Symposium:
Current Concepts of Calcium Absorption
Vesicular Calcium Transport in Chick Intestine12 Uniuersity of California, Riverside, CA 92512
ABSTRACT A critical question in transcellular calcium transport relates to the identity of the calcium carrier. Such a carrier is needed if for no other reason than to minimize toxicity due to a high intracellular concentra tion of the calcium ion. After briefly reviewing several of the carriers that have been proposed in the past, attention is directed to the endosomal-lysosomal trans port pathway. New findings are presented in support of the rapidity with which lysosomes might be mobi lized to transport calcium. New data demonstrate that these organdÃ-es can take up calcium even under con ditions of relatively low luminal levels of the divalent cation (0.9 mmol/L of CaCy. They may therefore play a role in the facilitated movement of calcium through the cell. J. Nutr. 122: 657-661, 1992. INDEXING KEY WORDS:
•7,25-driydroxycholecalciferol •calcium transport •lysosomes •intestine •chickens
Transepithelial calcium transport has been studied by many different approaches. It is generally accepted that the seco-steroid hormone 1,25-dihydroxycholecalciferol [1,25(OH)2D3] stimulates net absorption in the intestine. Moreover, due to the potential toxicity of transcellular calcium movement, a carrier or means of sequestering the divalent cation appears needed. Table 1 lists some of the proposed carriers and sum marizes arguments for and against their involve ment. The vitamin D-induced calcium binding protein [calbindin-D or CaBP (1)]has been studied extensively. It is believed by many to be a cytosolic protein that ferries calcium from the brush border to the basal lat eral membrane of the intestinal cell. Inhibitors of mRNA and protein synthesis abolish the induction of CaBP and inhibit l,25(OH)2D3-augmented calcium transport in the chick ileum (2), but not in duodenum (3). Experimental evidence likewise exists for the de tection of the protein before (4) or after (5) enhanced absorption. The mere presence of CaBP in the intes-
tinal epithelium is not sufficient to initiate calcium transport (6, 7). Calmodulin (CaM) levels in the intestine are not augmented by 1,25(OH)2D3 treatment (cf. 8). It has been proposed that this ubiquitous calcium-dependent regulatory protein, like CaBP, serves to buffer intra cellular calcium (9) in addition to its other functions. CaM has been found to be redistributed to the brush border in response to 1,25(OH)2D3 (6) and may serve to mediate entry of the divalent cation into epithelial cells. CaM antagonists, moreover, inhibit calcium transport (6). AJthough CaM has a lower affinity for calcium than does CaBP, this could actually facilitate transfer of the divalent cation to CaBP or other higher affinity binding sites in the cell enroute to eventual extrusion. Mitochondria can take up very large quantities of calcium in vitro and in vivo (10). However, recent work (11, 12) suggests that this may not occur in healthy cells. Methodological differences have also led to reports that either support (13) or refute (14) mitochondrial involvement in calcium transport through the cell. Biochemical studies, in which calcium uptake has been studied under in vitro conditions, have also suggested a carrier role for the Golgi apparatus (15) and endoplasmic reticulum (16). By compari son, biochemical studies performed after calcium absorption in vivo (12, 17, 18) and data from elec tron microscopy (14, 19) have suggested vesic ular carriers, particularly the endosomal-lysosomal pathway. 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. 1This work was supported in part by USPHS grant AM-09012 to Anthony W. Norman. 3To whom correspondence should be addressed: Department of Biochemistry, University of California, Riverside, CA 92521.
