Departments of 1 Surgery, 2 Anesthesiology, and 3 Human Physiology, University of California, Davis, California 95616-8644
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Much evidence supports the view that hypoxic/ischemic injury is largely due to increased intracellular Ca concentration ([Ca]i) resulting from 1) decreased intracellular pH (pHi), 2) stimulated Na/H exchange that increases Na uptake and thus intracellular Na (Nai), and 3) decreased Na gradient that decreases or reverses net Ca transport via Na/Ca exchange. The Na/H exchanger (NHE) is also stimulated by hypertonic solutions; however, hypertonic media may inhibit NHE's response to changes in pHi (Cala PM and Maldonado HM. J Gen Physiol 103: 1035-1054, 1994). Thus we tested the hypothesis that hypertonic perfusion attenuates acid-induced increases in Nai in myocardium and, thereby, decreases Cai accumulation during hypoxia. Rabbit hearts were Langendorff perfused with HEPES-buffered Krebs-Henseleit solution equilibrated with 100% O2 or 100% N2. Hypertonic perfusion began 5 min before hypoxia or normoxic acidification (NH4Cl washout). Nai, [Ca]i, pHi, and high-energy phosphates were measured by NMR. Control solutions were 295 mosM, and hypertonic solutions were adjusted to 305, 325, or 345 mosM by addition of NaCl or sucrose. During 60 min of hypoxia (295 mosM), Nai rose from 22 ± 1 to 100 ± 10 meq/kg dry wt while [Ca]i rose from 347 ± 11 to 1,306 ± 89 nM. During hypertonic hypoxic perfusion (325 mosM), increases in Nai and [Ca]i were reduced by 65 and 60%, respectively (P < 0.05). Hypertonic perfusion also diminished Na uptake after normoxic acidification by 87% (P < 0.05). The data are consistent with the hypothesis that mild hypertonic perfusion diminishes acid-induced Na accumulation and, thereby, decreases Na/Ca exchange-mediated Cai accumulation during hypoxia.
intracellular pH; myocardial hypoxia; nuclear magnetic resonance spectroscopy
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ION HOMEOSTASIS IS TIGHTLY controlled in most cells and tissues under normal conditions, whereas it has been demonstrated that ion homeostasis is disturbed in a wide variety of pathological conditions, including hypoxia and ischemia. The traditional view of hypoxia/ischemia-induced disruption in cellular ion homeostasis has been based on the notion that ATP depletion leads to impaired function of ATP-dependent transport systems, resulting in dissipation of ion gradients, e.g., those dependent on Na-K-ATPase (39, 45). Recent studies provide evidence that disturbances in ion homeostasis may be among the earliest alterations in cell function and may ultimately lead to cell injury and death (44). In particular, during myocardial hypoxia and ischemia, disturbances in intracellular pH (pHi), Na (Nai), and Ca (Cai) occur before ATP is depleted (1, 5). The discovery of robust intracellular proton-induced Na/H exchange in myocardium (27, 38), combined with earlier evidence for Na-dependent Ca transport (26), led to the development and testing of a unifying hypothesis linking changes in pHi, Nai, and Cai during hypoxia and ischemia (5, 10, 44, 47). Thus we and others have presented data consistent with the general hypothesis that, in the heart, hypoxia/ischemia results in the following chain of events: 1) anaerobic metabolism increases proton production and decreases pHi; 2) decreased pHi stimulates Na/H exchange, which increases Na uptake; 3) increased Na uptake increases Nai concentration ([Na]i); 4) increased [Na]i initially decreases and may ultimately reverse the force that normally drives net Ca efflux via Na/Ca exchange; and 5) decreased efflux or increased influx of Ca via Na/Ca exchange results in increased Cai concentration ([Ca]i). Although recent studies suggest that other Na transport pathways may also contribute to Nai accumulation after acidification and during hypoxia/ischemia (4, 14, 42), the relationship between Nai and Cai accumulation is well described by the hypothesis regardless of the Na uptake pathway. Furthermore, most evidence is consistent with the postulate that intracellular acidification stimulates Na/H exchange and that a major portion of acidification- and/or hypoxia/ischemia-induced Na uptake is via Na/H exchange (4, 5, 14, 29, 32, 37, 41, 48).
On the other hand, in numerous cell types, including cardiac myocytes, the Na/H exchanger is also stimulated by exposure to hypertonic solutions (12, 54). Yet operation in one mode (volume or pH regulation) may preclude functional response to a second stimulus. Briefly, previous studies from this laboratory have demonstrated that when Amphiuma red blood cells (RBCs) are shrunk by exposure to hypertonic solutions, the Na/H exchanger is stimulated to cause net uptake of Na, along with osmotically obligated water, until normal cell volume is restored (8). In these same cells, when pHi is decreased, the Na/H exchanger is stimulated to cause net Na uptake and proton efflux until normal pHi is restored (12). However, these two modes of stimulating the Na/H exchanger, either exposure to increased osmolarity or decreased pHi, appear to result in mutually exclusive responses (10-12). That is, after Amphiuma RBCs or cardiac myocytes are exposed to hypertonic media, the Na/H exchanger is capable of increasing pHi to a level higher than would occur in response to decreased pHi (12, 54); i.e., pH regulation is disrupted. Conversely, in Amphiuma RBCs, it has been demonstrated that when the Na/H exchanger is stimulated by lowering pHi, it is capable of increasing cell volume far above the level that would normally occur in response to exposure to hypertonic media (12); i.e., volume regulation is disrupted. Thus, with respect to exposure to hypertonic solution and decreased pHi, data from Amphiuma RBCs and cardiac myocytes suggest that the Na/H exchanger responds to the stimulus it receives first and "ignores" the other stimulus if it occurs later.
We therefore conducted the studies described here to further support the general hypothesis described above and to test the corollary or subhypothesis that modest increases in perfusate tonicity will limit Na uptake via the Na/H exchanger in response to decreased myocardial pHi. The results of the experiments reported here demonstrate that moderately hypertonic perfusion decreases Na accumulation after normoxic acidification and during hypoxia, and this decrease in Na accumulation is associated with a smaller increase in [Ca]i than observed during hypoxia under isotonic conditions.
