P-2-purinoreceptor evoked changes in intracellular calcium oscillations in single isolated human granulosa-lutein cells

In this study, we have demonstrated that P-2-purinoreceptor agonists evoke oscillatory intracellular calcium ([Ca2+](i)) responses in human granulosa-lutein cells (GLCs). Intracellular calcium was measured using microspectrofluorimetric techniques. ATP at concentrations of 1-100 mu M increased [Ca2+](i), whereas neither adenosine nor AMP evoked changes in [Ca2+](i). The nonhydrolysable ATP analogue, ATP gamma S, also elevated [Ca2+](i) with an efficacy similar to that of ATP, indicating that the changes in Ca2+ were not due to ATP hydrolysis, but that human GLCs possess functional P-2-purinoreceptors. Uridine triphosphate (UTP) was equipotent to ATP at stimulating [Ca2+](i), and both ATP and UTP were consistently more effective at eliciting a response than ADP, suggesting that human GLCs possess the P-2U class of purinergic receptors (ATP=UTP>>ADP>>AMP=adenosine). We have demonstrated that the purinergic agonist-induced changes in [Ca2+](i) involve both Ca2+ influx and Ca2+ mobilization from cytosolic stores. Prolonged ATP treatment in Ca2+-free buffer (1 mM EGTA) still evokes transient oscillatory changes in [Ca2+](i) in a pertussis toxin-insensitive manner. In Ca2+-containing conditions, the sustained phase of the response was generally unaffected by verapamil (10 mu M), suggesting that influx is not occurring through voltage-dependent Ca2+-channels. These findings are consistent with the hypothesis that ATP and other P-2-purinergic receptor agonists elicit changes in [Ca2+](i) in human ovarian cells and that these events are initiated by the release of Ca2+ from cytosolic stores, and sustained by extracellular calcium ([Ca2+](i)) influx. This is the first time that oscillatory patterns of [Ca2+](i) have been reported in human GLCs.