Neurochem Res (2014) 39:2234–2239 DOI 10.1007/s11064-014-1425-9
ORIGINAL PAPER
Zinc and Zinc Chelators Modify Taurine Transport in Rat Retinal Cells Asarı´ Ma´rquez • Mary Urbina • Lucimey Lima
Received: 20 June 2014 / Revised: 22 August 2014 / Accepted: 25 August 2014 / Published online: 3 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Zinc regulates Na?/Cl--dependent transporters, similar to taurine one, such as those for dopamine, serotonin and norepinephrine. This study examined the ex vivo effect of zinc (ZnSO4), N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino (TPEN) and diethylenetriaminepenta-acetic acid (DTPA), intracellular and extracellular zinc chelators, respectively, on rat retina [3H]taurine transport. Isolated cells were incubated in Locke solution with 100 nM of [3H]taurine for 25 s. Different concentrations of ZnSO4 (0.5–200 lM) were used. Low concentrations of ZnSO4 (30 and 40 lM) increased the transport, while higher concentrations (100, 150 and 200 lM) decreased it. Various concentrations of TPEN (1–200 lM) were added. Intermediate concentrations of TPEN (10–60 lM) significantly decreased [3H]taurine transport. The presence of TPEN, 20 lM, plus ZnSO4 reversed the effect of TPEN alone. Several concentrations of DTPA (1–500 lM) were also investigated. Reduction of transport took place at high concentrations of the chelator (100, 250 and 500 lM). DTPA, 500 lM, plus ZnSO4, did not modify the effect of it. These results indicate that zinc modulates taurine transport in a concentrationdependent manner, directly acting on the transporter or by forming taurine–zinc complexes in cell membranes. Keywords
Retina Taurine Taurine transport Zinc
A. Ma´rquez M. Urbina L. Lima (&) Laboratorio de Neuroquı´mica, Centro de Biofı´sica y Bioquı´mica, Instituto Venezolano de Investigaciones Cientı´ficas, Caracas, Venezuela e-mail: [email protected] A. Ma´rquez e-mail: [email protected]
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Abbreviations DTPA Diethylenetriaminepentaacetic acid io Intraocular TPEN N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino ZnSO4 Zinc sulfate
Introduction Taurine (2-aminoethane sulfonic acid) is a b-amino acid present at high concentrations in the retina of many vertebrates [1]. This amino acid is known to be involved in the mediation of multiple functions, such as osmoregulation, modulation of calcium fluxes, neuromodulation, antioxidation, modification of protein phosphorylation, membrane stabilization and regeneration of retinal cells after nerve lesion [2–6]. Zinc is highly concentrated in the retina [7], and it is believed to interact with taurine, to modify photoreceptor plasma membranes, to modulate synaptic transmission and to serve as an antioxidant [8]. Zinc deficiency in humans has been associated with abnormal dark adaptation, cataracts and blindness, as well as macular degeneration by decreasing antioxidants in the retina [7]. The role of zinc in the eye is associated with its coexistence with other molecules, such as taurine [7, 8]. Taurine system has been explored in the retina, such as synthesis, transport, storage [9–17], localization in various layers [18–21], and interaction with zinc in goldfish retina [21]. Despite accumulated evidences, there is no report concerning the effects of zinc on taurine transport in mammalian retina. Zinc regulates transporters with similar structure, such as those of dopamine, norepinephrine and serotonin [22–24]. Nusetti et al. [16] demonstrate that zinc causes a noncompetitive inhibition of high affinity taurine
Neurochem Res (2014) 39:2234–2239
transport in goldfish retinas, with an IC50 = 0.072 lM. Taurine transport inhibition did not reach 50 % with the concentrations used by these authors (0.001–500 lM), probably reflecting variable sensitivity of specific components or limited sites of zinc for exerting its effect. It might be that higher concentrations of zinc are needed to cause a greater inhibition [16]. It has been suggested that one of the mechanisms regulating taurine transporter (TAUT) occurs by transcription factors, as described, the promoter region of DNA for TAUT possesses several binding sites for different transcription factors, as tumor suppressor gene p53, transcriptional activator c-myb, gene viral homolog of avian myeloblastosis, activator-1 protein, Wills’1 tumor suppressor gene, and specificity protein 1 [15, 25]. For binding, p53 requires zinc to maintain structuredependent sequences of DNA [26, 27]. Wills’1 tumor suppressor gene and specificity protein 1 contain zinc finger motifs that directly bind to DNA to promote transcription [28, 29]. It has been suggested that specificity protein 1 is necessary for activating TAUT promoter DNA sequence [25], which might have a binding site to a response element for taurine, TREE, however, it has not been established yet [25]. TAUT has several putative phosphorylation sites for Ca2?/diacylglycerol-dependent protein kinase C [14, 15], a process dependent of calmodulin [14]. TAUT sequence also possesses phosphorylation sites for cAMP-dependent protein kinase [14]. Zinc affects taurine transport in goldfish retina [16], although the study of possible effects of zinc on taurine transport in mammalian retina is a novel area, and a line of interest for better understanding the interaction of zinc and taurine, and its relevance in physiopathological conditions.
