P-Glycoprotein Phosphorylation and Transport Activity in Rainbow Trout (Oncorhynchus mykiss) Hepatocytes.

Evidence suggests that P-glycoprotein (P-gp) transport activity can be post-transcriptionally regulated through protein kinase C-mediated phosphorylation. In the present study, P-gp efflux capacity and phosphorylation state were examined in rainbow trout hepatocytes, following protein kinase C (PKC) inhibition with 1-(5-isoquinolinyl-sulfonyl)-3-methyl-piperazine (H-7), or its activation following treatment with Phorbol 12-Myristate 13-Acetate (PMA). Decreased chemical efflux and increased accumulation following H-7 treatment were observed when rhodamine 123 (R123) was used as P-gp substrate, while PMA increased P-gp efflux and decreased the accumulation of daunorubicine (DNR). This substrate-dependent modulation suggests that phosphorylation affects P-gp’s ability to bind to different substrates or, more likely, to its co-substrate ATP. As well, these results raise the possibility that other transporters may be involved, particularly in R123 transport. Immunoblots of solubilized membrane fractions and whole cell extracts following phosphate-affinity SDS-PAGE showed that H-7 and PMA-treated cells produce different P-gp phosphorylation patterns compared to control cells; up to 5 different phosphoisoforms were visualized using the monoclonal antibody C-219. Differences in the distribution, number, and optical densities of bands were visualized, and can be interpreted as PKC activation or inhibition altering the relative abundance of P-gp phosphoisoforms in the cells. Hepatocytes were also treated with H-7 or PMA and radiolabelled in a media containing 32P, were immunoprecipitated, and subjected to conventional SDS-PAGE. The resulting gels confirmed that PMA increased the amount of 32P incorporated into P-gp as compared to controls, while H-7 decreased it, indicating that PKC inhibition prevents or reduces P-gp phosphorylation and the transport of certain substrates, while activation promotes phosphorylation and transport, also in a substrate-dependent manner.