The role of Ca ions and cAMP in ACTH lipolysis

Abstract

Ca ions and cAMP are known to be involved in the action of hormones. In this study, the actions of Ca ions and cAMP in ACTH lipolysis were investigated by comparing ACTH1-24, ACTH1-10 and ACTH11-24 with regard to: lipolytic activity, binding affinity to the fat cell membrane, changes in Ca ion binding to the fat cell membrane and cAMP formation. Rat adipocytes were used. Lipolytic activity was measured in terms of the amount of FFA released from fat cells into the medium after incubation with the ACTH analogs at 37 degrees C for 30 minutes. Binding affinity to the fat cell membrane was expressed as competitive binding affinity, determined by simultaneously incubating fat cell ghosts with 125I-ACTH1-24 and unlabelled ACTH analogs at 4 degrees for 40 minutes. Changes in Ca ion binding to the fat cell membrane was expressed as Ca binding capacity, measured by simultaneously incubating fat cell ghosts with 45Ca and ACTH analogs at 24 degrees C for 20 minutes. CAMP formation was measured by RIA as the amount of cAMP formed in fat cells incubated with 10(-6)M ACTH analogs at 37 degrees C for 0, 3, 5 and 10 minutes. FFA release after incubation with each of the ACTH analogs at 10(-6) M was 1.17 mEq/L/tube for ACTH1-24, 0.26 mEq/L/tube for ACTH1-10 and 0.25 mEq/L/tube for ACTH11-24, showing a high value only for ACTH1-24. The binding affinity to the fat cell membrane (high affinity constant) was 4.4 X 10(-8) M for ACTH1-24, 4.6 X 10(-8) M for ACTH1-10 and 4.6 X 10(-8) M for ACTH11-24. There were no significant differences among the binding affinities of these ACTH analogs. The binding capacity of 45Ca++ to the fat cell membrane (high affinity constant) was 3.64 X 10(-6) M for ACTH1-24, 4.49 X 10(-6) M for ACTH1-10 and 5.46 X 10(-6) M for ACTH11-24. There were no significant differences among these ACTH analogs. The increase in cAMP formation during the first 3 minutes of incubation was 35.5 fmol/mg dry weight of fat cells for ACTH1-24, 2.2 fmol/mg for ACTH1-10 and 1.8 fmol/mg for ACTH11-24. An increase was only observed for ACTH1-24. The fact that FFA release and cAMP formation were high only with ACTH1-24 among the ACTH analogs tested indicates that cAMP formation plays an important role in the lipolytic activity of ACTH in fat cells and that the three-dimensional structure of ACTH is important for cAMP formation.