Ultrastructural changes in the lysosomes of rachitic intestinal absorptive cells

In chicks maintained on a rachitogenic (vitamin D deficient) diet, the number of intestinal absorptive cell calcium-lysosomes is markedly decreased in comparison to normal animals. In addition, the majority (better than 50%) of these rachitic calcium-lysosomes are atypical in their fine structure resembling the lamellar bodies seen in certain diseases (Tay-Sachs disease, Duchenne's muscular dystrophy). Such atypical organelles are characterized by their internal membranous swirls reminiscent of myelin figures. This information appears to be a further indication that lysosomes are normally involved in calcium homeostatic mechanisms and therefore sensitive to circulating vitamin D levels.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

The immunocytochemical localization of superoxide dismutase in the enterocytes of the avian intestine: The effect of vitamin D3

Summary Both light microscopical and electron microscopical immunocytochemical techniques were utilized to localize CuZnsuperoxide dismutase (SOD) in the duodenum of normal, rachitic and vitamin-D₃-replete chicks. This enzyme catalyses the dismutation of the superoxide anion, a toxic free radical generated during the normal aerobic metabolism of most respiring cells. Light microscopy showed no SOD activity associated with the duodenal enterocytes of normal and rachitic chicks. However, in rachitic animals subsequently treated with vitamin D, i.e. vitamin-D-replete chicks, intense immunoreactivity for the enzyme was seen in association with the apical border of the duodenal absorptive cells. Immunostaining for SOD was not seen in goblet cells. With electron microscopy, immunostaining for SOD activity was identified in association with the apical microvilli and, to a lesser degree, with the terminal web, a well as in association with both lysosomes and peroxisomes. From this report it appears that there is a physiological relationship between vitamin D, SOD and the intestinal absorptive cell. However, the precise relationship must await further clarification.     

Effects of 1,25-dihydroxycholecalciferol administration on the rat renal vitamin K-dependent carboxylating system

We have shown previously that the in vitro activity of the renal vitamin K-dependent gamma-glutamyl carboxylase toward synthetic oligopeptide substrates is stimulated by administration of either parathyroid hormone (PTH) or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] to rats [(1983) J. Biol. Chem. 258, 12783-12786]. Here we report that administration of 1,25(OH)2D3 to rats increases their levels of endogenous carboxylase substrate as well. Rats fed a vitamin D-deficient diet had highly elevated serum PTH levels while vitamin D-replete animals had undetectable levels. Furthermore, since PTH increases 1,25(OH)2D3 levels by stimulating renal 25-hydroxyvitamin D-1 alpha-hydroxylase, it is very likely that the stimulatory effects of PTH on the renal vitamin K-dependent carboxylating system are mediated by 1,25(OH)2D3.     

Calcium containing lysosomes in the normal chick duodenum: a histochemical and analytical electron microscopic study.

Absorptive cells of the normal chick duodenum contain numerous supranuclear vesicular/vacuolar structures. By routine transmission electron microscopy, such structures are membrane bound and demonstrate a granular content. These vesicles appear to move laterally and eventually coalesce with the lateral plasma membrane (exocytosis). The granular contents are resistant to high temperature microincineration, thus revealing their mineral-containing nature. The granular vesicular matrix also stains intensely with osmium pyroantimonate EGTA chelation of pyroantimonate-stained vesicles selectively extracts the granules indicating a high concentration of calcium. X-ray microanalysis also demonstrates a significant intravesicular calcium localization. When tissues were incubated for the presence of acid phosphatase, the supranuclear vesicles were markedly positive for this lysosomal enzyme. A possible role for these calcium-containing lysosomes in the transcellular flux of calcium ions across the intestinal absorptive cell is discussed.     

Differentiation of the changes in alkaline phosphatase from calcium ion-activated adenosine triphosphatase activities associated with increased calcium absorption in chick intestine.

The pattern of response of the intestinal enzymes Ca2+-activated adenosine triphosphatase and alkaline phosphatase in the chick to 1,25-dihydroxycholecalciferol is consistent with a role for the former but not the latter enzyme in the vitamin D-dependent absorption of calcium.     

1,25-Dihydroxyvitamin D3-induced calcium-binding protein: localization in organ-cultured embryonic chick duodenum

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces de novo biosynthesis of a specific calcium-binding protein (CaBP) in embryonic chick duodenum in organ culture. Using a highly sensitive and specific, peroxidase-antiperoxidase immunocytochemical procedure, 1,25(OH)2D3-induced CaBP in the organ-cultured duodenum was found only in the cytoplasm of absorptive cells, corresponding to its localization in rachitic chick duodenal cells after a single injection of 1,25(OH)2D3 in vivo. This observation, along with evidence correlating CaBP with calcium transport, strongly supports the use of the embryonic chick duodenal organ culture system as a physiologically relevant model of the vitamin D-dependent calcium absorptive mechanism.     

