Protein kinase C inhibits Na⁺-K⁺-2Cl^- cotransporter activity in cultured rabbit nonpigmented ciliary epithelium

We examined the regulation of Na⁺-K⁺-2Cl^- cotransporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium, Na⁺-K⁺-2Cl^- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (⁸⁶Rb) transport. Phorbol 12, 13- dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide- sensitive potassium (⁸⁶Rb) uptake, with a half-maximal inhibitory concentration of ~0.1 μM. The inhibitory effect of PBDu on potassium uptake by the Na⁺-K⁺-2Cl^- cotransporter was abolished by PKC downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na⁺-K⁺-2Cl^- cotransporter-mediated inward potassium (⁸⁶Rb) transport by ~26% in control cells and by 40% in cells pretreated with ouabain. PKC activation also reduced the rate of bumetanide- sensitive potassium (⁸⁶Rb) efflux in ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na⁺- K⁺-2Cl^- cotransporter.