The overall objective of the proposed research is to evaluate the possible participation of calcium ion as an intracellular messenger mediating hormone action on adipose tissue cells. Earlier studies using fat cells and other cell systems have provided data suggesting an action of calcium prior to and antecedent to formation of cyclic AMP. In my laboratory, experiments to localize the site of calcium action on isolated fat cells are conducted by measuring cyclic AMP levels, rates of lipolysis and activity of the cyclic AMP dependent protein kinase in hamster adipocytes exposed to various stimuli and inhibitors of lipolysis. Subsequent studies will employ various pharmacologic agents which either promote (A23187 and X537A) or retard (verapamil and D600) calcium entry into cells. Another series of experiments are designed to prepare adipocyte-membrane "ghosts" filled with Ca ions -EGTA buffers which will "clamp" the intracellular calcium concentration at any desired level. By studying hormone activation of adenylate cyclase in such a preparation we hope to determine the relative roles of intra- and extracellular calcium in control of cyclic AMP formation. Lastly, studies are proposed to determine if hormones influence the distribution of calcium in adipocytes and if changes in the influx and/or the calcium into adipocytes is a necessary concomitant to hormone regulation of lipolysis and cyclic AMP formation.