The influence of sodium–calcium exchange inhibitors on rabbit lens ion balance and transparency

Experimental Eye Research 83 (2006) 1089e1095 www.elsevier.com/locate/yexer

The influence of sodiumecalcium exchange inhibitors on rabbit lens ion balance and transparency Shigeo Tamiya a, Nicholas A. Delamere a,b,* a

Department of Ophthalmology and Visual Sciences, University of Louisville, School of Medicine, Louisville, KY 40202, USA b Department of Pharmacology and Toxicology, University of Louisville, School of Medicine, Louisville, KY 40202, USA Received 24 January 2006; accepted in revised form 17 May 2006 Available online 12 July 2006

Abstract Calcium regulation is essential to the maintenance of lens transparency. To maintain cytoplasmic calcium concentration at the required low level the lens must export calcium continuously. Here, studies were conducted to test whether sodiumecalcium exchanger (NCX) inhibitors disturb calcium balance in the rabbit lens. Intact lenses were incubated up to 48 h in the presence or absence of the NCX inhibitor bepridil. Lens sodium, potassium and calcium content were determined by atomic absorption spectrophotometry. Fluo-4 was used to measure epithelial cell cytoplasmic calcium concentration in an intact lens preparation. NCX1 protein expression in lens epithelium was examined by western blot. NCX1 band density was similar in central and equatorial epithelium samples. Lenses exposed to bepridil (30 mM) lost transparency at the anterior and exhibited significant changes in electrolyte and water content. After 48 h, lens calcium content more than doubled, sodium increased four fold and potassium was significantly reduced. In contrast, lenses exposed to inhibitors of reverse mode calcium transport by NCX (KBR7943 or SN-6) remained transparent and the electrolyte and water content of the lens remained unchanged. The ability of bepridil to cause significant changes in lens transparency and electrolyte content points to an important role for NCX-meditated calcium export in the lens. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: NaeCa exchange; lens; calcium; sodium; hydration; transparency

1. Introduction Calcium regulation is essential to the maintenance of lens transparency. In human age-related cortical cataract, the lens calcium content increases dramatically and the magnitude of the calcium increase parallels the severity of the opacity (Duncan and Bushell, 1975). The mechanistic role of calcium in lens opacification is not fully understood but it is clear that multiple processes are involved. Binding of calcium to crystallins is thought to cause aggregation leading to light scatter (Jedziniak et al., 1972; Fein et al., 1979). Studies on selenite-induced cataract suggest that loss of transparency follows the activation of proteases by calcium * Corresponding author. Department of Ophthalmology and Visual Sciences, University of Louisville, School of Medicine, Louisville, KY 40202, USA. Tel.: þ1 502 852 5459; fax: þ1 502 852 7450. E-mail address: [email protected] (N.A. Delamere). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.05.014

(David and Shearer, 1984). Calcium accumulation is accompanied by more widespread disruption of lens electrolyte balance with an increase of sodium and loss of potassium. Such abnormal sodium and potassium levels disturb osmotic balance, causing the lens to gain water and swell. This too compromises transparency. Most calcium in the lens is sequestered and the free calcium concentration in the cytoplasm is <250 nM (Duncan et al., 1993; Collison et al., 2000). The calcium concentration in aqueous humor is w2 mM. Thus, the calcium gradient across the plasma membrane is steep. To maintain cytoplasmic calcium concentration at the required low level, the lens must export calcium continuously. In most cells, two mechanisms are available for calcium export: plasma membrane Ca-ATPase (PMCA) and NaeCa exchanger (NCX). The relative contribution of PMCA and NCX to calcium export is difficult to define because the situation can change (Sedova and Blatter, 1999). Thus, one

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mechanism may be dominant in a resting cell while the other mechanism comes into play when the cell is activated. In cultured porcine lens epithelium, the NCX inhibitor bepridil was found to cause a pronounced increase in the magnitude and duration of cytoplasmic calcium transients that occur in response to G protein-coupled receptor activation (Okafor et al., 2003). In contrast, bepridil caused only a modest increase in the baseline calcium concentration, suggesting that in resting lens epithelial cells the contribution of NCX to calcium export is minor. Based on studies with sodium-free medium, a minor contribution of NCX to calcium regulation also has been proposed in cultured bovine and human lens epithelium (Duncan et al., 1993). While the behavior of cultured cells may not reflect the situation in the intact lens, studies on rat lens (Tomlinson et al., 1991) demonstrated a pattern of 45Ca2þ fluxes consistent with sodiumecalcium exchange. The present study was conducted to test whether bepridil disturbs calcium balance in the intact rabbit lens. Experiments also were conducted to examine the response of the lens to KBR7943 and SN-6, compounds that selectively inhibit the reverse mode of NCX transport (Iwamoto et al., 2004). In reverse mode, NCX exports sodium and shifts calcium in the opposite direction, from the extracellular solution to the cytoplasm.

