Effects of adenosine 3′: 5′‐cyclic monophosphate and guanine nucleotides on calcium‐evoked ACTH release from electrically permeabilized AtT‐20 cells

Abstract

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 calciumevoked 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 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.

A direct inhibitory action of prostaglandins upon ACTH secretion at the late stages of the secretory pathway of AtT‐20 cells

“…This parallels the evidence for such a direct inhibitory control of exocytosis in chroma n cells (Gasman et al, 1997;Vitale et al, 1993; and insulin secreting cells (Lang et al, 1995). Our evidence suggests that a Ge involved in the stimulation of secretion in AtT-20 cells could also be a heterotrimeric Gprotein (Erlich et al, 1998;Guild, 1991;McFerran & Guild, 1995) indicating that there may be a dual regulation of ACTH secretion by G-proteins at the late stages of the exocytotic pathway with both a stimulatory Ges and an inhibitory Gei, (both possibly heterotrimeric) contributing to the control of the fusion machinery. The current study ®nds no late stage role for phospholipase A 2 or its products in mediating the stimulation of ACTH secretion by the calcium/ Ge system but does indicate a separate late stage inhibitory action of prostaglandins distal to the calcium/Ge point of control.…”

“…AtT-20 cells were prepared for permeabilization as previously described (Guild, 1991). In brief cells were liberated from the substrate, washed twice by centrifugation (2006g, 5 min)/resuspension in a balanced salt solution of the following composition (mM): NaCl 145, KCl 5.6, CaCl 2 0.5, glucose 5.6, HEPES 5, BSA 0.1% (w v 71 ): pH 7.4. and suspended at a density of 10 6 cells ml 71 and incubated for a further 30 min at 378C.…”

“…The cells were ®nally resuspended at a density of 10 7 cells ml 71 and electrically permeabilized by subjection to intense electric ®elds of brief duration (Knight & Baker, 1982). Optimum permeabilization parameters were determined as previously described (Guild, 1991). The standard permeabilization medium was essentially calcium free with a free calcium concentration of 1 nM.…”

“…It is an advantage in studies of stimulus-secretion coupling mechanisms to have homogenous populations of cells and such a population of corticotrophs can be provided by a pituitary tumour cell lines such as the AtT-20 cell line (Sabol, 1980). In this cell line signal-transduction studies have demonstrated a role for G-proteins, termed Ge (Gomperts, 1990), in mediating the ability of calcium ions to stimulate secretion (Guild, 1991;Erlich et al, 1998;Luini & Dematteis, 1988;1990;McFerran & Guild, 1995). One of the agents used to activate Ge in AtT-20 cells, mastoparan (McFerran & Guild, 1995;Erlich et al, 1998), has also been reported to directly activate phospholipase A 2 (Argiolas & Pisano, 1983).…”

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

“…CRF acts via the adenosine 3': 5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PKA) pathway in both anterior pituitary corticotrophs (Aguilera et al, 1983) and in a tumour cell line of the anterior pituitary, which consists of a homogeneous population of ACTH-secreting cells (Luini et al, 1985;. Cyclic AMP has a dual action both enhancing calcium influx into corticotrophs and potentiating the effect of such an increment in cytosolic calcium upon the secretory apparatus (Luini et al, 1985;Guild et al, 1986; Guild, 1991). The nature of AVP's signal-transduction pathway is less clear ' Author for correspondence.…”