Vitamin D and the dual processes of intestinal calcium absorption.
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Animals↗
Biomedical subjects
R H Wasserman
Publications and source records attributed to R H Wasserman.
Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development.
The immunolocalization of the plasma membrane calcium pump (PMCA) was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP) immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody SF10 from human erythrocyte plasma membrane cross-reacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.
Animals↗
Strontium as a tracer for calcium in biological and clinical research.
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Calcium↗
Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption.
A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)2D3 to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and 47Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope, 44Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the 44Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)2D3 was an increased tissue uptake of luminal 47Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2-4 h after the 1,25(OH)2D3 dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8-16 h, 47Ca absorption was maximal and 44Ca was sparsely distributed in the intestinal tissue. 47Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue 44Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)2D3 on the intestinal Ca absorption process. The ion microscopic images provided unique information on the specific time-dependent changes in the tissue localization of Ca during the process of its intestinal absorption as affected by 1,25(OH)2D3.
Animals↗
Vitamin-D-dependent transcriptional regulation of the intestinal plasma membrane calcium pump.
The vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was shown to increase intestinal plasma membrane calcium pump (PMCA) gene expression. The present study was done to determine whether gene transcription is involved in this process. Nuclei were isolated from duodena of vitamin-D-deficient chicks given 1,25(OH)2D3 intracardially at various times before experiment. The abundance of PMCA RNA in the nuclear and total cellular fractions, measured by a ribonuclease protection assay, was significantly increased above control values at 1.5 hr. and maximized at 3 hr. post-dose. As shown, cross-contamination of nuclear PMCA RNA by cytosolic RNA cannot account for these results. These studies are the first to show that 1,25(OH)2D3 regulates expression of a plasma membrane calcium pump gene by increasing the rate of transcription.
Animals↗
Distribution of calbindin-D28K in the brain of the fetal sheep in late gestation.
Immunocytochemical distribution of calbindin-D28K was examined in the brains of fetal sheep at 100-105 and 120-125 days of gestation (dGA) and at term in labor (term approximately 150 dGA) and compared to adult sheep. Brains were perfused, cut frozen and immunostained as freely floating sections with a polyclonal anti-calbindin-D28K antibody. Light microscopy revealed that calbindin-D28K immunogenicity in fetal cerebellum, rhombencephalon, mesencephalon, diencephalon and telencephalon was overall very similar to that seen in the brains of adult sheep. In addition even though fetal sheep peripheral plasma cortisol concentrations increase 10-20 times over the last 3-4 weeks of gestation to peak during labor and delivery, no glucocorticoid dependent increases in calbindin-D28K in the hippocampal formation could be seen in the sheep fetus as have been demonstrated for the adult rat given exogenous glucocorticoids. It is concluded that: (1) the basic pattern of calbindin-D28K distribution seen in adult sheep brains is established by at least 100 dGA in the sheep fetus; and (2) the pattern of calbindin-D28K expression in fetal sheep hippocampal formation is not related to peripheral plasma glucocorticoid concentrations.
Animals↗
Vitamin D and intestinal calcium transport: facts, speculations and hypotheses.
The intestinal absorption of Ca2+ occurs by both a saturable, transcellular process and a nonsaturable, paracellular path. The transcellular path is a multistep process, comprised of the transfer of luminal Ca2+ into the enterocyte, the translocation of Ca2+ from point of entry (the microvillus border or membrane) to the basolateral membrane, and the active extrusion from the cell into the circulatory system. Each step in the transcellular movement of Ca2+ has a vitamin D-dependent component. The paracellular path also appears to be affected by vitamin D status. This review emphasizes some aspects of the Ca2+ absorptive process that require resolution and/or further experimental support. The following are discussed: evidence for participation in the active transport of Ca2+ by all segments of the small intestine; a hypothetical model for the feedback control of entry of luminal Ca2+; the current views on vitamin D-dependent movement of Ca2+ through the cytosolic compartment of the enterocyte; the stimulated synthesis of the plasma membrane Ca2+ pump and its gene expression by vitamin D; and the vitamin D-dependency of the paracellular transfer of Ca2+ with a comment on the physiological significance of the rapid response of the Ca2+ absorptive system in vitamin D-replete animals to 1,25-dihydroxyvitamin D.
Animals↗
Modulation of the excitability of avian peripheral nerves by vitamin D: relation to calbindin-D28k, calcium status and lipid composition.
