Alterations in α1-Adrenergic Receptor-mediated Phosphatidylinositol Turnover in Hypoxic Cardiac Myocytes

The purpose of these studies was to examine the effects of hypoxia on α1-adrenergic receptor (α1AR) mediated phosphatidylinositol (PI) turnover in cultured neonatal rat cardiac myocytes. Cells were pre-labeled with [3H]-inositol and incubated for 1 h in either normoxia or hypoxia. Phenylephrine, an α1AR agonist, was added at various time intervals (0-60 min) before termination of the incubation. There was a time-dependent release of radioactivity from the lipid fraction to the aqueous fraction with α1AR stimulation. α1AR-mediated PI turnover was biphasic in normoxic cells and monophasic in hypoxic cells. Using ion-exchange chromatography, radioactivity in the inositol trisphosphate (IP3) peak was increased with acute phenylephrine stimulation (5 min) in the normoxic cells, while inositol phosphate (IP) and inositol bisphosphate (IP2) were increased with chronic stimulation (60 min). After 5 min of α1AR stimulation, hypoxia did not alter total aqueous radioactivity when compared to normoxia, but there was a significant increase in IP2. However, there was decreased PI turnover in chronically stimulated (30-60 min) hypoxic cells when compared to normoxic cells. Hypoxia had no effect on radioactivity in the IP3 fraction with either 0, 5, or 60 min of α1AR stimulation, but there was a significant increase in [1,4,5,]-IP3 in hypoxic cells with 30 s α1AR stimulation. With hypoxia, there was no difference in radioactivity in the phosphatidylinositols with either 0 or 5 min stimulation when compared to normoxia. However, after 60 min of α1AR stimulation, hypoxia resulted in increased PI and PIP, when compared to normoxic cells, but PIP2 radioactivity was unchanged. There was no effect of pertussis toxin on either the acute or chronic phase of PI turnover, negating involvement of Gi or Go. These data suggest that α1AR stimulation in neonatal rat cardiac myocytes is biphasic, and that hypoxia produces a slower monophasic response during extended α1-agonist exposure as would be found with ischemia.