C-type natriuretic peptide (CNP) effects in anterior pituitary cell lines: evidence for homologous desensitisation of CNP-stimulated cGMP accumulation in alpha T3-1 gonadotroph-derived cells.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclase-containing receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived alpha T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs.

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes.

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial.

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells.

The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size.

The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) actions on alphaT3-1 gonadotrophs show desensitization.

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived alphaT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation.

Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived alphaT3-1 cell line.

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation.

Desensitization of gonadotropin-releasing hormone action in alphaT3-1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization.

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%.

Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor.

Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated.

Ca2+ entry in gonadotrophs and alpha T3-1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells.

Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived alpha T3-1 cell line.

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of GnRH-stimulated gonadotropin release, but the mechanisms involved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not undergo rapid ( < 5 min) homologous desensitization in alpha T3-1 cells, and we have, therefore, used this cell line to address the question of whether desensitization occurs distal to PIC activity and/or in an intermediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulation of [3H]IPs (in cells stimulated in the presence of LiCl) increases linearly over 5-300 sec.