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Department of Surgery, University of Colorado, Denver, Colorado 80262
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Ischemic preconditioning (PC) attenuates cardiac
acidosis during global ischemia. This adaptation to
ischemia is detectable before other better known indexes of PC
are manifested. Clarification of the endogenous mechanisms may provide
insights into how protein kinase C (PKC) signaling might be linked to
altered intracellular biochemistry.
31P NMR studies of isolated, buffer-perfused rat heart
were performed to determine whether functionally cardioprotective PC by
cyclic ischemia (CI) and
1-adrenergic stimuli
[phenylephrine (PE)] attenuated acidosis during
ischemia and, if so, whether this
1) involves a PKC-dependent pathway
and is due to 2) decreased
glycolytic proton production, 3) an
increase in proton buffering, or 4)
proton extrusion. At the end of 20 min of global ischemia, both
CI-PC (pH = 6.86 ± 0.14) and PE-PC (pH = 6.90 ± 0.13)
attenuated end-ischemic acidosis (control pH = 6.54 ± 0.1). PKC
blockade with chelerythrine (Chel) prevented the attenuation of
ischemic acidosis by PC stimuli (end-ischemic pH: CI + Chel,
6.43 ± 0.06; PE + Chel, 6.17 ± 0.17). End-ischemic lactate
accumulation was decreased in CI-PC hearts (7.54 ± 0.5 vs. control,
14.61 ± 2.1 µmol/g wet wt) but not in those preconditioned
through the 1-adrenergic
receptor (12.25 ± 0.9 µmol/g wet wt). Physiologically relevant
buffers were not increased in the preconditioned groups. Blockade of
the
Na+/H+
exchanger [NHE; with
5-(N-ethyl-N-isopropyl)
amiloride (EIPA) or HOE-694] eliminated the attenuation of
ischemic acidosis seen with PC stimuli (pH: CI + EIPA, 6.5 ± 0.1;
PE + EIPA, 6.46 ± 0.2; PE + HOE-694, 6.26 ± 0.15; not
significantly different from control). We conclude that CI and
1-adrenergic PC stimuli
attenuate ischemic acidosis, and this may involve the cardiac
amiloride-sensitive NHE. The signaling pathways of both these two
stimuli appear to involve PKC.
myocardial ischemia; myocellular acidosis; sodium/hydrogen
exchange; 31P nuclear magnetic
resonance spectroscopy; cyclic ischemic preconditioning; 1-adrenergic agonist; phenylephrine
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE INITIAL DISCOVERY that cyclic ischemic preconditioning (CI-PC) delays lethal cell injury while decreasing both lactate production and ATP utilization during sustained ischemia was made by Murry and colleagues (18, 19). Preconditioning is now being investigated as a receptor-mediated cardioadaptation that is conserved in several mammalian species, including humans. Some of the unidentified endogenous mechanisms that lead to enhanced postischemic recovery may be transduced through protein kinase C (PKC) (4, 5, 11, 12, 16, 32).Although PKC can phosphorylate numerous myocardial proteins, its role in the regulation of ischemic physiology is unclear.
An attractive mechanism proposed for ischemic injury implicates acidosis as a trigger (26, 27). In this view, unbalanced H+ production in the ischemic myocardium stimulates Na+/H+ exchange, adding to Na+ influx. Increased intracellular Na+ concentration ([Na+]i) load can subsequently cause an increase in cytosolic free Ca2+ concentration ([Ca2+]i) by a variety of mechanisms, including both influx and endogenous release (22, 26, 27). Interventions that delay the rise in [Ca2+]i correspondingly delay the onset of myocardial necrosis.
An early and reproducible benefit of CI-PC that can be detected even before reperfusion is a reduction of intracellular acidosis during ischemia (1, 3, 25, 27, 35). On the basis of evidence of decreased glycolytic H+ production, Steenbergen et al. (27) proposed that by maintaining a higher intracellular pH (pHi) during ischemia, the Na+/H+ exchanger (NHE) would be less active. By maintaining lower [Na+]i until end ischemia, less Ca2+ should be exchanged into the cell. Therefore, the protected myocardium would exhibit diminished necrosis and improved mechanical function. Indeed, CI-PC prevents the deleterious increases in [Na+]i and [Ca2+]i that occur during ischemia (27, 31).
