Copyright © 2005, European Society of Cardiology
Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes
Servicio de Cardiología, Hospital Universitari Vall d'Hebron, Pg Vall d'Hebron 129, 08035 Barcelona, Spain
* Corresponding author. Tel.: +34 93 489 40 38; fax: +34 93 489 40 32. Email address: dgdorado{at}vhebron.net
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 (GCNO) 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 GCNO 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 β1-and 
1-subunits of GCNO and a similar proportion of GCNO activity were found in the particulate fraction. The dependence of GCNO activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187
[GenBank]
caused GCNO 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 GCNO, 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 GCNO activity but not with their effects on cytosol-associated GCNO.
Conclusion: These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GCNO differs from that of cytosolic GCNO, and 3) membrane association may have a crucial role in determining the response of cells to NO.
KEYWORDS Nitric oxide; Calcium (cellular); Signal transduction; Myocytes; Protein phosphorylation
* Ajay Shah (King's College, London) served as Guest Editor for this manuscript
Time for primary review 19 days
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