Departments of 1Biochemistry and 2Psychiatry, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands
Submitted 27 October 2003 ; accepted in final form 10 February 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
regulatory volume decrease; taurine; volume-regulated anion channels; chloride channel; protein kinase C
In a number of tissues, the release of small molecules such as taurine, betaine, and sorbitol contributes significantly to RVD. In excitable cells, these metabolites were found to be the major osmolytes released (41, 45). Taurine especially has been implicated as an osmolyte involved in volume regulation (1, 10, 19).
As is the case for volume-regulated anion channels (VRAC), the molecular identity of the organic osmolyte release pathway has not been elucidated yet (for review, see Ref. 42). In a number of cell types, the efflux of organic osmolytes and anion conductance was regulated similarly, suggesting that a single release pathway is involved [i.e., volume-sensitive organic osmolyte and anion channel (VSOAC)] (16, 2224, 40, 47). In other cells, however, distinct pathways and/or transporters have been proposed (40, 46). Indeed, in hippocampal slice preparations, at least two pathways for taurine release have been identified that differ in their kinetics of activation/inactivation and in their sensitivity for inhibitors (11).
Using intestine 407 cells, we investigated the hypotonicity-provoked release pathway for organic osmolytes and compared its properties with those of VRAC activation. This cell line was particularly suitable for this study because no plasma membrane Cl conductances other than VRAC, such as CFTR and voltage-sensitive or Ca2+-activated anion channels, are expressed (18, 55). In this article, we report that osmotic swelling of intestine 407 cells results in massive release of taurine after a distinct lag period. The results indicate that the efflux of taurine is regulated independently of VRAC and uses a signaling pathway involving protein kinase C (PKC).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. Intestine 407 cells were routinely grown as a monolayer in DMEM supplemented with 25 mM HEPES, 10% FCS, 1% nonessential amino acids, 40 mg/l penicillin, and 90 mg/l streptomycin in a humidified atmosphere of 95% O2 and 5% CO2 at 37°C. Before the experiments, cells were serum-starved overnight.
Radioisotope efflux assays. Confluent monolayers of intestine 407 cells were loaded with 5 µCi/ml 125I, 0.1 µCi/ml [3H]choline, or 0.1 µCi/ml [3H]taurine for 2 h in modified Meyler solution (108 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 20 mM NaHCO3, 0.8 mM Na2HPO4, 0.4 mM NaH2PO4, 20 mM HEPES, and 10 mM glucose, pH 7.40) and washed three times with isotonic buffer (66 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 123 mM mannitol, and 20 mM HEPES, pH 7.4). Hypotonic buffers were prepared by adjusting the concentration of mannitol. Full time courses were prepared by replacing the medium at 1- to 2-min intervals. For inhibitor studies, a single fraction of 8 min was collected. Radioactivity in the medium was determined by gamma (125I) or beta (3H) radiation counting and expressed as fractional efflux per minute as previously described (56).
The most abundant 3H-labeled osmolyte released after hyposmotic stimulation of [3H]choline-loaded cells was found to be phosphocholine (87% of 3H radioactivity), as determined by thin-layer chromatography (Silica Gel 60 plates; Merck, Darmstadt, Germany) using a methanol-1.2% NaCl-13.3 M NH4OH (10:10:1 vol/vol) solvent system (52). In addition to phosphocholine, low levels of [3H]choline and [3H]glycerophosphocholine (
12 and 1% of 3H radioactivity, respectively) were found. In contrast to the release of [3H]phosphocholine, which was increased dramatically after hyposmotic stimulation (isotonic: 184 ± 95 dpm, hypotonic: 1,778 ± 219 dpm), the release of [3H]choline and [3H]glycerophosphocholine (isotonic: 147 ± 22 and 14 ± 2 dpm, respectively; hypotonic: 253 ± 46 and 27 ± 3 dpm, respectively) was only moderately affected by hypotonicity.
Amino acid analysis.
Medium and lysate fractions of 250 µl were collected corresponding to 1- to 2-min stimuli. Taurine and -alanine contents of cell lysates and incubation medium were analyzed by reverse-phase HPLC after pre-column derivatization with o-phthaldialdehyde as previously described (9).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Organic release pathway does not involve VRAC. To date, the molecular nature of the organic anion release pathway has not been elucidated. In a number of cell models, the release of both Cl and organic osmolytes seems to be mediated by the same channel protein; in other models, however, the release of Cl and the release of taurine are apparently distinct processes. In an attempt to further discriminate between the release pathways for Cl and taurine in intestine 407 cells, we also studied the effects of pharmacological inhibitors on anion conductance and the release of taurine.
