The long-term objectives of this research is to understand the mechanisms by which glucocorticoid hormone excess, thyroid hormone excess and estrogen deficiency lead to osteopenia and magnesium deficiency leads to skeletal resistance to resorptive stimuli. In the proposed study we will test the hypothesis that these conditions induce changes in the regulatory properties of bone adenylate cyclase. Approximately two-thirds of this investigation will involve the detailed characterization of the adenylate cyclase present in membranes obtained from bones of normal adult guinea pigs. This will entail: 1) determination of the effects of PTH, isoproterenol, and prostaglandin E2, agonists which activate bone adenylate cyclase, on the Ka for Mg++ and on the Ki for calcium of the high and low affinity calcium inhibition sites previously identified in the enzyme complex; 2) identification of the sites within the enzyme complex where Mg++ binding activation occurs; and 3) identification of the sites within the enzyme complex where Ca++ binding inhibits adenylate cyclase activity and investigation of the mechanism by which Ca++ inhibition occurs. Reconstitution methodology employing plasma membranes from turkey erythrocytes and S49 cyc-murine lymphoma cells and cholate extracts from bone membranes will be used in these latter two studies. The possible role of the inhibitory guanine nucleotide regulatory protein, Ni, in the calcium inhibition of the adenylate cyclase will also be evaluated. Bone membranes will be pretreated with pertussis toxin to inactivate Ni and the parameters of calcium inhibition of the adenylate cyclase activity in the treated membranes compared to control membranes. Once this baseline data has been obtained, the regulatory characteristics of adenylate cyclase with respect to Mg++ and agonist activation and Ca++ inhibition will be examined in bone membranes obtained from animals exposed in vivo to: 1) glucocorticoid excess, 2) thyroid hormone excess, 3) estrogen deficiency, and 4) magnesium deficiency. In addition, the effects of glucocorticoid or thyroid hormone excess and estrogen on magnesium deficiency on the relative amounts of the stimulatory and inhibitory guanine nucleotide regulatory proteins, Ns and Ni, in the bone membranes will be determined.
