Symposium:
Current Concepts of Calcium Absorption
Intestinal Calcium Absorption: Calcium Entry12 Hew York State College of Veterinary Medicine, Department of Physiology, Cornell Uniuersity, Ithaca, NY 14853 ference from outside to inside the cell. The fact that calcium entry is saturable at high levels of luminal calcium suggests the involvement of calcium channels or a transporter, but there is little direct evidence for the existence of either. Vitamin D administration to vitamin D-deficient animals increases the movement of calcium across the brush border membrane complex both in vivo (2) and in vitro (3-6), providing a unique opportunity to study the specifics of entry. Many studies have focused on temporal correlations between vitamin D-induced changes in the brush border region and the uptake of calcium by various preparations. Byusing 1,25(OH)2D, it has been possible to shorten the lag period between entry of vitamin D into the body and its conversion to the dihydroxylated active form in the kidney (7). Over the years, a number of factors have been im plicated in the process of calcium entry. Several of these will now be briefly described. Alkaline phosphatase, known to be present in the brush border re gion of intestinal epithelial cells, hydrolyzes a number of organic pyro- and orthophosphates. Enzymatic ac tivity is reduced in vitamin D-deficient rats and chicks as compared with normal control animals and is sub stantially enhanced by the administration of vitamin D or 1,25(OH)2D (8, 9). A low affinity calcium, mag-
ABSTRACT 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 ca pacity 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 impli cated in intestinal calcium absorption. J. Nutr. 122: 644-650, 1992. INDEXING KEY WORDS:
•calcium •intestine •microuillus
Intestinal calcium transport (or absorption) may be regarded as a three-step process, consisting of 1) entry into the epithelial cell from the lumen; 2) transit through the cytosol, from the apical to the basolateral pole and 3) extrusion from the cell, across the baso lateral membranes, to the vascular supply in the lamina propria. The primary regulator of intestinal calcium transport is the vitamin D endocrine system, specifi cally the hormone, 1,25-dihydroxycholecalciferol [1,25(OH)2D]4. Experimental evidence exists to sup port multiple roles for this hormone, influencing, per haps independently and specifically, each of the three steps in the overall transport process (1). The entry of calcium into the intestinal cell across the microvillar or brush border membrane requires no metabolic energy, as calcium moves down a steep electrochemical gradient (Fig. 1). The concentration of calcium in the lumen is variable, but often in the millimole-per-liter range. Although total cellular cal cium is relatively high (mmol/L), the intracellular free calcium level is maintained at 10~7-10~6 mole/L. In addition to this 1,000-10,000-fold concentration dif ference, there is a significant negative potential dif-
1Presented as part of a symposium: Current Concepts of Calcium Absorption, given at the 75th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlanta, GA, April 22, 1991. The symposium was sponsored by the American Institute of Nutrition. Guest editor for this symposium was F. Bronner, De partment of BioStructure and Function, University of Connecticut Health Center, Farmington, CT. 2This work was supported by NIH Grant ES-04072 and in part by NIH Grants DK-04652 (R. H. Wasserman) and GM-24314 (G. H. Morrison). 3To whom correspondence should be addressed: NYSCVM-Department of Physiology, 721VRT, Cornell University, Ithaca, New York 14853. 4Abbreviations: 1,25(OH)2D, 1,25-dihydroxycholecalciferol; ATPase, adenosine triphosphatase; BBMV,brush border membrane vesicles; CaBC, calcium-binding complex; IMCal, intestinal mem brane calcium-binding protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
0022-3166/92 83.00 ©1992 American Institute of Nutrition.
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CURTIS S. FULLMER3
SYMPOSIUM: LUMEN
CURRENT
CONCEPTS
CELL
10~3M Co
10~6 - 10~7M Ca -50
mv
FIGURE 1 Schematic picture of the microvillus region (MV) of an intestinal epithelial absorptive cell. The move ment of calcium through this region from the lumen to the cytoplasm is characterized thermodynamically as a downhill event comprising significant chemical and electrical gra dients.
nesium-stimulated adenosine triphosphatase (ATPase) has also been shown to increase in response to vitamin D (8, 10). This latter enzyme is distinct from the high affinity Ca-ATPase located on the basolateral mem branes (11). A number of studies (10-14) suggest that the brush border alkaline phosphatase and low affinity Ca-ATPase activities are different expressions of the same molecular complex, but this viewpoint is not universal (15). It has been repeatedly noted that the difficulty in relating alkaline phosphatase activity to 1,25(OH)2Dstimulated calcium entry or absorption is the relatively long time lag associated with the response (1). How ever, Bachelet et al. (16) observed a significant increase in enzyme activity only 30 min after the onset of 1,25(OH)2D infusion into the intestinal blood supply. More recently, Nasr et al. (17) observed an early and biphasic response to a single intraperitoneal injection of 1,25(OH)2D to vitamin D-deficient rats. Brush bor der alkaline phosphatase activity was significantly en hanced at 10 min postdose, peaked at 45 min, declined at l h and gradually increased again over the ensuing 8-h period. These results provide a temporal coinci dence between enhanced brush border alkaline phos phatase activity and calcium entry. These results sug gest an early stimulation of enzyme activity rather than increased enzyme concentration. In this regard, Moriuchi and DeLuca (18) reported altered electrophoretic mobility of alkaline phosphatase shortly fol lowing 1,25(OH)2D administration, perhaps related to formation of the sialo enzyme (19). Pansu et al. (20) demonstrated that theophylline significantly inhibited alkaline phosphatase activity, but not calcium uptake, by isolated brush border membrane vesicles (BBMV), suggesting that the en zyme is not involved in calcium entry. Calbindin-D, the high affinity intestinal calciumbinding protein is absent in vitamin D-deficient
