Further evidence of an intravesicular Ca2+- pump in odontoblasts from rat incisors

Intracellular vesicles containing alkaline phosphatases were isolated from isolated odontoblasts using several centrifugation techniques, gradient media and filtering procedures. With a combined centrifugation technique using 0.32 M sucrose layered on 1.23 M sucrose, a fraction containing alkaline phosphatases with a 36-40-fold increased specific activity was obtained. This fraction also revealed a high Ca2+-accumulating ability. The vesicle fraction was totally free from mitochondria but to some extent contaminated by lysosomes. Characteristics of Ca2+-uptake were obtained. The Ca2+-uptake was maximal at 37-40 degrees C whereas no Ca2+-accumulated at 4 degrees C. Temperatures above 40 degrees C strongly inhibited Ca2+-uptake. ATP was the most potent stimulator of Ca2+-uptake whereas ITP, GTP, CTP, ADP, PPi and AMP also promoted Ca2+-uptake. Cysteine, EDTA and Triton X-100 were inhibitory to Ca2+-uptake. A correlation between alkaline phosphatases in intracellular vesicles as well as their relation to extracellular matrix vesicles and to the mineralization process is suggested.