Although this interpretation fits well with the data obtained, decreased intracellular H+ concentration ([H+]i) during global ischemia is possible as a result not only of decreasing production (reflected by lactate) but also of increasing intracellular buffering or proton extrusion (9, 26).
Our group and others have shown that
1-adrenergic PC protects
postischemic function in rat heart (4, 12, 30, 32). This mechanism
involves PKC (12, 16, 30).
1-Adrenoceptor agonists such as
phenylephrine (PE) produce intracellular alkalinization, possibly via
indirect stimulation of the NHE (22). We hypothesized that both CI or
PE preconditioning stimuli would attenuate ischemic acidosis, perhaps
by similar mechanisms mediated by PKC. Specifically, we determined
whether 1) CI and
1-adrenergic PC stimuli
attenuate ischemic acidosis, 2) both
stimuli limit ischemic acidosis via a PKC-dependent pathway,
3) CI and
1-adrenergic PC decrease
ischemic proton production (as measured by lactate accumulation),
4) CI- and
1-adrenergic-induced limitation
of ischemic acidosis involves buffering by producing increased
Pi, and
5) CI- and
1-adrenergic-induced limitation
of ischemic acidosis is due to proton extrusion by the NHE.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Materials. Male Sprague-Dawley rats (weight 300-350 g; Sasco, Omaha, NE) were fed a standard diet and quarantined in a quiet environment for 14 days before experimentation. The animal protocol was reviewed and approved by the Animal Care and Research Committee of the University of Colorado Health Sciences Center. PE was obtained from American Regent Laboratories (Shirley, NY). Chelerythrine chloride (Chel) was from LC Laboratories (Woburn, MA). 5-(N-ethyl-N-isopropyl) amiloride (EIPA) was obtained from Research Biochemicals International (Natick, MA). (3-Methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE-694) was a generous gift from Dr. H. J. Lang (Hoechst-Roussel Pharmaceuticals, Somerville, NJ). All other reagents utilized were obtained from Sigma Chemical (St. Louis, MO).
Isolated rat heart ischemia-reperfusion. After appropriate anesthesia (pentobarbital sodium, 60 mg/kg ip) and anticoagulant (heparin, 500 U ip) were administered, hearts were excised and placed in a 4°C buffer solution. Within 45 s, hearts were retrogradely perfused in the nonworking, isovolumetric Langendorff mode (80 mmHg, 37°C) with nonrecirculated phosphate-free Krebs-Henseleit buffer solution (116 mM NaCl, 11 mM glucose, 4.0 mM KCl, 24 mM NaHCO3, 1.2 mM CaCl2, and 1.19 mM MgSO4) saturated with 92.5% O2-7.5% CO2 and achieving a pH of 7.4, a PO2 of at least 450 mmHg, and a PCO2 of 40 mmHg.
A water-filled latex balloon was inserted through the left atrium into the left ventricle and adjusted to a left ventricular end-diastolic pressure (LVEDP) of 8-10 mmHg during the initial equilibration period. This preload volume was not adjusted after initial equilibration. Left ventricular developed pressure was measured with an intraventricular balloon catheter attached to a computerized bridge amplifier/digitizer (MacLab 8, ADInstruments, Milford, MA) and continuously recorded on a Macintosh Quadra 800 minicomputer (Apple Computers, Cupertino, CA) in millimeters of Hg. A 1-mm apical ventriculostomy was created. Global ischemia was induced by stopping flow of buffer to the heart, and the heart was held at 35 ± 0.4°C by flowing thermostatically heated air around the perfusion chamber while the probe was within the bore of the magnet. The experiment protocol is outlined in Fig. 1.
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-phosphate
resonance of ATP and was expressed as a percentage of the baseline ATP
concentration. PCr and Pi were
determined also by measuring the area under the curves corresponding to
their respective resonances.
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Experimental design.
A total of 67 animals were examined in this study. Eighteen rats were
utilized to assess functional data, and this group was divided into
three treatment groups (control, CI, and PE;
n = 6 per group) (Fig. 1). Control
hearts were perfused for 20 min and then subjected to normothermic
global ischemia for 20 min, followed by 40 min of reperfusion
(Fig. 1A). A three-way stopcock on
the aortic cannula was turned to halt perfusion and initiate global
ischemia. Normothermia was maintained by bathing the heart in
37.5°C perfusate during ischemia. Functional recovery of
initial developed pressure was utilized as the primary outcome
measurement. Preconditioning with CI was established with cessation of
perfusate flow for four periods of 5 min, each separated by 5 min of
perfusion, followed by 20 min of global ischemia and 40 min of
reperfusion (Fig. 1B). The
1-adrenergic preconditioned
hearts were equilibrated for 8 min and then exposed to PE (1.0 µmol/min) for 2 min, followed by 10 min of normal perfusion, 20 min
of global ischemia, and 40 min of reperfusion (Fig.
1C).
Statistical analysis. Measured parameters are presented as means ± SE. Differences were assessed by using a repeated-factor ANOVA model (with one between factor comprising the treatment and one repeated, within factor). Differences due to ischemic duration and interaction between factors (groups × time) were detected at P < 0.05 (SUPERANOVA, Abacus Concepts, Berkeley, CA). Selected linear contrasts were performed for group means (i.e., comparisons assessing treatment efficacy and blocker efficacy) with significance accepted at P < 0.001. Subsequently, the data were also reanalyzed using a one-factor ANOVA model [with less power but with the ability to perform Scheffé's post hoc test with significance preset at P < 0.05 (StatView 4.0.1, Abacus Concepts)]. The significance detected between groups was similar with both statistical approaches.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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PC stimuli.
On the basis of previous experience with the isolated, perfused rat
heart model (4, 16), we utilized 20 min of global, normothermic
ischemia, followed by 40 min of reperfusion. We first reproduced the functional protection that we previously reported (4,
16) with 1-adrenergic
stimulation and verified improved postischemic function after PC with
four cycles of ischemia (5 min of ischemia followed by
5 min of reperfusion) (18, 19, 27, 31). Although a transient decrement
in function followed the CI stimulus, function rapidly returned to the
equilibration baseline. No difference in preischemic function was noted
between groups. In untreated hearts, reperfusion after 20 min of
ischemia resulted in a recovery of 50.2 ± 6.1% of
preischemic baseline function. PE-treated hearts, however, recovered
75.3 ± 3.5% of initial function
(P < 0.05), whereas hearts
pretreated with CI achieved 86.2 ± 1.6% recovery
(P < 0.05). Pretreatment with either CI or PE improved LVEDP during postischemic reperfusion. At end reperfusion, LVEDP in untreated hearts was 34.8 ± 2.0 mmHg, in contrast to 17.2 ± 0.7 mmHg (P < 0.05) and 22.8 ± 1.4 mmHg (P < 0.05) for CI- and PE-treated hearts, respectively.
Regulation of ischemic acidosis. Pretreatment of hearts with either CI (Fig. 3) or PE (Fig. 4) attenuated ischemic acidosis. pHi at the onset of ischemia was similar between groups. The mean pHi in untreated hearts at the end of ischemia was 6.54 ± 0.10. In contrast, the end-ischemic pHi fell to only 6.90 ± 0.13 (P < 0.05 vs. control) in PE-treated hearts and 6.86 ± 0.14 (P < 0.05 vs. control) in CI-PC hearts. Indeed, preconditioned pHi curves diverged from control rapidly and became different after the initial 5 min of ischemia, which is in agreement with the findings of others (27, 35).
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1-adrenergic stimuli
disappeared when a PKC blocker was infused at the time of the
1-adrenergic stimulus (Fig. 4).
Similarly, Chel eliminated the protection against ischemic acidosis
conferred by CI pretreatment [end-ischemic pH: 6.43 ± 0.06, P < 0.05 vs. CI alone,
P = not significant (NS) vs.
control] (Fig. 3). Chel infusion had no effect on the normoxic
pHi. However, during
ischemia, the pH in hearts treated with a combination of PE + Chel fell rapidly to values more acidic than in untreated control
hearts (6.17 ± 0.17 by the end of the ischemic period,
P < 0.05 vs. PE alone or
control). This increased acidosis could be due to a nonspecific effect
of the PKC blocker or, more likely, could be peculiar to the PE
stimulus (because this difference was not significant vs. the drug
alone or the Chel + CI hearts at the end of ischemia).