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mg of CaCl2 in 0.4 mL, allowed to transport the cation for 3 or 30 min and then fixed. After 30 min of fixation, the tissue was washed twice for 10 min in phosphate buffered saline (PBS) and segments from each duo CarrierCalbindinCalmodulinMitochondriaGolgiEndoplasmicreticulumEndosomes/lysosomesForInduced denum within a group embedded together in O.C.T. medium (American Scientific products, Los Angeles, 1,25(OH)2D3Inhibitor by CA) for the preparation of 4 /urn-thick frozen sections. studiesTimecourse studiesTimecourse The sections were transferred to gelatin-coated mi studiesRedistribution studiesSynthesis croscope slides and stored at -80°Covernight. Before brushborder to inducedbynot by1,25|OH)2D3Affinity induced 1,25(OH)2D3Affinity immunolabeling, the sections were hydrated in PBS Ca2+Inhibitor for Ca2+Biochemical for for 10 min, treated with 0.5 g NaBH4/L for 5 min, and then washed twice for 5 min each in PBS-1 g/L studiesBiochemical studies,uptake studies,uptake bovine serum albumin (BSA).The sections were then overlaid, according to the manufacturer's suggestions, invitroElectron in vivo and vivoElectron in with primary antibody, sheep anti-cathepsin B (ICN microscopyBiochemical microscopyElectron Biomedicals, Costa Mesa, CA), for 30 min. After three studies,uptake 5-min washes in PBS-1 g/L BSA, the secondary anti vitroElectron in body, fluorescein-conjugated rabbit anti-sheep immicroscopyBiochemical microscopyElectron studies,uptake microscopy munoglobulin G (ICN Biomedicals) was applied for vitroElectron in 10 min. At the end of this time, the sections were microscopyBiochemical washed three times and mounted in 10% 0.1 mol/L of studies,uptake TRIS, pH 8.0, 90% glycerol. in vivoAgainstInhibitor Figure 1 depicts the results of indirect immunofluorescent labeling of sections with an antibody to cathepsin B. Cathepsin B is a marker enzyme for lyso Both biochemical and microscopic analyses have somes (12) and labeling of the protease should allow indicated that, under conditions of calcium absorption, visualization of the organellar distribution. In these lysosomes of intestinal epithelial cells contain high low magnification micrographs, tissue was prepared from l,25(OH)2D3-treated chicks either in the absence levels of calcium (12, 19). Thirty minutes after calcium was instilled into in situ ligated duodenal loops, sub of calcium transport (Fig. \A, B), or after 3 min of sequent biochemical analyses have shown that a ve calcium transport (Figs. 1C, D). The panels of the left represent phase-contrast micrographs, with corre sicular pathway is compatible with the time course of l,25(OH)2D3-augmented transport (17) and with dosesponding immunofluorescently labeled images on the response relationships between the seco-steroid hor right. In the absence of calcium absorption, cathepsin mone and intestinal calcium absorption (18). In the B immunoreactivity revealed a punctate distribution rare instances where either endocytic vesicles or ly within epithelial cells (Fig. IB). By comparison, 3 min sosomes were found to contain enhanced levels of cal of transport resulted in a rapid redistribution of an cium in the absence of net transport, hormone-me tigen to the basal-lateral membrane surface (Fig. ID). diated changes in microtubule isotypes may be the ex After 30 min of calcium transport, sections from both vitamin D-deficient (data not shown) and planation. Microtubules, cytoskeletal elements along l,25(OH)2D3-treated chicks (Fig. 1£,F) revealed sub which vesicles move, have been found to contain l,25(OH)2D3-regulated proteins (cf. 20). stantial cathepsin B immunofluorescence in the villus A more recent criticism of the vesicular calcium core. But whereas antigen depletion was noted in tissue transport pathway has been the absorption time chosen from rachitic birds (data not shown), the intensity of immunofluorescent staining in tissue from hormonefor analysis. Lee et al. (21) reported that cannulation of the mesenteric vein allows detection of calcium treated chicks had returned to pretransport levels (Fig. IF). In parallel studies, cathepsin B-specific enzyme transport within 5 min of introducing the divalent activity was also found to decline profoundly in lycation into the lumen of the intestine. To determine whether vesicular transport is rapid enough to account sosomal and endosomal fractions prepared from in for this observation, immunocytochemical analysis of testinal epithelium after 3 min of calcium absorption (22), with l,25(OH)2D3-mediated differences in re fixed, frozen sections was employed. Rachitic chicks (3-4 per group) received by intramuscular injection covery of epithelial activity after 30 min. The question has been raised whether cellular lyvehicle or l.Snmolof 1,25(OH)2D3 15 h before having their duodenal loop surgically exposed under ether sosomal and/or endosomal components can accumu late calcium when low concentrations are presented anesthesia. The loops were either immediately excised, to the brush border. In other words, is the 45Cafound slit longitudinally and placed in fixative (4% paraformaldehyde, 0.2% glutaraldehyde, 5 mmol/L of EGTA associated with lysosomal and endosomal organdÃ-es in phosphate buffered saline; 25°C)or injected with 4 in the presence of high absorptive calcium loads due TABLE 1 Proposed Ca'* carriers
