INTRODUCTION

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BRIEF INTERMITTENT PERIODS of ischemia and reflow, termed ischemic preconditioning, have been shown to have a protective effect during a subsequent prolonged period of ischemia. Preconditioning has been shown to reduce necrosis, postischemic contractile dysfunction, and the incidence of arrhythmias (16, 21). Preconditioning has also been shown to be associated with reduced ionic alterations during the prolonged period of ischemia; preconditioning attenuates the rise in intracellular H⁺ ([H⁺]), Na⁺ ([Na⁺]), and Ca²⁺ ([Ca²⁺]) concentrations during ischemia (21). In addition, preconditioning has been shown to reduce the rate of ATP utilization and the production of lactate during the prolonged period of ischemia (16). One explanation for the reduced acidification observed during ischemia in preconditioned hearts is decreased anaerobic glycolysis, secondary to reduced ATP utilization (16).

Several recent studies have suggested that increased proton extrusion might play a role in the reduced acidification observed in preconditioned hearts (19, 20). Ramasamy et al. (19) reported that ischemic preconditioning stimulates Na⁺/H⁺ exchange. -Adrenergic stimulation, which mimics preconditioning, is also suggested to stimulate Na⁺/H⁺ exchange via a protein kinase C (PKC)-dependent pathway (20), and inhibition of Na⁺/H⁺ exchange is reported to block the -adrenergic-induced reduction in intracellular pH during ischemia (20). However, it is difficult to reconcile the concept that the beneficial effects of preconditioning are mediated by stimulation of Na⁺/H⁺ exchange with the well-documented beneficial effect of inhibition of Na⁺/H⁺ exchange on ischemic injury. Numerous investigators have shown that inhibition of Na⁺/H⁺ exchange improves function, most likely by reducing Na⁺ and Ca²⁺ loading (12, 15, 22). When Na⁺/H⁺ exchange is reduced, there is less influx of Na⁺, thereby attenuating the rise in intracellular [Ca²⁺] due to Na⁺/Ca²⁺ exchange. Consistent with this, a recent study by Bugge and Ytrehus (2) has reported that inhibition of Na⁺/H⁺ exchange enhances the protective effects of preconditioning on infarct size. Thus it is unclear whether ischemic preconditioning affects Na⁺/H⁺ exchange flux and whether this is involved in the protective effect. Furthermore, previous studies have reported that although inhibition of Na⁺/H⁺ exchange reduces the rise in intracellular [Na⁺] during ischemia, it does not enhance the accumulation of H⁺, presumably because there are multiple pathways by which H⁺ can exit the cell.

In the present study, we investigated whether alterations in H⁺ efflux, and specifically Na⁺/H⁺ exchange, were responsible for the reduced acidification in preconditioned hearts and whether inhibition of Na⁺/H⁺ exchange would affect the improved recovery of function that is induced by preconditioning in this rat heart model. Although preconditioning has been shown to cause improvement in several end points, it is not clear that the mechanisms are necessarily the same. Thus the mechanism(s) involved in preconditioning may depend on the end point of preconditioning under study. To address the question of whether preconditioning enhances proton extrusion, we used ³¹P nuclear magnetic resonance (³¹P NMR) spectroscopy to monitor intracellular and extracellular pH. If the decrease in intracellular pH observed in preconditioned hearts is due to increased H⁺ efflux, rather than decreased production of protons, we would expect to see a greater decline in extracellular pH relative to intracellular pH in preconditioned hearts, and we would expect to see a larger decrease in extracellular pH in preconditioned compared with nonpreconditioned hearts. If increased H⁺ efflux is due to stimulation of Na⁺/H⁺ exchange, we would expect that inhibition of Na⁺/H⁺ exchange would result in a decrease in intracellular pH in preconditioned ischemic hearts to a level similar to that observed in nonpreconditioned ischemic hearts. However, we find no support for enhanced proton extrusion in preconditioned hearts and no evidence that inhibition of Na⁺/H⁺ exchange diminishes the protective effect of preconditioning on postischemic functional recovery. We do, however, observe less total acidification (intra- and extracellular pH) in preconditioned hearts than in nonpreconditioned hearts, consistent with less acid production in preconditioned hearts.

	MATERIALS AND METHODS

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Isolated rat heart preparation. Adult male Sprague-Dawley rats (Charles River; Raleigh, NC) weighing 270-330 g were anesthetized with pentobarbital sodium (80 mg/kg ip). The animals were given 100 U of heparin iv. The hearts were then rapidly excised and placed in ice-cold Krebs-Henseleit buffer, and the aortas were cannulated. Retrograde perfusion was begun from a reservoir suspended 90 cm above the heart. The nonrecirculating perfusate was a Krebs-Henseleit buffer containing (in mmol/l) 120 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.36 CaCl₂, 25 NaHCO₃, and 11 glucose. The buffer was aerated with 95% O₂-5% CO₂ and was maintained at a temperature of 37°C.

³¹P NMR. The studies were carried out using a Varian Unity Plus 400-MHz wide-bore NMR spectrometer with a variable temperature probe set to 37°C. A 20-mm ³¹P NMR probe (Cryomagnet Systems; Indianapolis, IN) was tuned to 161.9 MHz. The hearts were shimmed on the proton signal, giving typical nonspinning line widths at one-half height of 0.25 parts per million (ppm). Pulsing conditions employed include a 70° pulse angle, a 2-s delay, a 342-ms recycle time, a spectral width of ±3,600 Hz, 4,096 data points, and 128 acquisitions, which corresponded to 5-min blocks of data collection. The free induction decay was multiplied by an exponential function corresponding to 40-Hz line broadening preceding Fourier transformation.

Experimental procedures. Hearts were initially perfused for 15 min with normal Krebs-Henseleit buffer. Before NMR spectra were collected, hearts were perfused with a phosphate-free buffer for an additional 10 min; this allowed for the unambiguous assignment of the P_i peak to the intracellular space. The phosphate-free buffer also contained 15 mM PPA (pH adjusted to 7.4). Thus there were 25 min of preperfusion before acquisition of NMR spectra. Hearts were studied using NMR spectroscopy, which allowed the continuous monitoring of intracellular and extracellular pH and high-energy phosphates. The first part of the study consisted of a control group of four hearts that were monitored by ³¹P NMR under basal conditions for 10 min, followed by 20 min of ischemia and 20 min of reflow. Another group of four hearts was monitored by ³¹P NMR under basal conditions for 10 min and then subjected to a preconditioning protocol before the 20 min of ischemia and 20 min of reflow. The preconditioning protocol consisted of four cycles of 5 min of ischemia separated by 5 min of reflow. In the second part of the study, three hearts were preconditioned with addition of dimethylamiloride (DMA) during the fourth reflow of the preconditioning period, and an additional three hearts were preconditioned in the absence of DMA. DMA was not present during reperfusion after the 20-min period of sustained ischemia. We used DMA at a concentration of 50 µM and at a concentration of 300 µM. Results were similar with both concentrations. In these studies, we measured recovery of LVDP and intracellular pH.

	RESULTS

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Effects of PPA on cardiac function and metabolism. As shown in Table 1, addition of PPA resulted in no significant effect on LVDP, heart rate, or coronary flow. In addition, consistent with the observations of other investigators (5, 6, 9), PPA had no significant effect on ATP, creatine phosphate, or intracellular pH as measured by ³¹P NMR (data not shown).

