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Cardiovascular Research 1999 43(2):417-425; doi:10.1016/S0008-6363(99)00098-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Sodium influx via a non-selective pathway activated by the removal of extracellular divalent cations: possible role in the calcium paradox

Suzanne Bosteelsa, Peter Matejovicb, Willem Flamengb and Kanigula Mubagwab,*

aInterdisciplinair Research Centrum, KUL Campus Kortrijk, University of Leuven, Leuven, Belgium
bCentrum voor Experimentele Heelkunde en Anesthesiologie, University of Leuven, Leuven, Belgium

* Corresponding author. Tel.: +32-16-347-132; fax: +32-1634-7139 kanigula.mubagwa{at}kuleuven.ac.be

Received 4 September 1998; accepted 9 February 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Cation non-selective conductances which are induced upon removal of extracellular divalent cations have been identified in cardiac and other cells. We have examined whether the conductance identified in cardiac myocytes mediates an increase in intracellular Na+ (Nai+) and have tested the ability of drugs to prevent this influx. Methods: Rat single ventricular myocytes at 22°C were voltage-clamped in whole-cell mode to measure membrane currents or were loaded with SBFI to measure Nai+. Results: Removal of extracellular Ca2+ (Cao2+) and Mg2+ (Mgo2+), which induced a current with reversal potential of –10 mV, also caused an increase in SBFI fluorescence ratio (340/380 nm). These changes were reversible on repletion of Cao2+ and/or Mgo2+. They could not be prevented by nifedipine, indicating that they were not mediated by L-type Ca2+ channels. Both increases in non-selective conductance and in Nai+ were prevented by trivalent cations (Dyo3+, Gdo3+ or Lao3+; 100 µM) or reduced by the aminoglycoside gentamicin. Conclusion: A cation non-selective conductance, different from L-type Ca2+ channels, contributes to the Nai+ accumulation obtained during perfusion with Ca2+/Mg2+-free media, hence also to the Cai2+ overload and cellular damage upon Cao2+ repletion (the Ca2+ paradox).

KEYWORDS Experimental; Heart; Electrophysiology; Pathophysiology; Myocytes; Intra/extracellular ions; Ion transport; Membrane currents; Intracellular sodium; Calcium paradox


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Reperfusion of the heart with a Ca2+-containing solution after a period of extracellular Ca2+ (Cao2+)-free perfusion produces irreversible tissue damage in isolated heart preparations. This phenomenon is known as the Ca2+ paradox [1], and there has been a long debate about its underlying mechanisms. One of the major points of controversy has concerned the issue of whether an excessive intracellular Na+ (Nai+) accumulation during perfusion with Ca2+-free media is a prerequisite to the cell damage upon Cao2+ repletion [2–7]. According to the ‘Na+ hypothesis’, as formulated by Chapman and Tunstall [8], the following sequence of events take place in tissues as well as in single cells: (1) Upon Cao2+ removal, Na+ enters the cell via L-type Ca2+ channels, known to become permeable to monovalent cations and to escape inactivation when extracellular divalent cations are removed. This causes an increase of Nai+, the extent of which may be limited by an increased activity of the Na+/K+ pump. (2) Upon Cao2+ repletion, Ca2+ enters the cell in exchange for Na+, causing contracture and structural lesions. According to others [5,7,9–11], cell injury upon repletion of Cao2+ after a period in Cao2+-free solutions is not due to Nai+ overload although it may be aggravated by it.

Whatever the exact (critical or only aggravating) role played by the electrolyte imbalance which occurs during perfusion with Ca2+-free solutions, it is important to define its mechanisms. Evidence supporting the view that Nai+ increase upon perfusion with divalent cation-free solutions is due to Na+ entry via L-type Ca2+ channels has been reviewed [8] and includes the observation that Ca2+-antagonists attenuate the Nai+ elevation during perfusion with Ca2+-free solutions as well as the extent of damage caused by Cao2+-repletion. However, some Nai+ increase persists in divalent cation-free solutions in the presence of Ca2+-antagonists [3]. In addition, in many studies Ca2+-antagonists, even when given during the Cao2+-free period, failed to protect the heart against the Ca2+ paradox [12]. Finally, the Ca2+ paradox has been observed even in resting tissues or cells, in which action potentials were not induced to avoid the opening of L-type Ca2+ channels.

