Digestion 14: 77-84 (1976)
Cellular Localization of Intestinal Calcium-Binding Protein in Pig Duodenum1 B.M. Arnold, K. Kovacsand T.M. Murray Departments of Medicine and Pathology, St. Michael's Hospital and University of Toronto, Toronto, Ont.
Key Words. Binding proteins • Calcium • Immunologic Techniques • Intestine, small • Swine Abstract. The 7 -globulin fraction of a rabbit antiserum against porcine intestinal cal cium-binding protein (CaBP) was used in an immunoperoxidase method to study CaBP localization in porcine duodenal tissue. Specific immunostaining, indicative of the presence of CaBP, was noted within the cytoplasm of the duodenal epithelial cells. No CaBP was detected in goblet cells or in the subepithelial layers. When the specific antibody was replaced by either nonimmune rabbit 7 -globulin or when the specific antibody was pread sorbed with excess CaBP, no positive immunostaining was seen. Our studies lend support to the hypothesis that CaBP may function in intracellular calcium transport.
Introduction Wasserman and Taylor (1) observed that a calcium-binding protein (CaBP) was present in the duodenal mucosa of vitamin D-treated rachitic chicks. Subse quently, considerable evidence has been obtained which correlates duodenal CaBP concentration with intestinal calcium transport in the chick (2). Intestinal CaBP proteins have also been described in several other species (3, 4). in the chick, using fluorescent antibody techniques, CaBP was localized to the PASpositive goblet cells and microvillar surface of the intestinal cell giving rise to the hypothesis that CaBP in the brush border may be actively involved in transloca tion of calcium from the intestinal lumen to the interior of the absorptive cells (5). Others have suggested that CaBP may be an intracellular calcium transport 1 Supported in part by grants from the Medical Research Council of Canada (MA4515) and the Atkinson Charitable foundation.
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Received: July 12, 1975:accepted: November 12, 1975.
Arnold /Kovacs /Murray
78
protein, either as an integral transmucosal transport mechanism or to transport calcium to the serosal surface of the cell following mitochondrial uptake and release of calcium (6, 7). However, there has been no proof that CaBP is present in the cytoplasm. Information concerning the cytologic localization of CaBP would be valuable in elucidating the mechanism of vitamin D-induced intestinal calcium translocation. We have recently begun to study CaBP in the pig (8) and wish to report here the immunohistochemical localization of intestinal CaBP in porcine duodenum. Materials and Methods Preparation o f Antiserum Antisera were prepared in rabbits by immunization with pure porcine CaBP as previ ously reported (8 ). 7 -Globulin fractions from antiserum R3A and from normal rabbit serum were prepared by the method of Levy and Sober (9). The lyophilized 7 -globulin fractions were dissolved in normal saline and stored at 4 °C. We have recently reported in detail on the specificity of another antiserum (R2A) used in our newly developed radioimmunoassay for porcine intestinal CaBP ( 8). The specificity of antiserum R3A was tested using similar procedures; no immunoreactive components, other than CaBP, were found in duodenal extracts by radioimmunoassay of the eluted fractions from gel filtration or ion exchange chromatography in the presence of calcium or EDTA (8 ).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Immunohistological Techniques Small pieces of freshly obtained pig duodenum were fixed in 10 % neutral buffered formalin for 24 h, postfixed in 70 % ethanol for an additional 24 h, dehydrated in graded ethanol and embedded in paraffin. In addition, frozen sections of duodenum were prepared. Frozen sections were cut on a cryostat and either immunostained without any prior fixa tion, fixed in 10 % neutral buffered formalin for 1 h, or fixed in 1 % glutaraldehyde for 3 h and then immunostained as described below. Sections of 3 -5 Mm thickness were cut. The unlabelled antibody peroxidase-antiperoxidase complex method was used to localize CaBP in the duodenum (10). Both frozen and paraffin-embedded sections were washed in phos phate-buffered saline, pH 7.4, then exposed for 3 h to the specific antibody which was diluted 1:100 in phosphate-buffered saline, pH 7.4. Following thorough washing in phos phate-buffered saline, the sections were next exposed to goat-antirabbit 7 -globulin antise rum (also diluted 1:10 in phosphate-buffered saline, pH 7.4) for 5 min, thoroughly re washed in phosphate-buffered saline, then exposed to a 1:60 dilution of the peroxidaseantiperoxidase complex for 5 min. The sections were again thoroughly washed in phos phate-buffered saline. After washing the sections were exposed for 10 min to DAB solution (20 mg of 3,3'-diaminobenzidine in 10 ml Tris-buffered saline, 0.05 M, pH 7.6, to which were added three drops of 3% H ,0 2). Finally, the sections were washed in phosphatebuffered saline solution, dehydrated in graded ethanol, cleared in toluol and mounted. For control purposes the specific antibody was replaced by phosphate-buffered saline, pH 7.4, by normal nonimmune rabbit 7 -globulin, or else the specific antibody was preab sorbed with excess CaBP. Sections of kidney, liver, thyroid and pancreas were also made, using similar immunohistologic techniques. For orientation of few paraffin-embedded and frozen sections were also stained with hematoxylin-phloxine-saffron, hematoxylin and eosin, and with the PAS method. Finally, on a few frozen and paraffin-embedded immuno stained sections the PAS method was also applied.
