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CONJUGATED BILE ACID UPTAKE BY XENOPUS LAEVIS OOCYTES INDUCED BY MICROINJECTION WITH ILEAL POLY A + mRNA* Steven Sorscher, 1,z3 Jan Lillienau, 4.5 Judy L. Meinkoth, 3 Joseph H. Steinbach, 5 Claudio D. Schteingart, 5 James Feramisco, 3 and Alan F. Hofmann ~ Departments of Medicine and Pharmacology, University of California, San Diego La Jolla, California 92093 Received June 30, 1992
SUMMARY: Apical membranes of ileal enterocytes contain the major Na+/bile acid cotransporter activity in mammals. Microinjection of guinea pig ileal mucosal Poly A + mRNA (25 ng) into Xenopus oocytes resulted in 22,23-3H-cholyltaurine uptake at day 3 after injection (453 fmol/oocyte-hr), while control viral mRNA (25 ng) gave an uptake rate of 133 fmol/ oocyte-hr. The transport rate increased in direct relationship to the concentration of injected mRNA, cholyltaurine, or Na + in the incubation media. Uptake of cholyltaurine using rabbit ileal mucosal Poly A + mRNA was 3891 fmole/oocyte-hr compared to rabbit jejunal-mucosa Poly A + mRNA (control) injections inducing 728 fmol/oocyte-hr. Such expression of the ileal Na+/bile acid cotransporter may facilitate cloning of this key mammalian gene. e 1992 A c a d e m i c P r e s s , I n c .
Active bile acid transport by the terminal ileum is integral to the normal enterohepatic circulation of bile acids and is essential for normal gastrointestinal function and cholesterol metabolism. Active bile acid transport by ileal, but not jejunal tissue was shown by Lack and Weiner in 1961 (1), although observations of ileal bile acid absorption were made nearly a century earlier (2). Since then, the apical surface of enterocytes in the distal ileum has been
*This work was supported in part by NIH grants DK21506 and DK32130 (AFH) and funding from the Burroughs-Wellcome Company (AFH), as well as other NIH grants (JF). 1To whom correspondence should be addressed at present address: Department of Medicine, 0636, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636. 2Recipient of NIH Physician-Scientist Award. 3UCSD Cancer Center, Department of Medicine, University of California, San Diego, La Jolla, CA 92093. 4Visiting Fellow, Swedish Medical Research Council. Present address: Department of Physiological Chemistry, University of Lund, Lund, Sweden. SDivision of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0813.
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identified as the major site of Na ÷ dependent conjugated bile acid active transport in mammals (3,4,5). The functional properties (including Na ÷ dependency) of the transport system are fairly well described (3-5), but its molecular properties remain unclarified. Kramer and others used photolabile bile salt derivatives and photoaffinity labelling of brush border vesicles to identify a 99kD protein from the ileal brush border surface apparently involved in active bile acid transport (6). Photolysis of the ileal membrane vesicles in the presence of the photolabile bile salt derivative led to the incorporation of radioactivity into a 99kD protein. Transport of the photolabile bile salt derivative was stimulated by sodium and inhibited by taurocholate (7,8).
More recently, Shneider and coworkers have isolated a
100kD protein (present in mature ileal, but not jejunal tissue) using affinity chromatography with a conjugated bile acid agarose column (9). Since purified, transport protein (and protein sequence) or antibodies to the protein are unavailable, we have begun studies of this gene using expression of ileal mucosal Poly A ÷ mRNA in Xenopus oocytes as a possible approach to isolation of the gene. Xenopus oocytes have been used as an expression system to clone several genes (10,11), including the Na+/glucose cotransporter (12).