2. Methods KBR7943 and SN-6 were obtained from Tocris (Ellisville, MO). N-methyl-D-glucamine (NMDG), bepridil and other general chemicals were obtained from the Sigma Chemical Co. (St. Louis, MO). Medium 199 and sodium chloride-free/ calcium chloride-free Medium 199 were obtained from Invitrogen/Gibco (Carlesburg, CA). NMDG or, in some experiments choline chloride, was used as a substitute for sodium chloride in low-sodium M199. In solutions containing NMDG, pH was adjusted using HCl and thus the final concentration of chloride was unknown. Unless nominally calcium-free conditions were required, CaCl2 (final concentration 1.8 mM) was added to the low-sodium medium. Rabbit polyclonal NCX1 antibody was obtained from Swant (Bellinzona, Switzerland). Fluo-4-AM and Pluronic F127 were obtained from Molecular Probes (Eugene, OR). Albino Rabbit eyes were obtained from Pel Freez Biologicals (Rogers, AR) and were shipped to the laboratory overnight on ice. The lens was dissected from the eye by a posterior approach. Beginning with an incision at the optic nerve head, the eye was dissected open then the zonules were cut and the lens removed from the globe. The lenses were incubated for specified times in individual culture dishes containing 20 ml of medium M199 supplemented with 0.1% bovine serum albumin and penicillin/streptomycin (100 U per ml/0.1 mg per ml). The culture dish was placed in a humidified 36
C incubator equilibrated with 5% CO2/air. In some experiments, lenses were photographed using a digital camera. The culture dish containing incubated lens was placed on a dark background and illuminated from the side.

To analyze electrolyte and water content, lenses were first blotted gently on moist filter paper to remove adherent vitreous humor and culture medium. Each lens was then weighed, dried overnight in a 70
C oven and re-weighed. Weight loss upon drying was assumed to represent lens water content. Dried lenses were digested in 30% nitric acid for 4 h at 70
C. The acid digest was diluted and the concentration of sodium and potassium was measured by atomic absorption spectrofluorometry (Perkin Elmer, Elmwood, NY). Lanthanum chloride (1%) was added to an aliquot of the acid digest that was then used to measure calcium concentration by atomic absorption. Lanthanum improves calcium detection by quenching interference from other ions. For western blot studies, the lens capsule-epithelium was isolated and homogenized in RIPA buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate and protease inhibitors (2 mM antipain, 2 mM leupeptin, 1 mM pepstatin A, 1 mM phenylmethylsulfonylfluoride [PMSF] and 2 mg/ml aprotinin). The mixture was solubilized in Laemmli buffer and separated by electrophoresis on a 7.5% SDS-polyacrylamide mini-gel. Proteins were then transferred by electrophoresis to nitrocellulose membrane then blocked for 1 h with Odyssey blocking buffer (Licor, Lincoln, NE). After blocking, the nitrocellulose membrane was incubated overnight with anti-NCX antibody at 4
C in blocking buffer. The antibody was raised against canine cardiac sarcolemma NCX1. After five washes in TTBS (30 mM Tris, 150 mM NaCl, 0.5% (v/v) Tween-20 at pH 7.4), the nitrocellulose membrane was incubated for 1 h with secondary antibody conjugated with IRDye 800 (Rockland, Gilbertsville, PA). Protein bands were visualized by infra-red laser scanning (Licor Odyssey, Lincoln, NE). Cytoplasmic calcium concentration measurements were made using an intact lens preparation described first by Collison and Duncan (2001). Lenses were pre-incubated for 2 days under control conditions, then placed anterior upward in a plastic chamber containing artificial aqueous humor (AAH) (130 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 20 mM Hepes, 5 mM NaHCO3, 5 mM glucose, pH 7.3). The calcium-sensitive fluorescent dye Fluo-4-AM was first dissolved in a solution of Pluronic F127 (20%) in dimethylsulfoxide which was then added to the AAH at a final concentration of 5 mM Fluo-4-AM. The lens was exposed to Fluo-4-AM for 45 min and then the Fluo-4-AM solution was replaced with control AAH for 20 min to allow the dye to de-esterify to the Fluo-4 form. The chamber with the lens anterior facing upwards was positioned on the stage of an upright microscope equipped with a digital fluorescence imaging system (Zeiss/ Attofluor, Rockville MD). A water immersion objective was used to focus on the epithelium. The lens was continuously superfused with AAH and maintained close to 37
C. The emission wavelength was 520 nm and the excitation wavelength was 488 nm. Fluorescence was quantified continuously. The results are presented as fluorescence values (arbitrary units).

