EFFECTS OF ADENOSINE 3'-5'-CYCLIC MONOPHOSPHATE AND GUANINE-NUCLEOTIDES ON CALCIUM-EVOKED ACTH RELEASE FROM ELECTRICALLY PERMEABILIZED ATT-20 CELLS

1 The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the investigation of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2 AtT-20 cells were permeabilized by subjecting the cells to intense electric fields. Exposure of permeabilized cells to calcium (1 mM) in the external medium significantly stimulated ACTH secretion over the first 20 min of exposure. This calcium-stimulated ACTH secretion was dependent upon the presence of MgATP (5 mM). 3 The amount of ACTH secreted, in a 20 min incubation at 37-degrees-C, from permeabilized cells depended upon the free calcium concentration in the permeabilization medium. Calcium stimulated ACTH secretion from permeabilized cells in the concentration range of 10(-7) -10(-5) M (half maximal = 7 x 10(-7) M). Cyclic AMP(10(-4) M) increased the amount of ACTH secreted at each effective concentration of calcium. However, cyclic AMP did not alter the potency of calcium as a stimulant of ACTH secretion. 4 Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-4) M) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium-evoked ACTH secretion up to a maximum which could be stimulated by calcium acting singly. Guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 10(-4) M) inhibited calcium-evoked ACTH secretion from permeabilized cells. 5 GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner. The nucleotide significantly stimulated ACTH secretion at concentrations of 10(-5) M and above. Cyclic AMP (10(-4)) M) increased the amount of ACTH secretion evoked by effective concentrations of GTP-gamma-S. 6 The results of the present study support the hypothesis that, in AtT-20 cells, cyclic AMP is acting at some site, distal to calcium entry, which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. One such site may be a GTP-binding protein which the present study suggests may mediate the effects of calcium upon the secretory apparatus. These GTP-binding proteins may be a target for regulation by cyclic AMP.