Role of P2-purinergic receptors in rat Leydig cell steroidogenesis

The present study investigated the effects of extracellular ATP on the intracellular calcium ion concentration ([Ca²⁺]_i) and testosterone production in isolated adult rat Leydig cells. This nucleotide caused an increase in [Ca²⁺]_i, with a maximal effect at a concentration of 100 µM ATP, comprising a rapid initial spike followed by a long-lasting plateau. The first rapid spike was dependent on the release of Ca²⁺ from internal stores, since it also occurred in Ca²⁺-free medium and was abolished after depletion of internal stores with thapsigargin. The second, long-lasting, phase was dependent on the influx of Ca²⁺ from the extracellular medium. After 3 h of incubation, extracellular ATP stimulated testosterone secretion in a dose-dependent manner, with a maximal effect at 100 µM. Activation of steroidogenesis by ATP was fully dependent on the presence of Ca²⁺ in the external medium. Among different nucleotides, only ATP, adenosine 5´-[γ-thio]triphosphate, UTP, benzoylbenzoic-ATP and 2-methylthio-ATP were effective in inducing both the rise in [Ca²⁺]_i and testosterone secretion. These effects were blocked by preincubation of Leydig cells with oxidized ATP, an inhibitor of the P2Z-purinergic receptor subtype. These results show that rat Leydig cells possess P2-purinergic receptors whose activation triggers an increase in [Ca²⁺]_i due to the release of Ca²⁺ from internal stores and Ca²⁺ influx from the external medium. The stimulatory effect of extracellular ATP on testosterone secretion seems to be coupled to the influx of Ca²⁺ from the external medium.