Effects of preconditioning on intracellular and extracellular pH during ischemia. Figure 1 shows typical ³¹P NMR spectra under control (top trace) and ischemic conditions (bottom trace). These spectra illustrate the pH-dependent chemical shift of the P_i and PPA resonances in the ischemic hearts. The shift difference between P_i and PCr is a measure of intracellular pH, and the shift difference between PPA and PCr is a measure of extracellular pH (9). PPA shifts downfield (to the left), whereas P_i shifts upfield (to the right) with decreasing pH. As indicated in Fig. 1, during ischemia there is a downfield shift in PPA and an upfield shift in P_i consistent with a decrease in pH in both the intracellular and extracellular compartments. Figure 2A shows a plot of extracellular pH in preconditioned vs. nonpreconditioned hearts, and Fig. 2B shows intracellular pH in preconditioned vs. nonpreconditioned hearts. At the end of 20 min of ischemia, nonpreconditioned hearts showed an intracellular pH of 5.90 ± 0.08, whereas the hearts preconditioned before the 20-min ischemic period had an intracellular pH of 6.50 ± 0.06 (P < 0.05). The extracellular pH values after 20 min of ischemia for nonpreconditioned and preconditioned hearts were 5.51 ± 0.21 and 6.62 ± 0.06, respectively (P < 0.05). Thus the extracellular pH was similar to intracellular pH during ischemia in both preconditioned and nonpreconditioned hearts. The initial decline in extracellular pH is similar in preconditioned and nonpreconditioned hearts; however, by 10-15 min of ischemia, intracellular pH in preconditioned hearts leveled off, as did extracellular pH, whereas in the nonpreconditioned hearts, intracellular and extracellular pH both continued to fall. Also, preconditioned hearts appear to produce less acid than nonpreconditioned hearts because both intra- and extracellular pH were more alkaline in preconditioned hearts.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   31P nuclear magnetic resonance (31P NMR) spectra under control conditions (top trace) and after 20 min of ischemia (bottom trace). Intracellular pH was determined from the shift difference between Pi and phosphocreatine (PCr), whereas extracellular pH was determined from the shift difference between phenylphosphonic acid (PPA) and PCr. PCr resonance was set to 0 parts per million (ppm). ATP, ATP, and ATP peaks refer to , , and  phosphates of ATP.

View larger version (18K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig. 2.   Extracellular (A) and intracellular pH (B) in preconditioned hearts and nonpreconditioned hearts. Intra- and extracellular pH were determined as described in MATERIALS AND METHODS and Fig. 1. Hearts were preconditioned with 4 cycles of 5 min each of ischemia and reperfusion. At time 0, global ischemia was initiated by clamping the aortic inflow line. Reflow was begun after 20 min of global ischemia. Values are means ± SE.

	DISCUSSION

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During ischemia, intracellular pH falls to ~5.8-6.0 and levels off. The plateau of intracellular pH at ~6.0 has been attributed to inhibition of glyeraldehyde-3-phosphate dehydrogenase by the low pH and high NADH-to-NAD ratio (17), thereby inhibiting the further production of lactate. During ischemia, as H⁺ is extruded from the cell, it cannot be washed away and thus it reduces extracellular pH as well. Thus, during ischemia, intra- and extracellular pH both fall to ~5.8-6.0, and this low pH inhibits glycolysis and further generation of acid.

Numerous groups have reported that preconditioning reduces the decline in pH during ischemia, such that in preconditioned hearts pH falls to ~6.4-6.5 instead of the pH of 6.0 observed in nonpreconditioned hearts. The mechanism responsible for this reduction in acidification, as well as its role in preconditioning, has been debated (4, 19, 20, 25). There are two general hypotheses to account for the reduction in acidification in preconditioned hearts. Hypothesis 1 is that there is a reduction in the generation of H⁺, either because there is inhibition of or less stimulation for glycolysis or glycogenolysis (24) or because of glycogen depletion; this would reduce the generation of protons, resulting in less acidification during ischemia. However, because both inhibition of glycolysis (11) and glycogen depletion (7) have been reported to have detrimental rather than beneficial effects, it is likely that reduced demand for ATP is an important component of preconditioning. According to hypothesis 2, the reduction in acidification in preconditioned hearts is due to increased proton extrusion from the myocytes rather than a reduction in acid production. This could occur if preconditioning, possibly via activation of PKC, stimulates Na⁺/H⁺ exchange (19). In addition, agents such as phenylephrine that mimic preconditioning have been reported to reduce acidification during ischemia via a PKC-dependent mechanism, and this reduction in acidification is blocked by inhibition of Na⁺/H⁺ exchange (20).