The possibility that besides L-type Ca2+ channels, another pathway is involved in Nai+ loading during perfusion with Ca2+-free solutions was suggested by Bhojani & Chapman [3]. We showed in a previous study that in divalent cation-free media the membrane potential was depolarized and that this depolarization was nifedipine-insensitive. Voltage-clamp results suggested that removal of extracellular divalent cations unmasked a novel conductance pathway, permeable to monovalent cations, and unrelated to L-type Ca2+ channels [13]. Similar findings in smooth muscle cells have just been reported [14]. The aim of the present study was to further analyze how important this conductance pathway was for Nai+ loading in the presence of Ca2+ blockers. In addition, since it has been demonstrated by various techniques and in a variety of tissues that Nai+ rises in Ca2+-free media [2,3,8,15], while no Nai+ change was detected using NMR methods [7], we examined the possibility that the trivalent cation dysprosium (Dyo3+), the triethylenetetraminehexaacetate salt of which is added as shift agent in NMR studies, blocks the non-selective conductance pathway. Finally, we searched for other blockers of the conductance and demonstrate that gentamicin, a polycationic drug known to protect against the Ca2+ paradox [16], also partly inhibits the non-selective conductance induced in divalent cation-free media.

A preliminary report of this work has appeared in abstract form [17].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
Ca2+-tolerant ventricular myocytes were isolated from the hearts of Wistar rats (200–250 g) using a collagenase perfusion method. The hearts were rapidly removed and were Langendorff-perfused at 37°C at a constant flow rate of 5.5–8 ml min–1 with: (1) Ca2+-free Tyrode solution for 5 min; (2) Ca2+-free Tyrode solution containing collagenase (type A; Boehringer) and protease (type XIV; Sigma) for 3–10 min; (3) Ca2+-free Tyrode solution containing collagenase alone for 10–15 min (in most but not all cases); (4) 0.18-mM Ca2+ containing Tyrode for 3 min. After perfusion, the atria were discarded and the ventricular tissue was cut into chunks and cells were separated by gentle mechanical agitation. The residue was filtered through a mesh (200-µm hole diameter). In a few cases the filtrate was centrifuged twice at 8 g and resuspended to washout enzymatic medium and debris. Myocytes were stored in normal Tyrode solution at room temperature.

2.2 Electrophysiology
Methods for electrophysiological measurements are as described before [13]. Briefly, whole-cell membrane currents [18] were studied in single ventricular myocytes, using 1–5 M{Omega} borosilicate electrodes connected to an Axon 200-A amplifier, and the pClamp software (Axon Instruments). The holding potential was set at –80 mV and the command voltage consisted of 4-s ramps from –120 mV to +80 mV and back to –120 mV, given every 10 s. Currents were measured during the descending ramp from +80 mV to –120 mV.

2.3 Measurements of intracellular sodium
2.3.1 Cell loading with SBFI
In most experiments cells used for Nai+ measurements were loaded with the membrane permeable acetoxymethyl (AM) ester form of sodium benzofuran isophtalate (SBFI-AM; Molecular Probes) and were not voltage-clamped using a patch electrode to avoid loss of SBFI in the pipette solution. Thus, a separate group of cells was used for Nai+ measurements, and a different group of cells for current measurements. The dye-loading solution was made by adding 22 µl dimethylsulphoxide containing 3% Pluronic F-127 (Molecular Probes) to 50 µg SBFI-AM. The vial was sonicated and 11 µl of the content diluted in 1 ml standard Tyrode solution and added to 1 ml of cell suspension (final SBFI-AM concentration: 10 µM). Loading was carried out for 60–90 min at room temperature. After loading, the cells were kept in Tyrode solution for ≥30 min to allow completion of the SBFI-AM hydrolysis. Probenecid (0.3 mM) was added to all solutions after loading to minimize the loss of the indicator from cells and to reduce the degree of its subcellular compartmentalisation [19]. When SBFI fluorescence and voltage-clamp measurements were carried out simultaneously, the cell was loaded with the de-esterified form of SBFI (100 µM; from a stock in dimethylsulphoxide) via the patch electrode.