Calbindin-D28k (CaBP), previously localized in some of the cell bodies of ganglia of the avian intestinal (Remark's) nerve, was shown to be vitamin D-dependent. In the present studies, the effect of vitamin D3 on electrophysiological properties of this nerve was examined in vitro. Electrical stimulation of the nerve yielded a compound action potential with two primary components, Peaks I and II. Peak II, suppressed by hexamethonium bromide or Ca(2+)-free buffer, is synaptically mediated. The transit time between the two peaks was unaffected by vitamin D3. The apparent conduction velocity, defined as [(activation time + transit time)/nerve length], was increased by vitamin D-deficiency and decreased by vitamin D3 repletion, the latter decrease due entirely to an increase in activation time. Activation time after vitamin D-repletion was correlated with an increase in CaBP and plasma Ca2+ levels. However, normalization of plasma Ca2+ by supplementation of vitamin D-deficient diets with excess calcium (2.5 and 4.0%) also resulted in an increase in activation time, without affecting neuronal CaBP levels. Vitamin D3 also decreased the conduction velocity and increased CaBP of the vagus nerve and, by lipid analysis, was shown to increase and decrease its phosphatidylcholine and phosphatidylethanolamine content, respectively, and to decrease its phospholipid/cholesterol ratio. Modulation of peripheral nerve activity by vitamin D3 is related to calcium status and perhaps to changes in lipid composition. The functional role of CaBP in the behaviour of this complex nerve remains unknown.
Animals↗
Immunocytochemical localization of the plasma membrane calcium pump, calbindin-D28k, and parvalbumin in Purkinje cells of avian and mammalian cerebellum.
A monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA) was shown to react immunohistochemically with an epitope of the PMCA in avian and mammalian cerebellum. Western blot analysis of purified synaptosomes and homogenates from avian cerebellum revealed major immunoreactive proteins with molecular masses (130 kDa and 138 kDa) similar to those of purified erythrocyte PMCA. Dual-imaging confocal immunofluorescence microscopy of avian cerebellum showed that the PMCA antibody stained the periphery of the soma whereas calbindin-D28k was located in the cytosol. PMCA heavily stained the more distal dendrites of the Purkinje cells and, within the resolution of the fluorescence procedure, colocalized with calbindin-D28k. By using alkaline phosphatase-conjugated second antibody, PMCA was again localized to the peripheral soma, to a segmental pattern in dendrites, and to presumed spiny elements. The soma periphery and dendrites of Purkinje cells of the rat cerebellum were also prominently stained with anti-PMCA antibody and compared to parvalbumin localization. Dendritic depolarization and dendritic spiking behavior are significant Ca(2+)-dependent events of Purkinje cells. The rapid decline of intracellular free Ca2+ after the rapid rise time of Ca2+ transients is considered to be due to sequestration by Ca2+ buffers, uptake by intracellular stores, and Ca2+ extrusion mechanisms, the latter a function of PMCA now shown immunohistochemically to be a prominent feature of Purkinje cell dendrites.
Alkaline Phosphatase↗
Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression.
The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assessed. The DNA probe for Northern analysis was obtained by reverse transcription and PCR with intestinal poly(A)+ RNA, using two 20-mer oligonucleotide primers homologous to the 3' coding region of the human teratoma PMCA. An EcoRI restriction fragment of the PCR product was cloned into the pBluescript II KS(-) phagemid vector, and the chimeric plasmid was used to transform Escherichia coli. The amino acid sequence deduced from the nucleotide DNA sequence of the PCR product and the cloned DNA were 96% homologous with the teratoma sequence. Northern blots of intestinal poly(A)+ RNA with 32P-labeled DNA showed the presence of three major species of chicken PMCA mRNAs at about 6.6, 5.4, and 4.5 kb. Northern analysis with the chicken PMCA DNA indicated that repletion of vitamin D-deficient chickens with vitamin D increased PMCA mRNAs in the duodenum, jejunum, ileum, and colon. After injection of 1,25-dihydroxyvitamin D3 intravenously into vitamin D-deficient chickens, duodenal PMCA mRNA tended to increase by 2 hr, reached a maximum at about 16 hr, and returned to baseline levels at 48 hr. Adaptation of chickens to either a calcium- or phosphorus-deficient diet resulted in a 2- to 3-fold increase in duodenal PMCA mRNA. These results indicate that vitamin D and specific variables that affect calcium absorption through the vitamin D-endocrine system increase intestinal PMCA gene expression.
Amino Acid Sequence↗
Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin-D28k and the 1,25-dihydroxyvitamin D3 receptor.
We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.
Animals↗
Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine.
