REGULATION OF NA-K+-2CL(-) COTRANSPORT ACTIVITY IN RABBIT PROXIMAL TUBULE IN PRIMARY CULTURE()

The presence of Na+-K+-2Cl(-) cotransport in rabbit kidney proximal tu bule cells in primary culture was demonstrated by bumetanide-sensitive , ouabain-insensitive Rb-86(+) uptake studies. After addition of 10 mu M bumetanide, Rb-86(+) uptake was inhibited from 11.1 +/- 0.8 to 1.1 +/- 0.1 nmol.mg protein(-1).min(-1) under isotonic (300 mosM) conditio ns. 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 inhibite d, whereas increasing to 8.5 stimulated, transport at all osmolarities . Decreasing intracellular pH (pH(i)) by the NH4Cl pulse method showed similar results, suggesting a possible regula tory role of pH(i) on c otransport activity. Ca2+-free medium increased cotransport activity 3 5 and 20% at iso- and hypertonicity, respectively. At 300 mosM, ionomy cin (5 mu M) inhibited cotransport by 25%. The combination of forskoli n (10 mu M) and 3-isobutyl-1-methylxanthine (1 mM) resulted in inhibit ion of cotransport activity by 38% at hypertonic conditions. Calyculin (1 mu M) increased cotransport activity 134 and 128% at 150 and 300 m osM, respectively. In hypertonic medium calyculin did not influence co transport activity. Okadaic acid (1 mu M) had no effect on cotransport activity at all three osmolarities. NaF (10 mM) increased cotransport at all osmolarities tested.