ABSORPTION
645
chicks. It appears at a time after 1,25(OH)2D admin istration that coincides with the onset of enhanced calcium transport (2). Although most of the cellular calbindin resides in soluble form in the cytosol (21), a small fraction is membrane-bound and can be re leased only in the presence of detergent (22). Purified intestinal brush border preparations from vitamin Ddeficient chicks contain essentially no calbindin-D, whereas those isolated from vitamin D-replete chicks contain ~ 12% of the total cellular content of the pro tein (23). Two specific calbindin-D-binding proteins with molecular weights of ~66,000 and 14,000 were also identified in brush border preparations (23). In addition, it has been proposed that calbindin-D might interact with alkaline phosphatase in the brush border region (18, 24-26). Kowarski and Schachter (27) isolated a paniculate calcium-binding complex (CaBC) from isolated rat brush border preparations that exhibited vitamin Ddependent high affinity calcium-binding activity as well as para-nitrophenyl phosphatase and Ca-ATPase activities. The calcium-binding activity was subse quently dissociated from the enzymatic activities to yield a purified protein of ~200 kD with an apparent dissociation constant for calcium of 0.37 mmol/L (28). This protein, named IMCal (intestinal membrane cal cium-binding protein) was further dissociated by so dium dodecyl sulfate to yield a monomer of 20.5 kD, similar in size to a vitamin D-dependent protein iso lated by Miller et al. (29). It was proposed that CaBC or IMCal might function to mediate the transit of cal cium from the lumen, across the brush border mem brane, to the cytosol. Calcium-binding activity was generally well correlated with known features of cal cium transport, including dietary calcium level, rat age, intestinal distribution and vitamin D repletion (28). It is interesting to note that the original enzymatic activities associated with CaBC were apparently the result of an association between IMCal and the alka line phosphatase/Ca-ATPase complex. Bikle et al. (30) proposed that calmodulin may me diate calcium flux across the intestinal brush border membrane in response to 1,25(OH)2D administration. Although total cellular calmodulin concentrations are not increased by 1,25(OH)2D (30, 31 ), the level in iso lated BBMV is enhanced by the hormone and this cor responds nominally with stimulation of calcium up take. Calcium uptake is apparently not directly related to increased calcium-binding by calmodulin (30). Fur thermore, BBMV calcium uptake in response to 1,25(OH)2D was blocked by trifluoperazine and other specific calmodulin antagonists. Much of the calmod ulin in the brush border region is apparently associated with a 102-110 kD myosin-like protein (32, 33) that is brush border-specific. A number of other calmodulin-binding proteins have been detected in the brush border region (34), but their functional significance to calcium transport remains unknown. The time se-
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0 mv
OF CALCIUM
646
FULLMER
a significant action of 1,25(OH)2D in enhancing cal cium permeability is to alter the fluid state of the membrane. Initial attempts to detect vitamin D- or l,25(OH)2D-induced changes in membrane fluidity by electron spin resonance (38) or fluorescence polariza tion (39) techniques were unsuccessful. Brasitus et al. (40), recognizing the importance of probe selection and the existence of both static and dynamic components of fluidity, were successful in detecting a very rapid effect of 1,25(OH)2D. The dynamic component of membrane fluidity was diminished in vitamin D-de ficient compared with vitamin D-replete rats but was quickly restored to normal levels after the adminis tration of 1,25(OH)2D. Changes in membrane fluidity were correlated with fatty acyl alterations, providing some support for the liponomic theory. However, it was also observed (40) that the effects of 1,25(OH)2D on BBMV fluidity and phospholipid fatty acid altera tions (evident at 1-2 h after injection) preceded any demonstrable influence on calcium absorption in vivo (significantly elevated only at 5 h post-injection). This , lack of temporal correspondence indicates that the early changes in BBMV due to 1,25(OH)2D are prob ably not a major factor in calcium absorption. Several other changes have been observed in re sponse to 1,25(OH)2D administration, including very rapid increases in adenylate cyclase activity (41), ornithine decarboxylase activity (42) and reactivity of brush border membrane sulfhydryl groups (43). These responses, as well as some described above, occur so quickly after hormone administration that it is not clear if stimulation is primarily due to a direct action of 1,25(OH)2D or secondarily related to enhanced en try of calcium into the cell. Only further study will provide clarification of the events and their sequence. While isolated brush border preparations allow the examination of the initial calcium entry (and binding) step, they provide little contextual information con cerning the relative importance of this event in the overall process of transcellular transport. Furthermore, purified BBMV preparations are generally devoid of many microvillar components that may have signifi cant impact on calcium entry and subsequent transport in vivo. Wasserman et al. (2) employed the in situ ligated duodenal segment technique to examine intestinal calcium transport and tissue uptake in response to 1,25(OH)2D treatment of vitamin D-deficient chicks. In this system, direct comparisons may be made in a tissue that is fully capable of stimulated high capacity polarized calcium transport. The net accumulation of 47Caby mucosal tissue was significantly increased as early as l h after an intracardial injection of the hor mone. Intestinal calcium absorption, however, was not appreciably elevated until 4 h after 1,25(OH)2D ad ministration. This indicates again that cellular entry and transcellular transport are not necessarily linked.