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NaCl, 2.7 KCl, 2.1 K2HPO4, 0.95 KH2PO4, 2.7 sucrose and 2.5 HEPES, at 37 °C for 10 min, followed by mechanical separation with Pasteur pipette. The cells were washed with phosphate saline buffer Na?–K? (PBS) 0.1 M pH 7.4, centrifuged for 10 min at 2,000 rpm (300g), and counted in Neubauer chamber. Integrity of membrane was determined by 50 % Trypan blue exclusion ([96 %). Standardization of Taurine Transport in Isolated Rat Retinal Cells Taurine transport was determined at different incubation times of 10–120 s. Each tube had 100 ll of cell suspension (200,000 cells), 350 ll of Locke solution, 50 ll of [3H]taurine 15–165 nM final concentration (488 and 629 GBq), *298,839–2880,095 dpm per test tube. The cell preparation was preincubated for 5 min in Locke solution. Uptake was initiated by adding 100 ll of buffer containing radiolabeled substrate, [3H]taurine, incubation was done for 25 s at 37 °C. After the incubation, the process was stopped by rapid filtration through fiberglass filters (Watman GF/C), followed by two washed with 5 ml of cold Locke solution. The filters were placed in scintillation vials, dried and counted in 4 ml of toluene/omnifluor 0.04 % in a Packard scintillation counter Tricarb 1900 TR Model (efficiency 60–62 %) [11, 16]. [3H]Taurine uptake was also determined with dilutions of cell preparations, *62,500, 125,000, 250,000, 500,000 and 750,000 cells per tube. Na?-dependence was determined by incubating the cells in a modified Locke solution. Na? was substituted for N-methyl-glucamine 154 mM. Cells were incubated in the presence of b-alanine or hipotarurine, both at a concentration of 200 lM. The temperature dependence was evaluated by incubating cells in Locke solution at 0 and 37 °C for 25 s followed by filtration as described.
Materials and Methods Animals Male Sprague–Dawley rats (150–200 g) from the animal housing at Instituto Venezolano de Investigaciones Cient´ıficas had an adaptation period of at least 48 h in the experimental room of the Laboratory, food and water provided ad libitum. The animals were decapitated between 8:00 and 10:00 am, and the eyes were extracted from the orbit. Handling of animals was conducted following the standards of animal bioethics [30] and was approved by the Bioethics Committee for Animal Research of Instituto Venezolano de Investigaciones Cientı´ficas. Isolation of Rat Retinal Cells Retina was dissected and cells were isolated with 0.25 % trypsin in Locke buffer (500 ll), composed (in mM) of 154
Effect of Zinc, and Zinc Chelators Ex Vivo on the [3H]Taurine Transport All experiments were performed in duplicates, using *200,000 cells per tube, preincubated for 5 min and incubated for 25 s at 37 °C. To determine the effect of ZnSO4 and zinc chelators on [3H]taurine transport, each tube had 100 ll of cell suspension, 300 ll of Locke solution, 50 ll of [3H]taurine, and 50 ll of ZnSO4, N,N,N,NTetraquis-(2-pyridylmethyl) ethylendiamine (TPEN) or diethylenetriaminepenta-acetic acid (DTPA), at variable concentrations in dimethylsulfoxide (final \0.02 %). Solutions of substrate were prepared for each experiment and counted previously to performance in order to achieve the desired final concentration. Zinc sulphate was 0.5–200 lM. Concentrations of 40 or 100 lM were used for other experiments of [3H]taurine transport, according to results with the wide range. TPEN,
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Fig. 1 Effect of zinc sulfate (ZnSO4) ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of ZnSO4 for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
Neurochem Res (2014) 39:2234–2239
Taurine transport (fmol/106 cells)
2236 35 30
*
25
* *
20
*
*
15 10 5 0
ZnSO4 (µM)
intracellular chelator of zinc, was employed at various concentrations, 1–200 and 20 lM was considered for next experiments in combination with ZnSO4. DTPA, extracellular chelator of zinc, was utilized at several concentrations, 1–500 and 500 lM was used for further experiments in combination with ZnSO4. Preincubation and incubation in the presence of zinc or chelators were done as previously indicated. Equally, cells were washed and the process was stopped by filtration as already described. Statistical Analysis Each value represents mean ± standard error of the mean. The statistical significance of the specific data was determined by analysis of variance followed by Tukey test. Values of p \ 0.05 were considered significant.
