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Am J Physiol Heart Circ Physiol 277: H1817-H1822, 1999;
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Vol. 277, Issue 5, H1817-H1822, November 1999

Contribution of the Na+ channel and Na+/H+ exchanger to the anoxic rise of [Na+] in ventricular myocytes

B. N. Eigel and R. W. Hadley

Department of Pharmacology, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to quantify the contribution of the Na+/H+ exchanger (NHE) and the Na+ channel to the rise in cytosolic Na+ concentration ([Na+]) that is seen in anoxic guinea pig ventricular myocytes. [Na+] was measured with the use of microfluorometry and was found to rise to 44 mM after prolonged anoxia. This rise was partially sensitive to either TTX or HOE-642, selective inhibitors of the Na+ channel and NHE1, respectively. [Na+] did not significantly rise when both drugs were present, suggesting that other routes of Na+ entry were insignificant. However, the relative contributions of the NHE and the Na+ channel were found to be remarkably sensitive to ionic conditions expected to occur during ischemia. The Na+ channel was the dominant Na+ source during acidic anoxia. However, the NHE was the dominant Na+ source during both hyperkalemic anoxia and simulated ischemia (hyperkalemia, low pH, and anoxia). The data suggest that the NHE may prove to be the best pharmacological target to reduce Na+ entry during true ischemia and that inhibition of Na+ influx could contribute strongly to the cardioprotective effects of NHE inhibitors.

heart; sodium; sodium-binding benzofuran isophthalate; ischemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTERED IONIC HOMEOSTASIS is thought to have a vital role in producing myocardial injury in response to different models of metabolic stress, such as ischemia, hypoxia, or metabolic poisoning. Elevated cytosolic Ca2+ concentration ([Ca2+]) is reliably seen in response to ischemia or hypoxia (1, 11, 28) and is believed to be a central mediator in coupling ionic dyshomeostasis to myocardial injury and cell death. Cytosolic Na+ concentration ([Na+]) also increases during either ischemia or hypoxia in cardiac myocytes (3, 6, 7) and has been proposed to be an antecedent to the elevation in cytosolic [Ca2+] (6, 15, 20), because a rise in cytosolic [Na+] can be coupled to a rise in cytosolic [Ca2+] through the Na+/Ca2+ exchanger.

Earlier events in hypoxia- or ischemia-induced ionic dyshomeostasis are more uncertain. Intracellular acidification is commonly observed in cardiac myocytes during either hypoxia or metabolic poisoning (21, 23) and could be coupled to the rise in cytosolic [Na+] through the Na+/H+ exchanger (NHE) (2, 18, 29). Indeed, various NHE inhibitors have been found to be cardioprotective during ischemia (4, 25, 26), although other mechanisms besides that of diminishing the sequential rise in sodium and calcium ions could be involved. In addition, Na+-channel gating could be altered during hypoxia or ischemia (10, 27, 34); therefore, Na+ channels could serve as a source for excess Na+ entry. Evidence that Na+-channel blockers can be cardioprotective, or can diminish the rise in intracellular [Na+], has been reported in some experimental models (4, 7, 22), but not in others (23).

We sought to clarify several unresolved issues concerning the role of cytosolic [Na+] in myocardial injury during this study. First, we wanted to rigorously quantify the relative importance of the Na+ channel and the NHE to the rise in cytosolic [Na+] seen during anoxia or simulated ischemia in guinea pig ventricular myocytes. Second, we wanted to determine whether the Na+ channel and the NHE together could account for all of the excess entry of sodium ions in our model or whether other significant routes of Na+ entry existed. Finally, we wanted to test whether certain ionic conditions that might differ between tissue and cellular experimental models, such as extracellular acidification or hyperkalemia, could alter the relative importance of these Na+ entry routes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. Experiments were conducted in single guinea pig ventricular myocytes, which were isolated using an established Langendorff enzymatic digestion technique (5). Guinea pigs were anesthetized with a 50-mg intraperitoneal injection of pentobarbital sodium before the heart was removed. This method of euthanasia followed the guidelines of the American Veterinary Medical Association's Panel on Euthanasia and has been approved by the University of Kentucky's Institutional Animal Care and Use Committee.