2.3.2 Fluorescence measurement
Myocytes in the experimental chamber were continuously superfused with Tyrode solution. For radiometric fluorescence measurements, we used the PTI RF-D4012 model, which consists of a high-speed dual wavelength scanning illuminator coupled to an inverted microscope (Nikon IM35) via a bifurcated quartz fiber optic bundle. Illuminating light was obtained from a 75-W xenon arc lamp; light at 340-nm and 380-nm wavelength was alternately directed onto the cells placed above the 40x objective (under oil immersion; Dapo UV/340 Na 1.30 Olympus). The light emitted by the indicator within the cell was collected by the objective and passed via a dichroic mirror (long pass 400) to a photomultiplier equipped with a photon-counting detector. Light detected by the photomultiplier was restricted to that from a single cell by altering a rectangular diaphragm in the emission pathway. An interference filter centered on 510 nm with a half-bandwidth of 20 nm was placed in front of the photomultiplier. The different components of the set-up were computer-controlled and the Felix software was used for collection, analysis and presentation of data.

Before loading cells with the dye their autofluorescence was measured. The fluorescence signal after loading was usually 8–10 times larger than the original signal. With correction made for the autofluorescence, the fluorescence ratio (R) was calculated using the following formula:

Formula

To minimize photobleaching, illumination was switched on only during experimental manoeuvres and for short periods.

2.3.3 SBFI fluorescence signal calibration
We used an empirical ‘in situ’ calibration of SBFI fluorescence for Nai+. To avoid contracture before introducing calibration solutions, the cell was exposed to a Ca2+-free Tyrode solution containing 1 mM EGTA. We included ionophores (gramicidin, 10 µM; monensin, 14.5 µM; nigericin, 10 µM) and ouabain (100 µM) in the calibration solutions to create conditions that allow equilibration of Nai+ with Nao+ [20,21], changed Nao+ and established a calibration curve by plotting R as a function of the Na+ concentration. Although there were differences in the absolute R values between cells, the calibration curves from different cells were linear and had similar slopes between 0 and 15 mM. In six cells it was found that Nai+ changes predicted by the calibration curve from each cell were in good agreement (r=0.87; P=0.02) with values predicted from the average calibration curve obtained by pooling data from all six cells. For this reason the average calibration curve was used to estimate Nai+ changes in other cells, in which the experimental protocol was long and precluded an additional lengthy SBFI calibration procedure.

2.4 Solutions
The Tyrode solution used to perfuse the isolated hearts or to superfuse the cells contained (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, HEPES 10, glucose 10, pH 7.4 (titrated with NaOH). In voltage clamp experiments KCl was equimolarly replaced with CsCl to eliminate K+ currents. The pipette-filling solution contained (in mM): Cs-glutamate 130, TEA-Cl 25, MgCl2 1, EGTA 1, Na2ATP 5, HEPES 5; pH 7.2 (titrated with CsOH). The myocytes were initially superfused with standard K+-containing Tyrode solution. After obtaining the whole-cell configuration the superfusing solution was changed to the one containing Cs+. In experiments where SBFI fluorescence was measured, the K+-containing Tyrode solution was used. To obtain Ca2+- and/or Mg2+-free solutions, the divalent cations were simply omitted without substitution. All experiments were carried out at room temperature (22–23°C). Solutions which contained nifedipine (Sigma; 10–100 µM, from a stock in ethanol) were protected from light.

