Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

Abstract

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway—amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na + with various cations, were used to study MV Pi transport, which had been thought to be Na +-dependent. MVs induced rapid mineralization ∼3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required ∼1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC 50’s of 3–6 μM and ∼10 μM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were ∼1:30 for ACP, ∼1:20 for HAP, ∼1:3.3 for CPLX, and ∼1:2.0 for MVs. MV mineralization was not found to be strictly Na +-dependent: substitution of Li + or K + for Na + had minimal effect; while N-methyl d-glucamine (NMG +) was totally inhibitory, choline + was clearly stimulatory. Na + substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG + totally blocked and choline + stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.