The impetus for this work arose in part from our awareness of, and involvement with, previous studies in which it was shown that infusion of hypertonic solution has beneficial effects in shock resuscitation (24). Not all these effects can be explained on the basis of fluid shifts alone. The results described here provide a new and rational explanation, based on alterations in ion transport, for a portion of the benefits of hypertonic resuscitation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General
The methods have been modified slightly from those previously reported (5). New Zealand White rabbits were anesthetized with pentobarbital sodium (65 mg/kg) and heparinized (1,000 U/kg). Hearts were removed and perfused at a constant rate of 27-29 ml/min at 22-25°C. Control perfusate contained (mmol/l) 133 NaCl, 4.75 KCl, 1.25 MgCl2, 1.82 CaCl2, 20 HEPES, 8 NaOH, and 11.1 dextrose. Perfusate osmolarity was measured ±2 mosM by a freezing-point osmometer; control was 295 mosM. Perfusates were titrated to pH 7.35-7.45 and equilibrated with 100% O2 before and after hypoxia and with 100% N2 during hypoxia. Under these conditions, the partial pressure of O2 measured at the aorta was >550 Torr during normoxic perfusion and <20 Torr during hypoxic perfusion. To measure Nai, 15 mM dysprosium triethylenetetramine hexaacetic acid (DyTTHA) was substituted for osmotic equivalents of NaCl in the perfusate, and Ca was added to reach a concentration of 1.8-2 mM as measured by Ca electrode (3). To measure [Ca]i, hearts were loaded during the control interval (30-40 min) with perfusate containing 5 µM 5F-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA)-AM (5, 22, 30). FBAPTA was then washed out of the extracellular space with control solution for 15 min before measurement of [Ca]i. To discriminate between changes in Na uptake and Na efflux, Na efflux via Na-K-ATPase was inhibited by addition of 1 mM ouabain to, or removal of KCl from, the perfusate (43, 46) (osmotic substitution with sucrose) during the 60-min hypoxic interval in all hypoxic experiments except control. This allowed us to equate increases in Nai with net Na uptake (4, 5), as opposed to other studies that report changes in Nai without assessing Na uptake and Na efflux independently (17, 34, 37, 47, 53).Normoxic acidification was achieved using the NH4Cl
prepulse technique (7), in which 20 mM NH4Cl is added to
the perfusate for 45 min and then washed out of the heart (5). After
all perfusions were complete, hearts were weighed wet and dried for 48 h at 65°C to determine dry weight.
NMR Spectroscopy
Experiments were conducted using a GE Omega 300 horizontal bore system. 23Na, 19F, and 31P spectra were generated from the summed free induction decays of 1,000, 1,500, and 148 excitation pulses (90°, 45°, and 60°) with 2K, 2K, and 4K word data files and ±4,000-, ±5,000-, and ±4,000-Hz sweep widths, respectively. For all nuclei, data files were collected over 5-min intervals, and technical limitations required that data from only one nuclear resonance frequency were acquired from each heart. To improve signal-to-noise ratio for 19F measurement of [Ca]i, the free induction decays from two contiguous 5-min 19F files were added together. Because the NMR signal intensity reflects the time average for the interval over which data are collected, data are represented in time as corresponding to the midpoint of the appropriate 5- or 10-min acquisition interval.Nai (in meq/kg dry wt) was calculated from the calibrated area under the unshifted peak of the 23Na spectrum after subtraction of the extracellular peak (3, 29). [Ca]i (in nmol/l cell water) was calculated as the product of the 500 nM Ca-FBAPTA dissociation constant and the ratio of the areas of the Ca-bound and Ca-free peaks in the FBAPTA spectrum (5, 21). Because the NMR techniques measure Na as an amount and Ca as a concentration, results are reported as such. The pHi was determined from the chemical shift of the Pi resonance [with reference to control phosphocreatine (PCr)] calibrated at 25°C (5).
Unless otherwise stated, values are means ± SE, and ANOVA for repeated measures was used to test for differences among treatments. When differences among treatments were found, Tukey's test for multiple comparisons was used to determine the times at which differences between treatments occurred. Tukey's test was not used for comparisons across time, only across treatments for a particular time. For all comparisons, differences were considered significant when P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na Uptake vs. Na Efflux During Hypoxia
To test the hypothesis that hypoxia stimulates Na uptake (and thus increases Nai), hearts were exposed to hypoxia with and without the Na-K-ATPase inhibited by K-free perfusion. As shown in Fig. 1A, when the Na-K pump was allowed to function (normal K, open squares), Nai increased from 22 ± 1 to 32 ± 4 meq/kg dry wt during 60 min of hypoxia. In contrast, when Na efflux via Na-K-ATPase was inhibited by K-free perfusion, the increase in Nai was more than sevenfold greater (from 22 ± 1 to 100 ± 10 meq/kg dry wt) during hypoxia (P < 0.05, open vs. filled squares). Furthermore, when these data are taken together with those previously published (5), they demonstrate that although Na uptake and Na efflux via Na-K-ATPase are increased during hypoxia, uptake is increased more than efflux. In this case, the difference between the normal-K data (open squares) and the K-free data (filled squares) in Fig. 1A represents Na efflux via Na-K-ATPase. For data collected during the first 35 min of hypoxia, Nai increases almost linearly at a rate of ~1.65 meq · min
|
Figure 1A also shows the result of two experiments in which 100 µM ethylisopropylamiloride (EIPA; filled triangles) was added to the K-free perfusate 5 min before and during hypoxia. The results are consistent with previous reports that EIPA inhibits Na uptake during hypoxia/ischemia (5, 29).
Na Dependence of Cai Accumulation
Figure 1B shows how [Ca]i changes during hypoxia with and without Na-K-ATPase inhibition by K-free perfusion. During 60 min of hypoxia when the Na-K pump was allowed to function under normal-K conditions, [Ca]i rose from 407 ± 10 to 676 ± 55 nM; yet when the Na-K pump was inhibited by K-free hypoxic perfusion, [Ca]i rose from 347 ± 11 to 1,306 ± 89 nM. Thus, after 20 min of hypoxia and during reoxygenation, [Ca]i was significantly greater during the K-free (filled squares) than during the normal-K (open squares) protocol.Figure 1B also shows the effect of 100 µM EIPA on Cai accumulation during hypoxia. As shown previously (5), EIPA inhibits Na uptake and, as predicted, Cai accumulation (P < 0.05). Figure 1 supports the hypothesis that increases in [Ca]i during hypoxia are dependent on increases in Nai (presumably mediated by Na/Ca exchange).
Hypertonic Perfusion Decreases Nai and Cai Accumulation During K-Free Hypoxic Perfusion
Again, previous studies by this laboratory and others have demonstrated that the Na/H exchanger's response to changes in pHi is modified by exposure to hypertonic solutions (10-12, 54). To examine parallel behavior in the hypoxic heart, we added 10, 30, or 50 mmol/l of sucrose to the perfusate 5 min before hypoxic perfusion and throughout the remainder of the experiment. Figure 2 illustrates that hypoxia-induced changes in Nai are altered in a time- and osmolarity-dependent fashion by the imposed increases in perfusate osmolarity. That is, during the interval between 10 and 15 min of hypoxic perfusion, Nai was less for the 10 and 30 mosM hypertonic solutions than for the isotonic solution (P < 0.05). However, only the 30 mosM hypertonic solution limits Nai accumulation for the entire 60-min hypoxic interval (P < 0.05; see Fig. 3 for significant differences between isotonic and 30 mosM hypertonic experiments and see DISCUSSION for an explanation of the differences between responses to the 3 different tonicities).
|
|
Figure 3 provides further evidence that, during hypoxia, 1)
increases in [Ca]i are Na dependent and
2) increases in Nai and [Ca]i are diminished by hypertonic perfusion.