Fluorescent indicator loading and calibration. Fluorescence measurements of cytosolic [Na+] were made using sodium-binding benzofuran isophthalate (SBFI; Molecular Probes, Eugene, OR). Myocytes were loaded with the cell-permeable acetoxymethyl ester (AM) form of SBFI, which allows for minimal disruption of the intracellular environment. SBFI-AM was first dissolved in 20% Pluronic-DMSO (Sigma, St. Louis, MO). Cells were then loaded with 10 µM SBFI-AM for 50 min at 22°C.

Calibration of SBFI measurements was performed as previously described (21). Briefly, myocytes were incubated in a solution containing 10 µM gramicidin to allow for equilibration of intracellular and extracellular [Na+] and 1 mM ouabain to inhibit the Na+-K+ pump. Calibration solutions used [Na+] of 0-80 mM to generate a single calibration curve used throughout the study.

Fluorescence measurements. SBFI measurements were collected using a conventional inverted fluorescence microscope (Nikon Diaphot 200, Nikon, Melville, NY) with a 75-W xenon arc lamp serving as an excitation light source. Ratiometric measurements with SBFI were made using interference filters centered at 340 and 380 nm on the excitation side, with the emission filter centered at 540 nm. Fluorescence was measured using a photomultiplier detection system (Photon Technology International, South Brunswick, NJ). The output was filtered with a time constant of 50 ms and digitized on an 80486 computer at 1-2 kHz. Data were recorded and analyzed with specialized software written by one of the authors. Standard fluorescence background subtraction and ratiometric analysis techniques were used with all SBFI measurements.

Drugs and solutions. TTX (30 µM; Calbiochem, San Diego, CA) was used to inhibit TTX-sensitive Na+ channels, and 10 µM 4-isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (HOE-642, or cariporide; kindly supplied by Wolfgang Scholz and Andreas Weichert, Hoechst Marion Roussel, Frankfurt, Germany) was used to inhibit the NHE (25). TTX and HOE-642 were added to the anoxic bath solutions before myocytes were placed into the anoxia chamber.

Four separate anoxic bath solutions were used. The standard "normal" Tyrode solution contained the following (in mM): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.20, 22°C). The "acidic" Tyrode solution was similar to the normal solution except that the pH was set at 6.85 to mimic the extracellular acidification seen during moderate ischemia. The "hyperkalemic" Tyrode solution contained (in mM) 134 NaCl, 10 KCl, 2.5 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.20, 22°C), with KCl elevated to 10 mM to simulate a degree of hyperkalemia seen during moderate ischemia (33). The "simulated ischemic" Tyrode solution was identical to the hyperkalemic solution except that the pH was also lowered to 6.85 to combine the effects of both extracellular acidification and increased extracellular K+ concentration ([K+]). Glucose was not included in any of the anoxic bath solutions so that myocytes could not sustain ATP production through glycolysis.

Experimental conditions. Myocytes were made anoxic in a glass, gas-tight petri dish with a coverslip affixed to a hole drilled into the bottom of the chamber (21). The bath solution inside the chamber was bubbled with nitrogen for several minutes through a rubber septum before sodium hydrosulfite (1 mM; Sigma) was added to scavenge any remaining oxygen. Oxygen levels in the bath solution were undetectable using either the oxygen indicator resazurin or an oxygen electrode (Orion Research, model 97-08). Therefore, these solutions are referred to as "anoxic" because the oxygen content of the bath solution was reduced as much as feasible. A small volume (<30 µl) of SBFI-loaded myocytes was then placed on top of the chamber coverslip, and the chamber top was loosely sealed. A continuous flow of nitrogen was maintained above the solution throughout the course of anoxia to prevent oxygen entry into the chamber. This gassing procedure necessitated the use of HEPES for pH buffering, rather than NaHCO3, and would of course alter the activity of bicarbonate cotransport across the cell membrane. Only healthy heart cells, as defined by quiescence, rod shape retention, and initial cytosolic [Na+] of 7-13 mM, were selected for measurements.

The standard anoxia procedure consisted of maintaining anoxia for 60 min after the onset of rigor contracture. Cytosolic [Na+] was routinely measured and analyzed at the onset of anoxia, at the onset of rigor, and then at regular intervals after rigor contracture. The reason we chose to begin our statistical analysis at the onset of rigor is that there is considerable cell-to-cell variability in both the time to onset of rigor and in the initial time course of the rise in intracellular [Na+]. The significance of this is that the variability complicates statistical analysis at specific time points. On the other hand, the time course of the rise in cytosolic [Na+] becomes fairly consistent after a cell has reached rigor contracture, thereby facilitating quantitative analysis.

