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 apical membrane of the proximal tubule S2 segment from wild-type 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 regulatory mechanisms of drug-transporting P-gp activity, we isolated and perfused proximal tubule S2 segments from WT and KO mice, and measured luminal efflux of the intracellular fluorescence of rhodamine 123, a fluorescent substrate of P-gp. The decay half-time of the intracellular fluorescence (t1/2) was regarded as an index of the drug-transporting P-gp activity. In the WT mice, the t1/2 was 36+/-5 s (n=35) during the basal period, and was significantly increased to 440+/-45 s by the luminal addition of verapamil (an inhibitor of P-gp). The addition of phorbol 12-myristate 13-acetate (PMA) [a protein kinase C (PKC) activator] to the bath increased t1/2, but 4alpha-phorbol (the inactive form of PMA) did not. The PMA-induced increase in t1/2 was further increased by the luminal addition of verapamil, and was partially inhibited by co-treatment with staurosporine or H-7 (inhibitors of PKC). Pretreatment with wortmannin or LY294002 [inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase)] added to the bath also increased t1/2. The wortmannin- and LY294002-induced increase in t1/2 was also further increased by the luminal addition of verapamil. The effects of PMA on t1/2 were additive after cotreatment with wortmannin or LY294002. In the KO mice, t1/2 was 440+/-25 s (n=18) during the basal period, and was no longer affected by the addition of verapamil, staurosporine, H-7, wortmannin, or LY294002. Based on the use of pharmacological agents, we conclude that in the proximal tubule from WT mice, P-gp-mediated drug secretion occurs independently via PKC- and PI 3-kinase-dependent processes, whereas it is not present in KO mice, independently of PKC- and PI 3-kinase.
Tsuruoka
S
S
Department of Nephrology, Jichi Medical School, Minamikawachi, Tochigi, Japan.
Sugimoto
K
K
Fujimura
A
A
Imai
M
M
Asano
Y
Y
Muto
S
S
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D020112:Rhodamine 123; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C095122:multidrug resistance protein 3