Characteristics of the Na+-H+ antiporter in the intact renal proximal tubular cell.
The characteristics of the proximal tubular Na+-H+ antiporter were determined in isolated proximal tubular cells to ascertain whether the features of this transport system in intact cells are comparable with those previously described for isolated brush-border membrane vesicles. A method is described for the rapid isolation of a purified preparation of cells that demonstrate morphological and functional characteristics of the renal proximal tubule. The cells maintain their polarity while in suspension, and adenylate cyclase activity is enhanced by parathyroid hormone but not by arginine vasopressin. The cells display gluconeogenic function and Na+-dependent alpha-methyl-D-glucose and organic phosphate cotransport, processes that confirm their proximal tubule origin. O2 consumption rates and cytosolic adenosine triphosphate levels indicate functional integrity. Na+-H+ antiport activity was defined in these cells by measuring amiloride-sensitive Na+ uptake. At intracellular pH = 6.4 vs. extracellular pH = 7.4, KtNa was 10.1 +/- 2.8 mM, and maximal sodium flux was 0.89 +/- 0.13 nmol X 10(6) cells-1 X K0.5 for amiloride and ethyl-isopropyl amiloride, measured at an external Na+ concentration of 1 mM, was observed at 2.5 X 10(-5) M and 2.9 X 10(-6) M, respectively. The external and internal loci of the exchanger displayed asymmetric affinity for the hydrogen ion: the apparent pK for the external site was 7.20-7.26 vs. less than 6.5 for the internal site. The internal site demonstrated features of positive cooperativity. In summary, the Na+-H+ antiporter present in the luminal membrane of the renal proximal tubule has been characterized in the intact cell and displays functional and kinetic parameters closely resembling those described in isolated brush-border membrane vesicles.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D005943:Gluconeogenesis; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008855:Microscopy, Electron, Scanning; D010101:Oxygen Consumption; D010281:Parathyroid Hormone / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers; D013997:Time Factors
D002352:Carrier Proteins; D010281:Parathyroid Hormone; D010710:Phosphates; D017923:Sodium-Hydrogen Exchangers; D001127:Arginine Vasopressin; D000584:Amiloride; D000262:Adenylyl Cyclases; D005947:Glucose