Treating the cells with the purinoceptor antagonist and VRAC inhibitor suramin completely prevented the hypotonicity-induced release of taurine (Fig. 3), suggesting that the same channel and/or transporter is involved. In contrast, however, DIDS and millimolar concentrations of extracellular ATP, which efficiently inhibit VRAC activation (57), were unable to block the taurine efflux (Fig. 3).
|
Role of organic anion or cation transporters. Movement of taurine and other organic osmolytes across the plasma membrane is mediated by at least four different transport proteins (OATs 13 and Taut; Refs. 15, 27, 51). To investigate whether these transporters contribute to the swelling-induced release of taurine and phosphocholine, cells were treated with established inhibitors of each of these transport systems. As shown in Table 2, inhibitors of the OAT family of transporters, such as probenecid, methothrexate, fenobarbital, and tunicamycin, which lead to an intracellular retention of OATs, did not inhibit or only partially inhibited (2040% reduction) the hypotonicity-induced taurine efflux (20). In contrast, decynium-22, cyanine-863, and quinidine, inhibitors of organic cation transporters (OCTs; Refs. 28, 59), strongly diminished the efflux of both taurine and phosphocholine (Table 2). However, several OCT inhibitors (i.e., cimetidine, quinine, and verapamil) were found to be ineffective (Table 2). These combined pharmacological data argue against the concept of a single, "classical" organic osmolyte transporter (OAT or OCT) accounting for the cell swelling-induced release pathway.
|
In the presence of the specific PKC inhibitor GF109203X, marked inhibition of the cell swelling-induced taurine efflux was observed, and vice versa, activation of PKC by brief treatment of the cells with the phorbol ester PMA increased the response approximately twofold (Fig. 4). The potentiation by PMA was fully reversed in the presence of GF109203X, indicating that PKC activation is involved. Notably, PMA did not increase basal taurine efflux (basal: 8 ± 2%, PMA: 2 ± 4% of hypotonic control). Prolonged treatment of the cultures with PMA in an attempt to downregulate PKC resulted in reduced basal efflux but only slightly affected the hypotonicity-induced efflux. Notably, the volume-sensitive anion efflux was not affected by PKC activation or inactivation (54). Taken together, the results suggest the involvement of a Ca2+-insensitive PKC isoenzyme in the regulation of the cell swelling-induced release of organic osmolytes.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, at least two different mechanisms have been reported to be responsible for the release of organic osmolytes: a polyspecific one transporting both taurine and GABA and one selective for amino acids (25). The latter involves VSOAC (4), a pathway facilitating the release of both Cl and organic osmolytes (25, 26) and VSOC, a transporter of organic osmolytes only (11). The molecular identity of these pathways remains to be established. Our experiments clearly showed a difference in time course as well as in the threshold for activation between the release of 125I and [3H]taurine, suggesting that different pathways are involved. A differential sensitivity to DIDS of the Cl and taurine release from HeLa cells has been reported, also pointing to the existence of separate pathways (50). In contrast, the efflux of taurine was inhibited by chloride channel blockers, including DIDS and millimolar concentrations of ATP, in calf pulmonary endothelial (CPAE) cells (34). In intestine 407 cells, however, DIDS treatment did not affect hypotonicity-induced taurine efflux (Fig. 3). Recently, two different release pathways for taurinea "fast" one and a "slow" onehave been observed in hippocampal brain slices, with the slow one being sensitive to DIDS (11). It is therefore likely that only the fast release pathway is present in intestine 407 cells.
In an attempt to identify the transporters involved, cells were treated with a variety of OAT and OCT inhibitors. None of the OAT inhibitors tested were found to inhibit the release of taurine dramatically; however, a moderate (2040%) reduction in efflux was observed with some of the inhibitors tested. In contrast, taurine release was inhibited strongly by several OCT inhibitors but not by others. Because the taurine efflux was abolished almost completely after quinidine treatment, whereas the related and potent OCT inhibitor quinine (27) was ineffective, we think that a transport system other than the OCT is involved. This notion is supported by our observation that cimetidine and verapamil, inhibitors of all three OCT subtypes (58), did not reduce the hypotonicity-induced osmolyte efflux. However, we cannot completely rule out the possibility that a subset of transporters with different sensitivities for the inhibitors used may contribute to the observed organic osmolyte efflux.
