© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia
Physiologisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
* Corresponding author. Tel.: +49-641-994-7243; fax: +49-641-994-7239 Gerhild.Taimor{at}physiologie.med.uni-giessen.de
Objective: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia–reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. Methods: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. Results: Incubation of cardiomyocytes with the NO-donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 100 µM) induced apoptosis in 14.1±1.9% of the cells and necrosis in 24.4±4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 µM, 9.6±0.6% apoptotic cells) or YC-1 (75 µM, 14.6±2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9±0.7% or 13.4±2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. Conclusions: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection.
KEYWORDS Apoptosis; Cell culture/isolation; Myocytes; Nitric oxide; Second messengers
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