Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.
 ...
PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.
 ...
PMID:Inhibition of Bacillus cereus phospholipase C by univalent anions. 681 Aug 75

NO is related to the pathological condition acute renal failure, in which we previously observed that the level of soluble dipeptidase in urine was decreased. In this study the role of NO in the shedding of the glycosylphosphatidylinositol (GPI)-anchored form of renal dipeptidase (RDPase) was examined. The NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine rapidly inhibited the release of RDPase from porcine kidney proximal tubules. The substrate of NO synthase, l-Arg, also inhibited the release of RDPase, and this effect was reversed by the NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester. Western-blot analyses using antibodies raised against porcine RDPase and the inositol-1,2-cyclic monophosphate moiety formed on phospholipase C cleavage of the GPI anchor demonstrated that SNP mediated its inhibitory effect on the release of RDPase via a GPI-specific phospholipase C (GPI-PLC). Peroxynitrite scavengers (deferoxamine and superoxide dismutase) or reducing agent (dithiothreitol) did not affect SNP's inhibition of the release of RDPase. Exposure to the G-protein activator AlF(-)(4) mimicked the l-Arg effect in the presence of a low concentration of l-Arg, and the effect was completely reversed by U73122, an intracellular phosphatidylinositol-specific PLC (PI-PLC) inhibitor. These results suggest a signal-transduction pathway involving NO, which is produced by NO synthase(s) following activation of a G-protein-coupled PI-PLC, resulting in inhibition of the GPI-PLC that cleaves and releases RDPase. Therefore, this indicates a role for NO as an inhibitory regulator of the shedding of the GPI-anchored RDPase in acute renal failure.
 ...
PMID:Nitric oxide inhibits the shedding of the glycosylphosphatidylinositol-anchored dipeptidase from porcine renal proximal tubules. 1198 94

We examined the effects of intracellular Cl- concentration ([Cl-]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71-85, 2002). Treatment with cAMP (100 microM 8-cpt-cAMP plus 100 microM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl- secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in alpha-toxin-permeabilized monolayers to have the anion selectivity sequence NO3- > Br- > Cl- > I-. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS-Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl-] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl-] in the apical solution. This inhibitory effect of [Cl-]i occurred regardless of the presence or absence of net Cl- transport. However, from fluorometric measurements using the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl- secretion, produced a decrease of [Cl-]i from 76 +/- 14 to 36 +/- 8 mM (P = 0.03). Thus it might be expected that activation of Cl- secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl-]i from 15 to 145 mM decreased AS-Isc from 24.5 +/- 1.0 to 10.2 +/- 1.6 microA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 +/- 0.16 to 1.32 +/- 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 +/- 1.2 to 8.2 +/- 1.4 microA/fmol.
 ...
PMID:Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression. 1516 4

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.
 ...
PMID:Cl- interference with the epithelial Na+ channel ENaC. 1602 56