Here we provide evidence that
Xenopus laevis oocytes express bile acid transport activity after injection with total ileal mucosa Poly A ÷ mRNA from guinea pig and rabbit. This transport appears to be functional in that the activity was dependent upon the Na ÷ and bile acid concentration and the amount of injected mRNA. METHODS Radiochemicals, Biochemicals, and Animals: 22,23 3[H] cholyltaurine of high specific activity (58 Ci/mmole) was prepared as follows. Cholic acid was converted to its performyl derivative, 3~,7tz,12tz-triformyloxy-(513)-cholan-24-oic acid (13), which was treated with N-bromosuccinimide in a mixture of trifluoroacetic acid and trifluoroacetic anhydride at 23°C for 16 hs to give 23~-bromo-3~,7~,12~-triformyloxy-cholan-24-oic acid (14). This was esterified with diazomethane in ether and the resulting methyl ester was heated at 120°C in hexamethyl phosphoramide for 16 hs. Basic hydrolysis of the crude product and column chromatography purification gave (E)-3~,7~,12tz- trihydroxy-(5 ~)-cholen-22-en-24-oic acid. Conjugation with taurine by means of the coupling agent EEDQ (2-ethoxy-1- ethoxycarbonyl-l,2-dihydroquinolin) (15) gave sodium (E) A22-cholyltaurine. All compounds were satisfactorily characterized by 1H-NMR at 360 MHz. Sodium (E)-A22-taurocholate was reduced with pure tritium gas with 5% Pd on charcoal as catalyst in methanol at the National Tritium Facility (Berkeley, CA). The product was purified by reverse phase thin layer chromatography (KC18, Whatmann, Clifton, NJ) with methanol:water, 70:30, containing 5% w/v silver nitrate in the mobile phase. The band containing the product was eluted with methanol, and silver was eliminated by passage through a short Dowex 50-X4 exchange resin column (in the sodium form). Methanol was evaporated, the sample reconstituted with water, and desalted by means of a reverse phase cartridge (RPC18 Bond Elut, Varian, Harbor City, CA). The specific activity of the final 1456
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product was 58 Ci/mmol, and the position of the label was verified by 3H-NMR. Full details will be reported elsewhere. Poly A + mRNA was from ileal mucosa isolated using the "Fast Track" isolation kit (Invitrogen Corporation, San Diego, CA). A n in vitro translation kit utilizing rabbit reticulocyte lysate, 35S-methionine, and Brome mosaic viral m-RNA, was obtained from Promega Corp., Madison, WI. Guinea pigs, weighing 350-400 mg, were obtained from the Charles River Breeding Laboratories, Kingston, RI. In other experiments, rabbits obtained from Holbert's Rabbitry, Spring Valley, CA were used. Xenopus laevis frogs were purchased from Nasco Corp., Atkinson, WI and maintained under standard conditions at the Salk Research Institute, La Jolla, CA. Experimental Methods RNA Isolation. Guinea pigs or rabbits were used as a source of ileal mucosal Poly A + mRNA since studies from this laboratory have shown that the guinea pig or rabbit ileum actively transport conjugated bile acids using methodology developed for the rat (3). Guinea pigs were sacrificed by COz exposure, and rabbits were euthanized with pentobarbital administration. The last one quarter of the guinea pig or rabbit ileum or one quarter of the rabbit jejunum was removed, rinsed quickly with 0.9% saline, and its mucosa removed by scraping with the edge of a glass microscope slide. Scrapings were placed immediately on dry ice. Pooled scrapings from five animals were homogenized in 4M guanidinium thiocyanate containing 25 mmol/L sodium citrate pH 7, 0.5% (w/v) N-lauroylsarcosine, sodium salt (Sigma Chemical Co., St. Louis, MO), 0.1 mmol/L 13-mercaptoethanol, 1-2 drops Antifoam A emulsion (Sigma Chemical Co., St. Louis, MO). Total Poly A + mRNA was isolated as described by MacDonald (16), except that Poly A + mRNA was adsorbed to and eluted from oligo d(t) pellets. To assess the size and intactness of the Poly A + mRNA, Poly A + was analyzed by formaldehyde/agarose gel electrophoresis and in vitro translation (Promega, Madison, WI), and the yield of Poly A + mRNA was estimated by measuring the absorbance at 260 nm. Expression in Oocytes Ovaries were surgically removed from frogs anesthetized with ethyl 3-aminobenzoate. Oocytes were separated from ovarian debris and the follicular cell layer using Sigma Type I collagenase and then maintained in calcium free modified Barth's solution (88 mmol/L NaC1, 1 mmol/L KC1, 2.4 mmol/L NaHCO3, 15 mmol/L HEPES, 0.8 mmol/L MgSO4 containing 10 mg/L of sodium penicillin and 10 mg/L streptomycin sulfate). Stage 5 or 6 oocytes were individually selected by appearance (18-20) and placed in modified Barth's solution (88 mmol/L NaC1, 1 mmol/L KC1, 2.4 mmol/L NaHC03, 15 mmol/L HEPES, 0.3 mmol/L Ca (N03), 0.4 mmol/L CaCI2, 0.8 mmol/L Mg SO4). Each cell was injected with 0-100 ng of ileal or jejunal Poly A + mRNA or Brome mosaic viral mRNA in 25 nL H20 according to standard methods (18-20). Uninjected oocytes or oocytes injected with HzO alone were also used as controls. Bile Acid Uptake Modified Barth's solution was changed twice daily and cells that appeared degraded were discarded. Assay for bile acid uptake was performed at day 3 post injection. Except in "Na + free" experiments, cells were incubated in modified Barth's solution with 22,23 3[H] cholyltaurine (see above). Except where stated otherwise, the incubation solution was adjusted to 15 i.tmol/L cholyltaurine by adding chromatographically pure unlabelled cholyltaurine to 100 p.Ci of labelled taurocholate (see above) to allow measured counts of generally greater than 1000 dmp per oocyte. Incubation was for 30 min at 22°C. In "Na + free" experiments, cells were first washed in Na + free Barth's (88 mmol/L choline chloride, 1 mmol/L KC1, 2.4 rnmol/L choline bicarbonate, 15 mmol/L HEPES, 0.3 mmol/L Ca (N03)z, 0.4 mmol/L CaClz, 0.8 mmol/L Mg SO4) and incubated in 15 I~mol/L cholyltaurine in Na + free modified Barth's solution. After incubation, the cells were rinsed with 1 mmol/L cold cholyltaurine in Na + free modified Barth's solution to remove adsorbed 3H-cholyltaurine. Each oocyte was then 1457
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quickly examined under a dissecting microscope and discarded if not intact. Intact cells were placed in individual scintillation vials and solubilized with 350 p.L of 88% formic acid. Scintillation fluid (Scintiverse E, Fisher Scientific, Fairlawn, NJ) was added and radioactivity measured in a Beckman LS 3150T liquid scintillation analyzer. Uptake for noninjected, viral injected, jejunal and ileal Poly A ÷ mRNA injected cells was directly calculated from measured dpm. For each group of cells, a mean dpm was measured. Because of small variations in the measured dpm of incubation solutions, due to changes in volume secondary to buffer adhering to oocytes during transfer, the incubation solutions were standardized relative to the solution used to incubate the control cells. Measured mean dpm was divided by the product of this corrected incubation solution value and the specific activity of the solution. Given the inherent variability in the expression of functional protein by Xenopus oocytes after microinjection of Poly A ÷ mRNA, statistical analysis of the difference between treatment groups using nonparametric methods was used. In these experiments, approximately 10% of cells assayed showed profoundly increased cholyltaurine uptake; they were assumed to be dead cells, and data obtained from these cells were not used in uptake/oocyte-hr determinations. The average number of cells assayed for each experiment was 6.8.
RESULTS
Table 1 and Table 2 represent separate experiments summarizing the uptake rates of cholyltaurine when Xenopus oocytes were injected with guinea pig ileal Poly A + mRNA, Brome mosaic virus Poly A + mRNA, or when cells were not injected.
The uptake of
cholyltaurine was proportional to the amount of ileal Poly A + mRNA injected. These results indicate that ileal Poly A ÷ mRNA was expressed in a dose-dependent manner, and that the washing procedure eliminated non-specific uptake of cholyltaurine (Tables I and 2). Similar results were seen when rabbit ileal Poly A + mRNA uptake after injection was compared with uptake after injection with rabbit jejunal Poly A + mRNA.
When compared with
standardized control or jejunal Poly A + mRNA injected cells, micro-injected rabbit ileal Poly A + mRNA induced far greater concentration-dependent uptake (Table 3). Using Kruskal Wallis one way analysis of variance, there was a statistical difference (p < 0.05) between the groups represented in Table 1. With linear regression, the slope of the line (uptake versus
Table 1. Cholyltaurine uptake induced by injected ileal Poly A + mRNA versus controls (guinea pig) RNA Injected
Cholyltaurine Uptake* (fmoi/oocyte-hr)
Non-injected cells Brome mosaic virus (25 ng) Total ileal mRNA (50 ng)
132 133 453
"Average number of cells assayed was 8 for each experimental group. 1458
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Table 2.
Concentration of injected ileal Poly A + mRNA (guinea pig) and bile acid uptake
RNA Injected
Cholyltaurine Uptake* (fmol/oocyte-hr)
Viral mRNA (25 ng) Ileal mRNA (25 ng) Ileal mRNA (50 ng)
191 598 892
"Average number of cells assayed was 11 for each experimental group.
injected Poly A + RNA) for Table 2 was non-zero. Attempting to fit a quadratic formula, the coefficient of the second order term was not significantly different from zero. Thus, it appears that the uptake of bile acid is proportional to the amount of Poly A + R N A injected in these experiments.
Concentration dependence.
Table 4 shows the relationship between the medium
concentration of cholyltaurine and the uptake rate of guinea pig ileal Poly A + m R N A injected oocytes. Uptake increased in relationship to concentration (p < 0.05).
Again,
Kruskal Wallis one way analysis demonstrated a statistically significant difference (p < 0.05) between the groups.
Higher concentrations were not studied in order to conserve
radioactivity.
Sodium dependence. When Na + in the incubation medium was replaced by choline, uptake decreased by a factor of 1.6 and uptake in the absence of Na + did not differ significantly from uptake induced by viral m R N A (Table 5).
Table 3.
