THE ROLES OF ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE-A AND PROTEIN-KINASE-C IN STIMULUS-SECRETION COUPLING IN ATT-20 CELLS

The ACTH-secreting mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to study adenosine 3',5'-cyclic monophosphate (cAMP)-dep endent protein kinase A (PKA.) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (10 mu M), CRH-41 (100 n M), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 mu M) but no t mastoparan (10 mu M) stimulated cAMP accumulation. Measurement of AC TH secretion under identical incubation conditions revealed that GTP-g amma-S and mastoparan significantly stimulated ACTH secretion but fors kolin and CRH-41 did npt. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the ef fects of mastoparan and GTP-gamma-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nM to 1 mM) stimulated ACTH secre tion from electrically permeabilised cells in a concentration-dependen t manner. cAMP (100 mu M) and phorbol 12-myristate 13-acetate (PMA; 10 0 nM) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-depen dent pathway is necessary before any cAMP-mediated enhancement of secr etion is manifest. PMA, however, did stimulate ACTH secretion in the a bsence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Coincubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-4 1 (1 pM to 100 nM) and forskolin (1 nM to 100 mu M) stimulated ACTH se cretion from intact cells in a concentration-dependent manner. Go-incu bation with PMA (100 nM) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study ind icates that there are distinct roles for PKA and PKC in stimulus-secre tion coupling in AtT-20 cells. The PKA-dependent pathway, acting in co ncert with the calcium messenger system, serves as part of the stimulu s-secretion coupling pathway by which activation of CRH-41 receptors c ontrol ACTH secretion. The PKC-dependent pathway, in contrast, seems t o be independent of the calcium messenger system and may represent a s eparate control mechanism of ACTH secretion.