Regulation of Na(+)-K(+)-2Cl- cotransport activity in rabbit proximal tubule primary culture.

The presence of Na(+)-K(+)-2Cl- cotransport in rabbit kidney proximal tubule cells in primary culture was demonstrated by bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake studies. After addition of 10 microM bumetanide, 86RB+ uptake was inhibited from 11.1 +/- 0.8 to 1.1 +/- 0.1 nmol.mg protein-1.min-1 under isotonic (300 mosM) conditions. Na(+)-K(+)-2Cl- cotransport activities ranged from 2.2 +/- 0.3 to 13.2 +/- 1.0 nmol.mg protein-1.min-1 depending on the osmolarity of the medium (150-500 mosM). Decreasing extracellular pH to 6.5 inhibited, whereas increasing to 8.5 stimulated, transport at all osmolarities. Decreasing intracellular pH (pHi) by the NH4Cl pulse method showed similar results, suggesting a possible regulatory role of pHi on cotransport activity. Ca(2+)-free medium increased cotransport activity 35 and 20% at iso- and hypertonicity, respectively. At 300 mosM, ionomycin (5 microM) inhibited cotransport by 25%. The combination of forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM) resulted in inhibition of cotransport activity by 38% at hypertonic conditions. Calyculin (1 microM) increased cotransport activity 134 and 128% at 150 and 300 mosM, respectively. In hypertonic medium calyculin did not influence cotransport activity. Okadaic acid (1 microM) had no effect on cotransport activity at all three osmolarities. NaF (10 mM) increased cotransport at all osmolarities tested.