Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes

Objective: Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca2+-dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GC(NO)) to the cell membrane has been recently proposed to play a role. The aim of this study was to determine the possible functional relevance Of GC(NO) bound to the cardiomyocyte membrane. Methods: Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GCNO activity was assayed in parallel experiments. Results: In untreated cardiomyocytes, approximately 30% of beta(1)-and alpha(1)-subunits of GC(NO) and a similar proportion Of GC(NO) activity were found in the particulate fraction. The dependence of GC(NO) activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 caused GC(NO) to translocate to the sarcolemma, increased GCNO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca2+-dependent changes on the phosphorylation status of GC(NO), since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca2+/calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GC(NO) activity but not with their effects on cytosol-associated GC(NO). Conclusion: These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GC(NO) differs from that of cytosolic GC(NO), and 3) membrane association may have a crucial role in determining the response of cells to NO. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.