Isolation and partial characterization of intestinal calcium-binding proteins from the cow, pig, horse, guinea pig, and chick.

1. Intestinal calcium-binding proteins have been isolated in high purity from mucosal tissue of the cow, pig, horse, guinea pig, and chick. The proteins from all species exhibit rapid, although not identical, electrophoretic mobilities and possesses high affinities for calcium. 2. The intestinal calcium-binding proteins of mammalian origin exhibit a molecular size of approx. 11 000 by calibrated gel filtration and 9000 on the basis of amino acid composition. The analogous chick protein was found to be about 27 000-28 000 molecular weight by these methods. 3. The amino acid composition of each intestinal calcium-binding protein has been determined and indicates a considerable degree of similarity, especially among the mammalian species. 4. Immunoassay procedures have failed to show any species cross-reactivity when tested against antiserum prepared in response to either the bovine or chick intestinal calcium-binding protein.

The effect of calcium on the tryptic digestion of bovine intestinal calcium-binding protein.

The tryptic hydrolysis of bivine intestinal calcium-binding protein in the presence and absence of excess calcium has been investigated. Calcium-binding activity and immunological reactivity of the protein were not significantly affected in the presence of 1.0 mM CaCl2 following 24 h incubation at 38 degrees C with trypsin at ratios of 1:9 of enzyme to calcium-binding protein. Some modification of the protein did occur under these conditions, however, since analysis by analytical acrylamide gel electrophoresis indicated the formation of a more rapidly-migrating species from the slower-moving original protein band. Omission of added calcium from the incubation medium resulted in rapid and essentially complete destruction of calcium-binding activity and immunological reactivity, and the formation of peptides of low molecular weight. This provides evidence that the conformation of the calcium-binding protein in the presence of calcium differs from that in its absence.

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.

Comparison of the enhanced steady-state diffusion of calcium by calbindin-D9K and calmodulin: possible importance in intestinal calcium absorption.

The diffusion of calcium was measured using the unidirectional flux of 45Ca across an aqueous layer. The aqueous layer was bounded by two dialysis membranes and convection was eliminated by gelling the aqueous layer with agarose. The apparent self-diffusion coefficient was determined by the dependence of the tracer flux on the diffusion distance. The apparent self-diffusion coefficient increased linearly with the concentration of calbindin-D9K and calmodulin, but the effect of calmodulin was markedly less than that of calbindin-D9K. This difference is attributed to the lower association constant for calmodulin. The ion-exchange resin Chelex-100 also increased the steady-state of 45Ca, but the effect of Chelex-100 was much less efficient than the effect of calbindin-D9K. The mechanism of enhanced diffusion was attributed to an enhanced gradient of total 45Ca. These results indicate that the steady-state unidirectional calcium flux is a superposition of free calcium diffusion and bound calcium diffusion, with only a small contribution due to a 'bucket brigade' mechanism. We suggest that this phenomenon may be important in calcium absorption across the intestine.

Positive cotranscriptional regulation of intestinal calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 and glucocorticoids.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces expression of the gene encoding calbindin-D28K, a protein involved in intestinal Ca2+ transport. Glucocorticoids stimulate intestinal development and function, and presumed interaction with 1,25-(OH)2D3 has been intensively studied. Most studies involved administration of high doses of glucocorticoids in vivo, which inhibits intestinal Ca2+ transport by an unknown mechanism. However, it is now known from studies of the duodenal organ culture model that low concentrations of glucocorticoids enhance 1,25-(OH)2D3-dependent calbindin-D28K biosynthesis and Ca2+ transport. High concentrations mimic the action of administered glucocorticoids in vivo, suggesting that a distinct pharmacological or toxic mechanism causes inhibition of Ca2+ absorption. This report further shows that dexamethasone (DEX) rapidly enhanced calbindin-D28K gene expression, that is de novo calbindin-D28K mRNA biosynthesis. DEX also markedly reduced the actions of RNA and protein synthesis inhibitors on calbindin-D28K gene expression, although no evidence for an action of DEX or 1,25-(OH)2D3 at the translational level was obtained. Ca2+ transport activity was highly correlated with calbindin-D28K concentration regardless of treatment. Washout permitted complete reversal of inhibition, verifying the specificity of inhibitor activity. These results appear to show positive contranscriptional regulation of calbindin-D28K gene expression by 1,25-(OH)2D3 and glucocorticoids. The use of this model should continue to clarify the interactive roles of nuclear-acting hormones on the Ca2+ absorptive mechanism and on complex physiological and pathological processes in general.