In particular, Fig. 3A demonstrates that perfusion with
solutions made hyperosmotic (at 5 min) by addition of 30 mosM
NaCl (filled triangles) or sucrose (open triangles) limits
Nai accumulation during hypoxia (P < 0.05), and
there is no measurable difference between the two solutes. Thus the
response is dependent on tonicity, not solute type or ionic strength.
Figure 3B shows the changes in [Ca]i
that accompany the changes in Nai shown in Fig. 3A.
Compared with the isotonic control (filled squares),
[Ca]i is significantly decreased when perfusate
osmolarity is increased by 30 mosM (triangles) 5 min before K-free
hypoxia (P < 0.05). This is not only true for all data
collected during hypoxia after 20 min but also for data acquired during
the indicated intervals of recovery with normoxic, normal-K perfusion.
Again the results are consistent with the hypothesis that exposure to
mild hypertonic perfusion before and during hypoxia decreases
Nai accumulation and, therefore, limits increases in
[Ca]i mediated by Na/Ca exchange.
In addition, Fig. 3 includes data from experiments that demonstrate that addition of 30 mosM sucrose after 30 min of hypoxia (open squares) has no measurable effect on Nai and Cai accumulation. These data provide further support for the hypothesis that responses of the Na uptake pathway (presumably the Na/H exchanger) to volume or pH stimuli are mutually exclusive and that Cai accumulation is Na dependent.
Hypertonic Perfusion Decreases Na Accumulation After K-Free Normoxic Acidification
We have hypothesized that the sequence of events leading to increased Nai and [Ca]i during hypoxia is the result of increased intracellular H concentration ([H]i) stimulating pH-regulatory Na/H exchange and not a response to hypoxia per se (5). Circumstantial evidence supporting this view is given by our previous demonstration that, after normoxic acidification, pH recovery is associated with increased Na uptake as well as increased [Ca]i and that both are similar in magnitude and sensitivity to EIPA to those observed after exposure to hypoxia (5). If our corollary hypothesis that increased osmolarity inhibits acid-induced Na/H exchange is correct, hypertonic perfusion before and during normoxic acidification should similarly inhibit Na uptake. The results shown in Fig. 4 are consistent with this hypothesis and demonstrate that increasing osmolarity, by addition of 30 mosM sucrose (open triangles) 5 min before and during normoxic acidification (NH4Cl washout), decreases acid-induced Na uptake compared with isotonic perfusion (filled squares; P < 0.05).
|
Effect of Hypertonic Perfusion on Coronary Resistance During Hypoxia
Pharmacological inhibition of Nai accumulation has previously been shown to diminish increases in coronary resistance during hypoxia (5). Our hypothesis that hypertonic perfusion limits pH-regulatory Na/H exchange led us to predict that hypertonic perfusion would similarly decrease coronary resistance during hypoxia. Figure 5 shows mean perfusion pressure (as percentage of baseline) plotted vs. time before and during K-free hypoxia with and without hypertonic perfusion. As discussed above, the responses to addition of 30 mosM NaCl (filled triangles) or sucrose (open triangles) are not measurably different in terms of Na uptake. Figure 5 shows that both treatments significantly diminished the increase in perfusion pressure otherwise observed during hypoxia (filled squares; P < 0.05). Thus, under the constant-flow conditions used in this study, the results demonstrate that hypertonic perfusion decreases coronary resistance during hypoxia. Given that increased coronary resistance is strongly associated with cell damage (18, 23), these data provide evidence that hypertonic perfusion limits hypoxia-induced myocardial injury.
|
Effect of Hypertonic Perfusion on Hypoxic pHi and Energy Stores
We previously demonstrated that amiloride inhibition of Na uptake during hypoxia diminished the depletion of high-energy phosphate in the form of PCr (5). Presumably, limiting Na uptake diminishes ATP consumption by Na-K-ATPase. To test the hypothesis that hypertonic inhibition of pH-regulatory Na/H exchange would have similar effects, we repeated the hypertonic experiments while measuring pHi and high-energy phosphates. Figure 6 shows that increasing perfusate osmolarity by addition of 30 mosM NaCl decreased the fall in pHi otherwise observed during hypoxia (P < 0.05). During hypertonic perfusion (filled triangles), pHi was significantly greater than during isotonic perfusion (filled squares) 7.5, 12.5, 17.5, and 27.5 min after beginning hypoxia. On the other hand, hypertonic inhibition of Na uptake during hypoxia had no measurable effect on ATP or PCr (data not shown). Possible explanations for the relative increase in pHi and lack of effect on high-energy phosphates are included in the DISCUSSION.
|
Hypertonic Perfusion Decreases Nai Accumulation During Normal-K Hypoxic Perfusion
Most of the experiments described above were conducted using K-free perfusate to inhibit Na efflux via the Na-K-ATPase to assess the effect of hypertonic perfusion on Na uptake independent of efflux via the Na pump. However, it is now apparent that an Na-K-2Cl cotransporter is present in myocardium and may be active under hypoxic/acidotic conditions (4). If the Na-K-2Cl cotransporter were active, under K-free conditions it would mediate net Na, Cl, and K efflux from the cells and could contribute to the relative decrease in Na accumulation observed in response to hypertonic perfusion. To test for this effect, we therefore repeated some of the experiments summarized in Fig. 2 but substituted 4.75 mM K + 1 mM ouabain for K-free perfusion to inhibit the Na-K pump without initially causing the driving force for the Na-K-2Cl cotransporter to be directed out of the cell. The results of these experiments are shown in Fig. 7 and demonstrate that normal-K hypertonic perfusion similarly decreases Na accumulation (P < 0.05) during hypoxia. That is, with normal-K + 1 mM ouabain perfusate, mean Nai rises from 22 ± 1 to 80 ± 4 meq/kg dry wt in isotonic experiments but only from 23 ± 2 to 64 ± 3 meq/kg dry wt after addition of 30 mosM sucrose to the hypoxic perfusate. In this protocol, hypertonic perfusion significantly limited Nai accumulation early during hypoxia (P < 0.05 at 7.5 and 17.5 min), whereas the force driving the Na-K-2Cl cotransporter is predicted to be directed into the cell (4). Thus the result is consistent with the hypothesis that hypertonic perfusion is limiting Na uptake (presumably in large part via Na/H exchange) and not stimulating Na efflux.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypoxia Stimulates Na Uptake More Than Na Efflux
It has been previously demonstrated that increases in cardiac myocyte [Na]i are stimulated by hypoxia (5, 10, 39), ischemia (4, 35, 37, 47), and normoxic acidification (5, 38, 54). The consistency of this response, as well as its sensitivity to Na/H exchange inhibitors, has led many investigators to conclude that these increases in [Na]i are mediated in large part by Na/H exchange functioning in a pH-regulatory capacity (4, 5, 27, 37, 38, 47). However, although increases in [Na]i in response to hypoxia, ischemia, and normoxic acidification have been demonstrated, it remains controversial (particularly in the cases of hypoxia and ischemia) to what extent the increases in [Na]i are due to increases in Na uptake as opposed to decreases in Na efflux.With regard to the latter, increases in [Na]i under hypoxic/ischemic conditions have historically been attributed to decreased ATP production leading to a decrease in Na-K-ATPase activity and, therefore, a decrease in Na efflux (39, 45). However, in combination with our previous work (5), the data presented here (Fig. 1A) and data of others support the hypothesis that Na efflux via Na-K-ATPase is actually increased early during hypoxia (15). We recently reported a similar response to increased Na uptake during ischemia (4), and others have reported increased Na pump activity in heart cells after normoxic acidification (38) or exposure to hypotonic media (55).
