ca(2+)](i), we investigated membrane-resident P2Y(6) receptor functions using Cl(-) or K(+) channels blockers. Apical and basolateral UDP activation of I(SC) was inhibited by applying DIDS apically or TRAM-34 and clotrimazole basolaterally. Although both apical and basolateral UDP increased PKA activity, only apical UDP-induced I(SC) was sensitive to a CFTR inhibitor. These data demonstrate that P2Y agonists stimulate Ca(2+)-dependent Cl(-) secretion across human bronchial epithelia and that the cAMP/PKA pathway regulates apical but not basolateral P2Y(6) receptor-coupled ion transport in human bronchial epithelia.
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PMID:Apical versus basolateral P2Y(6) receptor-mediated Cl(-) secretion in immortalized bronchial epithelia. 1901 Nov 63