Cholyltaurine uptake induced by injected ileal Poly A + mRNA (rabbit) versus control
Cholyltaurine Uptake*
RNA Injected
(fmol/oocyte-hr) Non injected Jejunum (25 ng) Ileum (25 ng) lleum (50 ng)
778 728 3891 9085
"Average number of cells assayed was 9 for each experimental group. 1459
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Table 4. Bile acid uptake induced by xenopus oocytes Injected RNA/Incubation Solution Concentration (ixM)
Cholyltaurine Uptake* (fmol/oocyte-hr)
Virus/15 Virus/50 Ileal/15 Ileal/50 Ileal/100
132 506 453 932 1889
*Average number of cells assayed was 7 for each experimental group.
DISCUSSION These results indicate that ileal Poly A + m R N A of guinea pig and rabbit is translated in the Xenopus oocyte to form a functional Na+/bile acid cotransporter that is expressed in its surface membrane.
Uptake induced by injection of rabbit ileal Poly A + m R N A was
approximately ten times as great as that induced by injection of guinea pig ileal Poly A + mRNA, suggesting that the rabbit may be a more useful source of Poly A + m R N A in further expression cloning experiments. Hagenbuch and his colleagues have reported analogous studies with the Na + coupled bile acid transport protein of the basolateral (sinusoidal) membrane of the hepatocyte (17). However, based on substrate specificity of the transport system in the whole animal (22), this protein is unlikely to be the ileal bile acid transporter, at least in the rat (3). It may be possible to use membranes prepared from injected oocytes to prepare monoclonal antibodies to the transporter, as Tigyi et al. have reported for pinceau terminals
Table 5. Sodium dependency of bile acid uptake (fmol/oocyte-hr) mRNA injected
Non-injected Viral mRNA (25 ng) Ileal mRNA (50 ng)
Uptake from incubation solution containing Na ÷
Uptake from incubation solution lacking Na ÷
(fmol/oocyte-hr)
(fmol/oocyte-hr)*
656 621 1206
811 768
"Average number of cells assayed was 11 for each experimental group. 1460
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(21). It may also be possible to purify the Poly A + mRNA and then use the Xenopus system as a bioassay. Wright and his colleagues cloned the Na + coupled glucose transport protein of the rabbit jejunum in this manner (12) and subsequently defined its substrate specificity using Xenopus oocytes (23,24). The Tm,x of the bile acid transport system (3) is quite similar in magnitude to values calculated for the Tm~x of the jejunal glucose transporter (25), suggesting the bile acid transporter might constitute a major fraction of apical membrane protein in the ileum. Efforts to isolate the protein directly would appear reasonable as well as the molecular biological approach described here.
ACKNOWLEDGMENTS
We thank Dr. Alexander Susan of Sandoz for assistance in the tritiation of the cholyltaurine and Vincent Dionne and Ann O'Shea-Greenfield for technical assistance. This work was supported in part by NIH grants DK21506 and DK 32130 (AFH) and funding from the Burroughs-Wellcome Company (AFH) and other NIH grants (JF). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Lack, L., and Weiner, I.M. (1961) Am. J. Physiol. 200, 313-317. Tappeiner, A.J. (1867) Wien. Akad. Sitzber. 77, 281-304. Marcus, S.N., Schteingart, C.D., Marquez, M.L., Hofmann, A.F., Xia, Y., Steinbach, J.H., Ton-Nu, H-T., Lillienau, J., Angellotti, M.A., and Schmassmann, A. (1991) Gastroenterology 100, 212-221. Wilson, F.A. (1981) Am. J. Physiol. 241, G83-G92. Schwenk, M. (1985) In Enterohepatic Circulation of Bile Acids and Sterol Metabolism pp. 109-119. MTP Press Limited, Lancaster. Kramer, W., Burckhardt, G., Wilson, F., and Kurz, G. (1983) J. Biol. Chem. 258, 3623-3627. Burckhardt, G., Kramer, W., Kurz, G., and Wilson, F. (1983) J. Biol. Chem. 258, 3618-3622. Burckhardt, G., Kramer, W., Kurz, G., and Wilson, F. (1987) Biochem. Biophys. Res. Commun. 143, 1018-1023. Shneider, B.L., Ananthanarayanan, M., Moyer, M.S., Insler, N.F., and Suchy, F.J. (1990) Gastroenterology 100, A796. Hollman, M., O'Shea-Greenfield, A., Rogers, S., and Heinemann, S. (1989) Nature 342, 643-648. Kobilka, B., Matsui, H., Kobilka, T., Yang-Feng, T., Francke, U., Caron, M., Lefkowtiz, R., and Regan, J. (1987) Science 238, 650. Hediger, M., Coady, M., Ikeda, T., and Wright, E. (1987) Nature 330, 379-381. Tserng, K-Y., and Klein, P.D. (1977) Steroids 29, 635-648. Ranganathan, R.S., and Radhakrishna-Pillai, K.M. (1988) Third Chemical Conf. of North America, Toronto, Canada, Abstract #458. Tserng, K-Y., Hachey, D.L., and Klein, P.D. (1977) J. Lipid Res. 18, 404-407. MacDonald, R., Swift, G., Przybyla, A., and Chirgwin, J. (1987) In Methods in Enzymology: Guide to Molecular Cloning Techniques, pp. 219-227. Academic Press, Inc., San Diego, CA. 1461
