- Email: [email protected]
1,25-Dihydroxyvitamin D3 receptors in mouse colon
1,25-DIHYDROXYVITAMIN D3 RECEPTORS MOUSE COLON
IN
MARGARETA. HIRST and DAVID FELDMAN* Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, U.S.A. (Received 14 August 1980) SUMMARY Mouse colon possesses a cytoplasmic receptor-like binder for 1.25dihydroxyvitamin D, which appears similar to the receptors previously described in other more classical target organs. The properties of this receptor include a sedimentation coefficient of 3.2s in sucrose gradients prepared in 0.3 M KCI, an equilibrium dissociation constant of 0.2 nM and a competitive profile which demonstrates a substantial binding preference for 1,2%dihydroxyvitamin D, over other vitamin D3 analogues. The concentration of receptor sites was assessed along the entire gastrointestinal tract and found to be in the sequence: duodenum > ieiunum > ileum z cecum = proximal colon > distal colon; esophagus and stomach possessed only neghgible numbers of binding sites.
1,2S-(OH)2Vitamin Da (1,25-(OH),D& the active metabolite of vitamin D3, is a major regulator of cal-
cium and phosphorous metabolism in the body [l]. Considerable evidence has accumulated to indicate that the mechanism of action of this agent is analogous to the steroid hormones [Z-4]. In this respect, the initial intracellular step in 1,25-(OH),D, action is the binding of the hormone to a specific cytoplasmic receptor. Such receptors for 1,25-(OH)2D, have been described in chick intestine and other chick tissues [4-6]. More recently receptors have been extensively studied in mammalian species. In addition to small intestine [7-93, receptors have been biochemically characterized in several other mammalian organs including bone and bone cells [lo, 111, kidney [12, 133, parathyroid 1143, pituitary [15] and mammary gland and skin [ 163. Recently, similar receptors have also been described in two malignant cell lines, osteogenic sarcoma [ 171 and the MCF-7 breast cancer cells [18]. Although functions for the hormone have not been elucidated in many cases, the presence of I,25(OH),D, receptors in multiple sites suggests that the number of target organs responding to the hormone is greater than previously thought. However, a tissue which is believed to be a target organ for 1,25-(OH),D, but which has not been biochemically examined for the presence of receptors is the colon. Evidence has accumulated to indicate that, in addition to its action on small intestine, 1,25-(OH),D, *To whom correspondence should be sent. Abbreuiurions: 1.25(OH),D,, 1.25dihydroxyvitamin D,; 25(OH)D,, 25hydroxyvitamin Da; la-(OH)D3, Ir-hydroxyvitamin D,; 24,25-(OH),Do, 24,25-dihydroxyvitamin D3. SB. 14,4--A
also regulate calcium transport and calcium binding protein in the colon [ 19-211. We therefore felt it to be essential, for the receptor hypothesis to hold, that identifiable l,25-(OH)2D3 receptors be present in this putative target organ. We thought it would also be of interest to compare the 1,25-(OH)2D, receptor in small intestine and colon, the two locations in which the mediated function appears to be similar. In this report we present our findings which demonstrate the presence of 1,25-(OH)2D, receptors in colon. In addition, all areas of the gastrointestinal tract were examined and comparison made of the receptor content in the various segments. may
INTRODUCTION
315
MATERIALS AND METHODS Materials
1,25-(OH),[3H]D, (82 Ci/mmol) was obtained from Amersham Chemical Co. (Arlington Heights, IL). Crystalline 25-(OH)D,, 1,25_(0H)~D,, lol-(OH)Ds and 24-25-(OH)2D, were generous gifts from Dr M. Uskokovic of the Hoffman-LaRoche Co. (Nutley, NJ). Animals
Four to six week old male Swiss Webster mice (Simonsen Co., Gilroy, CA), fed a routine laboratory chow, were employed in all studies. Cytosol preparation
Colon, distal to the cecum and proximal to the rectum, was excised and rinsed in iced phosphatebuffered saline. All subsequent steps were carried out at 04°C. Mucosa was scraped from underlying muscle with a glass slide, the cells disrupted by sonication in KTEDM-buffer (300 mM KCl, 10 mM Tris, 1mM EDTA, 5mM dithiothreitol, 10 mM
MARGARETA. HIRST and DAVID FELDMAN
316
Other
methods
Protein concentration was determined by the Coomasie blue dye (Sigma) method of Bradford[22].
