ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
174, 738-743 (19%)
Embryonic Chick intestine in Organ Culture: Stimulation of Calcium Transport by Exogenous Vitamin D-Induced Calcium-Binding Protein’ R. A. CORRADINO, Department
of Physical
Biology,
C. S. FULLMER,
AND
R. H. WASSERMAN
New York State College of Veterinary Ithaca, New York 14853 Received
December
Medicine,
Cornell
Uniuersity,
12, 1975
Vitamin D:, or a potent metabolite, 1,25dihydroxycholecalciferol, induces calciumbinding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.
Vitamin D:, induces synthesis of a calcium-binding protein (CaBP)’ and enhances tissue uptake and mucosal-to-serosa1 transport of calcium in embryonic chick duodenum maintained in organ culture (1). In the same system, two metabolites of vitamin D:,, namely, 25hydroxyvitamin D, (2) and 1,25dihydroxyvitamin D:, (1,25-(OH),D,,) (31, also induce CaBP and stimulate tissue uptake of a”Ca with the latter compound being much more potent in its CaBP-inducing action (1, 4). Like vitamin D:, itself (5-7), 1,25-(OH),D,, (8-10) appears to act via stimulation of RNA and subsequent CaBP synthesis. Considerable correlative evidence supports a role for CaBP in the vitamin D-mediated transport of calcium by the intestine (11-13). Using two variations of a newly developed organ culture technique (Corradino, manuscripts in preparation’), we now report direct evidence that CaBP is probably
involved in the vitamin D-mediated, intestinal transport of calcium. Several intestinal “binding proteins” have been discovered (14-171, but this represents the first demonstration in an animal system that such a protein can stimulate transport of the bound substance. MATERIALS
’ Supported by NIH research grants, No. AM15355 and AM-04652. R.A.C. is the recipient of NIH Research Career Development Award No. AM00115. 2 Abbreviations used: CaBP, calcium-binding protein; 1,25-(OH)&,, 1,25-dihydroxyvitamin D:,; HEPES, 4.(2.hydroxyethyl)-l-piperazineethanesulfonic acid. ‘I Essential details are included in the present paper. 738 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
Duodena were excised from 20-day-old chick embryos and the pancreas removed from each. The duodena were then everted onto the 20-gauge wires of one or the other of the two types of culture apparatus shown in Figs. 1A and B. In initial experiments, the apparatus of Fig. 1A was employed. This apparatus allowed the continuous rotation (1 rpm) of seven everted duodena per treatment at the surface of the culture medium. This assured adequate gaseous exchange (5% CO,:5Oc11 O,:balance, air) and nutrient uptake. The duodena were maintained for 48 h at 37.5”C in 100 ml of serum-free McCoy’s 5A (modified) medium (Gibco) containing 1.25 mM calcium, 0.625 mM phosphate, and 100 units of nystatin/ml (1, 4). After culture, two of the duodena were homogenized and CaBP determined by radial immunoassay (1); ‘“Ca uptake and transmucosal transport were assessed, after a 30-min incubation in a lowsodium buffer solution containing ‘%a, as previously described (1). ‘“Ca uptake and transport data were initially calculated as “% of total %a in incubation medium taken up by the duodenum or transferred to the serosal solution per 100 mg tissue.” For simplicity, after statistical analyses, these data were then converted to “% of control” values.
CaBP STIMULATION
OF CALCIUM
TRANSPORT
739
FIG. 1. (A) Rotator apparatus (used in obtaining the data shown in Table 11 for the maintenance of embryonic chick doudena as intact, everted segments mounted on wire (20. gauge stainless-steel tubing) in organ culture. The duodena, simulated in the photo, are continuously rotated (1 rpm) at the surface of the culture medium (100 ml). The only parts of the apparatus in contact with the duodena and/or the medium are Plexiglas, Teflon, or stainless steel. The apparatus is sterilized with Et,0 (ethylene oxide) and is placed directly in the incubator during the culture period. The lid allows gaseous exchange but prevents contamination. (B) Roller tube system (used in obtaining the data shown in Table II) for the maintenance of embryonic chick duodena as intact, everted segments mounted on wire in organ culture. The duodena were mounted on 20-gauge stainless-steel tubing running the length of plastic roller tubes (16 x 150 mm) containing 9 ml of medium (described under Materials and Methods). The tubing is held in the center of the tube by a 24.gauge needle at the bottom and by running the wire through the center of the plastic top. The roller drum is set at a slight angle within the incubator and, as it rotates (0.2 rpm), only half of the duodenum is wetted at any given time. A small hole near the central wire in the plastic top allows gaseous exchange. Further experiments were carried out in the roller tube system shown in Fig. 1B. While essentially the same as the rotator apparatus in principle, this technique offers the distinct advantages of great simplicity and considerable increases in the numbers of treatments testable in a single experiment and in the number of replicates assayable per treatment. The medium employed with this system (9 ml per tube) was the same as described above but also contained 50 mM HEPES for greater buffering capacity. Other conditions were the same except that the culture period was reduced to 24 h since the pH drops rapidly after this time. After culture, CaBP and “Ca movement were assessed as described above. Vitamin D:,, albumin (bovine serum), poly-L-lysine, cr-casein (bovine milk), cu-amylase lBaci//us suhtilisl, and carbonic anhydrase (bovine erythrocytes) were all obtained from Sigma. Other substances were obtained as follows: fetal bovine serum iGibco), Fraction VI glycoprotein (Miles), ferritin iCalbiochem1; calsequestrin (18) was the generous
gift of Dr. D. H. MacLennan, and synthetic 1,25(OH)&, (19) was kindly provided by Dr. H. F. DeLuca. Bovine intestinal CaBP was isolated and purified as previously described (20). Chick intestinal CaBP was isolated as previously described (21) and checked for purity by analytical polyacrylamide disc gel electrophoresis immediately prior to use. Chick CaBP was “heat-treated” in a boiling-water bath for 2 h, after which its calcium binding and immunological activity disappeared. All proteins were dissolved in a Tris buffer (22) and filtered through a 0.22.pm Millipore filter prior to addition to the culture medium. The serum was exhaustively dialyzed against the same Tris buffer and filtered before use. Molecular weights (or estimates) of the various proteins obtained commercially were taken from several sources (2332.5). RESULTS
In initial experiments, a specially designed organ culture apparatus was used
740
CORRADINO.