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3. Results NCX protein expression was examined separately in central and equatorial regions of the lens epithelium (Fig. 1). A dense immunoreactive protein band was observed at approximately w160 kDa, the size reported for NCX in other tissues (Philipson et al., 1988). Band density was similar in central and equatorial epithelium samples. In both tissue samples, a second immunoreactive band appeared at w70 kDa, characteristic of the NCX cleaved form (Saba et al., 1999). Results from lens fibers were inconclusive due to non-specific antibody binding (data not shown). Intact cultured lenses were exposed to the NCX inhibitor bepridil at concentrations ranging from 1e30 mM. Over time, lenses that received 30 mM bepridil lost transparency. The opacity, which became first became detectable at 24 h, was restricted to the anterior of the lens (Fig. 2). The posterior of the lens remained transparent. Mild haze at the anterior of the lens was observed after incubation for 48 h in the presence of 5 mM or 10 mM bepridil. Lenses exposed to 1 mM bepridil remained transparent. Lenses exposed to 30 mM bepridil exhibited significant changes in electrolyte and water content. After 48 h, lens calcium content more than doubled (Fig. 3A) while sodium increased four fold (Fig. 3B). In contrast, lens potassium was significantly reduced. Changes in the electrolyte content were accompanied by a significant increase in water content of lenses that received 30 mM bepridil (Fig. 3C). To examine concentration dependence, lenses were exposed to bepridil at 1, 5, 10 and 30 mM. At concentrations of 10 mM or less, the electrolyte and water content of the lens remained similar to control values (Fig. 4).

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In some experiments, lenses were incubated in medium containing a reduced sodium concentration of 21 mM, with NMDG included as a sodium substitute. Under these conditions, marked lens opacification was observed (Fig. 5a). Unlike bepridil-treated lenses, both the anterior and posterior of the lens became opaque in lenses maintained in low sodium medium. Similar results were observed when choline chloride was used as a sodium substitute in the low sodium medium (data not shown). When calcium was absent from the low sodium culture medium the degree of opacity was diminished to a slight haze. Under low sodium conditions, lenses accumulated calcium (Fig. 5b). The calcium rise was not observed in lenses subject to incubation in calcium-free/low sodium medium. NCX is a bidirectional ion transport mechanism and bepridil is known to inhibit both forward and reverse transport modes (Stys et al., 1992). To examine the lens response to selective inhibition of reverse mode ion transport, lenses were exposed to KBR7943 or SN-6 at concentrations ranging from 1e30 mM. Under these conditions the electrolyte and water content of the lens remained similar to control values (Fig. 4). Lenses exposed to KBR7943 or SN-6 did not develop the pattern of anterior opacity observed in lenses cultured with 30 mM bepridil. On close on examination, however, KBR7943treated lenses displayed a slight haziness at the anterior surface (Fig. 2). SN-6-treated lenses remained fully transparent. In some experiments lens epithelial cells in an intact lens preparation (see Section 2) were loaded with the calcium sensitive fluorescent dye Fluo-4 then exposed to bepridil while fluorescence was monitored continuously. Bepridil was not found to cause a detectable increase in fluorescence (Fig. 6). In contrast, ATP (10 mM) added after bepridil elicited a transient fluorescence increase consistent with a rise in calcium concentration. 4. Discussion

Fig. 1. A representative western blot showing sodiumecalcium exchanger (NCX1) protein in the anterior (AE) and equatorial (EE) regions of rabbit lens epithelium. Epithelial lysates (40 mg protein per lane) were subjected to electrophoresis, transferred to nitrocellulose then probed with an antibody directed against canine cardiac NCX1. Arrows indicate the location of major bands at 160 and 70 kDa.

