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Inhibition of 1,25‐(OH) 2 D 3 ‐ and 24,25‐(OH) 2 D 3 ‐dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures
Author(s) -
Schwartz Z.,
Langston G.G.,
Swain L.D.,
Boyan Barbara D.
Publication year - 1991
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650060708
Subject(s) - alkaline phosphatase , ionophore , phospholipase a2 , vesicle , calcium , biochemistry , chemistry , stimulation , membrane , biology , endocrinology , medicine , enzyme , organic chemistry
This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A 2 activity by vitamin D 3 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 A 2 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 45 Ca 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 A 2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25‐(OH) 2 D 3 and in resting zone cells incubated with 24,25‐(OH) 2 D 3 . Phospholipase A 2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25‐(OH) 2 D 3 or 24,25‐(OH) 2 D 3 blocked the stimulation of alkaline phosphatase by the vitamin D 3 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 Ca 2+ influx per se are involved.