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Hamster molar tooth germs were tested in vitro for their capacity to incorporate 45Ca, 32PO4 and H14CO3 into their mineral. Concomitantly, the activities of alkaline phosphatase and carbonic anhydrase were measured in the cultured tooth germs. Incorporation of calcium and phosphate into the dental mineral increased with time in culture (0-10 days), whereas carbonate incorporation decreased slightly. Alkaline phosphatase and carbonic anhydrase increased with time. These results suggest that carbonic anhydrase is probably not involved in carbonate deposition, but in carbonate depletion of the dental mineral, carbonate being probably replaced by phosphate.

This study was designed to compare the various phosphatases and pyrophosphatases in bone with those in developing teeth. Moreover the alkaline phosphatase inhibitor 1-p-bromotetramisole (1-pBTM) was assessed for its ability to discriminate between the several phosphatase activities. Enzyme activities were determined over the pH range 3.50 to 11.25 in homogenates of calcifying hamster tibiae, and whole tooth germs using p-nitrophenyl-phosphate (pNPP) and pyrophosphate (PPi) as substrates. Characterization of alkaline phosphatase inhibition with 1-pBTM showed that in tooth germs the pI50 values of 1-pBTM for whole tooth germs, ectodermal and mesenchymal phosphatases were identical (pI50 = 5.36), which was slightly but significantly lower than that for bone alkaline phosphatase (pI50 = 5.64). In bone, two pNPP-ase activities were found with optima at pH 5.5 and pH 10.3 respectively. The alkaline phosphatase activity was completely inhibited by 10(-4) M 1-pBTM. Inhibition of the acid phosphatase was incomplete. With PPi as substrate, three activities were found with optima at pH 4.8, 7 and 8.7 respectively. All these PPi-ase activities were strongly inhibited by 1-pBTM in developing teeth, only one phosphatase activity was found, which exhibited an alkaline pH (10.3) optimum with both substrates. This activity was inhibited by 1-pBTM with both substrates although the effect on pNPP-ase activity was more marked. From these results we conclude that in bone there are insufficient differences in the extent of inhibition by 1-pBTM to distinguish between the various phosphatase activities when more physiological substrates such as pyrophosphate (PPi) are used.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of l and d-, p-bromotetramisole (pBTM) on alkaline phosphatase were studied in relation to 45Ca2+, 32phosphate and 3H-thymidine uptakes in non-mineralizing second (M2) and mineralizing first (M1) maxillary hamster molar tooth germs under the conditions of organ culture. At the concentration used in culture (10(-3)M), l-pBTM completely inhibited alkaline phosphatase activity in tooth germ homogenates. About 30% of the enzyme activity was inhibited by d-pBTM at the same concentration. In culture, there were no significant differences between the effects of l and d-pBTM isomers on all the parameters measured. In the non-mineralized M2 molars, l and d-pBTM significantly reduced both TCA-soluble and TCA-insoluble 32phosphate uptakes but not 45Ca2+. However, 3H-thymidine uptake was also significantly decreased. In M1 molars, the pBTM isomers significantly reduced the uptake of TCA-soluble 32phosphate and 45Ca2+ but not TCA-insoluble 32phosphate. Ouabain, a specific inhibitor of Na+-K+-ATPase (but not alkaline phosphatase), also significantly reduced 3H-thymidine uptake to the same extent as the pBTM isomers in the non-mineralizing M2 molars, but it did not significantly affect either 32phosphate (TCA-soluble and TCA-insoluble) or 45Ca2+ uptake. Although this inhibitor significantly reduced both 45Ca2+ and TCA-soluble 32phosphate uptake in the mineralizing M1 molars, this effect was much less dramatic than was the case with the pBTM isomers. The reduced 45Ca2+ uptake in the M1 molars is probably a consequence of reduced mineralization since in the non-mineralizing M2 molars calcium uptake was not significantly affected by the pBTM isomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnant CD1 Swiss albino mice were irradiated with 400 rads of whole body X-irradiation on the twelfth gestational day. The animals were then sacrificed beginning on day 14 through 20 of gestation by chloroform inhalation. The fetuses were extirpated via laparotomy and decapitated. The severed heads were rapidly frozen and sectioned in a cryostat. The sections were affixed to glass slides and incubated for succinic dehydrogenase activity according to the method of Nachlas et al. (1957) and counterstained in Safranin 0, routinely dehydrated and mounted. Data from observations indicated that siccinic dehydrogenase activity appeared normal in the tissue layers of the developing tooth germ when compared to control animals. When the experimental procedure had invoked damage to the developing tooth, succinic dehydrogenase activity was lessened relative to the degree of damage. Presumably the X-irradiation had affected the cellular maturation process thereby reducing the functional competency of the cells as illustrated by the reduced enzyme activity.