Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

M. A. Micci and Burgess N. Christensen

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The role of the Na+/Ca2+ exchanger in intracellular Ca2+ regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+ exchange was altered. In addition, the role of the Na+/Ca2+ exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1-10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1-2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]i and caffeine-stimulated Ca2+ release. Reduction of external Na+ concentration ([Na+]o) further and reversibly increased [Ca2+]i in ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]i promote net extracellular Ca2+ influx through a Na+/Ca2+ exchanger. Moreover, when [Na+]o was replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+ exchanger inhibitor 2',4'-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+ response 1 and 2 min after store depletion.

fluo 3; caffeine; ryanodine receptor; retina

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CALCIUM REGULATION plays a fundamental role in many cell functions. These include neurotransmitter release, neuromodulation, cell growth, electrical activity, excitation-contraction coupling, and muscle contraction (34). In addition, intracellular Ca2+ plays a central role in the phenomena of cell death and in cardiovascular function (11, 26, 33). Free intracellular Ca2+ is maintained around 10-7 M by various buffer systems, including mitochondrial and nonmitochondrial intracellular stores, plasma membrane voltage, and ligand-gated Ca2+ channels, Ca2+ pumps, and the Na+/Ca2+ exchanger (34).

The Na+/Ca2+ exchanger was first characterized in the squid giant axon (1) and extensively studied in cardiac tissue (28, 31). In many tissues, Na+/Ca2+ exchange is an electrogenic process that exchanges three Na+ for one Ca2+. It has a low affinity but a high capacity for Ca2+. In neurons, the Na+/Ca2+ exchanger has been implicated in the regulation of Ca2+ homeostasis at nerve terminals and in processes related to Ca2+ overload associated with cell injury and cell death (5, 26, 33). However, the physiological role of the Na+/Ca2+ exchanger in the regulation of Ca2+ homeostasis in neurons has not been unequivocally defined. Catfish retina cone horizontal cells express ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and non-NMDA] and L-type voltage-gated Ca2+ channels (30, 32). In addition, they contain the two main nonmitochondrial intracellular Ca2+ stores: the inositol 1,4,5-trisphosphate (IP3)-sensitive store (25) and the caffeine-sensitive store (24). Previous reports have shown that, in these cells, permeation of Ca2+ through glutamate-activated channels (NMDA and non-NMDA) leads to release of Ca2+ from caffeine-sensitive stores, resulting in an amplification of the Ca2+ signal (24). The presence of a Na+/Ca2+ exchanger has been previously reported in horizontal cells from carp and goldfish retinas (37). In the present study, we used the Ca2+-sensitive dye fluo 3, pharmacological agents, and the modification of the Na+ gradient to demonstrate the presence of a plasma membrane Na+/Ca2+ exchanger in catfish retina horizontal cells. Moreover, our results strongly suggest that, in these cells, the Na+/Ca2+ exchanger plays an important role in the refilling of caffeine-sensitive intracellular Ca2+ stores following release.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of Dissociated Retina Cells

Isolated cells were obtained from Texas channel catfish (Ictalurus punctatus) that were dark adapted to facilitate the separation of the retina from the pigment epithelium. The eyecup, prepared by removal of the cornea and lens, was placed for 3 min in low-Ca2+ catfish saline (CFS) containing (in mM) 126 NaCl, 4 KCl, 0.3 CaCl2, 1 MgCl2, 15 dextrose, and 10 HEPES (pH 7.4), to which 0.1 mg/ml papain (activity 2,723 U/mg papain; Calbiochem, La Jolla, CA) and hyaluronidase had been added to remove the vitreous humor.

The eyecup was then washed in low-Ca2+ CFS, and the retina was gently stripped from the pigment epithelium. The retina was cut into small pieces (~1.5 × 1.5 mm) and stored in normal CFS (NCFS, same as above but with 3 mM CaCl2). To obtain isolated cells, a piece of retina was incubated for 6 min in low-Ca2+ CFS containing 0.084 mg/ml papain (activity 20 U/mg papain; Worthington Biochemicals, Freehold, NJ) that had been previously activated with 2 mM L-cysteine for 30 min and then was washed in NCFS. Individual cells were obtained by a process of gentle mechanical dissociation that consisted of repeatedly passing the tissue through fire-polished Pasteur pipettes of decreasing bore size in cold saline.

