Expression of purinergic receptor channels and their role in calcium signaling and hormone release in pituitary gonadotrophs - Integration of P-2 channels in plasma membrane- and endoplasmic reticulum-derived calcium oscillations

The role of ATP as a positive feedback element in Ca2+ signaling and secretion was examined in female rat pituitary gonadotrophs. ATP and ADP, but not AMP or adenosine, induced a dose- and extracellular Ca2+-dependent rise in [Ca-2+](i) in identified gonadotrophs in a Mg2+- and suramin-sensitive manner. ATP, adenosine-5'-O-(3-thiotriphosphate), adenosine-5'-O-(1-thiotriphosphate), 2-methylthio-ATP, and 3'-O-(4-benzoyl)benzoyl-ATP. were roughly equipotent in rising [Ca2+](i) in gonadotrophs, while ADP was effective only at submillimolar concentration range, and none of these compounds permeabilized the cells. On the other hand, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, and UTP were unable to induce any rise in [Ca2+](i). This pharmacological profile is consistent with expression of P2X(2) and/or P2X(5) purinergic receptor channels. Patch-clamp experiments showed that ATP induced an inward depolarizing current in gonadotrophs clamped at -90 mV, associated with an increase in [Ca2+](i). The ATP-induced [Ca2+](i) response was partially inhibited by nifedipine, a blocker of voltage-sensitive Ca2+ channels (VSCC), but was not affected by tetrodotoxin, a blocker of voltage-sensitive Na+ channels. Thus, the P-2-depolarizing current itself drives Ca2+ into the cell, but also activates Ca2+ entry through VSCC. In accord with this, low [ATP] induced plasma membrane-dependent [Ca2+](i) oscillations in quiescent cells, and increased the frequency of spiking in spontaneously active cells. ATP-induced Ca2+ influx also affected agonist-induced and InsP(3)-dependent [Ca2+](i) oscillations by increasing the frequency, base line, and duration of Ca2+ spiking. In addition, ATP stimulated gonadotropin secretion and enhanced agonist-induced gonadotropin release. ATP was found to be secreted by pituitary cells during agonist stimulation and was promptly degraded by ectonucleotidase to adenosine. These observations indicate that ATP represents a paracrine/autocrine factor in the regulation of Ca2+ signaling and secretion in gonadotrophs, and that these actions are mediated by P-2 receptor channels.