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to nonspecific uptake from the cytoplasm? Can these organdÃ-essequester calcium when the concentration of the divalent cation at the brush border is low? To study this question, vitamin D-deficient chicks were dosed with vehicle or 1.3 nmol of 1,25(OH)2D3 15 h before experimentation. Calcium transport was stud ied with the aid of in situ ligated duodenal loops that contained 0.9 mmol CaCl2/L. Fractions were prepared as previously described (12). Briefly, the mucosae were collected by scraping and homogenized in a buffered sucrose medium that contained ruthenium red and EGTA to prevent calcium redistribution. Subcellular fractionation was achieved by a combination of dif ferential and Percoli gradient centrifugation. Aliquots were removed for determination of 45Caand protein. The results were expressed as percent of gradient spe cific activity (12). Figure 2 illustrates the results of these studies. Although after 30 min of transport serum 45Ca2+ no longer increased linearly, the time was chosen for comparison with previous studies (12). At that time lysosomal fractions not only exhibited the largest 45Ca specific activity (Bq/mg protein), but also revealed a
l,25(OH)2D3-mediated enhancement in radionuclide levels (Fig.1A). When the area under the curves defined by lysosomal fractions 1-3 was expressed as a ratio, the +D/-D value was 1.88, equivalent to the ratio obtained when high levels of calcium were absorbed (17). Similar analyses of endosomal fractions (Fig. 2B) revealed unexpected results. Radionuclide levels in fraction 3 from rachitic chicks were substantially higher than those in the corresponding fraction from hormone-treated birds, although the latter gradient also evinced a significant level of 45Ca in fraction 17 (Fig. 2B). The lower density peak (fraction 17) could represent either endoplasmic reticulum or an endo somal compartment where recycling of the calcium receptor takes place (cf. 22). The results further suggest that mechanisms for vesicular flow and membrane re cycling are defective in the intestinal epithelium of rachitic chicks. The fact that the same vesicular transport pathway is used in intestinal epithelium regardless of calcium load has further implications for saturable and nonsaturable transport. It is conceivable that the difference between the two routes resides at the brush border
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FIGURE 1 Redistribution of cathepsin B activity during intestinal calcium transport, as monitored by immunofluorescence microscopy. The micrograph depicts tissue prepared from rachitic chicks dosed with 1.3 nmol of 1,25(OH)2D3 15 h before experimentation. Under ether anesthesia, tissue was removed to fixative either in the absence of calcium transport (A) and (J5), or after 3 min of absorption (C) and [D] or after 30 min of absorption (£)and (F). Fixed frozen sections were prepared and labeled with anti-cathepsin B primary antibody followed by fluorescein-conjugated secondary antibody. Left panels depict phase-contrast micrographs, with corresponding fluorescence micrographs shown in the right panels. Adapted from Ref. 22.
660
NEMERE A
20 Lytoiom««
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FRACTION NUMBER B
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FIGURE 2 Subcellular distribution of 45Ca2+after absorption of 0.9 mmol/L of lumenal CaCl2. Percoli gradient fractions were prepared from intestinal mucosa of vitamin D-deficient chicks dosed with vehicle (O O) or 1.3 nmol of 1,25(OH)2D3 (• •)for 15 h after 30 min of absorption (A) or 20 min of absorption (A). G A, Golgi apparatus; BLM, basal lateral membrane; ER, endoplasmic reticulum.
membrane: low levels of lumenal calcium (saturable transport) may be internalized into endocytic vesicles through a specific calcium receptor, whereas nonsaturable transport in the presence of high calcium may involve formation of nonspecific endocytic vesicles, which nevertheless fuse with lysosomes, before exo-
cytosis. The avidity of these membrane-delimited organelles for calcium may be explained by the recent observation that they contain substantial levels of immunoreactive CaBP (cf. 20). Thus, calbindin-D28k in chick intestine may facilitate the transcellular move ment of calcium by mechanisms beyond diffusion.
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— o 1+
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LITERATURE CITED
regulated events in the intestinal brush border. J. Cell Biol. 100: 754-763. 10. Hamilton, J W. & Holdsworth, E. S. (1975) The location of calcium during its transport by the small intestine of chick. Aust. J. Exp. Biol. Med. Sci. 53: 453-468. 11. Demon, R. M., McCormack, J. G. &.Edgell, N. J. (1980) Role of calcium ions in the regulation of intramitochondrial metab olism. Biochem. J. 190: 107-117.
