EXTRACELLULAR ATP ACTIVATES DIFFERENT SIGNALING PATHWAYS IN RAT SERTOLI CELLS

1. The present study describes effects of extracellular ATP (ATP(e)) o n plasma membrane potential and cytoplasmic Ca2+ concentrations ([Ca2](i)) in rat Sertoli cells. Sertoli cells in suspension were stimulate d with ATP(e) and other nucleotides and ionic changes were monitored u tilizing the fluorescent dyes bis-oxonol and fura-2/AM. ATP(e) induced a prompt plasma membrane depolarization which was dependent on Na+ in flux from the extracellular medium, since it was abolished by omission of extracellular Na+. Depolarization was independent of [Ca2+](i) ris e as it also occurred in the absence of extracellular Ca2+ and after i ntracellular Ca2+ stores were discharged with thapsigargin. ATP(e) als o stimulated a rapid and biphasic increase in [Ca2+](i):a prompt spike was followed by a prolonged sustained plateau. The initial spike was dependent on Ca2+ release from intracellular stores since it was also present when cells were incubated in EGTA-supplemented Ca2+-free mediu m and was abolished by pretreatment with ionomycin and thapsigargin, a gents that discharge intracellular Ca2+ stores. The sustained phase wa s dependent on Ca2+ influx from the extracellular medium as it was abo lished when cells were incubated in EGTA-supplemented Ca2+-free medium . Ca2+ influx was due to activation of voltage-operated calcium channe ls (VOCCs) since it was abolished by the VOCC inhibitors verapamil and nifedipine or incubation in sucrose medium, an experimental condition which precludes plasma membrane depolarization by ATP(e). 2. ATP(e)-i nduced rises in intracellular Ca2+ concentration and plasma membrane d epolarization were reduced by pretreatment with pertussis toxin, sugge sting that ATP(e)-activated transduction mechanisms are in part under the control of pertussis toxin-sensitive G-proteins. These data show t hat Sertoli cells possess P2-purinergic receptor subtypes coupled to i nflux of Na+ and release of Ca2+ from intracellular stores and provide evidence for an activation of different pathways by extracellular ATP (e). Activation of these receptors induces Na+ influx that causes a ra pid plasma membrane depolarization. Furthermore, ATP(e) also triggers Ca2+ release from intracellular stores and Ca2+ influx from extracellu lar space via dihydropyridine-sensitive VOCCs.