Effects of barbiturates on the expression of the inducible nitric oxide synthase in vascular smooth muscle

Certain cytokines stimulate the expression of the inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) and in many other cell types. The large amounts of nitric oxide (NO) generated by iNOS in the vascular wall contribute to the unrelenting hypotension in septic shock. Because septic patients are often treated with barbiturates, we examined the effect of these anesthetic agents on the expression of iNOS in VSMCs. The induction of iNOS was elicited either in cultured rat aortic SMCs [interleukin-1 beta (IL-1 beta), 60 U/ml for 24 h] or in endothelium-denuded segments of the rabbit carotid artery [IL-1 beta (100 U/ml) for 7 h]. The activity of iNOS was assessed by the accumulation of nitrite in the conditioned medium of cultured VSMCs and by the hyporeactivity of carotid arteries to phenylephrine. Moreover. iNOS protein abundance was determined by Western blot analysis, iNOS messenger RNA (mRNA) by reverse transcription followed by the polymerase chain reaction (PCR), and activation of the transcription factor NF-kappa B by gel electrophoretic mobility-shift analysis of nuclear extracts from VSMCs. The IL-1 beta-stimulated increase in nitrite formation, iNOS protein, and mRNA abundance in VSMCs was significantly augmented in the presence of thiopental (100 mu M), whereas methohexital, hexobarbital, pentobarbital, and phenobarbital were without effect. The potentiating effect of thiopental was observed only when the barbiturate was administered during the first 2 h of the 24-h incubation period of cultured VSMCs with IL-1 beta. Thiopental did not affect the IL-1 beta-stimulated activation of NF-kappa B in VSMCs. This barbiturate also significantly augmented the hyporeactivity to phenylephrine in carotid arteries exposed to IL-1 beta, an effect that was abolished by N-G-nitro-L-arginine. Exposure of either cultured or native VSMCs to thiopental alone did not stimulate iNOS expression. These findings demonstrate that the thiobarbiturate thiopental, but not oxybarbiturates, augments the IL-1 beta-stimulated synthesis of NO in both cultured and native VSMCs. This effect of. thiopental is the result of an increased expression of iNOS, involving most likely mechanisms distinct from NF-kappa B activation. The use of thiopental for long-term treatment of septic patients might possibly potentiate the biosynthesis of NO in the vascular wall and thus cause a further deterioration of the hemodynamic state.