SIMVASTATIN AND INTRACELLULAR PH REGULATION BY THE NA-VIRUS-TRANSFORMED HUMAN MRC5 FIBROBLASTS( H+ ANTIPORT OF SV40)

1. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by simvastat in leads to inhibition of both cell growth and Na+/H+ antiport activit y. The effect of simvastatin on intracellular pH and Na+/H+ antiport a ctivity was therefore studied on an adherent cell line, the SV40-virus -transformed MRC5 human fibroblast. 2. Simvastatin led to a dose-depen dent decrease in intracellular pH, attributed to a reduction in Na+/H exchange, together with a rounding of cell shape. Mevalonate (1 mmol/ l) prevented these effects of simvastatin, and when added after inhibi tion of the antiport by simvastatin, reversed these changes within 1-2 h. 3. The phenomenon of mevalonate reversal of antiport inhibition by simvastatin was not sensitive to cycloheximide, indicating its post-t ranslational nature. This was also consistent with the short period of incubation with mevalonate leading to reversal of antiport inhibition (1-2h). These changes in intracellular pH regulation were not due to alterations in cell cholesterol content. 4. A variety of inhibitors of post-translational processes, such as N-linked glycosylation (tunicam ycin), phosphorylation (staurosporine), isoprenylation (farnesol, limo nene), and of pertussis-toxin-sensitive G-proteins or calmodulin (W7), had no effect on the reversal by mevalonate of simvastatin-induced ch anges in Na+/H+ antiport activity. 5. N-Ethylmaleimide (50 mumol/l for 5 min) prevented mevalonate reversing the effects of simvastatin, sug gesting the importance of thiol groups in the phenomenon of reversal o f the inhibition of Na+/H+ antiport activity by simvastatin. Furthermo re, concurrent incubation of simvastatin-treated cells with dithiothre itol (1 mmol/l) and N-ethylmaleimide restored the ability of mevalonat e to reverse the inhibitory effects of simvastatin on Na+H+ antiport a ctivity.