Department of Physiology and Membrane Biology, University of California, Davis, California
Submitted 24 August 2004 ; accepted in final form 20 September 2004
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ABSTRACT |
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myocardial ischemia; Na+/H+ exchange; Na+/Ca2+ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation
Given the aforementioned evidence that E2 stimulates release of NO and that NO inhibits NHE1 in cardiac myocytes (22), we tested the hypothesis that acute exposure of isolated rat hearts to E2 limits Na+ uptake during ischemia-reperfusion and thus limit Na+-dependent increases in [Ca2+]i as well as associated ischemia-reperfusion injury. The hypothesis further predicts that this effect of E2 is inhibited by the NO synthesis inhibitor N-nitro-L-arginine methyl ester (L-NAME).
In addition, to test more directly the hypothesis that E2 inhibits NHE1, we used the NH4Cl washout protocol (8) to acidify the heart under normoxic, normal perfusion, HEPES-buffered conditions with or without E2. This protocol allowed us to assess the response of NHE1 to intracellular acidification similar in magnitude to that which occurs during ischemia, but with fewer uncontrolled parameters (28).
The results are consistent with the hypothesis that E2 inhibits NHE1 in a NO-dependent manner. That is, in the HEPES-buffered NH4 washout protocol, E2 diminished the rate of proton efflux. In the ischemia protocol, E2 limited cytosolic ,
, and
accumulation during ischemia. Furthermore, these effects of acute E2 improved recovery of function and limited lactate dehydrogenase (LDH) release during reperfusion. However, E2 limitation of increases in [Ca2+]i during ischemia is greater than can be accounted for by the thermodynamic effect of the Na+ gradient on NCE, suggesting that E2 also attenuates NCE activity.
Thus the results of these studies provide new insights into the mechanisms of gender- or female hormone-dependent effects on myocardial ischemia-induced accumulation and injury. They go beyond previous studies to provide strong evidence that acute E2 inhibits NHE1 during ischemia and more generally after intracellular acidification. Furthermore, thermodynamic arguments provide strong circumstantial evidence that E2 also inhibits NCE independently of changes in [Na+]i during ischemia. While the evidence supports the conclusion that E2 inhibits NHE1 via NO, how it might inhibit NCE remains to be investigated. These results were previously reported in abstract form (23).
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METHODS |
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The normoxic acidification protocol was conducted using HEPES-buffered perfusate (HR), which was identical to the perfusate described above, except that 20 mM HEPES acid plus 8 mM NaOH were substituted for 25 mM NaHCO3, and the perfusate was equilibrated with 100% O2 and titrated to pH 7.40 ± 0.05. The protocol consisted of 20-min baseline perfusion with HR, 35-min perfusion with HR + 10 mM NH4Cl, followed by 30-min perfusion with HR (NH4Cl washout). For acute E2 and L-NAME experiments, 1 nM E2 and/or 1 µM L-NAME was added to both the NH4Cl solution (5 min before NH4Cl washout) and the NH4Cl washout solution. 31P NMR was used to measure pHi as described below. Proton efflux rates were calculated as the product of the change in pHi during the first 5 min of recovery after acidification multiplied by cardiac myocyte buffer capacity. The buffer capacity (mM/pH) equals 28 (pHi) + 222.6 (28), where pHi
is the pHi at the beginning of the recovery interval.
The control ischemia protocol consisted of 20-min baseline perfusion, followed by 40-min ischemia, followed by 40-min reperfusion. To limit glycolysis (1), 5.5 mM 2-deoxy-D-glucose was substituted for dextrose 10 min before ischemia. The E2 protocol was identical, except that 1 nM E2 was added to the perfusate used during the baseline and reperfusion intervals. Each of the protocols was also conducted with the addition of 1 µM L-NAME to the perfusate. When L-NAME was added, it was added together with E2 or when E2 would have been added. Initiation of 40 min of ischemia was designated t = 0 min. 23Na, 31P, and 19F NMR were used to measure , pHi and high-energy phosphates, and [Ca2+]i, respectively. To measure
, 7.5 mmol/l dysprosium triethylenetetraminehexaacetic acid was substituted for NaCl in the perfusate isosmotically, and Ca2+ was added until the perfusate concentration reached 1.82 mmol/l as measured using Ca2+ electrode (31). To measure [Ca2+]i, hearts were perfused for 30 min with perfusate containing the acetoxymethyl ester of 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) at 8 µmol/l (6). FBAPTA was then washed out of the extracellular space with control solution for 15 min before baseline measurement of [Ca2+]i was performed. After perfusions were complete, hearts used to measure 23Na were weighed wet and dried to constant weight (at least 48 h) at 65°C to determine dry weight. Their wet and dry weights were 1.22 ± .04 and 0.19 ± 0.01 g, respectively, and there were no significant differences between treatments.