In several selected cell models, the hypotonicity-provoked taurine release was found to be sensitive to inhibitors of protein tyrosine phosphorylation as well as to wortmannin, an inhibitor of PtdIns 3-kinase (11, 37). In Intestine 407 cells, however, in clear contrast to the activation of VRAC (53, 55), the release of taurine was found to be independent of tyrosine kinases and PtdIns 3-kinase and did not involve p21rho. A similar low sensitivity to genistein has been reported for isolated rat supraoptic astrocytes (6). Taken together, these results indicate not only that the release of taurine does not involve VRAC in intestine 407 cells but also that distinct signaling pathways are involved in the regulation of Cl and taurine efflux (54).
An increase in intracellular free Ca2+ has been reported to be involved in the activation of taurine efflux for several cell types, including erythroleukemia cells (21), cultured rat astrocytes (33), and rat cerebral cortex (30). In astrocytes and cerebral granule cells, however, the hypotonicity-provoked taurine efflux was independent of [Ca2+]i or required basal Ca2+ levels (36, 38, 40). With the use of intestine 407 cells, modulation of [Ca2+]i by BAPTA-AM loading, extracellular EGTA, Ca2+-mobilizing hormones, the Ca2+ channel blocker verapamil, or the Ca2+ ionophore A-23187 did not affect the volume-sensitive taurine efflux. However, we cannot exclude the possibility that a minimal basal [Ca2+]i is required, as observed previously for astrocytes (36).
Brief treatment of intestine 407 cells with the phorbol ester PMA potentiated the cell swelling-induced taurine efflux, whereas addition of the PKC inhibitor GF109203X largely inhibited the response, suggesting a major role for PKC as an activator of this process. The PKC family of serine kinases consists of a large group of isoenzymes playing crucial roles in cellular signaling and involved in a variety of biological processes (for review, see Ref. 35). The group can be divided into subgroups on behalf of their behavior and their protein sequence: the classical (,
,
), novel (
,
,
,
), and atypical (
,
) PKCs and the PKC-related kinases (PRK1, PRK2, PRK3). Whereas activation of PKC-
, -
, and -
requires binding of phosphatidylserine, both classical and novel PKCs are activated by the phorbol ester PMA. Because binding of phosphatidylserine occurs in a Ca2+-dependent manner, the permissive role of Ca2+ (36) in the activation of hyposmotically triggered taurine efflux might be explained by the activation of these isoforms of PKC. In CPAE cells, however, the release of organic osmolytes was found to be independent of PKC (34).
To summarize, the results of this study indicate that in intestine 407 cells, the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves an activation pathway that includes PKC. These observations are not generally applicable, however, because in several other model systems, a close relationship between Cl conductance and release of organic osmolytes has been established (16, 2224, 40, 47). Therefore, a definite answer to the question of whether the release pathways are the same or different awaits the molecular identification of the transporters involved.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Burg MB. Molecular basis of osmotic regulation. Am J Physiol Renal Fluid Electrolyte Physiol 268: F983F996, 1995.
3. Burg MB and Garcia-Perez A. How tonicity regulates gene expression. J Am Soc Nephrol 3: 121127, 1992.[Abstract]
4. Cannon CL, Basavappa S, and Strange K. Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel. Am J Physiol Cell Physiol 275: C416C422, 1998.
5. Carton I, Trouet D, Hermans D, Barth H, Aktories K, Droogmans G, Jorgensen NK, Hoffmann EK, Nilius B, and Eggermont J. RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells. Am J Physiol Cell Physiol 283: C115C125, 2002.
6. Deleuze C, Duvoid A, Moos FC, and Hussy N. Tyrosine phosphorylation modulates the osmosensitivity of volume-dependent taurine efflux from glial cells in the rat supraoptic nucleus. J Physiol 523: 291299, 2000.
7. Eggermont J, Trouet D, Carton I, and Nilius B. Cellular function and control of volume-regulated anion channels in bovine endothelial cells. Cell Biochem Biophys 35: 263274, 2001.[ISI][Medline]
8. Estevez AY, Bond T, and Strange K. Regulation of ICl,swell in neuroblastoma cells by G protein signaling pathways. Am J Physiol Cell Physiol 281: C89C98, 2001.
