Hypoxia inverts the negative chronotropic response to norepinephrine in normoxia in cultured neonatal rat cardiac myocytes: role of the alpha 1 adrenergic signal transduction system

Abstract

In the present study, we investigated the effect of hypoxia on the chronotropic response to norepinephrine (NE) of cultured neonatal rat ventricular myocytes. We measured beating of myocytes with the Fotonic sensor, using a newly developed method for a noncontact displacement measurement. The beating rate counted with the sensor had a high correlation coefficient with that counted visually under a microscope (r = 0.997, P < 0.01). NE concentrations of 10(-8) - 10(-4) M caused negative chronotropy dose dependently in the presence of 5 x 10(-7) M propranolol. NE-induced chronotropy was completely antagonized by 10(-6) M prazosin. Three hours hypoxia decreased the spontaneous beating rate 40% (P < 0.01). Negative chronotropy induced by 10(-4) M NE in normoxia was inverted to positive and was antagonized by prazosin. Hypoxia increased the basal level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to 190% (P < 0.01), while NE-stimulated Ins(1,4,5)P3 production was significantly suppressed. Immunoblotting analysis of G protein subunits demonstrated no quantitative changes in Gi alpha, Gq alpha, Go alpha and G beta common subunits in hypoxia. In a saturation binding assay with [3H]prazosin, Kd values were increased to 152% by hypoxia (P < 0.05) without significant change in Bmax. Basal activity of low Km-GTPase was increased to 122% by hypoxia (P < 0.05). These results suggest that the hypoxia-induced increase in low-Km GTPase activity, which could stimulate phospholipase C by an activated alpha GTP subunit of G protein and consequently induce receptor-independent increase in Ins(1,4,5)P3, may be responsible for the inversion of the NE-induced negative chronotropic response in normoxia.