Intestinal CaBP
79
Results The duodenal tissue possessed normal histological features when sections were stained with hematoxylin-eosin or with hematoxylin-phloxine-saffron (fig. 1). Immunostaining of the paraffin-embedded and frozen sections of the duodenum showed brown granular deposits, indicating the presence of CaBP. The brown deposits were abundantly present in the cytoplasm of the majority of the mucosal epithelial cells (fig. 2, 3). There was no preferential localization of the granular deposits in different areas of the cytoplasm, although in some cells the brown granules seemed to accumulate around the nucleus. In some areas cytoplasmic immunostaining was less intense than in others. In these areas immunostaining was more marked in the brush border, although in most of the sections it was not possible to distinguish immunostaining in the brush border from the granular deposits in the cytoplasm. There was no positivity in the goblet cells (fig. 2, 3), either on the paraffinembedded or frozen sections. Immunoperoxidase-stained sections subsequently
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Fig. I. A formalin-fixed, paraffin-embedded section of the mucosa of pig duodenum stained with hematoxylin-phloxine-saffron, demonstrating normal histologic features. Ap proximately X 250.
Arnold/Kovacs/Murray
80
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Fig. 2. A similar section stained by the immunoperoxidase method. Positive immunostaining is evident in the surface epithelium (arrows). No staining is present in the goblet cells. Approximately x 400. Fig. 3. Positive immunostaining in cross-section of the mucosal glands of the pig duode num (arrows). Approximately X 400.
Intestinal CaBP
81
Fig. 4. No deposits are apparent when the specific antibody was preabsorbed by anti gen excess. Approximately X 400.
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
stained with PAS demonstrated PAS positivity of the goblet cells without any immunostaining. Granular deposits were not apparent in other components of the intestinal wall such as smooth muscle, or connective tissue. In general, the quality of the frozen sections was somewhat inferior to that of the paraffinembedded sections, but the distribution of immunostaining was similar. No positive staining was noted in the mucosal epithelial cells when the specific antibody was replaced by either phosphate-buffered saline or normal nonimmune rabbit serum, or when the specific antibody was previously ab sorbed by excess antigen (fig. 4). When normal nonimmune rabbit 7 -globulin was used instead of the specific antibody, only nonspecific background staining was seen. No differences in the amount of immunostaining were noted when crypt cells were compared with cells at the tips of the villi. However, the methods we have used do not have sufficient resolution to make such comparisons effec tively. No positive immunostaining was found in the kidneys, liver, thyroid or in the pancreas, organs previously shown to contain immunoreactive CaBP in much lower amounts than duodenum (11).
Arnold/Kovacs/Murray
82
Discussion
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
The results of our studies, using a specific antibody to pig intestinal CaBP, clearly localized CaBP to the cytoplasm of the absorptive cells in the duodenum. CaBP was not demonstrable in the goblet cells. These findings are at variance with those of Taylor and Wasserman (5) who found, using immunofluorescent techniques, that in chick duodenum CaBP was localized to the goblet cells and to the microvillar surface of the epithelial cells. The difference between our findings may indicate a difference in the cellular biochemistry of the intestinal absorptive process between the chick and the pig. On the other hand, some recent evidence suggests that fluorescence of goblet cells in intestinal tissue may be a nonspecific finding (12). There have been some recent studies of cellular localization of CaBP in human intestinal cells (12). However, the antiserum used in these studies was prepared against renal CaBP (molecular weight greater than 21,500) (13). There is reason to believe that these studies are localizing renal CaBP in intestinal tissue rather than intestinal CaBP which in the human (and in the pig) has a molecular weight of about 12,000-13,000 by gel filtration (4). Recent studies in the pig using our radioimmunoassay show that the major CaBPs in kidney are chemi cally and immunochemically different from intestinal CaBP (14). It is important, therefore, that the antiserum used in our studies was raised against highly puri fied pig intestinal CaBP, rather than antigen from the kidney (8). CaBP has been shown to be present at the microvillar surface of the absorp tive cell in clucks (5). At this site, the transfer of calcium from the lumen to mucosa is significantly stimulated by the presence of vitamin D (15). This has led to the hypothesis that CaBP at the brush border is involved in the transfer of calcium from the lumen to the cytoplasm (3). However, different mechanisms may operate in the transfer of calcium across different parts of the cell mem brane; studies in the rat, using the everted gut sac technique, indicate that calcium transfer at the basal and lateral membranes of the cell is related to sodium concentration whilst calcium translocation of the brush border is so dium-independent (16). Although we saw preferential staining in the brush bor der in some sections, CaBP was not demonstrated in the lateral and basal plasma membranes. Our findings, therefore, are not at variance with the hypothesis that CaBP is associated with the movement of calcium across the brush border from gut lumen to cytoplasm. However, the striking finding in our study was the presence of CaBP within the cytoplasm of the absorptive cell. This is not surprising, since CaBP arises, in the chick at least, by ribosomal synthesis (17). However, intestinal CaBP has not been previously demonstrated in the cytoplasm. Our finding of CaBP in the cytoplasm lends some support to earlier hypotheses about intestinal calcium transport (6, 7, 18, 19). These hypotheses required that CaBP be present and
Intestinal CaBP
83
active in the cytoplasm of the absorptive cell. CaBP may participate at the brush border in the transfer of calcium from lumen to cytoplasm. In addition, it may then act as an intracellular transport protein, transferring calcium from the bmsh border to the serosal surface. Alternatively, following cellular absorption, cal cium is taken up by mitochondria; CaBP may then play a role in the release of calcium from mitochondria (19). However, these proposed mechanisms of main taining intracellular calcium homeostasis and transport need not be mutually exclusive, especially in the light of our findings that CaBP is present both in the cytoplasm and at the brush border. Further studies utilizing electron-microscop ic immunohistochemistry obviously would be of great interest. Such studies would give more precise subcellular localization of CaBP, and might provide information on the role of CABP in intestinal calcium absorption.
1 2 3 4 5 6
7 8
9 10
11
12
13
Wassennan, R.H. and Taylor, A.N.: Vitamin D, -induced calcium binding protein in chick intestinal mucosa. Science, N.Y. 152: 791 793 (1966). Wasserman, R.H. and Corradino, R.A.: Vitamin D, calcium, and protein synthesis. Vitams Horm. 31: 43 (1973). Wasserman, R.H.: Vitamin D-dependent calcium-binding protein; in The fat soluble vitamins (University of Wisconsin Press, Madison 1969). Hitchman, A.J. W. and Harrison, J.E.: Calcium binding proteins in the duodenal mucosa of the chick, rat, pig and human. Can. J. Biochem. 50: 758 765 (1972). Taylor, A.N. and Wasserman, R.H.: Immunofluorescent localization of vitamin D-dependent calcium binding protein. J. Histochem. Cytochcm. 18: 107 115 (1970). Del.uca, H.F.: The functional metabolism of vitamin D; in Calcium parathyroid hor mone and the calcitonins (Excerpta Medica, Amsterdam 1972). Kodicek, E.: The story of vitamin D from vitamin to hormone. Lancet i: 325-329 (1974). Murray, T.M.: Arnold, B.M.; Tam, W.H.; Hitchman, A.J.W., and Harrison, J.E.: A radioimmunoassay for a porcine intestinal calcium binding protein. Metabolism 23: 829-837 (1974). Levy, H. and Sober, A.H.: A simple method for preparation of gamma globulin. Proc. Soc. exp. Biol. Med. 103: 250-252 (1960). Stemberger, L.A.: Hardy, P.H.: Cuculis, F.F., and Meyer, H.G.: The unlabelled anti body enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identi fication of spirochetes. J. Histochem. Cytochem. 18: 315-333 (1970). Arnold. B.M.; Murray, T.M.: Hitchman, A.J. W., and Harrison, J.E.: Radioimmunoassay of intestinal calcium binding protein (CaBP) in pig tissues and blood. Clin. Res. 22: 460A (1974). Helmke, K.: Fererlin, K.; Piazolo, P.; Stroder, J.; Jeschke, R., and Franz, H.E.: Local ization of calcium-binding protein in intestinal tissue by immunofluorescence in nor mal, vitamin D-deficient and uremic subjects. Gut 15: 875-879 (1974). Piazolo, P.; Schleyer, M., and Franz, H.E.: Isolation and purification of a calcium binding protein from human tissues. Hoppe-Seyler’s Z. physiol. Chem. 352: 1480-1486 (1971).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
References
A mold/Kovacs/Murray
84
14 Arnold, B.M.; Kuttner, M.; Hitchman, A.J. W.: Harrison, J.E., and Murray, T.M.: Chem ical and immunochemical differentiation of intestinal calcium-binding protein (CaBP) from calcium-binding proteins in parathyroid and kidney. Clin. Res. 22: 749A (1974). 15 Wasserman, R.H. and Taylor, A.N.: Some aspects of the intestinal absorption of cal cium with special reference to vitamin D; in Mineral metabolism. An advanced treatise (Academic Press, New York 1969). 16 Martin, P.L. and DeLuca. H.F.: Influence of sodium on calcium transport by the rat small intestine. Am. J. Physiol. 216: 1351 -1359 (1969). 17 Emtage, J.S.; Lawson, D.E.M., and Kodicek, E.: Vitamin D-induced synthesis of mRNA for calcium-binding protein. Nature, Lond.246: 100-101 (1973). 18 DeLuca, H.F.: Metabolism and function of vitamin D; in The fat soluble vitamins (University of Wisconsin Press, Madison 1969). 19 Hamilton, J. W. and Holdsworth, E.S.: The release o f 45Ca from mitochondria of chick en intestinal mucosa by calcium binding protein. Biochem. biophys. Res. Commun. 40: 1325 1330 (1970).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Dr. T.M. Murray, MD, Room 6356, Medical Sciences Building. University of Toronto, Toronto M5S 1A8 (Canada)
Cellular Localization of Intestinal Calcium-Binding Protein in Pig Duodenum1 B.M. Arnold, K. Kovacsand T.M. Murray Departments of Medicine and Pathology, St. Michael's Hospital and University of Toronto, Toronto, Ont.
Key Words. Binding proteins • Calcium • Immunologic Techniques • Intestine, small • Swine Abstract. The 7 -globulin fraction of a rabbit antiserum against porcine intestinal cal cium-binding protein (CaBP) was used in an immunoperoxidase method to study CaBP localization in porcine duodenal tissue. Specific immunostaining, indicative of the presence of CaBP, was noted within the cytoplasm of the duodenal epithelial cells. No CaBP was detected in goblet cells or in the subepithelial layers. When the specific antibody was replaced by either nonimmune rabbit 7 -globulin or when the specific antibody was pread sorbed with excess CaBP, no positive immunostaining was seen. Our studies lend support to the hypothesis that CaBP may function in intracellular calcium transport.
Introduction Wasserman and Taylor (1) observed that a calcium-binding protein (CaBP) was present in the duodenal mucosa of vitamin D-treated rachitic chicks. Subse quently, considerable evidence has been obtained which correlates duodenal CaBP concentration with intestinal calcium transport in the chick (2). Intestinal CaBP proteins have also been described in several other species (3, 4). in the chick, using fluorescent antibody techniques, CaBP was localized to the PASpositive goblet cells and microvillar surface of the intestinal cell giving rise to the hypothesis that CaBP in the brush border may be actively involved in transloca tion of calcium from the intestinal lumen to the interior of the absorptive cells (5). Others have suggested that CaBP may be an intracellular calcium transport 1 Supported in part by grants from the Medical Research Council of Canada (MA4515) and the Atkinson Charitable foundation.
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Received: July 12, 1975:accepted: November 12, 1975.
