Modulation of cytosolic calcium levels in osteoblast-like osteosarcoma cells by olpadronate and its amino-derivative IG-9402.
The molecular mechanisms as well as the structure/activity relationships involved in the antiresorptive actions of bisphosphonates on bone cells are still not clear. Replacement of the R1-hydroxyl by an NH2 group in olpadronate (OPD) abolishes its antiresorptive activity. We show here that in the rat osteosarcoma-derived osteoblast-like ROS 17/2.8 cell line, OPD and IG-9402 (NH2-OPD; [3-(N,N-dimethylamine)-1-aminopropylidene bisphosphonate]), similar to 1,25(OH)2-vitamin D3, rapidly modulate cytosolic calcium levels ([Ca2+]i). As for the steroid hormone, the osteosarcoma cell Ca2+i response to OPD was rapid (30 sec) and sustained (>5 min), exhibiting a biphasic profile. The response to IG-9402 was also fast but smaller than that of OPD and 1,25(OH)2D3, and rapidly declined to levels near basal. The effect of these bisphosphonates on [Ca2+]i was dose-dependent, being maximal at 10(-8) M and was not observed in non-bone cellular systems, e.g., skeletal muscle and breast cells. Pretreatment of the ROS 17/2.8 cells with the Ca2+ channel blockers nifedipine and verapamil markedly reduced (>70%) the influx phase of the response to OPD and almost completely inhibited that of IG-9402, indicating the participation of voltage-dependent Ca2+ channels in the action of both compounds. Moreover, preincubation with the phospholipase C inhibitors U73122 and neomycin or depletion of inner stores with thapsigargin completely blocked the response to either olpadronate or its amino-derivative. Both OPD and IG-9402 significantly increased osteocalcin release into the culture medium of osteosarcoma cells. The results support the involvement of the Ca2+ signaling pathway as part of the mechanism by which bisphosphonates induce bone cellular responses.
Vazquez
G
G
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, (8000) Bahía Blanca, Argentina.
Santillan
G
G
Boland
R
R
Roldán
E
E
Pérez-Lloret
A
A
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects*; D002642:Chick Embryo; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004963:Estrenes / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004164:Diphosphonates; D004963:Estrenes; D011760:Pyrrolidinones; D015675:Osteocalcin; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019284:Thapsigargin; C055774:olpadronic acid; D014700:Verapamil; D010738:Type C Phospholipases; D002117:Calcitriol; D009355:Neomycin; D009543:Nifedipine