9. Fekkes D, van Dalen A, Edelman M, and Voskuilen A. Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated precolumn derivatization with o-phthaldialdehyde. J Chromatogr B Biomed Appl 669: 177186, 1995.[CrossRef][Medline]
10. Forster RP and Goldstein L. Amino acids and cell regulation. Yale J Biol Med 52: 497515, 1979.[ISI][Medline]
11. Franco R, Torres-Marquez ME, and Pasantes-Morales H. Evidence for two mechanisms of amino acid osmolyte release from hippocampal slices. Pflügers Arch 442: 791800, 2001.[CrossRef][ISI][Medline]
12. Gelband CH, Greco PG, and Martens JR. Voltage-dependent chloride channels: invertebrates to man. J Exp Zool 275: 277282, 1996.[CrossRef][ISI][Medline]
13. Grundemann D, Schechinger B, Rappold GA, and Schomig E. Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter. Nat Neurosci 1: 349351, 1998.[CrossRef][ISI][Medline]
15. Hagenbuch B, Meier PJ. The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 118, 2003.[ISI][Medline]
16. Hand M, Morrison R, and Strange K. Characterization of volume-sensitive organic osmolyte efflux and anion current in Xenopus oocytes. J Membr Biol 157: 916, 1997.[CrossRef][ISI][Medline]
17. Haussinger D. The role of cellular hydration in the regulation of cell function. Biochem J 313: 697710, 1996.[ISI][Medline]
18. Hazama A, Shimizu T, Ando-Akatsuka Y, Hayashi S, Tanaka S, Maeno E, and Okada Y. Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive Cl channels. J Gen Physiol 114: 525533, 1999.
19. Hoffmann EK and Lambert IH. Amino acid transport and cell volume regulation in Ehrlich ascites tumour cells. J Physiol 338: 613625, 1983.[Abstract]
20. Hooiveld GJ, van Montfoort JE, Meijer DK, and Muller M. Function and regulation of ATP-binding cassette transport proteins involved in hepatobiliary transport. Eur J Pharm Sci 12: 525543, 2001.[CrossRef][Medline]
21. Huang CC, Chang CB, Liu JY, Basavappa S, and Lim PH. Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells. J Cell Physiol 189: 316322, 2001.[CrossRef][ISI][Medline]
22. Jackson PS and Madsen JR. Identification of the volume-sensitive organic osmolyte/anion channel in human glial cells. Pediatr Neurosurg 27: 286291, 1997.[ISI][Medline]
23. Jackson PS, Morrison R, and Strange K. The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding. Am J Physiol Cell Physiol 267: C1203C1209, 1994.
24. Jackson PS and Strange K. Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am J Physiol Cell Physiol 265: C1489C1500, 1993.
25. Kimelberg HK and Mongin AA. Swelling-activated release of excitatory amino acids in the brain: relevance for pathophysiology. Contrib Nephrol 123: 240257, 1998.[ISI][Medline]
26. Kirk K and Kirk J. Volume-regulatory taurine release from a human lung cancer cell line: evidence for amino acid transport via a volume-activated chloride channel. FEBS Lett 336: 153158, 1993.[CrossRef][ISI][Medline]
27. Koepsell H, Gorboulev V, and Arndt P. Molecular pharmacology of organic cation transporters in kidney. J Membr Biol 167: 103117, 1999.[CrossRef][ISI][Medline]
28. Kristufek D, Rudorfer W, Pifl C, and Huck S. Organic cation transporter mRNA and function in the rat superior cervical ganglion. J Physiol 543: 117134, 2002.
29. Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247306, 1998.
30. Law RO. Taurine efflux and the regulation of cell volume in incubated slices of rat cerebral cortex. Biochim Biophys Acta 1221: 2128, 1994.[CrossRef][ISI][Medline]
31. Lee VHL. Membrane transporters. Eur J Pharm Sci 11: S41S50, 2000.[CrossRef][ISI][Medline]
32. Lepple-Wienhues A, Szabo I, Laun T, Kaba NK, Gulbins E, and Lang F. The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes. J Cell Biol 141: 281286, 1998.[Abstract]
33. Li G, Liu Y, and Olson JE. Calcium/calmodulin-modulated chloride and taurine conductances in cultured rat astrocytes. Brain Res 925: 18, 2002.[CrossRef][ISI][Medline]
34. Manolopoulos VG, Voets T, Declercq PE, Droogmans G, and Nilius B. Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells. Am J Physiol Cell Physiol 273: C214C222, 1997.
35. Mellor H and Parker PJ. The extended protein kinase C superfamily. Biochem J 332: 281292, 1998.[ISI][Medline]
36. Mongin AA, Cai Z, and Kimelberg HK. Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin. Am J Physiol Cell Physiol 277: C823C832, 1999.