It is important to reiterate the assertion that increases in [Na]i during hypoxia are a direct consequence of an increase in Na uptake. That is, changes in Nai may be due, in general, to changes in Na uptake and/or efflux, and increases in Nai only reflect increased uptake to the extent that efflux via the Na-K pump is unchanged. Having conducted our studies under conditions where Na efflux via the Na-K pump is near zero (K-free or plus 1 mM ouabain perfusion), we have met this criterion. Similarly, to evaluate the effects of inhibiting the hypothesized Na uptake pathway (with EIPA or hypertonic perfusion), Na efflux must be inhibited or quantified. Unlike previous reports in which Na efflux was not assessed (14, 47, 48, 53), the studies reported here have allowed us to determine how each of the protocols alters Na uptake as well as the way in which [Ca]i responds to these changes.
Hypertonic Perfusion Inhibits Na Accumulation During Hypoxia
We and others have presented evidence that the increase in myocardial [Na]i during hypoxia and after normoxic acidification is largely due to increased Na/H exchange stimulated by decreased pHi (5, 38, 41, 54). This laboratory has further presented evidence that in Amphiuma RBCs the Na/H exchanger can also be stimulated by decreases in cell volume. Recent studies in cardiac myocytes conclude that this is also true for heart muscle (54). At least in Amphiuma cells, however, the data suggest that the responses of the Na/H exchanger to decreased cell volume and pH may be mutually exclusive (12).More specifically, when Amphiuma RBCs are shrunk in hypertonic media, Na/H exchange is stimulated to take up Na and osmotically obliged water to restore cell volume (8). However, as a consequence of obligated proton efflux, pHi increases to levels much higher than those required to inactivate Na/H exchange stimulated by decreased pHi. In contrast, when Na/H exchange is stimulated by acidification, Na and water uptake and proton efflux continue until normal pHi is restored; yet, as a consequence, the cells swell to volumes much higher than those required to inactivate Na/H exchange stimulated by cell shrinkage. Briefly, the volume and pH dependence of the Na/H exchanger in Amphiuma RBCs are somehow prioritized and mutually exclusive (10-12), such that the pathway responds to the stimulus (volume or pH) presented first and is unresponsive to the other stimulus (volume or pH) if it is presented subsequently.
These results led us to question whether cardiac myocytes might respond similarly. That is, if hearts were first exposed to hypertonic perfusion, would the cardiac myocyte Na/H exchanger lose its normal response to decreases in pHi that occur during hypoxia and after normoxic acidification? Figure 2 shows that solutions made hypertonic by addition of 10 or 30 mosM sucrose initially demonstrate decreased Na uptake during hypoxia, but only the 30 mosM hypertonic solution decreases Nai accumulation after 20 min of hypoxia (Fig. 3). Possible reasons for the latter are discussed in Perspectives. Nevertheless, the data in Fig. 2 illustrate a response similar to that of pharmacological Na/H exchange inhibition (5, 38, 41, 54) and are thus consistent with the hypothesis that hypertonic perfusion also inhibits hypoxia-induced increases in Na/H exchange.
Role of Na-K-2Cl Cotransport in the Hypertonic Effect
As noted above, most of the data presented here were acquired using the K-free perfusion protocol to inhibit Na-K-ATPase (5) to determine the effects of hypertonic perfusion on Na accumulation in the absence of Na efflux via the Na pump. One potential drawback in using this protocol is that K-free perfusion will cause the force driving the Na-K-2Cl cotransporter to be directed out of the cell (4). To the extent that it is active, the Na-K-2Cl cotransporter will then act as an Na efflux pathway, and Na accumulation will underestimate Na uptake. To assess the importance of this effect, we repeated experiments shown in Fig. 2 but used normal-K perfusate (4.75 mM) + 1 mM ouabain instead of K-free perfusate to inhibit the Na-K-ATPase. The results demonstrate that, with normal-K perfusate, hypertonic perfusion limits Nai accumulation during hypoxia (Fig. 7). These results are of further interest, because they show that hypertonic perfusion even limits Nai accumulation early during hypoxia (P < 0.05 at 7.5 and 17.5 min) while the force driving the Na-K-2Cl cotransporter remains directed into the cell. That is, with the assumption that decreases in intracellular K concentration approximate increases in [Na]i, as previously measured (5), and intracellular Cl concentration increases during hypoxia, as previously measured during ischemia (4, 25, 40), one can calculate the force driving the cotransporter (4). For the conditions of these experiments, the force driving the Na-K-2Cl cotransporter will not be directed out of the cell until Nai rises above 50 meq/kg dry wt (after 20 min). Thus not only does mild hypertonic perfusion limit Nai accumulation during hypoxia under K-free (Fig. 2) and normal-K (Fig. 7) conditions, but if the calculated driving force is correct, for normal-K conditions the significant hypertonic effect observed before 20 min could not be mediated by net efflux via the Na-K-2Cl cotransporter (i.e., the driving force is in the wrong direction). Again, this result is consistent with the hypothesis that, compared with isotonic perfusion, hypertonic perfusion diminishes Na uptake during hypoxia.Hypertonic Perfusion Decreases Ca Accumulation During Hypoxia
The results shown in Fig. 3B provide evidence that hypertonic perfusion before hypoxia decreases Ca accumulation during and after hypoxia. As shown for Nai, increasing the perfusate osmolarity by 30 mosM 5 min before hypoxia decreases Cai accumulation regardless of whether the added solute is sucrose or NaCl. Thus the data in Fig. 3 lend further support to the hypothesis that hypertonic perfusion (introduced before hypoxia) decreases Na uptake, which, through its effect on the force driving Na/Ca exchange, decreases Cai accumulation. The hypothesis that the responses of the Na/H exchanger to volume and pH disturbances are mutually exclusive further predicts that hypertonic perfusion will have no effect on hypoxia-induced Na uptake and Cai accumulation if exposure to hypertonic media occurs after exposure to hypoxia. The results shown in Fig. 3 are consistent with this prediction, because when hearts are exposed to hypertonic perfusate after 30 min of hypoxic perfusion (30 mosM sucrose added), Nai and [Ca]i are greater than when exposed to hypertonic perfusate before hypoxia (P < 0.05) and not significantly different from isotonic hypoxic perfusion.Hypertonic Perfusion, pHi, High-Energy Phosphates, and Coronary Resistance
With respect to the effects of hypertonic perfusion on pHi, we and others previously reported that inhibition of the Na/H exchanger by amiloride analogs decreases the ability of the heart to regulate pH after normoxic acidification (5, 14, 49), during hypoxia (28), and after ischemia (48). On this basis, one might predict that inhibition of pH-regulatory Na/H exchange by hypertonic perfusion would cause a greater decrease in pHi during hypoxia. Instead, hypertonic perfusion diminishes the fall in pHi otherwise observed during isotonic hypoxic perfusion (cf. filled triangles and squares in Fig. 6). Although this response requires further study, we suggest that, in part, it is due to the effect of hypertonic perfusion on Cai accumulation during hypoxia (cf. filled triangles and squares in Fig. 3B). The data in Figs. 6 and 3B demonstrate clearly that hypoxia induces increases in [Ca]i and [H]i (decreased pHi) and that the +30 mosM hypertonic treatment decreases this response in [Ca]i and [H]i. At least two scenarios consistent with this response have been reported. First, during hypoxia the Na-dependent increase in [Ca]i will tend to increase ATP consumption by such Ca-dependent processes as muscle contraction (51) and thus increase proton production (13). Second, increased [Ca]i will tend to increase [H]i by displacing protons from intracellular buffers (52). Thus, by inhibiting Na uptake and, therefore, Cai accumulation during hypoxia, hypertonic perfusion is likely to diminish proton production and release and, thereby, diminish the fall in pHi.Recent studies consistent with this interpretation demonstrate that inhibition of Na/H exchange does not decrease and may increase pHi during ischemia (29, 37), whereas it consistently preserves or improves the recovery of the cell's high-energy phosphate stores during and after hypoxia/ischemia (5, 37) and improves recovery of function after ischemia (29, 32, 33, 47). In contrast, we find that inhibition of Na uptake by hypertonic perfusion during hypoxia has no measurable effect on high-energy phosphates (PCr and ATP; data not shown). Perhaps the simplest explanation for this difference is that amiloride analogs are more effective than hypertonic perfusion in inhibiting Na uptake.