Results Effect of Zinc on [3H]Taurine Transport The incubation of cell preparations with ZnSO4 had a biphasic effect on [3H]taurine transport. In the presence of 30 and 40 lM there was a significant increased of the transport, concentrations of 100, 150 and 200 lM of ZnSO4 reduced it (Fig. 1). Effect of N,N,N,N-tetrakis-(2-piridilmetil) Ethylenediamine on [3H]Taurine Transport TPEN, at low and high concentrations, resulted in no significant effect on transport compared to the control group (Fig. 2). Intermediate concentrations of TPEN (10, 20, 30, 40, 50 and 60 lM), significantly decreased [3H]taurine transport (Fig. 2). [3H]Taurine transport was significantly reduced by 20 lM TPEN, incubating the cells with TPEN plus zinc, 100 lM, did not significantly affect it, and the presence of zinc in the incubation medium significantly decreased it (Fig. 3).
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Effect of Diethylenetriaminepentaacetic Acid on [3H]Taurine Transport High concentrations of DTPA significantly decreased and low concentrations did not produce significant effects on [3H]taurine transport (Fig. 4). [3H]Taurine transport significantly decreased in the presence of 500 lM DTPA, incubation of the cells in the presence of DTPA plus zinc, 40 lM, did not affect [3H]taurine transport modified by the chelator, and the presence of ZnSO4, 40 lM, in the incubation medium significantly increased [3H]taurine transport (Fig. 5).
Discussion Beside the regulation of Na?/Cl--dependent transporters [22–24], other transporters, like those for glutamate and histidine, are also modulated by zinc [31, 32]. Norregaard et al. [33] showed that zinc is a noncompetitive inhibitor of dopamine transporter in synaptosomes and increases the binding of cocaine analogs to it. These effects occur due to binding to a particular transporter site affecting translocation of dopamine. The tertiary structure of the transporter is modified, involving His193, His375, and Glu396 residues, which are close together [34]. Moreover, zinc facilitates the formation of oligomeric complexes of the transporter, which affect translocation of the monoamine [35]. Stockner et al. [36], by mutagenesis and structural changes, confirmed the presence of these residues interacting with zinc. The changes of transporter structure could influence its sensitivity to other regulatory processes, such as phosphorylation. Zinc has a direct quick and irreversible inhibitory effect on glutamate transporter in Mu¨ller cells and cones of salamander retina [37]. These authors conclude that zinc changes the affinity for glutamate. By mutagenesis and molecular models, binding sites of zinc to His and Cys residues of serotonin transporter have been identified, which are located near the extracellular ends of the transmembrane helices I and III [38]. Increased histamin uptake
Neurochem Res (2014) 39:2234–2239 Fig. 2 Effect of the intracellular chelator of zinc, TPEN, ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of TPEN for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
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Taurine transport fmol/10 6 cells
120 100 80
*
*
*
*
*
*
60 40 20 0
TPEN (µM)
Taurine transport fmol/10 6 cells
30 25
*
*
20 15 10 5 0 Control
TPEN
TPEN+ZINC
ZINC
3
Fig. 3 Effect of TPEN and ZnSO4 ex vivo on [ H]taurine transport. Cells (*200,000) were preincubated in Locke solution in the absence or in the presence of TPEN, 20 lM, of ZnSO4, 100 lM, or both for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
by elevating Vmax, but unaltered Kt, is produced by zinc ex vivo in cultured endothelial and astroglial cells from brain [32]. The above evidence indicates that zinc affects various transport systems by modulating different subtypes of transporters, including that of taurine, as shown by our findings in the retina. The decrease of taurine transport caused by zinc, 100 lM, (Figs. 1, 3) could indicate moderate affection or specific target of one of TAUT, TAUT-1 or TAUT-2. However, the presence of 40 lM of zinc caused a significant increase of taurine transport (Figs. 1, 5). The
25
Taurine transport (fmol/10 6 cells)
Fig. 4 Effect of the extracellular chelator of zinc, DTPA, ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of DTPA for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
