Purinergic regulation of cytosolic calcium and phosphoinositide metabolism in rat osteoblast-like osteosarcoma cells.
We have shown that ATP increases cytosolic Ca2+ in UMR-106 cells through P2-purinergic receptor stimulation (Calcif Tissue Int 45:251-254). This response was further characterized using cells loaded with indo-1/AM or prelabeled with [3H]inositol. ATP elicited a rapid transient increase in Ca2+ from 148 to 540 nM, followed by a biphasic decline (first rapid and then slower) to basal within 1 minute and then a late slow rise to 200 nM by 4 minutes. ADP also elicited a rapid transient increase, but this was followed by a second smaller transient and a later, slow increase above basal Ca2+. These transient increases in Ca2+ induced by ATP and ADP were dose dependent, detected at 10(-6)M ATP and 10(-7)M ADP, and saturated at 10(-4)M with both nucleotides. The maximum increase in Ca2+ was 20% greater with ATP than ADP. EGTA chelation of extracellular Ca2+ abolished the biphasicity of the ATP-induced Ca2+ transient, the second ADP-induced transient, and all late slower increases in Ca2+. Desmethoxyverapamil pretreatment attenuated the biphasicity of the ATP-induced transient and the second peak elicited by ADP. Elevated extracellular Ca2+ (5 mM) prevented the return to the basal level that normally follows the ATP-induced Ca2+ transient and amplified the sustained increase in Ca2+ but had little effect on the response to ADP. IP3 and IP4 increased rapidly after addition of ATP, with I(1,4,5)P3 increasing before I(1,3,4)P3. These data indicate that P2-purinergic stimulation of UMR-106 cells causes three consecutive responses in cytosolic Ca2+: (1) a transient increase due to IP3-mediated mobilization of intracellular Ca2+; (2) a transient increase due in part to influx, probably associated with a Ca2+ channel; and (3) a later sustained increase that requires extracellular calcium.
Kumagai
H
H
Laboratory of Biological Chemistry, National Institute of Aging, Baltimore, Maryland.
Sacktor
B
B
Filburn
C R
CR
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D009711:Nucleotides; D010716:Phosphatidylinositols; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium