- Email: [email protected]
Expression of Human Polyspecific Renal Organic Cation Transport Activity in Xenopus laevis Oocytes
NOTES
Expression of Human Polyspecific Renal Organic Cation Transport Activity in Xenopus laevis Oocytes JOANNE K. CHUN, MICHELINE PIQUETTE-MILLER, LEI ZHANG,
AND
KATHLEEN M. GIACOMINIX
Received February 11, 1996, from the Departments of Biopharmaceutical Sciences and Cellular and Molecular Pharmacology, Schools of Pharmacy and Medicine, 513 Parnassus Avenue, S-926, Box 0446, University of California, San Francisco, CA 94143-0446. Final revised manuscript received March 3, 1997. Accepted for publication March 9, 1997X. Abstract 0 Polyspecific organic cation transporters in the basolateral and brush border membrane of the kidney play a role in the elimination of many clinically important drugs and endogenous compounds. In this study we report the functional expression of organic cation transport activity in Xenopus laevis oocytes injected with poly(A)+RNA (mRNA) isolated from human kidney. Uptake of [14C]tetraethylammonium (TEA) was measured in mRNA-injected or water-injected oocytes, 4 days after injection. In oocytes injected with 50 ng of mRNA isolated from human renal cortex, the uptake of [14C]TEA was significantly increased in comparison with water-injected oocytes (7.2 ± 0.6 and 3.5 ± 0.3 pmol/ oocyte/h, respectively). Injection of 20 ng of an enriched size-fraction (fraction C) of mRNA (mean size of 2.3 kb) resulted in further enhancement of [14C]TEA uptake: [14C]TEA uptake was enhanced sixto seven-fold in oocytes injected with fraction C (23.7 ± 3.7 pmol/oocyte/ h) in comparison with water-injected oocytes. The uptake of TEA in mRNA-injected oocytes was significantly inhibited by 5 mM of unlabeled TEA, cimetidine, and N1-methylnicotinamide. These data suggest that polyspecific organic cation transport activity can be successfully expressed in Xenopus laevis oocytes injected with mRNA isolated from human kidney.
Organic cation transporters in the renal proximal tubules are involved in the elimination of various types of clinically used drugs, including antiarrhythmics, antihistamines, opiates, and sedatives, as well as endogenous amines, such as choline, dopamine, and epinephrine.1-7 The elimination of these cationic drugs and endogenous molecules involves transport across the two membranes of the epithelial cells of the renal proximal tubules: the basolateral membrane and the brush border membrane. The mechanisms of organic cation transport across each of these membranes have been studied in isolated renal plasma membrane vesicles from a number of species using the model compounds N1-methylnicotinamide (NMN) and tetraethylammonium (TEA).2,8,9 In general, the studies have shown that TEA and NMN are transported from the blood into the cell across the basolateral membrane via a facilitative, but passive, electrogenic carriermediated system.2,10-12 The driving force is the insidenegative membrane potential and/or an organic cationorganic cation exchange mechanism. Subsequently, TEA and NMN are transported into the tubule lumen across the brush border membrane via a secondarily active, organic cationproton antiporter.10,11,13-15 Recent studies from this laboratory have demonstrated the presence of an organic cation-proton antiporter that transports TEA in renal brush border membrane vesicles isolated from human kidney.16,17 X
Abstract published in Advance ACS Abstracts, May 1, 1997.
© 1997, American Chemical Society and American Pharmaceutical Association
Until recently, little was known about the molecular characteristics of the organic cation transporters at either the basolateral or brush border membrane of the renal proximal tubules. However, in 1992, Hori and co-workers demonstrated that Xenopus laevis oocytes could be successfully used as an expression system for TEA transporter(s) in rat kidney.18 Xenopus laevis oocytes are an excellent expression system for exogenous mRNA and have been used successfully to express and clone several membrane transporters.19-24 This approach was recently used to express and clone polyspecific organic cation transport proteins from rat kidney.21,24 To date, polyspecific organic cation transporters from human kidney have not been cloned. In the present study, we demonstrate that renal organic cation transport activity can be expressed in Xenopus laevis oocytes injected with poly(A)+RNA (mRNA) from human kidney. The results suggest that Xenopus laevis oocytes may be used in cloning of organic cation transporter(s) from human kidney and in studying the functional characteristics of the cloned transporters. In addition, the data provide the first indication of the size of the cDNA transcripts which encode functionally active human renal organic cation transporter(s).