RESULTS
Sedimentation
analysis
We first examined colon cytosol on sucrose density gradients to characterize the sedimentation properties of the binding moieties present. As seen in Fig. 1 cytosol labelled with 1.3 nM I,25(OH),[‘H]D3 exhibited a 3.2s peak which was competed away by 250-fold excess radioinert 1,25-(OH),Ds. Addition of either excess radioinert 25-(OH)D, or 1,25-(OH),D, showed that this putative receptor preferentially bound 1,25-(OH),D, (data not shown). Note that cytosol incubated with 1,25-(OH),C3H]D3 did not exhibit a binding peak in the 5 6 S region where the characteristic 25-(OH)D, binder [23,24] is found to sediment. TOP
20
15
10
FRACTION
NUMBER
5
Fig. I. Sucrose gradient analysis of colon cytosol. Cytosol was incubated with 1.3 nM 1.25-(OH),[3H]D3 f 250-fold radioinert 1.25(OH&D3 to determine non-specific binding After free steroid was removed by charcoal treatment. aliquots of bound cytosol were layered onto 5-20x sucrose gradients prepared in KTEDM buffer and centrifuged at 257,OOOgfor 18 h. Fractions were collected by puncturing the bottom of the tube. [‘4C]-Bovine serum albumin was used as the 4.4s internal marker.
Na,MoO,, pH 7.4) and cytosol prepared by centrifugation at 100,OOOg for 1 h. For the distribution studies, tissues were divided as follows: esophagus, stomach, duodenum (first 1Ocm distal to stomach), jejunum (next 25 cm). ileum (25 cm of remaining small intestine up to cecal valve), cecum (next 4 cm distal to cecal valve), proximal colon (first half of colon proximal to the rectum) and distal colon (last half of colon proximal to rectum). Cytosols from these tissues were prepared as described above for colon. Binding
assay
1,25-(OH)2[3H]D3 f appropriateradioinert metabolites were added to sample tubes, dried under nitrogen and resolubilized in 10~1 of ethanol. Cytosols were added in 200 ~1 aliquots and incubated for 3 h at 0°C. Bound steroid was separated from free by serumcoated charcoal as previously described [9]. Sedimentation
analysis
Linear gradients (4.2 ml) of 5520% sucrose (w/v) in KTEDM-buffer were prepared with a Buchler gradient maker. Cytosol was incubated with 1,25(OH),C3H]D3 for 3 h at O’C. and treated with charcoal to remove free steroid. Aliquots (200~1) of this prebound cytosol were layered onto the gradients and centrifuged at 2°C for 18 h at 257,000 g in a Beckman SW 60 rotor.
