The effect of extracellularly applied divalent cations on cytosolic Ca2+ in murine Leydig cells: evidence for a Ca2+-sensing receptor

1. The effect of extracellularly applied divalent cations upon cytosolic Ca 2+ levels ([Ca2+]) was investigated in fura-2-loaded mouse Leydig (TM3) cel ls. 2. The extracellular application of Ca2+ (2.5-15 mM) or Ni2+ (0.5-5 mM) eli cited concentration-dependent elevations in cytosolic [Ca2+] that were foll owed by decays to baseline levels. Extracellular Mg2+ (0.8-15 mM) failed to influence cytosolic [Ca2+]. 3. Conditioning applications of Ca2+ (2.5-10 mM), Mg2+ (2.5-15 mM) or Ni2(0.5-5 mM) all attenuated the cytosolic Ca2+ response to a subsequent test application of 5 mM [Ni2+]. 4. The amplitude of Ni2+-induced cytosolic Ca2+ signals remained constant i n low-Ca2+ solutions. Such findings suggest a participation of Ca2+ release from intracellular stores. In parallel, depletion of Ca2+ stores by either ionomycin (5 mu M, in low-Ca2+ solutions) or thapsigargin (4 mu M) abolish ed or attenuated Ni2+-induced Ca2+ transients. 5. Ionomycin (5 mu M) elevated cytosolic [Ca2+] in Ca2+-free solutions even after prior Ni2+ application, indicating the presence of Ni2+-insensitive stores. 6. Caffeine (250 and 500 mu M) elevated cytosolic [Ca2+] and attenuated Ni2 +-induced Ca2+ release. Furthermore, TM3 cells stained intensely with a spe cific anti-ryanodine receptor antiserum, Ab(34). These findings suggest tha t Ca2+ release is regulated by ryanodine receptors. 7. Both membrane depolarization and hyperpolarization, brought about by cha nges in extracellular [K+] ([K+](e)) in the presence of valinomycin (5 mu M ), altered the waveform of the Ni2+-induced cytosolic Ca2+ signal. Hyperpol arization, in addition, diminished the response magnitude. Such voltage-ind uced response modulation localizes the regulatory events to the Leydig cell plasma membrane. 8. We propose the existence of a cell surface divalent cation (Ca2+) recept or in Leydig cells, the activation of which triggers Ca2+ fluxes through ry anodine receptors.