Materials and Methods Isolation of Poly(A)+RNA and Size-FractionationsTotal cellular RNA was isolated from human kidney by phenol and guanidinium isothiocyanate methods with TRIzol Reagent (Gibco BRL, Life Technologies, Inc., Eugene, OR) according to the protocol provided by the manufacturer. Poly(A)+RNA (mRNA) was selected by affinity chromatography on an oligo(dT) cellulose spin column (5 Prime f 3 Prime, Inc., Boulder, CO) and stored in water (1 µg/µL) at -80 °C until used. For size-fractionation, 80-150 µg of total mRNA was heated at 70 °C for 5 min, chilled on ice, and then layered on the top of a 6-20% (w/w) continuous sucrose gradient containing 1 mM EDTA and 10 mM Tris, pH 7.4. The sucrose gradient was centrifuged on a Beckman Ultracentrifuge (Beckman SW41Ti rotor) at 100 000g at 4 °C for 16 h. At the end of centrifugation, 500-700-µL fractions were collected in 2-mL Eppendorf tubes and ethanol-precipitated. The amount of mRNA in each fraction was quantitated by spectrophotometry at 260 nm. The molecular size ranges of the mRNA fractions were determined by gel electrophoresis of aliquots (3 µL) of each fraction on a 1% agarose/1.1% formaldehyde gel (at a constant voltage of 5 V/cm for 2 h at room temperature) followed by ethidium bromide staining. RNA molecular weight standards (Gibco BRL, Life Technologies, Inc., Eugene, OR) were also loaded on the gel and used as reference to estimate the molecular size range of each mRNA fraction. Oocytes and MicroinjectionsOocytes were harvested from Xenopus laevis (Xenopus, Ann Arbor, MI) and the follicular layer was removed with collagenase D treatment (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a calcium-free OR II solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4) at room temperature as previously described.22,23 Healthy looking stage V and VI oocytes were selected and maintained in modified Barth’s
S0022-3549(96)00509-6 CCC: $14.00
Journal of Pharmaceutical Sciences / 753 Vol. 86, No. 6, June 1997
Table 1sInhibition of [14C]TEA Uptake in Xenopus laevis Oocytes by Organic Cations
14
Figure 1sEnhanced [ C]TEA uptake in Xenopus laevis oocytes injected with human kidney mRNA. The uptake of [14C]TEA (500 µM) in oocytes injected with 50 ng of mRNA or 50 nL of water was determined 4 days after injection, at 25 °C. The uptake values are expressed as mean ± SE obtained in a representative experiment from 5−9 oocytes. solution (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.4 mM CaCl2, 0.33 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES/Tris, pH 7.4) overnight at 18 °C. These oocytes were then injected with 50 ng of mRNA or 20 ng of size-fractionated mRNA with a semi-automatic injector (PL1-188, Nikon, Melville, NY). Control oocytes were injected with 50 nL of water. The injected oocytes were maintained in modified Barth’s solution containing gentamicin at 10 mg/L for 4 days at 18 °C prior to uptake experiments. Tetraethylammonium UptakesThe uptake of [14C]TEA (53 mCi/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO) in oocytes was measured by conventional radiotracer techniques previously described.22,23 Briefly, groups of 9-10 injected oocytes were washed twice with 3 mL of 1X NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4) prior to being incubated for 60 min at 25 °C in 200 µL of reaction mixture containing [14C]TEA (500 µM) in 1X NaCl buffer. For inhibition studies, 5 mM of various unlabeled organic cations were also included in the reaction mixture. At the end of the incubation time, the oocytes were washed four times with ice-cold 1X NaCl buffer (3.5 mL). Each oocyte was placed separately into a scintillation vial and dissolved with 100 µL of 10% sodium dodecyl sulfate (SDS). The radioactivity was assayed by scintillation counting. Data AnalysissUptake values in oocytes are expressed as pmol/ oocyte/h and presented as means ( SE of data obtained in a minimum of 5-10 oocytes. Statistical differences were determined by analysis of variance and Student’s t test. Probability at the 0.05 level was considered significant.