Effector mechanisms.
The changes in tissue lactate and alanine after 20 min of
ischemia are shown in Fig. 5. At
the end of the ischemic period, untreated hearts produced 14.61 ± 2.09 µmol/g wet wt of lactate. CI-PC hearts generated significantly
less lactate (7.54 ± 0.54 µmol/g wet wt,
P < 0.05). However, pretreatment
with an 1-adrenergic stimulus
did not reduce ischemic lactate accumulation (12.25 ± 0.91 µmol/g
wet wt, P = NS). Alanine levels were
small and did not differ between any group.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In different mammalian species, PC protects hearts from postischemic cardiac injury. Although it appears that the proximal signaling pathways might involve PKC (4, 5, 11, 12, 16, 32), the distal effector mechanisms remain elusive. The roles of PKC in cardiac physiology and PC are also under investigation. The PKC family of kinases is itself regulated by combinations of factors and can phosphorylate a wide variety of protein targets in the cell (4, 5, 32). The variety of locations, regulation, and targets suggest that these kinases might comprehend a spectrum of effects.
One of the earliest differences that can be noted between control and PC hearts is the preservation of pHi during ischemia (1, 25, 27, 35). In this study we examine the prevailing explanation for this observation. We analyze the intracellular H+ pool during global ischemia as a balance of competing metabolic production and elimination processes (Fig. 6). These mechanisms could be regulated by PKC and other PC signals (4, 10, 24). We hypothesize that analysis of this early adaptation against ischemic acidosis might further our general understanding of how PC and PKC regulate ischemic physiology.
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In this discussion, we first dissect the role of decreased ischemic acidosis as an early effect of PC signals from the potential regulatory roles that pH and PC might play in affecting subsequent developments (ischemic and postischemic) in the rat heart. Next, we evaluate the role of three antiacidosis mechanisms and the role of PKC on NHE-driven H+ extrusion within the context of global ischemia. Finally, we consider the ramifications of pH extrusion by NHE and the clues pointing toward unsuspected PKC functions in PC.
Role of pH in PC. Steenbergen et al. (27) originally investigated ischemic acidosis as a potential mechanism of protection in rat heart preconditioned with cyclic ischemic episodes (CI-PC). Murry et al. (19) had previously shown that CI-PC dog hearts produced less lactate during regional ischemia. H+ efflux conducted by the NHE is believed to trigger Na+ entry into acidified myocytes (22, 26). There is substantial data implicating the subsequent rise in [Ca2+]i in mediating ischemic injury, myocellular dysfunction, and death (22, 26). Therefore, Steenbergen et al. (27) suggested that decreased [H+]i production from glycolysis might account for decreased Na+ entry via Na+/H+ exchange and, consequently, might lessen Ca2+ overload. Measurements of tissue Na+ and Ca2+ in preconditioned hearts suggest that this is indeed the case (27, 30, 31). Therefore, the attenuation of ischemic acidosis, and hence Ca2+ overload, was perceived as an attractive mechanism to explain the postischemic protection induced by PC.