Bepridil was found to cause significant accumulation of calcium by the organ-cultured rabbit lens. The calcium increase was accompanied by a progressive deterioration of lens transparency that involved opacification at the lens anterior. The changes were observed by the bepridil concentration of 30 mM which is close to the Ki reported for bepridil in cardiac myocytes (Garcia et al., 1988). Lower concentrations of bepridil that might be expected to cause partial NCX inhibition did not cause lens calcium accumulation or opacification although 5 mM and 10 mM bepridil caused mild anterior haze. Sensitivity of rabbit lens calcium balance to bepridil fits with evidence of NCX protein expression in the lens epithelium. Western analysis revealed a dense immunoreactive protein band w160 kDa as well as a less dense band w70 kDa that is likely to represent the cleaved form of NCX which is found in a number of different tissues (Philipson et al., 1988; Saba et al., 1999). A similar NCX band pattern was observed in Western blot analysis of porcine lens epithelium (Okafor et al., 2003). Due to non-specific antibody binding to rabbit lens fiber membrane preparations we were not able to confirm NCX protein expression in the lens cortex. NCX

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Fig. 2. Representative dark-field images of organ-cultured rabbit lenses exposed to bepridil (30 mM), KBR7943 (10 mM) and SN-6 (10 mM) for 48 h. The concentration of bepridil, KBR7943 and SN-6 was chosen to be close to the Ki for the inhibitory effect of each compound on sodiumecalcium exchange. The test compounds were dissolved in DMSO (0.06% final concentration). Control lenses received DMSO alone.

and PMCA are the two mechanisms available for calcium export. In a previous study, the protein expression pattern of PMCA was found to be different in the central (anterior) and equatorial epithelium (Tamiya et al., 2003). This was not the

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case with NCX which was detected with similar immunoblot band density in the two regions. NCX is a bi-directional ion transporter. By convention, the forward-mode of transport refers to calcium export coupled to sodium import while reverse-mode transports shifts sodium and calcium in the opposite directions. The direction and the rate of NCX-mediated ion transport is dependent on the sodium gradient, calcium gradient and electrical potential across

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Fig. 3. Time course of electrolyte and water changes in rabbit lenses cultured in the presence of 30 mM bepridil. (A) calcium; (B) sodium and potassium; and (C) water content. The data are presented mean  SD. (n ¼ 6e16 lenses). (*p < 0.01 from control).

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Fig. 4. Concentration dependence for the effect of bepridil, KBR7943 and SN-6 on (A) lens calcium; (B) sodium and potassium; and (C) water content. The data are presented as mean  SD. (n ¼ 3e16 lenses). (*p < 0.01 from control).

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Fig. 5. Panel (a) shows representative dark field images of lenses cultured 48 h in modified M199 medium that contained a reduced sodium concentration of 21 mM and either a normal calcium concentration (Low Na) or no calcium chloride (nominally calcium-free) (Low Na, Ca Free). To maintain osmolarity of the medium, NMDG was added as a substitute for sodium chloride. Panel (b) shows the calcium content of lenses incubated under the same conditions. The results are presented as mean  SD. (n ¼ 4 lenses). Calcium in the two groups is significantly different (*p < 0.01).

the plasma membrane (see Blaustein and Lederer, 1999 for review). Bepridil inhibits both forward and reverse modes of transport. In recent years, compounds have been developed that with selective inhibitory effects on reverse-mode NCX

Fig. 6. A representative trace showing changes in cytoplasmic calcium concentration-dependent Fluo-4 fluorescence in lens epithelial cells exposed to 30 mM bepridil. Epithelial cells in the intact lens were pre-loaded with Fluo4 and superfused initially with control AAH. Bepridil (30 mM) was introduced as indicated by the bar above the trace. At the end of the experiment, ATP (10 mM) was introduced to the superfusate as a control to confirm viability of the preparation. Previous studies have shown ATP-induced calcium signaling in lens epithelium (Collison and Duncan, 2001). The trace represents the combined results from >30 cells in a single lens. Similar results were obtained in 4 lenses.

