Sohan S. Jande

Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chickens

SummaryCalcium-binding protein (CaBP) has been localized with the immunoperoxidase method using antiserum against purified chick duodenal CaBP. Different preparative procedures were employed to investigate the experimental conditions possibly responsible for the contradictory reports in the literature of the precise cellular localization of CaBP. Freeze substitution, frozen sections followed by fixation and coagulant and non-coagulant fixatives were used with appropriate control sections to demonstrate that the true localization of CaBP in the chick duodenum is in the absorptive cell cytoplasm. The goblet cell localization reported in the literature seems to be a diffusion artifact due to inadequate fixation. CaBP was also localized in several other tissues. In the hen uterus, the tubular glands beneath the surface epithelium showed intense reaction. In the kidney, CaBP was present in the cells of the straight and convoluted segments of distal tubules. The cortex of the chick cerebellum showed the CaBP in Purkinje cells. The entire dendritic trees contained the reaction product. No other neurons in the molecular or the granular layer were stained. In the deep cerebellar nuclei, all neurons were negative and these were outlined by deeply staining axons of the Purkinje cells and their synaptic endings.

Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks

These studies show that the changes in the fine structure of the osteocytes with their age are correlated with the alterations in the periosteocytic bone matrix. These changes seem to be functionally significant. A young, bone-forming cell gradually changes into a mature bone-resorbing cell. Finally it degenerates. The osteocyte population is thus heterogeneous and is constituted by functionally different cells. The peripheral region of the newly formed pericellular bone matrix is recognizable as perilacunar matrix and becomes mineralized. The matrix near the cell, remains as osteoid and is the pericellular space. Later these regions are occupied by either a completely broken down or a partially broken down (modified matrix) bone matrix, both devoid of mineral. The breakdown product as shown by colloidal iron and colloidal thorium staining techniques seems to be acid mucopolysaccharide in nature.

Target Cells of Vitamin D in the Vertebrate Retina

Using PAP technique, cellular localization of vitamin D-dependent calcium-binding protein (D-CaBP) was investigated in vertebrate retina with monospecific antisera against chick duodenal D-CaBP. In the chick retina, the receptor cells were positive. In the inner nuclear layer, horizontal cells and some bipolar cells were also positive. Some amacrine cells as well as different levels of the inner plexiform layer were also positive for D-CaBP. A few interspersed ganglion cells were positive but their axons forming the optic tract were negative. Müllers cells were negative. In 1-day-old chicks and 4-week-old rachitic chicks there was paucity and absence, respectively, of D-CaBP staining in horizontal cells. In the mouse, rat, and rabbit the receptors had only trace amounts of reaction product in their outer segment and pedicle. Horizontal cells were densely positive throughout their cellular body and processes. Some amacrine cells in the inner nuclear layer were positive. In the mouse and rat three horizontal levels of the outer plexiform layer were very prominent because of their dense staining for D-CaBP. Many ganglion cells were also positive along with their axons forming the optic nerve. In the rabbit, no positive layers were seen in the inner plexiform layer, and ganglion cells with their fibers were negative. In the frog retina there were smaller amounts of D-CaBP in the receptor cells and horizontal cells than that of the chick retina. Also, the fibers of the ganglionic cells were positive for D-CaBP. In all species studied, some amacrine cells were stained for D-CaBP. Because of its possible roles in membrane calcium transport and intracellular Ca++ regulation, it has perhaps similar functions in these positive cells. The synthesis of D-CaBP is dependent upon vitamin D. These positive cells are thus target cells of vitamin D.

Immunocytochemical Demonstration of Two Vitamin D-Dependent Calcium-Binding Proteins in Mammalian Kidney

Distribution of vitamin D-dependent calcium-binding proteins (CaBPs) were studied in four mammalian species using monospecific antibodies raised against chick duodenal CaBP (D-CaBP), human cerebellar CaBP (L-CaBP), and rat duodenal CaBP (S-CaBP). The immunoperoxidase technique of unlabelled antibodies was employed. The distribution of D-CaBP/L-CaBP was identical in all the species studied except for the monkey. In the rat, pig, and human nephrons, D-CaBP/L-CaBP was seen in the cytoplasm of the cells of the distal convoluted tubules, initial segments of the collecting ducts and interspersed cells of the collecting ducts. Proximal convoluted tubules, glomeruli and maculae densae were negative. In the monkey, in addition to the cells of the distal convoluted tubules, the cells along the entire length of the collecting ducts were also strongly positive. S-CaBP was found to be species-specific, and hence positive results were obtained only in the rat nephron. The strongest positive reaction for S-CaBP was seen in the cells of the distal convoluted tubules. These same cells were also positive for D-CaBP/L-CaBP. S-CaBP was also detected in the cells of the thick ascending limb of the loop of Henle, along the entire length of the collecting ducts and in smaller amounts in cells of the macula densa. Intracellularly the S-CaBP was present only in the apical cytoplasm of positive cells. D-CaBP/L-CaBP stained the entire cytoplasm but the staining in the apical cytoplasm was denser.