FLJLLMER
which allowed the continuous rotation of everted embryonic duodena at the surface of the culture medium (Fig. 1A) assuring adequate gaseous exchange and nutrient uptake. After culture, CaBP and 4”Ca uptake and transport were measured. As shown in Table I, in the presence of vitamin D, in the culture medium for 48 h, CaBP was induced in the duodena, tissue uptake of radiocalcium was increased 2.8 times, and transmucosal transport was increased 9.3 times over control values. These responses (“%a movement), while not surprising, were considerably greater than those seen using earlier culture techniques (1, 4, 7). Of greatest interest, however, was the observation that, when highly purified chick intestinal CaBP (21) was included in the culture medium for 48 h, a highly significant rise in tissue uptake (1.7 x control) and a fivefold increase in transmucosal transport occurred (Table I). There was the suggestion of a correlation between the amount of CaBP associated with the tissue and tissue uptake and mucosal-to-serosal transport of ?a observed. Specific note should be made of several facts: First, experiments conducted with crude supernatants of duodenal mucosal homogenates containing CaBP or with less than highly purified CaBP, e.g., not carried through the entire purification procedure (21), were unsuccessful; second, in TABLE
I
STIMULATION OF CALCIUM TRANSPORT IN EMBRYONIC CHICK DUODENUM BY VITAMIN D:, OR EXOGENOUS CaBP USING A SPECIALLY DESIGNED ROTATOR DEVICE” CaBP ( wgi 100 mg of duodenurn)
‘“Ca movement
D:,
0 1.8
100 276 i 41*
934 If- 222*
(2.86
0.6
165 ir 24*
481 t 64*
Added to medium
Control Vitamin
Tis;;sueup-
(%’ of control) Mucosal-toserosal transport 100
(26 PM) CaBP iuM)
(I Values are mean -+ SE; values followed by an asterisk are significantly greater than the control value at the 1% level (Student’s t test). Vitamin D:, or CaBP was present in the culture medium for the entire 48-h period. See Fig. 1A for photograph of rotator device.
AND
WASSERMAN
earlier experiments using simpler culture techniques and reported briefly elsewhere,3 exogenous CaBP at 2.86 PM (which, incidentally, appears to be close to the minimal effective concentration) stimulated calcium transport even when in contact with the duodena for only the last 2 h of a 48-h culture period; and, third, the small amounts of CaBP found in the tissue (Table I), either by endogenous production induced by vitamin D:, or associated with the tissue after incubation with exogenous CaBP, cannot account for the amounts of calcium accumulated by the tissue or transported into the serosal fluid by simple irreversible binding of calcium (26). The maximum amount of calcium that could have been bound by CaBP was only about 0.2% of the actual amount transported. To check the specificity of the stimulation of calcium transport by exogenous CaBP, other experiments were performed using a roller tube technique (Fig. 1B) which, while essentially similar to the rotator device in effect (Fig. lA), allows greater replication and number of treatments. Using this technique, a number of proteins of varying binding affinities for several metals, was tested for their ability to stimulate calcium transport (Table II). Among these were two that bind calcium with high affinity, the bovine intestinal calcium-binding protein (20) and calsequestrin, from rabbit sarcoplasmic reticulum (18). Each sterol or protein was present in the medium for the entire 24-h culture period. None of the tested proteins had any significant effect on calcium transport, except purified chick CaBP which again stimulated both tissue uptake and transmucosal transport of ““Ca. Note also the effectiveness of 1,25-(OH),D,, at 6.5 x lO-1” M in inducing CaBP and stimulating calcium transport in this sytem. This enormously greater potency of 1,25(OH),D,, versus vitamin Dzi, especially for CaBP induction, in this system has been observed before (1, 4). Again, there appeared to be some relationship between CaBP concentration, whether endogenously produced in red Corradino, R. A., and Wasserman, Biophys. Sot. Abstr. 11, 276a.