Statistical analysis. Differences between means were analyzed using a two-way analysis of variance (general linear model). Student-Newman-Keuls multiple comparisons testing was used for post hoc significance testing between appropriate groups. Variance is described as standard errors of the mean.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The intent of this study was to determine the cellular mechanisms of excess Na+ entry in isolated guinea pig ventricular myocytes during anoxia and simulated ischemia. This objective was pursued by using selective pharmacological inhibitors (HOE-642 and TTX) under four different extracellular ionic conditions to determine what effect, if any, extracellular [K+] and/or pH might have on anoxic Na+ entry mechanisms.

Normal Tyrode solution and anoxic Na+ entry. Cytosolic [Na+] was measured with the fluorescent indicator SBFI in guinea pig ventricular myocytes under anoxic, glucose-free conditions in various modified Tyrode bath solutions. Figure 1 shows measurements made in cardiac myocytes bathed in an anoxic, normal Tyrode solution. The purpose of the experiments summarized in Fig. 1 was to determine whether the observed increase in cytosolic [Na+] during anoxia depended on Na+ entry through the Na+ channel and/or the NHE and, if so, whether these two mechanisms could account for essentially all Na+ entry in this experimental model. Anoxic guinea pig ventricular myocytes showed a pronounced rise in cytosolic [Na+], both before and after rigor contracture, as previously reported (21). Figure 1 shows that either TTX or HOE-642 alone significantly suppressed (P < 0.05) the rise in cytosolic [Na+] at rigor and at all time points postrigor versus the rise seen in drug-free myocytes (21.7 ± 2.0 and 24.4 ± 3.8 vs. 43.9 ± 4.6 mM, respectively, at 60 min postrigor). The combination of both TTX and HOE-642 also significantly suppressed (P < 0.05) anoxic intracellular [Na+] at rigor and at all times postrigor versus that in drug-free myocytes (13.5 ± 1.6 vs. 43.9 ± 4.6 mM at 60 min postrigor). However, cytosolic [Na+] at 60 min postrigor in either TTX- or HOE-642-treated cells was still significantly greater than the initial intracellular [Na+] (TTX: 21.7 ± 2.0 vs. 11.5 ± 0.6 mM; HOE-642: 24.4 ± 3.8 vs. 9.5 ± 0.8 mM). These data suggest that neither the Na+ channel nor the NHE alone can account for all of the rise in cytosolic [Na+] because neither TTX nor HOE-642 alone could prevent a significant rise in anoxic intracellular [Na+]. However, the combination of TTX and HOE-642 suppressed essentially all of the rise in cytosolic [Na+] because intracellular [Na+] at 60 min postrigor was not significantly different from initial cytosolic [Na+] (13.5 ± 1.6 vs. 8.3 ± 0.6 mM).


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  		Fig. 1.   Anoxic myocytes, bathed in normal Tyrode solution, showed a significant rise in cytosolic Na+ concentration ([Na+]) that was completely sensitive to inhibition by HOE-642 and TTX. Fluorescence measurements of cytosolic [Na+] were made in isolated guinea pig ventricular myocytes [loaded with sodium-binding benzofuran-isophthalate acetoxymethyl ester (SBFI-AM)] exposed to anoxia beginning at the "initial" time point and continuing until 60 min postrigor contracture (see METHODS AND MATERIALS). HOE-642 (10 µM), TTX (30 µM), or a combination of both drugs was used to selectively inhibit the Na+/H+ exchanger (NHE) and TTX-sensitive Na+ channel, respectively. Normal Tyrode solution had [K+] set to 4 mM and pH to 7.20. Each time point represents mean ± SE of 8 cells. * P < 0.05 vs. drug-free value at same time point. In addition, cytosolic [Na+] in drug-free or TTX- or HOE-642-treated myocytes at 60 min postrigor anoxia was significantly greater than each respective initial cytosolic [Na+]. However, combined treatment of TTX and HOE-642 showed no significant increase in cytosolic [Na+] throughout anoxia compared with initial cytosolic [Na+].