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Hagenbuch, B., Lubbart, H., Stieger, B., and Meier, P. (1990) J. Biol. Chem. 265, 5357. Jagus, R. (1987) In Methods in Enzymology: Guide to Molecular Cloning Techniques, pp. 296-306. Academic Press, Inc., San Diego, CA. Marcus-Sekura, C.J., and Hitchcock, M.J.M. (1987) In Methods in Enzymology: Guide to Molecular Cloning Techniques, pp. 284-287. Academic Press, Inc., San Diego. Melton, D.A. In Methods in Enzymology: Guide to Molecular Cloning Techniques, pp. 288-295. Academic Press, Inc., San Diego, CA. Tigyi, G., Matute, C., and Miledi, R. (1990) Proc. Natl. Acad. Sci. USA 2. 528-532. Shneider, B., Ananthanarayanan, M., Schteingart, C., Michaud, G., Hagenbuch, B., Meier, P., Hofmann, A., and Suchy, F. (1992) Gastroenterology 102, A888 [abstract]. Ikeda, T.S., Hwang, E.S., Coady, M.J., Hirayama, B.A., Hediger, M.A., and Wright, E.M. (1989) J. Membrane Biol. 110, 87-95. Wright, E., Hediger, M.A., Coady, M.J., Hirayama, B., and Turk, E. (1989) Biochem. Soc. Transactions 17, 810-811. Pappenheimer, J.R. (1990) Am. J. Physiol. 259, G290-G299.
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CONJUGATED BILE ACID UPTAKE BY XENOPUS LAEVIS OOCYTES INDUCED BY MICROINJECTION WITH ILEAL POLY A + mRNA* Steven Sorscher, 1,z3 Jan Lillienau, 4.5 Judy L. Meinkoth, 3 Joseph H. Steinbach, 5 Claudio D. Schteingart, 5 James Feramisco, 3 and Alan F. Hofmann ~ Departments of Medicine and Pharmacology, University of California, San Diego La Jolla, California 92093 Received June 30, 1992
SUMMARY: Apical membranes of ileal enterocytes contain the major Na+/bile acid cotransporter activity in mammals. Microinjection of guinea pig ileal mucosal Poly A + mRNA (25 ng) into Xenopus oocytes resulted in 22,23-3H-cholyltaurine uptake at day 3 after injection (453 fmol/oocyte-hr), while control viral mRNA (25 ng) gave an uptake rate of 133 fmol/ oocyte-hr. The transport rate increased in direct relationship to the concentration of injected mRNA, cholyltaurine, or Na + in the incubation media. Uptake of cholyltaurine using rabbit ileal mucosal Poly A + mRNA was 3891 fmole/oocyte-hr compared to rabbit jejunal-mucosa Poly A + mRNA (control) injections inducing 728 fmol/oocyte-hr. Such expression of the ileal Na+/bile acid cotransporter may facilitate cloning of this key mammalian gene. e 1992 A c a d e m i c P r e s s , I n c .
Active bile acid transport by the terminal ileum is integral to the normal enterohepatic circulation of bile acids and is essential for normal gastrointestinal function and cholesterol metabolism. Active bile acid transport by ileal, but not jejunal tissue was shown by Lack and Weiner in 1961 (1), although observations of ileal bile acid absorption were made nearly a century earlier (2). Since then, the apical surface of enterocytes in the distal ileum has been
*This work was supported in part by NIH grants DK21506 and DK32130 (AFH) and funding from the Burroughs-Wellcome Company (AFH), as well as other NIH grants (JF). 1To whom correspondence should be addressed at present address: Department of Medicine, 0636, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636. 2Recipient of NIH Physician-Scientist Award. 3UCSD Cancer Center, Department of Medicine, University of California, San Diego, La Jolla, CA 92093. 4Visiting Fellow, Swedish Medical Research Council. Present address: Department of Physiological Chemistry, University of Lund, Lund, Sweden. SDivision of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0813.
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identified as the major site of Na ÷ dependent conjugated bile acid active transport in mammals (3,4,5). The functional properties (including Na ÷ dependency) of the transport system are fairly well described (3-5), but its molecular properties remain unclarified. Kramer and others used photolabile bile salt derivatives and photoaffinity labelling of brush border vesicles to identify a 99kD protein from the ileal brush border surface apparently involved in active bile acid transport (6). Photolysis of the ileal membrane vesicles in the presence of the photolabile bile salt derivative led to the incorporation of radioactivity into a 99kD protein. Transport of the photolabile bile salt derivative was stimulated by sodium and inhibited by taurocholate (7,8).