R. H. (1971)
CaBP STIMULATION
OF CALCIUM TABLE
II
SPECIFICITY ok STIMULATION OF CALCIUM TRANSPORT IN EMBRYONIC CHICK TUBE CULTURE TECHNIQUE" Added to medium
741
TRANSPORT
Concentration (M)
DUODENUM
CaBP (pg/ 100 mg duodenurn)
USING
“Ca movement control) Tis;;;eup-
Control Vitamin D:; 1,25-(OH),D:, CaBP (chick) CaBP (chick; “heat-treated”) CaBP (bovine) Calsequestrin (rabbit muscle) Fetal bovine serum (dialyzed) Albumin (bovine serum) Fraction VI glycoprotein (bovine serum) Poly-L-lysine cu-Casein (bovine milk) Ferritin (horse spleen) cu.Amylase iB. suhtilisi Carbonic anhydrase (bovine erythrocytes)
2.6 x 10 > 6.5 x lo-“’ 2.86 x 10 I’ 2.86 x lo-” 2.86 x lo-” 2.86 x 10 ‘I 1.8% 2.86 x 10 Ii 2.86 x IO-” 2.86 x IO-” 2.86 x 10 +I 2.86 x lo-” 2.86 x 10 Ii 2.86 x lo-”
0 3.3 t 0.3 4.3 t 0.1 0.9 2 0.2 0 0 0 0 0 0 0 0 0 0 0
A RoLLER
100 372 i 6* 282 k 14* 192 i 22* 102 t 8 109 i 10 100 2 4 97 + 6 103 +- 4 114 2 9 98 k 9 108 -+ 11 101 t 3 112 2 7 115 * 5
(r/c of
Mucosalto-serosal transport 100 820 k 4a* 172 i 32* 225 i 23* 108 2 9 111 i 16 89 k 11 94 t 12 95 t- 6 85 + 13 86 t 7 115 5 12 97 -+ 14 110 ? 6 113 -c 7
” Values are means t SE; values followed by an asterisk are greater than control value at the 1% level (Student’s t test). The test sterol or protein was present in the culture medium for the entire 24-h culture period. See Fig. 1B for a photograph of the roller tube apparatus.
sponse to vitamin D:, or exogenously supplied in the culture medium, and “Ca movement. However, in the case of 1,25(OH&D,,, no such relationship was observed. While CaBP was present in the tissue at a concentration actually higher than that induced by a 40,000 times greater concentration of vitamin D:,, ““Ca movement was lower; indeed, transmucosal transport was only about one-fourth as great as that produced with vitamin D:,. This observation has an in uiuo counterpart in that, after a low dose of 1,25(OH),D,, to rachitic chicks, CaBP synthesis and duodenal calcium absorption rose rapidly and in parallel through 16 h after injection. Thereafter, CaBP continued to rise to a plateau which persisted through 72 h. However, calcium absorption reached a peak at 16 h and declined rapidly thereafter until it reached the untreated control level at 72 h (27). There is no clear explanation for this apparent discrepancy but two possibilities might be noted: First, CaBP is measured immunologically which may not accurately reflect its functional status under all conditions,
and, second, CaBP synthesized at a rapid rate, as after 1,25-(OH),D,, treatment, may not all reach its functional site within a given time span. DISCUSSION
There is already some evidence that several binding proteins may be involved in transport processes in bacteria (28-31). The major element of proof in ascribing a transport function to a binding protein is the so-called “reconstitution” experiment. As defined by Pardee (321, to achieve reconstitution of a transport system one must be able to “add the [binding] protein to cells incapable of transport and obtain transport.” In bacteria, one approach has been to osmotically shock the cells releasing the binding protein into the shock fluid. When transferred to fresh medium, the bacteria are incapable of transport of the bound substance. Alternatively, specific binding protein-free strains of bacteria, incapable of transport of the bound substrate, can be selected. In either case, addition of the binding protein to such cells results in increased substrate uptake,
742
CORRADINO.
FULLMER
which has been interpreted as “reconstitution” (28-31). In the case of intestinal CaBP, of course, such procedures are not possible. However, another possibility does exist. It is known that vitamin D-deficient rachitic chicks contain no intestinal CaBP and exhibit reduced calcium absorption (22). Following vitamin D:, administration to such animals, CaBP is biosynthesized and calcium absorption is increased (22). One could, therefore, attempt to restore calcium transport by introduction of isolated and purified CaBP into the lumen of ligated segments of rachitic chick intestine ilz situ. This in fact has been attempted; unsuccessfully, for a number of possible reasons, including too-brief contact, digestion of the protein, etc. Two factors were involved in the successful stimulation of calcium transport reported here: first, the observation that the embryonic chick duodenum, as in the rachitic chick, does not contain CaBP (33) but does respond to vitamin D:, and its metabolites (1, 4) and, unlike the duodenum from the severely rachitic chick, is normal in every respect; and, second, the development of novel organ culture techniques which permits the study of vitamin D (or CaBPI-mediated calcium transport for relatively lengthy periods (up to 48 h) under strictly controlled in vitro conditions, in a serum-free medium, completely isolated