mechanism by which zinc is affecting taurine transport remains unknown, thus, the present results are evidences of modifications that needs further approaches. Zinc may interact with a site of the transporter that affects the binding and translocation of taurine or form taurine-zinc complexes resulting in modifications of transporter function. Probably TAUT contains His residues, as found in serotonin and dopamine transporters, which, as mentioned, belong to the same family of Na?/ Cl--dependent transporters [34, 38]. However, there are no reports demonstrating the presence of crucial His residues in the structure of TAUT. Studying TAUT sequence is of great interest, since it will contribute to determining zinc binding sites and mechanisms of modulating its function. Dual effect of zinc, as demonstrated, has also been shown for dopamine transporter, which is stimulated at concentrations in the nM and is inhibited in lM range [22, 34, 39]. In addition, glycine receptors in ganglion cells of rat retina are stimulated by low concentrations of zinc (\2 lM) and inhibited at high concentrations ([10 lM) [40]. Nusetti et al. [16, 17] showed that zinc produces a decrease in the capacity of taurine transport without changes in affinity, by non-competitive inhibition of high affinity transport in goldfish retinal cells, but, up to now, there is no evidence in mammalian retina. Observing the biphasic effect of zinc on taurine transport (Fig. 1), it could be possible that conformation of the transporter affected by
20
*
*
*
15 10 5 0
DTPA (µM)
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2238
Neurochem Res (2014) 39:2234–2239
Taurine transport (fmol/106cells)
30
*
25
**
20
*
15 10 5 0 Control
DTPA
DTPA+ZINC
ZINC
Fig. 5 Effect of DTPA and ZnSO4 ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution in the absence or in the presence of DTPA, 500 lM, of ZnSO4, 40 lM, or both for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control. **P \ 0.05 respect to DTPA
zinc results in an optimal structure or might be acting in particular stages of transporter cycle during the process of penetration to the internal compartment. The wide range of explored concentrations of TPEN and DTPA allowed to observe variable effects. This might indicate that fluxes of zinc reaching optimity inside or outside the cell are relevant for modulation of taurine transport. The potency of both chelators, as expected, are in relation to target compartments and own affinity for zinc, which differentially affect transport. The present results contribute to enrich evidences about the interaction of zinc and taurine in the retina, probably in relation to structure-function of the tissue, although the mechanisms involved are not completely clear at the present. Moreover, the role of both molecules in central nervous system, such as brain and retina, and the relation to neurodegenerative diseases are challenges for the future. Acknowledgments Asarı´ Ma´rquez was a PhD Student of Centro de Estudios Avanzados of Instituto Venezolano de Investigaciones Cientı´ficas. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this work.
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5. Lima L (1999) Taurine and its trophic effect in the retina. Neurochem Res 24:1333–1338 6. Nusetti S, Obrego´n F, Quintal M, Benzo Z, Lima L (2005) Taurine and zinc modulate outgrowth from goldfish retinal explants. Neurochem Res 30:1483–1492 7. Ugarte M, Osborne N (2001) Zinc in the retina. Prog Neurobiol 64:219–249 8. Grahn B, Paterson P, Gottschall-Pass K, Zhang Z (2001) Zinc and the eye. J Am Coll Nutr 20:106–118 9. Lima L, Matus P, Drujan B (1991) Differential taurine uptake in central and peripheral regions of goldfish retina. J Neurosci Res 28:422–427 10. Militante J, Lombardini J (1999) Taurine uptake activity in the rat retina: protein kinase C-independent inhibition by chelerythrine. Brain Res 818:368–374 11. Guerra A, Urbina M, Lima L (2000) Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum, Effect of optic crush and axotomy. Amino Acids 19:1–17 12. Lima L, Cubillos S, Guerra A (2000) Regulation of high affinity taurine transport in goldfish and rat retinal cells. Adv Exp Med Biol 483:431–440 13. Hillenkamp J, Hussain A, Jackson T, Constable P, Cunningbam J, Marshall J (2004) Compartmental analysis of taurine transport to the outer retina in the bovine eye. Invest Ophthalmol Vis Sci 45:4099–4105 14. Tappaz M (2004) Taurine biosynthetic enzymes and taurine transporter: molecular identification and regulations. Neurochem Res 29:83–96 15. Han X, Patters A, Jones D, Zellicovik I, Chesney R (2006) The taurine transporter: mechanisms of regulation. Acta Physiol (Oxf) 187:61–73 16. Nusetti S, Urbina M, Lima L (2010) Effects of zinc ex vivo on taurine uptake in goldfish retinal cells. J Biomed Sci 17(Suppl 1):S13. doi:10.1186/1423-0127-17-S1-S13 17. Nusetti S, Urbina M, Obrego´n F, Quintal M, Benzo Z, Lima L (2010) Effects of zinc ex vivo and intracelular zinc chelator in vivo on taurine uptake in goldfish retina. Amino Acids 38:1429–1437 18. Orr H, Cohen A, Lowry O (1976) The distribution of taurine in the vertebrate retina. J Neurochem 26:609–611 19. Nag TC, Jotwani G, Wadhwa S (1998) Immunohistochemical localization of taurine in the retina of developing and adult human and adult monkey. Neurochem Int 33:195–200 20. Pow DV, Sullivan R, Reye P, Hermanussen S (2002) Localization of taurine transporters, taurine, and 3H taurine accumulation in the rat retina, pituitary, and brain. Glia 37:153–168 21. Nusetti S, Salazar V, Lima L (2009) Localization of taurine transporter, taurine, and zinc in goldfish retina. Adv Exp Med Biol 643:233–242 22. Scholze P, Norregaard L, Singer E, Freissmuth M, Gether U, Sitte H (2002) The role of zinc ions in reverse transport mediated by monoamine transporters. J Biol Chem 277:21505–21513 23. Garcı´a-Colunga J, Reyes-Haro D, Godoy-Garcı´a U, Miledi R (2005) Zinc modulation of serotonin uptake in the adult rat corpus callosum. J Neurosci Res 80:145–149 24. Norgaard-Nielsen K, Gether U (2006) Zn2? modulation of neurotransmitter transporters. Handb Exp Pharmacol 175:1–22 25. Chesney R, Patters A, Han X (2012) Taurine in the kidneys. In: El Idrissi A, L´Amoreaoux W (eds) Taurine in health and disease. Transworld Research Network, Kerala, India, pp 75–99 26. Hainaut P, Mann K (2001) Zinc binding and redox control of p53 structure and function. Antioxid Redox Signal 3:611–623 27. Butler JS, Loh SN (2003) Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain. Biochemistry 42:2396–2403