Oocyte
[14C]TEA Uptake (pmol/oocyte/h)a
Water-injected control Poly(A)+RNA-injected Control TEA Cimetidine NMN
3.48 ± 0.14 5.88 ± 0.28 3.94 ± 0.13b 2.96 ± 0.27b 4.09 ± 0.16b
a The uptake of [14C]TEA (500 µM), at 1 h, was measured in 1X NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4), in water-injected (50 nL) oocytes, and in mRNA-injected (50 ng) oocytes in the absence (control) and presence of 5 mM TEA, cimetidine, and NMN. Uptake values are expressed as mean ± SE obtained in a representative experiment from 9−10 oocytes. b Significantly different from the poly(A)+RNA-injected oocytes (Control; p < 0.05).
Figure 2sEnhanced [14C]TEA uptake in Xenopus laevis oocytes injected with enriched size-fractions of human kidney mRNA. The uptake of [14C]TEA (500 µM) in oocytes injected with 50 ng of mRNA, 20 ng of size-fractionated mRNA (fractions A-G), or 50 nL of water was determined 4 days after injection, at 25 °C. The uptake values are expressed as mean ± SE obtained in a representative experiment from 5−9 oocytes.
Results and Discussion Injection of mRNA (50 ng) into Xenopus laevis oocytes resulted in a significant enhancement of [14C]TEA uptake (7.2 ( 0.6 pmol/oocyte/h) compared with that obtained in waterinjected oocytes (3.5 ( 0.3 pmol/oocyte/h) (Figure 1). The [14C]TEA uptake in the mRNA-injected oocytes was significantly inhibited by (5 mM) cimetidine, TEA, and NMN (Table 1); these results are consistent with previous studies demonstrating that cimetidine, NMN, and TEA are substrates of both the potential sensitive basolateral membrane organic cation transporter and the brush border membrane organic cationproton antiporter.2,5,6,8-17 To estimate the molecular size range of the mRNA species encoding the transporter(s) in human kidney, we fractionated the mRNA by size using sucrose density gradient centrifugation and injected the various fractions into the oocytes. Injection of mRNA from fractions A-C resulted in significantly higher (four- to seven-fold) [14C]TEA uptake compared with that in water-injected oocytes (Figure 2). The maximum [14C]TEA uptake (six- to seven-fold) was obtained in oocytes injected with mRNA from fraction C. The molecular size of fraction C mRNA ranged between 1.5 and 3.5 kb (mean size of 2.3 kb; Figure 3). This size is slightly larger than the size of the cDNA encoding the rat renal basolateral membrane organic cation transporter (1.8 kb)21 and within the size range 754 / Journal of Pharmaceutical Sciences Vol. 86, No. 6, June 1997
Figure 3sRepresentative agarose formaldehyde gel electrophoresis of sizefractionated mRNA from human kidney. Aliquots (3 µL) of the mRNA size-fractions (fractions A-G, Lanes 2−8) and total mRNA (Lane 9) were electrophoresed in a denaturing agarose gel with ethidium bromide staining. RNA molecular weight standards are shown on the first lane to the left (Lane 1). The size range of each mRNA fraction was estimated by reference to the RNA molecular weight standards (Lane 1), and the molecular size of fraction C (Lane 4) was estimated as 1.5−3.5 kb.
obtained previously for the mRNA fractions from rat kidney producing maximal enhancement of TEA uptake in oocytes.18 This size comparison suggests that the functionally expressed transporter(s) for TEA in human kidney is slightly larger in size than those which are expressed in rat kidney. Organic cation transporters in the kidney play a critical role in the elimination of many endogenous compounds as well as xenobiotics. The mechanisms of organic cation transport at each of the polar membranes of the proximal tubule have been examined in a number of studies in isolated brush border and basolateral membrane vesicles from dog,2,10 rat,8 and rabbit1,6,9,13,14 kidney. Studies from this laboratory have provided
the first direct evidence that an organic cation transporter is present in human renal brush border membranes.16,17 The expressed transport activity obtained in oocytes injected with either the total or fractionated mRNA from human kidney is likely to reflect a co-expression of various functional polyspecific organic cation transporters. Ultimately, by expressing the individual cloned transporters in the oocytes, the functional characteristics, including the driving force and specificity of each cloned transporter, can be examined. Although two polyspecific organic cation transporters from rat kidney have been cloned,21,24 nothing is known currently about the molecular structure of organic cation transporters in human kidney. None of the transporters have been isolated, sequenced, or cloned. This study provides the first demonstration that human renal organic cation transporters can be functionally expressed in Xenopus laevis oocytes. Xenopus laevis oocytes provide a functional assay to clone and, importantly, to ascertain the functional characteristics of organic cation transporters for TEA in human kidney.