NMR spectroscopy. Experiments were conducted using a Bruker AMX400 spectrometer. 23Na, 31P, and 19F spectra were generated from the summed free induction decays of 1,000, 148, and 1,500 excitation pulses (90°, 60°, and 45°) using 2-, 4-, and 2-kilobyte word data files and ±4,000-, ±4,000-, and ±5,000-Hz sweep widths, respectively. Data files were collected over 5-min intervals, but to improve signal-to-noise ratio for 19F measurement of [Ca2+]i, two 5-min files were added together. Because the NMR signal intensity reflected the average for the interval over which data were collected, data in the figures correspond to the midpoint of the appropriate 5- or 10-min acquisition interval. NMR data reflect the signal collected from the entire heart, including both ventricles and both atria.
(in meq/kg dry wt) was calculated from the calibrated area of the "unshifted" intracellular peak after subtracting the shifted extracellular peak as previously described (3, 5, 31). This method measures
as an amount, and to minimize assumptions, we report it as such. [Ca2+]i (in nmol/l cytosolic water) was calculated as the product of the Ca2+-FBAPTA dissociation constant (Kd) and the ratio of the areas of the Ca2+-bound and Ca2+-free peaks in the FBAPTA spectrum (6). The Kd for Ca2+-FBAPTA was 294 nM. pHi was determined from the chemical shift of the inorganic phosphate (Pi) resonance (with reference to control phosphocreatine) calibrated at 37°C (2). High-energy phosphates are reported as percentage of control peak intensity (6).
To assess ischemia-reperfusion injury, total LDH released was measured from timed collections of perfusate leaving the heart for 40 min of reperfusion as previously described for creatine kinase (43). LDH (in IU/g dry wt) was measured spectrophotometrically (Pointe Scientific, Lincoln Park, MI).
Unless otherwise stated, results are reported as means ± SE. For the ischemia experiments, analysis of variance for repeated measures was used to test for differences between treatments. When differences between treatments were found, the Student-Newman-Keuls multiple-comparison test was used to determine which treatments were different and the times at which differences between treatments occurred. Only the latter are indicated in figures. For the NH4Cl washout experiments, the Bonferroni correction was used to compare proton efflux rates. Because overpowered studies are wasteful of resources (17) and/or raise serious ethical issues when animals are used (30), sample sizes were limited to those required to reject the null hypotheses with P < 0.05.
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RESULTS |
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DISCUSSION |
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Because the balance of proton production and efflux is so difficult to predict and control during ischemia, it is very hard to unequivocally determine the cause-and-effect relationships between changes in pHi and [Na+]i using that model. Therefore, we tested the hypothesis that E2 will inhibit NHE1 under normoxic, normal flow conditions of intracellular acidification using the NH4Cl prepulse technique under HEPES-buffered conditions (8, 22). Under these conditions, a known amount or bolus of protons can be "injected" into the cell, and the rate of proton efflux during pHi recovery can be ascribed to flux via NHE1 (28). The results of these experiments provide strong evidence that E2 inhibits NHE1 (Fig. 1), and because the effect of E2 is diminished by L-NAME, this result is consistent with the postulate that the E2 effect is NO mediated.
Also consistent with these hypotheses are that acute E2 limits and
accumulation during ischemia and that the effect of E2 is diminished by L-NAME, indicating that it is mediated by NO (Figs. 3 and 4).
The observation that E2 limits the fall in pHi during ischemia might initially seem contradictory to our hypothesis that E2, via NO, inhibits NHE1. However, investigators at our laboratory (31) and others (42) have shown that NHE1 inhibitors either have no effect on or limit the fall in pHi during ischemia. Furthermore, inhibiting ischemia-induced Na+ uptake via other pathways also limits ischemia-induced acidification (9, 24, 34). This is because ischemia-induced and
accumulation stimulates ATP hydrolysis (3, 21, 32) and thus proton production (18). In other words, during ischemia, a positive feedback loop is created whereby increased proton production increases
and
accumulation, which stimulates more proton production. If this occurs, inhibiting accumulation of any of the ions will have a tendency to diminish the accumulation of the others; in particular, limiting increases in [Na+]i and [Ca2+]i limits proton production and thereby ischemia-induced acidification (31). The data presented in Fig. 2 fit this paradigm in that acute E2 limitation of
and
accumulation (see Figs 3 and 4) is associated with a smaller decrease in pHi during ischemia, and, again, this effect of E2 was diminished by L-NAME. We cannot rule out the possibility that E2 inhibits proton production, e.g., by stimulating ATP-dependent K+ channels (29), independently of that due to inhibition of NHE1 (and thus limits stimulation of NHE1), but the data in Fig. 1 provide strong evidence that E2 inhibits the response of NHE1 to acidification under conditions in which the acidification is not E2 dependent.