Tissue-specific regulation of shell gland calbindin D28K biosynthesis by estradiol in precociously matured, vitamin D-depleted chicks.

Provision of Ca2+ for egg shell calcification in the avian uterus [egg shell gland (ESG)] derives mostly from vitamin D-dependent intestinal Ca2+ absorption from the diet. Ca2+ absorption is strongly linked to the intestinal vitamin D-dependent calbindin D28K (D28K) concentration. The laying hen ESG also contains D28K, and again, Ca2+ transport into the shell appeared to be linked to the ESG D28K concentration. However, evidence is now presented that ESG D28K synthesis may be estradiol (E2) dependent and vitamin D independent under certain conditions. One-day-old female chicks fed a vitamin D-free diet for as long as 6 weeks and then repeatedly injected im with E2 for up to 3 more weeks developed frank rickets, but possessed precociously matured reproductive tracts. While the tiny presumptive ESGs of nonestrogenized vitamin D-depleted chicks were devoid of D28K, the highly developed ESG, including the isthmus, of estrogenized chicks contained D28K. The ESGs of nonestrogenized, vitamin D-replete chicks also exhibited no development or detectable D28K. Regardless of whether vitamin D depleted or replete, estrogenized chick ESG contained similar D28K and D28K mRNA concentrations. Immunohistochemical techniques showed that the endometrial cellular localization of both D28K and Ca(2+)-ATPase (Ca2+ pump) in estrogenized chicks was similar to that in mature laying hens. There was no trace of D28K, nor was there any stimulation of Ca2+ absorption, in duodenum of vitamin D-free, immature chicks regardless of E2 treatment. As expected, both D28K and D28K mRNA were present in vitamin D-replete chick duodenum. We conclude that in E2-treated chicks, ESG D28K gene expression may be vitamin D independent and E2 dependent. This is the first clear demonstration of hormone-dependent tissue-specific D28K gene expression in the chick.

Modulation of chick intestinal and renal calbindin gene expression by dietary vitamin D3, 1,25-dihydroxyvitamin D3, calcium and phosphorus.

Synthetic oligonucleotide probes complementary to chick calbindin-28 kDa-mRNA were used to study the latter's regulation and relationship to calbindin in the chick. The effects of vitamin D3 sources and dietary alteration on the genomic expression were characterized by Northern blot and solution hybridization. Intestinal calbindin and its mRNA were almost absent in vitamin D-deficient chicks and were not affected by dietary alteration. Renal calbindin and its mRNA were lower in the vitamin D-deficient than in vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-fed chicks. In the same animal, renal calbindin mRNA and calbindin were higher than intestinal. In vitamin D3-fed chicks, dietary calcium (Ca) or phosphorus (P) restriction induced, and high dietary Ca inhibited, intestinal calbindin and its mRNA synthesis. In the same chicks, dietary P restriction induced renal calbindin mRNA and calbindin synthesis. In 1,25-(OH)2D3-fed chicks, dietary P restriction induced and high dietary Ca inhibited the synthesis of intestinal and renal calbindin. The results suggest that: (a) most of the changes in renal and intestinal calbindin could be attributed to the changes in the mRNA; (b) the adaptation to dietary Ca and P alterations requires vitamin D metabolites; (c) high dietary Ca affects intestinal and renal calbindin-mRNA and calbindin via mechanisms independent of kidney 1-hydroxylase; and (d) plasma Ca and renal calbindin or its mRNA tend to change together in vitamin D-deficient or vitamin D3-fed, but not in 1,25(OH)2D3-fed chicks.

Intestinal interactions of lead and calcium.

