Involvement of multiple protein kinase C isozymes in 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 protein kinase C (PKC)-mediated enhancement of calcium-and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2 A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the a, P, a and C isoforms of PKC.3 PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 0.05 nM, which activates all isozymes of PKC, except the C isozyme), thymeleatoxin (TMX, EC50 = 10 ± 0.5 nM, which activates the a, P and y isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 ± 0.5 nm, a PI-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4 Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentrationrange of 100 nM to 10 4M. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM) significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is nM). 5 GTP-y-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 1 gM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP-y-S investigated.6 The PKC inhibitor, chelerythrine chloride (10 gM), blocked the PMA (100 nM)-evoked enhancement of calcium-and GTP-y-S-stimulated ACTH secretion but did not significantly alter calcium-or GTP-y-S-evoked secretion itself. 7 The present paper indicates that AtT-20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The a and e isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations 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 ACTH secretion. The isozyme of PKC may act at a stage early in the secretory pathway to regulate the cytosolic calcium concentration.

Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells

“…In an attempt to delineate the level at which glucocorticoids block PMA‐mediated signalling we examined whether PMA‐induced translocation of PKC isoforms to membrane fractions was affected in glucocorticoid‐treated cells. Previous studies in the AtT20 D16:16 cell line reported expression of the α‐, β‐ and ε‐ (PMA sensitive) as well as the ζ‐ (PMA insensitive) PKC isoforms (McFerran et al 1995). Western blotting for the PMA‐sensitive isoforms revealed robust expression of α and ε isoforms (Fig.…”

“…In AtT20 mouse corticotroph cells phorbol‐ester‐mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage‐dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al 1992; Clark & Kempainen, 1994; McFerran et al 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC‐induced calcium influx results, at least in part, from inhibition of TEA‐sensitive potassium conductances.…”

“…As TEA‐sensitive BK channels act as immediate negative feedback regulators of voltage‐dependent calcium influx in several systems (Robitaille et al 1993; Yazejian et al 1997) and PKC‐mediated calcium influx is dependent upon inhibition of a TEA‐sensitive conductance in AtT20 cells (Reisine & Guild, 1987; Reisine, 1989), we have addressed whether BK channels are an important cellular target for PKC action in this system. Furthermore, as glucocorticoids block PKC‐stimulated ACTH release (Phillips & Tashjian, 1982; Woods et al 1992; Clark & Kempainen, 1994; McFerran et al 1995) and antagonize PKA‐mediated inhibition of BK channels in this system (Shipston et al 1996; Tian et al 1998) we have addressed the question as to whether glucocorticoids also block PKC‐mediated regulation of BK channels in AtT20 D16:16 corticotroph cells.…”

Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms

“…PKC-was identified in the insulin-secreting cell line RINm5F (49), and recently the presence of PKC-has been demonstrated in the MIN6 ␤-cell line (52). PKC-␣, -␦, -⑀, and -predominate in the FRTL-5 thyroid cell line (32,54), and PKC-␣, -␤, -⑀, and -are found in AtT-20 cells, a mouse anterior pituitary tumor cell line (36). In primary cultures of human antral G cells, Moore et al (38) demonstrated expression of the PKC isoforms ␣, ␥, , ⑀, , and .…”

Human Cardiac Inwardly-Rectifying K + Channel Kir 2.1b Is Inhibited by Direct Protein Kinase C-Dependent Regulation in Human Isolated Cardiomyocytes and in an Expression System

“…As an activator of the conventional (cPKC ␣, ␤ 1/2 , and ␥) and novel type PKC isoenzymes (nPKC ␦, ⑀, , and ), OAG at a concentration of 10 mol/L was used, thereby inhibiting the current at Ϫ120 mV significantly to 44.9Ϯ2.0% (nϭ4) after 30 minutes (Figure 3). Thymeleatoxin (100 nmol/L), a specific activator of the conventional protein kinase C isoenzymes, 19 was even more effective and …”