The proposal that attenuated acid production is not a perfect explanation of the ionic cascade leading to injury was recently provided by two of the original proponents of the hypothesis (2, 34). Asimakis (2) found that low glycogen levels during the diurnal cycle did not correlate with protection. Similarly, a collaboration that included Steenbergen and co-workers (34) reported that glycogen depletion (with glucagon) reduced lactate formation (a surrogate for ischemic H+ production) but not lethal ischemic injury. Schaefer et al. (25) have also reported that glycogen depletion (by hypoxic episodes), followed by a delay, decreases acidosis but does not relate infallibly to postischemic recovery. These studies emphasize that decreasing H+ production (by interventions other than CI-PC) is not intrinsically sufficient to confer protection. Using different approaches, we show that PC mechanisms might decrease ischemic acidosis by extrusion mechanisms that are independent of H+ production (Figs. 3-5). Other studies offer further evidence to dissociate ischemic acidosis from postischemic developments. Several studies report that (in nonadapted hearts) the pH at end ischemia does not predict postischemic recovery (8, 13). Investigations in our laboratories (15) also indicate that certain PC stimuli protect postischemic function (after modest ischemia) despite severe ischemic acidosis. However, a closer analysis shows that only those stimuli that prevent ischemic acidosis protect tissue viability (in addition to function) (15). Furthermore, we have shown (15) that phorbol 12-myristate 13-acetate (PMA), which does not protect postischemic function, prevents ischemic acidosis and also protects tissue viability (15). Conversely, studies that pharmacologically inhibit postischemic protection and ischemic pH preservation [see Fig. 4 and our previous reports (4, 15, 16)] concurrently associate, but do not establish, causality between acidosis and postischemic developments. In another interesting study, Murphy et al. (17) showed that lipoxygenase inhibitors eliminated functional protection induced by CI-PC but not protection against acidosis. This could imply that PC engages separate mechanisms of protection against both acidosis and stunning (4, 15). Alternatively, distal lipoxygenase-related mechanisms might synergize together with preserved pH to diminish postischemic injury (4, 17). Therefore, without a better understanding of the mechanism of pH regulation induced by PC, the distal interactions within the host of ischemic and PC factors cannot be easily separated into causes (necessary vs. sufficient) and effects (protection against infarction, apoptosis, stunning, etc.). Another approach presumes that ischemic acidosis itself may be a lesser problem compared with the consequences of NHE-driven Na+ entry. Indeed, pharmacological inhibition of NHE-1 (particularly during the active pH-restitution phase of early reperfusion) appears therapeutically useful (22, 26). This leads to the prospect that the endogenous adaptation mechanism of CI-PC and inhibition of NHE (particularly during reperfusion) might both provide similar coverage against [Na+]i-driven ionic overload and other postischemic injury end points. However, these two protective strategies affect the development of ischemic acidosis (which precedes subsequent reperfusion events) in distinct ways. Typically, during ischemia (in nonconditioned hearts) extracellular acidification and ATP depletion quickly retard H+ efflux by the NHE (22, 26). Thus, in these hearts, NHE blockers do not significantly affect the development of ischemic acidosis (20). However, after protracted ischemia, NHE inhibitors may actually increase acidosis (14). Our data show that PC of rat hearts additionally prevents acidosis from the beginning of ischemia. (The significations of this adaptation are considered below.) Taken together, these data indicate that if acidosis is relieved by NHE (notably during early reperfusion), then the ensuing Na+ influx can trigger subsequent ionic exchanges and injury. Interruptions of this cascade at any of the several intervening steps can decouple the usual detrimental association between ischemic acidosis and postischemic injury. Such a decoupling could be achieved by either pharmacological inhibitors or by signal-induced cellular adaptation. Conversely, although the attenuation of acidosis may be an useful component of some PC strategies, it is not sufficient. Indeed, certain protective maneuvers such as NHE inhibition or hypothermia may operate independently of mechanisms that preserve pH during ischemia. Therefore, the regulation of pH during ischemia by PC stimuli is likely to be most useful for identifying PKC-regulated targets and other ancillary mechanisms that cooperate together to produce observable adaptations during and after ischemia.Effect of PC on regulation of ischemic acidosis. Numerous studies have shown that ischemic PC attenuates acidosis during sustained ischemia. The best-studied case is the globally ischemic rat heart, but the effect has been reproduced in rabbit and pig hearts. In rats, Murphy and co-workers (7) found that CI-PC prevented ischemic acidosis and also protected postischemic function, both by PKC-dependent mechanisms. Furthermore, they found that direct PKC stimulation with diacylglycerol preserved ischemic acidosis and postischemic function, albeit weakly. Surprisingly, the prototypical PKC stimulant PMA, which is effective in protective postischemic viability in rabbits, did not improve either outcome. In agreement with the previous study (7), we have reported (15) that, in rats, the nonphysiological PKC stimulant PMA does not induce protection of postischemic function but does protect cell viability at higher doses.
In contrast, physiological signaling by either ischemic PC or1-adrenergic receptors
apparently induces the antiacidotic adaptation through PKC mechanisms
(Figs. 3 and 4). This suggests that phorbol esters and endogenous PKC
stimuli engage different second messengers and activate PKC isoforms in
distinct ways (4, 15). Although several studies (2, 8, 25, 34) have surmised that decreased production of
H+ from the glycolytic pathway is
unrelated to the protective adaptations induced by CI-PC, this is the
first report to investigate the mechanisms for regulating ischemic
acidosis in these preconditioned hearts. Figures 3 and 4 show that,
quite independent of H+
production, H+ extrusion through
the NHE is a prominent part of the adaptive mechanism responsible for
regulating ischemic pH after CI- or 1-adrenergic PC.