from systemic influences (1). Despite the apparently specific CaBP stimulation of calcium transport reported here, there are at least two reasons why caution should be exercised in interpreting the results as unequivocally demonstrating “reconstitution” or concluding finally that CaBP is a transport protein. First, nothing is as yet known of the kinetics of CaBP-“reconstituted” calcium transport nor of the ,mechanism whereby CaBP, whether endogenously produced or exogenously supplied, stimulates calcium transport. What is known from these experiments is that CaBP can stimulate transmucosal calcium flux over the control value. Considering the 30-min time period during which calcium movement is allowed, it seems likely that what was meas-
AND
WASSERMAN
ured was net mucosal-to-serosal flux since back flux, i.e., from serosal-to-mucosal compartments, was probably occurring. Highly desirable would be additional experiments in which the kinetics of CaBPstimulated unidirectional fluxes are independently measured. Second, the localization of exogenously supplied CaBP during stimulation of calcium transport is likewise unknown. It is known that CaBP in the vitamin D:,treated chick intestinal mucosa is localized in goblet cells and in the brush bordermicrovillar region of absorptive cells (34). The present findings are consistent with the association of exogenously supplied CaBP with the latter site, a known locus of vitamin D-mediated calcium transport (35) but are not conclusive. With these two reservations in mind, it is still justified, on the basis of the results reported here, to conclude that the vitamin D-induced CaBP is involved in intestinal calcium transport and may, in fact, be a calcium transport protein. ACKNOWLEDGMENTS Thanks are due Francis Davis, Frances Heistand, Sue Travis, Karen Bucher Ni, and H. Donald Hinman for technical assistance. REFERENCES 1. CORRADINO, R. A. (19733 J. Cell Biol. 58, 64-78. 2. DELUCA, H. F. (1969) Fed. Proc. 28, 1678-1689. 3. LAWSON, D. E. M., FRASER, D. R., KODICEK, E., MORRIS, H. R., AND WILLIAMS, D. H. (1971) Nature (London) 230, 228-230. 4. CORRADINO, R. A. (1973) Science 179,402-405. 5. CORRADINO, R. A., AND WASSERMAN, R. H. (1968) Arch. Biochem. Biophys. 125, 957-960. 6. MACGREGOR, R. R., HAMILTON, J. W., AND COHN, D. V. (1970) Biochim. Biophys. Acta 222, 482-490. 7. CORRADINO, R. A., AND WASSERMAN, R. H. (1971) Science 172, 731-733. 8. CORRADINO, R. A. (1973) Nature (London/ 243, 41-43. 9. TSAI, H. C., MIDGETT, R. J., AND NORMAN, A. W. (1973) Arch. Biochem. Biophys. 157, 339-347. 10. EMTACE, J. S., LAWSON, D. E. M., AND KODICEK, E. (1974) Biochem. J. 140, 239-247. 11. WASSERMAN, R. H., AND CORRADINO, R. A. (1973) Vitam. Harm. 31, 43-103. 12. WASSERMAN, R. H., CORRADINO, R. A., FULI~ MER, C. S., AND TAYLOR, A. N. (1974) Vitam.
CaBP Horm.
STIMULATION
OF CALCIUM
32, 299-324.
13. EMTAGE, J. S., LAWSON, D. E. M., AND KODICEK, E. (1974) Biochem. J. 144, 339-346. 14. WASSERMAN, R. H., CORRADINO, R. A., AND TAYLOR,
A. N. (1969) J. Gen. Physiol.
54, 114s
26.
A., AND HOWELL, K. E. (1972) Gastroenterology 62, 647-667. VAN CAMPEN, D. (1974) Fed. Proc. 33, 100-105. KOWARSKI, S., BLAIR-STANEK, C. S., AND SCHACTER, D. (1974). Amer. J. Physiol. 226, 401-407. MACLENNAN, D. H., AND WONG, P. T. S. (1971) Proc. Nat. Acad. Sci. USA68, 1231-1235. SEMMLER, E. J., HOLICK, M. F., SCHNOES, H. K., AND DELUCA, H. F. (1972) Tetrahedron Lett. 40, 4147-4150. FULLMER, C. S., AND WASSERMAN, R. H. (1975) Biochim. Bioph.ys. Acta 393, 134-142. WASSERMAN, R. H., CORRADINO, R. A., AND TAYLOR, A. N. (1968) J. Biol. Chem. 243, 397% 3986. WASSERMAN, R. H., AND TAYLOR, A. N. (1966) Science 152, 791-793. GURD, F. R. N., AND WILCOX, P. E. (1956) Aduun. Prot. Chem. 11, 311-427. TRISTRAM, G. R., AND SMITH, R. H. (1963) Ad-
15. EICHOLZ,
27.
16. 17.
28.
18. 19.
20. 21.
22. 23. 24.
29.
30. 31. 32. 33. 34. 35.
743
mm. Prot. Chem. 18, 227-316. B. L., AND WACKER, E. C. 11970) in The Proteins (Neurath, H., ed.), 2nd ed., Vol. 5, p. 25, Academic Press, New York. BREDDERMAN, P. J., AND WASSERMAN, R. H. (1974) Biochemistry 13, 1687-1694. WASSERMAN, R. H., TAYLOR, A. N., AND FULL MER, C. S. (1974) Biochem. Sot. Spec. Puhl. 3, 55-74. MEDVECZKY, N., AND ROSENBERG, H. (1970) Biochim. Bioph,ys. Acta 211, 158-168. WEINER, J. H., FURLONG, C. E., AND HEPPEL, L. A. (1971) Arch. Biochem. Biophys. 142, 715717. AMES, G. F., AND LEVER, J. E. (1972) J. Biol. Chem. 247, 4309-4316. PARNES, J. R., AND Boos, W. (1973) J. Biol. Chem. 248, 4436-4445. PARDEE, A. B. (1968) Science 162, 632-637. CORRADINO, R. A., TAYLOR, A. N., AND WASSERMAN, R. H. (1969) Fed. Proc. 28, 760. TAYLOR, A. N., AND WASSERMAN, R. H. (1970). J. Histochem. C.ytochem. 18, 107-115. WASSERMAN, R. H., AND TAYLOR, A. N. (1969) in Mineral Metabolism: An Advanced Treatise (Comar, C. L., and Bronner, F., eds.), Vol. 3, p. 321, Academic Press, New York.
25. VALLEE,
134s.
