STIMULATION OF ALPHA(2)-ADRENERGIC RECEPTORS INCREASES NA+-K+-ATPASE ACTIVITY IN DISTAL CONVOLUTED TUBULE CELLS

The influence of alpha-adrenoceptor subtypes on Na+ transport in distal convoluted tubules (DCT) has not been examined due to the difficulty of isolating and quantifying responses in this segment. These experiments were designed to test the hypothesis that alpha-adrenergic receptors stimulate Na+ absorption in DCT cells. Norepinephrine and epinephrine increased Na-22+ uptake into immortalized mouse DCT cells by 49 and 55% compared with basal uptake. Selective alpha2-agonists (guanabenz, clonidine, and B-HT 933) stimulated Na-22+ uptake by 39-45%, but alpha1-agonists had no effect. Alpha2-agonist-stimulated Na-22+ uptake was abolished with alpha2-antagonists (yohimbine, idazoxan). The entry pathways for alpha2-agonist-stimulated Na-22+ uptake were determined with the NaCl cotransport inhibitor, chlorothiazide (10(-4) M), and the Na+ channel blocker, amiloride (10(-6) M). Agonist-stimulated Na-22+ uptake was inhibited 42 +/- 5 with chlorothiazide and 47 +/- 7% with amiloride. These results suggested that alpha2-receptors may activate Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase), resulting in an increased driving force for luminal Na+ entry through both pathways. Ouabain-suppressible Rb-86 uptake and intracellular Na+ activity ([Na+]i; measured in single cells on glass cover slips loaded with the fluorescent probe sodium-binding benzofuran isophthalate) were used to measure Na+-K+-ATPase activity. Alpha2-agonists significantly increased Rb-86 uptake within 30 s. After 2.5 min, epinephrine and B-HT 933 decreased [Na+]i from a control level of 13 +/- 1 to 5 +/- 1 mM. Calphostin C, a specific inhibitor of protein kinase C, abolished alpha2-receptor-stimulated Na-22+ and Rb-86 uptake. In summary, activation of alpha2-receptors on immortalized DCT cells increases Na+-K+-ATPase activity via a protein kinase C-dependent pathway that results in a decrease of [Na+]i and in turn enhances the driving force for Na+ influx through chlorothiazide- and amiloride-sensitive pathways.