Appavoo Rengasamy

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

Background 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.

Inhalational anesthetic effects on rat cerebellar nitric oxide and cyclic guanosine monophosphate production

Background Inhalational anesthetics interact with the nitric oxide‐cyclic guanosine monophosphate (NO‐cGMP) pathway in the central nervous system (CNS) and attenuate excitatory neurotransmitter‐induced cGMP concentration. The site of anesthetic action on the NO‐cGMP pathway in the CNS remains controversial. This study investigated the effect of inhalational anesthetics on N‐methyl‐D‐aspartate (NMDA)‐stimulated NO synthase activity and cyclic cGMP production in rat cerebellum slices. Methods The interaction of inhalational anesthetics with NO synthase activation and cGMP concentration was determined in cerebellum slices of 10‐day‐old rats. Nitric oxide synthase activity in cerebellum slices was assessed by measuring the conversion of L‐[sup 3 H]arginine to L‐[sup 3 H]citrulline. The cGMP content of cerebellum slices was measured by radioimmunoassay. Results Isoflurane at 1.5% and 3% enhanced the NMDA‐stimulated NO synthase activity by two times while halothane at 1.5% and 3% produced no significant effect. However, the NMDA‐stimulated cGMP production was inhibited by both anesthetic agents. The anesthetic inhibition of cGMP accumulation was not significantly altered by a mixture of superoxide dismutase and catalase or by glycine, a coagonist of the NMDA receptor. Conclusions The enhancement of NMDA‐induced NO synthase activity by isoflurane and the inhibition of NMDA‐stimulated cGMP production by halothane and isoflurane suggests that inhalational anesthetics interfere with the neuronal NO‐cGMP pathway. This inhibitory effect of anesthetics on cGMP accumulation is not due to either their interaction with the glycine binding site of the NMDA receptor or to the action of superoxide anions.

Determination of nitric oxide synthase activity by measurement of the conversion of l-arginine to l-citrulline

Abstract Nitric oxide (NO), first recognized for its importance as an endothelium-dependent vasodilator, is now recognized as an important messenger molecule for the activation of soluble guanylate cyclase in a wide variety of tissues, including the central and the peripheral nervous system. In addition, NO by itself may have multiple functions independent of cyclic GMP, ranging from cytotoxicity to ADP ribosylation. In brain tissue, NO is produced enzymatically from l -arginine by NO synthase, a constitutive, calcium-, calmodulin-, and NADPH-dependent enzyme. In this article we describe several methods for the quantitation of NO synthase activity based on the measurement of l -citrulline, the stable by-product of NO synthesis from l -arginine. This method is sensitive, rapid, and reliable and can be performed with a simple experimental setup that is convenient for quantitating NO production of a large number of samples at a time. The l -citrulline methodology is compared to several other methods of quantifying NO and NO synthase activity, and its advantages and disadvantages are discussed.

Endothelium-Derived Relaxing Factor (EDRF)

Recent investigations have greatly improved our understanding of the chemical nature of endothelium-derived relaxing factor (EDRF) and the regulation and metabolic pathway of its production. EDRF is a potent but labile relaxing factor with a biologic half-life of between 6.3 and 50 seconds in an oxygenated aqueous medium.1,2 The production of EDRF from the endothelium requires an increase in intracellular calcium.3,6 Following its production and release from the endothelial cell, EDRF is transferred to the vascular smooth muscle (VSM) where it activates soluble guanylate cyclase resulting in an increase in smooth muscle cyclic GMP concentration, which correlates with its relaxing action.7–13 Extremes of both high and low oxygen tension inhibit the production or stability of EDRF.14–15 Early investigations into the chemical nature of EDRF implicated an unstable, non-prostanoid oxidation product of arachidonic acid or some type of free radical.1,16–18 A great deal of recent evidence, however, suggests that EDRF is nitric oxide or a similar nitrogen oxide species.19–29 EDRF can be formed from L-arginine by a pathway involving a calcium-, calmodulin-and NADPH-dependent enzyme.30–39 EDRF synthesis has now been described in a wide range of cell types in addition to the endothelium, and indeed EDRF may be the second messenger responsible for the activation of guanylate cyclase in most cells containing the enzyme.34,35,38–43 This manuscript will present data from our laboratory which support these and other pharmacologic characteristics of EDRF.