TRANSPORT
OF
BIOCHEMISTRY
AND
BIOPHYSICS
174, 738-743 (19%)
Embryonic Chick intestine in Organ Culture: Stimulation of Calcium Transport by Exogenous Vitamin D-Induced Calcium-Binding Protein’ R. A. CORRADINO, Department
of Physical
Biology,
C. S. FULLMER,
AND
R. H. WASSERMAN
New York State College of Veterinary Ithaca, New York 14853 Received
December
Medicine,
Cornell
Uniuersity,
12, 1975
Vitamin D:, or a potent metabolite, 1,25dihydroxycholecalciferol, induces calciumbinding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.
Vitamin D:, induces synthesis of a calcium-binding protein (CaBP)’ and enhances tissue uptake and mucosal-to-serosa1 transport of calcium in embryonic chick duodenum maintained in organ culture (1). In the same system, two metabolites of vitamin D:,, namely, 25hydroxyvitamin D, (2) and 1,25dihydroxyvitamin D:, (1,25-(OH),D,,) (31, also induce CaBP and stimulate tissue uptake of a”Ca with the latter compound being much more potent in its CaBP-inducing action (1, 4). Like vitamin D:, itself (5-7), 1,25-(OH),D,, (8-10) appears to act via stimulation of RNA and subsequent CaBP synthesis. Considerable correlative evidence supports a role for CaBP in the vitamin D-mediated transport of calcium by the intestine (11-13). Using two variations of a newly developed organ culture technique (Corradino, manuscripts in preparation’), we now report direct evidence that CaBP is probably
involved in the vitamin D-mediated, intestinal transport of calcium. Several intestinal “binding proteins” have been discovered (14-171, but this represents the first demonstration in an animal system that such a protein can stimulate transport of the bound substance. MATERIALS
’ Supported by NIH research grants, No. AM15355 and AM-04652. R.A.C. is the recipient of NIH Research Career Development Award No. AM00115. 2 Abbreviations used: CaBP, calcium-binding protein; 1,25-(OH)&,, 1,25-dihydroxyvitamin D:,; HEPES, 4.(2.hydroxyethyl)-l-piperazineethanesulfonic acid. ‘I Essential details are included in the present paper. 738 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
Duodena were excised from 20-day-old chick embryos and the pancreas removed from each. The duodena were then everted onto the 20-gauge wires of one or the other of the two types of culture apparatus shown in Figs. 1A and B. In initial experiments, the apparatus of Fig. 1A was employed. This apparatus allowed the continuous rotation (1 rpm) of seven everted duodena per treatment at the surface of the culture medium. This assured adequate gaseous exchange (5% CO,:5Oc11 O,:balance, air) and nutrient uptake. The duodena were maintained for 48 h at 37.5”C in 100 ml of serum-free McCoy’s 5A (modified) medium (Gibco) containing 1.25 mM calcium, 0.625 mM phosphate, and 100 units of nystatin/ml (1, 4). After culture, two of the duodena were homogenized and CaBP determined by radial immunoassay (1); ‘“Ca uptake and transmucosal transport were assessed, after a 30-min incubation in a lowsodium buffer solution containing ‘%a, as previously described (1). ‘“Ca uptake and transport data were initially calculated as “% of total %a in incubation medium taken up by the duodenum or transferred to the serosal solution per 100 mg tissue.” For simplicity, after statistical analyses, these data were then converted to “% of control” values.
CaBP STIMULATION
OF CALCIUM
TRANSPORT
739
FIG. 1. (A) Rotator apparatus (used in obtaining the data shown in Table 11 for the maintenance of embryonic chick doudena as intact, everted segments mounted on wire (20. gauge stainless-steel tubing) in organ culture. The duodena, simulated in the photo, are continuously rotated (1 rpm) at the surface of the culture medium (100 ml). The only parts of the apparatus in contact with the duodena and/or the medium are Plexiglas, Teflon, or stainless steel. The apparatus is sterilized with Et,0 (ethylene oxide) and is placed directly in the incubator during the culture period. The lid allows gaseous exchange but prevents contamination. (B) Roller tube system (used in obtaining the data shown in Table II) for the maintenance of embryonic chick duodena as intact, everted segments mounted on wire in organ culture. The duodena were mounted on 20-gauge stainless-steel tubing running the length of plastic roller tubes (16 x 150 mm) containing 9 ml of medium (described under Materials and Methods). The tubing is held in the center of the tube by a 24.gauge needle at the bottom and by running the wire through the center of the plastic top. The roller drum is set at a slight angle within the incubator and, as it rotates (0.2 rpm), only half of the duodenum is wetted at any given time. A small hole near the central wire in the plastic top allows gaseous exchange. Further experiments were carried out in the roller tube system shown in Fig. 1B. While essentially the same as the rotator apparatus in principle, this technique offers the distinct advantages of great simplicity and considerable increases in the numbers of treatments testable in a single experiment and in the number of replicates assayable per treatment. The medium employed with this system (9 ml per tube) was the same as described above but also contained 50 mM HEPES for greater buffering capacity. Other conditions were the same except that the culture period was reduced to 24 h since the pH drops rapidly after this time. After culture, CaBP and “Ca movement were assessed as described above. Vitamin D:,, albumin (bovine serum), poly-L-lysine, cr-casein (bovine milk), cu-amylase lBaci//us suhtilisl, and carbonic anhydrase (bovine erythrocytes) were all obtained from Sigma. Other substances were obtained as follows: fetal bovine serum iGibco), Fraction VI glycoprotein (Miles), ferritin iCalbiochem1; calsequestrin (18) was the generous
gift of Dr. D. H. MacLennan, and synthetic 1,25(OH)&, (19) was kindly provided by Dr. H. F. DeLuca. Bovine intestinal CaBP was isolated and purified as previously described (20). Chick intestinal CaBP was isolated as previously described (21) and checked for purity by analytical polyacrylamide disc gel electrophoresis immediately prior to use. Chick CaBP was “heat-treated” in a boiling-water bath for 2 h, after which its calcium binding and immunological activity disappeared. All proteins were dissolved in a Tris buffer (22) and filtered through a 0.22.pm Millipore filter prior to addition to the culture medium. The serum was exhaustively dialyzed against the same Tris buffer and filtered before use. Molecular weights (or estimates) of the various proteins obtained commercially were taken from several sources (2332.5). RESULTS
In initial experiments, a specially designed organ culture apparatus was used
740
CORRADINO.
