To investigate the mechanisms of ß3-adrenergic eNOS activation, we performed Western blot and immunohistochemical examinations of eNOS translocation and phosphorylation after incubation of isolated trabeculae of human left ventricular failing myocardium (AHA/ESC classification D, orthotopic heart transplantations, n= 12) with the preferential ß3-adrenoceptor agonist BRL 37344 (10 mM, 5 min.).

BRL did not alter eNOS-translocation, eNOS Thr495- and Ser635 phosphorylation in cardiomyocytes. BRL decreased eNOS phosphorylation at Ser1177 and Ser114. Since these alterations correspond to a decreased NO-liberation, we investigated the protein expression and the subcellular distribution of the ß3- adrenoceptor. In human failing left ventricular myocardium, a prominent location of the ß3-adrenoceptor was present in endothelial rather than in myocardial cells. In conclusion, in human failing myocardium ß3-adrenergic stimulation decreases eNOS activation in the cardioyocytes. The NO-dependent negative inotropic effect observed after ß3-adrenergic stimulation in human failing myocardium may be due to a paracrine effect via NO-liberation from the cardiac endothelial cells. Thus, the present paper points towards a paracrine mechanism by which the microcirculation may influence cardiac contractility after ß3- adrenergic stimulation.