Arnold /Kovacs /Murray
78
protein, either as an integral transmucosal transport mechanism or to transport calcium to the serosal surface of the cell following mitochondrial uptake and release of calcium (6, 7). However, there has been no proof that CaBP is present in the cytoplasm. Information concerning the cytologic localization of CaBP would be valuable in elucidating the mechanism of vitamin D-induced intestinal calcium translocation. We have recently begun to study CaBP in the pig (8) and wish to report here the immunohistochemical localization of intestinal CaBP in porcine duodenum. Materials and Methods Preparation o f Antiserum Antisera were prepared in rabbits by immunization with pure porcine CaBP as previ ously reported (8 ). 7 -Globulin fractions from antiserum R3A and from normal rabbit serum were prepared by the method of Levy and Sober (9). The lyophilized 7 -globulin fractions were dissolved in normal saline and stored at 4 °C. We have recently reported in detail on the specificity of another antiserum (R2A) used in our newly developed radioimmunoassay for porcine intestinal CaBP ( 8). The specificity of antiserum R3A was tested using similar procedures; no immunoreactive components, other than CaBP, were found in duodenal extracts by radioimmunoassay of the eluted fractions from gel filtration or ion exchange chromatography in the presence of calcium or EDTA (8 ).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Immunohistological Techniques Small pieces of freshly obtained pig duodenum were fixed in 10 % neutral buffered formalin for 24 h, postfixed in 70 % ethanol for an additional 24 h, dehydrated in graded ethanol and embedded in paraffin. In addition, frozen sections of duodenum were prepared. Frozen sections were cut on a cryostat and either immunostained without any prior fixa tion, fixed in 10 % neutral buffered formalin for 1 h, or fixed in 1 % glutaraldehyde for 3 h and then immunostained as described below. Sections of 3 -5 Mm thickness were cut. The unlabelled antibody peroxidase-antiperoxidase complex method was used to localize CaBP in the duodenum (10). Both frozen and paraffin-embedded sections were washed in phos phate-buffered saline, pH 7.4, then exposed for 3 h to the specific antibody which was diluted 1:100 in phosphate-buffered saline, pH 7.4. Following thorough washing in phos phate-buffered saline, the sections were next exposed to goat-antirabbit 7 -globulin antise rum (also diluted 1:10 in phosphate-buffered saline, pH 7.4) for 5 min, thoroughly re washed in phosphate-buffered saline, then exposed to a 1:60 dilution of the peroxidaseantiperoxidase complex for 5 min. The sections were again thoroughly washed in phos phate-buffered saline. After washing the sections were exposed for 10 min to DAB solution (20 mg of 3,3'-diaminobenzidine in 10 ml Tris-buffered saline, 0.05 M, pH 7.6, to which were added three drops of 3% H ,0 2). Finally, the sections were washed in phosphatebuffered saline solution, dehydrated in graded ethanol, cleared in toluol and mounted. For control purposes the specific antibody was replaced by phosphate-buffered saline, pH 7.4, by normal nonimmune rabbit 7 -globulin, or else the specific antibody was preab sorbed with excess CaBP. Sections of kidney, liver, thyroid and pancreas were also made, using similar immunohistologic techniques. For orientation of few paraffin-embedded and frozen sections were also stained with hematoxylin-phloxine-saffron, hematoxylin and eosin, and with the PAS method. Finally, on a few frozen and paraffin-embedded immuno stained sections the PAS method was also applied.
Intestinal CaBP
79
Results The duodenal tissue possessed normal histological features when sections were stained with hematoxylin-eosin or with hematoxylin-phloxine-saffron (fig. 1). Immunostaining of the paraffin-embedded and frozen sections of the duodenum showed brown granular deposits, indicating the presence of CaBP. The brown deposits were abundantly present in the cytoplasm of the majority of the mucosal epithelial cells (fig. 2, 3). There was no preferential localization of the granular deposits in different areas of the cytoplasm, although in some cells the brown granules seemed to accumulate around the nucleus. In some areas cytoplasmic immunostaining was less intense than in others. In these areas immunostaining was more marked in the brush border, although in most of the sections it was not possible to distinguish immunostaining in the brush border from the granular deposits in the cytoplasm. There was no positivity in the goblet cells (fig. 2, 3), either on the paraffinembedded or frozen sections. Immunoperoxidase-stained sections subsequently
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Fig. I. A formalin-fixed, paraffin-embedded section of the mucosa of pig duodenum stained with hematoxylin-phloxine-saffron, demonstrating normal histologic features. Ap proximately X 250.
