Na-H exchange in myocardium: effects of hypoxia and acidification

on Na and Ca

STEVEN E. ANDERSON, ELIZABETH MURPHY, CHARLES STEENBERGEN, ROBERT E. LONDON, AND PETER M. CALA Department of Human Physiology, University of California, Davis, California 95616; Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park 27709; and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

ANDERSON, STEVEN E., ELIZABETH MURPHY, CHARLES STEENBERGEN, ROBERT E. LONDON, AND PETER M. CALA. Na-H exchange in myocardium: effects of hypoxia and acidification on Na and Ca. Am. J. Physiol. 259 (Cell Physiol. 28): C940-C948, 1990.-Historically, increases in cell Na content during ischemic and hypoxic episodes were thought to result from impaired ATP production causing decreased Na’-K’ATPase activity. Here we report the results of testing the alternate hypothesis that hypoxia-induced Na uptake is 1) the result of increased entry, as opposed to decreased extrusion 2) via Na-H exchange operating in a pH regulatory capacity and that cell Ca accumulation occurs via Na-Ca exchange secondary to collapse of the Na gradient. We used ‘:jNa-, lgF-, and :‘lPnuclear magnetic resonance (NMR) to measure intracellular Na content (Na;), Ca concentration ([Cali), pH (pHi), and highenergy phosphates in Langendorff-perfused rabbit hearts. When the Na+-K’-ATPase was inhibited by ouabain and/or K-free perfusion, hearts subjected to hypoxia gained Na at a rate X0 times that of normoxic controls [during the first 12.5 min Na; increased from 7.9 ? 5.8 to 34.9 t 11.0 (SD) meq/kg dry wt compared with 11.1 t 16.3 to 13.6 t 9.0 meq/kg dry wt, respectively]. When normoxic hearts were acidified using a 20 mM NH&l prepulse technique, pHi rapidly fell from 7.27 t 0.24 to 6.63 t 0.12 but returned to 7.07 t 0.10 within 20 min, while Na uptake was similar in rate and magnitude to that observed during hypoxia (24.5 k 13.4 to 132.1 t 17.7 meq/kg dry wt). During hypoxia and after NH&l washout, increases in [Cal; were similar in time course to those observed for Nai. Increases in Nai were insensitive to benzamil (50 PM) and bumetanide (10 PM), whereas increases in Nai as well as pHi regulation (after NH&l washout) and increases in [Cal; were inhibited by amiloride (1 mM) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 100 PM). EIPA and amiloride also decreased changes in coronary resistance and phosphocreatine measured after 60 min of hypoxic perfusion (P < 0.05). These results are consistent with our hypothesis. amiloride; sodium-potassium-adenosinetriphosphatase; gendorff-perfused rabbit hearts

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(18, 29) and increased intracellular Na (30) are commonly associated with myocardial hypoxia, ischemia, and reperfusion. Historically, these sequelae of myocardial ischemia were ascribed to the effect of hypoxia on ATP production leading to diminished Na+-K’ATPase (pump) activity and progress toward Donnan equilibrium. In a more general sense as well, ATP depletion was considered to be a primary reason for cellular CELL SWELLING

dysfunction under hypoxic and/or ischemic conditions (28). Recent studies, however, have begun providing evidence that alterations in intracellular ion concentrations [most notably H (11) and Ca (35)] occur before or concomitantly with decreases in ATP (1) and may play a primary role in cell dysfunction and damage, independent of ATP availability. Our awaren .essof the fact that i.ntracellular acidification has been shown to stimulate Na-H exch .ange in a variety of cell types (8) including chick myocytes (14,27) and sheep Purkinje fibers (12) suggested the hypothesis that myocardial hypoxia/ischemia, as a result of increased anaerobic metabolism, leads to decreased intracellular pH (pHi), which in turn stimulates Na-H exchange in a pH regulatory mode and therefore increased Na uptake. If Na efflux (primarily mediated by Na+-K+ATPase) does not increase to match increased Na influx, increases in intracellular Na content (Na;) and concentration ( [Na]i) will occur. Finally, an increase in [Na]i will not only increase ATP utilization by Na+-K+-ATPase but will also result in decreased energy for Ca extrusion via Na-Ca exchange and lead to an increase in intracellular free Ca concentration ( [Ca] ;). The results of studies we report here using the isolated crystalloid-perfused rabbit heart and nuclear magnetic resonance (NMR) (25, 35) are consistent with our hypothesis. In this study, under conditions in which Na’K+-ATPase is inhibited (perfusate K removal and/or ouabain addition), the mean increase in intracellular Na is XO-fold greater during the first 15 min of hypoxic perfusion than during normoxic perfusion. We also show that this Na uptake is sensitive to amiloride and the more specific Na-H exchange blocker 5-( N-ethyl-N-isopropyl)amiloride (EIPA; Ref. 14) yet not affected by benzamil, a more selective inhibitor of Na-Ca exchange and conductive Na transport (32), or bumetanide, which blocks Na-Cl and/or Na-K-2Cl cotransport pathways (16). Using the NH&l prepulse technique to decrease pHi under normoxic conditions (7), we show that intracellular acidification is associated with increased Na uptake, which is similar in time course, magnitude, and pharmacological sensitivity to that observed during hypoxic perfusion. The latter studies also show that Na uptake is associated with H extrusion and that net Na uptake ceases when pHi has been regulated back to normal (pHi =L:7.05). Thus pH regulatory Na-H exchange

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is a feature of intact myocardium. Furthermore, both during hypoxia and during normoxic intracellular acidification, the measured increase in Na; is associated with an increase in [Cal;, and the increase in [Cal; does not occur when Na uptake is inhibited by EIPA. Taken together, these data suggest that Na-H exchange is the primary effector increasing cell Na uptake during hypoxia and, by inference, ischemia and early postischemic reperfusion. This increased Na uptake has serious implications for Na-Ca exchange, Ca homeostasis (1, 20, 34, 35, 37), and cellular energy consumption (by Na’-K’-ATPase) which, taken together with our experimental results and their implications, leads us to postulate that Na-H exchange plays a central role in cell damage during hypoxia, ischemia, and reperfusion. In contrast to commonly accepted explanations for cellular Na accumulation subsequent to hypoxic/ischemic episodes, our results suggest that increased myocyte uptake of Na (via H-stimulated Na-H exchange) causes an increase in energy consumption rather than being the effect of diminished available energy. Portions of the data presented here have been reported previously (3, 5, 9).

METHODS

General. New Zealand White rabbits weighing 2-3 kg were anesthetized with pentobarbital sodium (50 mg/kg) and heparinized (1,000 units/kg) by injection into the marginal ear vein. The heart was isolated and perfused using a modified Langendorff technique (4) at a constant flow of 27-29 ml/min at 23-25°C with a Gilson Minipuls 2 peristaltic pump. Perfusion pressure was continuously monitored by means of a perfusate-filled cannula connecting the aortic cannula with a Statham P23 Db strain gauge transducer and a Hewlett-Packard 7402A oscillographic recorder. The heart was inserted to hang freely within an NMR tube (30 mm OD, 28 mm ID) which in turn was lowered into the bore of the magnet used for our experiments and centered in the active volume of a custom-built cylindrical window probe for “‘Na and ‘lP measurement or a modified saddle coil for ‘“F (to measure [Cal;). In “‘Na and “‘P experiments, perfusate flowing from the pulmonary artery bathed the heart and was withdrawn from the NMR tube above the heart to be recirculated by the perfusion pump. In “F experiments perfusate was withdrawn from below the heart so the heart was not bathed. The control perfusate consisted of (in mM) 127 NaCl, 4.75 KCl, 1.25 MgC12, 1.82 CaC12, 24 NaHCOzj [or 20 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 8 NaOH], and 11.1 dextrose. (Significant differences between HEPESand HCOybuffered experiments were not observed.) To measure changes in Na; using NMR it was necessary to separate intra- and extracellular Na resonance by substituting 15 mM dysprosium triethylene tetraminehexaacetic acid (DyTTHA; Ref. 25) in the perfusate (and thereby the extracellular space) for osmotic equivalents of NaCl. Because DyTTHA chelates Ca (2), Ca was added to perfusates containing DyTTHA to reach a concentration of 1.8-2.0 mM as measured by Ca electrode. Perfusate

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pH was adjusted to 7.35-7.45 after equilibrium with 95% 02-5% CO:! (or 100% Oa) for normoxic experiments or 95% Nz-5% CO, (or 100% N2) for hypoxic experiments. Perfusate Na and K concentrations were measured by flame photometry from samples collected periodically from the inflow (arterial) line as well as the withdrawal (venous) line. We used two basic protocols to test the hypothesis that hypoxia and, more specifically, intracellular acidification stimulate Na-H exchange in the perfused heart. These were hypoxia-induced acidosis and acidosis induced using the NH&l “prepulse” technique (7). Both protocols called for an initial control perfusion of 45-60 min while the heart stabilized and the NMR probe and magnet were tuned and shimmed. Because functional Na+-K’-ATPase masked changes in Na entry, unless otherwise stated, measurements of cellular ion changes were done under conditions in which Na+-K+-ATPase was inhibited by removal of K from the perfusate (NaCl substituted for KCl). To test for the extent of Na+-K+ATPase inhibition, we conducted some experiments with K-free perfusion plus 1 mM ouabain. The NH&l prepulse protocol consisted of control perfusion, 40 min of K-free perfusion with added 20 mM NH&l (to permit NH, uptake), and 30 min of K-free perfusion without NH&l (allowing NH:, to rapidly exit the cells leaving excess H behind). Sucrose was added to osmotically balance perfusates used during the NH&l prepulse protocol. Na;, [Cal;, and pHi were measured at the end of the control perfusion and every 5 or 10 min during 1 h of normoxic or hypoxic K-free perfusion or after NH&l washout. Heart K loss during the last 40 min of K-free hypoxic or normoxic perfusion was calculated as the product of the volume recirculated over that interval and the change in K concentration in that volume. At the end of perfusion, hearts were examined for appearance, weighed wet after light blotting, and dried for 48 h at 65°C before dry weight was measured. Unless otherwise stated all results are reported as means t SD. NMR spectroscopy: ““Na and “‘P. ““Na and “lP experiments were conducted using a Nicolet NT 200 spectrometer equipped with an Oxford 4.7-T vertical-bore superconducting magnet operating in the pulsed Fourier transform mode to generate spectra. For both nuclei, data were gathered over 5-min intervals using a &4,000-Hz sweep width. “‘{Na spectra were generated from the summed free induction decays of 1,000 excitation pulses (75 ps) at 278-ms intervals using 2,048 word data files, and “P spectra were generated similarly using 148 excitation pulses (45 ps) at 2-s intervals using 4,096 word data files. NMR data in figures and text represent the signal average for the corresponding acquisition interval. pHi was calculated from Pi resonance shift [with reference to phosphocreatine (PCr)] calibrated over the anticipated pH range at 25OC. Calibration data points (pH vs. Pi shift) were fit by a Henderson-Hasselbalchtype equation with pK equal to 6.75 and asymptotes of 5.641 and 3.236. Na; was calculated from the area under the unshifted peak of the ““Na spectra. The area under the peak corresponding to Na; was determined by using standard

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Nicolet 1280 routines to reverse and subtract each spectrum from itself after adjusting the resonance offset so as to minimize the portion of the difference spectrum that arose from extracellular Na (Na, = the sum of all Na in the NMR probe’s active volume residing outside the cell: bath + vascular + interstitium). All spectra (forward and reverse) were scaled identically before Na, was subtracted out. Nicolet software was then used to integrate over the Na; peak. Na; in milliequivalents was calculated by multiplying the area under the Na; peak by a calibration term equal to the quotient of the flame photometer determined amount of Na in control perfusate contained in a heart-sized phantom divided by the area under the unshifted peak when the phantom was bathed in perfusate containing DyTTHA (13). This value was divided by dry heart weight to express Na; as milliequivalents per kilogram dry weight. Because this analysis is very sensitive to asymmetry in the large Na, peak, NMR Na measurements in some hearts required baseline adjustments to compensate for minor asymmetry likely to arise from static field inhomogeneity. In these hearts, a constant factor was subtracted from all Nai spectral areas such that the baseline (zero-time acquisition) Na; value was between 0 and 10 meq/kg dry wt. Although the method directly measures Na content, we were able to measure changes in cell volume and [Na]; indirectly (and less accurately) from measured changes in Na,. Na, (meq/kg dry wt) was calculated from the Na spectrum as the calibrated difference between the total spectral area and the Na; peak area. Because the heart resides completely within the active volume of the NMR probe and is bathed in perfusate, the sum of the extracellular volume and the intracellular volume is constant. Assuming the extracellular Na concentration ( [NalO) is homogeneous and equal to that measured in the perfusate by flame photometry, we were able to estimate the extracellular volume (V,) as Na,/[Na],,. Further assuming that intracellular volume under control conditions (Vi) equals 2.5 liters/kg dry wt (4), we calculated that [Na]i equals Na; divided by the intracellular fluid volume (the sum of Vi and the negative value of the change in V,). NMR spectroscopy: ‘“F measurement of [Cali using “F-BAPTA. To measure [Cal;, the acetoxymethyl ester of “F-l,Z-bis(Z-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA; Ref. 35) was added to the control perfusate at a final concentration of 5 PM. The heart was loaded with “F-BAPTA for 30 min during which time the ester entered the cells and was cleaved by intracellular esterases. This left the negatively charged ‘FBAPTA trapped within the cells. After loading, perfusion was switched back to control solution. Thus any ‘FBAPTA remaining in the extracellular space would be washed out of the heart. Perfusion protocols used to measure changes in [Cal; were the same as those used to measure 2:1Naand ‘lP except that they were conducted using a Nicolet NT 360 spectrometer equipped with an 8.5-T magnet or a GE Omega CSI system equipped with an 8-T magnet. The magnet was shimmed on protons to provide a water line width of lO times greater than during normoxic K-free perfusion. (See text for details.)

Hypoxic stimulation of Na uptake. Figure 2 summarizes the results from eight hearts, each of which was exposed to either normoxic or hypoxic perfusion with K-free media with or without 1 mM ouabain. Control values for Nai in hearts exposed to the normoxic or hypoxic protocols were 11.1 t 16.3 and 7.9 t 5.8 (SD) meq/kg dry wt, respectively. [This relatively large variability in control values for Nai is representative and reflects the insensitivity of the NMR technique (see DISCUSSION). Nevertheless, because the induced changes in Na uptake are large relative to the variability in mean values, the effects of the described protocols are easily identified.] Here the greatest difference in rate of myocyte Na uptake occurs between the first two acquisition intervals after

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initiating K-free perfusion where mean Nai increases from 11.1 t 16.3 to 13.6 t 9.0 meq/kg dry wt during normoxia and from 7.9 t 5.8 to 34.9 t 11.0 meq/kg dry wt during hypoxia. Thus the rate of Na uptake by hypoxic cells is X0 times greater than normoxic cells during this interval. Furthermore, after 70 min of hypoxic perfusion, the mean increase in Na; was nearly three times that of hearts perfused with normoxic media. [Na]i is calculated to increase from 3.0 t 2.2 to 41.6 t 8.3 meq/liter (1.9 t 10.0% increase in cell volume) during 70 min of hypoxic K-free perfusion but only from 4.3 t 6.5 to 20.8 t 12.7 meq/liter (6.7 t 11.1% decrease in cell volume) during normoxic K-free perfusion. Addition of ouabain did not significantly increase net Na uptake above that observed with K-free perfusion alone, indicating that for both the normoxic and hypoxic protocols, K-free perfusion essentially inhibits the Na efflux via the Na-K pump completely. Thus the increased Na; in these hypoxic hearts reflects increased dissipative entry not decreased extrusion by Na’-K’-ATPase. Inhibition of Na+-K’-ATPase was also demonstrated by net cellular K loss measured during K-free normoxic and hypoxic perfusion. Cell K loss was 121 t 9 and 114 t 39 meq/kg dry wt during the last 40 min of hypoxia and normoxia, respectively. Pharmacological probes of the Na uptake route. Figure 3 shows net Na uptake during hypoxic K-free perfusion after addition of 50 PM benzamil or 100 PM EIPA to the perfusate. With benzamil in the perfusate, mean Na; increased from 1.5 to 83.5 meq/kg dry wt in 60 min, not different from hypoxia without benzamil. In contrast, under the same conditions, mean Na; in hearts perfused with EIPA increased from 8.0 to 11.5 meq/kg dry wt, a decrease in Na uptake of >95% relative to that observed in the absence of EIPA. Clearly, EIPA inhibited Na uptake during hypoxic perfusion. Furthermore, there was no measurable difference between normoxic and hypoxic Na; when EIPA was included in the K-free perfusate nor

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minutes FIG. 3. Pharmacological sensitivity of Na uptake in the perfused heart during hypoxia. Intracellular Na (meq/kg dry wt) is plotted vs. minutes of hypoxic K-free perfusion. Benzamil (50 PM; n , n = 2) had no significant effect on Na uptake, whereas 100 PM 5-(N-ethyl-Nisopropyl)amiloride (EIPA; q , n = 2) inhibited Na uptake.

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t 0.24 to 6.63 t 0.12 during the first 5-min interval and was regulated back to control levels (7.07 t 0.10) within 15 min. Over the same time interval Nai rose from a control level of 24.5 t 13.4 to 132.1 t 17.7 meq/kg dry wt, where it plateaued in temporal coincidence with the recovery of pHi. If, at this point, the heart was reperfused with ouabain-free K-containing perfusate, Na; diminished toward control levels and the heart resumed beating (see Fig. 6). Further support for the notion that acidactivated Na uptake and H extrusion are via Na-H exchange was obtained from the NH&l washout performed in the presence of 100 PM EIPA, in which increases in Na; as well as pHi regulation were inhibited (data not shown). Relationship between Na uptake and Ca uptake. Figure 5 shows the results of [Cali measurements for the protocol described for Fig. 1 plus and minus the Na-H exchange inhibitor EIPA. Although quantitative comparisons between Na; and Cai cannot be made between hearts, the qualitative changes are comparable. Without EIPA [Cal; rose during 60 min of normoxic K-free perfusion from 515 t 231 to 861 t 279 nM, rose further during the subsequent 60 min of hypoxic K-free perfusion to 1,216 t 137 nM, and finally returned to 795 t 101 nM after 40 min of reperfusion with normoxic (control) perfusate. Addition of 100 PM EIPA to the K-free perfusates is shown to inhibit the [Cal; increase observed during hypoxia. Similarly, Fig. 6 shows the results of two experiments measuring changes in Nai and [Cali for the normoxic NH&l prepulse protocol including return to normal K perfusate after the pH regulatory phase had been completed. Again, the changes in Na; and [Cal; are qualitatively similar and have the same time course in response to the perturbations shown. Furthermore, just as in response to hypoxia after normoxic acidification,

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minutes FIG. 4. Na uptake and intracellular pH (pHi) regulation from 7 separate perfused hearts after normoxic intracellular acidification. Intracellular Na (0, meq/kg dry wt, n = 3) and pH (H, n = 4) (means t SE) on the left and right ordinates, respectively, are plotted vs. minutes after NH&l washout. Hearts were perfused with K-free solution at all times shown. These data support the hypothesis that decreased pHi stimulates Na uptake via Na-H exchange functioning in a pH regulatory role.

was there a measurable difference between Nai in hearts perfused with 1 mM amiloride or 100 PM EIPA. In a third set of experiments (data not shown) net Na uptake during hypoxic K-free perfusion was unaffected by addition of 10 PM bumetanide to the perfusate. Relationship between Na uptake and H extrusion. Having shown that hypoxia stimulated myocyte Na uptake, we proceeded to test the hypothesis that intracellular H stimulates Na uptake under normoxic conditions using the NH&l prepulse. Figure 4 shows the results of seven experiments measuring Nai and pHi after a 20 mM NH&l prepulse. After NH&l washout, pHi fell from 7.27

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minutes 5. EIPA inhibits increases in intracellular Ca concentration during K-free hypoxic perfusion. Intracellular Ca (nmol/liter, means + SD) with (0, n = 3) and without (m, n = 3) addition of 100 PM EIPA is plotted vs. time. K was removed from the perfusate at zero time, O2 was removed at 60 min, and normal-K normal-O2 perfusion was resumed at 120 min. FIG.

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of variance confirmed that the three treatments were different (P < 0.05). The Tukey multiple-comparison test showed that under conditions of constant-flow perfusion EIPA prevents the increase in coronary resistance otherwise observed during hypoxia (P c 0.05).

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FIG. 6. Na uptake and increased intracellular Ca after intracellular acidification in 2 separate perfused hearts. Intracellular Na (meq/kg dry wt, left ) and free Ca (nmol/liter, right ) are plotted vs. minutes of perfusion. Intracellular acidification occurs rapidly after NH&l washout (see Fig. 4). Note both Na and Ca decrease after Na-K pump function is restored by replacing 4.75 mM KC1 in the perfusate (prior solutions shown were K free).

the Na-H exchange blocker EIPA (100 PM) prevented changes in [Cal;. It is important to note that reperfusion with normoxic, normal K solution causes a decrease in [Cal; after pHi regulation in this protocol. As is the case for Na;, this provides strong evidence th .at the changes in [Cal; are the result of reversible changes in Ca transport as opposed to membrane damage. Inhibition of changes in [Cal; by EIPA (previously shown to prevent Na uptake; see Fig. 3) as well as the decreases in [Cali that follow stimulation of the Na pump by perfusate K show that these changes are Na; dependent and implicate Na-Ca exchange as the responsible pathway. Effects of amiloride on heart preservat ion. The results of our experiments suggested that inhibition of Na and therefore Ca uptake would diminish the rate of highenergy phosphate consumption during hypoxia. To test that hypothesis we perfused hearts with perfusate containing normal K under hypoxic conditions with (n = 4) and without (n = 4) 1 mM amiloride while monitoring high-energy phosphates using “‘P-NMR. Figure 7 summarizes the results of these experiments. PCr and the beta phosphate of ATP (bATP) are expressed as percent of their control peak intensities (means t SE) vs. time in which hypoxic perfusion begins immediately after the control acquisition at zero time and normoxic perfusion 1s reinstated at 85 min. The Stude nt’s t test showed that at the end of the hypoxic in.terval PCr was significantly greater (P < 0.05) when the perfusate contained amiloride than when it did not (Fig. 7, left). Although this protocol did not show a significant effect of amiloride on bATP, Fig. 7, right, shows that the trend for ATP is similar to that of PCr except the ATP does not recover with normoxic reperfusion after hypoxia. The effects of EIPA on coronary perfusion pressure are summarized in Fig. 8 where perfusion pressure (cmH20) is plotted vs. time for normoxic hearts and hypoxic hearts (presented previously in Figs. 2 and 3) with and without EIPA during K-free perfusion. Analysis

Our results show that hypoxia stimulates cellular Na uptake and [Cal; increase when isolated rabbit hearts are perfused with K-free crystalloid solution. Furthermore, the cells are able to extrude the Na gained during hypoxia as well as decrease [Cali upon restoration of perfusate K and O2 and therefore Na-K pump function. In addition, we show that Na uptake, simultaneous H extrusion, and increased [Cali can be induced by decreased pHi and, further, that acid-induced Na uptake and [Cali increase (normoxic conditions) are similar in rate and magnitude to that observed during hypoxia. Finally, we have shown that amiloride and its more Na-H exchange-specific analogue EIPA inhibit hypoxia- and acid-induced Na uptake and [Cal; increase as well as blocking H extrusion after NH&l washout. In light of the similarity in magnitude and rate of hypoxia- and acid-induced Na uptake and the pharmacological sensitivity of the Na uptake pathway, we conclude that both hypoxia- and H-activated Na uptake are via the Na-H exchanger. The arguments that implicate Na-H exchange as the pathway responsible for increased Na uptake may be extended to implicate Na/Ca as the pathway responsible for changes in [Cal; secondary to hypoxia- and pH-induced Na-H exchange. However, the [Cal; data reported here are at least in part consistent with other interpretations, and further studies will be required to clearly elucidate the mechanisms responsible for the hypoxia- and H-induced changes in [Cal;. The results reported here are qualitatively similar to those previously reported using NMR to measure Nai in myocardium (13, 25). Although DyTTHA shift reagents used in the above and present experiments have proven useful in cellular and isolated organ NMR determination of Na;, because of their Ca-chelating abilities (2) and susceptibility to enzyme degradation (23), they have found only marginal success in in vivo application. We have previously shown in red blood cells, however, that the NMR techniques accurately measure changes in Nai known to be the result of Na transport by Na-H exchange (2). Those studies showed that DyTTHA shift reagents used to separate intra- and extracellular Na resonance did not affect normal Na transport, lending further credence to the quantitative measurements we report here for the perfused heart. Hypoxia and acid induce Na uptake. Our results show that when isolated rabbit hearts are perfused with Kfree crystalloid solution, hypoxia stimulates Na accumulation which, in the first 15 min, is X0 times greater in rate than that under normoxic conditions. In addition, intracellular acidification (NH&l prepulse) under normoxie conditions stimulates Na accumulation that is similar in rate and magnitude to that observed after exposure to hypoxic perfusion. Given that K-free perfusion completely inhibits measurable net Na efflux via

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FIG. 7. Amiloride sensitivity of phosphocreatine (PCr) and ATP in the perfused heart during hypoxia and reoxygenation. PCr and ATP are plotted as percent of zero-time NMR peak height vs. minutes of perfusion. Hypoxic perfusion was begun after zero-time data were acquired, and reoxygenation occurred after the 80-min data acquisition. Mean values for highenergy phosphates during perfusion with (open symbols, n = 4) and without (closed symbols, n = 4) 1 mM amiloride are shown (*SE). The t test showed that after 80 min of hypoxic perfusion PCr was significantly greater (P < 0.05) after treatment with amiloride than without.

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minutes FIG. 8. EIPA sensitivity of perfusion pressure in the heart during hypoxic K-free perfusion. Perfusion pressure (cmHzO) at constant flow is plotted vs. minutes of K-free perfusion. Mean values during K-free normoxic (0, n = 4), hypoxic (H, IZ = 4), and hypoxic plus 100 PM EIPA (A, n = 4) perfusion are shown (&SE). After analysis of variance, the Tukey test showed that EIPA prevented the increase in coronary resistance otherwise observed after 20 min of hypoxic perfusion (P < 0.05).

the Na-K pump (confirmed by no additional effect of 1 mM ouabain on Nai; Fig. 2), we conclude that hypoxia and acidification initially increase Na uptake as opposed to decreasing Na extrusion via Na+-K+-ATPase. Furthermore, on the basis of the arguments below, the hypoxia-induced Na uptake appears to be mediated by Na-H exchange. Because there is sustained H production due to anaerobic metabolism during perfusion with hypoxic buffer containing glucose, so too is Na-H exchange flux sustained with the result that the cells load with Na. Pharmacological evidence implicates Na uptake via NaH exchange. Pharmacological agents known to block Na-