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12. Nemere, I., Leathers, V. L. & Norman, A. W. (1986) 1,25Dihydroxyvitamin D3-mediated intestinal calcium transport: Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-Dî8|J.J. Biol. Chem. 261: 16106-16114. 13. Sampson, H. W., Matthews, J. L., Martin, J. H. & Kunin, J. 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. 14. Warner, R. R. & Coleman, J. R. (1975) Electron probe analysis of calcium transport by small intestine. J. Cell Biol. 64: 54-74. 15. MacLaughlin, J. A., Weiser, M. M. & Freedman, R. A. (1980) Biphasic recovery of vitamin D-dependent Ca+2+ uptake by rat intestinal Golgi membranes. Gastroenterology 78: 325-332. 16. Rubinoff, M. J. & Nellans, H. N. (1985) Active calcium se questration by intestinal microsomes. Stimulation by increased calcium load. J. Biol. Chem. 260: 7824-7828. 17. Nemere, I. a Norman, A. W. (1988) 1,25-Dihydroxyvitamin D3-mediated vesicular transport of calcium in intestine: Time course studies. Endocrinology 122: 2962-2969. 18. Nemere, I. & Norman, A. W. (1989) 1,25-Dihydroxyvitamin D3-mediated vesicular transport of calcium in intestine: Doseresponse studies. Mol. Cell. Endocrinol. 67: 47-53. 19. Davis, W. L., Jones, R. G. & Hagler, H. K. (1979) Calcium containing lysosomes in the normal chick duodenum: A histochemical and analytical electron microscopic study. Tissue & Celili: 127-138. 20. Nemere, I. (1990) Intestinal calcium transport: Vesicular car riers, noncytoplasmic calbinding D 28K, and non-nuclear effects of 1,25-dihydroxyvitamin D3. In: Calcium Transport and Intracellular Calcium Homeostasis (Pansu, D. &. Bronner, F., eds.), pp. 233-240, Springer-Verlag, New York, NY. 21. Lee, Y. S., Reimers, T. J., Cowan, R. G., Fullmer, C. S. & Was serman, R. H. (1988) Calcium-dependent translocation of calbindin-D28k from intestine to blood. Arch. Biochem. Biophys. 261:27-34. 22. Nemere, I. & Norman, A. W. (1991) Redistribution of cathepsin B activity from the endosomal-lysosomal pathway in chick intestine within 3 min of calcium absorption. Mol. Cell Endo crinol 76: 7-16.
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1. Wasserman, R. H. & Taylor, A. N. |1966) Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science 152: 791-793. 2. Tsai, H. C. & Norman, A. W. (1973) Studies on the model of action of calciferol VI: Effect of 1,25-dihydroxy-vitamin D3 on RNA synthesis in the intestinal mucosa. Biochem. Biophys. Res. Commun. 54: 622-627. 3. Bikle, D. D., Zolock, D. T., Morrissey, R. L. & Herman, R. H. |1978) Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis. /. Biol. Chem. 253: 484-488. 4. Bishop, C. W., Kendrick, N. C. & DeLuca, H. F. (1983) Induc tion of calcium-binding protein before 1,25-dihydroxyvitamin D3 stimulation of duodenal calcium uptake. /. Biol. Chem. 258: 1305-1310. 5. Spencer, R., Sharman, M. a Lawson, D. E. M. (1978) The re lationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken. Biochem. J. 170: 93-101. 6. Bikle, D. D., Zolock, D. T. & Munson, S. (1984) Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: A comparison of calcium uptake, calcium binding protein, and alkaline phosphatase ac tivity. Endocrinology 115: 2077-2084. 7. Krisinger, J., Strom, M., Darwish, H. D., Perlman, K..,Smith, C. & DeLuca, H. F. (1991) Induction of calbindin-D 9k mRNA but not calcium transport in rat intestine by 1,25-dihydroxy vitamin D3 24-homologs. J. Biol. Chem. 266: 1910-1913. 8. Bikle, D. D., Munson, S. & Chafouleas, J. (1984) Calmodulin may mediate 1,25-dihydroxyvitamin D-stimulated intestinal calcium transport. FEES Lett. 174: 30-33. 9. Glenney, J. R., Jr. &. Glenney, P. (1985) Comparison of Ca++-
OF CALCIUM
Current Concepts of Calcium Absorption