Neurochem Res (2014) 39:2234–2239 28. Laity JH, Lee BM, Wright PE (2001) Zinc finger proteins: new insights into structural and functional diversity. Curr Opin Struct Biol 11:39–46 29. Ito T, Azumano M, Uwatoko C, Itoh K, Kuwahara K (2009) Role of zinc finger structure in nuclear localization of transcription factor Sp1. Biochem Biophys Res Commun 380:28–32 30. Jayo MJ, Cisneros FJ (1996) Guı´a para el Cuidado y Uso de los Animales de Laboratorio. Copyright National Academy Press, Washington, DC 31. Vanderberg RJ, Mitrovic AD, Johnston GA (1998) Molecular basis for differential inhibition of glutamate transporter subtypes by zinc ions. Mol Pharmacol 54:189–196 32. Huszti Z, Horva´th-Szklai A, Nosza´l B, Madara´sz E, Deli A (2001) Enhancing effect of zinc on astroglial and cerebral endotelial histamine uptake. Biochem Pharmacol 62:1491–1500 33. Norregaard L, Frederiksen D, Nielsen EO, Gether U (1998) Delineation of an endogenous zinc-binding site in the human dopamine transporter. EMBO J 17:4266–4273 34. Norgaard-Nielsen K, Norregaard L, Hastrup H, Javitch JA, Gether U (2002) Zn27 site engineering at the oligomeric interface of the dopamine transporter. FEBS Lett 524:87–91
2239 35. Giros B, Caron MG (1993) Molecular characterization of the dopamine transporter. Trends Pharmacol Sci 14(2):43–49 36. Stockner T, Montgomery TR, Kudlacek O, Weissensteiner R, Ecker GF, Freissmuth M, Sitte HH (2013) Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model. PLoS Comput Biol 9:e1002909. doi:10.1371/journal.pcbi.1002909 37. Spiridon M, Kamm D, Billups B, Mobbs P, Attwell D (1998) Modulation by zinc of the glutamate transporters in glial cells and cones isolated from the tiger salamander retina. J Physiol 506:363–376 38. White KJ, Kiser PD, Nichols DE, Barker EL (2006) Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter. Protein Sci 15:2411–2422 39. Loland CJ, Norregaard L, Gether U (1999) Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous zn21-binding site. J Biol Chem 274:36928–36934 40. Han Y, Wu SM (1999) Modulation of glycine receptors in retinal ganglion cells by zinc (neurotransmitterysynaptic transmission). Proc Natl Acad Sci USA 96:3234–3238
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ORIGINAL PAPER
Zinc and Zinc Chelators Modify Taurine Transport in Rat Retinal Cells Asarı´ Ma´rquez • Mary Urbina • Lucimey Lima
Received: 20 June 2014 / Revised: 22 August 2014 / Accepted: 25 August 2014 / Published online: 3 September 2014 Ó Springer Science+Business Media New York 2014
Abstract Zinc regulates Na?/Cl--dependent transporters, similar to taurine one, such as those for dopamine, serotonin and norepinephrine. This study examined the ex vivo effect of zinc (ZnSO4), N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino (TPEN) and diethylenetriaminepenta-acetic acid (DTPA), intracellular and extracellular zinc chelators, respectively, on rat retina [3H]taurine transport. Isolated cells were incubated in Locke solution with 100 nM of [3H]taurine for 25 s. Different concentrations of ZnSO4 (0.5–200 lM) were used. Low concentrations of ZnSO4 (30 and 40 lM) increased the transport, while higher concentrations (100, 150 and 200 lM) decreased it. Various concentrations of TPEN (1–200 lM) were added. Intermediate concentrations of TPEN (10–60 lM) significantly decreased [3H]taurine transport. The presence of TPEN, 20 lM, plus ZnSO4 reversed the effect of TPEN alone. Several concentrations of DTPA (1–500 lM) were also investigated. Reduction of transport took place at high concentrations of the chelator (100, 250 and 500 lM). DTPA, 500 lM, plus ZnSO4, did not modify the effect of it. These results indicate that zinc modulates taurine transport in a concentrationdependent manner, directly acting on the transporter or by forming taurine–zinc complexes in cell membranes. Keywords
Retina Taurine Taurine transport Zinc
A. Ma´rquez M. Urbina L. Lima (&) Laboratorio de Neuroquı´mica, Centro de Biofı´sica y Bioquı´mica, Instituto Venezolano de Investigaciones Cientı´ficas, Caracas, Venezuela e-mail: [email protected] A. Ma´rquez e-mail: [email protected]
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Abbreviations DTPA Diethylenetriaminepentaacetic acid io Intraocular TPEN N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino ZnSO4 Zinc sulfate
