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.

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.

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.

Vitamin D, with parathyroid hormone and calcitonin, is an essential factor in the homeostatic regulation of systemic calcium in most vertebrate species. Targets for this aspect of vitamin D action, through its biologically active metabolites, are primarily the intestine, kidney and bone. Each of these tissues or organs are stimulated by 1,25(OH)2D3 to increase the transport calcium into the extracellular fluid compartment when plasma calcium levels are below normal and/or when there is a greater need for calcium to meet the requirements of physiological processes, such as growth, gestation and lactation. During such periods, the efficiency of the absorption of calcium from the intestine increases, the resorption of calcium salts from bone is stimulated, and the efficiency of the reabsorption of filtered calcium by the renal tubule is increased. In addition to the homeostatic function of vitamin D, there is an increasing amount of evidence that vitamin D has important effects on tissues and organs other than those concerned with calcium homeostasis. With regard to the intestinal epithelial system, the genomic effect of 1,25(OH)2D3 was shown several years ago when the de novo synthesis of a specific vitamin D-induced calcium-binding protein (CaBP, calbindin-D) was demonstrated. In our view, this appears to be an essential factor in the well-documented enhancement of calcium absorption by vitamin D. The function of calbindin-D, a high affinity calcium-binding protein, in the absorptive process is not precisely known but currently considered to act as an intracellular facilitator of the diffusion of calcium from the microvillar pole of the enterocyte to the basal-lateral membrane. There is evidence that vitamin D influences another step in the absorptive process. This step appears to be associated with the entrance of luminal calcium into the enterocyte, the first step in the transepithelial transport process. This response appears to occur relatively early (1 h or less) after 1,25(OH)2D3 is given to vitamin D-deficient animals, whereas the de novo synthesis of transport proteins has a much longer lag time (about 4 h). The in vitro absorption studies of Nemere et al (1984) and the in vivo experiments of our group (Wasserman et al, 1982) accentuate this point. However, the more rapid reaction, i.e., the possible modification of the permeability properties of the brush border membrane, does not result in a substantive increase in overall calcium absorption unless the enterocyte had been "primed" by previous exposure to vitamin D. The "priming" reaction might represent the synthesis of CaBP or some other intracellular component.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Injection of 1,25 dihydroxycholecalciferol (1,25(OH)2D3, 10 micrograms) directly into the in situ ligated duodenal loop of rachitic chicks significantly elevated the tissue accumulation of 47Ca within 20-30 min. The transfer of 47Ca from lumen to blood, during the same time period, was not increased nor was there any measurable intestinal calcium-binding protein synthesized. Lesser amounts of 1,25(OH)2D3 (1 or 5 micrograms) did not result in any statistically significant elevation of 47Ca tissue accumulation, nor did they have any effect on 47Ca transfer from lumen to blood (transmural). Ten micrograms of 1,24R,25(OH)3D3 was similarly effective in elevating tissue accumulation, whereas 24R,25(OH)2D3 and 25(OH)D3 were not. These results provide additional evidence for an early and direct action of 1,25(OH)2D3 in altering intestinal epithelial membrane transport prior to the induction of synthesis of specific transport proteins.

The effects of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or both, on the relationship among calcium absorption, vitamin D-induced calcium-binding protein (CaBP), and phospholipid metabolism were examined. When 1,25(OH)2D3 was injected intracardially into D3-deficient chicks, both the stimulation of calcium absorption and the induction of the synthesis of CaBP occurred 2-4 hr later. When 1,25(OH)2D3 was injected into chicks partially repleted with D3, an earlier increase in calcium absorption was observed without a significant change in the concentration of CaBP already present in the duodenal mucosa. Other early events were an increased uptake of calcium by the intestinal tissue and an alteration in phospholipid metabolism. These and other observations support the proposal that at least two phases of calcium absorption are influenced by 1,25(OH)2D3--permeation of calcium across the brush border, and transfer of calcium through and out of the cell. The first phase responds more rapidly to 1,25(OH)2D3 than does the second phase, correlates with changes in phospholipid metabolism, and might not be dependent on de novo protein synthesis. The second phase correlates with CaBP synthesis and therefore is dependent on protein synthesis. Either the first phase or the second phase can constitute the limiting step in calcium absorption.

Calcium-binding proteins from the chick intestine and uterus of the laying hen (Gallus domesticus) were isolated and purified by identical methods. Proteins from each source were shown to exhibit identical electrophoretic mobility and were immunologically identical. Estimation of molecular size by gel filtration indicated a value of approximately 28,000 daltons for both the chick intestinal and hen uterine calcium-binding proteins. The amino acid compositions of chick intestinal and hen uterine calcium-binding proteins were essentially identical.

A protein has been detected in chick brain which is immunologically identical to the vitamin D-induced calcium-binding protein found in intestinal tissue. It is present in highest concentration in the cerebellum and at lower levels in the remainder of the brain. Brain calcium-binding protein has the same molecular weight and electrophoretic properties as intestinal calcium-binding protein. Although the synthesis of intestinal calcium-binding protein is totally dependent upon a source of vitamin D, this has not yet been shown for brain calcium-binding protein. The total calcium-binding protein content of the cerebellum of chicks fed a vitamin D-free diet continued to increase during growth from 1 to 5 weeks, and is not responsive to exogenous vitamin D or 1,25-dihydroxyvitamin D.