tetramisole and Rickets

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.

Topics: Adenosine Triphosphate; Animals; Calcinosis; Calcium Pyrophosphate; Cartilage; Cartilage Diseases; Detergents; Extracellular Matrix; Octoxynol; Pyrophosphatases; Rats; Rickets; Skull; Spectroscopy, Fourier Transform Infrared; Tetramisole

To clarify the role of alkaline phosphatase (ALP) and ATPase in skeletal mineralization, we studied the effect of the anthelmintic drug, l-tetramisole (levamisole), a stereospecific inhibitor of ALP activity, and its inactive isomer, d-tetramisole (dexamisole), on in vitro calcification of rachitic rat cartilage. ALP activity in homogenized rachitic rat proximal tibiae was inhibited by l-tetramisole in a dose-dependent manner. Histochemical and electron microscopic cytochemical analysis of intact epiphyseal plate rachitic rat cartilage showed that 5 x 10(-2) M and greater concentrations of l-tetramisole (1) virtually abolished ALP activity, (2) moderately reduced ATPase activity, and (3) prevented in vitro cartilage calcification, while preserving the structural integrity of the matrix vesicles. Concentrations of d-tetramisole as high as 1 x 10(-1) M failed to inhibit ALP activity in tibial homogenates by more than 10 per cent and did not alter histochemical staining of enzyme activity or calcification in intact cartilage slices. Heating the cartilage slices destroyed ALP activity, prevented calcification, and disrupted matrix vesicle integrity. These data show that there is a close association between ALP activity and in vitro calcification of rachitic rat cartilage. In the absence of ALP activity, intact matrix vesicles do not promote calcification. Our data also suggest that some ATPase activity of rachitic rat cartilage may be distinct from ALP activity.

Topics: Adenosine Triphosphatases; Alkaline Phosphatase; Animals; Calcinosis; Cartilage; Cell Membrane; Male; Rats; Rickets; Stereoisomerism; Tetramisole