If preconditioning enhances proton efflux during ischemia, the most likely mechanism is by a change in Na⁺/H⁺ exchanger. Stimulation of Na⁺/H⁺ activity (increasing V_max) would not be expected to alter the plateau value of intracellular pH during sustained ischemia, but increasing the V_max of the Na⁺/H⁺ exchanger might increase the relative proportion of H⁺ that is extruded via Na⁺/H⁺ exchange vs. Cl/ exchange and hence alter the intracellular [Na⁺]. Consistent with this, inhibition of Na⁺/H⁺ exchange does not alter intracellular pH during ischemia, presumably because H⁺ exits via other mechanisms; however, Na⁺/H⁺ exchange inhibitors do reduce the rise in intracellular [Na⁺] during ischemia (15, 18). For altered Na⁺/H⁺ exchange to be the mechanism responsible for the higher intracellular pH in preconditioned hearts, a change in the cytosolic K_m for protons would be required, and there are reports that PKC can alter the K_m as well as increase the V_max of Na⁺/H⁺ and Cl/ exchangers (23). If PKC or preconditioning were to alter the K_m for the Na⁺/H⁺ exchanger, this might alter the plateau of intracellular pH during ischemia. Furthermore, if the K_m were altered, addition of Na⁺/H⁺ exchange inhibitors would be expected to block the altered pH during ischemia in preconditioned hearts, similar to the effect reported for phenylephrine-treated hearts (20). To test this hypothesis, we added the Na⁺/H⁺ exchange inhibitor, DMA, during the fourth reflow of the preconditioning protocol, with continued presence of DMA during the sustained period of ischemia. Addition of DMA to preconditioned hearts did not affect the fall in intracellular pH during ischemia or recovery of function during reflow. We did not observe an additive protective effect with preconditioning and DMA, but this may reflect the excellent recovery of function in the preconditioned hearts in the absence of DMA, which would make it very difficult to detect any additional protective effect.

To test the more general hypothesis that the reduced acidification in preconditioned hearts is due to increased proton extrusion (by any extrusion mechanism), we measured intra- and extracellular pH. Consistent with earlier studies (1, 13, 21, 25), the data in this study clearly show that there is a significant attenuation of intracellular acidification during ischemia in preconditioned hearts. We reasoned that if this decreased intracellular acidification in preconditioned hearts was due to increased proton extrusion rather than decreased production of protons, then the extracellular pH in preconditioned hearts should be more acidic than intracellular pH in preconditioned hearts and also more acidic than extracellular pH in nonpreconditioned hearts. However, this result was not observed. As shown in Fig. 2, in preconditioned hearts, during ischemia the extracellular pH is similar to the intracellular pH. Furthermore, preconditioned hearts have less intra- and extracellular acidification than nonpreconditioned hearts. If preconditioned hearts generate the same amount of acid as nonpreconditioned hearts, for preconditioned hearts to have less intracellular H⁺ (a higher intracellular pH), it would be necessary for these hearts to have increased extracellular H⁺ (a lower extracellular pH). Instead we found that preconditioned hearts had less extracellular H⁺ and less intracellular H⁺ (a higher extracellular pH and a higher intracellular pH, respectively) compared with nonpreconditioned hearts. These data strongly suggest that preconditioned hearts produce less H⁺ than nonpreconditioned hearts. However, it is possible that preconditioning stimulates some other cell types (e.g., smooth muscle cell or endothelium) to accumulate or neutralize the extruded H⁺. However, if there were markedly different levels of intracellular pH in different cell types that constitute a significant percentage of tissue volume and have high levels of cytosolic P_i during ischemia, we should have observed a split in the P_i peak in the ³¹P NMR spectra, reflecting P_i in two different pH environments, and this splitting was not observed.

Buffering capacity does not appear to account for the difference in intracellular pH between preconditioned and nonpreconditioned rat hearts. P_i, an important pH buffer, can be monitored using ³¹P NMR, and we find no difference between [P_i] in preconditioned versus nonpreconditioned hearts (21). de Albuquerque et al. (8) also find that there is no significant difference in intracellular buffering capacity between preconditioned and nonpreconditioned rat hearts. Extracellular buffering capacity does not appear to be responsible for pH differences either, because the buffer is the same in preconditioned and nonpreconditioned hearts.

These studies demonstrate that, compared with nonpreconditioned hearts, preconditioned hearts exhibit a more alkaline intra- and extracellular pH during ischemia. This more alkaline intracellular pH in preconditioned hearts is unlikely to be due to increased proton extrusion because we observed less, rather than more, extracellular acidification in preconditioned versus nonpreconditioned hearts. The data in this study show that the reduced acidification in preconditioned hearts is due to less acid production. This reduction in proton production is supported by the reduced levels of lactate in preconditioned hearts (16). The mechanism(s) responsible for decreased glycolysis might include reduced metabolic demand, loss of glycogen (25), or inhibition of glycolysis or glycogenolysis.