Age-related decline in the activitities of erythrocyte membrane adenylate cyclase and protein kinase

Abstract

Erythrocytes from normal male CD rats were separated into four age groups by centrifugation on discontinuous Stractan (a polyarabinogalactan, 30,000 molecular weight) gradients. Age separation was confirmed by the measurement of resistance to osmotic lysis in each group. As the cell density increased, their resistance to lysis decreased. Reticulocyte counts in the four fractions were 9.2, 3.5, 1.6, and 0.4%, the youngest (lightest) to oldest cells, respectively. Membranes from cells in each age group were prepared by hypotonic lysis and washing out of hemoglobin. Basal level of erythrocyte membrane adenylate cyclase activity decreased with increasing cell age from 7.05 ± 0.64 pmol cAMP/mg membrane protein/10 min in the youngest to 0.34 ± 0.31 in the oldest cells. Fluoride-stimulated activity (10 m m F −) also decreased with increasing cell age from 347 ± 39 to 63 ± 3. The ratio of F −-stimulated to basal activity did not change as a function of cell age, indicating that these two activities decreased at a similar rate. Isoproterenol (10 −4 m, Iso)-stimulated adenylate cyclase activity decreased from 683 ± 45 to 179 ± 13. In contrast to F −-stimulated/basal activity, the ratio of Iso-stimulated to basal activity decreased as a function of cell age. Thus, hormone sensitivity changes at a different rate than basal and F −-stimulated activity of erythrocyte membrane adenylate cyclase. This suggests that either the number of Iso receptors on the erythrocyte surface or the coupling of receptors to the enzyme changes with cell age. Protein kinase activity in membranes prepared from age-separated erythrocytes decreased with increasing cell age from 690 ± 25 to 284 ± 44 pmol/mg membrane protein/5 min. Stimulation of this enzyme by exogenous cAMP could not be demonstrated, probably due to a significant level of endogenous cAMP production. In addition to showing a decrease in adenylate cyclase and protein kinase activities with cell age, we have developed a relatively simple method for obtaining a highly hormone sensitive mammalian erythrocyte system.