Simulated ischemic Tyrode solution and anoxic Na+ entry. The primary limitation of this isolated cellular model of anoxia is that, compared with true ischemia, it does not allow for the accumulation of extracellular species such as protons or potassium ions. Therefore, the purpose of the next experimental series was to investigate how anoxic Na+ entry might be altered by the combination of moderate extracellular acidification (pH = 6.85) and moderate extracellular hyperkalemia ([K+] = 10 mM). Figure 2 shows the effects of anoxia and simulated ischemic Tyrode solution on cytosolic [Na+] in isolated myocytes. Drug-free cells in simulated ischemic Tyrode solution showed an increase in cytosolic [Na+] at 60 min postrigor (36.0 ± 3.7 mM) similar to that in drug-free myocytes bathed in normal Tyrode solution (43.9 ± 4.6 mM; Fig. 1). However, in simulated ischemic Tyrode solution (Fig. 2), TTX did not significantly inhibit the rise in cytosolic [Na+] at any time point during anoxia in comparison with that in drug-free myocytes (40.2 ± 5.8 vs. 36.0 ± 3.7 mM at 60 min postrigor). However, HOE-642 did significantly (P < 0.05) reduce cytosolic [Na+] at rigor and at all time points postrigor compared with that in drug-free myocytes (13.3 ± 1.8 vs. 36.0 ± 3.7 mM at 60 min postrigor). Furthermore, cytosolic [Na+] in HOE-642-treated myocytes at 60 min postrigor was not significantly different from the initial cytosolic [Na+] (13.3 ± 1.8 vs. 11.2 ± 0.6 mM). These data show that in the presence of moderate hyperkalemia and acidosis anoxic Na+ influx could only be significantly inhibited by HOE-642 and that this inhibition was largely complete.


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  		Fig. 2.   Na+ influx during simulated ischemia occurs predominantly through a HOE-642-sensitive pathway. Cytosolic [Na+] measurements (n = 6), drug concentrations, and statistics were as described in Fig. 1. Simulated ischemic Tyrode solution had [K+] set to 10 mM and pH to 6.85. * P < 0.05 vs. drug-free myocytes. Further statistical testing showed that cytosolic [Na+] in HOE-642-treated myocytes was never significantly greater than the initial intracellular [Na+].

Hyperkalemic Tyrode solution and anoxic Na+ entry. The experiments in Figs. 3 and 4 were conducted to address which component of the simulated ischemic Tyrode solution was responsible for the altered drug efficacy seen in Fig. 2. Figure 3 shows the effect of hyperkalemia and anoxia on Na+ entry. TTX did not significantly inhibit the rise of cytosolic [Na+] at any time point during anoxia compared with that in drug-free myocytes in hyperkalemic Tyrode solution (31.3 ± 2.5 vs. 36.1 ± 4.4 mM at 60 min postrigor). On the other hand, cytosolic [Na+] in HOE-642-treated myocytes was significantly lower (P < 0.05) at rigor and at all time points postrigor compared with the intracellular [Na+] of drug-free cells (16.2 ± 2.4 vs. 36.1 ± 4.4 mM at 60 min postrigor). Despite an upward trend, cytosolic [Na+] in HOE-642-treated myocytes at 60 min postrigor was not significantly different from the initial cytosolic [Na+] (16.2 ± 2.4 vs. 8.9 ± 0.9 mM). These data suggest that under hyperkalemic conditions only HOE-642 could significantly reduce anoxic Na+ entry.


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  		Fig. 3.   Anoxic Na+ influx occurs through a HOE-642-sensitive pathway in hyperkalemic Tyrode solution. Cytosolic [Na+] measurements (n = 6), drug concentrations, and statistics were as described in Fig. 1. Hyperkalemic Tyrode solution had an extracellular [K+] of 10 mM and a pH of 7.20. * P < 0.05 vs. drug-free myocytes. Only HOE-642-treated cells had a cytosolic [Na+] that never significantly increased during anoxia compared with the initial intracellular [Na+].

Acidic Tyrode solution and anoxic Na+ entry. Figure 4 shows the effect of acidic Tyrode solution and anoxia on Na+ accumulation. In contrast to the results with hyperkalemic and simulated ischemic Tyrode solutions, HOE-642 did not significantly inhibit the rise in anoxic cytosolic [Na+] at any time point during anoxia compared with that in drug-free myocytes in acidic Tyrode solution (32.5 ± 1.6 vs. 36.9 ± 6.7 mM at 60 min postrigor). However, TTX was able to significantly inhibit Na+ entry at all postrigor time points compared with that in drug-free myocytes (12.8 ± 0.9 vs. 36.9 ± 6.7 mM at 60 min postrigor). Furthermore, cytosolic [Na+] in TTX-treated cells at 60 min postrigor was not significantly different from the initial resting cytosolic [Na+] (12.8 ± 0.9 vs. 10.6 ± 1.1 mM). These data suggest that under acidic conditions, and in the absence of elevated extracellular [K+], TTX-sensitive Na+ channels alone are responsible for the significant rise in cytosolic [Na+] during anoxia.


