Molecular Pharmacology, Vol 13, 786-795, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

The Role of Guanyl Nucleotides in the Expression of Catecholamine-Responsive Adenylate Cyclase during Maturation of the Rat Reticulocyte

¹ Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, and Section on Mineral Metabolism, Metabolic Diseases Branch, National Institute of Arthritis, Metabolic, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

Adenylate cyclase in membranes prepared from rat reticulocytes and control erythrocytes was studied in relation to sensitivity to isoproterenol and the guanyl nucleotides GTP and 5'-guanylylimidodiphosphate [Gpp(NH)p]. In reticulocyte preparations, isoproterenol alone activates adenylate cyclase to a rate equivalent to 70% of that obtained with isoproterenol plus GTP or Gpp(NH)p. In contrast, only 20% of maximal enzyme activity [isoproterenol plus Gpp(NH)p] in control membranes is sensitive to isoproterenol alone. Maximal adenylate cyclase activity in reticulocytes can be achieved by the use of isoproterenol and either GTP or Gpp(NH)p. Maximal adenylate cyclase activity in control membranes occurs only with isoproterenol and Gpp(NH)p. As the reticulocyte differentiates to the mature erythrocyte, maximal enzyme stimulation becomes more dependent upon the presence of Gpp(NH)p. In both membrane preparations, Gpp(NH)p decreases by 10-fold the concentration of isoproterenol required for half-maximal activity (K_A) and increases by 5-fold the concentration of propranolol required for half-maximal inhibition (I₅₀). GTP has no effect upon the K_A for isoproterenol or the I₅₀ for propranolol in either set of membranes. Gpp(NH)p but not GTP allows development of the holocatalytic state. Even in the presence of Gpp(NH)p, however, there is still much less catecholamine-responsive adenylate cyclase in control membranes than in reticulocyte membranes. Direct detection of beta adrenergic receptors with the specific ligand [¹²⁵I]hydroxybenzylpindolol showed that neither GTP nor Gpp(NH)p alters receptor number or affinity for agonist or antagonist. It is concluded that the loss of catecholamine-sensitive adenylate cyclase in control membranes involves both a decrease in catalytic units of adenylate cyclase and also an uncoupling of the beta receptor. Gpp(NH)p recouples the beta receptor and adenylate cyclase by mechanisms that do not perturb hormone-receptor interaction. It is suggested that the distinct effects of the two guanyl nucleotides upon coupled reticulocyte membranes and uncoupled control membranes are due to differing metabolic fates of the guanyl nucleotides, possibly at specific sites on the adenylate cyclase complex for these nucleotides.

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