Finally, our hypothesis that hypertonic perfusion inhibits hypoxia-induced Na/H exchange predicts that hypertonic perfusion will have the same effect on vascular resistance during hypoxia as pharmacological Na/H exchange inhibitors. The results of experiments summarized in Fig. 5 are consistent with the prediction as well as previous studies in which it was shown that inhibiting Na/H exchange diminishes hypoxia/ischemia-induced myocardial injury (29, 32, 41, 47, 53).
Limitations of NMR Methods and Pharmacological Probes
The NMR methods used to measure Nai and [Ca]i have been evaluated and used previously by numerous investigators (6, 22, 31, 37, 44). Although their limitations may be significant, they do not preclude the conclusions we have drawn from our experiments. When blood or perfusate [Ca] is maintained within the normal range (3), no significant effects of DyTTHA on myocardial physiology have been reported in vivo or in vitro (4-6, 37). The most serious limitation in using the DyTTHA method for measuring Nai with NMR is its relatively low resolution. Because the differences between treatments reported in this study are large, we were not limited by this concern.Marban et al. (31) critically evaluated the FBAPTA method for measuring [Ca]i in myocardium and concluded that it offers advantages over fluorescent indicators loaded by the AM method, in that compartmentalization into intracellular organelles is negligible, FBAPTA-AM is promptly and completely deesterified, and signals from endothelial cells do not dominate the spectra. They also report that FBAPTA-loaded hearts have normal high-energy phosphates, and their metabolic response to ischemia is similar to hearts without FBAPTA (31). The most serious drawback in using FBAPTA is that it was designed with a dissociation constant in the range of resting [Ca]i and, as a significant Ca buffer, tends to limit changes from that value (22). To the extent that this occurs in our experiments, FBAPTA would limit increases in [Ca]i during hypoxia. However, even if the magnitude of the changes in [Ca]i we report was limited by FBAPTA, this potential artifact would have no effect on the validity of our conclusions, since they are predicated on the observation that in hearts with identical FBAPTA loading (i.e., same baseline FBAPTA buffer capacity), during hypoxia, [Ca]i is significantly less for the hypertonic group than for the isotonic control.
Finally, we have used EIPA as an inhibitor of Na uptake. Although EIPA is a potent inhibitor of Na/H exchange (5, 14, 27, 29, 48), on the basis of the fact that it also inhibits veratridine-induced hypercontracture and Cai loading, Haigney et al. (16) suggested that EIPA inhibits noninactivating Na channels during hypoxia and ischemia. Thus we cannot rule out the possibility that Na uptake during hypoxia is mediated by pathways other than Na/H exchange.
Perspectives
Hypertonic perfusion and Na uptake. PREDICTED RESPONSE. To reiterate, exposure of cardiac myocytes to hypertonic solutions stimulates Na/H exchange (54), and this response has been shown to be volume regulatory in Amphiuma RBCs (8). If cardiac myocytes are perfect osmometers and volume regulation is accomplished by Na/H exchange exclusively, the volume-regulatory Na uptake in milliequivalents per liter cell water will be approximately equal to the imposed increase in perfusate osmolarity. However, to the extent that Na uptake and proton efflux via volume-regulatory Na/H exchange entrain net flux of additional ions through other pathways (e.g., Cl uptake via Cl/HCO3 exchange), the Na uptake required to regulate cell volume will be diminished (9). For example, one-to-one coupling of Na/H exchange with Cl/HCO3 exchange would result in no change in pHi, but for each cycle of the Na/H exchanger the net osmotically active ion uptake would be two ions. That is, Na and Cl would be taken up while H and HCO3 could be recycled as CO2 and water (9). Because it has been reported that intracellular HCO3 concentration is ~0.5 mM in HEPES-buffered sheep Purkinje fibers and the Michaelis-Menten constant for HCO3 to support Cl uptake via Cl/HCO3 exchange is ~1 mM (50), we cannot rule out the possibility that Na/H and Cl/HCO3 exchange are functionally coupled in our studies of the heart. If they are coupled one to one, volume regulation could thus be achieved by increasing [Na]i and intracellular Cl concentration equally, and the sum of the increases in concentrations of these two ions would be approximately equal to the imposed increase in perfusate osmolarity. (Accordingly the increase in [Na]i would be about one-half the imposed increase in perfusate osmolarity.)