Arnold/Kovacs/Murray
80
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Fig. 2. A similar section stained by the immunoperoxidase method. Positive immunostaining is evident in the surface epithelium (arrows). No staining is present in the goblet cells. Approximately x 400. Fig. 3. Positive immunostaining in cross-section of the mucosal glands of the pig duode num (arrows). Approximately X 400.
Intestinal CaBP
81
Fig. 4. No deposits are apparent when the specific antibody was preabsorbed by anti gen excess. Approximately X 400.
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
stained with PAS demonstrated PAS positivity of the goblet cells without any immunostaining. Granular deposits were not apparent in other components of the intestinal wall such as smooth muscle, or connective tissue. In general, the quality of the frozen sections was somewhat inferior to that of the paraffinembedded sections, but the distribution of immunostaining was similar. No positive staining was noted in the mucosal epithelial cells when the specific antibody was replaced by either phosphate-buffered saline or normal nonimmune rabbit serum, or when the specific antibody was previously ab sorbed by excess antigen (fig. 4). When normal nonimmune rabbit 7 -globulin was used instead of the specific antibody, only nonspecific background staining was seen. No differences in the amount of immunostaining were noted when crypt cells were compared with cells at the tips of the villi. However, the methods we have used do not have sufficient resolution to make such comparisons effec tively. No positive immunostaining was found in the kidneys, liver, thyroid or in the pancreas, organs previously shown to contain immunoreactive CaBP in much lower amounts than duodenum (11).
Arnold/Kovacs/Murray
82
Discussion
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
The results of our studies, using a specific antibody to pig intestinal CaBP, clearly localized CaBP to the cytoplasm of the absorptive cells in the duodenum. CaBP was not demonstrable in the goblet cells. These findings are at variance with those of Taylor and Wasserman (5) who found, using immunofluorescent techniques, that in chick duodenum CaBP was localized to the goblet cells and to the microvillar surface of the epithelial cells. The difference between our findings may indicate a difference in the cellular biochemistry of the intestinal absorptive process between the chick and the pig. On the other hand, some recent evidence suggests that fluorescence of goblet cells in intestinal tissue may be a nonspecific finding (12). There have been some recent studies of cellular localization of CaBP in human intestinal cells (12). However, the antiserum used in these studies was prepared against renal CaBP (molecular weight greater than 21,500) (13). There is reason to believe that these studies are localizing renal CaBP in intestinal tissue rather than intestinal CaBP which in the human (and in the pig) has a molecular weight of about 12,000-13,000 by gel filtration (4). Recent studies in the pig using our radioimmunoassay show that the major CaBPs in kidney are chemi cally and immunochemically different from intestinal CaBP (14). It is important, therefore, that the antiserum used in our studies was raised against highly puri fied pig intestinal CaBP, rather than antigen from the kidney (8). CaBP has been shown to be present at the microvillar surface of the absorp tive cell in clucks (5). At this site, the transfer of calcium from the lumen to mucosa is significantly stimulated by the presence of vitamin D (15). This has led to the hypothesis that CaBP at the brush border is involved in the transfer of calcium from the lumen to the cytoplasm (3). However, different mechanisms may operate in the transfer of calcium across different parts of the cell mem brane; studies in the rat, using the everted gut sac technique, indicate that calcium transfer at the basal and lateral membranes of the cell is related to sodium concentration whilst calcium translocation of the brush border is so dium-independent (16). Although we saw preferential staining in the brush bor der in some sections, CaBP was not demonstrated in the lateral and basal plasma membranes. Our findings, therefore, are not at variance with the hypothesis that CaBP is associated with the movement of calcium across the brush border from gut lumen to cytoplasm. However, the striking finding in our study was the presence of CaBP within the cytoplasm of the absorptive cell. This is not surprising, since CaBP arises, in the chick at least, by ribosomal synthesis (17). However, intestinal CaBP has not been previously demonstrated in the cytoplasm. Our finding of CaBP in the cytoplasm lends some support to earlier hypotheses about intestinal calcium transport (6, 7, 18, 19). These hypotheses required that CaBP be present and
Intestinal CaBP
83
active in the cytoplasm of the absorptive cell. CaBP may participate at the brush border in the transfer of calcium from lumen to cytoplasm. In addition, it may then act as an intracellular transport protein, transferring calcium from the bmsh border to the serosal surface. Alternatively, following cellular absorption, cal cium is taken up by mitochondria; CaBP may then play a role in the release of calcium from mitochondria (19). However, these proposed mechanisms of main taining intracellular calcium homeostasis and transport need not be mutually exclusive, especially in the light of our findings that CaBP is present both in the cytoplasm and at the brush border. Further studies utilizing electron-microscop ic immunohistochemistry obviously would be of great interest. Such studies would give more precise subcellular localization of CaBP, and might provide information on the role of CABP in intestinal calcium absorption.