H exchange blocked both hypoxia- and acid-induced Na and Ca accumulation. That is, Na uptake during K-free hypoxic and normoxic perfusion as well as that subsequent to normoxic cytoplasmic acidification (pH regulation) were not affected by agents known to preferentially inhibit conductive Na transport and Na-Ca exchange (32) or Na-K-2Cl (and Na-Cl) cotransport (16). In contrast, both Na and Ca accumulation were inhibited by amiloride and its more Na-H exchange-specific analogue EIPA (14). Furthermore, the fact that EIPA prevents measurable Na; accumulation under both hypoxic and normoxic conditions suggests that the increase in Na uptake associated with hypoxia and intracellular acidification occurs via Na-H exchange. Cell pH regulation and maintenance. The data show that both hypoxia and acid pHi stimulate Na uptake, which appears to be coupled to H extrusion. These results and the widespread pH regulatory role of the Na-H exchange pathway (12, 14, 27) lead us to infer that increased H, produced by anaerobic metabolism in cells subjected to hypoxia, stimulates Na-H exchange to function at an increased rate to regulate cell pH. In the perfused heart we observed that after exposure to hypoxic perfusion, pHi becomes slightly acid and remains nearly steady during Na uptake but falls rapidly as Na uptake ceases. We interpret this to reflect Na uptake and H extrusion in the face of continuous metabolic H production during hypoxia. In contrast, after pHi is altered by a discreet H load (NH&l prepulse), amiloride-inhibitable Na-dependent H extrusion appears to regulate pHi. That is, Na-H exchange is capable of regulating pHi after the bolus acid load provided by the NH&l prepulse, but not during hypoxia when anaerobic metabolism continues to produce H. Extrusion of H by Na-H exchange may in fact entrain further H production by removing the negative feedback of low pHi on phosphofructokinase. Thus we speculate that during hypoxia metabolic H production

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and Na/H-dependent H extrusion initially proceed at similar rates and thereby maintain a nearly steady pHi. However, for reasons that remain unclear (but are likely to be related to increased [Cali; Ref. lo), Na-H exchange cannot be maintained after -20 min of hypoxia, and pHi falls as Na uptake ceases. Nevertheless, the observation that as Na uptake during hypoxic perfusion slowed pHi decreased more rapidly is consistent with our hypothesis and suggests that hypoxia-induced Na uptake is linked to H extrusion. While the Na-H exchanger is greatly stimulated to perform what appears to be a pH regulatory function during hypoxia or in response to NH&l prepulse, our data are also consistent with this role for the pathway under normoxic conditions. That is, when the Na-K pump is inhibited during normoxic perfusion, the subsequent increase in cell Na content (Fig. 2) can be blocked completely by the presence of amiloride or EIPA. From this we infer that Na-H exchange is functional under normoxic conditions and, further, that in the steady state, measurable net Na leak flux is via Na-H exchange, presumably fulfilling a pH regulatory function. Increased Na uptake increases energy consumption. In the process of developing the protocols we used to test the hypothesis, we performed a number of experiments without inhibiting the Na-K pump. We found that, in “nonworking” hearts at 23-25°C changes in Na; during more than one hour of hypoxic perfusion or ischemia were modest (at the limit of NMR resolution) unless the perfusate was K free or contained ouabain. Taken together with data in Figs. 1-3, this observation suggests that, unless it is inhibited by ouabain or K-free perfusion, Na+-K+-ATPase is able to partially compensate for Na gained by hypoxia-induced Na-H exchange. Inferentially then, during the first hour of ischemia in this model, ATP levels remain sufficient to fuel the Na-K pump at elevated rates. Recent reports of a two- to threefold increase in Na; during the first 30 min of ischemia at 37°C (26, 31) are not, however, inconsistent with our results. Because it is likely that the change in H production and therefore Na-H exchange and Na uptake would be less during 25°C ischemia than at 37OC, it is not surprising that Na uptake during ischemia at 25°C is at the limit of resolution in our system. Assuming that the error in our Na; measurement is approximated by the variability in control Nai, we could only reliably measure changes in Na; that were >3 meq/kg dry wt (or a 1.75 fold increase). Furthermore, the plateau in Na; reported after 20-30 min of 37°C ischemia is similar to the plateau we commonly observe in individual hearts after 20 min of K-free hypoxic perfusion at 25°C and may reflect a common mechanism preventing further Na; accumulation, i.e., Cai inhibition of Na-H exchange (10). The above observations are consistent with previous reports that during ischemia marked changes in mechanical function and ion distribution occur before significant changes in cell ATP levels (1). Our direct measurements during hypoxia illustrate that while the maximum gain in cell Na is occurring, ATP levels remain at 60-70% of those in normoxic cells (compare the time courses of Figs. 2 and 7). Furthermore, our finding that amiloride inhibition of Na uptake and [Cal; increase preserves