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quence of calmodulin redistribution to the brush bor der region in response to 1,25(OH)2D has not been reported. Intestinal BBMV preparations have been a popular system for studying calcium entry. They are comprised of an outer continuous layer of microvillar membrane with an entrapped vesicular space, providing the op portunity to examine entry without the complicating influences of other cellular constituents and transport components. Rasmussen and colleagues (3) were among the first to have used purified BBMVto evaluate the effect of 1,25(OH)2D on calcium uptake. Vesicles from l,25(OH)2D-dosed chicks showed a more rapid initial rate of calcium uptake as compared with vita min D-deficient chicks. At longer uptake intervals there was no difference due to treatment, showing that the hormone is responsible for an enhanced rate, but not capacity, of calcium entry. Vesicular calcium up take was nonenergy dependent and saturable. Miller and Bronner (4) reported similar results for calcium uptake by BBMV isolated from rat intestine and fur ther resolved calcium uptake into two components: calcium-binding to the membrane and calcium trans port into the vesicular space. Matsumoto et al. (35) and, later, Bikle et al. (6) showed that 1,25(OH)2Dstimulated calcium uptake by BBMV is a very early intestinal event, occurring within 2 h of hormone ad ministration. Rasmussen and colleagues (5, 35) and O'Doherty (36) showed that a very early response to 1,25(OH)2D administration to vitamin D-deficient animals is a significant alteration in membrane lipids. For instance, phospholipase A2 activity, as well as lysophosphatidyl choline acetyltransferase activity, was increased twoto threefold within 3 h of 1,25(OH)2D administration in intestinal cells from rats (36). Max et al. (37) dem onstrated a l,25(OH)2D-related increase in membrane phospholipid phosphorus and changes in the fatty acid composition of the phosphatidylcholine (PC) fraction in purified BBMV from chicks. Matsumoto et al. (35) reported that pretreatment of chicks with 1,25(OH)2D stimulated the migration of labeled choline into PC and diminished the incorporation of ethanolamine into phosphatidylethanolamine (PE)in BBMV. The re sult was a significant increase in the labeled PC/PE ratio, which was closely correlated with the time course of BBMV calcium uptake after an injection of 1,25(OH)2D to vitamin D-deficient chicks. In addi tion, 1,25(OH)2D administration increased the incor poration of [3H]arachidonic, but not [3H]palmitic acid into PC, but not diacylglycerol (35). These results led to the so-called "liponomic theory," which proposed that the primary effect of 1,25(OH)2D is to alter the lipid structure of the membrane, thereby increasing calcium permeability (entry) and transcellular calcium transport. The observed changes in phospholipid character of the microvillar membrane led to the proposal (5) that
647
SYMPOSIUM: CURRENT CONCEPTS OF CALCIUM ABSORPTION
300 250
I
200
E 'o o u
150
O—-O •
100
Absorbed
•Retained
.o-
O
50
100
50 O
o 20
40
Luminal Co (mM)
FIGURE 2 Duodenal absorption and tissue retention of luminally administered calcium in vitamin D-deficient chicks. Hatchling chicks were fed a vitamin D-deficient diet (2) for 28 d and fasted for 16 h before having calcium trans port assessed by the in situ ligated segment technique (2). The intraluminal dosing solution (0.5 mL) contained the in dicated levels of ""Ça,3.76 GBq 47Cain 145 mmol/L of NaCl, pH 7.0. After a 20-min absorption period, the animals were killed and the intact duodenal segments were removed and the remaining radioactivity was determined. The segments were then drained and rinsed with cold 145 mmol/L of NaCl; the mucosa was removed, weighed and ashed before deter mination of tissue calcium by atomic absorption spectrophotometry. Calcium absorption was calculated from the residual amount of 47Cain the segment. Triangles indicate calcium absorption (open), tissue retention (closed) in com parable deficient animals that received 12.5 ¿ig vitamin D3. Each point represents the mean ±SEMfor six chicks.
'/
b
b
b
/f""""'J ° uAOoà Ç5!j5040302010na°T /i
\ \ fsL/_
__.¡b•^ • — •Absorbed o— -o Retained
-/
0124
8 Time after 1,25(OH)2
12
16
D (hours)
FIGURE 3 Duodenal absorption and tissue retention of luminally administered 47Cain vitamin D-deficient chicks at varying times after a single intravenous injection of 1,25(OH)2D (1.0/ig)- Chicks and absorption technique were identical to those described in the legend for Figure 2, except that the intraluminal dosing solution contained 20 mmol/L of Ca. Data are presented as percentage of the original ra dioactivity absorbed or retained by the tissue. Each point represents the mean ±SEMfor six chicks, (a) Denotes sig nificant difference (P > 0.05) in 47Catissue retention due to 1,25(OH)2D. (b) Denotes significant difference (P > 0.05) in 47Caabsorption due to 1,25(OH)2D.
step(s). Some years ago, Sampson et al. (44) employed microincineration and tissue fixation techniques in conjunction with 45Ca autoradiography and electron microscopy in an attempt to trace the movement of calcium through the epithelial cell. In the rachide rat, calcium (administered orally) was shown to pass across the limiting cell membrane and associate primarily with elements within the microvilli. There was no in dication of mass calcium movement from this apical region into the cytosol proper or to the intracellular organdÃ-es. Vitamin D treatment resulted in dimin ished calcium accumulation in the microvillar region and its appearance in the mitochondria located more basally. This indicates that mobilization of microvillarbound calcium requires vitamin D, whereas the translocation across the luminal-facing membrane and sub sequent binding to the microvilli do not. The fixation techniques employed in these studies, however, did not preclude loss or possible redistribution of calcium that was not bound tightly to cellular elements. Recently, we have developed a procedure to study calcium transport that combines ion microscopic im aging and stable isotopie analyses with improved cryoscopic and embedding techniques (45). Ion mi croscopy is a method based on secondary ion mass spectrometry, which produces visual images of the el emental distribution within a sample in relation to tissue morphology (46). Because ions to be examined are selected on the basis of mass to charge ratio, dif ferent isotopes of the same element may be imaged sequentially. By employing enriched 44Ca as the sole luminal species, it was possible to discriminate be-
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Recently, we have expanded this comparison. As shown in Figure 2, luminal calcium accumulates to a considerable extent in the mucosal tissue of vitamin D-deficient chicks, essentially doubling as the luminal calcium concentration is increased from 2 to 20 mmol/ L. Though it remains constant at luminal concentra tions up to 50 mmol/L. This indicates that the capacity to accumulate calcium from the lumen, while consid erable, is saturable. Figure 3 shows the time course for in situ tissue accumulation and absorption of calcium when 1,25(OH)2D was administered to vitamin D-deficient chicks. Within 30 min of 1,25(OH)2D administration, mucosal 47Caaccumulation was markedly and signif icantly enhanced but had declined by 2 and 4 h postdose, a time when transcellular transport had com menced in the treated but not in the deficient chicks. These results confirm that 1,25(OH)2D acts rapidly to enhance cellular calcium entry, but that considerable time elapses between entry and subsequent transport. They also demonstrate the absolute vitamin D depen dence of transcellular calcium transport. One difficulty associated with whole-organ trans port systems has been the inability to identify the spa tial distribution of calcium during transit and that re tained by the tissue. Such information might provide insight as to the location of the limiting transport
648
FULLMER
tween calcium in transit (44Ca)and ambient tissue cal cium (40Ca).This procedure was employed to compare and contrast the movement of luminally administered calcium in vitamin D-deficient and -replete chicks under physiological conditions. Preliminary radioisotope (47Ca)data showed that luminal calcium rapidly and extensively becomes as sociated with the mucosal tissue but is transferred to the blood (absorbed) at a very slow rate in vitamin Ddeficient chicks. Vitamin D-repletion results in a greater initial increase in tissue uptake coinciding with transepithelial transport, followed by diminished tis sue levels as the bulk of luminal calcium is transported to the blood (45). The corresponding 44Ca images al lowed the visualization of these processes. In the vi tamin D-deficient case, luminal 44Ca rapidly crossed the cell membrane, and high concentrations became localized in the microvillar/terminal web region. After absorption had proceeded for a time, accumulation
continued with a steep, inwardly directed gradient, but there was little apparent transcellular movement. In the vitamin D-replete situation, luminal 44Carap idly entered the cell, perhaps accumulating transiently in the microvillar region, but continued to move through the cytosol in a basal direction, appearing very quickly in the lamina propria. Figure 4 compares the tissue distribution of 44Ca and 40Ca at 20 min after the intraluminal injection of 44Ca to vitamin D-deplete and -replete chicks. These studies provide the first visualization of ep ithelial calcium transport under physiological condi tions. They demonstrate graphically that the luminal limiting cell membrane does not represent a substan tial barrier to calcium entry into the cell. Inwardly bound calcium is initially sequestered in the apical cell region, perhaps by cytoskeletal elements and/or calmodulin. Under the influence of vitamin D and presumably due to the presence of calbindin (see réf.
