REVERSIBLE S-NITROSATION OF CREATINE-KINASE BY NITRIC-OXIDE IN ADULT-RAT VENTRICULAR MYOCYTES

We have previously demonstrated that the nitric oxide (NO) donor S-nit roso-N-acetylcysteine (SNAC) reversibly decreases the activity of crea tine kinase (CK) in an isolated rat heart preparation, markedly suppre ssing myocardial contractile responsiveness to an inotropic challenge. We wished to further examine the role of exogenous and endogenous sou rces of NO species on S-nitrosation of GK and subsequent enzyme activi ty in adult rat ventricular myocytes (ARVM), Two S-nitrosothiol groups were formed in the GK dimer after nitrosation of rabbit skeletal musc le CK in solution. CK inactivation due to S-nitrosation was time-and c oncentration-dependent in solution and in ARVM lysate for both NO dono rs S-nitroso-N-acetylpenicillamine (SNAP) and SNAG and was rapidly rev ersible with the sulfhydryl dithiothreitol (DTT). Similarly, SNAG or S NAP dose-dependently decreased CK activity in intact ARVM, which was f urther attenuated by increasing the metabolic activity of the cells wi th electrical pacing for 1 h. Co-cultures of ARVM with interleukin 1 b eta (IL-1 beta)- and interferon gamma (IFN gamma) pretreated cardiac m icrovascular endothelial cells (CMEC) caused no detectable decline in myocyte CK activity. Increasing GSH levels attenuated the decline in m yocyte CK activity with SNAG, while decreases in myocyte GSH levels en hanced the inhibitory effect of SNAG on intact myocyte CK activity. Th ese data indicate that the degree of inhibition of cardiac myocyte CK by NO is dependent on the extent of myocyte metabolic activity and the intracellular GSH content. (C) 1998 Academic Press Limited.