Introduction Taurine (2-aminoethane sulfonic acid) is a b-amino acid present at high concentrations in the retina of many vertebrates [1]. This amino acid is known to be involved in the mediation of multiple functions, such as osmoregulation, modulation of calcium fluxes, neuromodulation, antioxidation, modification of protein phosphorylation, membrane stabilization and regeneration of retinal cells after nerve lesion [2–6]. Zinc is highly concentrated in the retina [7], and it is believed to interact with taurine, to modify photoreceptor plasma membranes, to modulate synaptic transmission and to serve as an antioxidant [8]. Zinc deficiency in humans has been associated with abnormal dark adaptation, cataracts and blindness, as well as macular degeneration by decreasing antioxidants in the retina [7]. The role of zinc in the eye is associated with its coexistence with other molecules, such as taurine [7, 8]. Taurine system has been explored in the retina, such as synthesis, transport, storage [9–17], localization in various layers [18–21], and interaction with zinc in goldfish retina [21]. Despite accumulated evidences, there is no report concerning the effects of zinc on taurine transport in mammalian retina. Zinc regulates transporters with similar structure, such as those of dopamine, norepinephrine and serotonin [22–24]. Nusetti et al. [16] demonstrate that zinc causes a noncompetitive inhibition of high affinity taurine
Neurochem Res (2014) 39:2234–2239
transport in goldfish retinas, with an IC50 = 0.072 lM. Taurine transport inhibition did not reach 50 % with the concentrations used by these authors (0.001–500 lM), probably reflecting variable sensitivity of specific components or limited sites of zinc for exerting its effect. It might be that higher concentrations of zinc are needed to cause a greater inhibition [16]. It has been suggested that one of the mechanisms regulating taurine transporter (TAUT) occurs by transcription factors, as described, the promoter region of DNA for TAUT possesses several binding sites for different transcription factors, as tumor suppressor gene p53, transcriptional activator c-myb, gene viral homolog of avian myeloblastosis, activator-1 protein, Wills’1 tumor suppressor gene, and specificity protein 1 [15, 25]. For binding, p53 requires zinc to maintain structuredependent sequences of DNA [26, 27]. Wills’1 tumor suppressor gene and specificity protein 1 contain zinc finger motifs that directly bind to DNA to promote transcription [28, 29]. It has been suggested that specificity protein 1 is necessary for activating TAUT promoter DNA sequence [25], which might have a binding site to a response element for taurine, TREE, however, it has not been established yet [25]. TAUT has several putative phosphorylation sites for Ca2?/diacylglycerol-dependent protein kinase C [14, 15], a process dependent of calmodulin [14]. TAUT sequence also possesses phosphorylation sites for cAMP-dependent protein kinase [14]. Zinc affects taurine transport in goldfish retina [16], although the study of possible effects of zinc on taurine transport in mammalian retina is a novel area, and a line of interest for better understanding the interaction of zinc and taurine, and its relevance in physiopathological conditions.
2235
NaCl, 2.7 KCl, 2.1 K2HPO4, 0.95 KH2PO4, 2.7 sucrose and 2.5 HEPES, at 37 °C for 10 min, followed by mechanical separation with Pasteur pipette. The cells were washed with phosphate saline buffer Na?–K? (PBS) 0.1 M pH 7.4, centrifuged for 10 min at 2,000 rpm (300g), and counted in Neubauer chamber. Integrity of membrane was determined by 50 % Trypan blue exclusion ([96 %). Standardization of Taurine Transport in Isolated Rat Retinal Cells Taurine transport was determined at different incubation times of 10–120 s. Each tube had 100 ll of cell suspension (200,000 cells), 350 ll of Locke solution, 50 ll of [3H]taurine 15–165 nM final concentration (488 and 629 GBq), *298,839–2880,095 dpm per test tube. The cell preparation was preincubated for 5 min in Locke solution. Uptake was initiated by adding 100 ll of buffer containing radiolabeled substrate, [3H]taurine, incubation was done for 25 s at 37 °C. After the incubation, the process was stopped by rapid filtration through fiberglass filters (Watman GF/C), followed by two washed with 5 ml of cold Locke solution. The filters were placed in scintillation vials, dried and counted in 4 ml of toluene/omnifluor 0.04 % in a Packard scintillation counter Tricarb 1900 TR Model (efficiency 60–62 %) [11, 16]. [3H]Taurine uptake was also determined with dilutions of cell preparations, *62,500, 125,000, 250,000, 500,000 and 750,000 cells per tube. Na?-dependence was determined by incubating the cells in a modified Locke solution. Na? was substituted for N-methyl-glucamine 154 mM. Cells were incubated in the presence of b-alanine or hipotarurine, both at a concentration of 200 lM. The temperature dependence was evaluated by incubating cells in Locke solution at 0 and 37 °C for 25 s followed by filtration as described.