More recently, Shneider and coworkers have isolated a
100kD protein (present in mature ileal, but not jejunal tissue) using affinity chromatography with a conjugated bile acid agarose column (9). Since purified, transport protein (and protein sequence) or antibodies to the protein are unavailable, we have begun studies of this gene using expression of ileal mucosal Poly A ÷ mRNA in Xenopus oocytes as a possible approach to isolation of the gene. Xenopus oocytes have been used as an expression system to clone several genes (10,11), including the Na+/glucose cotransporter (12).
Here we provide evidence that
Xenopus laevis oocytes express bile acid transport activity after injection with total ileal mucosa Poly A ÷ mRNA from guinea pig and rabbit. This transport appears to be functional in that the activity was dependent upon the Na ÷ and bile acid concentration and the amount of injected mRNA. METHODS Radiochemicals, Biochemicals, and Animals: 22,23 3[H] cholyltaurine of high specific activity (58 Ci/mmole) was prepared as follows. Cholic acid was converted to its performyl derivative, 3~,7tz,12tz-triformyloxy-(513)-cholan-24-oic acid (13), which was treated with N-bromosuccinimide in a mixture of trifluoroacetic acid and trifluoroacetic anhydride at 23°C for 16 hs to give 23~-bromo-3~,7~,12~-triformyloxy-cholan-24-oic acid (14). This was esterified with diazomethane in ether and the resulting methyl ester was heated at 120°C in hexamethyl phosphoramide for 16 hs. Basic hydrolysis of the crude product and column chromatography purification gave (E)-3~,7~,12tz- trihydroxy-(5 ~)-cholen-22-en-24-oic acid. Conjugation with taurine by means of the coupling agent EEDQ (2-ethoxy-1- ethoxycarbonyl-l,2-dihydroquinolin) (15) gave sodium (E) A22-cholyltaurine. All compounds were satisfactorily characterized by 1H-NMR at 360 MHz. Sodium (E)-A22-taurocholate was reduced with pure tritium gas with 5% Pd on charcoal as catalyst in methanol at the National Tritium Facility (Berkeley, CA). The product was purified by reverse phase thin layer chromatography (KC18, Whatmann, Clifton, NJ) with methanol:water, 70:30, containing 5% w/v silver nitrate in the mobile phase. The band containing the product was eluted with methanol, and silver was eliminated by passage through a short Dowex 50-X4 exchange resin column (in the sodium form). Methanol was evaporated, the sample reconstituted with water, and desalted by means of a reverse phase cartridge (RPC18 Bond Elut, Varian, Harbor City, CA). The specific activity of the final 1456
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product was 58 Ci/mmol, and the position of the label was verified by 3H-NMR. Full details will be reported elsewhere. Poly A + mRNA was from ileal mucosa isolated using the "Fast Track" isolation kit (Invitrogen Corporation, San Diego, CA). A n in vitro translation kit utilizing rabbit reticulocyte lysate, 35S-methionine, and Brome mosaic viral m-RNA, was obtained from Promega Corp., Madison, WI. Guinea pigs, weighing 350-400 mg, were obtained from the Charles River Breeding Laboratories, Kingston, RI. In other experiments, rabbits obtained from Holbert's Rabbitry, Spring Valley, CA were used. Xenopus laevis frogs were purchased from Nasco Corp., Atkinson, WI and maintained under standard conditions at the Salk Research Institute, La Jolla, CA. Experimental Methods RNA Isolation. Guinea pigs or rabbits were used as a source of ileal mucosal Poly A + mRNA since studies from this laboratory have shown that the guinea pig or rabbit ileum actively transport conjugated bile acids using methodology developed for the rat (3). Guinea pigs were sacrificed by COz exposure, and rabbits were euthanized with pentobarbital administration. The last one quarter of the guinea pig or rabbit ileum or one quarter of the rabbit jejunum was removed, rinsed quickly with 0.9% saline, and its mucosa removed by scraping with the edge of a glass microscope slide. Scrapings were placed immediately on dry ice. Pooled scrapings from five animals were homogenized in 4M guanidinium thiocyanate containing 25 mmol/L sodium citrate pH 7, 0.5% (w/v) N-lauroylsarcosine, sodium salt (Sigma Chemical Co., St. Louis, MO), 0.1 mmol/L 13-mercaptoethanol, 1-2 drops Antifoam A emulsion (Sigma Chemical Co., St. Louis, MO). Total Poly A + mRNA was isolated as described by MacDonald (16), except that Poly A + mRNA was adsorbed to and eluted from oligo