Halothane and Isoflurane Inhibit Endothelium-derived Relaxing Factor-dependent Cyclic Guanosine Monophosphate Accumulation in Endothelial Cell-Vascular Smooth Muscle Co-cultures Independent of an Effect on Guanylyl Cyclase Activation

Abstract

Interaction of inhalational anesthetics with the nitric oxide signaling pathway and the mechanism of such effects are controversial. The aim of this study was to clarify the sites and mechanism of inhalational anesthetic interaction with the vascular nitric oxide and guanylyl cyclase signaling pathway. To specifically study the mechanism of anesthetic interaction with the nitric oxide-guanylyl cyclase pathway, cultured vascular smooth muscle and endothelial cell-vascular smooth muscle (EC-VSM) co-culture models were chosen. Monolayer cultures of VSM with or without cultured endothelial cells grown on microcarrier beads were preequilibrated with anesthetic and stimulated with agonists. The effect of inhalational anesthetics on cyclic guanosine monophosphate (GMP) content of unstimulated VSM and of VSM in which soluble guanylyl cyclase had been activated by the endothelium-independent nitrovasodilators, sodium nitroprusside, nitroglycerin, or nitric oxide was determined. Experiments were also performed to assess the effect of inhalational anesthetics on unstimulated endothelial cell-vascular smooth muscle co-cultures and on co-cultures in which nitric oxide synthase and subsequent cyclic GMP production had been activated by the receptor-mediated agonists bradykinin and adenosine triphosphate and by the non-receptor-mediated calcium ionophore A23187. Increasing concentrations of halothane and isoflurane from 0.5 to 5% had no effect on basal cyclic GMP concentrations in cultured VSM alone or in endothelial cell-vascular smooth muscle co-cultures, and had no effect on sodium nitroprusside, nitroglycerin, or nitric oxide stimulated cyclic GMP accumulation in cultured VSM. In agonist-stimulated co-cultures, however, halothane and isoflurane significantly (P < 0.05) inhibited increases in cyclic GMP concentration in response to both receptor- and non-receptor-mediated nitric oxide synthase activating agents. Inhalational anesthetics do not stimulate or inhibit basal cyclic GMP production in co-cultures or VSM, suggesting that inhalational anesthetics do not activate soluble or particulate guanylyl cyclase and do not activate nitric oxide synthase. Inhalational anesthetics do not inhibit nitrovasodilator-induced cyclic GMP formation, suggesting a lack of interference with soluble guanylyl cyclase activation. Inhalational anesthetics inhibit both agonist and calcium ionophore-stimulated nitric oxide-dependent cyclic GMP accumulation in endothelial cell-vascular smooth muscle co-cultures. Consistent with previous vascular ring studies, anesthetics appear to inhibit nitric oxide-guanylyl cyclase signaling distal to receptor activation in the endothelial cell and proximal to nitric oxide activation of guanylyl cyclase.