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  		Fig. 4.   Anoxic Na+ influx occurs through a TTX-sensitive pathway in acidic Tyrode solution. Cytosolic [Na+] measurements (n = 6), drug concentrations, and statistics were as described in Fig. 1. Acidic Tyrode solution had an extracellular [K+] of 4 mM and a pH of 6.85. * P < 0.05 vs. drug-free myocytes. Only TTX-treated cells had a cytosolic [Na+] at 60 min postrigor that was not significantly increased compared with the initial intracellular [Na+].

Anoxic Na+ entry before rigor contracture. Inspection of Figs. 1-4 reveals that the pharmacological sensitivity of anoxic Na+ entry under each of our specific experimental conditions is very similar at almost all durations of anoxia. However, cytosolic [Na+] at the onset of rigor is the first anoxia data shown in each of Figs. 1-4. This was done because the time between the onset of anoxia and the development of rigor, as well as the time course of the prerigor rise in intracellular [Na+], was quite variable, as described in MATERIALS AND METHODS. We did attempt to analyze cytosolic [Na+] at even earlier time points, specifically at 10 and 15 min postanoxia, to determine whether the pharmacological sensitivity of the initial rise in cytosolic [Na+] was similar to that seen postrigor. However, the pharmacological sensitivity of anoxic Na+ entry could only be evaluated at 15 min postanoxia, because the rise in cytosolic [Na+] at 10 min postanoxia was too small to allow for statistical differences to be established among groups.

The pharmacological sensitivity of Na+ entry at 15 min postanoxia under each experimental condition was very similar to that previously described at 60 min postrigor. TTX (13.8 ± 1.6 mM), HOE-642 (13.2 ± 1.2 mM), and TTX plus HOE-642 (9.9 ± 1.3 mM) each significantly decreased cytosolic [Na+] in normal Tyrode solution compared with that in drug-free myocytes (22.3 ± 3.1 mM). However, only HOE-642 significantly decreased intracellular [Na+] compared with that in drug-free myocytes in simulated ischemic Tyrode solution (12.6 ± 1.9 vs. 20.2 ± 1.8 mM) or in hyperkalemic Tyrode solution (11.0 ± 1.7 vs. 22.7 ± 2.5 mM). As expected, only TTX significantly decreased cytosolic [Na+] at 15 min postanoxia in acidic Tyrode solution (12.5 ± 0.7 vs. 17.4 ± 1.2 mM). Therefore, it appears that the pharmacological sensitivity of anoxic Na+ entry does not vary much over a wide range of anoxic time intervals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Routes of anoxic Na+ entry vary depending on ionic conditions. This study was designed to define the routes of excess Na+ entry in ventricular myocytes during anoxia and simulated ischemia. Our data suggest that both the Na+ channel and NHE are available routes for Na+ entry in this single-cell experimental model. Anoxic Na+ entry through the NHE could be enhanced because of the profound and rapid cytosolic acidification that occurs in this experimental model (21). Na+-channel opening has been reported to be enhanced by hypoxia (10) and by lipid metabolites thought to be produced during hypoxia (27, 30, 34), but not by metabolic inhibition (13). It is also possible that moderate depolarization, occurring as a result of decreased Na+-K+-pump activity during anoxia, might enhance Na+ channel activity.

Whereas the exact mechanism of enhanced Na+-channel function is uncertain, the main point of our experiments is that, together, the Na+ channel and the NHE can account for essentially all of the excess Na+ that enters anoxic guinea pig ventricular myocytes. Because we have explored the effect of different experimental variables known to occur during true ischemia, it should be possible to extrapolate some of our results to true ischemia. The main qualification that we would like to point out, in regard to these conclusions, is that our experimental design depends on the specificity of TTX and HOE-642. At present these drugs are regarded as the most selective drugs available for inhibiting the Na+ channel and the NHE1 isoform (16, 24), which is the NHE isoform predominantly expressed in cardiac tissue (17).

