Alpha 1 adrenoceptor mediated signal transduction in neonatal rat ventricular myocytes: effects of prolonged hypoxia and reoxygenation.

ABSTRACT

STUDY OBJECTIVE: The aim was to study the effects of prolonged hypoxia and reoxygenation on alpha 1 adrenoceptors and inositol phosphate accumulation in neonatal rat ventricular myocytes maintained in culture for 6-8 d. DESIGN: Neonatal rat ventricular myocytes were subjected to 2 h hypoxia followed by 2 h reoxygenation. Cells were harvested at various times during hypoxia and after reoxygenation and measurements of alpha 1 adrenoceptor density and affinity and determinations of basal and (-)-noradrenaline stimulated inositol phosphate accumulation were carried out. EXPERIMENTAL MATERIAL: A neonatal rat ventricular myocyte preparation almost completely free of contaminating non-myocytes was used. Cells were grown in serum containing medium for 5 d before experiments were performed. alpha 1 Adrenoceptors were measured using the radioligand 125I-HEAT and inositol phosphates were measured by anion exchange chromatography after incubation with 1 microM (-)-noradrenaline for 5 min. MEASUREMENTS AND MAIN RESULTS: Hypoxia resulted in an increase in alpha 1 adrenoceptor density which was reversed by reoxygenation. There were no changes in antagonist affinity. (-)-Noradrenaline stimulated inositol phosphate production was increased at 1 h hypoxia but declined to control levels after 2 h hypoxia, while basal levels increased significantly at this time. This pattern was similar for all inositol phosphates measured inositol-1-phosphate, inositol bisphosphate, and the putative second messenger, inositol trisphosphate. Displacement by (-)-noradrenaline of 125I-HEAT binding was significantly shifted to the right after 2 h hypoxia. CONCLUSIONS: Prolonged hypoxia in neonatal rat ventricular myocytes increases alpha 1 adrenoceptor density without change in antagonist affinity. Inositol phosphates follow a biphasic response, increasing after 1 h and decreasing after 2 h hypoxia in response to (-)-noradrenaline stimulation. This second messenger response and the rightward shift of the (-)-noradrenaline displacement curve suggests that after 2 h hypoxia there is a decrease in agonist affinity for the alpha 1 adrenoceptor consistent with uncoupling of the alpha 1 adrenoceptor from its effector.