Inducible nitric oxide synthase in vascular smooth muscle cells from prehypertensive spontaneously hypertensive rats

Abstract

The inducible nitric oxide synthase (iNOS) present in vascular smooth muscle cells (VSMC) may play a role in the generation of nitric oxide (NO) in the vascular wall, regulating blood vessel tone in normotension and hypertension. In this study the effect of interleukin (IL)-1β, a cytokine that induces iNOS, on NO generation (measured as nitrite), cyclic guanosine monophosphate (cGMP) generation, and steady-state abundance of iNOS mRNA were examined in VSMC from 3 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, during the period preceding the elevation of blood pressure. With cell density dependent variations in nitrite production eliminated, VSMC from SHR and WKY did not differ in NO generation except after prolonged incubation (30 h), when SHR cells produced less NO. However, cGMP concentrations associated with IL-1β stimulation were significantly smaller in SHR VSMC than in cells from WKY. IL-1β stimulation resulted in increased abundance of iNOS mRNA to the same extent in both WKY and SHR VSMC. Inhibitors of NOS, N G-monomethyl- l-arginine (L-NMMA) and N ω-nitro- l-arginine methyl ester (L-NAME), did not block the induction of iNOS mRNA, although nitrite production and cGMP generation were inhibited. The protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin-D almost completely blocked the production of nitrite in cells from both strains of rats. Actinomycin-D completely blocked the induction of iNOS mRNA by IL-1β in cells from both strains of rats, whereas cycloheximide partially blocked its synthesis in WKY, but had no significant effect on IL-1β induced expression of iNOS mRNA in SHR VSMC. Thus, IL-1β controls iNOS gene expression at the transcriptional level, and an intermediate labile protein, whose synthesis is inhibited by cycloheximide, is required for IL-1β stimulated induction of iNOS mRNA transcription in WKY cells but not in SHR. We conclude that although iNOS is expressed to similar extent in VSMC of prehypertensive SHR and WKY and similar amounts of NO are initially generated, there are differences between the VSMC of SHR and WKY in the regulation of the transcription of iNOS mRNA, there is a lower sustained production of NO, and there is a reduced generation of cGMP in response to IL-1β stimulated NO production. These differences between VSMC from prehypertensive SHR and WKY may indicate a pathophysiological role of iNOS in early blood pressure elevation in SHR.