Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells.
We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis.
Baldi
Carolina
C
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, (8000) Bahía Blanca, Argentina.
Vazquez
Guillermo
G
Boland
Ricardo
R
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002463:Cell Membrane Permeability; D025802:Electric Capacitance; D005682:Gadolinium / Q000737:chemistry / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism; D051381:Rats; D013324:Strontium / Q000737:chemistry / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D008345:Manganese; D019284:Thapsigargin; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002117:Calcitriol; D002118:Calcium; D013324:Strontium