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FIGURE 4 Ion microscopic images showing the distribution in the villus of tissue *°Ca [upper panels) and of intraluminally administered 44Ca (lower panels) in vitamin D-deficient (left panels) and vitamin D-replete (right panels) chicks. Note that the intraluminally administered calcium did not spread through the cells of the deficient chicks. For further details, see text and réf. 45.
SYMPOSIUM: CURRENT CONCEPTS OF CALCIUM ABSORPTION
ACKNOWLEDGMENT The author acknowledges the excellent secretarial assistance of Norma Jayne.
LITERATURE CITED 1. Wasserman, R. H. a Chandler, J. S. (1985) Molecular mech anisms of intestinal calcium absorption. In: Bone and Mineral Research (Peck, W. A., ed.), pp. 181-211, Elsevier Science Pub lishers, Amsterdam, The Netherlands. 2. Wasserman, R. H., Brindak, M. E., Meyer, S. A. & Fullmer, C. S. (1982) Evidence for multiple effects of vitamin D3 on cal cium absorption: Response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3. Proc. Nati. Acad. Sci. USA 79: 7939-7943. 3. Rasmussen, H., Fontaine, O., Max, E. & Goodman, D. B. P. (1979) The effect of la-hydroxyvitamin D3-administration on calcium transport in chick intestine brush border membrane vesicles. J. Biol. Chem. 254: 2993-2999. 4. Miller, A. &. Bronner, F. (1981) Calcium uptake in isolated brush border vesicles from rat small intestine. Biochem. J. 196: 391-401. 5. Fontaine, O., Matsumoto, T., Goodman, D. B. P. & Rasmussen, H. (1981) Liponomic control of Ca2+ transport: Relationship to mechanism of action of 1,25-dihydroxyvitamin D3. Proc. Nati. Acad. Sci. USA 78: 1751-1754. 6. Bikle, D. D., Munson, S. & Zolock, D. T. (1983) Calcium flux across chick duodenal brush border membrane vesicles: Regu lation by 1,25-dihydroxyvitamin D. Endocrinology 113: 20722080. 7. Norman, A. W. (1975) Hormone-like action of 1,25-dihydroxycholecalciferol. Vitam Horm 32: 325-384. 8. Holdsworth, E. S. (1970) The effects of vitamin D on the en zyme activities in the mucosal cells of the chick small intestine. J. Membr. Biol. 3: 43-53. 9. Norman, A. W., Mircheff, A. K., Adams, T. H. & Spielvogel, A. (1970) Studies on the mechanisms of action of calciferol; III. Vitamin D-mediated increase of intestinal brush border al kaline phosphatase activity. Biochim. Biophys. Acta 215: 348359. 10. Haussler, M. R., Nagode, L. A. & Rasmussen, H. (1970) Induction of intestinal brush border alkaline phosphatase and identity with Ca-ATPase. Nature 228: 1199-1201. 11. Ghijsen, W. E. J. M., Dejonge, M. D. & Van Os, C. H. (1980) Dissociation between Ca2+-ATPase and alkaline phos phatase activities in plasma membranes of rat duodenum. Biochim. Biophys. Acta 599: 538-551. 12. Forstner, G. G., Sabesin, S. M. & Isselbacher, K. J. (1968) Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem. J. 106: 381-390. 13. Oku, T. & Wasserman, R. H. (1978) Properties of the vitamin D-stimulated calcium-dependent adenosine triphosphatase and alkaline phosphatase in chick intestinal brush border. Fed. Proc. 37: 408.
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47), the calcium that has entered the cell can move through the cytoplasm to the basolateral pole where it is extruded by the calcium pump. Thus, although vitamin D or 1,25(OH)2D may enhance the calcium entry rate, its primary action is to provide the neces sary transport elements.
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32.
34.
35. 36.
37.
38.
3?.
in their distribution and in the effect of vitamin D steroids on their concentrations. FEES Lett. 127: 13-16. Bikle, D. D. & Munson, S. (1985) 1,25-dihydroxyvitamin D increases calmodulin binding to specific protein in the chick duodenal brush border membrane. J. Clin. Invest. 76: 23132316. Bilde, D. D., Munson, S. & Mancianti, M. L. (1991) Limited tissue distribution of the intestinal brush border myosin I pro tein. Gastroenterology 100: 395-402. GlenneyJ. R.& Weber, K. (1980) Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells. J. Biol. Chem. 255: 1055110554. Matsumoto, T., Fontaine, O. & Rasmussen, H. (1981) Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. J. Biol. Chem. 256: 3354-3360. O'Doherty, P. J. A. (1978) l,25-DihydroxyvitaminD3 increases the activity of the intestinal phosphatidylcholine deacylationreacylation cycle. Lipids 14: 75-77. Max, E. E., Goodman, D. B. P. & Rasmussen, H. (1978) Purification and characterization of chick intestine brush border membrane. Biochim. Biophys. Acta 511: 224-239. Putkey, J. A., Spielvogel, A. M., Sauerheber, R. D., Sunlap, C. S. & Norman, A. W. (1982) Effects of essential fatty acid deficiency and spin label studies of enterocyte membrane lipid fluidity. Biochim. Biophys. Acta 688: 177-190. Bikle, D. D., Whitney, J. & Munson, S. (1984) The relationship of membrane fluidity to calcium flux in chick intestinal brush border membranes. Endocrinology 114: 260-267.