37. Morales-Mulia S, Cardin V, Torres-Marquez ME, Crevenna A, and Pasantes-Morales H. Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons. Neurochem Int 38: 153161, 2001.[ISI][Medline]
38. Morales-Mulia S, Vaca L, Hernandez-Cruz A, and Pasantes-Morales H. Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes. J Neurochem 71: 23302338, 1998.[ISI][Medline]
40. Motais R, Fievet B, Borgese F, and Garcia-Romeu F. Association of the band 3 protein with a volume-activated, anion and amino acid channel: a molecular approach. J Exp Biol 200: 361367, 1997.
41. Nakanishi T, Uyama O, and Sugita M. Osmotically regulated taurine content in rat renal inner medulla. Am J Physiol Renal Fluid Electrolyte Physiol 261: F957F962, 1991.
42. Nilius B and Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 14151459, 2001.
43. Nilius B, Eggermont J, and Droogmans G. The endothelial volume-regulated anion channel, VRAC. Cell Physiol Biochem 10: 313320, 2000.[CrossRef][ISI][Medline]
44. Nilius B, Voets T, Prenen J, Barth H, Aktories K, Kaibuchi K, Droogmans G, and Eggermont J. Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells. J Physiol 516: 6774, 1999.
45. Pasantes-Morales H, Franco R, Torres-Marquez ME, Hernandez-Fonseca K, and Ortega A. Amino acid osmolytes in regulatory volume decrease and isovolumetric regulation in brain cells: contribution and mechanisms. Cell Physiol Biochem 10: 361370, 2000.[CrossRef][ISI][Medline]
46. Perlman DF and Goldstein L. Organic osmolyte channels in cell volume regulation in vertebrates. J Exp Zool 283: 725733, 1999.[CrossRef][ISI][Medline]
47. Rutledge EM, Mongin AA, and Kimelberg HK. Intracellular ATP depletion inhibits swelling-induced d-[3H]aspartate release from primary astrocyte cultures. Brain Res 842: 3945, 1999.[CrossRef][ISI][Medline]
48. Strange K. Molecular identity of the outwardly rectifying, swelling-activated anion channel: time to reevaluate pICln. J Gen Physiol 111: 617622, 1998.
49. Strange K, Emma F, and Jackson PS. Cellular and molecular physiology of volume-sensitive anion channels. Am J Physiol Cell Physiol 270: C711C730, 1996.
50. Stutzin A, Torres R, Oporto M, Pacheco P, Eguiguren AL, Cid LP, and Sepúlveda FV. Separate taurine and chloride efflux pathways activated during regulatory volume decrease. Am J Physiol Cell Physiol 277: C392C402, 1999.
51. Sweet DH, Bush KT, Nigam SK. The organic anion transporter family: from physiology to ontogeny and the clinic. Am J Physiol Renal Physiol 281: F197F205, 2001.
52. Tijburg LBM, Nishimaki-Mogami T, and Vance DE. Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes. Biochim Biophys Acta 1085: 167177, 1991.[ISI][Medline]
53. Tilly BC, Edixhoven MJ, Tertoolen LG, Morii N, Saitoh Y, Narumiya S, and de Jonge HR. Activation of the osmo-sensitive chloride conductance involves p21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton. Mol Biol Cell 7: 14191427, 1996.[Abstract]
54. Tilly BC, Edixhoven MJ, van den Berghe N, Bot AG, and de Jonge HR. Ca2+-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in intestine 407 cells. Am J Physiol Cell Physiol 267: C1271C1278, 1994.
55. Tilly BC, van den Berghe N, Tertoolen LG, Edixhoven MJ, and de Jonge HR. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances. J Biol Chem 268: 1991919922, 1993.
56. Vaandrager AB, Bajnath R, Groot JA, Bot AGM, and De Jonge HR. Ca2+ and cAMP activate different chloride efflux pathways in HT-29.cl19A colonic epithelial cell line. Am J Physiol Gastrointest Liver Physiol 261: G958G965, 1991.
57. Van der WijkT, de Jonge HR, and Tilly BC. Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels. Biochem J 343: 579586, 1999.[CrossRef][ISI][Medline]
58. Wessler I, Roth E, Deutsch C, Brockerhoff P, Bittinger F, Kirkpatrick CH, and Kilbinger H. Release of non-neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters. Br J Pharmacol 134: 951956, 2001.
59. Wieland A, Hayer-Zillgen M, Bonisch H, and Bruss M. Analysis of the gene structure of the human (SLC22A3) and murine (Slc22a3) extraneuronal monoamine transporter. J Neural Transm 107: 11491157, 2000.[CrossRef][ISI]