Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH(3) cells

Okadaic acid as well as other, structurally different, inhibitors of serine /threonine phosphatases 1 and 2A induce apoptosis in pituitary GH(3) cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytoto xic effect; of this agent. After about 18 months cells were selected that s urvived at 300 nM okadaic acid, which is about 30 times the initially letha l concentration. This study revealed that a major pharmaco-kinetic mechanis m underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the m dr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled b y a decreased accumulation of rhodamine 123,a fluorescent dye which is a su bstrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like ver apamil and reserpine but not by the leukotriene receptor antagonist MK571 w hich is a modulator of the multidrug resistance-associated protein (MRP). O kadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and acti nomycin D, bur not to non-substrates of mdr1, e.g. cytosine arabinoside. Th us, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pit uitary GH(3) cells. Maintenance of resistance after withdrawal of okadaic a cid as well as metaphase spreads of 100 nM okadaic acid-resistant cells sug gested a stable MDR genotype without indications for the occurrence of extr achromosomal amplifications, e.g. double minute chromosomes.