Lowering of Cytosolic Free Ca²⁺by Carbachol, a Muscarinic Cholinergic Agonist, in Clonal Pituitary Cells (GH₃Cells)*

Abstract

Muscarinic cholinergic agonists have been shown to inhibit PRL secretion in normal and tumor-derived pituitary cells. Evidence from experiments with the fluorescent Ca2+ probe quin 2 shows that carbachol, acting through muscarinic acetylcholine receptors, lowers the cytosolic free Ca2+ concentration ([Ca2+]i), in GH3 cells. A decrease in [Ca2+]i is observed rapidly after carbachol addition, the lowered steady state [Ca2+]i is maintained, and upon the addition of atropine [Ca2+]i returns to the initial basal value. The lowering from a basal [Ca2+]i, averaging 110 +/- 2 nM (+/- SEM, n = 9), to a steady state [Ca2+]i of 63 +/- 4 nM (+/- SEM, n = 5) at 10 micron carbachol is dose dependent, a significant decrease from basal [Ca2+]i being observed at 0.1 micron. Carbachol does not prevent TRH-induced mobilization of Ca2+ but attenuates the resulting rise in [Ca2+]i. The lowering of steady state [Ca2+]i and the attenuation of the rise in [Ca2+]i provoked by stimulators of PRL secretion could explain the inhibition of both basal and stimulated PRL secretion. Concomitantly with the action on [Ca2+]i, carbachol causes hyperpolarization of GH3 cells. Together with the established inhibition of adenylate cyclase by muscarinic cholinergic agonists, these findings suggest a relation between changes in trans-membrane Ca2+ fluxes and cAMP generation.

Pertussis toxin selectively abolishes hormone induced lowering of cytosolic calcium in GH3 cells

“…[Ca"]i is lowered in GH3 cells in response to carbachol [8] and somatostatin [7]. The [Ca2']i response to these inhibitors of adenylate cyclase can be demonstrated in a single experiment, as is shown in fig.lA_ After the addition of carbachol, [Ca2"]i is lowered from a basal level averaging 116 nM (table 1) to about 70 nM, A&opine, an inhibitor of the muscarinic acetylcholine receptor, reverses the action of carbachol such that {Ca**]i returns within 2 min to the original basal levels.…”

“…bachol, a muscarinic cholinergic agonist, lower the [Ca2']i and cause hyperpolarization of GH3 cells [7,8], a cell line derived from rat pituitary tumors [9]. In this study we use PT to probe for the involvement of Ni [4] in mediation of the effects of somatostatin and carbachol on [Ca2+]i and membrane potential.…”

Cholinergic Stimulation of Inositol Phosphate Production in Cultured Anterior Pituitary Cells

“…The functional importance of this metabolite, however, is not to mobilize intracellular cal cium and in fact its role has not been established. The hy pothesis for a mechanism of action of acetylcholine and carbachol at the pituitary is in apparent contrast with the lowering of cytosolic free Ca2+ by carbachol observed in GH, cells [27], Several possibilities may, at least partially, account for this discrepancy.…”

Conditionally expressed Gα 15 couples to endogenous receptors in GH 3 cells

“…In non-induced GH 3 cells, activation of muscarinic receptors transiently decreased [Ca 2+ ] i (Fig. 5;Schlegel et al 1985), a process believed to be based on the G o -mediated inhibition of voltage-dependent Ca 2+ channels (Schlegel et al 1985;Kleuss et al 1991;Offermanns et al 1991). Interestingly, while incubation of cells with carbachol elicited very pronounced stimulation of inositol phosphate production after induction of Gα 15 expression, this increase in inositol phosphate production was not accompanied by a change in the course of free [Ca 2+ ] i usually observed after addition of carbachol (Fig.…”