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-DERIVED AND ENDOPLASMIC RETICULUM-DERIVED CALCIUM OSCILLATIONS

The role of ATP as a positive feedback element in Ca2+ signaling and s ecretion was examined in female rat pituitary gonadotrophs. ATP and AD P, but not AMP or adenosine, induced a dose- and extracellular Ca2+-de pendent rise in [Ca-2+](i) in identified gonadotrophs in a Mg2+- and s uramin-sensitive manner. ATP, adenosine-5'-O-(3-thiotriphosphate), ade nosine-5'-O-(1-thiotriphosphate), 2-methylthio-ATP, and 3'-O-(4-benzoy l)benzoyl-ATP. were roughly equipotent in rising [Ca2+](i) in gonadotr ophs, while ADP was effective only at submillimolar concentration rang e, and none of these compounds permeabilized the cells. On the other h and, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, and UTP were unable to induce any rise in [Ca2+](i). This pharmacological profile i s consistent with expression of P2X(2) and/or P2X(5) purinergic recept or channels. Patch-clamp experiments showed that ATP induced an inward depolarizing current in gonadotrophs clamped at -90 mV, associated wi th an increase in [Ca2+](i). The ATP-induced [Ca2+](i) response was pa rtially inhibited by nifedipine, a blocker of voltage-sensitive Ca2+ c hannels (VSCC), but was not affected by tetrodotoxin, a blocker of vol tage-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 [Ca 2+](i) oscillations in quiescent cells, and increased the frequency of spiking in spontaneously active cells. ATP-induced Ca2+ influx also a ffected agonist-induced and InsP(3)-dependent [Ca2+](i) oscillations b y increasing the frequency, base line, and duration of Ca2+ spiking. I n addition, ATP stimulated gonadotropin secretion and enhanced agonist -induced gonadotropin release. ATP was found to be secreted by pituita ry cells during agonist stimulation and was promptly degraded by ecton ucleotidase to adenosine. These observations indicate that ATP represe nts a paracrine/autocrine factor in the regulation of Ca2+ signaling a nd secretion in gonadotrophs, and that these actions are mediated by P -2 receptor channels.