The biological interactions between lead and calcium are complex and not well-understood, but can be demonstrated in virtually every tissue. High affinity lead-binding to intracellular calcium receptor and transport proteins as well as the involvement of lead in calcium-activated and calcium-regulating processes may provide a molecular basis for the broad spectrum cellular and systemic effects. The intestinal absorptive cells are responsible for transporting the entire body complement of calcium and most of the body lead burden. They represent, therefore, the first critical step in maintaining systemic and cellular calcium homeostasis, as well as the first line of defense against lead poisoning. Any interactions which occur at this level, either to enhance the body burden of lead or to diminish the transport of calcium, may have serious health-related repercussions. This article reviews research concerning those interactions involving calcium, lead and the vitamin D endocrine system which ultimately influence intestinal function, calcium homeostasis and body lead retention.

On the molecular mechanism of intestinal calcium transport.

The intestinal absorption of calcium is certainly a complex process, dependent on several factors of which vitamin D, via 1,25(OH)2D3, is the major controlling hormone. The efficiency of calcium absorption is a function of calcium status and calcium need. As the body's demand for calcium increases, the process commonly termed, adaptation, is activated in which the synthesis of 1,25(OH)2D3 from precursor is increased, resulting in the stimulation of the rate of calcium absorption. The increased demand for calcium might result from the ingestion of a diet deficient in calcium, from growth, pregnancy, lactation and egg shell formation in the laying hen. Accomapanying the change in calcium absorptive efficiency are molecular modifications of the transporting enterocytes, some mentioned herein and elsewhere (Wasserman & Chandler, 1985; Wasserman, 1980; Wasserman et al., 1984). Highly correlated with the rate of calcium absorption under a wide variety of conditions is the concentration of the vitamin D-induced calcium-binding protein, calbindin-D28K (avian type) and calbindin-D9K (mammalian intestinal type). The role of calbindin-D in this transport process is not precisely known but is considered to act at the present time as a cytosolic facilitator of Ca2+ diffusion from the brush border membrane to the basolateral membrane. In addition to the induction of calbindin-D synthesis, 1,25(OH)2D3 exerts other effects on the intestinal epithelium that can have consequences on the calcium absorptive process. Some of these effects are summarized in Figure 14. Vitamin D-dependent reactions might be either direct effects of 1,25(OH)2D3 or indirect effects due to elevated intracellular Ca2+ concentrations. These include changes in the fluidity of the brush border membrane, an increase in microvillar alkaline phosphatase-low affinity Ca-activated ATPase activity, an association of calmodulin with the 105 kD brush border cytoskeletal protein and, following calbindin D synthesis, the binding of calbindin D to a 60 kD brush border protein and to microtubules. The latter has been suggested to be related to the proposed transfer of Ca2+ by an endocytotic-exocytotic mechanism. In addition, a vitamin D-dependent intestinal membrane calcium-binding protein has been identified (Kowarski & Schachter, 1980). Playing into this multi-component system is a stimulation of cyclic nucleotide synthesis by 1,25(OH)2D3 which, through activation of cyclic nucleotide-dependent protein kinases, might modify membrane Ca2+ "channels" by phosphorylation reactions.4+ Intracellular organelles, i.e., the endoplasmic reticulum, mitochondria, the Golgi apparatus, are potent sequesters of Ca2+ and could contribute to the protection of the cell from excessively high Ca2+ concentrations by transiently storing absorbed Ca2+.

Intestinal calcium and lead absorption: effects of dietary lead and calcium.

The combined effects of dietary calcium (Ca) and lead (Pb) status on intestinal Ca and Pb absorption and related parameters were investigated in young growing chicks. Dietary Pb intake resulted in two remarkable, apparently independent and essentially opposite effects on intestinal Ca and Pb absorption, depending on dietary Ca and Pb levels and duration of treatment. The initial response (1 week) to Ca deficiency was stimulated Ca absorption and calbindin-D level, regardless of dietary Pb intake. The later response (2 weeks) was a reversal, by Pb, of the early phase stimulation. Intestinal Pb absorption was similarly enhanced by Ca deficiency initially, and this response was also inhibited by prolonged dietary Pb intake. Ingestion of Pb by chicks fed adequate Ca resulted in generally elevated intestinal Ca absorption and calbindin-D levels after both 1 and 2 weeks. Intestinal Pb absorption was also increased in the adequate Ca situation, but only after 2 weeks at the lower levels of dietary Pb. The results underscore the complicated nature of Pb-Ca interactions and demonstrate the importance of thorough characterization of the animal model system.

