Endothelin-1 and phenylephrine-induced activation of the phosphoinositide cycle increases cell injury of cultured cardiomyocytes exposed to hypoxia/reoxygenation

Abstract

We explored the effect of glucose-free hypoxia/reoxygenation of cultured neonatal rat ventricular myocytes on endothelin-1 and alpha 1-adrenoceptor induced activity of the phosphoinositide cycle. At the same time the influence of these agonists on depletion of energy-rich phosphates and cellular damage was assessed. Glucose-free hypoxia did not lead to an increase in basal phospholipase C activity. However, endothelin-1 (10(-8) M) and phenylephrine (10(-5) M) evoked activation of phospholipase C was attenuated after 60 min of hypoxia and declined to 38% and 30% respectively of normoxic values after 90 min of hypoxia. During glucose-free hypoxia, phosphatidylinositol 4,5-bisphosphate, the substrate for phospholipase C, but not phosphatidylinositol or phosphatidylinositol 4-monophosphate was seen to decline to 59% of normoxic values which was independent of activation of phospholipase C by agonists. ATP levels decreased after 30 min of hypoxia and declined to 29% relative to normoxic control after 90 min of hypoxia. Total adenine nucleotide levels showed a similar pattern. The presence of 10(-8) M endothelin-1 during hypoxia did not influence the magnitude of ATP depletion. However, after 15 min of reoxygenation, by itself not significantly leading to recovery of ATP levels, ATP levels were decreased by endothelin-1 as compared to hypoxia/reoxygenation without phospholipase C agonist. Cellular damage as determined by lactate dehydrogenase leakage was not observed during 90 min hypoxia. Reoxygenation resulted in a three-fold increase in enzyme release relative to normoxic control. In the presence of endothelin-1 or phenylephrine this reoxygenation-induced damage was respectively 1.7 and 3.0-fold increased. We conclude that the agonist-induced activity of the phosphoinositide cycle is decreased in time during glucose-free hypoxia, partially through a decrease in phosphatidylinositol 4,5-bisphosphate level. However, the remaining activity may give rise to increased cellular damage. As endothelin-1 and alpha 1-adrenergic amines are known to be released during myocardial ischemia, stimulation of the phosphoinositide cycle by these agonists might be an important factor in determining the magnitude of myocardial injury.