THE INVITRO EFFECT OF HYPOXIA ON THE TROUT ERYTHROCYTE BETA-ADRENERGIC SIGNAL TRANSDUCTION SYSTEM

We have investigated the effects of acute in vitro hypoxia on trout (Oncorhynchus mykiss) erythrocytes in order to elucidate the mechanism(s) by which increased catecholamine responsiveness of the Na+/H+ antiporter is achieved. Blood was withdrawn from cannulated trout and maintained in vitro under normoxic (P(O2) = 1 7.9+/-0.2 kPa, N=12) or hypoxic (P(O2) = 2.8+/-0.1 kPa, N=12) conditions for 30 min prior to exposure to concentrations of noradrenaline, forskolin or 8-bromo-cyclic AMP ranging from 0 to 10(-6) mol l-1, 10(-7) to 10(-5) mol l-1 or 10(-4) to 10(-2) mol l-1, respectively. Na+/H+ exchange activity was quantified as the maximal reduction in whole-blood pH (pHe) after addition of the various Na+/H+ antiporter activators. Erythrocyte intracellular cyclic AMP contents were also determined after addition of noradrenaline or forskolin. To complete the investigation, radioreceptor binding assays were conducted on separate blood samples to characterize the numbers and affinities of the surface population of beta-adrenoceptors of erythrocytes maintained under normoxic or hypoxic conditions. Exposure of erythrocytes to noradrenaline, forskolin or 8-bromo-cyclic AMP resulted in dose-dependent reductions in pHe as a result of Na+/H+ antiporter activation. In all cases, the effects were significantly more pronounced under hypoxic than normoxic conditions. Hypoxia significantly increased the production of cyclic AMP in the presence of noradrenaline but did not affect the forskolin-induced production of cyclic AMP. Blood oxygen status also affected the number of beta-adrenoceptors expressed at the erythrocyte surface; hypoxic erythrocytes possessed 880.7+/-28.6 (N=6) receptors per cell whereas normoxic erythrocytes possessed 532.6+/-43.2 (N=6) receptors per cell. These results suggest that in vitro exposure of trout erythrocytes to hypoxic conditions results in at least two significant alterations in the catecholamine signal transduction system: (1) an enhancement in erythrocyte cyclic AMP production, in part by virtue of an increase in the number of surface beta-adrenoceptors, and (2) a hypoxia-induced increase in the cyclic AMP sensitivity of one or more of the steps culminating in Na+/H+ antiporter activation. These events ultimately increase the responsiveness of the erythrocyte Na+/H+ antiporter to catecholamines during hypoxia.