Saturation
analysis
Since the gradient profile indicated a single 1,25-(OH)J3H]D3 binding species at 3.2S we proceeded to characterize this binder by saturation analysis and Scatchard plot. As shown in Fig. 2A, specific l,25-(OH)2[3flD3 binding saturated at approximately 1 nM whereas non-specific binding was a linear function of ligand concentration. In Fig. 2B is shown the Scatchard analysis of the specific binding data. The straight line indicates a single class of noninteracting binding sites. The mean apparent equilibrium dissociation constant (Kd) was 0.22 + 0.03 nM in three such experiments. The concentration of binding sites (N,,,) was 206 f 5 fmol/mg cytosol protein. Competitive
binding
analysis
Colon cytosol was incubated with 1.3 nM 1,25-(OH)J3H]D3 + radioinert competitor analogues to determine the relative affinity of a variety of related steroids for the receptor sites. As shown in Fig. 3, 1,25-(OH),D, was the best competitor reducing the binding of 1,25-(OH)J3H]D3 to 500/, at approximately an equivalent concentration of radioinert steroid. The potency of the other metabolites relative to 1,25-(OH),D, was 25-(OH)D3 5 2.7x, 1r-(OH)D3 2 1.3% and 24,25-(OH),D, 5 0.4%. Distribution
analysis
The distribution of 1,25-(OH),D, receptors was assessed throughout the gastrointestinal tract and the data are shown in Table 1. Since the 5 6 S 25-(OH)D3 binding protein was not detected in our scraped and washed cells, a single point assay at a saturating concentration of 1,25-(OH)2D3 was employed in these experiments. The highest concentration of receptor sites was seen in duodenum followed by jejunum and ileum Although less than small intestine, colon had substantial numbers of sites: the cecum and proximal colon had the same content while distal colon was
1,25-(OH)2Da receptors in colon
(B) ‘:.
0.32
2 5 E
317
Kd = 0.18 nM
: :
N max = 203 fmollmg
0.24
d 0” iz “I ,N
0.16
8 fi ; 0.08
-. :.
I 0
0.2
1.25-(OH)2[3HI
0.4
0.6
0.8
0 0
1.0
O3 CONCENTRATION
20
40
1.25-(OH)213HI
(nM)
60
80
100
D3 BOUND (fmolhnl)
Fig. 2. A. Saturation analysis of 1,25-(OH)2[3H]Da binding in colon cytosol. Cytosol was incubated with increasing concentrations of 1,25-(OH)2[3H]Da (0.13 to 0.95 nM) + 250-fold radioinert 1.25-(OH)zD, to determine non-specific binding. B. Scatchard plot of specific binding data from Fig. 2A. The regression line was calculated by least squares.
somewhat lower. Esophagus only negligible
and stomach
exhibited
binding.
DISCUSSION
The data indicate the presence of a high affinity binder in mouse colon with properties similar to the
1,25-(OH),D, receptor previously demonstrated in other mammalian organs including small intestine [7-181. These properties include a 3.2s sedimen-
_.
1.3
13
tation coefficient in gradients prepared in hypertonic buffers, an affinity of 0.2 nM for l,25-(OH)2D3 and a pronounced preference to bind l,25-(OH),D, compared to other vitamin D analogues. The presence of receptors in small intestine and colon prompted us to examine the entire gastro intestinal tract for receptors. The results (Table 1) indicate a selective distribution of 1,25-(OH)*D3 receptors. All segments of small and large intestine possess receptors with the greatest concentration being present in duodenum and decreasing numbers of sites found as
130
COMPETITOR CONCENTRATION
1300 [nMl
Fig. 3. Competitive binding assay of 1,25-(OH),[3H]Da binding in colon cytosol. Cytosol was incubated with 1.3 nM 1.25~(OH)J3H]D, + competitors at the indicated concentrations. Specific binding in the absence of competitors was taken as the control or 100% value and averaged 200 & 20 fmol/mg cytosol protein. Values shown are means + SE.
318
MARGARETA. HIRST and DAVIDFELDMAN Table 1. Specific 1,25-(OH),Ds sites along the gastrointestinal
Zone Esophagus Stomach Duodenum Jejunum Ileum Cecum Proximal colon Distal colon
binding tract
Specific binding (fmol/mg protein) 8 (2.4-10.1) 12(7.1-16) 527 (481-581) 384 (33&430) 344 (285405) 274 (224-340) 269 (23 l-296) 160(156167)
Binding was assessed with a single point assay at a saturating concentration (1.3 nM) of l.25-(OH)z[3H]D,. Corrections were made for non-specific binding. The values shown represent means and ranges of three experiments.
