Adenosine 5'-triphosphate (ATP) receptors induce intracellular calcium changes in mouse Leydig cells

Cytoplasmic calcium ([Ca²⁺](i)) changes evoked by adenosine 5'-triphosphate (ATP) were recorded in cultured individual Leydig cells within 10-18 h after cell dispersion. [Ca²⁺](i) was monitored using Fura-2AM loaded cells with a digital ratio imaging system. Five micromolars ATP induced biphasic [Ca²⁺](i) responses in most cells (94%, n = 100), characterized by a fast increase from a basal level (126 ± 5 nM SE, n = 60 cells) to a peak (5-7 times above basal levels) within seconds, followed by a slow decrease toward a plateau level (2-3 times above basal) within 5 min. The peak phase of the [Ca²⁺](i) response increased with ATP concentrations (1-100 μM ATP) in a dose-dependent manner with an IC₅₀ of 5.9 ± 1.2 μM, and it desensitized in a reversible manner with repeated application of 5 μM ATP at < 5-min intervals. The [Ca²⁺](i) peak response was dependent on Ca²⁺ release from an intracellular pool, whereas the plateau phase was dependent on extracellular [Ca²⁺]. ATP did not appear to induce formation of nonspecific membrane pores, since stimulation for 10 min with ATP (10-100 μM) in the presence of extracellular Lucifer yellow (LY) (5 mg/mL) did not result in dye loading of the cells. [Ca²⁺](i) transients were elicited by other adenosine nucleotides with an order of potencies (ATP > Adenosine diphosphate [ADP] > Adenosine > Adenosine monophosphate [AMP]) that was compatible with the expression of P₂ receptors. [Ca²⁺](i) responses were suppressed by the purinergic P₂ receptor antagonist, suramin. These results provide functional evidence for the expression of purinergic P₂ receptors in Leydig cells.