2.5 Data presentation
SBFI data were not sampled continuously but only for short periods (to avoid SBFI bleaching). Data during each sampling period were averaged and are presented as one figure point. Average results are expressed as mean±standard error of the mean (SEM).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Fig. 1A shows the time course of changes in membrane currents upon perfusion with Ca2+-free Tyrode, in the absence or in the presence of the L-type Ca2+ channel blocker nifedipine. Mgo2+ had been omitted from the beginning without any effect. Further removing Cao2+ in the absence of nifedipine caused a large increase in outward and inward currents recorded at +80 mV and –80 mV, respectively. Upon addition of nifedipine, the current change at +80 mV was largely suppressed whereas the change at –80 mV was unaffected. The effect of Cao2+ removal was reversible upon repletion of Cao2+. Upon a second Cao2+ removal, the above changes at –80 mV were reproducible whereas those at +80 mV were prevented by the continued presence of nifedipine. Removing Cao2+ alone (in the presence of Mgo2+) had no effect (not illustrated). This result suggests that removal of extracellular divalent cations either induces two different conductances differentially blocked by nifedipine or a single conductance blocked voltage-dependently by the Ca2+ antagonist. Fig. 1B shows the relationship between membrane currents and potential, measured using ramp voltage pulses. Since Cs+ replaced K+ in the extracellular and intracellular media to block K+ channels, the relationship under control conditions (1.8 mM Cao2+) was linear over most of the voltage range, with some outward rectification at positive potentials. Perfusion with Ca2+-free solution shifted the current–voltage relationship inward at potentials negative to +15 mV, and outward above this level. The relationship now showed inward rectification with a marked negative slope-conductance between –40 mV and –15 mV. Given the known permeability of L-type Ca2+ channels to monovalent cations in the absence of extracellular divalent cations [22–24], the negative slope-conductance could be due to Na+ movement via L-type Ca2+ channels. That this was the case is demonstrated by the fact that addition of nifedipine (100 µM) eliminated the negative conductance and shifted the current–voltage relation outward between –50 mV and +45 mV, and inward above +45 mV. No effect of nifedipine was present negative to –50 mV. The nifedipine-sensitive and insensitive components of the current induced in Ca2+- and Mg2+-free solution are illustrated in Fig. 1C. The nifedipine-sensitive current was only activated at potentials >–50 mV, reached inward maximum at about –10 mV and had its reversal potential at +45 mV (i.e. close to ENa). The nifedipine-insensitive current showed inward rectification and its reversal potential was at –10 mV, indicating that it is a highly non-selective current as recently characterized [13]. These data suggest that the increase in current obtained in Ca2+- and Mg2+-free solution was partly due to ion flow through L-type Ca2+ channels, and that a major current component was carried through another pathway.


Figure 1
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  		Fig. 1 Two current components induced by the removal of extracellular divalent cations: A. Time course of changes in currents at +80 mV (unfilled circles) and at –80 mV (filled circles), during removal and repletion of Cao2+. Mgo2+ had been omitted from the beginning. Cao2+-removal induced an outward current at +80 mV and an inward current at –80 mV. Nifedipine (100 µM) blocked the outward current but had no effect on the inward current: B. Current–voltage relationships obtained using ramps from +80 to –120 mV in the presence of 1.8 mM Cao2+ ({diamond}), in the absence of Cao2+ without nifedipine (O), and in the absence of Cao2+ with nifedipine ({square}). The effect of nifedipine is restricted to a potential range (>–50 mV) where L-type Ca2+ channels are activated: C. The two components of the conductance induced in divalent cation-free solution. The nifedipine-sensitive component was obtained as difference between currents in 0 mM Cao2+ but in the absence of nifedipine and currents in 0 mM Cao2+ but in the presence of 100 µM nifedipine (O-{square} of B). The nifedipine-insensitive component was obtained as difference between currents in 0 mM Cao2+ in the presence of nifedipine and currents in 1.8 mM Cao2+ ({square}-{diamond} of B).

