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390 DEPENDENCY OF ENDOTHELIAL NITRIC OXIDE SYNTHASE-ACTIVATION MECHANISMS ON INTRACELLULAR CA2+-CONCENTRATION IN HUMAN ATRIAL MYOCARDIUM
  1. C. Pott,
  2. W. Bloch1,
  3. D. Steinritz1,
  4. K. Brixius,
  5. U. Mehlhorn,
  6. C. Ziskoven,
  7. R. H.G. Schwinger
  1. Klinik III for Internal Medicine, 1Institute I for Anatomy, 2Clinic for Thoracic and Cardiovascular Surgery, University of Cologne

Abstract

We have recently shown two ways of endothelial nitric oxide synthase (eNOS)-activation in human myocardium (Brixius et al. 2004): 1) a translocation and 2) an Akt-dependent phosphorylation of the enzyme at Ser1177. The present study investigates the Ca2+ dependency of these two mechanisms. Methods and Results: Human right atrial tissue was obtained from patients undergoing coronary bypass-operation. In immunohistochemical experiments, the translocated form of eNOS as well as the (Ser1177)-phosphorylated eNOS (peNOS) and the phosphorylated form of Akt (pAkt) were labelled using specific antibodies. eNOS-translocation was measured in the absence and presence of the Ca2+ chelator BAPTA after application of BRL 37344 (BRL), a preferential β3-adrenoceptor agonist, which has been shown to increase eNOS activity in human right atrial myocardium. In the absence of BAPTA, BRL (10 μM) time-dependently increased staining intensity of translocated eNOS (+BRL, t=5 min, +53.52±15.21%; n=6; p≤0.05), whereas in the presence of BAPTA, this effect was blunted (20 μM; +BRL (10 μM; t=5 min): -42.11±11.26%; n=4; p(0.05). In contrast, BRL 37344 significantly increased staining of peNOS (+BRL 37344 (10 μM; t=5 min): +156.6±26.4%; n=3; p≤0.05) and pAkt in the presence of BAPTA. Using the NO-sensitive dye diaminofluorescein (DAF), we could show that BRL induced a strong release of NO (+BRL 37344 (10 μM): +237.1±70.4%). This effect however was completely abolished in the presence of BAPTA. Though Ca2+ dependent, the translocation state of myocardial eNOS was not changed by the adenylate cyclase activator forskolin (0.3 μM) (+forskolin (t=5 min): -20.2±12.3%; n=3; p(0.05) nor was NO-release induced (n=2). Conclusions: 1) In human myocardium, BRL induced eNOS-translocation is dependent on intracellular Ca2+ whereas eNOS-phosphorylation is not. 2) At least in atrial myocardium, eNOS-translocation and not eNOS-phosphorylation generates bulk NO. 3) Though Ca2+ dependent, eNOS-translocation and NO-release could not be mimicked by adenylate cyclase activation as a mediator of β-adrenergic stimulation.

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