1 2 3 4 5 6
7 8
9 10
11
12
13
Wassennan, R.H. and Taylor, A.N.: Vitamin D, -induced calcium binding protein in chick intestinal mucosa. Science, N.Y. 152: 791 793 (1966). Wasserman, R.H. and Corradino, R.A.: Vitamin D, calcium, and protein synthesis. Vitams Horm. 31: 43 (1973). Wasserman, R.H.: Vitamin D-dependent calcium-binding protein; in The fat soluble vitamins (University of Wisconsin Press, Madison 1969). Hitchman, A.J. W. and Harrison, J.E.: Calcium binding proteins in the duodenal mucosa of the chick, rat, pig and human. Can. J. Biochem. 50: 758 765 (1972). Taylor, A.N. and Wasserman, R.H.: Immunofluorescent localization of vitamin D-dependent calcium binding protein. J. Histochem. Cytochcm. 18: 107 115 (1970). Del.uca, H.F.: The functional metabolism of vitamin D; in Calcium parathyroid hor mone and the calcitonins (Excerpta Medica, Amsterdam 1972). Kodicek, E.: The story of vitamin D from vitamin to hormone. Lancet i: 325-329 (1974). Murray, T.M.: Arnold, B.M.; Tam, W.H.; Hitchman, A.J.W., and Harrison, J.E.: A radioimmunoassay for a porcine intestinal calcium binding protein. Metabolism 23: 829-837 (1974). Levy, H. and Sober, A.H.: A simple method for preparation of gamma globulin. Proc. Soc. exp. Biol. Med. 103: 250-252 (1960). Stemberger, L.A.: Hardy, P.H.: Cuculis, F.F., and Meyer, H.G.: The unlabelled anti body enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identi fication of spirochetes. J. Histochem. Cytochem. 18: 315-333 (1970). Arnold. B.M.; Murray, T.M.: Hitchman, A.J. W., and Harrison, J.E.: Radioimmunoassay of intestinal calcium binding protein (CaBP) in pig tissues and blood. Clin. Res. 22: 460A (1974). Helmke, K.: Fererlin, K.; Piazolo, P.; Stroder, J.; Jeschke, R., and Franz, H.E.: Local ization of calcium-binding protein in intestinal tissue by immunofluorescence in nor mal, vitamin D-deficient and uremic subjects. Gut 15: 875-879 (1974). Piazolo, P.; Schleyer, M., and Franz, H.E.: Isolation and purification of a calcium binding protein from human tissues. Hoppe-Seyler’s Z. physiol. Chem. 352: 1480-1486 (1971).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
References
A mold/Kovacs/Murray
84
14 Arnold, B.M.; Kuttner, M.; Hitchman, A.J. W.: Harrison, J.E., and Murray, T.M.: Chem ical and immunochemical differentiation of intestinal calcium-binding protein (CaBP) from calcium-binding proteins in parathyroid and kidney. Clin. Res. 22: 749A (1974). 15 Wasserman, R.H. and Taylor, A.N.: Some aspects of the intestinal absorption of cal cium with special reference to vitamin D; in Mineral metabolism. An advanced treatise (Academic Press, New York 1969). 16 Martin, P.L. and DeLuca. H.F.: Influence of sodium on calcium transport by the rat small intestine. Am. J. Physiol. 216: 1351 -1359 (1969). 17 Emtage, J.S.; Lawson, D.E.M., and Kodicek, E.: Vitamin D-induced synthesis of mRNA for calcium-binding protein. Nature, Lond.246: 100-101 (1973). 18 DeLuca, H.F.: Metabolism and function of vitamin D; in The fat soluble vitamins (University of Wisconsin Press, Madison 1969). 19 Hamilton, J. W. and Holdsworth, E.S.: The release o f 45Ca from mitochondria of chick en intestinal mucosa by calcium binding protein. Biochem. biophys. Res. Commun. 40: 1325 1330 (1970).
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:06:32 PM
Dr. T.M. Murray, MD, Room 6356, Medical Sciences Building. University of Toronto, Toronto M5S 1A8 (Canada)