Vesicular Calcium Transport in Chick Intestine12 Uniuersity of California, Riverside, CA 92512
ABSTRACT A critical question in transcellular calcium transport relates to the identity of the calcium carrier. Such a carrier is needed if for no other reason than to minimize toxicity due to a high intracellular concentra tion of the calcium ion. After briefly reviewing several of the carriers that have been proposed in the past, attention is directed to the endosomal-lysosomal trans port pathway. New findings are presented in support of the rapidity with which lysosomes might be mobi lized to transport calcium. New data demonstrate that these organdÃ-es can take up calcium even under con ditions of relatively low luminal levels of the divalent cation (0.9 mmol/L of CaCy. They may therefore play a role in the facilitated movement of calcium through the cell. J. Nutr. 122: 657-661, 1992. INDEXING KEY WORDS:
•7,25-driydroxycholecalciferol •calcium transport •lysosomes •intestine •chickens
Transepithelial calcium transport has been studied by many different approaches. It is generally accepted that the seco-steroid hormone 1,25-dihydroxycholecalciferol [1,25(OH)2D3] stimulates net absorption in the intestine. Moreover, due to the potential toxicity of transcellular calcium movement, a carrier or means of sequestering the divalent cation appears needed. Table 1 lists some of the proposed carriers and sum marizes arguments for and against their involve ment. The vitamin D-induced calcium binding protein [calbindin-D or CaBP (1)]has been studied extensively. It is believed by many to be a cytosolic protein that ferries calcium from the brush border to the basal lat eral membrane of the intestinal cell. Inhibitors of mRNA and protein synthesis abolish the induction of CaBP and inhibit l,25(OH)2D3-augmented calcium transport in the chick ileum (2), but not in duodenum (3). Experimental evidence likewise exists for the de tection of the protein before (4) or after (5) enhanced absorption. The mere presence of CaBP in the intes-
tinal epithelium is not sufficient to initiate calcium transport (6, 7). Calmodulin (CaM) levels in the intestine are not augmented by 1,25(OH)2D3 treatment (cf. 8). It has been proposed that this ubiquitous calcium-dependent regulatory protein, like CaBP, serves to buffer intra cellular calcium (9) in addition to its other functions. CaM has been found to be redistributed to the brush border in response to 1,25(OH)2D3 (6) and may serve to mediate entry of the divalent cation into epithelial cells. CaM antagonists, moreover, inhibit calcium transport (6). AJthough CaM has a lower affinity for calcium than does CaBP, this could actually facilitate transfer of the divalent cation to CaBP or other higher affinity binding sites in the cell enroute to eventual extrusion. Mitochondria can take up very large quantities of calcium in vitro and in vivo (10). However, recent work (11, 12) suggests that this may not occur in healthy cells. Methodological differences have also led to reports that either support (13) or refute (14) mitochondrial involvement in calcium transport through the cell. Biochemical studies, in which calcium uptake has been studied under in vitro conditions, have also suggested a carrier role for the Golgi apparatus (15) and endoplasmic reticulum (16). By compari son, biochemical studies performed after calcium absorption in vivo (12, 17, 18) and data from elec tron microscopy (14, 19) have suggested vesic ular carriers, particularly the endosomal-lysosomal pathway. 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. 1This work was supported in part by USPHS grant AM-09012 to Anthony W. Norman. 3To whom correspondence should be addressed: Department of Biochemistry, University of California, Riverside, CA 92521.