40. Brasitus, T. A., Dudeja, P. K.., Eby, B. & Lau, K. (1986) Correction by 1,25-dihydroxycholecalciferol of the ab normal fluidity and lipid composition of enterocyte brush border membranes in vitamin D-deprived rats. J. Biol. Chem. 261: 16404-16409. 41. Corradino, R. A. (1974) Embryonic chick intestine in organ culture: Interaction of adenylate cyclase system and vitamin D3mediated calcium absorptive mechanism. Endocrinology 94: 1607-1614. 42. Shinki, T., Takahashi, N., Migaura, C., Samejima, K. Nishi, Y. & Suda, T. (1981) Ornithine decarboxylase activity in chick duodenum induced by la,25-dihydroxycholecalciferol. Biochem. J. 195: 685-690. 43. Mykkanen, H. M. & Wasserman, R. H. (1990) Reactivity of sulfhydryl groups in the brush border membranes of chick duo dena is increased by 1,25-dihydroxycholecalciferol. Biochem. Biophys. Acta 1033: 282-286. 44. Sampson, H. W., Matthews, J. L., Martin, J. H. &. Kunin, A. S. (1970) An electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin-D-treated rats. Calcif. Tissue Res. 5: 305-316. 45. Chandra, S., Fullmer, C. S., Smith, C. A., Wasserman, R. H. & Morrison, G. H. (1990) Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vi tamin D-replete chickens: A "Ca stable isotope study. Proc. Nati. Acad. Sci. USA 87: 5715-5719. 4e. Chandra, S. & Morrison, G. H. (1988) Ion microscopy in bi ology and medicine. Methods Enzymol. 158: 157-179. 47. Stein, W. D. (1992) Facilitated diffusion of calcium across the rat intestinal epithelial cell. J. Nutr. 122(suppl.): 000-000.
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33.
FULLMER
Current Concepts of Calcium Absorption
Intestinal Calcium Absorption: Calcium Entry12 Hew York State College of Veterinary Medicine, Department of Physiology, Cornell Uniuersity, Ithaca, NY 14853 ference from outside to inside the cell. The fact that calcium entry is saturable at high levels of luminal calcium suggests the involvement of calcium channels or a transporter, but there is little direct evidence for the existence of either. Vitamin D administration to vitamin D-deficient animals increases the movement of calcium across the brush border membrane complex both in vivo (2) and in vitro (3-6), providing a unique opportunity to study the specifics of entry. Many studies have focused on temporal correlations between vitamin D-induced changes in the brush border region and the uptake of calcium by various preparations. Byusing 1,25(OH)2D, it has been possible to shorten the lag period between entry of vitamin D into the body and its conversion to the dihydroxylated active form in the kidney (7). Over the years, a number of factors have been im plicated in the process of calcium entry. Several of these will now be briefly described. Alkaline phosphatase, known to be present in the brush border re gion of intestinal epithelial cells, hydrolyzes a number of organic pyro- and orthophosphates. Enzymatic ac tivity is reduced in vitamin D-deficient rats and chicks as compared with normal control animals and is sub stantially enhanced by the administration of vitamin D or 1,25(OH)2D (8, 9). A low affinity calcium, mag-
ABSTRACT 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 ca pacity 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 impli cated in intestinal calcium absorption. J. Nutr. 122: 644-650, 1992. INDEXING KEY WORDS:
•calcium •intestine •microuillus
Intestinal calcium transport (or absorption) may be regarded as a three-step process, consisting of 1) entry into the epithelial cell from the lumen; 2) transit through the cytosol, from the apical to the basolateral pole and 3) extrusion from the cell, across the baso lateral membranes, to the vascular supply in the lamina propria. The primary regulator of intestinal calcium transport is the vitamin D endocrine system, specifi cally the hormone, 1,25-dihydroxycholecalciferol [1,25(OH)2D]4. Experimental evidence exists to sup port multiple roles for this hormone, influencing, per haps independently and specifically, each of the three steps in the overall transport process (1). The entry of calcium into the intestinal cell across the microvillar or brush border membrane requires no metabolic energy, as calcium moves down a steep electrochemical gradient (Fig. 1). The concentration of calcium in the lumen is variable, but often in the millimole-per-liter range. Although total cellular cal cium is relatively high (mmol/L), the intracellular free calcium level is maintained at 10~7-10~6 mole/L. In addition to this 1,000-10,000-fold concentration dif ference, there is a significant negative potential dif-
1Presented as part of a symposium: Current Concepts of Calcium Absorption, given at the 75th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlanta, GA, April 22, 1991. The symposium was sponsored by the American Institute of Nutrition. Guest editor for this symposium was F. Bronner, De partment of BioStructure and Function, University of Connecticut Health Center, Farmington, CT. 2This work was supported by NIH Grant ES-04072 and in part by NIH Grants DK-04652 (R. H. Wasserman) and GM-24314 (G. H. Morrison). 3To whom correspondence should be addressed: NYSCVM-Department of Physiology, 721VRT, Cornell University, Ithaca, New York 14853. 4Abbreviations: 1,25(OH)2D, 1,25-dihydroxycholecalciferol; ATPase, adenosine triphosphatase; BBMV,brush border membrane vesicles; CaBC, calcium-binding complex; IMCal, intestinal mem brane calcium-binding protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
0022-3166/92 83.00 ©1992 American Institute of Nutrition.
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CURTIS S. FULLMER3
SYMPOSIUM: LUMEN
CURRENT
CONCEPTS
CELL
10~3M Co
10~6 - 10~7M Ca -50
mv
FIGURE 1 Schematic picture of the microvillus region (MV) of an intestinal epithelial absorptive cell. The move ment of calcium through this region from the lumen to the cytoplasm is characterized thermodynamically as a downhill event comprising significant chemical and electrical gra dients.