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function. Here, reverse-mode NCX inhibitors KBR 7943 and SN-6 did not cause detectable changes of calcium, sodium, potassium or water balance in the organ-cultured rabbit lens. KBR 7943 caused a very mild degree of anterior opacification. Lack of detectable electrolyte changes in KBR 7943-treated and SN-6-treated lens suggest that the electrolyte changes caused by 30 mM bepridil result from inhibition of forwardmode NCX-mediated ion transport. On this basis, bepridiltreated lenses may have an impaired ability to export calcium that enters the lens from the outside. To examine the link between lens opacification and external calcium, lenses were cultured for 48 h in calcium-free medium with or without bepridil. Calcium-free medium alone caused significant lens opacification (data not shown) meaning that results from the bepridil-treated calcium-free group (which also lost transparency) provide little useful information. In earlier studies it has been shown that reduction of external calcium concentration causes an increase of lens sodium permeability that leads to a progressive increase of lens sodium and water content (Delamere and Paterson, 1979). Such changes in lens composition may contribute to the observed loss of transparency in calcium-free medium. It is noteworthy that lens transparency did not deteriorate in lenses exposed to calcium-free medium with a low sodium concentration. In these lenses an increase in sodium permeability likely has less impact because the driving force for entry of extracellular sodium is low, thus lens sodium content cannot increase to a level that causes water gain and lens swelling. Under low sodium conditions that are likely to inhibit NCXmediated calcium export, lenses became severely opaque. This fits with a report by Tomlinson and coworkers (Tomlinson et al., 1991) who observed opacification in rat lenses incubated in sodium-free conditions. Transparency in the rabbit lens was lost at both anterior and posterior. We cannot rule out the possibility that NMDG or differences between chloride concentration in control and low sodium medium contributed to the observed loss of lens transparency. However, this seems unlikely because a similar pattern of lens changes was observed in low sodium experiments in which choline chloride was used as a sodium substitute and medium chloride concentration was fixed. Loss of transparency was calcium-dependent as indicated by near complete prevention of opacification by eliminating calcium from the low sodium bathing medium. The low sodium experiments should be interpreted with caution because the low sodium conditions inevitably will inhibit not only NCX but also NaeH exchange and all other co-transporter and counter transport mechanisms coupled to the sodium gradient. Thus amino acid uptake is likely to be compromised, leading in the long term to altered protein synthesis and metabolism. Moreover, in low sodium conditions NCX may reverse to carry calcium into the lens. However, in low sodium conditions there is little or no tendency for sodium entry so the sodium content of the lens will not increase to the point where it causes water gain and cell swelling. Bepridil-treated lenses gained sodium and water while lenses incubated in low sodium medium did not. The different sodium response may contribute to the difference in lens opacification observed in lenses subjected to

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bepridil or low sodium treatment. Thus, while the results of the low sodium experiment are consistent with the involvement of NCX in lens calcium homeostasis and transparency, it cannot be concluded that NCX inhibition (exclusive of inhibition of other mechanisms) is the cause of opacification in low sodium medium. We also cannot rule out the possibility in bepridil studies, that bepridil inhibits other calcium handling mechanisms in addition to NCX. It has been shown, for example, that bepridil alters mitochondrial calcium transport in some cells (Matlib, 1985; Storozhevykh et al., 1996). The effects of bepridil on lens calcium and transparency were accompanied by a marked increase of lens sodium content and a decrease of potassium content. These findings fit with earlier reports of sodium and potassium changes in lens that accumulate calcium. It has been suggested that Na,KATPase function is impaired when cytoplasmic calcium is allowed to rise (Delamere et al., 1993). Changes in cell sodium and potassium disturb osmotic balance and this fits with observation that lenses treated with 30 mM bepridil had significantly elevated water content. Tissue swelling due to water gain will disrupt the precise organization of lens cells and this could contribute to the observed deterioration of transparency in bepridil-treated lenses. The time course of the bepridil effect on lens electrolytes and lens transparency is noteworthy. Calcium accumulation was observed only after 48 h. At 24 h lens calcium content was slightly but significantly reduced and sodium was only slightly increased. The time course of the bepridil effect suggests that the lens may be able to use an alternate mechanism, probably PMCA, to export calcium over the short term but not for an extended period. Consistent with this notion, exposure of the lens to bepridil failed to cause an immediately detectable change of cytoplasmic calcium concentration, as evidenced by lack of a Fluo-4 fluorescence increase in the epithelium. While we are not able to rule out the possibility that bepridil caused a small change of cytoplasmic calcium that fell below the sensitivity threshold in the Fluo-4-loaded lens, a robust fluorescence increase was elicited by ATP. A transient cytoplasmic calcium rise in response to ATP is well documented in lens epithelium (Collison and Duncan, 2001). Experiments to study the role of PMCA were not possible due to lack of a selective PMCA inhibitor. Lack of an immediate effect of bepridil on cytoplasmic calcium may explain why the contribution of NCX-mediated calcium export was not seen in earlier studies in which calcium was found to remain normal in lenses subjected for 24 h to conditions that reduced the sodium gradient driving force for NCX (Delamere and Paterson, 1982; McGahan et al., 1983). The ability of bepridil to cause significant changes in lens transparency and electrolyte content points to an important role for NCX in the lens. There is evidence for NCX in both lens cell types. Studies in other laboratories have demonstrated NCX-mediated calcium transport in membrane vesicles obtained from rat, bovine and fish lens epithelium as well as fiber cells (Galvan and Louis, 1988; Wang et al., 1992; Ye and Zadunaisky, 1992a,b). While vesicle studies and western blot analysis have not confirmed NCX in rabbit lens fibers, studies

