Radioautography of rat incisor dentin as a continuous record of the incorporation of a single dose of ³H‐labeled proline and tyrosine

Abstract

After injection of labeled precursors such as 3H-proline or 3H-tyrosine into rats, the incisor dentin contains a continuous and stable record of precursor incorporation into labeled proteins. This record was visualized and quantitated with radioautography in order to evaluate the quantitative changes in enamel where newly secreted proteins randomize with older proteins and both are eventually lost. Up to 4 hours after injection, the pulse-dose was incorporated as a highly labeled band of predentin. The band was entirely within calcified dentin at 2 days and was further removed from new predentin by 4 and 8 days. Dentin which formed proximal to the heavily labeled band contained an amount of radioactivity reflecting the level of labeled precursor available at that time. A standardizing factor for experimental error was obtained by quantitating the reaction in the heavily labeled band, and a post-pulse incorporation factor was determined from the amount of radioactivity added per day as weakly labeled dentin. The variation within the heavily labeled band was assumed to reflect experimental error. The number of grains in the bands were averaged from 4 hours to 8 days to give the standardizing factor. This was multiplied by the ratio of enamel to dentin counts in the same section to obtain a corrected enamel count. In this way the coefficient of variation was improved from a high of 17.2% in uncorrected enamel counts to 2.4% in corrected counts. The post-pulse incorporation factor was higher with tyrosine than with proline. With proline it amounted to 5% increase per day from 1 to 4 days and 2.5% per day from 4 to 8 days after injection. In addition, with 3H-proline the incorporation into predentin increased from 30 minutes to 4 hours. With tyrosine, the counts increased from 30 minutes to 1 hour, but decreased by nearly one third from 1 to 4 hours. This was interpreted as a loss of short-lived matrix proteins including procollagen peptides produced during conversion from procollagen to tropocollagen in the predentin.

In vivo experimentation on rat incisor enamel organs through a surgical window

“…Because of the appositional formation of dentin by odontoblasts, it becomes a continuous and stable record of the avai1ability of labeled amino acids for incorporation into protein (Josephsen and Warshawsky, 1982). Although this study does not quantitate the arnount of radioactivity available to the enamel organ fo110wing the initial pulse introduced by the microinjection, it does show that concurrent with the diffusion of :lH-pro1ine into…”

The distribution of 3H‐proline in alveolar bone of the mouse as seen by radioautography

“…Formation and "migration" of the appositional band in bone are reported by several authors (Stallard, 1963;Crumley, 1964;Garant and Cho, 1979;Dreyer and Sampson, 1984) and are suggested to be useful as a method for determination of bone growth (Kenney and Ramfjord, 1969;Tonna, 1975Tonna, , 1976. This band is likely a direct reflection of the amount of label injected initially (Josephsen and Warshawsky, 1982). Grain counts over the appositional band were significantly higher than in new or old bone (P < .001) and represented a significant percentage of the total count (87.…”

“…This reaction has been used to calculate a postpulse incorporation factor in dentin which can be used to calculate a standardizing factor for experimental error. This technique is proven useful in the study of incorporation of radioactive label into dentin (Josephsen and Warshawsky, 1982).In general, previous studies were concerned with areas of initial bone formation and little attention was given to remodeling in other areas of the septum. The purpose of the present study is to document changes in the pattern of 3H-proline label relating to the remodeling of the interdental septum resulting from physiologic tooth movements.…”

Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse‐chase labeling with L‐[35S]‐ and L‐[methyl‐3H]‐methionine

“…SMITH ET AL. (Josephsen and Warshawsky, 1982). Such incorporation becomes less easy to detect beyond 8 hours when cells start to lose radiolabeling at a rate that is faster than the rate at which they incorporate new radiolabeling from the systemic circulation.…”