Immunocytochemical localization of vitamin D-dependent calcium-binding protein in the growing chick lymphoid organs.

Monospecific antiserum against chick duodenal vitamin D-dependent calcium-binding protein (D-CaBP) was used to localize this protein by the peroxidase-antiperoxidase method (PAP) in the thymus, spleen and bursa of Fabricius of normal growing chicks in 20 day old embryos; in normal growing chicks at 1, 2, 3, 4 and 6 weeks of age in chicks fed a rachitogenic diet for 4 weeks. In the normal chick thymus, D-CaBP was localized throughout the cytoplasm and in the nucleus of cortical epithelial reticular cells (ERC) and in Hassal's corpuscles of the medulla. In the normal spleen reticular cells of the marginal zones showed dense deposition of reaction product in the nucleus and throughout the cytoplasm. In the bursa of Fabricius, only a few scattered cells in the medulla showed some staining. Wide variation was encountered in D-CaBP staining in the thymus and spleen of 4 week old chicks from different broods. In 4 week old rachitic chicks, staining in the thymus and in the spleen was generally reduced in intensity and occasionally was entirely absent. The presence of D-CaBP in some reticular cells of the thymus, spleen and bursa of Fabricius, identifies these lymphoid organs as vitamin D3 targets. Thus, 1,25(OH)2D3 may have an important role in some aspects of the immune defence mechanism.

Calcium-binding protein immunocytochemistry in organotypic cultures of cerebellum.

Vitamin D-dependent calcium-binding protein is known to be present in specific classes of neurons including Purkinje cells of the rat and chick. Explant cultures of newborn mouse cerebellum consisting of cerebellar cortex and deep nuclear region were fixed at maturity (20 days in vitro). Paraffin sections were reacted with the antiserum to purified chick duodenal calcium-binding protein. The Purkinje cell of these cultures reacted in its entirety--neuronal soma, dendrite, axon and terminals (in the deep nuclear region). The results verified many of the findings of our previous morphological studies. Qualitatively similar results were obtained with cultures maintained either in medium containing serum and embryo-extract or in a defined medium. There is not yet sufficient data to indicate whether this protein in the neurons in culture is dependent upon an exogenous source of vitamin D. This immocytochemical marker should prove useful to identify a specific neuron-type in culture.

Electron microscopic detection of calcium binding to the duodenal absorptive cell plasma membranes.

Electron dense structures (EDS) were observed on the plasma membranes of duodenal absorptive cells of chicks of various physiological states regarding the calcium binding protein (CaBP). EDS were seen only when calcium was added to formal-glutaraldehyde fixative in cacodylate buffer and the material was post-osmicated in phosphate buffer. Microvillar, lateral and basal plasma membranes showed the EDS and these were seen always in the inner dark layer of the three-layered unit membrane. No EDS were seen in the embryonic material and some were present in material from chicks two days after hatching. There were more in that of 6-week-old normal and vitamin D replete animals. The absorptive cells from chicks, fed on a diet supplemented with cestrum powder, known to mimic the effects of 1,25-dihydroxycholecalciferol, showed the highest number of EDS. On the basis of this distinct correlation of EDS frequency with amounts of CaBP known to be present in chick duodenum, it seems that EDS, precipitated calcium phosphate, strongly suggest the sites of CaBP.

Effect of vitamin D3 on duodenal absorptive cell plasma membranes: freeze fracture replication study.

Vitamin D3 induced changes in the number of 5.8 nm sized randomly scattered particles on the EF faces of plasma membranes of chick duodenal absorptive cells. Their number increased as compared to those in rachitic animals. Also vitamin D3 seemed to alter the nature of the zonula occludens. These changes appear to be related to increased active absorption and passive diffusion of calcium respectively under vitamin D3 influence.

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 cytochalasin B and dihydrocytochalasin B on calcium transport by intestinal absorptive cells.

In vivo calcium absorption was studied in normal and rachitic chicks. Cytochalasin B (CB) at a concentration of 25 microgram/ml added to the medium inside the duodenal lumen inhibited calcium absorption (20 min) from 82.5 +/- 1.9% of calcium absorbed in the controls to 59.2 +/- 3% in normal and from 70.0 +/- 2.3% to 47.0 +/- 2.1% in rachitic chicks. In vitro studies by everted ileal sacs of young rabbits also showed an inhibition of active transport of calcium due to CB. Whereas in the controls the ratio of 45Ca concentrations in serosal and mucosal media (60 min) was 7.2 +/- 0.32, the ratios were 5.24 +/- 0.52; 4.40 +/- 0.36; 3.40 +/- 0.42; 5.77 +/- 0.52; 1.38 +/- 0.08; and 1.06 +/- 0.02 in the presence of CB at concentrations of 5, 10 and 25 microgram/ml; colchicine 10(-4)M, Na citrate 0.02M, and heat-devitalized conditions, respectively. 45Ca concentration in the mucosal scrapings was also affected. It showed an increase from controls (15,101 +/- 404 cpm/mg) and correlated with CB concentration: 17,378 +/- 489, 19,015 +/- 1000, and 20,201 +/- 362 at 5, 10, and 25 microgram/ml, respectively. Dihydrocytochalasin B also inhibited active calcium transport and caused an increase in 45Ca concentration in the mucosal scrapings. Correlated electron microscopic studies showed certain changes in the brush border, especially in some actin microfilaments in the terminal web region. It seems that these morphological alterations may be related to transcytoplasmic movement of calcium.

Ultrastructural observations on the chorionic epithelium, parathyroid glands and bones from chick embryos developed in shell-less culture.