FLJLLMER
which allowed the continuous rotation of everted embryonic duodena at the surface of the culture medium (Fig. 1A) assuring adequate gaseous exchange and nutrient uptake. After culture, CaBP and 4”Ca uptake and transport were measured. As shown in Table I, in the presence of vitamin D, in the culture medium for 48 h, CaBP was induced in the duodena, tissue uptake of radiocalcium was increased 2.8 times, and transmucosal transport was increased 9.3 times over control values. These responses (“%a movement), while not surprising, were considerably greater than those seen using earlier culture techniques (1, 4, 7). Of greatest interest, however, was the observation that, when highly purified chick intestinal CaBP (21) was included in the culture medium for 48 h, a highly significant rise in tissue uptake (1.7 x control) and a fivefold increase in transmucosal transport occurred (Table I). There was the suggestion of a correlation between the amount of CaBP associated with the tissue and tissue uptake and mucosal-to-serosal transport of ?a observed. Specific note should be made of several facts: First, experiments conducted with crude supernatants of duodenal mucosal homogenates containing CaBP or with less than highly purified CaBP, e.g., not carried through the entire purification procedure (21), were unsuccessful; second, in TABLE
I
STIMULATION OF CALCIUM TRANSPORT IN EMBRYONIC CHICK DUODENUM BY VITAMIN D:, OR EXOGENOUS CaBP USING A SPECIALLY DESIGNED ROTATOR DEVICE” CaBP ( wgi 100 mg of duodenurn)
‘“Ca movement
D:,
0 1.8
100 276 i 41*
934 If- 222*
(2.86
0.6
165 ir 24*
481 t 64*
Added to medium
Control Vitamin
Tis;;sueup-
(%’ of control) Mucosal-toserosal transport 100
(26 PM) CaBP iuM)
(I Values are mean -+ SE; values followed by an asterisk are significantly greater than the control value at the 1% level (Student’s t test). Vitamin D:, or CaBP was present in the culture medium for the entire 48-h period. See Fig. 1A for photograph of rotator device.
AND
WASSERMAN
earlier experiments using simpler culture techniques and reported briefly elsewhere,3 exogenous CaBP at 2.86 PM (which, incidentally, appears to be close to the minimal effective concentration) stimulated calcium transport even when in contact with the duodena for only the last 2 h of a 48-h culture period; and, third, the small amounts of CaBP found in the tissue (Table I), either by endogenous production induced by vitamin D:, or associated with the tissue after incubation with exogenous CaBP, cannot account for the amounts of calcium accumulated by the tissue or transported into the serosal fluid by simple irreversible binding of calcium (26). The maximum amount of calcium that could have been bound by CaBP was only about 0.2% of the actual amount transported. To check the specificity of the stimulation of calcium transport by exogenous CaBP, other experiments were performed using a roller tube technique (Fig. 1B) which, while essentially similar to the rotator device in effect (Fig. lA), allows greater replication and number of treatments. Using this technique, a number of proteins of varying binding affinities for several metals, was tested for their ability to stimulate calcium transport (Table II). Among these were two that bind calcium with high affinity, the bovine intestinal calcium-binding protein (20) and calsequestrin, from rabbit sarcoplasmic reticulum (18). Each sterol or protein was present in the medium for the entire 24-h culture period. None of the tested proteins had any significant effect on calcium transport, except purified chick CaBP which again stimulated both tissue uptake and transmucosal transport of ““Ca. Note also the effectiveness of 1,25-(OH),D,, at 6.5 x lO-1” M in inducing CaBP and stimulating calcium transport in this sytem. This enormously greater potency of 1,25(OH),D,, versus vitamin Dzi, especially for CaBP induction, in this system has been observed before (1, 4). Again, there appeared to be some relationship between CaBP concentration, whether endogenously produced in red Corradino, R. A., and Wasserman, Biophys. Sot. Abstr. 11, 276a.