Finally, the findings that acute E2 limits ischemia-reperfusion-induced decreases in recovery of LVDP (Fig. 5) and increases in LDH release during reperfusion (Fig. 6) are consistent with the hypothesis that E2-sensitive and
accumulation contribute to ischemia-reperfusion injury. Although the effects are statistically significant, in this model LVDP recovery is modest. Thus whether the effects of E2 observed in this protocol would be important in a clinical setting remains to be determined.
Although the results of these studies are consistent with the hypothesis that acute E2 limits and thereby
accumulation, it should also be noted that with respect to the data shown in Figs. 3 and 4, the E2-dependent limitation of cytosolic Ca2+ accumulation is greater than one would expect based on the reduction in
accumulation alone. That is, investigators at our laboratory (4) and others (38, 46) have shown that NCE remains near equilibrium under a variety of conditions and most notably during ischemia. This behavior is interpreted as support for the notion that increases in [Ca2+]i during ischemia are mediated by NCE. In other words, if NCE is the dominant Ca2+ transport pathway, the driving force for NCE will be at or near zero; incipient forces will dissipate because of NCE flux, and the pathway will be at equilibrium. If that is the case, the reversal potential ENCE will equal Em. Conversely, if ENCE is not equal to Em, NCE is not the dominant Ca2+ transport pathway. If ENCE is calculated at the end of ischemia after acute E2 (OVX+E2 from the data in Figs. 3 and 4), the result is ENCE = 126 mV (see APPENDIX at the end of this article). Again, this means Em would have to be 126 mV for NCE to be at equilibrium. The negative limit for Em under essentially all physiological conditions, including those in which K+ channels are stimulated, is the K+ equilibrium potential, which in this case is near 65 mV (35). [In fact, Em is likely to be nearer to 50 mV (46).] Because ENCE is far more negative than Em (Em ENCE
6176 mV), one may safely argue that after acute E2 treatment, NCE is no longer the dominant Ca2+ transport protein during ischemia.
Thus Occam's razor leads one to conclude that during ischemia after acute E2, NCE must be inhibited kinetically and/or that some other Ca2+ transport pathway is stimulated so that NCE is no longer dominant. One could argue that there may be errors in the values of [Na+] and [Ca2+] used in our calculations, but it is unlikely that the errors could be large enough to cause this large discrepancy between ENCE and Em. First, the error in our assumptions for extracellular [Na+] and [Ca2+] are likely to be <5%, and the error in our estimate of Em must be <20 mV (Em cannot be more negative than EK). Second, if the discrepancy were due to errors in measurement of or
, to account for the discrepancy would require an overestimate of at least 100% in our measurement of [Na+]i or an underestimate of [Ca2+]i >90%. Again, while the above discussion is based on formal thermodynamic arguments, the same conclusions can be drawn qualitatively from the data in Figs. 3 and 4, which show that during ischemia after acute E2 treatment, nominal increases in [Na+]i are associated with no change or with decreases in [Ca2+]i. This cannot happen if NCE is the dominant Ca2+ transport pathway.
As mentioned above, one could postulate that NCE is not dominant (at equilibrium), because E2 stimulates some other pathway to remove Ca2+ from the cytosol, e.g., Ca2+-ATPase at the sarcolemma or the sarcoplasmic reticulum (13). However, in order for the ischemia-induced changes in and
to be dissociated by E2 as observed in Figs. 3 and 4, a very large increase in efflux via some other pathways would be required to surpass flux via NCE unless the latter was inhibited. Thus, again, the simplest explanation for the data includes E2 inhibition of NCE activity. How this occurs requires further study.
The results of our experiments are consistent with a number of reports suggesting that female hormones play a role in Ca2+-mediated ischemic injury. These include the studies of Cross and colleagues (14, 15), who showed that in mutant mice that overexpressed either 2-adrenergic receptors or NCE, female mice were protected from ischemic injury, in contrast to males and ovariectomized females, and in the
2-adrenergic receptor overexpressors, the female resistance to ischemic injury was reduced by treatment with L-NAME. In contrast, using a simulated ischemia-reperfusion protocol with isolated rat astrocytes, Matsuda et al. (33) found that sodium nitroprusside and 8-bromo-cGMP exacerbated injury and concluded that NO and cGMP stimulated NCE. This is not consistent with our studies or with those conducted by Cross and colleagues. On the other hand, in a metabolic inhibition study using isolated mouse cardiac myocytes overexpressing NCE, Sugishita et al. (48) reported findings consistent with those reported by Cross and colleagues (14, 15) but included measurements of [Ca2+]i and [Na+]i that were consistent with our findings. That is, increases in [Na+]i and [Ca2+]i during metabolic inhibition were greater in male than in female transgenics, and gender differences were eliminated by treating the males with E2. In a more recent report, Sugishita et al. concluded that "the acute cardioprotective effect of estrogen during [metabolic inhibition] may be mediated by an ER-independent anti-oxidant action, which results in improved function of Na+-K+ ATPase" (47, p. 331). However, in the more recent study, the authors used a dose of E2 100 times greater than the dose that we used, which raises the question whether the anti-oxidant effect was physiological.
