Abstract
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, 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 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.
Highlights
The role of ATP as a positive feedback element in Ca2؉ signaling and secretion was examined in female rat pituitary gonadotrophs
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
Extracellular Ca2ϩ Dependence of ATP Actions in Gonadotrophs—Addition of ATP induced a rapid increase in [Ca2ϩ]i in single rat pituitary gonadotrophs, which were identified by addition of gonadotropin-releasing hormone (GnRH) at the end of recording (Figs. 1–5)
Summary
The role of ATP as a positive feedback element in Ca2؉ signaling and secretion was examined in female rat pituitary gonadotrophs. 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. In several cell types that secrete by exocytosis, including pituitary cells [11], spontaneous firing of action potentials (APs) at resting potential is associated with [Ca2ϩ]i oscillations, but the possible participation of P2 receptor channels in such Ca2ϩ spiking (termed the plasma membrane oscillator) has not been addressed These channels could modulate secretion by changing the frequency, amplitude, or duration of APs and their resulting increases in [Ca2ϩ]i.
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