Effect of L- and D-tetramisole on 32Pi and 45Ca uptake and mineralization by matrix vesicle-enriched fractions from chicken epiphyseal cartilage.

Alkaline phosphatase has been implicated in matrix vesicle-mediated calcification. We sought to clarify its role in this process by studying the effect of L-tetramisole, a strong uncompetitive inhibitor of this enzyme, and D-tetramisole, its inactive isomer, on the uptake of 32Pi and 45Ca by matrix vesicle-enriched microsomes obtained from chicken epiphyseal cartilage. Uptake of both 32Pi and 45Ca was inhibited in a dosage-dependent manner by the L-isomer; however, 32Pi uptake was selectively inhibited at low drug concentrations and at early time periods. With increasing incubation time, inhibition of uptake of both ions was lost. Apatite crystal growth was only weakly inhibited by both the D- and L-isomers of tetramisole. The kinetics of ion uptake by the vesicles was complex. 45Ca/32Pi uptake ratios, taken with the selective inhibiton of 32Pi uptake by L-tetramisole, indicated that early phases of vesicle ion uptake might be governed by alkaline phosphatase, a known Pi-binding protein. Later stages were clearly controlled by apatite formation. Although inhibition of vesicle uptake of 32Pi and 45Ca by graded levels of L-tetramisole paralleled inhibition of alkaline phosphatase activity, surprisingly, D-tetramisole was almost as inhibitory of ion uptake as the L-isomer. This finding, coupled with the fact that alkaline phosphatase substrates were not required for vesicle ion uptake, indicates that alkaline phosphatase hydrolase activity is not directly involved in vesicle-mediated calcification under these experimental conditions.