one proceeds distally. Esophagus and stomach exhibited negligii;le numbers of binding sites. These biochemical measurements of receptors correlate well with the recent studies of Stumpf et al. employing a radio-autographic approach [25]. Those workers demonstrated 1,25-(OH)J3H]D, localization in both small and large intestine with a somewhat stronger localization in the small intestine. Furthermore, they noted that the epithelial cells of the stomach were generally unlabelled except for dispersed individual cells in basal portions of gastric glands. The presence of receptors in only a small subpopulation of cells may explain our tinding of a low level of receptors when assayed in the total stomach epithelial layer. It has been shown that mammalian colon exhibits vitamin D dependent increases in calcium transport and calcium binding protein [19-211. However, it is of interest that although a rise in calcium binding protein was found in the cecum after vitamin D treatment, no increase in net calcium flux was detected in this segment [20] nor did 1,25-(OH),D, effect phosphate absorption in the proximal colon as it does in the small intestine [21]. These dissociated responses are intriguing in view of the similarity of the receptors in cecum and colon to the small intestine where both calcium and phosphate transport are stimulated. Multiple other examples exist where the effects of a steroid hormone are discrepant in various target organs despite an apparently identical receptor [26]. In conclusion, the colon is an additional organ which possesses receptors for 1,25-(OH),D,. The receptors appear similar in their properties to the receptors previously described in small intestine and other target organs. The gastrointestinal tract has a selective distribution of receptors for 1,25-(OH)zD3; the concentration is very low in esophagus and stomach and substantial in small and large intestine. The presence of receptors coupled with documented functions of 1,25-(OH),D, in colon indicates that this segment of the gastrointestinal tract is also a 1,25-(OH)zD, target organ.
Acknowledgements-Supported from the USPHS.
by NIH grant AG 01355
REFERENCES
1. Lawson D. E. M. (Editor) Vitamin D. Academic Press, London (1978). 2. DeLuca H. F. and Schnoes H. K.: Metabolism and mechanism of action of vitamin D. Ann. Reti. Biochem. 45 (1976) 631-666. 3. Haussler M. R. and McCain T. A.: Basic and clinical concepts related to vitamin D metabolism and action. N. Eng. J. Med. 297 (1977) 974-983. 4. Norman A. W. and Wecksler W. R.: Vitamin D. recentars and biologic responses. In Receptors and Hormone Action (Edited bv B. W. O’Mallev and L. Birnbaumer). Academic Press,
1.25(OH),D,
19.
20.
21.
22.
receptors in colon
cancer cell line (MCF 7 cells) Biochem. biophys. Rrs. Commun. 93 (1980) 9-l 5. Harrison H. C. and Harrison H. E.: Calcium transport by rat colon in uirro. Am. J. Physiol. 217 (1969) 121-125. Petith M. M., Wilson H. D. and Schedl H. P.: Vitamin D dependence of in ho calcium transport and mucosal calcium binding protein in rat large intestine. Gasrroenterology 76 (1979) 99-l 04. Lee D. B. N., Walling M. W., Gafter U., Silis V. and Coburn J. W.: Calcium and inorganic phosphate transport in rat colon. J. din. Inuest. 65 (1980) 1326133 I. Bradford M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biothem. 72 (I 976) 248-254.
319
23. Haddad J. G. and Birge S. J.: Widespread. specific binding of 25-hydroxycholecalciferol in rat tissues. J. biol. Chem. 250 (1975) 299-303. 24. VanBaelen H. V., Bouillon R. and DeMoor P.: Binding of 25-hydroxycholecalciferol in tissues. J. biol. Chem. 252 ( 1977) 25 15-25 18. 25. Stumpf W. E., Sar M., Reid F. A.. Tanaka Y. and DeLuca H. F.: Target cells for 1.25-dihydroxyvitamin D, in intestinal tract, stomach, kidney. skin. pituitary and parathyroid. Sckrnce 206 (1979) I 188-l 190. 26. Feldman D.: Glucocorticoid receptors and regulation of phosphoenolpyruvate-carboxykinase activity in rat kidney and adipose tissue. Ant. J. Physiol. 233 (1977) El477151.