in lenses from other species suggest it is likely to be present. Short term studies with the intact lens failed to provide evidence of sodium-dependent calcium regulation (Delamere and Paterson, 1982; McGahan et al., 1983). Here we show that long term exposure to the NCX inhibitor bepridil causes significant disruption of lens electrolyte balance and transparency. The damaging effect of bepridil is likely to result from inhibition of NCX-meditated calcium export either in lens epithelium, fibers, or both cell types. Selective inhibitors of reverse mode NCX transport, which shifts calcium into the cell, did not disrupt lens ion balance. It is noteworthy that reverse-mode NCX inhibitors are thought to have possible therapeutic value due their ability to suppress tissue calcium accumulation following hypoxic or ischemic damage (Hobai and O’Rourke, 2004; Iwamoto, 2004). Acknowledgement Supported by NIH Grant EY09532, Research to Prevent Blindness Inc. and the Kentucky Lions Eye Foundation. References Blaustein, M.P., Lederer, W.J., 1999. Sodium/calcium exchange: its physiological implications. Physiol. Rev. 79, 763e854. Collison, D.J., Coleman, R.A., James, R.S., Carey, J., Duncan, G., 2000. Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques. Invest. Ophthalmol. Vis. Sci. 41, 2633e2641. Collison, D.J., Duncan, G., 2001. Regional differences in functional receptor distribution and calcium mobilization in the intact human lens. Invest. Ophthalmol. Vis. Sci. 42, 2355e2363. David, L.L., Shearer, T.R., 1984. Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest. Ophthalmol. Vis. Sci. 25, 1275e1283. Delamere, N.A., Paterson, C.A., 1979. The influence of calcium-free solutions upon permeability characteristics of the rabbit lens. Exp. Eye Res. 28, 45e53. Delamere, N.A., Paterson, C.A., 1982. Studies on calcium regulation in relation to sodium-potassium balance in the rabbit lens. Ophthalmic. Res. 14, 230e240. Delamere, N.A., Paterson, C.A., Borchman, D., Manning Jr., R.E., 1993. The influence of calcium on the rabbit lens sodium pump. Invest. Ophthalmol. Vis. Sci. 34, 405e412. Duncan, G., Bushell, A.R., 1975. Ion analyses of human cataractous lenses. Exp. Eye Res. 20, 223e230. Duncan, G., Webb, S.F., Dawson, A.P., Bootman, M.D., Elliott, A.J., 1993. Calcium regulation in tissue-cultured human and bovine lens epithelial cells. Invest. Ophthalmol. Vis. Sci. 34, 2835e2842. Fein, T., Pande, A., Spector, A., 1979. Further investigation of the role of calcium in human lens protein aggregation. Invest. Ophthalmol. Vis. Sci. 18, 761e765. Galvan, A., Louis, C.F., 1988. Calcium regulation by lens plasma membrane vesicles. Arch. Biochem. Biophys. 264, 472e481. Garcia, M.L., Slaughter, R.S., King, V.F., Kaczorowski, G.J., 1988. Inhibition of sodiumecalcium exchange in cardiac sarcolemmal membrane vesicles. 2. Mechanism of inhibition by bepridil. Biochemistry 27, 2410e2415. Hobai, I.A., O’Rourke, B., 2004. The potential of Naþ/Ca2þ exchange blockers in the treatment of cardiac disease. Expert Opin. Investig. Drugs 13, 653e664. Iwamoto, T., 2004. Forefront of Naþ/Ca2þ exchanger studies: molecular pharmacology of Naþ/Ca2þ exchange inhibitors. J. Pharmacol. Sci. 96, 27e32.