 
The large nifedipine-insensitive inward current (at negative potentials) is due to Na+ influx since it is absent when Nao+ is replaced by a large cation such as NMDG+ [13,14]. Therefore, it is expected to cause an increase in Nai+. Fig. 2A illustrates the effect on SBFI fluorescence of superfusing with Ca2+- and Mg2+-free Tyrode while holding the membrane potential at –60 mV. Upon removal of both Cao2+ and Mgo2+, the holding current became more inward and the SBFI fluorescence ratio increased progressively. Due to uncertainties about the extent of dilution of Nai+ by the patch pipette, further such experiments were carried out in resting, non-clamped cells superfused with Tyrode containing nifedipine (10 µM). Qualitatively similar results were obtained (Fig. 2B): compared to the value under resting conditions, R rose by 0.027±0.003 after 1 min, and by 0.055±0.006 after 5 min (n=12). This increase was reversed upon repletion of the divalent cations and could be reproduced during a second Cao2+ and Mgo2+ removal. Increasing Cao2+ alone in the absence of Mgo2+, or increasing Mgo2+ alone in the absence of Cao2+, also reversed the Nai+ increase (not illustrated), a result consistent with the finding that either Cao2+ or Mgo2+ is sufficient for blocking the non-selective conductance. Since nifedipine did not prevent the increase in the SBFI fluorescence ratio, L-type Ca2+ channels are excluded as the major pathway for the Na+ influx.


Figure 2
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  		Fig. 2 Increase of intracellular Na+ induced by the removal of extracellular divalent cations. A: SBFI fluorescence ratio and membrane current measured simultaneously in a cell clamped at –60 mV during removal and repletion of Cao2+ and Mgo2+. B: SBFI fluorescence ratio in a resting, non-voltage-clamped cell during removal and repletion of Cao2+ and Mgo2+. Nifedipine (10 µM) had been added from the beginning. In A and B, Cao2+ and Mgo2+ were removed simultaneously.

 
We sought for tools to block or prevent the opening of the non-selective pathway for Na+ entry. We found that beside Gdo3+[13], other trivalent cations (Dyo3+ and Lao3+) could also block the pathway. Fig. 3A shows results from a cell initially superfused with 1.8 mM, in which Cao2+ removal reproducibly induced an inward current at –80 mV. Exposure to 100 µM Dyo3+ suppressed the current increase obtained in divalent cation-free solution. The current–voltage relationship with 100 µM Dyo3+ alone was nearly identical to the one obtained in its absence but in the presence of 1.8 mM Cao2+ (Fig. 3B), hence the Cao2+-sensitive and the Dyo2+-sensitive currents were practically indistinguishable. In four cells, Cao2+ and Mgo2+ removal increased the holding current at –80 mV from –78±36.2 pA to –1278±207.8 pA, and addition of Dyo3+ (100 µM) decreased the current to –98±25.3 pA. Complete block could also be obtained with 10 µM but not lower Dyo3+ concentrations. Lao3+ (100 µM) had similar effect. The block by trivalent ions was not removed on washout of Dyo3+, Lao3+ or Gdo3+(current: 118±8.3 pA at –80 mV after 20 min washout of Dyo3+ in divalent cation-free solution in the four cells mentioned above), in contrast to the block induced by divalent cations which is completely reversible [13,14]. Fig. 3C shows that Dyo3+ also prevented the increase in Nai+ obtained upon superfusion with divalent cation-free solution. The inhibitory effect of Dyo3+ on Nai+ changes was also irreversible.


Figure 3
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  		Fig. 3 Block by dysprosium (Dyo3+, 100 µM) of the non-selective current and inhibition of the increase of intracellular Na+ induced in divalent cation-free solutions: A. Time course of changes in currents at –80 mV during removal and repletion of Cao2+. Cao2+ removal induced an inward current. Dyo3+was added in the absence of Cao2+. Dyo3+ blocked the current, which did not recover on washout of the trivalent cation: B. Current–voltage relationships obtained using ramps from +80 to –120 mV in the presence of Cao2+ ({diamond}), in the absence of Cao2+ without Dyo3+ (O) and in the absence of Cao2+ with Dyo3+ ({square}). Dyo3+ completely suppressed the conductance increase caused by the removal of extracellular divalent cations: C. Data show the SBFI fluorescence ratio (in a resting, non-voltage-clamped cell) during removal and repletion of Cao2+ and Mgo2+, in the absence or in the presence of Dyo3+. Nifedipine (10 µM) had been added from the beginning. In A and B, Mgo2+ had been omitted before Cao2+ removal. In C, Cao2+ and Mgo2+ were removed simultaneously.

