Simvastatin and intracellular pH regulation by the Na+/H+ antiport of SV40-virus-transformed human MRC5 fibroblasts.
1. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by simvastatin leads to inhibition of both cell growth and Na+/H+ antiport activity. The effect of simvastatin on intracellular pH and Na+/H+ antiport activity was therefore studied on an adherent cell line, the SV40-virus-transformed MRC5 human fibroblast. 2. Simvastatin led to a dose-dependent 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 inhibition of the antiport by simvastatin, reversed these changes within 1-2h. 3. The phenomenon of mevalonate reversal of antiport inhibition by simvastatin was not sensitive to cycloheximide, indicating its post-translational nature. This was also consistent with the short period of incubation with mevalonate leading to reversal of antiport inhibition (1-2 h). 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 (tunicamycin), phosphorylation (staurosporine), isoprenylation (farnesol, limonene), and of pertussis-toxin-sensitive G-proteins or calmodulin (W7), had no effect on the reversal by mevalonate of simvastatin-induced changes in Na+/H+ antiport activity. 5. N-Ethylmaleimide (50 mumol/l for 5 min) prevented mevalonate reversing the effects of simvastatin, suggesting the importance of thiol groups in the phenomenon of reversal of the inhibition of Na+/H+ antiport activity by simvastatin. Furthermore, concurrent incubation of simvastatin-treated cells with dithiothreitol (1 mmol/l) and N-ethylmaleimide restored the ability of mevalonate to reverse the inhibitory effects of simvastatin on Na+H+ antiport activity.
Department of Pharmacology, Leicester Royal Infirmary, U.K.
J E
JE
Davies
L L
LL
Ng
D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002784:Cholesterol / Q000032:analysis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D066298:In Vitro Techniques; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008798:Mevalonic Acid / Q000494:pharmacology; D019821:Simvastatin; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D017923:Sodium-Hydrogen Exchangers; D002784:Cholesterol; D008148:Lovastatin; D019821:Simvastatin; D008798:Mevalonic Acid