References and Notes 1. Rafidazeh, C.; Manganel, M.; Roch-Ramel, F.; Schali, C. Pflugers Arch. 1986, 407, 404-408. 2. Kinsella, J. L.; Holohan, P. D.; Pessah, N. I.; Ross, C. R. J. Pharmacol. Exp. Ther. 1979, 209, 443-450. 3. Inui, K.-I.; Takano, M.; Okano, T.; Hori, R. J. Pharmacol. Exp. Ther. 1985, 233, 181-85. 4. McKinney, T. D.; Kunnemann, M. E. Am. J. Physiol. 1985, 249, F532-541. 5. McKinney, T. D.; Kunnemann, M. E. Am. J. Physiol. 1987, 252, F525-535. 6. Gisclon, L. G.; Wong, F. M.; Giacomini, K. M. Am. J. Physiol. 1987, 253, F141-150. 7. Besseghir, K.; Roch-Ramel, F. Renal Physiol. 1987, 10, 221241. 8. Takano, M.; Inui, K.-I.; Okano, T.; Saito, H.; Hori, R. Biochim. Biophys. Acta 1984, 773, 113-124. 9. Wright, S. H.; Wunz, T. M. Am. J. Physiol. 1987, 253, F10401050.
10. Holohan, P. D.; Ross, C. R. J. Pharmacol. Exp. Ther. 1981, 216, 294-298. 11. Pritchard, J. B.; Miller, D. S. Physiol. Rev. 1993, 73, 765-796. 12. Sokol, P. P.; McKinney, T. D. Am. J. Physiol. 1990, 258, F15991607. 13. Hsyu, P.-H.; Gisclon, L. G.; Hui, A. C.; Giacomini, K. M. Am. J. Physiol. 1988, 254, F56-61. 14. Dantzler, W. H.; Brokl, O. H.; Wright, S. H. Am. J. Physiol. 1989, 256, F290-297. 15. Miyamoto, Y.; Tiruppathi, C.; Ganapathy, V.; Leibach, F. H. Am. J. Physiol. 1989, 256, F540-548. 16. Ott, R. J.; Hui, A. C.; Yuan, G.; Giacomini, K. M. Am. J. Physiol. 1991, 261, F443-451. 17. Chun, J. K.; Zhang, L.; Piquette-Miller, M.; Lau, E.; Tong, L.Q.; Giacomini, K. M., unpublished results. 18. Hori, R.; Hirai, M.; Katsura, T.; Takano, M.; Yasuhara, M.; Kaneko, S.; Satoh, M. Biochem. J. 1992, 283, 409-411. 19. Hediger, M. A.; Coady, M. J.; Ikeda, T. S.; Wright, E. M. Nature 1987, 330, 379-381. 20. Magagnin, S.; Werner, A.; Markovich, D.; Sorribas, V.; Stange, G.; Biber, J.; Murer, H. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5979-5983. 21. Grundemann, D.; Gorboulev, V.; Gambaryan, S.; Veyhl, M.; Koepsell, H. Nature 1994, 372, 549-552. 22. Giacomini, K. M.; Markovich, D.; Werner, A.; Biber, J.; Wu, X.; Murer, H. Pflugers Archiv. 1994, 427, 381-383. 23. Werner, A.; Biber, J.; Forgo, J.; Palacin, M.; Murer, H. J. Biol. Chem. 1990, 265, 12331-12336. 24. Okuda, M.; Saito, H.; Urakami, Y.; Takano, M.; Inui, K.-I. Biochem. Biophys. Res. Commun. 1996, 224, 500-507.
Note Added in ProofsRecently, we cloned the first polyspecific organic cation transporter from human liver (Zhang et al., Mol. Pharmacol. In press, 1997). The mRNA transcript of this transporter is present in low abundance in human kidney.
Acknowledgments We are grateful for support from the National Institute of Health (GM36780).