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mg of CaCl2 in 0.4 mL, allowed to transport the cation for 3 or 30 min and then fixed. After 30 min of fixation, the tissue was washed twice for 10 min in phosphate buffered saline (PBS) and segments from each duo CarrierCalbindinCalmodulinMitochondriaGolgiEndoplasmicreticulumEndosomes/lysosomesForInduced denum within a group embedded together in O.C.T. medium (American Scientific products, Los Angeles, 1,25(OH)2D3Inhibitor by CA) for the preparation of 4 /urn-thick frozen sections. studiesTimecourse studiesTimecourse The sections were transferred to gelatin-coated mi studiesRedistribution studiesSynthesis croscope slides and stored at -80°Covernight. Before brushborder to inducedbynot by1,25|OH)2D3Affinity induced 1,25(OH)2D3Affinity immunolabeling, the sections were hydrated in PBS Ca2+Inhibitor for Ca2+Biochemical for for 10 min, treated with 0.5 g NaBH4/L for 5 min, and then washed twice for 5 min each in PBS-1 g/L studiesBiochemical studies,uptake studies,uptake bovine serum albumin (BSA).The sections were then overlaid, according to the manufacturer's suggestions, invitroElectron in vivo and vivoElectron in with primary antibody, sheep anti-cathepsin B (ICN microscopyBiochemical microscopyElectron Biomedicals, Costa Mesa, CA), for 30 min. After three studies,uptake 5-min washes in PBS-1 g/L BSA, the secondary anti vitroElectron in body, fluorescein-conjugated rabbit anti-sheep immicroscopyBiochemical microscopyElectron studies,uptake microscopy munoglobulin G (ICN Biomedicals) was applied for vitroElectron in 10 min. At the end of this time, the sections were microscopyBiochemical washed three times and mounted in 10% 0.1 mol/L of studies,uptake TRIS, pH 8.0, 90% glycerol. in vivoAgainstInhibitor Figure 1 depicts the results of indirect immunofluorescent labeling of sections with an antibody to cathepsin B. Cathepsin B is a marker enzyme for lyso Both biochemical and microscopic analyses have somes (12) and labeling of the protease should allow indicated that, under conditions of calcium absorption, visualization of the organellar distribution. In these lysosomes of intestinal epithelial cells contain high low magnification micrographs, tissue was prepared from l,25(OH)2D3-treated chicks either in the absence levels of calcium (12, 19). Thirty minutes after calcium was instilled into in situ ligated duodenal loops, sub of calcium transport (Fig. \A, B), or after 3 min of sequent biochemical analyses have shown that a ve calcium transport (Figs. 1C, D). The panels of the left represent phase-contrast micrographs, with corre sicular pathway is compatible with the time course of l,25(OH)2D3-augmented transport (17) and with dosesponding immunofluorescently labeled images on the response relationships between the seco-steroid hor right. In the absence of calcium absorption, cathepsin mone and intestinal calcium absorption (18). In the B immunoreactivity revealed a punctate distribution rare instances where either endocytic vesicles or ly within epithelial cells (Fig. IB). By comparison, 3 min sosomes were found to contain enhanced levels of cal of transport resulted in a rapid redistribution of an cium in the absence of net transport, hormone-me tigen to the basal-lateral membrane surface (Fig. ID). diated changes in microtubule isotypes may be the ex After 30 min of calcium transport, sections from both vitamin D-deficient (data not shown) and planation. Microtubules, cytoskeletal elements along l,25(OH)2D3-treated chicks (Fig. 1£,F) revealed sub which vesicles move, have been found to contain l,25(OH)2D3-regulated proteins (cf. 20). stantial cathepsin B immunofluorescence in the villus A more recent criticism of the vesicular calcium core. But whereas antigen depletion was noted in tissue transport pathway has been the absorption time chosen from rachitic birds (data not shown), the intensity of immunofluorescent staining in tissue from hormonefor analysis. Lee et al. (21) reported that cannulation of the mesenteric vein allows detection of calcium treated chicks had returned to pretransport levels (Fig. IF). In parallel studies, cathepsin B-specific enzyme transport within 5 min of introducing the divalent activity was also found to decline profoundly in lycation into the lumen of the intestine. To determine whether vesicular transport is rapid enough to account sosomal and endosomal fractions prepared from in for this observation, immunocytochemical analysis of testinal epithelium after 3 min of calcium absorption (22), with l,25(OH)2D3-mediated differences in re fixed, frozen sections was employed. Rachitic chicks (3-4 per group) received by intramuscular injection covery of epithelial activity after 30 min. The question has been raised whether cellular lyvehicle or l.Snmolof 1,25(OH)2D3 15 h before having their duodenal loop surgically exposed under ether sosomal and/or endosomal components can accumu late calcium when low concentrations are presented anesthesia. The loops were either immediately excised, to the brush border. In other words, is the 45Cafound slit longitudinally and placed in fixative (4% paraformaldehyde, 0.2% glutaraldehyde, 5 mmol/L of EGTA associated with lysosomal and endosomal organdÃ-es in phosphate buffered saline; 25°C)or injected with 4 in the presence of high absorptive calcium loads due TABLE 1 Proposed Ca'* carriers