Dietary calcium levels and treatment interval determine the effects of lead ingestion on plasma 1,25-dihydroxyvitamin D concentration in chicks.

The combined effects of dietary calcium level and lead level on several indices of vitamin D endocrine function were examined in young, growing chicks. Day-old animals fed a nutritionally adequate diet for 2 wk were fed diets either adequate (1.2%) or low (0.1%) in calcium, and containing 0, 0.2 or 0.8% lead for an additional 1 or 2 wk. In the calcium-adequate group, lead ingestion significantly elevated intestinal calbindin-D28k protein and mRNA levels as well as plasma 1,25-dihydroxyvitamin D concentration compared with the control animals fed a lead-free diet. The effect was apparent after 1 wk of treatment and continued through wk 2. In the calcium-deficient group, the early (1 wk) increases in plasma 1,25-dihydroxyvitamin D and calbindin-D28k protein and mRNA were significantly reversed by lead ingestion over the 2-wk trial period in a dose-dependent fashion. In these circumstances, vitamin D endocrine function is severely compromised. Therefore, lead ingestion may result in either enhanced or diminished circulating 1,25-dihydroxyvitamin D concentrations and ensuing intestinal responses, depending of dietary calcium level and the duration of lead intake. These results provide possible explanations for several apparently conflicting sets of observations regarding lead-calcium interactions.

Lead-calcium interactions: involvement of 1,25-dihydroxyvitamin D.

Interactions between dietary calcium (Ca) and lead (Pb) which influence serum levels of the vitamin D hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D), intestinal Ca and Pb absorption, and body Pb retention were investigated in chicks. In a 5 x 5 (levels of Ca and Pb) design, response surface modeling was used to describe and compare the various interactions. Lead ingestion and Ca deficiency alone or in combination generally increased serum 1,25(OH)2D levels over most of the range of dietary Ca and Pb. However, in severe Ca deficiency, Pb ingestion resulted in marked decreases in hormone concentration. Overall similarities in response profiles for 1,25(OH)2D, intestinal Ca absorption and calbindin-D suggest that major interactions between Pb and Ca are mediated via changes in circulating 1,25(OH)2D concentration, rather than direct effects on the intestine. The response profiles for Ca and Pb absorption differed, in part, suggesting that intestinal transport of the two cations may not be identical. Kidney and bone Pb content also differed in response to varying Ca and Pb levels, providing evidence for additional tissue-specific interactions not related to 1,25(OH)2D. The present study provides a comprehensive basis on which to interpret the results of previous clinical and experimental results.

Intestinal lead and calcium absorption: effect of 1,25-dihydroxycholecalciferol and lead status.

This study was designed to investigate, in some detail, the relative effects of the hormonal form of vitamin D (1,25-dihydroxycholecalciferol) on duodenal Pb and Ca absorption as a function of dietary Pb level. When cholecalciferol-deficient chicks were chronically repleted with physiologic levels of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), as the sole source of the vitamin, 203Pb and 47Ca absorption were enhanced over 4- and 8-fold, respectively. Ingestion of Pb during the repletion period had no significant effect on the intestinal Ca absorption response to 1,25-(OH)2D3 even at a very high dietary Pb level. The efficiency of intestinal 203Pb absorption was, however, significantly diminished by dietary Pb, in an apparent dose-dependent fashion. The results indicate that the extent to which systemic Ca homeostatic mechanisms influence intestinal Pb absorption is dependent, in large part, on Pb status.

Regulation of intestinal calbindin-D28K gene expression: a solution hybridization study.

