guanosine-triphosphate and mevastatin

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

Topics: ADP Ribose Transferases; Bacterial Toxins; Botulinum Toxins; Cells, Cultured; Cytosol; Cytotoxins; Endothelium, Vascular; Escherichia coli Proteins; Gene Expression Regulation, Enzymologic; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Lipoproteins, LDL; Lovastatin; Membrane Proteins; Mevalonic Acid; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Polyisoprenyl Phosphates; ras Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA Processing, Post-Transcriptional; RNA, Messenger; Sesquiterpenes

The role of isoprenylation in formyl peptide receptor-mediated G protein activation was studied using plasma membranes isolated from normal HL-60 granulocytes and from cells in which isoprenylation was inhibited with mevastatin. Plasma membrane expression of formyl peptide receptors and G protein beta subunits, but not alpha i2 and alpha i3, was significantly reduced by inhibition of isoprenylation. This reduced expression resulted in impaired basal and fMet-Leu-Phe-stimulated G protein activation.

Topics: Adenosine Diphosphate Ribose; Anticholesteremic Agents; Cell Membrane; Cholera Toxin; Granulocytes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Lovastatin; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; NAD; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Tumor Cells, Cultured; Virulence Factors, Bordetella