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 Ca2+ levels ([Ca2+]) was investigated in fura-2-loaded mouse Leydig (TM3) cells. 2. The extracellular application of Ca2+ (2.5-15 mM) or Ni2+ (0.5-5 mM) elicited concentration-dependent elevations in cytosolic [Ca2+] that were followed 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 in low-Ca2+ solutions. Such findings suggest a participation of Ca2+ release from intracellular stores. In parallel, depletion of Ca2+ stores by either ionomycin (5 microM, in low-Ca2+ solutions) or thapsigargin (4 microM) abolished or attenuated Ni2+-induced Ca2+ transients. 5. Ionomycin (5 microM) elevated cytosolic [Ca2+] in Ca2+-free solutions even after prior Ni2+ application, indicating the presence of Ni2+-insensitive stores. 6. Caffeine (250 and 500 microM) elevated cytosolic [Ca2+] and attenuated Ni2+-induced Ca2+ release. Furthermore, TM3 cells stained intensely with a specific anti-ryanodine receptor antiserum, Ab34. These findings suggest that Ca2+ release is regulated by ryanodine receptors. 7. Both membrane depolarization and hyperpolarization, brought about by changes in extracellular [K+] ([K+]e) in the presence of valinomycin (5 microM), altered the waveform of the Ni2+-induced cytosolic Ca2+ signal. Hyperpolarization, in addition, diminished the response magnitude. Such voltage-induced response modulation localizes the regulatory events to the Leydig cell plasma membrane. 8. We propose the existence of a cell surface divalent cation (Ca2+) receptor in Leydig cells, the activation of which triggers Ca2+ fluxes through ryanodine receptors.
Adebanjo
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Center for Skeletal Aging and Osteoporosis, Veterans Affairs Medical Center and Medical College of Pennsylvania-Hahnemann School of Medicine, Allegheny University of the Health Sciences and University of Pennsylvania, Philadelphia, PA 19104, USA.
Igietseme
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Huang
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CL
Zaidi
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D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002413:Cations, Divalent / Q000008:administration & dosage / Q000494:pharmacology*; D002460:Cell Line; D003213:Conditioning, Psychological; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009532:Nickel / Q000494:pharmacology; D009994:Osmolar Concentration; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology
D002413:Cations, Divalent; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D019837:Ryanodine Receptor Calcium Release Channel; D009532:Nickel; D002118:Calcium