Protein kinase C and phosphatidylinositol 3-kinase independently contribute to P-glycoprotein-mediated drug secretion in the mouse proximal tubule

We have recently reported that the typical membrane of the proximal tubule S2 segment from wildtype mice (WT mice) has the capacity for P-glycoprotein - (P-gp-) mediated drug efflux, whereas mice in which both mdr1a and mdr1b genes are disrupted (KO mice) do not. To examine the intracellular regulato ry mechanisms of drug-transporting P-gp activity, we isolated and perfused proximal tubule S2 segments from WT and KO mice, and measured luminal efflu x of the intracellular fluorescence of rhodamine 123, a fluorescent substra te of P-gp. The decay half-time of the intracellular fluorescence (t(1/2)) was regarded as an index of the drug-transporting P-gp activity. In the WT mice, the t(1/2) was 36 +/-5 s (n=35) during the basal period, and was sign ificantly increased to 440 +/- 45 s by the luminal addition of verapamil (a n inhibitor of P-gp). The addition of phorbol 12-myristate 13-acetate (PMA) [a protein kinase C (PKC) activator] to the bath increased t(1/2), but 4 a lpha -phorbol (the inactive form of PMA) did not. The PMA-induced increase in t(1/2) was further increased by the luminal addition of verapamil, and w as partially inhibited by co-treatment with staurosporine or H-7 (inhibitor s of PKC). Pretreatment with wortmannin or LY294002 [inhibitors of phosphat idylinositol 3-kinase (PI 3-kinase)] added to the bath also increased t(1/2 ). The wortmannin- and LY294002-induced increase in t(1/2) was also further increased by the luminal addition of verapamil. The effects of PMA on t(1/ 2) were additive after cotreatment with wortmannin or LY294002. In the KO m ice, t(1/2) was 440 +/- 25 s (n-18) during the basal period, and was no lon ger affected by the addition of verapamil, staurosporine, H-7, wortmannin, or LY294002. Based on the use of pharmacological agents, we conclude that i n the proximal tubule from WT mice, P-gp-mediated drag secretion occurs ind ependently via PKC- and PI 3-kinase-dependent processes, whereas it is not present in KO mice, independently of PKC- and PI 3-kinase.