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myocardial high-energy phosphates during hypoxic perfusion (Fig. 7) suggests that the measured increases in Na; and [Cal; are not a consequence but a cause of energy consumption (38). This is in sharp contrast to the more traditional view that during hypoxia/ischemia increased Na; is a result of decreased Na-K pump function due to ATP depletion (28, 36). Although amiloride has been shown to decrease oxygen consumption (33), the net effect of amiloride during hypoxic perfusion is to limit depletion of high-energy phosphate residing in PCr. However, because our studies were performed on nonworking hearts at 25OC, further study will be necessary to evaluate the extent of relevance of the present observation to the in vivo situation during hypoxia/ischemia. Speculation: Na, Ca, cell damage, and perfusion pressure. We noted above that during hypoxia, perfusion pressure increases as a function of time. Other investigators have shown an increase in myocyte volume accompanying Na; increase after exposure to hypoxia or ischemia and suggested that increased coronary resistance is dependent on myocyte volume increase (29). Although our data do not address the issue directly, they do not support that interpretation, since cell volume, inferred from NMR Na measurements, did not increase while coronary resistance increased. (Cell volume increase predicted from Nai increases are compensated by K loss; in all K-free-perfused hearts K loss was greater than or equal to Na gain.) An alternate hypothesis suggests that the increase in coronary resistance reflects alterations in myocyte and/ or vascular smooth muscle tone. That is, increased myocyte and vascular smooth muscle Na; will decrease the energy in the Na gradient and impair Ca extrusion via Na-Ca exchange. As a consequence, myocytes and vascular smooth muscle cells may accumulate Ca and contract (6), thereby increasing coronary resistance and perfusion pressure under constant-flow conditions. A third hypothesis suggests that increased coronary resistance observed during hypoxic perfusion is secondary to myocardial contracture due to lack of ATP (1). The data in Fig. 8 which show that EIPA inhibition of cellular Na uptake is associated with decreased coronary resistance during hypoxia are consistent with the latter hypotheses in that EIPA decreases Cai accumulation (Fig. 5) and high-energy phosphate loss (Fig. 7) in this model. Finally, the changes in [Cal; reported here for hypoxic rabbit hearts are consistent with those reported for ischemic hearts (22,34,35,37). If increased Cai subsequent to increased Na; (15) interacts with or potentiates any of a variety of pathological cellular responses to increased Cai (i.e., lipid oxidation, peroxide generation, mitochondrial uncoupling, protease activation, etc.; Refs. 17, 24, 39), amiloride and its analogues may help prevent cell damage and death associated with myocardial hypoxia, ischemia, and reperfusion (19, 34, 37). In conclusion, these studies show that hypoxia and intracellular acidification stimulate Na and Ca uptake in the isolated perfused rabbit heart. Both parallel changes in pHi and Nai as well as pharmacological sensitivity provide evidence that the increased Na uptake is via NaH exchange acting in a pH regulatory mode. These results are consistent with the hv-pothesis that H-stim-

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C948

NA/H,

NA,

AND

CA

IN

ulated Na-H exchange is the primary effector of increases in Nai and [ Ca]i and therefore myocardial damage following hypoxia and ischemia. We express our appreciation to John C. Klein and Jennifer K. Lawlor for technical assistance and to Phoebe Ling for assistance with word processing. This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-21179 (to P. M. Cala), National Institutes of Health Regional Resource Grant RR-02479, and a Grant-in-Aid from the American Heart Association, California Affiliate, with funds contributed by the American Heart Association, Golden Empire Chapter (to S. E. Anderson). Address for reprint requests: S. E. Anderson, Dept. of Human Physiology, Univ of California, Davis, CA 95616. Received

11 December

1989; accepted

in final

form

10 August

HYPOXIC

1. ALLEN, D. G., AND C. H. ORCHARD. Myocardial contractile function during ischemia and hypoxia. Circ. Res. 60: 153-168, 1987. 2. ANDERSON, S. E., J. S. ADORANTE, AND P. M. CALA. Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na content. Am. J. Physiol. 254 (Cell Physiol. 23): C466C474, 1988. 3. ANDERSON, S. E., P. M. CALA, AND E. J. CRAGOE, JR. Hypoxic stimulation of Na-H exchange measured by “‘Na-NMR in perfused rabbit hearts. Physiologist 29: 126, 1986. 4. ANDERSON, S. E., AND J. A. JOHNSON. Tissue-fluid pressure measured in perfused rabbit hearts during osmotic transients. Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H1127-H1137, 1987. 5, ANDERSON, S. E., C. STEENBERGEN, R. LONDON, P. CALA, E. CRAGOE, AND E. MURPHY. “F measurement of Cai during hypoxia: role of Na/H and Na/Ca exchange (Abstract). Circulation 78, Suppl. II: 216, 1988. 6. BLAUSTEIN, M. P. Sodium ions, calcium ions, blood pressure regulation and hypertension: a reassessment and a hypothesis. Am. J. Physiol. 232 (Cell Physiol. 1): C165-C173, 1977. 7. BORON, W. F., AND P. DE WEER. Intracellular pH transients in squid giant axons caused by COz, NHII, and metabolic inhibitors. J. Gen. Physiol. 67: 91-112, 1976. 8. CALA, P. M. Volume regulation by Amphiuma red blood cells: the membrane potential and its implications regarding the nature of the ion-flux pathways. J. Gen. Physiol. 76: 683-708, 1980. 9. CALA, P. M., S. E. ANDERSON, AND E. J. CRAGOE, JR. Na/H exchange-dependent cell volume and pH regulation and disturbances. Comp. Biochem. Physiol. A Comp. Physiol. 90: 551-555, 1988. 10. CALA, P. M., L. J. MANDEI,, AND E. MURPHY. Volume regulation by Amphiuma red blood cells: cytosolic free Ca and alkali metal/H exchange. Am. J. Physiol. 250 (Cell Physiol. 19): C423C429, 1986. 11. COBBE, S. M., AND P. A. POOLE-WILSON. The time of onset and severity of acidosis in myocardial ischemia. J. Mol. CeZZ. Cardiol. 12: 745-760, 1980. 12. DIETMER, J. W., AND D. ELLIS. Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibres. J. Physiol. Lond. 304: 471-488, 1980. 13. FOSSEL, E. T., AND H. HOEFELER. Observation of intracellular potassium and sodium in the heart by NMR: a major fraction of potassium is “invisible.” Magn. Reson. Med. 3: 534-540, 1986. 14. FRELIN, C., P. VIGNE, AND M. LAZDUNSKI. The role of the Na/H exchange system in cardiac cells in relation to the control of the internal Na concentration. J. Biol. Chem. 259: 8880-8885, 1984. 15. GRINWALD, P. M., AND C. BROSNAHAN. Sodium imbalance as a cause of calcium overload in post-hypoxic reoxygenation injury. J. Mol. Cell. Cardiol. 19: 487-495, 1987. 16. HAAS, M., AND B. FORBUSH III. [“Hlbumetanide binding to duck red cells. J. Biol. Chem. 261: 8434-8441, 1986. 17. HEARSE, D. J., AND A. TOSAKI. Free radicals and calcium: simultaneous interacting triggers as determinants of vulnerability to reperfusion-induced arrhythmias in the rat heart. J. Mol. Cell. Cardiol. 20: 213-223, 1988. 18. JENNINGS, R. B., J. SCHAPER, M. L. HILL, C. STEENBERGEN, JR., AND K. A. REIMER. Effect of reperfusion late in the phase of reversible ischemic injury. Circ. Res. 56: 262-278. 1985.