IN
MARGARETA. HIRST and DAVID FELDMAN* Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, U.S.A. (Received 14 August 1980) SUMMARY Mouse colon possesses a cytoplasmic receptor-like binder for 1.25dihydroxyvitamin D, which appears similar to the receptors previously described in other more classical target organs. The properties of this receptor include a sedimentation coefficient of 3.2s in sucrose gradients prepared in 0.3 M KCI, an equilibrium dissociation constant of 0.2 nM and a competitive profile which demonstrates a substantial binding preference for 1,2%dihydroxyvitamin D, over other vitamin D3 analogues. The concentration of receptor sites was assessed along the entire gastrointestinal tract and found to be in the sequence: duodenum > ieiunum > ileum z cecum = proximal colon > distal colon; esophagus and stomach possessed only neghgible numbers of binding sites.
1,2S-(OH)2Vitamin Da (1,25-(OH),D& the active metabolite of vitamin D3, is a major regulator of cal-
cium and phosphorous metabolism in the body [l]. Considerable evidence has accumulated to indicate that the mechanism of action of this agent is analogous to the steroid hormones [Z-4]. In this respect, the initial intracellular step in 1,25-(OH),D, action is the binding of the hormone to a specific cytoplasmic receptor. Such receptors for 1,25-(OH)2D, have been described in chick intestine and other chick tissues [4-6]. More recently receptors have been extensively studied in mammalian species. In addition to small intestine [7-93, receptors have been biochemically characterized in several other mammalian organs including bone and bone cells [lo, 111, kidney [12, 133, parathyroid 1143, pituitary [15] and mammary gland and skin [ 163. Recently, similar receptors have also been described in two malignant cell lines, osteogenic sarcoma [ 171 and the MCF-7 breast cancer cells [18]. Although functions for the hormone have not been elucidated in many cases, the presence of I,25(OH),D, receptors in multiple sites suggests that the number of target organs responding to the hormone is greater than previously thought. However, a tissue which is believed to be a target organ for 1,25-(OH),D, but which has not been biochemically examined for the presence of receptors is the colon. Evidence has accumulated to indicate that, in addition to its action on small intestine, 1,25-(OH),D, *To whom correspondence should be sent. Abbreuiurions: 1.25(OH),D,, 1.25dihydroxyvitamin D,; 25(OH)D,, 25hydroxyvitamin Da; la-(OH)D3, Ir-hydroxyvitamin D,; 24,25-(OH),Do, 24,25-dihydroxyvitamin D3. SB. 14,4--A
also regulate calcium transport and calcium binding protein in the colon [ 19-211. We therefore felt it to be essential, for the receptor hypothesis to hold, that identifiable l,25-(OH)2D3 receptors be present in this putative target organ. We thought it would also be of interest to compare the 1,25-(OH)2D, receptor in small intestine and colon, the two locations in which the mediated function appears to be similar. In this report we present our findings which demonstrate the presence of 1,25-(OH)2D, receptors in colon. In addition, all areas of the gastrointestinal tract were examined and comparison made of the receptor content in the various segments. may
INTRODUCTION
315
MATERIALS AND METHODS Materials
1,25-(OH),[3H]D, (82 Ci/mmol) was obtained from Amersham Chemical Co. (Arlington Heights, IL). Crystalline 25-(OH)D,, 1,25_(0H)~D,, lol-(OH)Ds and 24-25-(OH)2D, were generous gifts from Dr M. Uskokovic of the Hoffman-LaRoche Co. (Nutley, NJ). Animals
Four to six week old male Swiss Webster mice (Simonsen Co., Gilroy, CA), fed a routine laboratory chow, were employed in all studies. Cytosol preparation
Colon, distal to the cecum and proximal to the rectum, was excised and rinsed in iced phosphatebuffered saline. All subsequent steps were carried out at 04°C. Mucosa was scraped from underlying muscle with a glass slide, the cells disrupted by sonication in KTEDM-buffer (300 mM KCl, 10 mM Tris, 1mM EDTA, 5mM dithiothreitol, 10 mM
MARGARETA. HIRST and DAVID FELDMAN
316
Other
methods
Protein concentration was determined by the Coomasie blue dye (Sigma) method of Bradford[22].