d(t) pellets. To assess the size and intactness of the Poly A + mRNA, Poly A + was analyzed by formaldehyde/agarose gel electrophoresis and in vitro translation (Promega, Madison, WI), and the yield of Poly A + mRNA was estimated by measuring the absorbance at 260 nm. Expression in Oocytes Ovaries were surgically removed from frogs anesthetized with ethyl 3-aminobenzoate. Oocytes were separated from ovarian debris and the follicular cell layer using Sigma Type I collagenase and then maintained in calcium free modified Barth's solution (88 mmol/L NaC1, 1 mmol/L KC1, 2.4 mmol/L NaHCO3, 15 mmol/L HEPES, 0.8 mmol/L MgSO4 containing 10 mg/L of sodium penicillin and 10 mg/L streptomycin sulfate). Stage 5 or 6 oocytes were individually selected by appearance (18-20) and placed in modified Barth's solution (88 mmol/L NaC1, 1 mmol/L KC1, 2.4 mmol/L NaHC03, 15 mmol/L HEPES, 0.3 mmol/L Ca (N03), 0.4 mmol/L CaCI2, 0.8 mmol/L Mg SO4). Each cell was injected with 0-100 ng of ileal or jejunal Poly A + mRNA or Brome mosaic viral mRNA in 25 nL H20 according to standard methods (18-20). Uninjected oocytes or oocytes injected with HzO alone were also used as controls. Bile Acid Uptake Modified Barth's solution was changed twice daily and cells that appeared degraded were discarded. Assay for bile acid uptake was performed at day 3 post injection. Except in "Na + free" experiments, cells were incubated in modified Barth's solution with 22,23 3[H] cholyltaurine (see above). Except where stated otherwise, the incubation solution was adjusted to 15 i.tmol/L cholyltaurine by adding chromatographically pure unlabelled cholyltaurine to 100 p.Ci of labelled taurocholate (see above) to allow measured counts of generally greater than 1000 dmp per oocyte. Incubation was for 30 min at 22°C. In "Na + free" experiments, cells were first washed in Na + free Barth's (88 mmol/L choline chloride, 1 mmol/L KC1, 2.4 rnmol/L choline bicarbonate, 15 mmol/L HEPES, 0.3 mmol/L Ca (N03)z, 0.4 mmol/L CaClz, 0.8 mmol/L Mg SO4) and incubated in 15 I~mol/L cholyltaurine in Na + free modified Barth's solution. After incubation, the cells were rinsed with 1 mmol/L cold cholyltaurine in Na + free modified Barth's solution to remove adsorbed 3H-cholyltaurine. Each oocyte was then 1457
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quickly examined under a dissecting microscope and discarded if not intact. Intact cells were placed in individual scintillation vials and solubilized with 350 p.L of 88% formic acid. Scintillation fluid (Scintiverse E, Fisher Scientific, Fairlawn, NJ) was added and radioactivity measured in a Beckman LS 3150T liquid scintillation analyzer. Uptake for noninjected, viral injected, jejunal and ileal Poly A ÷ mRNA injected cells was directly calculated from measured dpm. For each group of cells, a mean dpm was measured. Because of small variations in the measured dpm of incubation solutions, due to changes in volume secondary to buffer adhering to oocytes during transfer, the incubation solutions were standardized relative to the solution used to incubate the control cells. Measured mean dpm was divided by the product of this corrected incubation solution value and the specific activity of the solution. Given the inherent variability in the expression of functional protein by Xenopus oocytes after microinjection of Poly A ÷ mRNA, statistical analysis of the difference between treatment groups using nonparametric methods was used. In these experiments, approximately 10% of cells assayed showed profoundly increased cholyltaurine uptake; they were assumed to be dead cells, and data obtained from these cells were not used in uptake/oocyte-hr determinations. The average number of cells assayed for each experiment was 6.8.
RESULTS
Table 1 and Table 2 represent separate experiments summarizing the uptake rates of cholyltaurine when Xenopus oocytes were injected with guinea pig ileal Poly A + mRNA, Brome mosaic virus Poly A + mRNA, or when cells were not injected.
The uptake of
cholyltaurine was proportional to the amount of ileal Poly A + mRNA injected. These results indicate that ileal Poly A ÷ mRNA was expressed in a dose-dependent manner, and that the washing procedure eliminated non-specific uptake of cholyltaurine (Tables I and 2). Similar results were seen when rabbit ileal Poly A + mRNA uptake after injection was compared with uptake after injection with rabbit jejunal Poly A + mRNA.