KARMAZYN, M. Amiloride enhances postischemic ventricular recovery: possible role of Na’-H’ exchange. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H608-H615, 1988. KIM, D., E. J. CRAGOE, JR., AND T. W. SMITH. Relation among sodium pump inhibition, Na-Ca and Na-H exchange activities, and Ca-H interaction in cultured chick heart cells. Circ. Res. 60: 185193, 1987. KIRSCHENLOHR, H. L., J. C. METCALFE, P. G. MORRIS, G. C. RODRIGO, AND G. A. SMITH. Ca”’ transient, Mg”‘, and pH measurements in the cardiac cycle by “F NMR. Proc. Natl. Acad. Sci. USA 85: 9017-9021, 1988. LEE, H., R. MOHABIR, N. SMITH, AND W. T. CLUSIN. Effect of ischemia on calcium-dependent fluorescence transients in rabbit hearts containing Indo 1. Circulation 78: 1047-1059, 1988. MATWIOFF, N. A., C. GASPAROVIC, R. WENK, J. D. WICKS, AND A. RATH. “‘P and ““Na NMR studies of the structure and lability of the sodium shift reagent, bis( tripolyphosphate)dysprosium( III) ion, and its decomposition in the presence of rat ([DyU’:&)17-) muscle. Magn. Reson. Med. 3: 164-168, 1986. MCCORD, J. M. Free radicals and myocardial ischemia: overview and outlook. Free Radical Biol. & Med. 4: 9-14, 1988. PIKE, M. M., J. C. FRAZER, D. F. DEDRICK, J. S. INGWALL, P. D. ALLEN, C. S. SPRINGER, JR., AND T. W. SMITH. “‘{Na and “‘K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intraand extracellular ions using a shift reagent. Biophys. J. 48: 159-173, 1985. PIKE, M. M., M. KITAKAZE, AND E. MARBAN. Increase in intracellular free sodium concentration during ischemia revealed by ““Na NMR in perfused ferret hearts (Abstract). Circulation 78, Suppl. II: 11-151, 1988. PIWNICA-WORMS, D., R. JACOB, C. R. HORRES, AND M. LIEBERMAN. Na/H exchange in cultured chick heart cells. J. Gen. Physiol. 85: 43-64, 1985. POOLE-WILSON, P. A., AND M. A. TONES. Sodium exchange during hypoxia and on reoxygenation in the isolated rabbit heart. J. Mol. Cell. Cardiol. 20, Suppl. II: 15-22, 1988. POWERS, E. R., D. R. DIBONA, AND W. J. POWELL, JR. Myocardial cell volume and coronary resistance during diminished coronary perfusion. Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H467H477, 1984. REGAN, T. J., L. BROISMAN, B. HAIDER, C. EADDY, AND H. A. OLDEWURTEL. Dissociation of myocardial sodium and potassium alteration in mild versus severe ischemia. Am. J. Physiol. 238 (Heart Circ. Physiol. 7): H575-H580, 1980. SHERRY, A. D., C. F. GERALDES, M. M. CASTRO, F. M. JEFFREY, AND C. R. MALLOY. A New ““Na magnetic resonance shift agent for perfused hearts (Abstract). Circulation 78, Suppl. II: 11-152, 1988. SIEGL, P. K. S., E. J. CRAGOE, JR., M. J. TRUMBLE, AND G. J. KACZOROWSKI. Inhibition of Na’/Ca” exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride. Proc. Natl. Acad. Sci. USA 81: 3238-3242, 1984. SOLTOFF, S. P., E. J. CRAGOE, JR., AND L. J. MANDEL. Amiloride analogues inhibit proximal tubule metabolism. Am. J. Physiol. 250 (Cell Physiol. 19): C744-C747, 1986. STEENBERGEN, C., R. E. LONDON, ANP, E. MURPHY. Calcium during ischemia: a role of Na/Ca exchange (Abstract). Circulation 80, Suppl. II: 11-237, 1989. STEENBERGEN, C., E. MURPHY, L. LEVY, AND R. E. LONDON. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ. Res. 60: 700-707, 1987. STEWART, L. C., L. VANDER ELST, AND J. S. INGWALL. Inhibition of Na’ pump in ischemic guinea pig myocardium (Abstract). Biophys. J. 55: 510a, 1989. TANI, M., AND J. R. NEELY. Role of intracellular Na’ in Ca’ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Circ. Res. 65: 1045-1056, 1989. VAUGHAN-JONES, R. D. Regulation of intracellular pH in cardiac muscle. In: Proton Passage Across Cell Membranes. Chichester, UK: Wiley, 1988, p. 23-46. (Ciba Found. Symp.) VER DONCK, L., J. VAN REEMPTS, G. VANDEPLASSCHE, AND M. BORGERS. A new method to study activated oxygen species induced damage in cardiomyocytes and protection by Ca’+-antagonists. J. Mol. Cell. Cardiol. 20: 811-823, 1988.

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Na-H exchange in myocardium: effects of hypoxia and acidification on Na and Ca.

Historically, increase in cell Na content during ischemic and hypoxic episodes were thought to result from impaired ATP production causing decreased N...
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