Intracellular Ca2+ Measurement

Freshly dissociated catfish retina cells were plated on a 25-mm glass coverslip glued to a 35-mm Falcon culture dish from which the plastic bottom had been removed. The cells were loaded with the Ca2+-sensitive fluorescent dye fluo 3 by incubating them for 30 min at room temperature in NCFS containing 3.16 µM fluo 3-AM. At the end of the incubation, the cells were then washed with fresh NCFS and viewed with a confocal laser-scanning microscope system (Noran Instruments, Middleton, WI), utilizing an argon ion laser (488 nm) coupled to an inverted Nikon Diaphot microscope. The Ca2+-sensitive dye fluo 3 was chosen because its long excitation wavelength made it suitable for use with the argon ion laser available in our system. Because fluo 3 fluorescence increases when it is bound to intracellular free Ca2+, we monitored fluorescence changes and plotted these changes as brightness intensity vs. time. Measurements were made from the entire cell soma and proximal dendrites. The single-cell technique allowed us to monitor the intracellular free Ca2+ in individual horizontal cells before and after pharmacological manipulations.

A potential problem associated with the use of esterified dyes is their tendency to be sequestered in intracellular compartments (for a review, see Ref. 17). Whether release or uptake of fluo 3 from one intracellular compartment to another occurs in fluo 3-loaded horizontal cells is a difficult question to test. However, because compartmentalization is a time-dependent phenomenon, one should expect to observe a decrease in the dye sensitivity to Ca2+ (i.e., smaller Ca2+ responses) with time, if such a problem does exist. In the present work, test caffeine applications were used at the beginning and end of each experiment to check for possible rundown of the Ca2+ response due to dye compartmentalization, leakage, or other dye-related artifacts. Complete recovery of the caffeine-induced Ca2+ response was always observed at the end of each experimental session, indicating that dye compartmentalization did not represent a problem in our system. Moreover, under control conditions, we did not observe a baseline drift over time, thus indicating that photo bleaching and/or dye leakage was absent or minimal.

Drug Application

Drugs were diluted from stock solutions to their final concentration in NCFS. Solutions were exposed to horizontal cells by completely exchanging the bathing solution in the dish. Nifedipine was included in most experiments to block the L-type Ca2+ channel, because isolated cone horizontal cells are capable of remaining depolarized under control conditions (32, 35).

Modulation of Intracellular Ca2+

Ca2+ release from the caffeine-sensitive store was produced by application of caffeine (20 mM dissolved in Ca2+-free CFS) from a caffeine-containing pipette placed near the cell and connected to a compressed air source (Picospritzer, General Valve, Fairfield, NJ). Ejection of caffeine was controlled by regulating the air pressure applied to the pipette. Time of single caffeine applications was 1 or 10 s.

The following protocol was used to study the refilling of the caffeine-sensitive store. A single horizontal cell was repeatedly challenged with 1- or 10-s applications of caffeine until no further release of Ca2+ from the caffeine-sensitive store was observed. One minute after the end of the last caffeine application, Ca2+ release from the caffeine-sensitive store was stimulated by a 10-s application of caffeine (indicated in Figs. 1-6 as percentage of caffeine-induced Ca2+ release compared with the first control response). Caffeine-induced Ca2+ release was measured again 2 min after the end of this last caffeine application.

Materials and Solutions for Imaging Experiments

Fluo 3-AM was purchased from Molecular Probes (Eugene, OR). 2',4'-Dimethylbenzamil (DMB) was provided by Research Biomedical International (Natick, MA) as part of the Chemical Synthesis Program of the National Institute of Mental Health (contract N01-MH-30003). Caffeine, ouabain, and N-methyl-D-glucamine (NMDG) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were analytical grade or the highest purity available.

The standard physiological salt solution (NCFS) contained (in mM) 126 NaCl, 4 KCl, 3 CaCl2, 1 MgCl2, 15 dextrose, and 10 HEPES (pH 7.4). For Ca2+-free solutions, the CaCl2 was omitted and 1 mM EGTA was added to chelate trace amounts of Ca2+. After the addition of EGTA, pH was adjusted to 7.4. In the low-Na+ solution, NMDG or LiCl isosmotically replaced NaCl. Stock solutions of 6.3 mM fluo 3-AM were prepared in DMSO immediately before use. Stock solutions of 20 mM nifedipine were prepared in absolute ethanol and stored at -20°C. DMB was prepared in DMSO at 10 mM concentration and diluted to its final concentration in NCFS. DMSO at 0.5% concentration did not produce any effect on the Ca2+ measurements (data not shown).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Free Intracellular Ca2+ Concentration in Dissociated Catfish Retina Horizontal Cells

Effect of caffeine. Figure 1A shows changes in intracellular Ca2+ concentration ([Ca2+]i) measured from a representative catfish horizontal cell following repetitive applications of caffeine (20 mM) by pressure ejection from a nearby pipette. In the presence of 3 mM extracellular Ca2+, subsequent applications of caffeine at intervals shorter than 1 min gave rise to progressively smaller increases in [Ca2+]i due to emptying of caffeine-sensitive intracellular Ca2+ stores as previously reported by Linn and Christensen (24) (Fig. 1Aa). The extracellular solution was then changed to Ca2+-free CFS, and Ca2+ release was again stimulated by caffeine. Two minutes after extracellular Ca2+ removal, caffeine evoked only a small Ca2+ response, and subsequent applications did not stimulate any further release (Fig. 1Ab). When extracellular Ca2+ was reintroduced, the caffeine-evoked Ca2+ response recovered even in the presence of the voltage-gated L-type Ca2+ channel blocker nifedipine (Fig. 1Ac). Figure 1B shows the time course of the refilling of caffeine-sensitive Ca2+ stores in horizontal cells. Cells were incubated in NCFS in the presence of 20 µM nifedipine, and Ca2+ release from the caffeine-sensitive store was induced by challenging each cell with a 10-s application of caffeine. One minute after store depletion, a subsequent application of caffeine produced a Ca2+ response that was ~67% of the control response (Fig. 1B). Complete refilling of the store was observed after 2 min from store depletion (Fig. 1B). These data demonstrate that, after depletion, the caffeine-sensitive stores are rapidly refilled. Refilling requires extracellular Ca2+, indicating that an influx mechanism is necessary for refilling. Influx cannot be attributed in this case to voltage-dependent Ca2+ channels because refilling occurs in the presence of nifedipine (20 µM).


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Fig. 1.   Intracellular Ca2+ levels in a dissociated horizontal cell in response to repeated exposures to caffeine. A: intracellular Ca2+ changes in a single fluo 3-loaded horizontal cell were plotted as fluorescence intensity [in arbitrary units (A.U.)] vs. time. Caffeine (20 mM, 1 s) was applied from a caffeine-containing pipette placed near the cell at times indicated. Horizontal bars (top) indicate periods during which the external conditions were changed. Numbers above each Ca2+ transient represent percent of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). a: In the presence of 3 mM extracellular Ca2+, subsequent applications of caffeine at intervals shorter than 1 min gave rise to progressively smaller increases in intracellular Ca2+ ([Ca2+]i). b: When extracellular Ca2+ was removed, caffeine evoked only a small Ca2+ response, and subsequent applications did not produce any further release of Ca2+. c: When extracellular Ca2+ was reintroduced, caffeine-evoked Ca2+ response recovered even in the presence of the voltage-gated L-type Ca2+ channel blocker nifedipine. B: composite data from experiments similar to those described in A, showing recovery of the caffeine-induced Ca2+ response in 8 horizontal cells incubated in normal catfish saline (NCFS) containing 20 µM nifedipine. Caffeine-sensitive stores were depleted by challenging the cells with a 10-s application of 20 mM caffeine. One minute after depletion, cells were challenged again with a 10-s application of 20 mM caffeine, to test for refilling. Caffeine-induced Ca2+ response was recorded and is indicated as percentage of caffeine-induced Ca2+ release relative to first control response. Same protocol was repeated 2 min after this second caffeine application. Error bars indicate SD.

Effect of the Na+ electrochemical gradient on [Ca2+]i and caffeine-induced Ca2+ response. Free intracellular Ca2+ is maintained around 10-7 M by various buffer systems, including mitochondrial and nonmitochondrial intracellular stores, plasma membrane voltage- and ligand-gated Ca2+ channels, Ca2+ pumps, and the Na+/Ca2+ exchanger. Although Na+/Ca2+ exchange is believed to be mainly a Ca2+ efflux pathway, the direction of exchange is freely reversible, and there is evidence that, depending on membrane potential and Na+/Ca2+ ionic gradients, Na+/Ca2+ exchange may be important for the elevation of intracellular Ca2+ in several systems including neurons (6, 7). A common test for the presence of a plasma membrane Na+/Ca2+ exchanger is to determine the effect of raising the intracellular Na+ concentration ([Na+]i) or of lowering the external Na+ concentration ([Na+]o) on resting [Ca2+]i.

EFFECT OF LOW EXTRACELLULAR SODIUM CONCENTRATION. The effects of lowering [Na+]o on [Ca2+]i and on the caffeine-induced Ca2+ response are illustrated in Fig. 2A. Incubation of horizontal cells in low-Na+ CFS (NMDG substituted for Na+), in the presence of 20 µM nifedipine, produced an increase in resting [Ca2+]i and the caffeine-stimulated Ca2+ response (Fig. 2Ab). Both of these effects reversed on reintroduction of 126 mM Na+ to the extracellular medium (Fig. 2Ac). The effect of low [Na+]o on the caffeine-induced Ca2+ responses in horizontal cells is summarized in Fig. 2B. After depletion of caffeine-sensitive Ca2+ stores, subsequent applications of caffeine at 1 and 2 min gave rise to significantly higher caffeine-evoked Ca2+ responses in the low-Na+ solution compared with control (Fig. 2B). Complete recovery to control responses was measured on return to NCFS.


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Fig. 2.   Effect of low external Na+ concentration ([Na+]o) on resting [Ca2+]i and caffeine-induced Ca2+ response in dissociated horizontal cells. A: caffeine (20 mM, 10 s) was applied at times indicated to a single horizontal cell. Horizontal bars (top) indicate periods during which the external conditions were changed. Numbers above each Ca2+ transient represent percentage of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). Nifedipine (nif; 20 µM) was included in all bathing solutions. a: Control responses recorded in 126 mM Na+-catfish saline (CFS) demonstrate typical release and refilling of intracellular caffeine-sensitive Ca2+ pools. b: Reducing [Na+]o [N-methyl-D-glucamine (NMDG) substituted for Na+] produced an increase in resting [Ca2+]i and caffeine-evoked Ca2+ response. c: When the normal Na+ gradient was reintroduced across the plasma membrane, both baseline [Ca2+]i and caffeine-evoked Ca2+ response returned to control levels. CB, control baseline. B: composite data from experiments similar to those described in A, indicating percentage of caffeine-induced Ca2+ release from 3 dissociated horizontal cells incubated in normal CFS (CTRL), 6 mM Na+-CFS (low-Na+; NMDG substituted for Na+), and after reintroduction of normal 126 mM CFS (recovery). Caffeine-induced Ca2+ release from intracellular stores was measured at 1 and 2 min after store depletion (as described in MATERIALS AND METHODS) and indicated as percentage of Ca2+ released compared with control response. Errors bars indicate SD. * P < 0.05 vs. control; ** P < 0.01 vs. control (paired Student's t-test).

EFFECT OF HIGH INTRACELLULAR SODIUM CONCENTRATION. To increase [Na+]i, we exposed freshly dissociated catfish retina horizontal cells to 3 mM ouabain (a Na+-K+ pump blocker). As shown in Fig. 3, an 18-min incubation in 3 mM ouabain in the presence of the L-type Ca2+ channel blocker nifedipine produced small increases in resting intracellular Ca2+ [Fig. 3Aa, ouabain baseline (OB)] and caffeine-stimulated Ca2+ responses. Moreover, in the same cell, reducing [Na+]o in the presence of ouabain produced further increases in resting [Ca2+]i and the caffeine-induced Ca2+ response. Resting [Ca2+]i temporarily reversed to OB when normal [Na+]i was reintroduced. Longer incubation in ouabain in NCFS did in fact produce a further increase in cytoplasmic Ca2+ (Fig. 3Ac, last trace). The effect of ouabain on the caffeine-induced Ca2+ response is summarized in Fig. 3B. Caffeine-induced Ca2+ responses recorded in the presence of ouabain and ouabain plus low-Na+ CFS, in the presence of 20 µM nifedipine, were significantly larger than control responses.


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Fig. 3.   Effect of [Na+]o reduction and ouabain on resting [Ca2+]i and caffeine-evoked Ca2+ response. A: caffeine (20 mM, 10 s) was applied at times indicated to a single horizontal cell. Horizontal bars (top) indicate periods during which the external conditions were changed. Nifedipine (20 µM) was included in all bathing solutions. Numbers above each Ca2+ transient represent percentage of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). a: A 13-min incubation in 126 mM Na+-CFS containing 3 mM ouabain produced an increase in the caffeine-evoked Ca2+ response and, after an 18-min incubation, an increase in the baseline [Ca2+]i (OB, ouabain baseline). b: In the presence of 3 mM ouabain, decreasing extracellular Na+ produced a further increase in baseline [Ca2+]i and in the caffeine-evoked Ca2+ response. c: [Ca2+]i temporarily reversed to OB on incubation of the cell in 126 mM Na+-CFS. CB, control baseline. B: composite data from experiments similar to those described in A, showing caffeine-induced Ca2+ responses obtained from 3 horizontal cells incubated in ouabain for 15 min (ouabain) and then transferred to ouabain and 6 mM Na+-CFS (ouabain + low-Na+). Increases in Ca2+ release were compared with responses measured in NCFS. Error bars indicate SD. * P < 0.05 vs. control, ** P < 0.01 vs. control (paired Student's t-test).

EFFECT OF VANADATE. To determine whether the Ca2+-ATPase pump on the plasma membrane plays a role in the extrusion of Ca2+ entering the cell through the Na+/Ca2+ exchanger, we tested the effects of vanadate (an ATPase inhibitor, Ref. 9) on resting [Ca2+]i and the caffeine-induced Ca2+ response in freshly dissociated catfish retina horizontal cells. Cells were incubated in NCFS (3 mM Ca2+), and the Ca2+ baseline was recorded (Fig. 4A). A typical Ca2+ release and refilling of the caffeine-sensitive store are also shown (Fig. 4A). Cells were then incubated in 1 mM vanadate in the absence of extracellular Ca2+ for 30 min to ensure its entry in the cell, a process reported to have a half time of 4 min (8). At the beginning of the incubation with vanadate, caffeine was applied to the cell to deplete the intracellular Ca2+ stores (Fig. 4B). Under these conditions, vanadate did not produce an increase in resting [Ca2+]i, even after a 30-min incubation (Fig. 4B, last trace). When Ca2+ was reintroduced to the extracellular medium, resting cytosolic Ca2+ significantly increased even in the presence of the L-type Ca2+ channel blocker nifedipine (20 µM) (Fig. 4C). Moreover, under these conditions, caffeine induced an increase in Ca2+ that was significantly larger compared with the control response (Fig. 4C). Because vanadate blocks the Na+-K+-ATPase (8), these data suggest that in the presence of vanadate [Na+]i increases in the cells, thus driving influx of Ca2+ through the Na+/Ca2+ exchanger. Moreover, because vanadate also blocks the plasma membrane Ca2+-ATPase pump (13, 36), Ca2+ entering the cell through the exchanger accumulates in the cytoplasm, thus resulting in an increase in [Ca2+]i that is significantly higher than the one observed in the presence of ouabain (which does not block the Ca2+-ATPase pump) (Fig. 3). Moreover, in the presence of vanadate, recovery of [Ca2+]i after the caffeine-induced Ca2+ release was significantly slower than the recovery observed under control conditions (Fig. 4A) or at the beginning of the vanadate incubation, when presumably vanadate had not entered the cell (Fig. 4B). In addition, Fig. 4D shows that, when extracellular Ca2+ is subsequently removed, in the presence of vanadate, resting [Ca2+]i slowly decreases, suggesting that under this condition the Na+/Ca2+ exchanger might contribute to the extrusion of excess Ca2+. Moreover, in the absence of extracellular Ca2+ (still in the presence of vanadate), the recovery of [Ca2+]i after the caffeine pulse is faster than the one observed in the presence of 3 mM Ca2+ (Fig. 4C). Taken together, these results suggest that catfish retina horizontal cells possess a large capacity for Ca2+ buffering and that the recovery of [Ca2+ ]i after caffeine-induced Ca2+ release requires both the Na+/Ca2+ exchanger and the plasma membrane Ca2+-ATPase pump. In addition, our data show that, in catfish retina horizontal cells, vanadate does not block the Ca2+-ATPase on the caffeine-sensitive Ca2+ store because refilling of the store occurs in the presence of vanadate (Fig. 4D).


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Fig. 4.   Effect of vanadate on resting [Ca2+]i. Caffeine (20 mM, 10 s) was applied to a single horizontal cell at times indicated. Horizontal bars (top) indicate periods during which the external conditions were changed. Numbers above each Ca2+ transient represent percentage of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). Nifedipine (20 µM) was included in all the bathing solutions. A: control responses recorded in NCFS containing 3 mM Ca2+ demonstrate typical release and refilling of the caffeine-sensitive Ca2+ pool. B: the cell was then incubated with 1 mM vanadate in 0 Ca2+-CFS, and caffeine was applied to deplete the caffeine-sensitive store. C: when extracellular Ca2+ was reintroduced, both resting [Ca2+ ]i and the caffeine-induced Ca2+ release increased. D: when extracellular Ca2+ was removed (still in the presence of 1 mM vanadate), resting [Ca2+]i slowly decreased, suggesting that under this condition the Na+/Ca2+ exchanger might contribute to the extrusion of excess intracellular Ca2+. Moreover, in the presence of vanadate, Ca2+ released from the caffeine-sensitive store was slowly taken up into the store, as demonstrated by the fact that a subsequent application of caffeine produced a response that was similar in amplitude to the previous one. Similar results were obtained in 5 other horizontal cells.

Effect of Na+/Ca2+ Exchange Inhibition on the Caffeine-Evoked Ca2+ Response

Effect of Li+. Replacing extracellular Na+ with Li+, which readily permeates through Na+ channels (7) but does not substitute for Na+ on the Na+/Ca2+ exchanger (2, 3), results in the depletion of intracellular Na+ and consequently in the block of the Na+/Ca2+ exchanger. Figure 5 illustrates the changes in [Ca2+]i under these conditions. Control responses recorded in NCFS demonstrate typical release and refilling of the Ca2+ pool (Fig. 5A). When Na+ was replaced by Li+, a test application of caffeine induced an initial Ca2+ response that was not different from the control response. However, 1 min after store depletion, subsequent applications of caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store (Fig. 5B). Moreover, when, in the same cell, [Ca2+]i was transiently increased by depolarizing the cell with K+, ~55% of the caffeine-induced Ca2+ response was recovered (Fig. 5C). Complete recovery of the caffeine-evoked Ca2+ response was observed on washout of Li+ and reintroduction of extracellular Na+ (Fig. 5D).