Thus, for the studies described here, volume-regulatory responses mediated by functionally coupled Na/H and Cl/HCO3 exchangers would be expected to cause changes in [Na]i between one-half and one times the imposed change in perfusate osmolarity. In response to 10, 30, and 50 mosM increases in perfusate osmolarity, we predict that volume-regulatory Na/H exchange would mediate increases in [Na]i with upper limits of 10, 30, and 50 mM, respectively. With the assumption that control cell water is 2.5 l/kg dry wt (5), Nai would increase by up to 25, 75, and 125 meq/kg dry wt to achieve volume regulation for the three respective hypertonic perfusates. On the other hand, if Na uptake via volume-regulatory Na/H exchange is coupled one to one with Cl uptake via Cl/HCO3 exchange, the corresponding increases in Nai would be decreased by 50%. As described below, data shown in Fig. 2 are qualitatively consistent with the latter prediction. OBSERVED RESPONSE. After perfusate osmolarity is increased by 10 mosM (Fig. 2), Nai increases slowly during the first 30 min of hypoxia and then increases more rapidly. To achieve volume regulation during 10 mosM hypertonic perfusion, an increase in Nai of 12.5-25 meq/kg dry wt is predicted. This is close to the increase of 12 (from 21 to 33) meq/kg dry wt observed in the first 25 min of hypertonic hypoxic perfusion. We postulate that after volume regulation had been completed the Na/H exchanger will begin to function in its pH-regulatory mode. Indeed, Fig. 2 shows that, after 25 min of hypoxic perfusion with 10 mosM hypertonic solution, Na uptake began to proceed at a more rapid rate, similar to that observed during isotonic hypoxic perfusion (Fig. 2). After perfusate osmolarity is increased by 30 mosM [sucrose (open triangles) and NaCl (filled triangles) in Fig. 3A], Nai increases by 23-27 meq/kg dry wt during 60 min of hypoxic perfusion. Because an increase in Nai of 37.5-75 meq/kg dry wt was predicted to achieve volume regulation under these conditions, we postulate that, for this protocol, volume regulation is not achieved within 60 min. Therefore, the Na/H exchanger remains in its volume-regulatory mode and does not respond to the decrease in pHi that occurs during hypoxic perfusion. This hypothesis is further supported by the fact that addition of the Na/H exchange inhibitor EIPA (50-100 µM) to the 30 mM hypertonic solution decreases Na uptake by 44% compared with the same treatment without EIPA (Nai rises from 21 to 30 meq/kg dry wt with hypertonic EIPA; ANOVA for two treatments: +15 mM NaCl ± EIPA, P = 0.0365; data not shown). That is, if Nai uptake during hypertonic perfusion is mediated by Na/H exchange, it should be, and is, inhibited by EIPA. After perfusate osmolarity is increased by 50 mosM (open circles in Fig. 2), Nai increases by 54 (from 24 to 78) meq/kg dry wt during 60 min of hypoxia. Because we predicted an increase in Nai of 62.5-125 meq/kg dry wt to achieve volume regulation under these conditions, we postulate that, for this protocol, volume regulation likewise was not achieved within 60 min. However, because the increase in Nai observed in response to hypoxia alone (78 meq/kg dry wt; filled squares in Fig. 2) is within the range predicted to achieve volume regulation during 50 mosM hypertonic perfusion, we would not expect the magnitude of Na uptake in response to this level of hypertonic perfusion to be different from hypoxia alone. On the other hand, if our hypothesis is correct and the response of the Na/H exchanger to decreased cell volume and pH are prioritized by the order of the stimulus and mutually exclusive, Na uptake after exposure to hypertonic perfusion will be independent of subsequent exposure to acidifying conditions (e.g., hypoxia). Preliminary studies are consistent with this prediction. When hearts were perfused under hypertonic normoxic conditions, 60 min after addition of 58.5 mM sucrose + 1 mM ouabain to the perfusate, Nai had increased by 48 (from 27 ± 2 to 75 ± 8) meq/kg dry wt (n = 3), indicating that hypertonic perfusion stimulated Na uptake under normoxic conditions [data not shown; P < 0.05 vs. isotonic normoxic perfusion with K-free perfusate (5)]. However, this Na uptake is not significantly different from that observed during 60 min of isotonic hypoxic perfusion (filled squares in Fig. 2) or 50 mM hypertonic hypoxic perfusion (open circles in Fig. 2). Thus, as expected, if responses are prioritized and mutually exclusive, responses to pH and volume stimuli were not additive. In summary, our data are consistent with the interpretation that, for hypertonic perfusion to decrease Na uptake during hypoxia, the increase in osmolarity must be large enough (>10 mosM) to maintain the Na/H exchanger in the volume-regulatory mode throughout the hypoxic interval but small enough (<50 mosM) so that the Na uptake required for volume regulation is less than would otherwise occur during isotonic hypoxic perfusion. This interpretation also explains our observation that moderately hypertonic (+30 mosM) perfusion decreases Na uptake after normoxic acidification (Fig. 4), whereas other investigators have shown that increasing perfusate osmolarity by 100-150 mosM increases Na uptake after normoxic acidification (54). For the latter studies, the Na uptake required to achieve volume regulation would be up to 375 meq/kg dry wt (3-15 times greater than calculated for the 10-50 mosM hypertonic solutions used here), thus causing Na uptake to be greater than that required to regulate pHi.Hypertonic perfusion and cell volume. Although the experiments reported here were not designed to test the hypothesis that the response of the Na/H exchanger to hypertonic perfusion is volume regulatory, our Na data allow measurement of changes in cell volume associated with hypertonic perfusion, and these are consistent with the arguments presented above [volume changes were calculated from changes in the area of the extracellular Na peak (5); data not shown]. Briefly, after 2.5 min of K-free hypoxic perfusion, cell volumes for all hypertonic protocols were nominally less than the isotonic control, and those obtained from hearts exposed to 50 mosM hypertonic perfusate were significantly less (P < 0.05 by paired t-test). Furthermore, after 52.5 min of K-free hypoxic perfusion, the cell volumes for the hypertonic protocols had increased to levels not significantly different from control or from each other. However, the nominal extent of recovery (percentage of control volume) was inversely related to the tonicity of the perfusate. (Relative to their own isotonic prehypoxia cell volumes, they were 100 ± 3, 96 ± 8, 92 ± 11, and 87 ± 11% for the isotonic and +10, +30, and +50 mosM perfusions, respectively.) This is noteworthy, since it is a characteristic of a graded response, such as volume regulation (8), and consistent with our contention that cells exposed to +30 and +50 mosM hypertonic perfusion had not achieved volume regulation by the end of the hypoxic interval.
Hypertonic resuscitation. Finally, these studies provide a new explanation for some of the observed beneficial effects of hypertonic solutions used to treat hypovolemia in trauma care. That is, although a portion of the effects are undoubtedly due to transfer of volume from the intracellular to the extracellular compartment (24), additional effects consistent with our hypothesis have been reported, e.g., increases in myocardial pHi and contractility (2, 54). In addition, our results provide a rational explanation for the finding that the optimum dose for hypovolemic resuscitation with hypertonic solutions causes an increase in extracellular osmolarity on the order of 25-30 mosM (36).
Conclusions
Over the last few years, data have accumulated in support of the hypothesis that myocardial injury due to hypoxia and ischemia is largely the result of the following chain of events: 1) increased anaerobic metabolism increases proton production, which decreases pHi; 2) decreased pHi stimulates pH-regulatory Na/H exchange; 3) increased Na/H exchange increases cell Na uptake; 4) increased Na uptake increases [Na]i; 5) increased [Na]i decreases or reverses the force driving Na/Ca exchange; 6) altered Na/Ca exchange causes an increase in [Ca]i; and 7) increased [Ca]i causes a cascade of Ca-dependent processes that lead to cell injury and death.This study presents further evidence consistent with the hypothesis. More specifically, we have demonstrated that increases in [Na]i during hypoxia (step 4) are due to increased Na uptake (step 3) and not to decreased Na efflux via Na-K-ATPase. We provide further support for the hypothesis by demonstrating that initiating hypertonic perfusion before hypoxia attenuates the hypoxia-induced increase in Na uptake and that decreased Na uptake is associated with a relative decrease in [Ca]i (predicted converse of steps 5 and 6). Finally, the results demonstrate that hypertonic perfusion decreases Na and Na-dependent Cai accumulation during hypoxia only if cells are exposed to hypertonic perfusion before hypoxia and not if the order of exposure is reversed.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-Related Disease Research Program of the University of California (Grant 3RT-0112) and National Heart, Lung, and Blood Institute Grant HL-21179. NMR spectrometer expense was funded in part by National Institutes of Health Grant RR-02511 and National Science Foundation Grant PCM-8417289.