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to nonspecific uptake from the cytoplasm? Can these organdÃ-essequester calcium when the concentration of the divalent cation at the brush border is low? To study this question, vitamin D-deficient chicks were dosed with vehicle or 1.3 nmol of 1,25(OH)2D3 15 h before experimentation. Calcium transport was stud ied with the aid of in situ ligated duodenal loops that contained 0.9 mmol CaCl2/L. Fractions were prepared as previously described (12). Briefly, the mucosae were collected by scraping and homogenized in a buffered sucrose medium that contained ruthenium red and EGTA to prevent calcium redistribution. Subcellular fractionation was achieved by a combination of dif ferential and Percoli gradient centrifugation. Aliquots were removed for determination of 45Caand protein. The results were expressed as percent of gradient spe cific activity (12). Figure 2 illustrates the results of these studies. Although after 30 min of transport serum 45Ca2+ no longer increased linearly, the time was chosen for comparison with previous studies (12). At that time lysosomal fractions not only exhibited the largest 45Ca specific activity (Bq/mg protein), but also revealed a
l,25(OH)2D3-mediated enhancement in radionuclide levels (Fig.1A). When the area under the curves defined by lysosomal fractions 1-3 was expressed as a ratio, the +D/-D value was 1.88, equivalent to the ratio obtained when high levels of calcium were absorbed (17). Similar analyses of endosomal fractions (Fig. 2B) revealed unexpected results. Radionuclide levels in fraction 3 from rachitic chicks were substantially higher than those in the corresponding fraction from hormone-treated birds, although the latter gradient also evinced a significant level of 45Ca in fraction 17 (Fig. 2B). The lower density peak (fraction 17) could represent either endoplasmic reticulum or an endo somal compartment where recycling of the calcium receptor takes place (cf. 22). The results further suggest that mechanisms for vesicular flow and membrane re cycling are defective in the intestinal epithelium of rachitic chicks. The fact that the same vesicular transport pathway is used in intestinal epithelium regardless of calcium load has further implications for saturable and nonsaturable transport. It is conceivable that the difference between the two routes resides at the brush border
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FIGURE 1 Redistribution of cathepsin B activity during intestinal calcium transport, as monitored by immunofluorescence microscopy. The micrograph depicts tissue prepared from rachitic chicks dosed with 1.3 nmol of 1,25(OH)2D3 15 h before experimentation. Under ether anesthesia, tissue was removed to fixative either in the absence of calcium transport (A) and (J5), or after 3 min of absorption (C) and [D] or after 30 min of absorption (£)and (F). Fixed frozen sections were prepared and labeled with anti-cathepsin B primary antibody followed by fluorescein-conjugated secondary antibody. Left panels depict phase-contrast micrographs, with corresponding fluorescence micrographs shown in the right panels. Adapted from Ref. 22.
660
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(PERCE Co
0
1
2
3
4
S
B
7
B
9
10 11 12 13 14 15 1B 17 1B 19
FRACTION
NUMBER
FIGURE 2 Subcellular distribution of 45Ca2+after absorption of 0.9 mmol/L of lumenal CaCl2. Percoli gradient fractions were prepared from intestinal mucosa of vitamin D-deficient chicks dosed with vehicle (O O) or 1.3 nmol of 1,25(OH)2D3 (• •)for 15 h after 30 min of absorption (A) or 20 min of absorption (A). G A, Golgi apparatus; BLM, basal lateral membrane; ER, endoplasmic reticulum.
membrane: low levels of lumenal calcium (saturable transport) may be internalized into endocytic vesicles through a specific calcium receptor, whereas nonsaturable transport in the presence of high calcium may involve formation of nonspecific endocytic vesicles, which nevertheless fuse with lysosomes, before exo-
cytosis. The avidity of these membrane-delimited organelles for calcium may be explained by the recent observation that they contain substantial levels of immunoreactive CaBP (cf. 20). Thus, calbindin-D28k in chick intestine may facilitate the transcellular move ment of calcium by mechanisms beyond diffusion.
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— o 1+
SYMPOSIUM:
CURRENT
CONCEPTS
LITERATURE CITED
regulated events in the intestinal brush border. J. Cell Biol. 100: 754-763. 10. Hamilton, J W. & Holdsworth, E. S. (1975) The location of calcium during its transport by the small intestine of chick. Aust. J. Exp. Biol. Med. Sci. 53: 453-468. 11. Demon, R. M., McCormack, J. G. &.Edgell, N. J. (1980) Role of calcium ions in the regulation of intramitochondrial metab olism. Biochem. J. 190: 107-117.