S. Tamiya, N.A. Delamere / Experimental Eye Research 83 (2006) 1089e1095 Iwamoto, T., Inoue, Y., Ito, K., Sakaue, T., Kita, S., Katsuragi, T., 2004. The exchanger inhibitory peptide region-dependent inhibition of Naþ/Ca2þ exchange by SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a novel benzyloxyphenyl derivative. Mol. Pharmacol. 66, 45e55. Jedziniak, J.A., Kinoshita, J.H., Yates, E.M., Hocker, L.O., Benedek, G.B., 1972. Calcium-induced aggregation of bovine lens alpha crystallins. Invest. Ophthalmol. 11, 905e915. Matlib, M.A., 1985. Action of bepridil, a new calcium channel blocker on oxidative phosphorylation, oligomycin-sensitive adenosine triphosphatase activity, swelling, Caþþ uptake and Naþ-induced Caþþ release processes of rabbit heart mitochondria in vitro. J. Pharmacol. Exp. Ther. 233, 376e381. McGahan, M.C., Chin, B., Bentley, P.J., 1983. Calcium metabolism of the rabbit lens. Exp. Eye Res. 36, 57e66. Okafor, M., Tamiya, S., Delamere, N.A., 2003. Sodiumecalcium exchange influences the response to endothelin-1 in lens epithelium. Cell Calcium 34, 231e240. Philipson, K.D., Longoni, S., Ward, R., 1988. Purification of the cardiac NaþeCa2þ exchange protein. Biochim. Biophys. Acta. 945, 298e306. Saba, R.I., Bollen, A., Herchuelz, A., 1999. Characterization of the 70 kDa polypeptide of the Na/Ca exchanger. Biochem. J. 338 (Pt 1), 139e145. Sedova, M., Blatter, L.A., 1999. Dynamic regulation of [Ca2þ]i by plasma membrane Ca(2þ)-ATPase and Naþ/Ca2þ exchange during capacitative

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Ca2þ entry in bovine vascular endothelial cells. Cell Calcium 25, 333e343. Storozhevykh, T.P., Sorokina, E.G., Vinskaya, N.P., Pinelis, V.G., Vergun, O.V., Fayuk, D.A., Sobolevskiy, A.I., Khodorov, B.I., 1996. Bepridil exacerbates glutamate-induced deterioration of calcium homeostasis and cultured nerve cell injury. Int. J. Neurosci. 88, 199e214. Stys, P.K., Waxman, S.G., Ransom, B.R., 1992. Ionic mechanisms of anoxic injury in mammalian CNS white matter: role of Naþ channels and Na(þ)eCa2þ exchanger. J. Neurosci. 12, 430e439. Tamiya, S., Dean, W.L., Paterson, C.A., Delamere, N.A., 2003. Regional distribution of Na,K-ATPase activity in porcine lens epithelium. Invest. Ophthalmol. Vis. Sci. 44, 4395e4399. Tomlinson, J., Bannister, S.C., Croghan, P.C., Duncan, G., 1991. Analysis of rat lens 45Ca2þ fluxes: evidence for Na(þ)eCa2þ exchange. Exp. Eye Res. 52, 619e627. Wang, Z., Hess, J.L., Bunce, G.E., 1992. Calcium efflux in rat lens: Na/Caexchange related to cataract induced by selenite. Curr. Eye Res. 11, 625e632. Ye, J., Zadunaisky, J.A., 1992. Study of the Ca2þ/Naþ exchange mechanism in vesicles isolated from apical membranes of lens epithelium of spiny dogfish (Squalus acanthias) and bovine eye. Exp. Eye Res. 55, 243e250. Ye, J.J., Zadunaisky, J.A., 1992. Ca2þ/Naþ exchanger and Naþ,Kþ2Cl cotransporter in lens fiber plasma membrane vesicles. Exp. Eye Res. 55, 797e804.