Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT‐20 cells

Abstract

1 The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12-myristate 13-acetate (PMA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2 PMA stimulated ACTH secretion from intact cells in a concentration-dependent manner. Other phorbol esters; phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol analogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG) also stimulated ACTH release from intact AtT-20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from 3 Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration- 6 GTP-y-S (108-1O'-M) stimulated ACTH secretion from permeabilized cells either in the presence or absence of ATP (5 mM) indicating that its effects on secretion are ATP-independent. 7 The results of the present study support the hypothesis that, in AtT-20 cells, PMA is acting at some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. This site of action is either at the level of or at some stage distal to a GTP-binding protein which mediates the effects of calcium upon secretion. 8 PMA, unlike adenosine 3':5'-cyclic monophosphate (cyclic AMP) (Guild, 1991), can stimulate ACTH secretion from permeabilized cells in the absence of added calcium and guanine nucleotides which suggests that PMA and cyclic AMP are acting through distinct mechanisms at this post calcium site of action.

Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT‐20 cells

“…conventional PKC's (cPKC's) and the calcium-independent or novel PKC's (nPKC's) (Ohno et al, 1991). Several isoforms of PKC have been specifically implicated as having The present study adds to the considerable body of evidence supporting an involvement of PKC in the stimulation of ACTH release from both the normal corticotroph (AbouSamra et al, 1986) and the model for this secretory cell, the AtT-20 cell line Reisine, 1989;McFerran & Guild, 1994). PKC interacts with the ACTH secretory pathway at multiple points as shown by its 'pre-' and 'post-' calcium site of action to regulate calcium influx and the effect of particular cytosolic calcium concentrations upon ACTH secretion McFerran & Guild, 1994).…”

“…; human ACTH antiserum and human ACTH for standards was the gift of the National Hormone and Pituitary programme, Baltimore, MD, USA. Polyclonal antisera to PKC-o, -P, ', a were prepared as previously described (Strulovici et al, 1989;1991 Reisine, 1989;McFerran & Guild, 1994). TMX (10 pM-1 jAM) and dPPA (10 pM-1 tiM) both stimulated ACTH secretion in a concentration-dependent manner (EC50= 10± 0.5 nM (n = 3) and 3 ± 0.5 nM (n = 3) respectively and significant (P <0.05) at concentrations of TMX and dPPA of 10 pM and above) (Figure 2 (Figure 4).…”

“…The ability of activators of protein kinase C to stimulate ACTH secretion from intact AtT-20 cells remaining attached to the culture dishes was measured as previously described McFerran & Guild, 1994 (Guild, 1991). At this point, the zero time samples were centrifuged (200 g, 5 min) and an aliquot of the supernatant was stored for subsequent measurement of ACTH content.…”

“…Activation of these two protein kinases influences the calcium messenger system and both have a 'pre-' and 'post-' calcium site of action to regulate calcium influx and the effect of particular cytosolic calcium concentrations upon ACTH secretion Guild, 1991;McFerran & Guild, 1994).…”

“…Interestingly, PKA and PKC act through distinct mechanisms to influence cytosolic calcium concentrations in AtT-20 cells Reisine, 1989) and both have distinct 'post-calcium' sites of action at a late stage in the secretory process, potentiating the effects of calcium and guanine nucleotides upon the secretory apparatus (Guild, 1991;McFerran & Guild, 1994).…”