Materials and Methods Animals Male Sprague–Dawley rats (150–200 g) from the animal housing at Instituto Venezolano de Investigaciones Cient´ıficas had an adaptation period of at least 48 h in the experimental room of the Laboratory, food and water provided ad libitum. The animals were decapitated between 8:00 and 10:00 am, and the eyes were extracted from the orbit. Handling of animals was conducted following the standards of animal bioethics [30] and was approved by the Bioethics Committee for Animal Research of Instituto Venezolano de Investigaciones Cientı´ficas. Isolation of Rat Retinal Cells Retina was dissected and cells were isolated with 0.25 % trypsin in Locke buffer (500 ll), composed (in mM) of 154
Effect of Zinc, and Zinc Chelators Ex Vivo on the [3H]Taurine Transport All experiments were performed in duplicates, using *200,000 cells per tube, preincubated for 5 min and incubated for 25 s at 37 °C. To determine the effect of ZnSO4 and zinc chelators on [3H]taurine transport, each tube had 100 ll of cell suspension, 300 ll of Locke solution, 50 ll of [3H]taurine, and 50 ll of ZnSO4, N,N,N,NTetraquis-(2-pyridylmethyl) ethylendiamine (TPEN) or diethylenetriaminepenta-acetic acid (DTPA), at variable concentrations in dimethylsulfoxide (final \0.02 %). Solutions of substrate were prepared for each experiment and counted previously to performance in order to achieve the desired final concentration. Zinc sulphate was 0.5–200 lM. Concentrations of 40 or 100 lM were used for other experiments of [3H]taurine transport, according to results with the wide range. TPEN,
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Fig. 1 Effect of zinc sulfate (ZnSO4) ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of ZnSO4 for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
Neurochem Res (2014) 39:2234–2239
Taurine transport (fmol/106 cells)
2236 35 30
*
25
* *
20
*
*
15 10 5 0
ZnSO4 (µM)
intracellular chelator of zinc, was employed at various concentrations, 1–200 and 20 lM was considered for next experiments in combination with ZnSO4. DTPA, extracellular chelator of zinc, was utilized at several concentrations, 1–500 and 500 lM was used for further experiments in combination with ZnSO4. Preincubation and incubation in the presence of zinc or chelators were done as previously indicated. Equally, cells were washed and the process was stopped by filtration as already described. Statistical Analysis Each value represents mean ± standard error of the mean. The statistical significance of the specific data was determined by analysis of variance followed by Tukey test. Values of p \ 0.05 were considered significant.
Results Effect of Zinc on [3H]Taurine Transport The incubation of cell preparations with ZnSO4 had a biphasic effect on [3H]taurine transport. In the presence of 30 and 40 lM there was a significant increased of the transport, concentrations of 100, 150 and 200 lM of ZnSO4 reduced it (Fig. 1). Effect of N,N,N,N-tetrakis-(2-piridilmetil) Ethylenediamine on [3H]Taurine Transport TPEN, at low and high concentrations, resulted in no significant effect on transport compared to the control group (Fig. 2). Intermediate concentrations of TPEN (10, 20, 30, 40, 50 and 60 lM), significantly decreased [3H]taurine transport (Fig. 2). [3H]Taurine transport was significantly reduced by 20 lM TPEN, incubating the cells with TPEN plus zinc, 100 lM, did not significantly affect it, and the presence of zinc in the incubation medium significantly decreased it (Fig. 3).
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Effect of Diethylenetriaminepentaacetic Acid on [3H]Taurine Transport High concentrations of DTPA significantly decreased and low concentrations did not produce significant effects on [3H]taurine transport (Fig. 4). [3H]Taurine transport significantly decreased in the presence of 500 lM DTPA, incubation of the cells in the presence of DTPA plus zinc, 40 lM, did not affect [3H]taurine transport modified by the chelator, and the presence of ZnSO4, 40 lM, in the incubation medium significantly increased [3H]taurine transport (Fig. 5).