R. H. (1971)
CaBP STIMULATION
OF CALCIUM TABLE
II
SPECIFICITY ok STIMULATION OF CALCIUM TRANSPORT IN EMBRYONIC CHICK TUBE CULTURE TECHNIQUE" Added to medium
741
TRANSPORT
Concentration (M)
DUODENUM
CaBP (pg/ 100 mg duodenurn)
USING
“Ca movement control) Tis;;;eup-
Control Vitamin D:; 1,25-(OH),D:, CaBP (chick) CaBP (chick; “heat-treated”) CaBP (bovine) Calsequestrin (rabbit muscle) Fetal bovine serum (dialyzed) Albumin (bovine serum) Fraction VI glycoprotein (bovine serum) Poly-L-lysine cu-Casein (bovine milk) Ferritin (horse spleen) cu.Amylase iB. suhtilisi Carbonic anhydrase (bovine erythrocytes)
2.6 x 10 > 6.5 x lo-“’ 2.86 x 10 I’ 2.86 x lo-” 2.86 x lo-” 2.86 x 10 ‘I 1.8% 2.86 x 10 Ii 2.86 x IO-” 2.86 x IO-” 2.86 x 10 +I 2.86 x lo-” 2.86 x 10 Ii 2.86 x lo-”
0 3.3 t 0.3 4.3 t 0.1 0.9 2 0.2 0 0 0 0 0 0 0 0 0 0 0
A RoLLER
100 372 i 6* 282 k 14* 192 i 22* 102 t 8 109 i 10 100 2 4 97 + 6 103 +- 4 114 2 9 98 k 9 108 -+ 11 101 t 3 112 2 7 115 * 5
(r/c of
Mucosalto-serosal transport 100 820 k 4a* 172 i 32* 225 i 23* 108 2 9 111 i 16 89 k 11 94 t 12 95 t- 6 85 + 13 86 t 7 115 5 12 97 -+ 14 110 ? 6 113 -c 7
” Values are means t SE; values followed by an asterisk are greater than control value at the 1% level (Student’s t test). The test sterol or protein was present in the culture medium for the entire 24-h culture period. See Fig. 1B for a photograph of the roller tube apparatus.
sponse to vitamin D:, or exogenously supplied in the culture medium, and “Ca movement. However, in the case of 1,25(OH&D,,, no such relationship was observed. While CaBP was present in the tissue at a concentration actually higher than that induced by a 40,000 times greater concentration of vitamin D:,, ““Ca movement was lower; indeed, transmucosal transport was only about one-fourth as great as that produced with vitamin D:,. This observation has an in uiuo counterpart in that, after a low dose of 1,25(OH),D,, to rachitic chicks, CaBP synthesis and duodenal calcium absorption rose rapidly and in parallel through 16 h after injection. Thereafter, CaBP continued to rise to a plateau which persisted through 72 h. However, calcium absorption reached a peak at 16 h and declined rapidly thereafter until it reached the untreated control level at 72 h (27). There is no clear explanation for this apparent discrepancy but two possibilities might be noted: First, CaBP is measured immunologically which may not accurately reflect its functional status under all conditions,
and, second, CaBP synthesized at a rapid rate, as after 1,25-(OH),D,, treatment, may not all reach its functional site within a given time span. DISCUSSION
There is already some evidence that several binding proteins may be involved in transport processes in bacteria (28-31). The major element of proof in ascribing a transport function to a binding protein is the so-called “reconstitution” experiment. As defined by Pardee (321, to achieve reconstitution of a transport system one must be able to “add the [binding] protein to cells incapable of transport and obtain transport.” In bacteria, one approach has been to osmotically shock the cells releasing the binding protein into the shock fluid. When transferred to fresh medium, the bacteria are incapable of transport of the bound substance. Alternatively, specific binding protein-free strains of bacteria, incapable of transport of the bound substrate, can be selected. In either case, addition of the binding protein to such cells results in increased substrate uptake,
742
CORRADINO.
FULLMER
which has been interpreted as “reconstitution” (28-31). In the case of intestinal CaBP, of course, such procedures are not possible. However, another possibility does exist. It is known that vitamin D-deficient rachitic chicks contain no intestinal CaBP and exhibit reduced calcium absorption (22). Following vitamin D:, administration to such animals, CaBP is biosynthesized and calcium absorption is increased (22). One could, therefore, attempt to restore calcium transport by introduction of isolated and purified CaBP into the lumen of ligated segments of rachitic chick intestine ilz situ. This in fact has been attempted; unsuccessfully, for a number of possible reasons, including too-brief contact, digestion of the protein, etc. Two factors were involved in the successful stimulation of calcium transport reported here: first, the observation that the embryonic chick duodenum, as in the rachitic chick, does not contain CaBP (33) but does respond to vitamin D:, and its metabolites (1, 4) and, unlike the duodenum from the severely rachitic chick, is normal in every respect; and, second, the development of novel organ culture techniques which permits the study of vitamin D (or CaBPI-mediated calcium transport for relatively lengthy periods (up to 48 h) under strictly controlled in vitro conditions, in a serum-free medium, completely isolated from systemic influences (1). Despite the apparently specific CaBP stimulation of calcium transport reported here, there are at least two reasons why caution should be exercised in interpreting the results as unequivocally demonstrating “reconstitution” or concluding finally that CaBP is a transport protein. First, nothing is as yet known of the kinetics of CaBP-“reconstituted” calcium transport nor of the ,mechanism whereby CaBP, whether endogenously produced or exogenously supplied, stimulates calcium transport. What is known from these experiments is that CaBP can stimulate transmucosal calcium flux over the control value. Considering the 30-min time period during which calcium movement is allowed, it seems likely that what was meas-