RESULTS
Sedimentation
analysis
We first examined colon cytosol on sucrose density gradients to characterize the sedimentation properties of the binding moieties present. As seen in Fig. 1 cytosol labelled with 1.3 nM I,25(OH),[‘H]D3 exhibited a 3.2s peak which was competed away by 250-fold excess radioinert 1,25-(OH),Ds. Addition of either excess radioinert 25-(OH)D, or 1,25-(OH),D, showed that this putative receptor preferentially bound 1,25-(OH),D, (data not shown). Note that cytosol incubated with 1,25-(OH),C3H]D3 did not exhibit a binding peak in the 5 6 S region where the characteristic 25-(OH)D, binder [23,24] is found to sediment. TOP
20
15
10
FRACTION
NUMBER
5
Fig. I. Sucrose gradient analysis of colon cytosol. Cytosol was incubated with 1.3 nM 1.25-(OH),[3H]D3 f 250-fold radioinert 1.25(OH&D3 to determine non-specific binding After free steroid was removed by charcoal treatment. aliquots of bound cytosol were layered onto 5-20x sucrose gradients prepared in KTEDM buffer and centrifuged at 257,OOOgfor 18 h. Fractions were collected by puncturing the bottom of the tube. [‘4C]-Bovine serum albumin was used as the 4.4s internal marker.
Na,MoO,, pH 7.4) and cytosol prepared by centrifugation at 100,OOOg for 1 h. For the distribution studies, tissues were divided as follows: esophagus, stomach, duodenum (first 1Ocm distal to stomach), jejunum (next 25 cm). ileum (25 cm of remaining small intestine up to cecal valve), cecum (next 4 cm distal to cecal valve), proximal colon (first half of colon proximal to the rectum) and distal colon (last half of colon proximal to rectum). Cytosols from these tissues were prepared as described above for colon. Binding
assay
1,25-(OH)2[3H]D3 f appropriateradioinert metabolites were added to sample tubes, dried under nitrogen and resolubilized in 10~1 of ethanol. Cytosols were added in 200 ~1 aliquots and incubated for 3 h at 0°C. Bound steroid was separated from free by serumcoated charcoal as previously described [9]. Sedimentation
analysis
Linear gradients (4.2 ml) of 5520% sucrose (w/v) in KTEDM-buffer were prepared with a Buchler gradient maker. Cytosol was incubated with 1,25(OH),C3H]D3 for 3 h at O’C. and treated with charcoal to remove free steroid. Aliquots (200~1) of this prebound cytosol were layered onto the gradients and centrifuged at 2°C for 18 h at 257,000 g in a Beckman SW 60 rotor.