Discussion Beside the regulation of Na?/Cl--dependent transporters [22–24], other transporters, like those for glutamate and histidine, are also modulated by zinc [31, 32]. Norregaard et al. [33] showed that zinc is a noncompetitive inhibitor of dopamine transporter in synaptosomes and increases the binding of cocaine analogs to it. These effects occur due to binding to a particular transporter site affecting translocation of dopamine. The tertiary structure of the transporter is modified, involving His193, His375, and Glu396 residues, which are close together [34]. Moreover, zinc facilitates the formation of oligomeric complexes of the transporter, which affect translocation of the monoamine [35]. Stockner et al. [36], by mutagenesis and structural changes, confirmed the presence of these residues interacting with zinc. The changes of transporter structure could influence its sensitivity to other regulatory processes, such as phosphorylation. Zinc has a direct quick and irreversible inhibitory effect on glutamate transporter in Mu¨ller cells and cones of salamander retina [37]. These authors conclude that zinc changes the affinity for glutamate. By mutagenesis and molecular models, binding sites of zinc to His and Cys residues of serotonin transporter have been identified, which are located near the extracellular ends of the transmembrane helices I and III [38]. Increased histamin uptake
Neurochem Res (2014) 39:2234–2239 Fig. 2 Effect of the intracellular chelator of zinc, TPEN, ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of TPEN for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
2237
Taurine transport fmol/10 6 cells
120 100 80
*
*
*
*
*
*
60 40 20 0
TPEN (µM)
Taurine transport fmol/10 6 cells
30 25
*
*
20 15 10 5 0 Control
TPEN
TPEN+ZINC
ZINC
3
Fig. 3 Effect of TPEN and ZnSO4 ex vivo on [ H]taurine transport. Cells (*200,000) were preincubated in Locke solution in the absence or in the presence of TPEN, 20 lM, of ZnSO4, 100 lM, or both for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
by elevating Vmax, but unaltered Kt, is produced by zinc ex vivo in cultured endothelial and astroglial cells from brain [32]. The above evidence indicates that zinc affects various transport systems by modulating different subtypes of transporters, including that of taurine, as shown by our findings in the retina. The decrease of taurine transport caused by zinc, 100 lM, (Figs. 1, 3) could indicate moderate affection or specific target of one of TAUT, TAUT-1 or TAUT-2. However, the presence of 40 lM of zinc caused a significant increase of taurine transport (Figs. 1, 5). The
25
Taurine transport (fmol/10 6 cells)
Fig. 4 Effect of the extracellular chelator of zinc, DTPA, ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution and various concentrations of DTPA for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control
mechanism by which zinc is affecting taurine transport remains unknown, thus, the present results are evidences of modifications that needs further approaches. Zinc may interact with a site of the transporter that affects the binding and translocation of taurine or form taurine-zinc complexes resulting in modifications of transporter function. Probably TAUT contains His residues, as found in serotonin and dopamine transporters, which, as mentioned, belong to the same family of Na?/ Cl--dependent transporters [34, 38]. However, there are no reports demonstrating the presence of crucial His residues in the structure of TAUT. Studying TAUT sequence is of great interest, since it will contribute to determining zinc binding sites and mechanisms of modulating its function. Dual effect of zinc, as demonstrated, has also been shown for dopamine transporter, which is stimulated at concentrations in the nM and is inhibited in lM range [22, 34, 39]. In addition, glycine receptors in ganglion cells of rat retina are stimulated by low concentrations of zinc (\2 lM) and inhibited at high concentrations ([10 lM) [40]. Nusetti et al. [16, 17] showed that zinc produces a decrease in the capacity of taurine transport without changes in affinity, by non-competitive inhibition of high affinity transport in goldfish retinal cells, but, up to now, there is no evidence in mammalian retina. Observing the biphasic effect of zinc on taurine transport (Fig. 1), it could be possible that conformation of the transporter affected by
20
*
*
*
15 10 5 0
DTPA (µM)
123
2238
Neurochem Res (2014) 39:2234–2239
Taurine transport (fmol/106cells)
30
*
25
**
20
*
15 10 5 0 Control
DTPA
DTPA+ZINC
ZINC
Fig. 5 Effect of DTPA and ZnSO4 ex vivo on [3H]taurine transport. Cells (*200,000) were preincubated in Locke solution in the absence or in the presence of DTPA, 500 lM, of ZnSO4, 40 lM, or both for 5 min at 37 °C. Transport was initiated by adding [3H]taurine and incubated for 25 s. N = 4, *P \ 0.05 respect to Control. **P \ 0.05 respect to DTPA
zinc results in an optimal structure or might be acting in particular stages of transporter cycle during the process of penetration to the internal compartment. The wide range of explored concentrations of TPEN and DTPA allowed to observe variable effects. This might indicate that fluxes of zinc reaching optimity inside or outside the cell are relevant for modulation of taurine transport. The potency of both chelators, as expected, are in relation to target compartments and own affinity for zinc, which differentially affect transport. The present results contribute to enrich evidences about the interaction of zinc and taurine in the retina, probably in relation to structure-function of the tissue, although the mechanisms involved are not completely clear at the present. Moreover, the role of both molecules in central nervous system, such as brain and retina, and the relation to neurodegenerative diseases are challenges for the future. Acknowledgments Asarı´ Ma´rquez was a PhD Student of Centro de Estudios Avanzados of Instituto Venezolano de Investigaciones Cientı´ficas. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this work.
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