![]() |
FOOTNOTES |
---|
The results of the experiments described here have been presented in abstract form (19, 20).
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: S. E. Anderson, Dept. of Human Physiology, University of California, One Shields Ave., Davis, CA 95616-8644 (E-mail: seanderson{at}ucdavis.edu).
Received 3 June 1999; accepted in final form 30 November 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, DG,
and
Orchard CH.
Myocardial contractile function during ischemia and hypoxia.
Circ Res
60:
153-168,
1987[Abstract].
2.
Allen, DG,
and
Smith GL.
The effects of hypertonicity on tension and intracellular calcium concentration in ferret ventricular muscle.
J Physiol (Lond)
383:
425-439,
1987[Abstract].
3.
Anderson, SE,
Adorante JS,
and
Cala PM.
Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na+ content.
Am J Physiol Cell Physiol
254:
C466-C477,
1988
4.
Anderson, SE,
Dickinson CZ,
Liu H,
and
Cala PM.
Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion.
Am J Physiol Cell Physiol
270:
C608-C618,
1996
5.
Anderson, SE,
Murphy E,
Steenbergen C,
London RE,
and
Cala PM.
Na/H exchange in myocardium: effects of hypoxia and acidification on Na and Ca.
Am J Physiol Cell Physiol
259:
C940-C948,
1990
6.
Balschi, JA,
Bittl JA,
Springer CS, Jr,
and
Ingwall JS.
31P and 23Na NMR spectroscopy of normal and ischemic rat skeletal muscle. Use of a shift reagent in vivo.
NMR Biomed
3:
47-58,
1990[Medline].
7.
Boron, WF,
and
De Weer P.
Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors.
J Gen Physiol
67:
91-112,
1976[Abstract].
8.
Cala, PM.
Volume regulation by Amphiuma red blood cells: characteristics of volume-sensitive K/H and Na/H exchange.
J Mol Physiol
8:
199-214,
1985.
9.
Cala, PM.
Volume-sensitive ion fluxes in Amphiuma red blood cells: general principles governing Na/H and K/H exchange transport and Cl/HCO3 exchange coupling.
In: Current Topics in Membranes and Transport, edited by Benos DJ,
and Mandel LJ.. New York: Academic, 1986, vol. 27, p. 193-218.
10.
Cala, PM,
Anderson SE,
and
Cragoe EJ.
Na/H exchange-dependent cell volume and pH regulation and disturbances.
Comp Biochem Physiol A Physiol
90:
551-555,
1988[ISI].
11.
Cala, PM,
Maldonado H,
and
Anderson SE.
Cell volume and pH regulation by the Amphiuma red blood cell: a model for hypoxia-induced cell injury.
Comp Biochem Physiol A Physiol
102:
603-608,
1992[ISI].
12.
Cala, PM,
and
Maldonado HM.
pH regulatory Na/H exchange by Amphiuma red blood cells.
J Gen Physiol
103:
1035-1054,
1994[Abstract].
13.
Dennis, SC,
Gevers W,
and
Opie LH.
Protons in ischemia: where do they come from; where do they go to?
J Mol Cell Cardiol
23:
1077-1086,
1991[ISI][Medline].
14.
Grace, AA,
Kirschenlohr HL,
Metcalfe JC,
Smith GA,
Weissberg PL,
Cragoe EJ, Jr,
and
Vandenberg JI.
Regulation of intracellular pH in the perfused heart by external HCO3 and Na+-H+ exchange.
Am J Physiol Heart Circ Physiol
265:
H289-H298,
1993
15.
Grinwald, PM.
Sodium pump failure in hypoxia and reoxygenation.
J Mol Cell Cardiol
24:
1393-1398,
1992[ISI][Medline].
16.
Haigney, MC,
Lakatta EG,
Stern MD,
and
Silverman HS.
Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading.
Circulation
90:
391-399,
1994[Abstract].
17.
Haigney, MC,
Miyata H,
Lakatta EG,
Stern MD,
and
Silverman HS.
Dependence of hypoxic cellular calcium loading on Na+-Ca2+ exchange.
Circ Res
71:
547-557,
1992[Abstract].
18.
Hearse, DJ,
Maxwell L,
Saldanha C,
and
Gavin JB.
The myocardial vasculature during ischemia and reperfusion: a target for injury and protection.
J Mol Cell Cardiol
25:
759-800,
1993[ISI][Medline].
19.
Ho, HS,
Anderson SE,
Holcroft JW,
and
Cala PM.
Intracellular ionized calcium and Na-H exchange in hypoxic myocardium (Abstract).
Circulation
84:
171,
1991.
20.
Ho, HS,
Anderson SE,
Holcroft JW,
and
Cala PM.
The effects of hypertonic perfusion on intracellular sodium, calcium, and hydrogen ion concentrations during hypoxia (Abstract).
Surg Forum
43:
32-35,
1992.
21.
Kirschenlohr, HL,
Grace AA,
Clarke SD,
Shachar-Hill Y,
Metcalfe JC,
Morris PG,
and
Smith GA.
Calcium measurements with a new high-affinity NMR indicator in the isolated perfused heart.
Biochem J
293:
407-411,
1993[ISI][Medline].
22.
Kirschenlohr, HL,
Metcalfe JC,
Morris PG,
Rodrigo GC,
and
Smith GA.
Ca2+ transient, Mg2+, and pH measurements in the cardiac cycle by 19F NMR.
Proc Natl Acad Sci USA
85:
9017-9021,
1988[Abstract].
23.
Kolocassides, KG,
Galinanes M,
and
Hearse DJ.
Ischemic preconditioning, cardioplegia or both?
J Mol Cell Cardiol
26:
1411-1414,
1994[ISI][Medline].
24.
Kramer, GC,
English TP,
Gunther RA,
and
Holcroft JW.
Physiological mechanisms of fluid resuscitation with hyperosmotic/hyperoncotic solutions.
In: Perspectives in Shock Research: Metabolism, Immunology, Mediators, and Models. New York: Liss, 1989, p. 311-320.
25.
Lai, ZF,
and
Nishi K.
Intracellular chloride activity increases in guinea pig ventricular muscle during simulated ischemia.
Am J Physiol Heart Circ Physiol
275:
H1613-H1619,
1998
26.
Langer, GA.
Sodium-calcium exchange in the heart.