JS960509L
Journal of Pharmaceutical Sciences / 755 Vol. 86, No. 6, June 1997
Expression of Human Polyspecific Renal Organic Cation Transport Activity in Xenopus laevis Oocytes JOANNE K. CHUN, MICHELINE PIQUETTE-MILLER, LEI ZHANG,
AND
KATHLEEN M. GIACOMINIX
Received February 11, 1996, from the Departments of Biopharmaceutical Sciences and Cellular and Molecular Pharmacology, Schools of Pharmacy and Medicine, 513 Parnassus Avenue, S-926, Box 0446, University of California, San Francisco, CA 94143-0446. Final revised manuscript received March 3, 1997. Accepted for publication March 9, 1997X. Abstract 0 Polyspecific organic cation transporters in the basolateral and brush border membrane of the kidney play a role in the elimination of many clinically important drugs and endogenous compounds. In this study we report the functional expression of organic cation transport activity in Xenopus laevis oocytes injected with poly(A)+RNA (mRNA) isolated from human kidney. Uptake of [14C]tetraethylammonium (TEA) was measured in mRNA-injected or water-injected oocytes, 4 days after injection. In oocytes injected with 50 ng of mRNA isolated from human renal cortex, the uptake of [14C]TEA was significantly increased in comparison with water-injected oocytes (7.2 ± 0.6 and 3.5 ± 0.3 pmol/ oocyte/h, respectively). Injection of 20 ng of an enriched size-fraction (fraction C) of mRNA (mean size of 2.3 kb) resulted in further enhancement of [14C]TEA uptake: [14C]TEA uptake was enhanced sixto seven-fold in oocytes injected with fraction C (23.7 ± 3.7 pmol/oocyte/ h) in comparison with water-injected oocytes. The uptake of TEA in mRNA-injected oocytes was significantly inhibited by 5 mM of unlabeled TEA, cimetidine, and N1-methylnicotinamide. These data suggest that polyspecific organic cation transport activity can be successfully expressed in Xenopus laevis oocytes injected with mRNA isolated from human kidney.
Organic cation transporters in the renal proximal tubules are involved in the elimination of various types of clinically used drugs, including antiarrhythmics, antihistamines, opiates, and sedatives, as well as endogenous amines, such as choline, dopamine, and epinephrine.1-7 The elimination of these cationic drugs and endogenous molecules involves transport across the two membranes of the epithelial cells of the renal proximal tubules: the basolateral membrane and the brush border membrane. The mechanisms of organic cation transport across each of these membranes have been studied in isolated renal plasma membrane vesicles from a number of species using the model compounds N1-methylnicotinamide (NMN) and tetraethylammonium (TEA).2,8,9 In general, the studies have shown that TEA and NMN are transported from the blood into the cell across the basolateral membrane via a facilitative, but passive, electrogenic carriermediated system.2,10-12 The driving force is the insidenegative membrane potential and/or an organic cationorganic cation exchange mechanism. Subsequently, TEA and NMN are transported into the tubule lumen across the brush border membrane via a secondarily active, organic cationproton antiporter.10,11,13-15 Recent studies from this laboratory have demonstrated the presence of an organic cation-proton antiporter that transports TEA in renal brush border membrane vesicles isolated from human kidney.16,17 X
Abstract published in Advance ACS Abstracts, May 1, 1997.
© 1997, American Chemical Society and American Pharmaceutical Association
Until recently, little was known about the molecular characteristics of the organic cation transporters at either the basolateral or brush border membrane of the renal proximal tubules. However, in 1992, Hori and co-workers demonstrated that Xenopus laevis oocytes could be successfully used as an expression system for TEA transporter(s) in rat kidney.18 Xenopus laevis oocytes are an excellent expression system for exogenous mRNA and have been used successfully to express and clone several membrane transporters.19-24 This approach was recently used to express and clone polyspecific organic cation transport proteins from rat kidney.21,24 To date, polyspecific organic cation transporters from human kidney have not been cloned. In the present study, we demonstrate that renal organic cation transport activity can be expressed in Xenopus laevis oocytes injected with poly(A)+RNA (mRNA) from human kidney. The results suggest that Xenopus laevis oocytes may be used in cloning of organic cation transporter(s) from human kidney and in studying the functional characteristics of the cloned transporters. In addition, the data provide the first indication of the size of the cDNA transcripts which encode functionally active human renal organic cation transporter(s).