 
We also searched for organic agents to block the divalent cation-sensitive non-selective pathway. Since in other cells the divalent cation-sensitive current seems to occur via gap junction channels [25,26], we examined whether blockers of gap-junction channels [27] could inhibit the current observed under our experimental conditions. Of heptanol (2 mM; n=4), octanol (3 mM; n=1) and halothane (2 mM; n=4) none was effective in blocking the non-selective conductance. Since aminoglycoside antibiotics such as gentamicin have been reported to block non-selective channels [26,28] and to prevent the Ca2+ paradox [16], we also tested whether these drugs affect the non-selective conductance and the Nai+ increase induced by the removal of divalent cations. Fig. 4A shows the time course of current changes induced by Cao2+ removal at –80 mV, with or without added gentamicin. The increase in inward current in divalent cation-free solutions was reversibly and reproducibly reduced in the presence gentamicin (660 mg/l). In five preparations, gentamicin blocked the current at –80 mV incompletely (to ca. 45% of its control value in the absence of the antibiotic). A similar result could be obtained with another aminoglycoside antibiotic, neomycin (see Fig. 4A). The effect of gentamicin (Fig. 4B) or neomycin involved a decrease of total conductance without any change in reversal potential. Fig. 4C shows results of experiments carried out to test whether gentamicin has an effect on the Nai+ increase in divalent cation-free solutions. Upon addition of gentamicin in normal Tyrode there was a very rapid increase in basal SBFI fluorescence. The magnitude of the SBFI fluorescence change induced in Ca2+- and Mg2+-free solution was smaller in the presence of gentamicin and returned to a higher value after washout of the drug (while the increase in basal fluorescence was not reversible). In the presence of gentamicin R change upon removal of extracellular divalent cations was 48±9.7% of the control level (n=6).


Figure 4
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  		Fig. 4 Block by the aminoglycoside antibiotics of the non-selective current and inhibition of the increase of intracellular Na+ induced by divalent cation-free solutions: A. Time course of changes in currents at –80 mV during removal and repletion of Cao2+. Gentamicin (660 mg/l) or neomycin (3 mM) were added in the absence of Cao2+. Cao2+-removal induced an inward current. Gentamicin or neomycin partially blocked the current, which fully recovered on washout of the antibiotics: B. Current–voltage relationships obtained using ramps from +80 to –120 mV in the presence of 1.8 mM Cao2+ ({diamond}), in the absence of Cao2+ without gentamicin (O), and in the absence of Cao2+ with gentamicin ({square}): C. Data show the SBFI fluorescence ratio (in a resting, non-voltage-clamped cell) during removal and repletion of Cao2+ and Mgo2+, in the absence or in the presence of gentamicin. Nifedipine (10 µM) had been added from the beginning. Notice that gentamicin caused an increase in basal fluorescence, which did not recover on washout of the antibiotic. In A and B, Mgo2+ had been omitted before Cao2+ removal. In C, Cao2+ and Mgo2+ were removed simultaneously.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Two pathways for Na+ influx in divalent cation-free solutions
In the present study we have shown that in the absence of L-type Ca2+ channel blockers two current components can be induced upon perfusion with extracellular divalent cation-free solutions. One current component has a reversal potential near +45 mV, is blockable by nifedipine and is probably due to monovalent cation flow via L-type Ca2+ channels [22–24]. Another component has a reversal potential near –10 mV, is insensitive to nifedipine and is probably due to a cation non-selective conductance. Since previous results [13,14] show that the inward component of the non-selective conductance disappears in Na+-free solutions, it can be concluded that Na+ is the major carrier of the inward current. This Na+ influx which takes place during Cao2+ depletion will depolarise the cell and could cause Na+ overload. In 14 cells, we estimated that the current density carried by the non-selective component was 2 pA/pF at –40 mV [13]. For an average cell of 100–150 pF, corresponding to a volume of about 50 pL [15], this will cause an molar influx of 0.075 mmol l–1 s–1 or >4 mmol l–1 min–1. The expected increase in Nai+ will be partially offset by an increased activity of the Na+/K+ pump, which is regulated by Nai+. Bhojani and Chapman [3] have shown that Nai+ (measured with Na+-sensitive microelectrodes) increases in cells incubated in 0–mM Cao2+ solution and that this increase was most pronounced when ouabain is included in the medium. Our results confirm the finding that Nai+ increases during perfusion with Cao2+- and Mgo2+-free solutions. While in previous studies the Nai+ increase was attributed nearly exclusively to ion movement through L-type Ca2+ channels, our data, obtained while inhibiting L-type Ca2+ channels with nifedipine, implicate a different, non-selective conductance as an additional pathway for Na+ entry into cells superfused with divalent cation-free solutions (see also [14]). The relative importance of Na+ entry via the non-selective conductance vs. via L-type Ca2+ channels was not systematically investigated in the present study, and it remains possible that the two pathways could be differently affected by temperature.

