Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.