Saturation
analysis
Since the gradient profile indicated a single 1,25-(OH)J3H]D3 binding species at 3.2S we proceeded to characterize this binder by saturation analysis and Scatchard plot. As shown in Fig. 2A, specific l,25-(OH)2[3flD3 binding saturated at approximately 1 nM whereas non-specific binding was a linear function of ligand concentration. In Fig. 2B is shown the Scatchard analysis of the specific binding data. The straight line indicates a single class of noninteracting binding sites. The mean apparent equilibrium dissociation constant (Kd) was 0.22 + 0.03 nM in three such experiments. The concentration of binding sites (N,,,) was 206 f 5 fmol/mg cytosol protein. Competitive
binding
analysis
Colon cytosol was incubated with 1.3 nM 1,25-(OH)J3H]D3 + radioinert competitor analogues to determine the relative affinity of a variety of related steroids for the receptor sites. As shown in Fig. 3, 1,25-(OH),D, was the best competitor reducing the binding of 1,25-(OH)J3H]D3 to 500/, at approximately an equivalent concentration of radioinert steroid. The potency of the other metabolites relative to 1,25-(OH),D, was 25-(OH)D3 5 2.7x, 1r-(OH)D3 2 1.3% and 24,25-(OH),D, 5 0.4%. Distribution
analysis
The distribution of 1,25-(OH),D, receptors was assessed throughout the gastrointestinal tract and the data are shown in Table 1. Since the 5 6 S 25-(OH)D3 binding protein was not detected in our scraped and washed cells, a single point assay at a saturating concentration of 1,25-(OH)2D3 was employed in these experiments. The highest concentration of receptor sites was seen in duodenum followed by jejunum and ileum Although less than small intestine, colon had substantial numbers of sites: the cecum and proximal colon had the same content while distal colon was
1,25-(OH)2Da receptors in colon
(B) ‘:.
0.32
2 5 E
317
Kd = 0.18 nM
: :
N max = 203 fmollmg
0.24
d 0” iz “I ,N
0.16
8 fi ; 0.08
-. :.
I 0
0.2
1.25-(OH)2[3HI
0.4
0.6
0.8
0 0
1.0
O3 CONCENTRATION
20
40
1.25-(OH)213HI
(nM)
60
80
100
D3 BOUND (fmolhnl)
Fig. 2. A. Saturation analysis of 1,25-(OH)2[3H]Da binding in colon cytosol. Cytosol was incubated with increasing concentrations of 1,25-(OH)2[3H]Da (0.13 to 0.95 nM) + 250-fold radioinert 1.25-(OH)zD, to determine non-specific binding. B. Scatchard plot of specific binding data from Fig. 2A. The regression line was calculated by least squares.
somewhat lower. Esophagus only negligible
and stomach
exhibited
binding.
DISCUSSION
The data indicate the presence of a high affinity binder in mouse colon with properties similar to the
1,25-(OH),D, receptor previously demonstrated in other mammalian organs including small intestine [7-181. These properties include a 3.2s sedimen-
_.
1.3
13
tation coefficient in gradients prepared in hypertonic buffers, an affinity of 0.2 nM for l,25-(OH)2D3 and a pronounced preference to bind l,25-(OH),D, compared to other vitamin D analogues. The presence of receptors in small intestine and colon prompted us to examine the entire gastro intestinal tract for receptors. The results (Table 1) indicate a selective distribution of 1,25-(OH)*D3 receptors. All segments of small and large intestine possess receptors with the greatest concentration being present in duodenum and decreasing numbers of sites found as
130
COMPETITOR CONCENTRATION
1300 [nMl
Fig. 3. Competitive binding assay of 1,25-(OH),[3H]Da binding in colon cytosol. Cytosol was incubated with 1.3 nM 1.25~(OH)J3H]D, + competitors at the indicated concentrations. Specific binding in the absence of competitors was taken as the control or 100% value and averaged 200 & 20 fmol/mg cytosol protein. Values shown are means + SE.
318
MARGARETA. HIRST and DAVIDFELDMAN Table 1. Specific 1,25-(OH),Ds sites along the gastrointestinal
Zone Esophagus Stomach Duodenum Jejunum Ileum Cecum Proximal colon Distal colon
binding tract
Specific binding (fmol/mg protein) 8 (2.4-10.1) 12(7.1-16) 527 (481-581) 384 (33&430) 344 (285405) 274 (224-340) 269 (23 l-296) 160(156167)
Binding was assessed with a single point assay at a saturating concentration (1.3 nM) of l.25-(OH)z[3H]D,. Corrections were made for non-specific binding. The values shown represent means and ranges of three experiments.
