DIFFERENTIAL MODULATION OF P-GLYCOPROTEIN EXPRESSION BY DEXAMETHASONEAND 3-METHYCHOLANTHRENE IN RAT HEPATOCYTE PRIMARY CULTURES

Spontaneous and culture condition-dependent changes in P-glycoprotein expression and activity have been monitored in primary cultures of rat hepatocytes by using immunoblotting, PCR and fluorimetric techniques. In hepatocytes cultured in basal medium without addition of dexametha sone or 3-methylcholanthrene, mdr mRNA and P-glycoprotein increased pr ogressively throughout a 72 h culture period, in concert with an enhan cement in the ability to extrude the fluorescent dye Rhodamine-123. Ad dition of 1 mu M dexamethasone to the culture medium slowed down the i ncrease in mdr mRNA and P-glycoprotein, while inducing a significant i ncrease in the efficiency of R-123 efflux. Addition of either 100 nM o r 10 mu M DEX produced different changes in mdr mRNA and protein, unre lated to the rate of Rhodamine-123 extrusion. When 50 mu M 3-methylcho lanthrene was added to the culture medium in the absence of any hormon e supplementation, no significant changes in P-glycoprotein activity a nd expression took place, in comparison with control cultures. On the contrary, in the presence of dexamethasone (100 nM and 1 mu M), 3-meth ylcholanthrene induced an increase in mdr mRNA and in the amount of im munoblottable protein during culture, without producing any concomitan t increase in the efficiency to extrude Rhodamine-123. The last phenom enon resulted to be an artefact, since 3-methylcholanthrene was shown to inhibit Rhodamine-123 transport competitively. These results indica te that rat hepatocyte P-glycoprotein may be variously modulated in vi tro, by supplementing culture medium with hormones and/or xenobiotics. Functional activity of the P-glycoprotein is not necessarily related with protein amount and/or mdr RNA.