(Received for publication, February 14, 1996, and in revised form, June 4, 1996)

Melanija Tomi , Richard M. Jobin , Leoncio A. Vergara and Stanko S. Stojilkovic

From the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda Maryland 20892

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 [Ca²⁺]_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, ,-methylene-ATP, ,-methylene-ATP, and UTP were unable to induce any rise in [Ca²⁺]_i. This pharmacological profile is consistent with expression of P2X₂ and/or P2X₅ 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 P₂-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 InsP₃-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 P₂ receptor channels.

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