The role of calmodulin in the responses to adrenocorticotropin of plasma membranes from adrenal cells.

Inhibitors of calmodulin [trifluoperazine, chlorpromazine, pimozide, and calmidazolium N-(6-aminohexyl)5-chloro-1-napthalenesulphonamide (W7)] and calmodulin antibodies were used to investigate the role of calmodulin in the response of Y-1 mouse adrenal cells to ACTH, with particular reference to events in the plasma membrane. In whole cells it was found that two responses (production of steroids and cAMP) to two stimulating agents (ACTH and forskolin) were inhibited by trifluoperazine at concentrations consistent with those involved in binding of the inhibitor to pure calmodulin (10-25 microM). The steroidogenic responses were also inhibited by the three other inhibitors of calmodulin (chlorpromazine, calmidazolium, and W-7). Trifluoperazine and pimozide (1-500 microM) did not inhibit binding of an [125I]ACTH analog to highly purified plasma membranes of Y-1 cells or to the cells themselves. With Y-1 plasma membranes it was found that trifluoperazine, pimozide, W-7, and calmodulin antibodies inhibited the increase in adenylate cyclase activity in response to ACTH, but not the cyclase responses to cholera toxin or forskolin. Moreover, the effect of cholera toxin on the ADP-ribosylation of specific membrane substrates was independent of the presence or absence of endogenous and/or exogenous Ca2+/calmodulin. The response of adenylate cyclase to ACTH was also decreased in plasma membranes from which calmodulin was removed by washing, and exogenous calmodulin partly reversed this decrease. Anti-calmodulin immunoglobulin inhibited the stimulation of adenylate cyclase produced in plasma membrane by ACTH, but was without effect on the responses to cholera toxin and forskolin. Exogenous calmodulin partly reversed the inhibition of stimulation by ACTH of adenylate cyclase produced by the antibody. It is concluded that calmodulin influences the events taking place in the plasma membrane in response to ACTH, after the binding of the hormone to its receptor and before the action of the G protein (Gs). That is, calmodulin is involved in coupling the occupied receptor to Gs. The effects of inhibitors of calmodulin in whole cells must involve some additional effect(s) requiring the intact cell.