Materials and Methods Isolation of Poly(A)+RNA and Size-FractionationsTotal cellular RNA was isolated from human kidney by phenol and guanidinium isothiocyanate methods with TRIzol Reagent (Gibco BRL, Life Technologies, Inc., Eugene, OR) according to the protocol provided by the manufacturer. Poly(A)+RNA (mRNA) was selected by affinity chromatography on an oligo(dT) cellulose spin column (5 Prime f 3 Prime, Inc., Boulder, CO) and stored in water (1 µg/µL) at -80 °C until used. For size-fractionation, 80-150 µg of total mRNA was heated at 70 °C for 5 min, chilled on ice, and then layered on the top of a 6-20% (w/w) continuous sucrose gradient containing 1 mM EDTA and 10 mM Tris, pH 7.4. The sucrose gradient was centrifuged on a Beckman Ultracentrifuge (Beckman SW41Ti rotor) at 100 000g at 4 °C for 16 h. At the end of centrifugation, 500-700-µL fractions were collected in 2-mL Eppendorf tubes and ethanol-precipitated. The amount of mRNA in each fraction was quantitated by spectrophotometry at 260 nm. The molecular size ranges of the mRNA fractions were determined by gel electrophoresis of aliquots (3 µL) of each fraction on a 1% agarose/1.1% formaldehyde gel (at a constant voltage of 5 V/cm for 2 h at room temperature) followed by ethidium bromide staining. RNA molecular weight standards (Gibco BRL, Life Technologies, Inc., Eugene, OR) were also loaded on the gel and used as reference to estimate the molecular size range of each mRNA fraction. Oocytes and MicroinjectionsOocytes were harvested from Xenopus laevis (Xenopus, Ann Arbor, MI) and the follicular layer was removed with collagenase D treatment (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a calcium-free OR II solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4) at room temperature as previously described.22,23 Healthy looking stage V and VI oocytes were selected and maintained in modified Barth’s
S0022-3549(96)00509-6 CCC: $14.00
Journal of Pharmaceutical Sciences / 753 Vol. 86, No. 6, June 1997
Table 1sInhibition of [14C]TEA Uptake in Xenopus laevis Oocytes by Organic Cations
14
Figure 1sEnhanced [ C]TEA uptake in Xenopus laevis oocytes injected with human kidney mRNA. The uptake of [14C]TEA (500 µM) in oocytes injected with 50 ng of mRNA or 50 nL of water was determined 4 days after injection, at 25 °C. The uptake values are expressed as mean ± SE obtained in a representative experiment from 5−9 oocytes. solution (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.4 mM CaCl2, 0.33 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES/Tris, pH 7.4) overnight at 18 °C. These oocytes were then injected with 50 ng of mRNA or 20 ng of size-fractionated mRNA with a semi-automatic injector (PL1-188, Nikon, Melville, NY). Control oocytes were injected with 50 nL of water. The injected oocytes were maintained in modified Barth’s solution containing gentamicin at 10 mg/L for 4 days at 18 °C prior to uptake experiments. Tetraethylammonium UptakesThe uptake of [14C]TEA (53 mCi/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO) in oocytes was measured by conventional radiotracer techniques previously described.22,23 Briefly, groups of 9-10 injected oocytes were washed twice with 3 mL of 1X NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4) prior to being incubated for 60 min at 25 °C in 200 µL of reaction mixture containing [14C]TEA (500 µM) in 1X NaCl buffer. For inhibition studies, 5 mM of various unlabeled organic cations were also included in the reaction mixture. At the end of the incubation time, the oocytes were washed four times with ice-cold 1X NaCl buffer (3.5 mL). Each oocyte was placed separately into a scintillation vial and dissolved with 100 µL of 10% sodium dodecyl sulfate (SDS). The radioactivity was assayed by scintillation counting. Data AnalysissUptake values in oocytes are expressed as pmol/ oocyte/h and presented as means ( SE of data obtained in a minimum of 5-10 oocytes. Statistical differences were determined by analysis of variance and Student’s t test. Probability at the 0.05 level was considered significant.