nesium-stimulated adenosine triphosphatase (ATPase) has also been shown to increase in response to vitamin D (8, 10). This latter enzyme is distinct from the high affinity Ca-ATPase located on the basolateral mem branes (11). A number of studies (10-14) suggest that the brush border alkaline phosphatase and low affinity Ca-ATPase activities are different expressions of the same molecular complex, but this viewpoint is not universal (15). It has been repeatedly noted that the difficulty in relating alkaline phosphatase activity to 1,25(OH)2Dstimulated calcium entry or absorption is the relatively long time lag associated with the response (1). How ever, Bachelet et al. (16) observed a significant increase in enzyme activity only 30 min after the onset of 1,25(OH)2D infusion into the intestinal blood supply. More recently, Nasr et al. (17) observed an early and biphasic response to a single intraperitoneal injection of 1,25(OH)2D to vitamin D-deficient rats. Brush bor der alkaline phosphatase activity was significantly en hanced at 10 min postdose, peaked at 45 min, declined at l h and gradually increased again over the ensuing 8-h period. These results provide a temporal coinci dence between enhanced brush border alkaline phos phatase activity and calcium entry. These results sug gest an early stimulation of enzyme activity rather than increased enzyme concentration. In this regard, Moriuchi and DeLuca (18) reported altered electrophoretic mobility of alkaline phosphatase shortly fol lowing 1,25(OH)2D administration, perhaps related to formation of the sialo enzyme (19). Pansu et al. (20) demonstrated that theophylline significantly inhibited alkaline phosphatase activity, but not calcium uptake, by isolated brush border membrane vesicles (BBMV), suggesting that the en zyme is not involved in calcium entry. Calbindin-D, the high affinity intestinal calciumbinding protein is absent in vitamin D-deficient
ABSORPTION
645
chicks. It appears at a time after 1,25(OH)2D admin istration that coincides with the onset of enhanced calcium transport (2). Although most of the cellular calbindin resides in soluble form in the cytosol (21), a small fraction is membrane-bound and can be re leased only in the presence of detergent (22). Purified intestinal brush border preparations from vitamin Ddeficient chicks contain essentially no calbindin-D, whereas those isolated from vitamin D-replete chicks contain ~ 12% of the total cellular content of the pro tein (23). Two specific calbindin-D-binding proteins with molecular weights of ~66,000 and 14,000 were also identified in brush border preparations (23). In addition, it has been proposed that calbindin-D might interact with alkaline phosphatase in the brush border region (18, 24-26). Kowarski and Schachter (27) isolated a paniculate calcium-binding complex (CaBC) from isolated rat brush border preparations that exhibited vitamin Ddependent high affinity calcium-binding activity as well as para-nitrophenyl phosphatase and Ca-ATPase activities. The calcium-binding activity was subse quently dissociated from the enzymatic activities to yield a purified protein of ~200 kD with an apparent dissociation constant for calcium of 0.37 mmol/L (28). This protein, named IMCal (intestinal membrane cal cium-binding protein) was further dissociated by so dium dodecyl sulfate to yield a monomer of 20.5 kD, similar in size to a vitamin D-dependent protein iso lated by Miller et al. (29). It was proposed that CaBC or IMCal might function to mediate the transit of cal cium from the lumen, across the brush border mem brane, to the cytosol. Calcium-binding activity was generally well correlated with known features of cal cium transport, including dietary calcium level, rat age, intestinal distribution and vitamin D repletion (28). It is interesting to note that the original enzymatic activities associated with CaBC were apparently the result of an association between IMCal and the alka line phosphatase/Ca-ATPase complex. Bikle et al. (30) proposed that calmodulin may me diate calcium flux across the intestinal brush border membrane in response to 1,25(OH)2D administration. Although total cellular calmodulin concentrations are not increased by 1,25(OH)2D (30, 31 ), the level in iso lated BBMV is enhanced by the hormone and this cor responds nominally with stimulation of calcium up take. Calcium uptake is apparently not directly related to increased calcium-binding by calmodulin (30). Fur thermore, BBMV calcium uptake in response to 1,25(OH)2D was blocked by trifluoperazine and other specific calmodulin antagonists. Much of the calmod ulin in the brush border region is apparently associated with a 102-110 kD myosin-like protein (32, 33) that is brush border-specific. A number of other calmodulin-binding proteins have been detected in the brush border region (34), but their functional significance to calcium transport remains unknown. The time se-
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0 mv
OF CALCIUM
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FULLMER
a significant action of 1,25(OH)2D in enhancing cal cium permeability is to alter the fluid state of the membrane. Initial attempts to detect vitamin D- or l,25(OH)2D-induced changes in membrane fluidity by electron spin resonance (38) or fluorescence polariza tion (39) techniques were unsuccessful. Brasitus et al. (40), recognizing the importance of probe selection and the existence of both static and dynamic components of fluidity, were successful in detecting a very rapid effect of 1,25(OH)2D. The dynamic component of membrane fluidity was diminished in vitamin D-de ficient compared with vitamin D-replete rats but was quickly restored to normal levels after the adminis tration of 1,25(OH)2D. Changes in membrane fluidity were correlated with fatty acyl alterations, providing some support for the liponomic theory. However, it was also observed (40) that the effects of 1,25(OH)2D on BBMV fluidity and phospholipid fatty acid altera tions (evident at 1-2 h after injection) preceded any demonstrable influence on calcium absorption in vivo (significantly elevated only at 5 h post-injection). This , lack of temporal correspondence indicates that the early changes in BBMV due to 1,25(OH)2D are prob ably not a major factor in calcium absorption. Several other changes have been observed in re sponse to 1,25(OH)2D administration, including very rapid increases in adenylate cyclase activity (41), ornithine decarboxylase activity (42) and reactivity of brush border membrane sulfhydryl groups (43). These responses, as well as some described above, occur so quickly after hormone administration that it is not clear if stimulation is primarily due to a direct action of 1,25(OH)2D or secondarily related to enhanced en try of calcium into the cell. Only further study will provide clarification of the events and their sequence. While isolated brush border preparations allow the examination of the initial calcium entry (and binding) step, they provide little contextual information con cerning the relative importance of this event in the overall process of transcellular transport. Furthermore, purified BBMV preparations are generally devoid of many microvillar components that may have signifi cant impact on calcium entry and subsequent transport in vivo. Wasserman et al. (2) employed the in situ ligated duodenal segment technique to examine intestinal calcium transport and tissue uptake in response to 1,25(OH)2D treatment of vitamin D-deficient chicks. In this system, direct comparisons may be made in a tissue that is fully capable of stimulated high capacity polarized calcium transport. The net accumulation of 47Caby mucosal tissue was significantly increased as early as l h after an intracardial injection of the hor mone. Intestinal calcium absorption, however, was not appreciably elevated until 4 h after 1,25(OH)2D ad ministration. This indicates again that cellular entry and transcellular transport are not necessarily linked.