AND
WASSERMAN
ured was net mucosal-to-serosal flux since back flux, i.e., from serosal-to-mucosal compartments, was probably occurring. Highly desirable would be additional experiments in which the kinetics of CaBPstimulated unidirectional fluxes are independently measured. Second, the localization of exogenously supplied CaBP during stimulation of calcium transport is likewise unknown. It is known that CaBP in the vitamin D:,treated chick intestinal mucosa is localized in goblet cells and in the brush bordermicrovillar region of absorptive cells (34). The present findings are consistent with the association of exogenously supplied CaBP with the latter site, a known locus of vitamin D-mediated calcium transport (35) but are not conclusive. With these two reservations in mind, it is still justified, on the basis of the results reported here, to conclude that the vitamin D-induced CaBP is involved in intestinal calcium transport and may, in fact, be a calcium transport protein. ACKNOWLEDGMENTS Thanks are due Francis Davis, Frances Heistand, Sue Travis, Karen Bucher Ni, and H. Donald Hinman for technical assistance. REFERENCES 1. CORRADINO, R. A. (19733 J. Cell Biol. 58, 64-78. 2. DELUCA, H. F. (1969) Fed. Proc. 28, 1678-1689. 3. LAWSON, D. E. M., FRASER, D. R., KODICEK, E., MORRIS, H. R., AND WILLIAMS, D. H. (1971) Nature (London) 230, 228-230. 4. CORRADINO, R. A. (1973) Science 179,402-405. 5. CORRADINO, R. A., AND WASSERMAN, R. H. (1968) Arch. Biochem. Biophys. 125, 957-960. 6. MACGREGOR, R. R., HAMILTON, J. W., AND COHN, D. V. (1970) Biochim. Biophys. Acta 222, 482-490. 7. CORRADINO, R. A., AND WASSERMAN, R. H. (1971) Science 172, 731-733. 8. CORRADINO, R. A. (1973) Nature (London/ 243, 41-43. 9. TSAI, H. C., MIDGETT, R. J., AND NORMAN, A. W. (1973) Arch. Biochem. Biophys. 157, 339-347. 10. EMTACE, J. S., LAWSON, D. E. M., AND KODICEK, E. (1974) Biochem. J. 140, 239-247. 11. WASSERMAN, R. H., AND CORRADINO, R. A. (1973) Vitam. Harm. 31, 43-103. 12. WASSERMAN, R. H., CORRADINO, R. A., FULI~ MER, C. S., AND TAYLOR, A. N. (1974) Vitam.
CaBP Horm.
STIMULATION
OF CALCIUM
32, 299-324.
13. EMTAGE, J. S., LAWSON, D. E. M., AND KODICEK, E. (1974) Biochem. J. 144, 339-346. 14. WASSERMAN, R. H., CORRADINO, R. A., AND TAYLOR,
A. N. (1969) J. Gen. Physiol.
54, 114s
26.
A., AND HOWELL, K. E. (1972) Gastroenterology 62, 647-667. VAN CAMPEN, D. (1974) Fed. Proc. 33, 100-105. KOWARSKI, S., BLAIR-STANEK, C. S., AND SCHACTER, D. (1974). Amer. J. Physiol. 226, 401-407. MACLENNAN, D. H., AND WONG, P. T. S. (1971) Proc. Nat. Acad. Sci. USA68, 1231-1235. SEMMLER, E. J., HOLICK, M. F., SCHNOES, H. K., AND DELUCA, H. F. (1972) Tetrahedron Lett. 40, 4147-4150. FULLMER, C. S., AND WASSERMAN, R. H. (1975) Biochim. Bioph.ys. Acta 393, 134-142. WASSERMAN, R. H., CORRADINO, R. A., AND TAYLOR, A. N. (1968) J. Biol. Chem. 243, 397% 3986. WASSERMAN, R. H., AND TAYLOR, A. N. (1966) Science 152, 791-793. GURD, F. R. N., AND WILCOX, P. E. (1956) Aduun. Prot. Chem. 11, 311-427. TRISTRAM, G. R., AND SMITH, R. H. (1963) Ad-
15. EICHOLZ,
27.
16. 17.
28.
18. 19.
20. 21.
22. 23. 24.
29.
30. 31. 32. 33. 34. 35.
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mm. Prot. Chem. 18, 227-316. B. L., AND WACKER, E. C. 11970) in The Proteins (Neurath, H., ed.), 2nd ed., Vol. 5, p. 25, Academic Press, New York. BREDDERMAN, P. J., AND WASSERMAN, R. H. (1974) Biochemistry 13, 1687-1694. WASSERMAN, R. H., TAYLOR, A. N., AND FULL MER, C. S. (1974) Biochem. Sot. Spec. Puhl. 3, 55-74. MEDVECZKY, N., AND ROSENBERG, H. (1970) Biochim. Bioph,ys. Acta 211, 158-168. WEINER, J. H., FURLONG, C. E., AND HEPPEL, L. A. (1971) Arch. Biochem. Biophys. 142, 715717. AMES, G. F., AND LEVER, J. E. (1972) J. Biol. Chem. 247, 4309-4316. PARNES, J. R., AND Boos, W. (1973) J. Biol. Chem. 248, 4436-4445. PARDEE, A. B. (1968) Science 162, 632-637. CORRADINO, R. A., TAYLOR, A. N., AND WASSERMAN, R. H. (1969) Fed. Proc. 28, 760. TAYLOR, A. N., AND WASSERMAN, R. H. (1970). J. Histochem. C.ytochem. 18, 107-115. WASSERMAN, R. H., AND TAYLOR, A. N. (1969) in Mineral Metabolism: An Advanced Treatise (Comar, C. L., and Bronner, F., eds.), Vol. 3, p. 321, Academic Press, New York.
25. VALLEE,
134s.
TRANSPORT
