The tissue distribution, developmental changes, and the vitamin D dependence in the rat of two calcium-binding proteins [CaBPs, 28,000 and 10,000 Mr (28 and 10 K)] were examined. The radioimmunoassays used employed specific antibodies to either the human cerebellar CaBP (28 K protein) or to the smaller rat intestinal CaBP (10 K protein). The assay for the 28 K CaBP may be used to detect this protein in a number of mammalian species and tissues, whereas the 10 K CaBP assay appears to be specific only for the rat intestinal CaBP. This report demonstrates that the tissue distribution of the two CaBPs is different in the rat. High levels of the 28 H protein were found in the cerebellum and kidney, whereas the smaller CaBP was concentrated in the duodenum, jejunum, and cecum. Many other organs and tissues contained small quantities of both CaBPs. Developmental studies indicated some variability in the concentration of the CaBPs. Duodenum, kidney, and cerebellum all contained small amounts of one of the CaBPs prior to birth. Adult levels in all three tissues were already reached at 30 postpartum days. Levels of both CaBPs began to decline in rats older than 2 mo. The vitamin D dependence appeared to reflect cell turnover in that the duodenal and kidney CaBPs showed a vitamin D dependence not observed for the cerebellar protein.

The effect of long-term diabetes mellitus on bone and mineral metabolism was studied in BB rats. Diabetic rats were treated with 1 U of long-acting insulin every other day for 12 wk and compared with nondiabetic littermates. Urinary calcium excretion was increased greater than 10-fold, but serum total and diffusible calcium remained normal. Serum concentrations of both 1 alpha, 25-dihydroxyvitamin D3 and vitamin D-binding protein were significantly decreased in diabetic rats. The intestinal calbindin-D 9K concentration was decreased by nearly 50%, and active duodenal calcium absorption was totally abolished. Trabecular bone volume measured in the tibial metaphysis was decreased by 44%, and the osteoblast and osteoid surfaces were less than 10% of values observed in control rats, whereas the osteoclast surface was unchanged by diabetes. The daily bone formation (bone mineral apposition rate) measured by labeling twice with calcein was decreased by 86% in diabetic rats. The serum concentration of osteocalcin, a biochemical marker of osteoblast function, was similarly decreased (mean +/- SE 23 +/- 3 and 62 +/- 4 micrograms/L in diabetic [n = 15] and nondiabetic [n = 15] rats, respectively). Serum osteocalcin was significantly correlated with the serum concentration of insulinlike growth factor I (r = 0.89, P less than 0.001). Bone strength measured as the energy needed to fracture the femur was markedly decreased (5.3 +/- 1.4 and 8.4 +/- 1.3 N.m.degree in diabetic and nondiabetic rats, respectively; P less than 0.01). These histological, chemical, and biomechanical data clearly indicate that long-standing diabetes in BB rats results in severe low-turnover osteoporosis probably related to decreased osteoblast recruitment and/or function.

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In the present study the early phases of the development of the inferior olive were examined by using immunocytochemical techniques. We observed that, from embryonic day 16 onward, antibodies against the calcium binding proteins parvalbumin and calbindin and the calcitonin gene related peptide stain partially overlapping territories of the inferior olive. This staining delimits a biochemical zonation of the inferior olive which is combinatory and transient. We have previously observed a biochemical parcellation of the cerebellar Purkinje cells which, like that of the inferior olive, is first observed at E16, involves the combined expression of marker proteins and is also transient. In order to know whether the biochemical compartmentations of the cerebellum and inferior olive arise independently, the time course of the development of the olivocerebellar projection was studied by anterograde and retrograde in vitro axonal tracing by using the fluorescent carbocyanine dye DiI. The olivocerebellar axons were found to reach the limit of the cerebellar plate at E16 and to enter it at E17. Even at this age the great majority of the climbing fibers are tightly fasciculated, which minimizes their interactions with the PC clusters. These observations indicate that the topographical heterogeneity of Purkinje cells and inferior olive neurons arise independently. The transient biochemical individualization of subgroups of neurons during development could contribute to recognition mechanisms.