In the present study it is not possible to provide an exact measure of the increase in Nai+ that takes place during perfusion with divalent cation-free solutions. As mentioned in Methods, it was difficult to obtain a calibration of the SBFI fluorescence ratio (R) as a function of the Na+ concentration for every cell. Using the average calibration curve obtained from six cells, the mean slope of R change with Na+ is 0.00948±0.001 mM–1. With a mean R increase of about 0.027 after 1 min in Ca2+- and Mg2+-free Tyrode, the estimated increase of Nai+ is of about 2.8 mM. This value is much in accord with the increase predicted from the density of the non-selective current. Beyond 1 min the rate of Nai+ increase became smaller probably since the Na+/K+ pump (not inhibited in our experiments), which is largely dependent on Nai+, should become more and more activated. After 5 min in Ca2+- and Mg2+-free Tyrode, the estimated increase of Nai+ was of about 5.8 mM. It should also be noted that the above method will underestimate large increases in Nai+, given the fact that resting Nai+ in rat myocytes is about 10 mM (see Ref. [29]) and that the slope of R change with Na+ was highest below 15 mM but decreased above this level.

4.2 Possible role in the Ca2+ paradox
The data suggest that opening of a Na+-permeable conductance is an important component of cellular events occurring during perfusion with divalent cation-free solutions. Perfusion with Ca2+-free media is known to predispose the heart to the Ca2+ paradox upon repletion of Cao2+. As mentioned in the Introduction, one important issue concerning the mechanisms underlying the Ca2+ paradox has been whether an increase of Nai+ occurs in Cao2+-free solution and is a prerequisite for the cellular damage occurring on Cao2+ repletion. Whereas a variety of techniques utilized to measure Nai+ changes during perfusion with Ca2+-free media (including mass spectroscopy, Na+-sensitive electrodes, Na+-sensitive fluorescent dyes) all have indicated that Nai+ increases upon Cao2+ removal, NMR spectroscopy studies failed to show such an increase [7]. Van Echteld et al. [7,10] have pointed to the fact that studies which demonstrated a Nai+ increase upon perfusion with Ca2+-free solutions were usually carried out in the absence of Mgo2+ or in the presence of very low Mgo2+ concentrations. This is consistent with our findings since the non-selective conductance and the increase in SBFI fluorescence occurred only when both Cao2+ and Mgo2+ were omitted. Since Nai+ increase was absent even in cells which later underwent the Ca2+ paradox, it has been proposed that a rise in Nai+ during Cao2+ depletion is not required for cell injury to develop upon Cao2+ repletion [5,7]. Our data do not allow to resolve this issue. Nevertheless there is a consensus that an increase in Nai+ will constitute an aggravating condition for injury associated with the Ca2+ paradox.

