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

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.