Oocyte
[14C]TEA Uptake (pmol/oocyte/h)a
Water-injected control Poly(A)+RNA-injected Control TEA Cimetidine NMN
3.48 ± 0.14 5.88 ± 0.28 3.94 ± 0.13b 2.96 ± 0.27b 4.09 ± 0.16b
a The uptake of [14C]TEA (500 µM), at 1 h, was measured in 1X NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.4), in water-injected (50 nL) oocytes, and in mRNA-injected (50 ng) oocytes in the absence (control) and presence of 5 mM TEA, cimetidine, and NMN. Uptake values are expressed as mean ± SE obtained in a representative experiment from 9−10 oocytes. b Significantly different from the poly(A)+RNA-injected oocytes (Control; p < 0.05).
Figure 2sEnhanced [14C]TEA uptake in Xenopus laevis oocytes injected with enriched size-fractions of human kidney mRNA. The uptake of [14C]TEA (500 µM) in oocytes injected with 50 ng of mRNA, 20 ng of size-fractionated mRNA (fractions A-G), or 50 nL of water was determined 4 days after injection, at 25 °C. The uptake values are expressed as mean ± SE obtained in a representative experiment from 5−9 oocytes.
Results and Discussion Injection of mRNA (50 ng) into Xenopus laevis oocytes resulted in a significant enhancement of [14C]TEA uptake (7.2 ( 0.6 pmol/oocyte/h) compared with that obtained in waterinjected oocytes (3.5 ( 0.3 pmol/oocyte/h) (Figure 1). The [14C]TEA uptake in the mRNA-injected oocytes was significantly inhibited by (5 mM) cimetidine, TEA, and NMN (Table 1); these results are consistent with previous studies demonstrating that cimetidine, NMN, and TEA are substrates of both the potential sensitive basolateral membrane organic cation transporter and the brush border membrane organic cationproton antiporter.2,5,6,8-17 To estimate the molecular size range of the mRNA species encoding the transporter(s) in human kidney, we fractionated the mRNA by size using sucrose density gradient centrifugation and injected the various fractions into the oocytes. Injection of mRNA from fractions A-C resulted in significantly higher (four- to seven-fold) [14C]TEA uptake compared with that in water-injected oocytes (Figure 2). The maximum [14C]TEA uptake (six- to seven-fold) was obtained in oocytes injected with mRNA from fraction C. The molecular size of fraction C mRNA ranged between 1.5 and 3.5 kb (mean size of 2.3 kb; Figure 3). This size is slightly larger than the size of the cDNA encoding the rat renal basolateral membrane organic cation transporter (1.8 kb)21 and within the size range 754 / Journal of Pharmaceutical Sciences Vol. 86, No. 6, June 1997
Figure 3sRepresentative agarose formaldehyde gel electrophoresis of sizefractionated mRNA from human kidney. Aliquots (3 µL) of the mRNA size-fractions (fractions A-G, Lanes 2−8) and total mRNA (Lane 9) were electrophoresed in a denaturing agarose gel with ethidium bromide staining. RNA molecular weight standards are shown on the first lane to the left (Lane 1). The size range of each mRNA fraction was estimated by reference to the RNA molecular weight standards (Lane 1), and the molecular size of fraction C (Lane 4) was estimated as 1.5−3.5 kb.
obtained previously for the mRNA fractions from rat kidney producing maximal enhancement of TEA uptake in oocytes.18 This size comparison suggests that the functionally expressed transporter(s) for TEA in human kidney is slightly larger in size than those which are expressed in rat kidney. Organic cation transporters in the kidney play a critical role in the elimination of many endogenous compounds as well as xenobiotics. The mechanisms of organic cation transport at each of the polar membranes of the proximal tubule have been examined in a number of studies in isolated brush border and basolateral membrane vesicles from dog,2,10 rat,8 and rabbit1,6,9,13,14 kidney. Studies from this laboratory have provided
the first direct evidence that an organic cation transporter is present in human renal brush border membranes.16,17 The expressed transport activity obtained in oocytes injected with either the total or fractionated mRNA from human kidney is likely to reflect a co-expression of various functional polyspecific organic cation transporters. Ultimately, by expressing the individual cloned transporters in the oocytes, the functional characteristics, including the driving force and specificity of each cloned transporter, can be examined. Although two polyspecific organic cation transporters from rat kidney have been cloned,21,24 nothing is known currently about the molecular structure of organic cation transporters in human kidney. None of the transporters have been isolated, sequenced, or cloned. This study provides the first demonstration that human renal organic cation transporters can be functionally expressed in Xenopus laevis oocytes. Xenopus laevis oocytes provide a functional assay to clone and, importantly, to ascertain the functional characteristics of organic cation transporters for TEA in human kidney.