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Fig. 5.   Effect of intracellular Na+ removal on the caffeine-induced Ca2+ response. Caffeine (20 mM, 10 s) was applied to a single horizontal cell at times indicated. Horizontal bars (top) indicate the period during which the external conditions were changed. Numbers above each Ca2+ transient represent percentage of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). A: control responses recorded in 126 mM Na+-CFS demonstrate typical release and refilling of the caffeine-sensitive Ca2+ pool. B: when Li+ replaced Na+, a test application of caffeine induced a Ca2+ response equal to the control response. However, subsequent applications of caffeine failed to release Ca2+ from the caffeine-sensitive store. C: application of K+ (126 mM substituted for Na+) from a nearby pipette produced an increase in [Ca2+]i due to Ca2+ influx through voltage-gated Ca2+ channels. With subsequent applications of caffeine, Ca2+ release decreased. D: complete recovery of the caffeine-evoked Ca2+ response was observed on washout of Li+ and reintroduction of extracellular Na+. Similar results were obtained in 5 other horizontal cells.

Effect of DMB. The Na+/Ca2+ exchanger is inhibited by the amiloride analog DMB with an inhibition constant of ~10 µM (22, 27). Figure 6 shows the effect of various concentrations of DMB on caffeine-evoked Ca2+ responses in a representative horizontal cell. Dissociated horizontal cells were incubated in NCFS, and Ca2+ release from intracellular caffeine-sensitive stores was stimulated by application of caffeine, as described in MATERIALS AND METHODS. Control responses recorded in NCFS demonstrate typical release and refilling of caffeine-sensitive intracellular Ca2+ stores (Fig. 6A, top left). The addition of 5 µM DMB to the extracellular medium did not affect the caffeine-induced Ca2+ response (Fig. 6A, top right), whereas 20 µM DMB produced a reduction of the amount of Ca2+ released from the caffeine-sensitive store (Fig. 6A, bottom left). Complete recovery of the caffeine-induced Ca2+ response was observed on DMB washout (Fig. 6A, bottom right). Figure 6B summarizes the effect of DMB on the caffeine-induced Ca2+ response during a 10-s application of caffeine. Caffeine-induced Ca2+ responses were recorded 1 and 2 min after depletion under control conditions, in the presence of 5, 20, and 50 µM DMB, and after DMB washout. As shown in Fig. 6B, 20 and 50 µM DMB significantly reduced the amplitude of the caffeine-induced Ca2+ response. Complete recovery of the caffeine-induced Ca2+ response was observed after DMB washout.


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Fig. 6.   Effect of 2',4'-dimethylbenzamil (DMB) on caffeine-induced Ca2+ response. A: effect of 5 and 20 µM DMB on the caffeine-induced Ca2+ response in a single horizontal cell. Caffeine (20 mM, 10 s) was applied at times indicated. Horizontal bars (top) indicate periods during which the external conditions were changed. Numbers above each Ca2+ transient represent percentage of Ca2+ released from the caffeine-sensitive store compared with the first Ca2+ response (indicated by *). Nifedipine (20 µM) was included in all bathing solutions. Top left: control responses recorded in 126 mM Na+-CFS demonstrate typical release and refilling of the caffeine-sensitive Ca2+ pool. Top right: in the same cell, 1 min after store depletion, the caffeine-induced Ca2+ response measured in the presence of 5 µM DMB was not different from the response recorded in NCFS 1 min after depletion (67% of the first control response). Complete recovery of the caffeine-induced Ca2+ response was observed 2 min after depletion in the presence of 5 µM DMB, thus indicating that at this concentration DMB did not affect the refilling of the caffeine-sensitive Ca2+ store. Bottom left: incubation of the same cell in 20 µM DMB resulted in a dramatic decrease of the caffeine-induced Ca2+ response at both 1 and 2 min after store depletion. Bottom right: complete recovery of the caffeine-induced Ca2+ response was observed on washout of DMB. Similar results were observed in 5 other cells. B: composite data from experiments similar to the one described in A, showing the effect of 5, 20, and 50 µM DMB on the recovery of the caffeine-induced Ca2+ response at 1 and 2 min after store depletion in 5 horizontal cells. Error bars indicate SD. * P < 0.01 vs. control (paired Student's t-test).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we used the Ca2+-sensitive dye fluo 3, pharmacological agents, and the modification of the Na+ gradient to demonstrate the presence of a plasma membrane Na+/Ca2+ exchanger in catfish retina horizontal cells. In many tissues, it has been demonstrated that the Na+/Ca2+ exchanger is an electrogenic process and that it exchanges three Na+ for one Ca2+ (for a review, see Refs. 6, 18). Depending on membrane potential and Na+/Ca2+ ionic gradients, the Na+/Ca2+ exchanger will carry intracellular Ca2+ out of the cell or, in the reverse mode, will bring extracellular Ca2+ into the cell. Modulation of resting [Ca2+]i and Ca2+ store refilling by altering either intracellular or extracellular Na+ provides support for the presence of the Na+/Ca2+ exchanger in catfish retina cone horizontal cells. In particular, our data show that the ouabain-induced reductions in the Na+ electrochemical gradient produce small increases in resting [Ca2+]i (Fig. 3A) and an increase in the amount of Ca2+ sequestered (and releasable) from the caffeine-sensitive store (Fig. 3B). The increase in free [Ca2+]i after ouabain treatment suggests that reduction of the Na+ electrochemical gradient mediates net Ca2+ influx via the Na+/Ca2+ exchanger. The effect of ouabain on the amount of Ca2+ released from the caffeine-sensitive store is suggestive of an important contribution of the intracellular Ca2+ stores in the maintenance of Ca2+ homeostasis in horizontal cells.

