TY - JOUR
T1 - Inhibition of 1,25‐(OH)2D3‐ and 24,25‐(OH)2D3‐dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures
AU - Schwartz, Z.
AU - Langston, G. G.
AU - Swain, L. D.
AU - Boyan, Barbara D.
PY - 1991/7
Y1 - 1991/7
N2 - This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth‐passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01‐10 μM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 μM A23187 in resting zone cells and at 0.1 and 1 μM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25‐(OH)2D3 and in resting zone cells incubated with 24,25‐(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25‐(OH)2D3 or 24,25‐(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose‐dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity in isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.
AB - This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth‐passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01‐10 μM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 μM A23187 in resting zone cells and at 0.1 and 1 μM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25‐(OH)2D3 and in resting zone cells incubated with 24,25‐(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25‐(OH)2D3 or 24,25‐(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose‐dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity in isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.
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U2 - 10.1002/jbmr.5650060708
DO - 10.1002/jbmr.5650060708
M3 - Article
C2 - 1659121
AN - SCOPUS:0025900356
VL - 6
SP - 709
EP - 718
JO - Journal of Bone and Mineral Research
JF - Journal of Bone and Mineral Research
SN - 0884-0431
IS - 7
ER -