Our data suggest that in addition to L-type Ca2+ channels, which are a recognized pathway for Na+ entry during perfusion with divalent cation-free solutions, a non-selective conductance also plays an important role. This pathway could account for the observed incomplete inhibition of Nai+ increase in Ca2+-free solutions by Ca2+-antagonists [3]. In a few studies, Ca2+-antagonists either were without an effect on the Nai+ increase during perfusion with 0-mM Cao2+ solutions or failed to attenuate the tissue damage induced upon Cao2+ repletion (e.g. [12]). This implies that a Ca2+-antagonist-insensitive pathway, presumably the non-selective conductance, was responsible for Na+ influx in these cases. This is made more likely by the observation that in the studies where the Ca2+ paradox was insensitive to Ca2+-antagonists either both Cao2+ and Mgo2+ were removed (or complexed by EDTA) or Mgo2+ was kept to a level so low that it could not replace Cao2+ to fully block the Ca2+-antagonist-insensitive pathway. It is therefore fortunate that Mgo2+ is usually increased in many cardioplegic solutions [30,31] where Cao2+ is to be kept low: this allows closing of the non-selective conductance by Mgo2+ while at the same time avoiding the direct deleterious effects of Cao2+.

Cao2+ repletion damage was not present in our experiments, which were carried at room temperature, and in which Cao2+/Mgo2+ depletion/repletion could be repeated up to three times in one cell. We did not test for reversibility after prolonged (≥15 min) depletion periods. We have already mentioned in our previous study [13] that irreversible cell injury occurred during short Cao2+/Mgo2+ depletion/repletion experiments carried out at 37°C. Additional experiments at this temperature (not shown) confirmed these previous findings. It is interesting to mention that cell death at 37°C often occurred even during the time of superfusion with divalent cation-free solution and was not restricted to the Cao2+ repletion period. Since the non-selective conductance is inhibited by Cao2+ with a K0.5 of about 60 µM [13], it is possible that Na+ starts entering via this pathway and is exchanged with Cao2+ during the period Cao2+ is at submillimolar levels before its extreme depletion. In addition, it is still unclear whether the non-selective conductance may show some limited permeability to Ca2+ at high temperature. For cells which survived the divalent cation depletion period at 37°C, contracture and cell death occurred upon Cao2+ repletion in all cases.

4.3 Blockers of the non-selective pathway
An attempt was made to identify blockers of the non-selective conductance. We found that in addition to divalent cations, trivalent cations such as Gdo3+, Lao3+ and Dyo3+ also block the non-selective conductance. The nature of this block seems different from that produced by divalent cations since washout of the trivalent cations was not accompanied by a restoration of the non-selective conductance. This suggests that the block by trivalent cations may involve trapping of the ions within or into the mouth of the channel. This hypothesis is supported by the observation that the block could sometimes be removed by large depolarizing pulses which probably drove the cation out of the channel (our unpublished results). We were more interested in identifying organic blockers of the conductance. Unfortunately only the antibiotics gentamicin and neomycin showed a partial effect, and this at quite high concentrations. The mechanisms of action of these drugs on the conductance remains unclear. Since the drugs are polycationic, we thought that their effect was related to the presence of positive charges and tested this possibility by applying another polycation, protamine, but found no effect of this latter drug (not shown). Therefore factors others than drug electrical charge have to be involved. Other substances tried include blockers of the gap-junction channel but none of them was effective. This finding is by itself important since it indicates that the conductance opened in cardiac cells by the removal of extracellular divalent cations is unlikely to consist of gap-junction hemichannels. This is in contrast with oocytes, where the divalent cation-sensitive non-selective conductance was suppressed in cells injected with an antisense oligonucleotide sequence for connexin-38 [26], a finding which suggests that in those cells gap-junction channels were involved [25].

Time for primary review 26 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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