1,25-dihydroxycholecalciferol: the metabolite of vitamin D responsible for increased intestinal calcium transport

1,25-Dihydroxycholecalciferol was prepared from [26,27-³H]-25-hydroxycholecalciferol and from [1,2-³H]-25-hydroxycholecalciferol enzymatically and purified chromatographically. Injection of 62.5 pmoles of 1,25-dihydroxycholecalciferol intravenously into vitamin D-deficient chicks resulted in the accumulation of a maximum of 5.9% of the dose in the intestine. During the 12 hr period following injection, this radioactivity was found almost entirely as 1,25-dihydroxycholecalciferol. It has previously been shown that intestinal calcium absorption is initiated by 1,25-dihydroxycholecalciferol during this period. These results provide strong evidence that the 1,25-dihydroxycholecalciferol is not metabolized further before it initiates intestinal calcium absorption.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.     

Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption

The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli.     

Calcium lysosomes in rachitic and vitamin D3 replete chick duodenal absorptive cells

Calcium-containing lysosomes were described in a previous communication in this series (Davis et al., 1979). Their potential role in intestinal calcium uptake and transcellular transport was hypothesized. To further this notion, the effects of a rachitogenic diet and vitamin D₃ repletion were investigated. Intestinal absorptive cells from chicks maintained on a vitamin D deficient diet were characterized by decreased numbers of supranuclear calcium lysosomes. In contrast, intestinal cells from chicks given vitamin D₃ (cholecalciferol) subsequent to the rachitogenic diet showed numerous large compound supranuclear calcium lysosomes. Since other steroid hormones are known to effect lysosomes, it is tempting to speculate that vitamin D, itself a steroid hormone, may activate lysosomes which themselves might be involved in calcium homeostatic mechanisms.     

The effect of age and 1,25-dihydroxyvitamin D3 treatment on the intestinal calcium-binding protein of suckling rats.

The intestinal level of the vitamin D-dependent duodenal calcium-binding protein was assayed by an equilibrated column technique in rat embryos, neonates, and pups. Calcium-binding protein was undetectable in unborn, newborn, and 1- to 2-day-old rats i.e., the level was lower than in severely vitamin D-deficient animals. Calcium-binding protein was detected after the animals were 5-days old and thereafter rose monotonically as a function of body weight. Treatment with 1,25-dihydroxyvitamin D₃ failed to raise the calcium-binding protein levels of newborn or 1-day-old rats, but doubled the level in 11- or 12-day-old pups. Plasma calcium was raised in all treated animals. The failure to detect calcium-binding protein in vitamin D-replete suckling animals provides evidence for a dissociation between calcium absorption and calcium binding protein.     

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

Effects of vitamin D3 on the uptake and release of calcium by isolated intestinal mitochondria

Administration of vitamin D3 or 1,25 (OH)2D3 to rachitic chicks produces a decrease of 45Ca uptake by mitochondria from intestinal mucosa. This effect of vitamin D3 shows tissue specificity, since it was not observed in liver mitochondria from the same animals. The Km values were similar (about 10 microM) for intestinal mitochondria from rachitic and treated animals. The Ca2+ efflux in previously loaded mitochondria was not changed by treatment. The Ca content of recently isolated mitochondria was strikingly lower after vitamin D3 administration. It is concluded that vitamin D3 may participate in the mechanism which regulates the intramitochondrial Ca concentration.     

Synergistic effects of 1,25(OH)2D3 and 24,25(OH)2D3 on duodenal CaBP in rachitic chicks and on eggshell weight in Japanese quails

The role of 24,25(OH)2D3 in calcium homeostasis is still controversial. In the present study the administration of low doses of 1,25(OH)2D3 and of higher doses of 24,25(OH)2D3 either alone or in conjunction with each other, were studied in rachitic chicks and in Japanese quails. Whereas 24,25(OH)2D3 alone had no significant effect on duodenal CaBP and on alkaline phosphatase in chick serum, it increased the influence of 1,25(OH)2D3 on these two parameters strongly. Also, when 1,25(OH)2D3 and 24,25(OH)2D3 were given simultaneously to Japanese quails, calcium excretion via the egg shell was clearly higher than when either metabolite had been administered alone. These results indicate that 1,25(OH)2D3 and 24,25(OH)2D3 exert a strong synergistic effect in rachitic animals.     

Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor

Cartilage calcification at specific sites is a key event that leads to skeletal development and growth. To obtain insights into the control of cartilage calcification, we examined whether cells distributed in permanent cartilage regions might have the ability to express the calcification-related phenotype in a permissive environment. Chondrocytes were isolated from the permanent and growth plate cartilages of 4-week-old rabbit ribs. They were seeded as a pelleted mass in a centrifuge tube and cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. These cells proliferated for several generations, and then synthesized large amounts of proteoglycans, yielding a cartilage-like tissue in 16 days. Cultures from the permanent and growth plate cartilages showed similar time courses for increases in DNA synthesis and proteoglycan production that reached similar maximal levels. Thereafter, they initiated the syntheses of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induced matrix calcification without additional phosphate. The increases in alkaline phosphatase, 1,25-dihydroxycholecalciferol receptor, and calcium contents in cultures from the permanent cartilage were consistently delayed for 4-7 days relative to the growth plate-derived cells, but caught up by Day 28. The maximal levels of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor in the cultures from the permanent cartilage were 40- to 100-fold higher than that of the in vivo permanent cartilage. These results provide evidence that permanent cartilage cells in postnatal young rabbit ribs have the capacity to express alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induce calcification in a permissive environment, although they never express these calcification-related phenotypes in vivo.