ABSORPTION
661
12. Nemere, I., Leathers, V. L. & Norman, A. W. (1986) 1,25Dihydroxyvitamin D3-mediated intestinal calcium transport: Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-Dî8|J.J. Biol. Chem. 261: 16106-16114. 13. Sampson, H. W., Matthews, J. L., Martin, J. H. & Kunin, J. 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. 14. Warner, R. R. & Coleman, J. R. (1975) Electron probe analysis of calcium transport by small intestine. J. Cell Biol. 64: 54-74. 15. MacLaughlin, J. A., Weiser, M. M. & Freedman, R. A. (1980) Biphasic recovery of vitamin D-dependent Ca+2+ uptake by rat intestinal Golgi membranes. Gastroenterology 78: 325-332. 16. Rubinoff, M. J. & Nellans, H. N. (1985) Active calcium se questration by intestinal microsomes. Stimulation by increased calcium load. J. Biol. Chem. 260: 7824-7828. 17. Nemere, I. a Norman, A. W. (1988) 1,25-Dihydroxyvitamin D3-mediated vesicular transport of calcium in intestine: Time course studies. Endocrinology 122: 2962-2969. 18. Nemere, I. & Norman, A. W. (1989) 1,25-Dihydroxyvitamin D3-mediated vesicular transport of calcium in intestine: Doseresponse studies. Mol. Cell. Endocrinol. 67: 47-53. 19. Davis, W. L., Jones, R. G. & Hagler, H. K. (1979) Calcium containing lysosomes in the normal chick duodenum: A histochemical and analytical electron microscopic study. Tissue & Celili: 127-138. 20. Nemere, I. (1990) Intestinal calcium transport: Vesicular car riers, noncytoplasmic calbinding D 28K, and non-nuclear effects of 1,25-dihydroxyvitamin D3. In: Calcium Transport and Intracellular Calcium Homeostasis (Pansu, D. &. Bronner, F., eds.), pp. 233-240, Springer-Verlag, New York, NY. 21. Lee, Y. S., Reimers, T. J., Cowan, R. G., Fullmer, C. S. & Was serman, R. H. (1988) Calcium-dependent translocation of calbindin-D28k from intestine to blood. Arch. Biochem. Biophys. 261:27-34. 22. Nemere, I. & Norman, A. W. (1991) Redistribution of cathepsin B activity from the endosomal-lysosomal pathway in chick intestine within 3 min of calcium absorption. Mol. Cell Endo crinol 76: 7-16.
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1. Wasserman, R. H. & Taylor, A. N. |1966) Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science 152: 791-793. 2. Tsai, H. C. & Norman, A. W. (1973) Studies on the model of action of calciferol VI: Effect of 1,25-dihydroxy-vitamin D3 on RNA synthesis in the intestinal mucosa. Biochem. Biophys. Res. Commun. 54: 622-627. 3. Bikle, D. D., Zolock, D. T., Morrissey, R. L. & Herman, R. H. |1978) Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis. /. Biol. Chem. 253: 484-488. 4. Bishop, C. W., Kendrick, N. C. & DeLuca, H. F. (1983) Induc tion of calcium-binding protein before 1,25-dihydroxyvitamin D3 stimulation of duodenal calcium uptake. /. Biol. Chem. 258: 1305-1310. 5. Spencer, R., Sharman, M. a Lawson, D. E. M. (1978) The re lationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken. Biochem. J. 170: 93-101. 6. Bikle, D. D., Zolock, D. T. & Munson, S. (1984) Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: A comparison of calcium uptake, calcium binding protein, and alkaline phosphatase ac tivity. Endocrinology 115: 2077-2084. 7. Krisinger, J., Strom, M., Darwish, H. D., Perlman, K..,Smith, C. & DeLuca, H. F. (1991) Induction of calbindin-D 9k mRNA but not calcium transport in rat intestine by 1,25-dihydroxy vitamin D3 24-homologs. J. Biol. Chem. 266: 1910-1913. 8. Bikle, D. D., Munson, S. & Chafouleas, J. (1984) Calmodulin may mediate 1,25-dihydroxyvitamin D-stimulated intestinal calcium transport. FEES Lett. 174: 30-33. 9. Glenney, J. R., Jr. &. Glenney, P. (1985) Comparison of Ca++-
OF CALCIUM