References and Notes 1. Rafidazeh, C.; Manganel, M.; Roch-Ramel, F.; Schali, C. Pflugers Arch. 1986, 407, 404-408. 2. Kinsella, J. L.; Holohan, P. D.; Pessah, N. I.; Ross, C. R. J. Pharmacol. Exp. Ther. 1979, 209, 443-450. 3. Inui, K.-I.; Takano, M.; Okano, T.; Hori, R. J. Pharmacol. Exp. Ther. 1985, 233, 181-85. 4. McKinney, T. D.; Kunnemann, M. E. Am. J. Physiol. 1985, 249, F532-541. 5. McKinney, T. D.; Kunnemann, M. E. Am. J. Physiol. 1987, 252, F525-535. 6. Gisclon, L. G.; Wong, F. M.; Giacomini, K. M. Am. J. Physiol. 1987, 253, F141-150. 7. Besseghir, K.; Roch-Ramel, F. Renal Physiol. 1987, 10, 221241. 8. Takano, M.; Inui, K.-I.; Okano, T.; Saito, H.; Hori, R. Biochim. Biophys. Acta 1984, 773, 113-124. 9. Wright, S. H.; Wunz, T. M. Am. J. Physiol. 1987, 253, F10401050.
10. Holohan, P. D.; Ross, C. R. J. Pharmacol. Exp. Ther. 1981, 216, 294-298. 11. Pritchard, J. B.; Miller, D. S. Physiol. Rev. 1993, 73, 765-796. 12. Sokol, P. P.; McKinney, T. D. Am. J. Physiol. 1990, 258, F15991607. 13. Hsyu, P.-H.; Gisclon, L. G.; Hui, A. C.; Giacomini, K. M. Am. J. Physiol. 1988, 254, F56-61. 14. Dantzler, W. H.; Brokl, O. H.; Wright, S. H. Am. J. Physiol. 1989, 256, F290-297. 15. Miyamoto, Y.; Tiruppathi, C.; Ganapathy, V.; Leibach, F. H. Am. J. Physiol. 1989, 256, F540-548. 16. Ott, R. J.; Hui, A. C.; Yuan, G.; Giacomini, K. M. Am. J. Physiol. 1991, 261, F443-451. 17. Chun, J. K.; Zhang, L.; Piquette-Miller, M.; Lau, E.; Tong, L.Q.; Giacomini, K. M., unpublished results. 18. Hori, R.; Hirai, M.; Katsura, T.; Takano, M.; Yasuhara, M.; Kaneko, S.; Satoh, M. Biochem. J. 1992, 283, 409-411. 19. Hediger, M. A.; Coady, M. J.; Ikeda, T. S.; Wright, E. M. Nature 1987, 330, 379-381. 20. Magagnin, S.; Werner, A.; Markovich, D.; Sorribas, V.; Stange, G.; Biber, J.; Murer, H. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5979-5983. 21. Grundemann, D.; Gorboulev, V.; Gambaryan, S.; Veyhl, M.; Koepsell, H. Nature 1994, 372, 549-552. 22. Giacomini, K. M.; Markovich, D.; Werner, A.; Biber, J.; Wu, X.; Murer, H. Pflugers Archiv. 1994, 427, 381-383. 23. Werner, A.; Biber, J.; Forgo, J.; Palacin, M.; Murer, H. J. Biol. Chem. 1990, 265, 12331-12336. 24. Okuda, M.; Saito, H.; Urakami, Y.; Takano, M.; Inui, K.-I. Biochem. Biophys. Res. Commun. 1996, 224, 500-507.
Note Added in ProofsRecently, we cloned the first polyspecific organic cation transporter from human liver (Zhang et al., Mol. Pharmacol. In press, 1997). The mRNA transcript of this transporter is present in low abundance in human kidney.
Acknowledgments We are grateful for support from the National Institute of Health (GM36780).
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Journal of Pharmaceutical Sciences / 755 Vol. 86, No. 6, June 1997