It is known that in most cells a large fraction of intracellular Ca2+ is buffered and/or sequestered (4, 15) to maintain free [Ca2+]i near 100 nM. It has been shown that in these cells reductions in the Na+ gradient might in fact induce net Ca2+ gain through a Na+/Ca2+ exchanger, but only a small fraction would be expected to appear as free cytosolic Ca2+ unless the buffer systems are overwhelmed. Our data support this observation and demonstrate that in catfish retina horizontal cells net Ca2+ gain through the Na+/Ca2+ exchanger is normally attenuated by intracellular Ca2+ sequestration. In addition we have shown that in these cells the plasma membrane Ca2+-ATPase also plays a major role in Ca2+ homeostasis. In fact, our data show that, in the presence of extracellular Ca2+, vanadate (an ATPase inhibitor) induces a large increase in [Ca2+ ]i. Because the observed increase in [Ca2+]i does not occur in the absence of extracellular Ca2+, this suggests that vanadate (like ouabain) produces net Ca2+ influx through the Na/Ca2+ exchanger by decreasing the Na+ electrochemical gradient (by blocking the Na+-K+-ATPase). Moreover, because the increase in resting [Ca2+]i is larger in the vanadate-incubated cells than in the ouabain-incubated ones and because vanadate blocks the plasma membrane Ca2+-ATPase in horizontal cells, the plasma membrane Ca2+ pump may play an important role in the maintenance of Ca2+ homeostasis by extruding excess Ca2+ from the cytoplasm. In conclusion, our data strongly suggest that, in catfish retina horizontal cells, any condition that will reduce the Na+ electrochemical gradient will result in net Ca2+ influx through the Na+/Ca2+ exchanger.

We have also shown that Ca2+ entering the cell through the Na+/Ca2+ exchanger is rapidly buffered by the intracellular caffeine-sensitive Ca2+ store and extruded through the plasma membrane Ca2+ pump. In particular, our data show that when the Na+ electrochemical gradient is reduced the caffeine-sensitive store can sequester (and release) an amount of Ca2+ that is significantly larger (up to 200% in the presence of vanadate) than the amount of Ca2+ stored under resting conditions. A similar mechanism has recently been reported in astrocytes, where it has been shown that the Na+/Ca2+ exchanger modulates the Ca2+ contents of the two nonmitochondrial stores when the Na+ electrochemical gradient is reduced (16). Previous reports have shown that in catfish cone horizontal cells permeation of Ca2+ through glutamate-activated channels (NMDA and non-NMDA) leads to release of Ca2+ from caffeine-sensitive stores, resulting in an amplification of the Ca2+ signal (23, 24). Because release of Ca2+ from intracellular stores plays an important role in a variety of cellular responses (12), it could be hypothesized that, in the catfish retina, a plasma membrane Na+/Ca2+ exchanger may modulate horizontal cell responsiveness to photoreceptors by regulating the amount of Ca2+ stored in the caffeine-sensitive Ca2+ pool.

