The role of adrenergic agents in augmenting proximal tubular salt and water flux, was studied in a preparation of freshly isolated rabbit renal proximal tubular cells in suspension. Norepinephrine (NE, 10-5 M) increased sodium influx (JNa) 60±5% above control value. The alpha adrenergic antagonist, phentolamine (10-5 M), inhibited the NE-induced enhanced JNa by 90±2%, while the beta adrenergic antagonist, propranolol, had a minimal inhibitory effect (10±2%). The alpha adrenergic subtype was further defined. Yohimbine (10-5 M), an alpha2 adrenergic antagonist but not prazosin (10-5 M), an alpha, adrenergic antagonist completely blocked the NE induced increase in JN.. Clonidine, a partial alpha2 adrenergic agonist, increased JNS by 58±2% comparable to that observed with NE (10-5 M). Yohimbine, but not prazosin, inhibited the clonidine-induced increase in JN., confirming that alpha2 adrenergic receptors were involved. Additional alpha2 adrenergic agents, notably p-amino clonidine and alpha-methyl-norepinephrine, imparted a similar increase in JN.. The clonidineinduced increase in JNS could be completely blocked by the amiloride analogue, ethylisopropyl amiloride (EIPA, 10'-M). sites/cell and KD 5.4±1.4 nM. In summary, in the isolated rabbit renal proximal tubular cell preparation, alpha2 adrenergic receptors are the predominant expression of alpha adrenoceptors, and in the absence of organic Na'-cotransported solutes, alpha2 adrenergic agonists enhance 22Na influx into the cell by stimulating the brush border membrane Na'-H' exchange pathway.

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.

The adaptative response of the renal proximal tubule to a reduction of renal mass was studied in brush border membrane vesicles prepared from the solitary remnant kidney (RK) of subtotally nephrectomized rabbits. The in vivo acid-base status of RK and sham-operated controls (SK) was similar. In the absence of organic solutes, Na+ flux across the membrane demonstrated features of Na+-H+ antiport, i.e., stimulation by a transmembrane pH gradient and inhibition by amiloride. Kinetic parameters for Na+-H+ antiport were derived with different experimental protocols. In the presence of an opposing H+ gradient and over a limited range of Na+ concentration, JNamax was enhanced 65% in RK vesicles compared with SK vesicles and KtNa was unchanged. The enhanced JNamax was not apparent under H+ equilibrium conditions, Comparable values for JNamax and KtNa were obtained by studying RK vesicles at external Na+ concentrations of 0-200 mM and resolving uptake into a substrate component, representing Na+-H+ antiport, and a nonsaturable diffusive component. The apparent Na+ permeability (P'Na) of RK vesicles was identical to the P'Na of normal kidney vesicles, under both H+ gradient and H+ equilibrium conditions. H+ permeability, measured with acridine orange, was also the same in RK and SK vesicles. These studies demonstrate that in the remnant kidney model of chronic renal insufficiency there is an increase in the JNamax of the Na+-H+ antiporter in the luminal membrane of the proximal tubule that is revealed only under transmembrane H+ gradient conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Brush border membrane vesicles were used to investigate the pathways for Na+ uptake across the apical membrane of the renal proximal tubular cell. The kinetics of uptake in the absence of organic solutes were consistent with parallel saturable and nonsaturable pathways. At pH equilibrium (pHin = pHout = 7.5), the Jmax and Kt for saturable uptake were 41 +/- 15 (+/- SE) nmol X mg-1 X min-1 and 33 +/- 9, respectively, and the apparent permeability coefficient, P'Na, was 0.27 +/- 0.02 microliters X mg-1 X min-1. As the equilibrium pH was varied between 6.0 and 8.0, no consistent trend for Kt or P'Na was observed; Jmax varied up to twofold. In contrast, in the presence of an outward H+ gradient (pHin = 6.0 vs. pHout = 7.5), the Kt decreased by an order of magnitude, with little change in Jmax. At low sodium concentrations (1 mM) external Li+ and NH+4, and to a lesser extent K+, Rb+, and Cs+, inhibited Na+ uptake. Amiloride (10(-3) M) inhibited 1 mM Na+ uptake by 80% even in the absence of a H+ gradient. Uptake also varied with the anion composition at high sodium concentrations (100 mM), as predicted from the anion permeabilities. Sodium uptake was more sensitive to variations in membrane potential at high sodium concentrations than at low concentrations. On the basis of these experiments we suggest that the saturable Na+ uptake occurs via an electroneutral Na+-H+ antiporter and that the diffusive flux occurs through a conductive pathway.