Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.21 (CPT)
 4,580 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.
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PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

Proton transport pathways in isolated superfused rabbit cortical connecting (CNT) and collecting tubules (CCD) were determined using the fluorescent pH-sensitive dye BCECF following acid or base load by exposure to NH4Cl. Following removal of NH4Cl which results in a rapid decline in pHi two mechanisms appear to be responsible for pHi recovery, a Na-independent NEM-sensitive H efflux with a slow activity, which was virtually absent in 30% of the segments tested and a second rapid Na-dependent H efflux. In CCD this latter pathway was shown to have an apparent Km for (Na+)e of 38.2 +/- 0.4 mM (S.D., n = 7) and was sensitive to EIPA. Similar results were obtained with the CNT. With regard to the H pump in six out of ten CCD isoproterenol (200 nM) resulted in a 2-fold stimulation of H pump activity. These effects of isoprenaline were inhibited both by the non-specific beta-adrenoceptor antagonist propranolol as well as by the specific b1 antagonist metoprolol. Interestingly, these stimulatory effects of this beta agonist, which is known to stimulate cAMP formation in rabbit CCD, were not reproduced by the addition of exogenous cAMP analogues db cAMP (0.1 mM), CPT cAMP (0.1 mM), 8 Br-cAMP (0.1 mM) or the addition of forskolin (0.3 mM). In conclusion, these data obtained in isolated rabbit CNT and CCT demonstrate the presence of an active Na-H exchange which is for the most part responsible for the recovery of pHi. It should be noted also that the contribution of the H pump to pHi regulation appears to be negligible in these segments.
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PMID:Regulation of intracellular pH in rabbit cortical connecting tubule and cortical collecting duct. 166 Nov 63

The amiloride-sensitive epithelial sodium channel (ENaC) plays a key role in sodium reabsorption in the collecting ducts. We examined ENaC mRNA distribution along the nephron and acute effects of vasopressin and hyperosmolality on ENaC mRNA expression. ENaCalpha, beta, and gamma mRNA expressions were observed in cortical, outer medullary and initial inner medullary collecting ducts (CCD, OMCD and ilMCD, respectively). ENaCalpha mRNA expression was also observed in medullary and cortical thick ascending limbs (MAL and CAL, respectively), while ENaCbeta and gamma mRNA expressions were not observed. Furthermore, ENaCalpha mRNA expression in MAL but not in collecting ducts was stimulated by acute exposure to arginine vasopressin (AVP), 8-(4-chlorophenylthio) (CPT)-cAMP and hyperosmolality. However, the physiological significance of these effects is not known, since ENaC protein is reported to be absent in MAL. These data suggest that ENaCalpha mRNA expression in MAL but not in collecting ducts is acutely regulated by AVP and hyperosmolality. The absence of stimulation of ENaCalpha mRNA expression in collecting ducts suggests the physiological significance of ENaCbeta and gamma mRNA for acute regulation by vasopressin. Determining the physiological significance of the acute effect of vasopressin in MAL will require further investigations.
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PMID:Acute regulation of the epithelial sodium channel gene by vasopressin and hyperosmolality. 1456 2