We measured the effect of TTX and HOE-642 on the anoxic rise of cytosolic [Na+] under four general conditions: normal, acidic, hyperkalemic, and simulated ischemic (hyperkalemic and acidic) Tyrode solution. In the absence of drugs, cytosolic [Na+] increased during anoxia in a similar fashion in all four conditions. However, these conditions did produce pronounced changes in the sensitivity of anoxic Na+ entry to either TTX or HOE-642. In acidic Tyrode solution (Fig. 4), HOE-642 could not significantly reduce the rise in anoxic intracellular [Na+], whereas in TTX-treated myocytes, cytosolic [Na+] did not significantly increase during anoxia. In contrast, in both simulated ischemic (Fig. 2) and hyperkalemic (Fig. 3) Tyrode solutions, anoxic Na+ entry was not significantly inhibited by the addition of TTX. However, cytosolic [Na+] did not significantly increase in either condition during anoxia in the presence of HOE-642. Our interpretation of these results is that extracellular acidification leads to an inhibition of Na+ influx through the NHE, whereas both extracellular hyperkalemia and the combination of extracellular acidification and hyperkalemia (simulated ischemia) lead to an inhibition of Na+ entry through TTX-sensitive Na+ channels. However, the underlying mechanism(s) for the altered drug sensitivity of Na+ entry under acidic and/or hyperkalemic conditions was not explored in this study.

Comparison of the current results with previous studies in isolated myocytes. The current experiments provide a valuable, quantitative expansion of previous work focused on Na+ influx in models of hypoxia or metabolic poisoning in cardiac myocytes. R-56865, which can block Na+ channels, decreased the rise in cytosolic [Na+] in hypoxic myocytes (6, 7) and was also found to reduce cytosolic [Na+] in metabolically poisoned rat ventricular myocytes (22). TTX, the most selective Na+-channel blocker, also blocked the secondary elevation of cytosolic [Ca2+] in hypoxic myocytes (7), consistent with an effect of reducing cytosolic [Na+]. In contrast, TTX was reported to be ineffective in metabolically poisoned guinea pig ventricular myocytes (23). Finally, HOE-642 did not prevent a rise in cytosolic [Na+] in metabolically poisoned rat myocytes (22), although hexamethylene amiloride, another NHE inhibitor, did suppress cytosolic [Na+] accumulation in metabolically poisoned guinea pig myocytes (23).

Comparison of the current results with previous studies in isolated hearts. Pretreatment with TTX has been reported to protect ischemic rat hearts from injury (4), whereas lidocaine, a less selective Na+-channel blocker, has been reported to decrease cytosolic [Na+] in ischemic rat hearts (31). Derivatives of amiloride, known to be somewhat selective NHE inhibitors, have been reported to decrease the rise of intracellular [Na+] during myocardial ischemia in some studies (2, 9, 12, 14, 19) but not in others (8, 32).

Reconciliation of the current results with previous studies. Our study has expanded on this body of work by achieving a quantitative appraisal of the routes for Na+ entry in our experimental model, at least at the resolution that our methodology and pharmacological tools allow. Our results demonstrate that even moderate changes in extracellular pH or [K+] can lead to significant alterations in anoxic Na+ entry and also point out that quantitative, or even qualitative, differences in Na+ entry are to be expected in different experimental models. This latter observation could help to explain conflicting reports of Na+-channel and/or NHE involvement in mediating Na+ accumulation in various models of ischemia, hypoxia, or metabolic poisoning.

In conclusion, both the NHE and the Na+ channel can be sources of excess Na+ entry during hypoxia or simulated ischemia, but their relative importance will vary according to the specific experimental model. The NHE might be expected to be the dominant Na+ source in true ischemia, depending on the degree of hyperkalemia that exists. That could be fortunate, because it would provide an additional beneficial mechanism for the demonstrated cardioprotective effects of NHE1 inhibitors such as HOE-642 or HOE-694 (25, 26), in addition to other mechanisms, such as a beneficial effect on cytosolic pH during reperfusion (24).


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grant HL-56910 (to R. W. Hadley). We thank Hoechst Marion Roussel and especially Wolfgang Scholz and Andreas Weichert for the generous supply of HOE-642.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. W. Hadley, Dept. of Pharmacology, Univ. of Kentucky, College of Medicine, MS-371 UKMC, Lexington, KY 40536-0084 (E-mail: rhadley{at}pop.uky.edu).

Received 17 November 1998; accepted in final form 10 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(5):H1817-H1822
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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