Functional Coupling of Na+/Ca2+ Exchange to the Refilling of Intracellular Ca2+ Stores

In addition to modulating the amount of Ca2+ sequestered in the caffeine-sensitive store when the Na+ electrochemical gradient is reduced, we have shown that a plasma membrane Na+/Ca2+ exchanger plays an important role in the refilling of caffeine-sensitive Ca2+ stores under resting conditions. This observation is supported by our results showing that when Na+/Ca2+ exchange is blocked (in the absence of Na+ or in the presence of DMB) caffeine is able to stimulate only an initial Ca2+ release. Subsequent applications of caffeine fail to stimulate release of Ca2+ from the caffeine-sensitive intracellular Ca2+ stores. Substituting external Na+ with Li+ results in the depletion of intracellular Na+, inactivation of the Na+/Ca2+ exchanger (3), and, consequently, failure of caffeine-induced Ca2+ release. These data suggest that, in catfish retinal horizontal cells, refilling of the caffeine-sensitive Ca2+ stores requires influx of Ca2+ through the Na+/Ca2+ exchanger. Moreover, in the absence of Na+, ~50% of the caffeine-induced Ca2+ response is recovered when intracellular Ca2+ levels are elevated by stimulating Ca2+ influx through voltage-dependent Ca2+ channels (Fig. 5). Therefore, an indirect effect of Na+ removal and/or Li+ on the caffeine-sensitive Ca2+ store itself cannot account for the observed effect on Ca2+ store refilling.

Similarly, we have shown that DMB (a selective Na+/Ca2+ exchanger inhibitor; Refs. 22, 27) significantly impairs the refilling of the caffeine-sensitive Ca2+ store after depletion. Among the amiloride derivatives, DMB is considered one of the most selective inhibitors of Na+/Ca2+ exchange currently available. It interacts at the Na+ binding site on the exchanger in a competitive fashion and reversibly ties up the transporter in an inactive state. DMB has been shown to effectively block the Na+/Ca2+ exchanger in several cell preparations (21) without affecting other transport mechanisms. In catfish horizontal cells, the effect of DMB on the caffeine-evoked Ca2+ response is likely to be mediated through the Na+/Ca2+ exchanger because it occurs at a concentration of DMB consistent with the reported IC50 for this pathway (10 µM). Recently, a polypeptide [exchanger inhibitory peptide (XIP)] has been synthesized that resembles a putative calmodulin binding site of the canine cardiac Na+/Ca2+ exchanger with possible autoinhibitory properties (10). Because XIP has been shown to inhibit the exchanger only at the intracellular surface, it needs to be dialyzed in the cell and its effect is not reversible. Because our objective in the present study was to look at the effect of Na+/Ca2+ exchanger on the refilling of caffeine-sensitive intracellular Ca2+ stores, we have chosen to use an inhibitor like DMB, whose effect could be easily reversed, to allow us to study Ca2+ store refilling under different experimental conditions in the same cell.

Ca2+ influx through Ca2+ release-activated channels (CRAC) has been extensively studied and described in several systems, where it has been shown to be associated with depletion of IP3-sensitive Ca2+ stores (20, 29). It is, however, still unclear whether a similar coupling mechanism exists with stores that express ryanodine receptors (14). Our data show that, in our system, refilling of caffeine-sensitive stores requires Ca2+ influx through a plasma membrane Na+/Ca2+ exchanger. This could represent a novel and important mechanism for the refilling of intracellular Ca2+ stores under both resting and stimulated conditions. Whether CRAC channels are present in horizontal cells that could contribute to the refilling of the IP3-sensitive store is not presently known. However, since CRAC channels are not modulated by changes in the electrochemical Na+ gradient and are not blocked by Li+, our results in zero Na+ strongly suggest that such a mechanism, if it exists, does not play a major role in the refilling of the caffeine-sensitive store. Further studies are necessary to address this issue definitively.

In summary, our data show that, in catfish retina horizontal cells, Ca2+ turnover through a plasma membrane Na+/Ca2+ exchanger plays a fundamental role in the regulation of both Ca2+ homeostasis and the amount of Ca2+ stored in the intracellular caffeine-sensitive Ca2+ store. Recent morphological evidence has demonstrated the colocalization of the Na+/Ca2+ exchanger and intracellular stores in both astrocytes and neurons (5). Further studies need to be done to determine whether a similar pattern of colocalization is present in catfish retina horizontal cells. Such colocalization would add further support to the role played by intracellular caffeine-sensitive store and plasma membrane Na+/Ca2+ exchanger in the regulation of Ca2+ homeostasis in these cells.

    ACKNOWLEDGEMENTS

This research was supported by National Eye Institute Grant NEI-01897 and Univ. of Texas Medical Branch Small Grant 454670. 2',4'-Dimethylbenzamil was provided by Research Biomedical International (One Strathmore Road, Natick, MA 01760-2447) as part of the Chemical Synthesis Program of the National Institute of Mental Health (contract N01-MH-30003).

    FOOTNOTES

Address reprint requests to M. A. Micci.

Received 22 December 1997; accepted in final form 25 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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