A solution hybridization assay employing specific synthetic oligodeoxynucleotide probes was developed to study the regulation of intestinal calbindin-D28K mRNA. The technique is rapid and quantitative and eliminates the need for sample transfer, blotting, autoradiography, and densitometry. Following validation of the assay, chick intestinal calbindin-D and calbindin-D mRNA levels were compared under conditions known to stimulate intestinal calcium (Ca) transport. Both the protein and its mRNA were undetectable in 25-day-old vitamin D-deficient chicks. Following acute administration of vitamin D3, calbindin-D mRNA levels increased somewhat more rapidly than calbindin-D protein, but overall, the correlation was excellent. Chicks fed a nutritionally adequate diet for 14 days and then changed to a low Ca (0.1%) diet responded with increased calbindin-D and calbindin-D mRNA levels. Again the correlation was excellent over the ensuing 14-day experimental period. The combined effects of vitamin D repletion and dietary Ca status were also investigated with respect to calbindin D and its mRNA. Fourteen-day-old vitamin D-deficient chicks were changed to diets containing vitamin D and either adequate (1.2%) or low (0.3%) in Ca. The intestinal responses were measured at intervals up to 14 days. In the normal Ca situation, there were initial increases in both calbindin mRNA levels, which peaked at between 4 and 7 days, and calbindin protein levels, which peaked at 7 days. Both values subsequently declined during the remaining 7 days of the experimental period. In the low Ca situation, there were similar increases in calbindin mRNA and protein levels through 4 and 7 days respectively, but these levels remained high for the remainder of the 14-day experimental period. The present results demonstrate that intestinal tissue levels of calbindin D and its mRNA respond similarly to vitamin D repletion and dietary Ca restriction as well as the combination of these stimuli. There is no evidence to support significant post-transcriptional regulation of calbindin-D by Ca.

Intestinal calcium absorption: calcium entry.

Any consideration of calcium entry into the cell must recognize that it is the initial event in a complex sequentially integrated process. Any step in this process, when viewed individually and in isolation, may appear to be of overwhelming importance, but this need not be an accurate reflection of its relative role in the overall process. Calcium entry may be of substantial importance in terms of calcium transport rate or capacity under certain circumstances, but it is most likely not the sole limiting step in calcium absorption. The Symposium papers that follow stress the importance of additional factors and events that have been implicated in intestinal calcium absorption.

Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography.

A method is described for the facile identification of half-cystine residues in peptides from protein digests. Combined use of S-beta-(4-pyridylethylation) of cysteine residues with 4-vinyl pyridine and peptide mapping by reversed-phase high-performance liquid chromatography provides the basis for this procedure. Essentially simultaneous, in-line effluent monitoring at 220, 280, and 254 nm provides highly sensitive, specific, and instantaneous identification of all peptide peaks, as well as those containing tryptophan and half-cystine residues, respectively. The resultant cysteine-derivatized peptides are well suited to amino acid compositional and protein sequence analysis, and are protected from loss during enzymatic digestion and/or peptide mapping.

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.

Intestinal absorption of arsenate in the chick.

The intestinal absorption of arsenate(As(V)) has been investigated in the chick by means of the in situ ligated duodenal loop technique. By this procedure, it was observed that arsenate is rapidly and essentially completely absorbed (80-95%) from the lumen at As(V) concentrations up to 5 mM, declining to about 50% absorption at 50 mM. Transfer from the intestinal lumen to the mucosal cells at low As(V) concentration (0.1 mM) is rapid, while transfer from the mucosal cells to the body occurs more slowly. At stable As(V) concentrations greater than 1 mM, fractional mucosal cell accumulation of As(V) remains constant, while fractional transfer to the body declines. However, total mucosal accumulation of As(V) and that transferred to the body increase in a linear logarithmic fashion from 0.05 to 5 mm As(V). The results indicate that As(V) readily penetrates both the mucosal and serosal surfaces of the epithelial membrane. Furthermore, arsenate and phosphate do not appear to share a common transport pathway in the duodenum and no evidence was obtained for any interaction between the two at this level. Vitamin D3 administration to rachitic chicks was effective in significantly elevating duodenal arsenate absorption, acting primarily to enhance serosal transport.

Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins.

Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.