one proceeds distally. Esophagus and stomach exhibited negligii;le numbers of binding sites. These biochemical measurements of receptors correlate well with the recent studies of Stumpf et al. employing a radio-autographic approach [25]. Those workers demonstrated 1,25-(OH)J3H]D, localization in both small and large intestine with a somewhat stronger localization in the small intestine. Furthermore, they noted that the epithelial cells of the stomach were generally unlabelled except for dispersed individual cells in basal portions of gastric glands. The presence of receptors in only a small subpopulation of cells may explain our tinding of a low level of receptors when assayed in the total stomach epithelial layer. It has been shown that mammalian colon exhibits vitamin D dependent increases in calcium transport and calcium binding protein [19-211. However, it is of interest that although a rise in calcium binding protein was found in the cecum after vitamin D treatment, no increase in net calcium flux was detected in this segment [20] nor did 1,25-(OH),D, effect phosphate absorption in the proximal colon as it does in the small intestine [21]. These dissociated responses are intriguing in view of the similarity of the receptors in cecum and colon to the small intestine where both calcium and phosphate transport are stimulated. Multiple other examples exist where the effects of a steroid hormone are discrepant in various target organs despite an apparently identical receptor [26]. In conclusion, the colon is an additional organ which possesses receptors for 1,25-(OH),D,. The receptors appear similar in their properties to the receptors previously described in small intestine and other target organs. The gastrointestinal tract has a selective distribution of receptors for 1,25-(OH)zD3; the concentration is very low in esophagus and stomach and substantial in small and large intestine. The presence of receptors coupled with documented functions of 1,25-(OH),D, in colon indicates that this segment of the gastrointestinal tract is also a 1,25-(OH)zD, target organ.
Acknowledgements-Supported from the USPHS.
by NIH grant AG 01355
REFERENCES
1. Lawson D. E. M. (Editor) Vitamin D. Academic Press, London (1978). 2. DeLuca H. F. and Schnoes H. K.: Metabolism and mechanism of action of vitamin D. Ann. Reti. Biochem. 45 (1976) 631-666. 3. Haussler M. R. and McCain T. A.: Basic and clinical concepts related to vitamin D metabolism and action. N. Eng. J. Med. 297 (1977) 974-983. 4. Norman A. W. and Wecksler W. R.: Vitamin D. recentars and biologic responses. In Receptors and Hormone Action (Edited bv B. W. O’Mallev and L. Birnbaumer). Academic Press,
1.25(OH),D,
19.
20.
21.
22.
receptors in colon
cancer cell line (MCF 7 cells) Biochem. biophys. Rrs. Commun. 93 (1980) 9-l 5. Harrison H. C. and Harrison H. E.: Calcium transport by rat colon in uirro. Am. J. Physiol. 217 (1969) 121-125. Petith M. M., Wilson H. D. and Schedl H. P.: Vitamin D dependence of in ho calcium transport and mucosal calcium binding protein in rat large intestine. Gasrroenterology 76 (1979) 99-l 04. Lee D. B. N., Walling M. W., Gafter U., Silis V. and Coburn J. W.: Calcium and inorganic phosphate transport in rat colon. J. din. Inuest. 65 (1980) 1326133 I. Bradford M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biothem. 72 (I 976) 248-254.
319
23. Haddad J. G. and Birge S. J.: Widespread. specific binding of 25-hydroxycholecalciferol in rat tissues. J. biol. Chem. 250 (1975) 299-303. 24. VanBaelen H. V., Bouillon R. and DeMoor P.: Binding of 25-hydroxycholecalciferol in tissues. J. biol. Chem. 252 ( 1977) 25 15-25 18. 25. Stumpf W. E., Sar M., Reid F. A.. Tanaka Y. and DeLuca H. F.: Target cells for 1.25-dihydroxyvitamin D, in intestinal tract, stomach, kidney. skin. pituitary and parathyroid. Sckrnce 206 (1979) I 188-l 190. 26. Feldman D.: Glucocorticoid receptors and regulation of phosphoenolpyruvate-carboxykinase activity in rat kidney and adipose tissue. Ant. J. Physiol. 233 (1977) El477151.