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RECEPTORS AND SIGNAL TRANSDUCTION

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca²⁺ in human atrial myocardium

¹Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne; and ²Laboratory for Muscle Research and Molecular Cardiology, Department of Internal Medicine III and ³Department of Cardiothoracic Surgery, University of Cologne, Cologne; and ⁴Clinic II of Internal Medicine and Cardiology, Oberpfalz, Germany

Submitted 7 January 2005 ; accepted in final form 2 December 2005

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation.

₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation

This article has been cited by other articles:

				U. M. Fischer, R. Schindler, K. Brixius, U. Mehlhorn, and W. Bloch Extracorporeal Circulation Activates Endothelial Nitric Oxide Synthase in Erythrocytes Ann. Thorac. Surg., December 1, 2007; 84(6): 2000 - 2003. [Abstract] [Full Text] [PDF]