The basolateral membrane of the enterocyte was previously shown to contain an adenosine triphosphate-dependent calcium pump. Using immunological procedures, the localization of the Ca2+ pump in chick intestine, and the effect of dietary variables on the concentration of the pump, were studied. A monoclonal antibody produced against the human erythrocyte calcium pump was shown to cross-react with a chick intestinal Ca2+ pump epitope. The most intense staining of intestinal tissue, as determined immunohistochemically, occurred at the basolateral membrane of the duodenum, jejunum, ileum, and colon, with minor staining elsewhere. By the Western blotting procedure, vitamin D repletion of vitamin D-deficient chicks was shown to significantly increase the concentration of the Ca2+ pump epitope of duodenal, jejunal, and ileal mucosa by a factor of 2-3. Chicks were also fed diets deficient in calcium or phosphorus, a situation known to result in the stimulation of the synthesis of calbindin-D28k and an enhancement of the efficiency of Ca2+ absorption. Adaptation of the chicks to these deficient diets was verified by an increase in intestinal levels of calbindin-D28k, and is now shown to increase the Ca2+ pump epitope. From these immunological studies, it seems apparent that dietary variables that enhance intestinal Ca2+ absorption also increase the amount of the intestinal basolateral Ca2+ pump.
Animals↗
Intestinal calcium transport and calcium extrusion processes at the basolateral membrane.
The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.
Animals↗
Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption.
Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.
Absorption↗
Hormonal effects on the sulfhydryl groups associated with intestinal brush border membrane proteins.
Previous studies demonstrated that the administration of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) to cholecalciferol-deficient chicks rapidly increases the reactivity and amount of the sulfhydryl (HS-) groups in intestinal brush border membranes (BBM). In the present study, the tissue and hormonal specificity of this effect was investigated. The HS- groups of intestinal and renal BBM were enhanced by vitamin D-3 and/or 1,25(OH)2D3, but no change was noted in isolated intestinal mitochondria and purified intestinal basolateral membranes, cardiac sarcolemma and erythrocyte membranes. Other steroid hormones including estradiol, testosterone, aldosterone, cortisol, dexamethasone and progesterone, yielded a response similar to 1,25(OH)2D3 on BBM HS- groups. Triiodothyronine and retinoic acid also resulted in an increase in intestinal BBM HS- groups. In a kinetic approach, using a specific sulfhydryl fluorescent probe (N-7-dimethylamino-4-coumarin-3-yl-maleimide, DACM), the reactivity of the BBM HS- groups was increased by estrogen and testosterone, as was previously shown for 1,25(OH)2D3. Intestinal BBM proteins, labeled with DACM, were separated by gel electrophoresis. Fluorescence scans of the gel showed two heavily labeled bands, one of 110 kDa, putatively brush border myosin I, and one of 43 kDa, putatively actin. Labeling of the 110 kDa protein was increased by 1,25(OH)2D3 and estradiol. Further studies are required to elucidate the physiological meaning of these hormone-mediated increases in reactivity and amount of the BBM sulfhydryl groups, as well as the nature of the intermediate biochemical reactions involved in this response.
Animals↗
Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen.
The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.
Animals↗
Reactivity of sulfhydryl groups in the brush-border membranes of chick duodena is increased by 1,25-dihydroxycholecalciferol.
The total amount and reactivity of SH-groups were determined in isolated duodenal brush-border membranes from rachitic chicks given 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3) before isolation of the membranes. 1,25(OH)2D3 treatment significantly increased the total amount of SH groups (9.7 +/- 2.3 vs. 23.9 +/- 2.1, P less than 0.001, n = 6) in brush-border membranes solubilized in 1% sodium dodecyl sulfate. The rate of reaction (reactivity) of membrane-bound SH-groups, determined with the fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), was also significantly enhanced by the intravenous injection of various doses (0.005-0.10 microgram) of 1,25(OH)2D3 into vitamin D-deficient chicks. An increase in reactivity occurred as early as 10 min after dosing of the chicks with 1,25(OH)2D3. Fluorescence scanning of the membrane proteins labeled with DACM and separated by polyacrylamide gel electrophoresis revealed three major peaks of fluorescence and a generally higher degree of fluorescent labeling of these and many other proteins in the membranes isolated from the 1,25(OH)2D3-treated chicks. The physiological significance of the 1,25(OH)2D3-mediated increase in the reactivity and total amount of membrane-bound SH-groups in terms of vitamin-D-dependent epithelial transport and epithelial membrane properties needs to be further explored.
Animals↗
Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: a 44Ca stable isotope study.
The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.
Animals↗