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quence of calmodulin redistribution to the brush bor der region in response to 1,25(OH)2D has not been reported. Intestinal BBMV preparations have been a popular system for studying calcium entry. They are comprised of an outer continuous layer of microvillar membrane with an entrapped vesicular space, providing the op portunity to examine entry without the complicating influences of other cellular constituents and transport components. Rasmussen and colleagues (3) were among the first to have used purified BBMVto evaluate the effect of 1,25(OH)2D on calcium uptake. Vesicles from l,25(OH)2D-dosed chicks showed a more rapid initial rate of calcium uptake as compared with vita min D-deficient chicks. At longer uptake intervals there was no difference due to treatment, showing that the hormone is responsible for an enhanced rate, but not capacity, of calcium entry. Vesicular calcium up take was nonenergy dependent and saturable. Miller and Bronner (4) reported similar results for calcium uptake by BBMV isolated from rat intestine and fur ther resolved calcium uptake into two components: calcium-binding to the membrane and calcium trans port into the vesicular space. Matsumoto et al. (35) and, later, Bikle et al. (6) showed that 1,25(OH)2Dstimulated calcium uptake by BBMV is a very early intestinal event, occurring within 2 h of hormone ad ministration. Rasmussen and colleagues (5, 35) and O'Doherty (36) showed that a very early response to 1,25(OH)2D administration to vitamin D-deficient animals is a significant alteration in membrane lipids. For instance, phospholipase A2 activity, as well as lysophosphatidyl choline acetyltransferase activity, was increased twoto threefold within 3 h of 1,25(OH)2D administration in intestinal cells from rats (36). Max et al. (37) dem onstrated a l,25(OH)2D-related increase in membrane phospholipid phosphorus and changes in the fatty acid composition of the phosphatidylcholine (PC) fraction in purified BBMV from chicks. Matsumoto et al. (35) reported that pretreatment of chicks with 1,25(OH)2D stimulated the migration of labeled choline into PC and diminished the incorporation of ethanolamine into phosphatidylethanolamine (PE)in BBMV. The re sult was a significant increase in the labeled PC/PE ratio, which was closely correlated with the time course of BBMV calcium uptake after an injection of 1,25(OH)2D to vitamin D-deficient chicks. In addi tion, 1,25(OH)2D administration increased the incor poration of [3H]arachidonic, but not [3H]palmitic acid into PC, but not diacylglycerol (35). These results led to the so-called "liponomic theory," which proposed that the primary effect of 1,25(OH)2D is to alter the lipid structure of the membrane, thereby increasing calcium permeability (entry) and transcellular calcium transport. The observed changes in phospholipid character of the microvillar membrane led to the proposal (5) that
647
SYMPOSIUM: CURRENT CONCEPTS OF CALCIUM ABSORPTION
300 250
I
200
E 'o o u
150
O—-O •
100
Absorbed
•Retained
.o-
O
50
100
50 O
o 20
40
Luminal Co (mM)
FIGURE 2 Duodenal absorption and tissue retention of luminally administered calcium in vitamin D-deficient chicks. Hatchling chicks were fed a vitamin D-deficient diet (2) for 28 d and fasted for 16 h before having calcium trans port assessed by the in situ ligated segment technique (2). The intraluminal dosing solution (0.5 mL) contained the in dicated levels of ""Ça,3.76 GBq 47Cain 145 mmol/L of NaCl, pH 7.0. After a 20-min absorption period, the animals were killed and the intact duodenal segments were removed and the remaining radioactivity was determined. The segments were then drained and rinsed with cold 145 mmol/L of NaCl; the mucosa was removed, weighed and ashed before deter mination of tissue calcium by atomic absorption spectrophotometry. Calcium absorption was calculated from the residual amount of 47Cain the segment. Triangles indicate calcium absorption (open), tissue retention (closed) in com parable deficient animals that received 12.5 ¿ig vitamin D3. Each point represents the mean ±SEMfor six chicks.
'/
b
b
b
/f""""'J ° uAOoà Ç5!j5040302010na°T /i
\ \ fsL/_
__.¡b•^ • — •Absorbed o— -o Retained
-/
0124
8 Time after 1,25(OH)2
12
16
D (hours)
FIGURE 3 Duodenal absorption and tissue retention of luminally administered 47Cain vitamin D-deficient chicks at varying times after a single intravenous injection of 1,25(OH)2D (1.0/ig)- Chicks and absorption technique were identical to those described in the legend for Figure 2, except that the intraluminal dosing solution contained 20 mmol/L of Ca. Data are presented as percentage of the original ra dioactivity absorbed or retained by the tissue. Each point represents the mean ±SEMfor six chicks, (a) Denotes sig nificant difference (P > 0.05) in 47Catissue retention due to 1,25(OH)2D. (b) Denotes significant difference (P > 0.05) in 47Caabsorption due to 1,25(OH)2D.