Localization of vitamin D-dependent calcium binding protein in the electrosensory and electromotor system of high frequency gymnotid fish

Vitamin D-dependent calcium binding protein (D-CaBP) was localized in the brains of high frequency gymnotid fish. In birds and mammals this protein is seen in a variety of cell types including Purkinje cells, inferior olivary cells and CA1 pyramids of the hippocampus. This distribution has led us to speculate that D-CaBP may be important in buffering intracellular calcium, perhaps more specifically that calcium which enters the cell during dendritic calcium spikes. In the gymnotid fish D-CaBP was found in many of the same cell types in which it is also seen in birds and mammals. In addition, D-CaBP is specifically present in neurons which drive the electric organ (pacemaker and relay cells) and neurons within the electrosensory system which are phase-locked to the electric organ discharge (spherical and giant cells). Relay cells and giant cells have exceptionally high concentrations of D-CaBP. These cells do not exhibit calcium spikes and the role of their D-CaBP may be to regulate calcium released from intracellular stores.

Effects of demineralization in an ethanolic solution of triethylammonium EDTA on solubility of bone matrix components and on ultrastructural preservation

SummaryA solution of triethylammonium EDTA in 80% ethanol was evaluated as a demineralizing reagent for bone in comparison with aqueous solutions of EDTA. Biochemical analysis and acrylamide gel electrophoresis of extracts of finely powdered bovine bone showed that most of the macromolecular components of the organic matrix extractable in aqueous EDTA were retained when the triethylammonium EDTA reagent was used. Ultrastructural examination of chick tibias decalcified with the reagents showed a better preservation of cellular morphology, especially the membranous components, and more uniformly distributed ground substance, though slightly less in quantity, when the aqueous reagent was used. Use of the two reagents appears to be complementary, the alkylammonium reagent being more appropriate for use in studies of the organic matrix of bone, including immunohistochemical studies of bone glycoproteins. The aqueous reagent is more appropriate for use in studies of cellular ultrastructure.

Comparative histological study of the effects of high calcium diet and vitamin D supplements on epiphyseal plates of vitamin-D-deficient chicks

A comparative histological and microradiographic study of the tibial epiphyseal plates of chickens raised on: (1) a vitamin-D-deficient diet; (2) a vitamin-D-deficient diet supplemented with cholecalciferol, and (3) a vitamin-D-deficient diet to which extra calcium had been added, has revealed that a high-calcium diet did not normalize the epiphyseal plates completely. However, it restored the normal length and chondrocyte arrangement to the proliferative zone. The degenerative zone became elongated and this seems to be related to the hypophosphataemic condition which has developed as a result of the special diet.

Effects of parathormone on osteocytes and their surrounding bone matrix

SummaryYoung chicks were treated with 25 U.S.P. units of Parathormone (PTH, Eli Lilly and Co.) each day for three days. Osteocytes in the tibial mid-diaphyses were studied. As early as two hours after the treatment, there was an evident increase in the amount of affected matrix. However, it was only partially broken down (modified). Based on the relative number of osteocytes in the formative, resorptive, and degenerative phase and of dead osteocytes (empty lacunae), the effects of PTH on the osteocyte population have been quantitatively evaluated. At two hours after the treatment, there was a decrease in the number of formative osteocytes and an increase in the number of resorptive osteocytes. The number of formative cells continued to decrease. The resorptive osteocytes were maximal (76±4.5% of the population) at one day after the treatment. Later this number decreased with a somewhat corresponding increase in the degenerative osteocytes. It seems that PTH treatment promotes the maturation of osteocytes and decreases the formative but enhances the resorptive phase of the osteocytes.

Cytochemical localization of parathyroid hormone activated adenyl cyclase in rat kidney

SummaryPTH sensitive adenyl cyclase has been localized on the brush border of the cells of the proximal tubules only. Sodium fluoride activated adenyl cyclase has been observed at the same location as well as in association with interstitial cells, but not on distal tubules or collecting tubules. The technique is quite specific and gives reproducible results.

The influence of dihydroxylated vitamin D metabolites on bone formation in the chick

SummaryThe ability of 1,25(OH)2D3 and of 24,25(OH)2D3 to prevent or to heal rickets in chicks was evaluated by studies of plasma biochemistry, growth plate histology, bone morphometry and microradiography, and bone mineralization. 1,25(OH)2D3 at a dose of 100 ng/day produced fewest abnormalities compared with vitamin D3-treated control chicks. Bone growth was slightly greater than vitamin D3-treated controls in chicks given a lower dose of this metabolite; the reverse was observed in chicks given a higher dose. 24,25(OH)2D3 was less effective than 1,25(OH)2D3 in preventing rickets even at doses as high as 400 ng/day. Treatment of rachitic chicks with doses of 24,25(OH)2D3 up to 300 ng/day produced no healing effect on the bone lesions, in marked contrast to the beneficial effects observed with 1,25(OH)2D3.