Annu Rev Physiol
44:
435-449,
1982[ISI][Medline].
27.
Lazdunski, M,
Frelin C,
and
Vigne P.
The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH.
J Mol Cell Cardiol
17:
1029-1042,
1985[ISI][Medline].
28.
Liu, H,
Anderson SE,
and
Cala PM.
Effects of pH-regulatory Na/H exchange inhibition in hypoxic newborn rabbit myocardium (Abstract).
Circulation
86:
I-28,
1992.
29.
Liu, H,
Cala PM,
and
Anderson SE.
Ethylisopropylamiloride diminishes changes in intracellular Na, Ca, and pH in ischemic newborn myocardium.
J Mol Cell Cardiol
29:
2077-2086,
1997[ISI][Medline].
30.
Marban, E,
Kitakaze M,
Chacko VP,
and
Pike MM.
Ca2+ transients in perfused hearts revealed by gated 19F NMR spectroscopy.
Circ Res
63:
673-678,
1988[Abstract].
31.
Marban, E,
Kitakaze M,
Koretsune Y,
Yue DT,
Chacko VP,
and
Pike MM.
Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion.
Circ Res
66:
1255-1267,
1990[Abstract].
32.
Meng, H,
and
Pierce GN.
Protective effects of 5-(N,N-dimethyl)amiloride on ischemia-reperfusion injury in hearts.
Am J Physiol Heart Circ Physiol
258:
H1615-H1619,
1990
33.
Myers, ML,
Mathur S,
Li G,
and
Karmazyn M.
Sodium-hydrogen exchange inhibitors improve postischaemic recovery of function in the perfused rabbit heart.
Cardiovasc Res
29:
209-214,
1995[ISI][Medline].
34.
Neubauer, S,
Newell JB,
and
Ingwall JS.
Metabolic consequences and predictability of ventricular fibrillation in hypoxia. A 31P- and 23Na-nuclear magnetic resonance study of the isolated rat heart.
Circulation
86:
302-310,
1992[Abstract].
35.
Nishida, M,
Borzak S,
Kraemer B,
Navas JP,
Kelly RP,
Smith TW,
and
Marsh JD.
Role of cation gradients in hypercontracture of myocytes during simulated ischemia and reperfusion.
Am J Physiol Heart Circ Physiol
264:
H1896-H1906,
1993
36.
Pascual, JM,
Runyon DE,
Watson JC,
Clifford CB,
Dubick MA,
and
Kramer GC.
Resuscitation of hypovolemia in pigs using near-saturated sodium chloride solution in dextran.
Circ Shock
40:
115-124,
1993[ISI][Medline].
37.
Pike, MM,
Luo CS,
Clark MD,
Kirk KA,
Kitakaze M,
Madden MC,
Cragoe EJ, Jr,
and
Pohost GM.
NMR measurements of Na+ and cellular energy in ischemic rat heart: role of Na+-H+ exchange.
Am J Physiol Heart Circ Physiol
265:
H2017-H2026,
1993
38.
Piwnica-Worms, D,
Jacob R,
Shigeto N,
Horres CR,
and
Lieberman M.
Na/H exchange in cultured chick heart cells: secondary stimulation of electrogenic transport during recovery from intracellular acidosis.
J Mol Cell Cardiol
18:
1109-1116,
1986[ISI][Medline].
39.
Poole-Wilson, PA,
and
Tones MA.
Sodium exchange during hypoxia and on reoxygenation in the isolated rabbit heart.
J Mol Cell Cardiol
2:
15-22,
1988.
40.
Ramasamy, R,
Zhao P,
Gitomer WL,
Sherry AD,
and
Malloy CR.
Determination of chloride potential in perfused rat hearts by nuclear magnetic resonance spectroscopy.
Am J Physiol Heart Circ Physiol
263:
H1958-H1962,
1992
41.
Scholz, W,
and
Albus U.
Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy.
Cardiovasc Res
29:
184-188,
1995[ISI][Medline].
42.
Silverman, HS,
and
Stern MD.
Ionic basis of ischaemic cardiac injury: insights from cellular studies.
Cardiovasc Res
28:
581-597,
1994[ISI][Medline].
43.
Smith, TW,
Rasmusson RL,
Lobaugh LA,
and
Lieberman M.
Na+/K+ pump inhibition induces cell shrinkage in cultured chick cardiac myocytes.
Basic Res Cardiol
88:
411-420,
1993[ISI][Medline].
44.
Steenbergen, C,
Fralix TA,
and
Murphy E.
Role of increased cytosolic free calcium concentration in myocardial ischemic injury.
Basic Res Cardiol
88:
456-470,
1993[ISI][Medline].
45.
Stewart, LC,
Vander Elst L,
and
Ingwall JS.
Inhibition of Na+ pump in ischemic guinea pig myocardium (Abstract).
Biophys J
55:
510A,
1989.
46.
Stimers, JR,
Shigeto N,
and
Lieberman M.
Na/K pump current in aggregates of cultured chick cardiac myocytes.
J Gen Physiol
95:
61-76,
1990[Abstract].
47.
Tani, M,
and
Neely JR.
Role of intracellular Na+ and Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts.
Circ Res
65:
1045-1056,
1989[Abstract].
48.
Vandenberg, JI,
Metcalfe JC,
and
Grace AA.
Mechanisms of pHi recovery after global ischemia in the perfused heart.
Circ Res
72:
993-1003,
1993[Abstract].
49.
Vandenberg, JI,
Metcalfe JC,
and
Grace AA.
Intracellular pH recovery during respiratory acidosis in perfused hearts.
Am J Physiol Cell Physiol
266:
C489-C497,
1994
50.
Vaughan-Jones, RD.
An investigation of chloride-bicarbonate exchange in the sheep cardiac Purkinje fibre.
J Physiol (Lond)
379:
377-406,
1986[Abstract].
51.
Vaughan-Jones, RD.
Regulation of intracellular pH in cardiac muscle.
In: Proton Passage Across Cell Membranes. Chichester, UK: Wiley, 1988, p. 23-46. (Ciba Found. Symp. 139)
52.
Vaughan-Jones, RD,
Lederer WJ,
and
Eisner DA.
Ca2+ ions can affect the intracellular pH in mammalian cardiac muscle.
Nature
301:
522-524,
1983[ISI][Medline].
53.
Weiss, RG,
Lakatta EG,
and
Gerstenblith G.
Effects of amiloride on metabolism and contractility during reoxygenation in perfused rat hearts.
Circ Res
66:
1012-1022,
1990[Abstract].
54.
Whalley, DW,
Hemsworth PD,
and
Rasmussen HH.
Sodium-hydrogen exchange in guinea-pig ventricular muscle during exposure to hyperosmolar solutions.
J Physiol (Lond)
444:
193-212,
1991[Abstract].
55.
Whalley, DW,
Hool LC,
Ten Eick RE,
and
Rasmussen HH.
Effect of osmotic swelling and shrinkage on Na+-K+ pump activity in mammalian cardiac myocytes.
Am J Physiol Cell Physiol
265:
C1201-C1210,
1993