step(s). Some years ago, Sampson et al. (44) employed microincineration and tissue fixation techniques in conjunction with 45Ca autoradiography and electron microscopy in an attempt to trace the movement of calcium through the epithelial cell. In the rachide rat, calcium (administered orally) was shown to pass across the limiting cell membrane and associate primarily with elements within the microvilli. There was no in dication of mass calcium movement from this apical region into the cytosol proper or to the intracellular organdÃ-es. Vitamin D treatment resulted in dimin ished calcium accumulation in the microvillar region and its appearance in the mitochondria located more basally. This indicates that mobilization of microvillarbound calcium requires vitamin D, whereas the translocation across the luminal-facing membrane and sub sequent binding to the microvilli do not. The fixation techniques employed in these studies, however, did not preclude loss or possible redistribution of calcium that was not bound tightly to cellular elements. Recently, we have developed a procedure to study calcium transport that combines ion microscopic im aging and stable isotopie analyses with improved cryoscopic and embedding techniques (45). Ion mi croscopy is a method based on secondary ion mass spectrometry, which produces visual images of the el emental distribution within a sample in relation to tissue morphology (46). Because ions to be examined are selected on the basis of mass to charge ratio, dif ferent isotopes of the same element may be imaged sequentially. By employing enriched 44Ca as the sole luminal species, it was possible to discriminate be-
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Recently, we have expanded this comparison. As shown in Figure 2, luminal calcium accumulates to a considerable extent in the mucosal tissue of vitamin D-deficient chicks, essentially doubling as the luminal calcium concentration is increased from 2 to 20 mmol/ L. Though it remains constant at luminal concentra tions up to 50 mmol/L. This indicates that the capacity to accumulate calcium from the lumen, while consid erable, is saturable. Figure 3 shows the time course for in situ tissue accumulation and absorption of calcium when 1,25(OH)2D was administered to vitamin D-deficient chicks. Within 30 min of 1,25(OH)2D administration, mucosal 47Caaccumulation was markedly and signif icantly enhanced but had declined by 2 and 4 h postdose, a time when transcellular transport had com menced in the treated but not in the deficient chicks. These results confirm that 1,25(OH)2D acts rapidly to enhance cellular calcium entry, but that considerable time elapses between entry and subsequent transport. They also demonstrate the absolute vitamin D depen dence of transcellular calcium transport. One difficulty associated with whole-organ trans port systems has been the inability to identify the spa tial distribution of calcium during transit and that re tained by the tissue. Such information might provide insight as to the location of the limiting transport
648
FULLMER
tween calcium in transit (44Ca)and ambient tissue cal cium (40Ca).This procedure was employed to compare and contrast the movement of luminally administered calcium in vitamin D-deficient and -replete chicks under physiological conditions. Preliminary radioisotope (47Ca)data showed that luminal calcium rapidly and extensively becomes as sociated with the mucosal tissue but is transferred to the blood (absorbed) at a very slow rate in vitamin Ddeficient chicks. Vitamin D-repletion results in a greater initial increase in tissue uptake coinciding with transepithelial transport, followed by diminished tis sue levels as the bulk of luminal calcium is transported to the blood (45). The corresponding 44Ca images al lowed the visualization of these processes. In the vi tamin D-deficient case, luminal 44Ca rapidly crossed the cell membrane, and high concentrations became localized in the microvillar/terminal web region. After absorption had proceeded for a time, accumulation
continued with a steep, inwardly directed gradient, but there was little apparent transcellular movement. In the vitamin D-replete situation, luminal 44Carap idly entered the cell, perhaps accumulating transiently in the microvillar region, but continued to move through the cytosol in a basal direction, appearing very quickly in the lamina propria. Figure 4 compares the tissue distribution of 44Ca and 40Ca at 20 min after the intraluminal injection of 44Ca to vitamin D-deplete and -replete chicks. These studies provide the first visualization of ep ithelial calcium transport under physiological condi tions. They demonstrate graphically that the luminal limiting cell membrane does not represent a substan tial barrier to calcium entry into the cell. Inwardly bound calcium is initially sequestered in the apical cell region, perhaps by cytoskeletal elements and/or calmodulin. Under the influence of vitamin D and presumably due to the presence of calbindin (see réf.
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FIGURE 4 Ion microscopic images showing the distribution in the villus of tissue *°Ca [upper panels) and of intraluminally administered 44Ca (lower panels) in vitamin D-deficient (left panels) and vitamin D-replete (right panels) chicks. Note that the intraluminally administered calcium did not spread through the cells of the deficient chicks. For further details, see text and réf. 45.
SYMPOSIUM: CURRENT CONCEPTS OF CALCIUM ABSORPTION
ACKNOWLEDGMENT The author acknowledges the excellent secretarial assistance of Norma Jayne.
LITERATURE CITED 1. Wasserman, R. H. a Chandler, J. S. (1985) Molecular mech anisms of intestinal calcium absorption. In: Bone and Mineral Research (Peck, W. A., ed.), pp. 181-211, Elsevier Science Pub lishers, Amsterdam, The Netherlands. 2. Wasserman, R. H., Brindak, M. E., Meyer, S. A. & Fullmer, C. S. (1982) Evidence for multiple effects of vitamin D3 on cal cium absorption: Response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3. Proc. Nati. Acad. Sci. USA 79: 7939-7943. 3. Rasmussen, H., Fontaine, O., Max, E. & Goodman, D. B. P. (1979) The effect of la-hydroxyvitamin D3-administration on calcium transport in chick intestine brush border membrane vesicles. J. Biol. Chem. 254: 2993-2999. 4. Miller, A. &. Bronner, F. (1981) Calcium uptake in isolated brush border vesicles from rat small intestine. Biochem. J. 196: 391-401. 5. Fontaine, O., Matsumoto, T., Goodman, D. B. P. & Rasmussen, H. (1981) Liponomic control of Ca2+ transport: Relationship to mechanism of action of 1,25-dihydroxyvitamin D3. Proc. Nati. Acad. Sci. USA 78: 1751-1754. 6. Bikle, D. D., Munson, S. & Zolock, D. T. (1983) Calcium flux across chick duodenal brush border membrane vesicles: Regu lation by 1,25-dihydroxyvitamin D. Endocrinology 113: 20722080. 7. Norman, A. W. (1975) Hormone-like action of 1,25-dihydroxycholecalciferol. Vitam Horm 32: 325-384. 8. Holdsworth, E. S. (1970) The effects of vitamin D on the en zyme activities in the mucosal cells of the chick small intestine. J. Membr. Biol. 3: 43-53. 9. Norman, A. W., Mircheff, A. K., Adams, T. H. & Spielvogel, A. (1970) Studies on the mechanisms of action of calciferol; III. Vitamin D-mediated increase of intestinal brush border al kaline phosphatase activity. Biochim. Biophys. Acta 215: 348359. 10. Haussler, M. R., Nagode, L. A. & Rasmussen, H. (1970) Induction of intestinal brush border alkaline phosphatase and identity with Ca-ATPase. Nature 228: 1199-1201. 11. Ghijsen, W. E. J. M., Dejonge, M. D. & Van Os, C. H. (1980) Dissociation between Ca2+-ATPase and alkaline phos phatase activities in plasma membranes of rat duodenum. Biochim. Biophys. Acta 599: 538-551. 12. Forstner, G. G., Sabesin, S. M. & Isselbacher, K. J. (1968) Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem. J. 106: 381-390. 13. Oku, T. & Wasserman, R. H. (1978) Properties of the vitamin D-stimulated calcium-dependent adenosine triphosphatase and alkaline phosphatase in chick intestinal brush border. Fed. Proc. 37: 408.
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47), the calcium that has entered the cell can move through the cytoplasm to the basolateral pole where it is extruded by the calcium pump. Thus, although vitamin D or 1,25(OH)2D may enhance the calcium entry rate, its primary action is to provide the neces sary transport elements.
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33.
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