When compared with
standardized control or jejunal Poly A + mRNA injected cells, micro-injected rabbit ileal Poly A + mRNA induced far greater concentration-dependent uptake (Table 3). Using Kruskal Wallis one way analysis of variance, there was a statistical difference (p < 0.05) between the groups represented in Table 1. With linear regression, the slope of the line (uptake versus
Table 1. Cholyltaurine uptake induced by injected ileal Poly A + mRNA versus controls (guinea pig) RNA Injected
Cholyltaurine Uptake* (fmoi/oocyte-hr)
Non-injected cells Brome mosaic virus (25 ng) Total ileal mRNA (50 ng)
132 133 453
"Average number of cells assayed was 8 for each experimental group. 1458
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Table 2.
Concentration of injected ileal Poly A + mRNA (guinea pig) and bile acid uptake
RNA Injected
Cholyltaurine Uptake* (fmol/oocyte-hr)
Viral mRNA (25 ng) Ileal mRNA (25 ng) Ileal mRNA (50 ng)
191 598 892
"Average number of cells assayed was 11 for each experimental group.
injected Poly A + RNA) for Table 2 was non-zero. Attempting to fit a quadratic formula, the coefficient of the second order term was not significantly different from zero. Thus, it appears that the uptake of bile acid is proportional to the amount of Poly A + R N A injected in these experiments.
Concentration dependence.
Table 4 shows the relationship between the medium
concentration of cholyltaurine and the uptake rate of guinea pig ileal Poly A + m R N A injected oocytes. Uptake increased in relationship to concentration (p < 0.05).
Again,
Kruskal Wallis one way analysis demonstrated a statistically significant difference (p < 0.05) between the groups.
Higher concentrations were not studied in order to conserve
radioactivity.
Sodium dependence. When Na + in the incubation medium was replaced by choline, uptake decreased by a factor of 1.6 and uptake in the absence of Na + did not differ significantly from uptake induced by viral m R N A (Table 5).
Table 3.
Cholyltaurine uptake induced by injected ileal Poly A + mRNA (rabbit) versus control
Cholyltaurine Uptake*
RNA Injected
(fmol/oocyte-hr) Non injected Jejunum (25 ng) Ileum (25 ng) lleum (50 ng)
778 728 3891 9085
"Average number of cells assayed was 9 for each experimental group. 1459
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Table 4. Bile acid uptake induced by xenopus oocytes Injected RNA/Incubation Solution Concentration (ixM)
Cholyltaurine Uptake* (fmol/oocyte-hr)
Virus/15 Virus/50 Ileal/15 Ileal/50 Ileal/100
132 506 453 932 1889
*Average number of cells assayed was 7 for each experimental group.
DISCUSSION These results indicate that ileal Poly A + m R N A of guinea pig and rabbit is translated in the Xenopus oocyte to form a functional Na+/bile acid cotransporter that is expressed in its surface membrane.
Uptake induced by injection of rabbit ileal Poly A + m R N A was
approximately ten times as great as that induced by injection of guinea pig ileal Poly A + mRNA, suggesting that the rabbit may be a more useful source of Poly A + m R N A in further expression cloning experiments. Hagenbuch and his colleagues have reported analogous studies with the Na + coupled bile acid transport protein of the basolateral (sinusoidal) membrane of the hepatocyte (17). However, based on substrate specificity of the transport system in the whole animal (22), this protein is unlikely to be the ileal bile acid transporter, at least in the rat (3). It may be possible to use membranes prepared from injected oocytes to prepare monoclonal antibodies to the transporter, as Tigyi et al. have reported for pinceau terminals
Table 5. Sodium dependency of bile acid uptake (fmol/oocyte-hr) mRNA injected
Non-injected Viral mRNA (25 ng) Ileal mRNA (50 ng)
Uptake from incubation solution containing Na ÷
Uptake from incubation solution lacking Na ÷
(fmol/oocyte-hr)
(fmol/oocyte-hr)*
656 621 1206
811 768
"Average number of cells assayed was 11 for each experimental group. 1460
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(21). It may also be possible to purify the Poly A + mRNA and then use the Xenopus system as a bioassay. Wright and his colleagues cloned the Na + coupled glucose transport protein of the rabbit jejunum in this manner (12) and subsequently defined its substrate specificity using Xenopus oocytes (23,24). The Tm,x of the bile acid transport system (3) is quite similar in magnitude to values calculated for the Tm~x of the jejunal glucose transporter (25), suggesting the bile acid transporter might constitute a major fraction of apical membrane protein in the ileum. Efforts to isolate the protein directly would appear reasonable as well as the molecular biological approach described here.
ACKNOWLEDGMENTS
We thank Dr. Alexander Susan of Sandoz for assistance in the tritiation of the cholyltaurine and Vincent Dionne and Ann O'Shea-Greenfield for technical assistance. This work was supported in part by NIH grants DK21506 and DK 32130 (AFH) and funding from the Burroughs-Wellcome Company (AFH) and other NIH grants (JF). REFERENCES
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