The fact that the present study was conducted with a 100-fold lower dose of E2 (near peak estrus in the rat) and that the effect is diminished by L-NAME (but not by D-NAME) provides evidence that the response that we observed is not completely due to an anti-oxidant effect of E2. Furthermore, E2-dependent limitation of increases in during ischemia seem to be dependent on the presence of estrogen receptor-
. Zhai et al. (51) reported that in perfused hearts of male mice, estrogen receptor-
knockouts increased
accumulation, decreased nitrite production, and increased multiple indicators of injury during ischemia-reperfusion. Another study by investigators at the same laboratory (52) showed that in female OVX rats, chronic E2 treatment was associated with the opposite response to ischemia-reperfusion: decreased
accumulation, increased nitrite production, and decreased injury.
Recent studies in a number of organs are consistent with the postulate that NHE1 may be inhibited by signal transduction pathways associated with increases in NO and specifically the cascade of steps through NO, cGMP, cGMP-dependent kinase (cGK) and p38 MAPK (19). These include studies from isolated hepatocytes that suggest a preconditioning effect limiting increases in [Na+]i during hypoxia is mediated by NO through cGK and p38 MAPK (11) and furthermore that atrial natriuretic peptide diminishes hypoxia-induced increases in [Na+]i by a similar pathway that inhibits NHE1 (10). These results are also consistent with studies that suggest activation of p38 MAPK inhibits the response of NHE1 to angiotensin II in vascular smooth muscle cells by phosphorylating the NHE1 at serine/threonine residues located between amino acids 671 and 714 (27).
Finally, it has been suggested that KH or, more broadly, crystalloid perfused hearts are "hyperoxic" and thus more susceptible to oxidative stress because the perfusate is commonly equilibrated with 95% O2. As is the case for respiration, the pertinent factor is not PO2 per se but rather the oxygen concentration in the solution at the site of the reaction. At the level of cardiac myocytes in the KH-perfused heart, there are at least two arguments that suggest the cells are not hyperoxic. First, there is no change in the extravascular O2 transport pathway, except perhaps for interstitial edema, which would lengthen the pathway. Second, although the actual concentration of intracellular O2 is elusive and not agreed upon, NMR measurements of intracellular O2 in perfused and in situ myocardium indicate that O2 delivery to heart cells in KH-perfused hearts is not greater than in situ (26). Furthermore, when the critical level of intracellular O2 is measured (25), KH-perfused hearts are marginally closer than blood-perfused hearts to hypoxia (12). Nevertheless, to the extent that ischemia-reperfusion injury is the result of oxidative stress to the capillary endothelium, the crystalloid perfused heart model used in the present study may be more susceptible.
Conclusions.
The results of the present study are the first strong evidence that acute exposure to 1 nM E2 (20 min or less) inhibits the response of NHE1 to acidification. That is, acute E2 exposure decreases the rate of proton efflux after NH4 washout in HEPES medium. Furthermore, acute E2 exposure diminishes accumulation of cytosolic H,
, and
in hearts from OVX during ischemia. Limiting the accumulation of these ions was associated with improved recovery of LVDP and decreased release of LDH during reperfusion. In addition, E2-dependent effects on all ions were diminished by addition of 1 µM L-NAME. These results are consistent with the previously untested hypothesis that an NO-dependent effect of E2 inhibits NHE1 to limit Na+ and thereby Ca2+ uptake during ischemia. However, thermodynamic arguments also illustrate that the effect of E2 on [Ca2+]i is greater than can be accounted for by the effect of limiting
accumulation on sarcolemmal NCE and thus suggest that E2 inhibits NCE kinetically.
Finally, we speculate that regardless of findings related to chronic effects of E2 (e.g., those related to hormone replacement therapy), there may be a role for acute E2 treatment in therapies aimed at limiting myocardial ischemia-induced injury, including those designed for use in organ preservation or bypass surgery.
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APPENDIX |
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GRANTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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