Effects of catecholamines and prostaglandin E1 on cyclic AMP, cation fluxes, and protein phosphorylation in the frog erythrocyte.

Stimulation of the 8-adrenergic receptor of the intact frog erythrocyte resulted in increased cyclic AMP levels, but substantial effects could only be observed in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP were about 0.1 to 0.2 pmol/liter of cells; incubation for 20 min with isoproterenol (lo-‘ M) plus isobutylmethylxanthine (IBMX) M) resulted in an increase to 15 pmol/liter of cells. Prostaglandin El was also effective, in combination with isobutylmethylxanthine, in raising cyclic AMP levels. Both isoproterenol and prostaglandin El (in combination with IBMX) caused a severalfold increase in sodium and potassium influxes, and these effects were mimicked by the addition of exogenous cyclic AMP. The increased cation fluxes caused a net uptake of salt and water, resulting in an increase in cell volume of about 8%. Agents that stimulated cation influx also stimulated sodium efflux, but inhibited potassium efflux. When cells had been preincubated with 32Pi, the incorporation of 32P into a protein band with an apparent M, = 240,000 was stimulated by either isoproterenol or prostaglandin El (in combination with IBMX) in a dosedependent fashion that resembled the effects of these agents on cation influx. The influx of sodium and potassium induced by isoproterenol or prostaglandin El was transient; sodium influx increased during the first 30 min of incubation and then declined toward the basal level, while the rise and fall of potassium influx was somewhat slower. 32P incorporation into the M, = 240,000 protein occurred without an apparent lag after hormone addition and paralleled or preceded the rising phase of the cation transport response, but did not return to basal levels. Both isoproterenol and prostaglandin El also stimulated 32P incorporation into proteins with M, = 50,000 and M, = 18,000, but these effects were not apparent until 20 min and 60 min after hormone stimulation, respectively. It is uggested that phosphorylation of a protein with an apparent M, = 240,000 may be involved in the mechanism by which cyclic AMP affects cation permeability in frog erythrocytes. This protein may be homologous to a protein of similar molecular weight present in the turkey erythrocyte whose state of phosphorylation correlates with cyclic AMP-dependent increases in cation permeability.