Three-day-old chick embryos were cultured in Petri dishes until they reached developmental stages 37 or 39 (Hamburger & Hamilton, 1951). The electron microscopical study of the chorio-allantoic membranes showed that, even in the absence of the shell and the corresponding calcium supply, 'capillary-covering' and 'villus-cavity' cells differentiated well. The parathyroid glands from the cultured embryos showed ultrastructural signs indicative of active synthesis and secretion of the parathyroid hormone. This correlates well with the significant hypocalcemia (5.4 mg/100 ml) observed in these embryos. In all cultured embryos mineralization of bones was greatly reduced as shown by alizarin bulk staining and confirmed by histological and electron microscopical analysis. The ultrastructural characteristics of osteoblasts and osteocytes, as well as of the bone matrix, appeared normal. The defect in mineralization appeared thus to be due to a deficiency in the availability of calcium and not to a delay in bone differentiation. This implies that the yolk sac appears to lack calcium regulatory capacity since it cannot compensate for the absence of shell by increasing its own contribution in order to assure an adequate mineralization of the embryonic bones.

Radioisotopic and morphometric evaluation of the effects of beta-aminopropionitrile on chick bone matrix formation and its mineralization.

Bones from 2-week-old chicks, fed a diet containing 0.05% beta-aminopropionitrile (beta-APN), were studied for 3H-tetracycline and 3H-proline incorporation at 7, 14 and 21 days of the experiment. Liquid scintillation counts showed a comparable amount of 3H-proline incorporation in the controls and experimentals. However, the incorporation of 3H-tetracycline was significantly lower in experimentals at all intervals. Fluorescent microscopy after cold tetracycline injections at similar intervals showed a comparable amount of linear bone apposition in controls and beta-APN-fed chicks. The bone ash content in beta-APN-fed chicks was lower at all intervals whereas the serum calcium level was similar to the controls. Microradiography showed that cortical and marrow areas, in cross sections, were not significantly different in the controls and beta-APN-fed chicks. On the basis of these data it is suggested that beta-APN at the dose used does not inhibit the bone matrix synthesis but inhibits its mineralization.

Investigations of alkaline phosphatase Ca+2-ATPase and Na+, K+-ATPase during beta-APN-induced initial bone mineralization inhibition.

Tibias of 6-day-old white Leghorn chick embryos treated with beta-aminopropionitrile (beta-APN; 0.1 mg/egg/day) for 4 days and injected with 3H-proline or 3H-tetracycline on the 11th day were analyzed for incorporation of 3H-proline and 3H-tetracycline. The incorporation of 3H-proline was comparable in the controls and beta-APN-treated embryos. However, the incorporation of 3H-tetracycline was significantly lower in beta-APN-treated embryos. The bone ash contents were also lower in the latter group. Alkaline phosphatase and Ca+2-ATPase were found to be significantly lower in beta-APN-treated embryonic bones. There was, however, no difference in the activity of Na+, K+-ATPase. The histochemical examination showed the alkaline phosphatase to be present on osteoblasts and matrix vesicle plasma membranes at the periosteal surface. The chick embryonic liver tissue showed no significant differences in the activities of any of the above enzymes. The results suggest that beta-APN-induced inhibition of the bone mineralization may be due to the bone-specific inhibition of alkaline phosphatase and Ca+2-ATPase.

Effects of beta-aminoproprionitrile on formation and mineralization of rat bone matrix.

Bones of 4-week-old Sprague-Dawley rats fed a diet containing 0.25% beta-aminoproprionitrile (beta-APN) for 21 days and injected with 3H-proline, 3H-tetracycline, and cold tetracycline at 5-, 10-, and 20-day intervals were analyzed for incorporation of 3H-proline and 3H-tetracycline. Tibial cross sections were examined by fluorescent microscopy to determine the linear bone apposition between intervals and the distribution and intensity of tetracycline bands. The incorporation of 3H-proline was similar in beta-APN-treated and control rats. Incorporation of 3H-tetracycline, however, was significantly lower in beta-APN-treated rats. Fluorescent microscopy of tibial cross sections showed a comparable amount of linear matrix apposition in control and treated rats. It is suggested that beta-APN does not inhibit the bone matrix formation but does interfere with its mineralization.

A biochemical and morphological investigation of alkaline phosphatase and Ca+2-ATPase during initial mineralization in chick embryonic tibia.

Biochemical analysis of the enzymes alkaline phosphatase and Ca+2-ATPase in the membrane fraction obtained from chick embryonic tibias showed a positive correlation between the elevated activity of these two enzymes and the onset of mineralization. Histochemistry further showed an increased intensity of alkaline phosphatase at the time of the onset of mineralization. It seems that both of these enzymes are involved in the process of mineralization.

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

A 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.

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

Calcium-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.

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. Müller's 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.

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

The 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.

Effect of testosterone and 6-hydroxydopamine treatment on the metabolism of catecholamine and 5-hydroxytryptamine in methylcholanthrene-induced prostate carcinoma of rats.

The precursors tyrosine and tryptophan as well as the synthesizing and deaminating enzymes of catecholamines have been identified in methylcholanthrene-induced prostatic carcinoma of rats. Tyrosine hydroxylase, monoamine oxidase, catechol O-methyltransferase, dopamine, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid seemed to be neoplastic in origin, since electron microscopic studies failed to reveal the presence of any neuronal elements in this squamous epithelial cell carcinoma. Castration of rats significantly reduced the activity of tyrosine hydroxylase and the levels of tyrosine, dopamine, tryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in prostate tumors. The changes appeared to be androgen specific since reintroduction of testosterone restored several of these biochemical parameters virtually to control limits. Chemical sympathectomy induced by 6-hydroxydopamine failed to alter monoamine metabolism; however, the prostatic tumor grown in 6-hydroxydopamine-treated rats showed significantly (32%) less necrosis than those grown in normal animals.

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.