Evidence for a role of pituitary ATP receptors in the regulation of pituitary function.

Abstract

Despite a rapidly increasing acceptance for a role of ATP as an extracellular mediator in several biological systems, the present report shows The role of nucleotides in metabolism is well established, but recognition of their potential importance as extracellular transmitters, regulators or modulators has been very recent. Extracellular ATP has been shown to induce a variety of responses, such as stimulation of inositol phospholipid turnover and activation of membrane conductances in many cell types and preparations (1-4). These responses are now known to be mediated by a family of ATP receptors, designated P2 purinoceptors (5-7), some of which have been cloned (8-11). ATP has been shown to stimulate inositol phosphate accumulation and intracellular Ca2+ metabolism in pituitary cell cultures (12), and our own data from fetal hypothalamic neuron cultures suggests that ATP has a possible regulatory role in the neuroendocrine system (13). Recently we have shown that ATP and UTP act on ATP receptors to provoke a rapid and dramatic increase in cytosolic Ca2+ in pituitary gonadotropin-release hormone (GnRH)-responsive cells (14). Using a superfusion system to examine the functional effect of extracellular nucleotides on the kinetics of pituitary hormone secretion and using real-time dynamic bioluminescence measurements of ATP, we now demonstrate that ATP receptors can mediate significant release of pituitary gonadotropin and that ATP can be exocytotically released from pituitary cells.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.MATERIALS AND METHODS Materials. Collagenase was purchased from Serva; medium M199, Ca2+-free Hanks' balanced salt solution, and sera were from GIBCO/BRL; GnRH and A23187 were from Calbiochem; and 2-methylthio-ATP and adenosine 5'-[3,y-methylene]triphosphate (13,y-methylene-ATP) were from Research Biochemical International (Natick, MA). Bromo-A23187, highly purified nucleotides, and all others, unless otherwise stated, were obtained from Sigma. Superfusion of Pituitary Cells. Pituitary glands from4-week-old female Wistar rats were enzymatically dispersed in medium M199 containing bovine serum albumin (BSA; 0.3%), collagenase (1 mg/ml), hyaluronidase (0.5 mg/ml), and DNase (25 ,ug/ml) as described (15). Aliquots of the cell suspension containing -3-4 x 106 cells were plated out on a 12-well culture plate containing 15 mg of Cytodex I beads, which had been preswollen in phosphate-buffered saline and autoclaved. Superfusion experiments were performed as described (15). After 2 days in culture, cells and beads were transferred to superfusion columns (5 x 25 mm Bio-Rad Econocolumns) and superfused with medium M199 containing 0.3% BSA, 1.8 mM Ca2+, 2 mM glutamine, and 20 mM Hepes at 37°C at the rate of 0.65 ml/min. A 90-min superfusion was carried out to wash cells before the start of superfusate collection. Superfusate fractio...

Expression of P2Y receptors in the rat anterior pituitary

“…GnRH-induced stimulation of gonadotropin release was accompanied by elevation in basal ATP release, raising the possibility that ATP is stored in the secretory vesicles of these cells [2]. This is consistent with an earlier study showing calcium dependence of ATP release [23]. The roles of ATP as a neurotransmitter or cotransmitter are well established in the peripheral and central nervous systems [24].…”

Functional Role of Spliced Cytoplasmic Tails in P2X2-Receptor-Mediated Cellular Signaling

“…In pituitary cells, activation of P2Y receptors leads to increase in inositol phosphates turnovers associated with increase in [Ca 2+ ] i (8) and translocation of protein kinase C in gonadotrope-derived alpha T3-1 cells (9). On the other hand, P2X subunits were expressed in a cell typespecific manner.…”

Synchronized Spontaneous Ca2+ Transients in Acute Anterior Pituitary Slices

“…Since single action potentials seem to be efficient enough to trigger fast exocytosis in gland (adrenal) slices (44), a minute fraction of fast-acting factors (e.g. ATP, dopamine copacked with hormones in LDCV) (19,21) readily released upon single action potentials would quickly alter the firing frequency of any coactive cells (45) and thereby change the hierarchy of the propagation of electrical events. Finally, dyecoupled cells are not always within a single field at a single plane of focus suggesting that the cell phasing synchronization can be out of focus during optical recordings.…”