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


METHODS
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.


METHODS
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.


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.
Kagiya
T
T
Cardiology Section (IIIc), Veterans Affairs Medical Center, San Francisco, CA 94121.
Rocha-Singh
K J
KJ
Honbo
N
N
Karliner
J S
JS
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D002478:Cells, Cultured; D007295:Inositol Phosphates / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010100:Oxygen / Q000502:physiology*; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D007295:Inositol Phosphates; D011942:Receptors, Adrenergic, alpha; D010100:Oxygen; D009638:Norepinephrine