However, in rabbits, Bugge and Ytrehus (6) have suggested that NHE
blockade provides protection that is additive to that provided by
CI-PC. This suggests that, in rabbits, CI-PC-stimulated PKC isoform
mechanisms are different from those in rats (32) and thus might not
regulate ischemic pH through the NHE. Indeed, in isolated hypoxic
rabbit cardiomyocytes, direct H+
extrusion by another mechanism
(H+-ATPase) has been identified.
These investigators found that, in these rabbit myocytes, simulated
ischemic PC activated the H+-ATPase via PKC. This resulted
in protection against apoptosis over the next 25 h, providing a
rationale explaining why preservation of the pH can be important (11).
These studies of PC against ischemic acidosis emphasize that signaling
may stimulate subsets of PKC isoform(s) to conduct specific changes (4,
15). The interaction of PKC isoforms with specific targets appears to
include selected mechanisms for preserving pH during ischemia
acting with other independent mechanisms regulating postischemic
functional recovery and viability. The precise details of PKC-mediated
adaptation may differ among species (4, 32).
Mechanism of PC against ischemic acidosis in rats. Until recently, the view that CI-PC preserved the ischemic pH by decreasing H+ production was virtually unquestioned. In an isolated system such as the globally ischemic heart, decreased intracellular acidosis is due to either decreased H+ production, increased extrusion, or augmented buffering capacity (9) (Fig. 6). Many investigators have offered different types of circumstantial evidence to support the first mechanism, decreased H+ production during ischemia (1, 3, 27, 35). Thus CI-PC rat hearts invariably have less glycolytic flux, as indicated by accumulated lactate. Moreover, glycogen depletion (by hypoxic periods similar to the CI-PC protocol) reduced lactate accumulation and caused postischemic protection (1, 3, 27, 35). However, as noted earlier, reduced ischemic glycogen and lactate contents per se appear insufficient to confer protection (2, 8, 25, 34).
Our data are the first to show that decreased lactate accumulation does not explain decreased acidosis after PC in globally ischemic rat hearts. While investigating the ability of the1-adrenergic pathway to mimic
CI-PC (4, 16), we observed that, although both stimuli preserved pH
during ischemia, only CI-PC decreased accumulated lactate (Fig.
5). Thus, for CI-PC hearts, decreased glycolysis might well be one
mechanism contributing to preserving pH during ischemia (1, 3,
8, 27, 35). However, the 1-adrenergic PC mechanism must
have achieved cardiac antiacidosis by another process. We therefore
proceeded to examine the alternative possibilities of extrusion and
buffering. Upon inhibition of H+
extrusion by the cardiac NHE,
1-adrenergic PC did not
preserve pH any longer. This striking result was corroborated when two different NHE inhibitors (EIPA and HOE-694) also abolished the preservation of ischemic pH induced by CI-PC (Figs. 3 and 4). For both
PC stimuli, the antiacidotic effect was abolished by PKC blockade. This
suggests that, in the globally ischemic rat heart, PKC-dependent
mechanisms are available for modulating
H+ extrusion, thereby helping to
preserve the pH during ischemia.
Proton buffering does not appear to be a major mechanism of preserving
the ischemic pH in these preconditioned hearts.
Pi [with an acidic
dissociation constant
(pKa) of
6.9], bicarbonate, and histidyl residues of intracellular
proteins comprise the primary ischemic myocardial intracellular buffers
(9). At a pH >6.4, the buffering capacity of the ischemic heart
depends mainly on Pi. The
endogenous bicarbonate buffering system, with a
pKa of 6.1, is
not an optimal intracellular buffer and is unavailable during extended
global ischemia. Only at very low pH values do the
high-capacity,
low-pKa (5.2)
intrinsic protein buffers become significant (1, 9, 33). During
ischemia, PCr is broken down to sustain cellular ATP,
accumulating Pi as a by-product of
this reaction (Table 1), which could increase intracellular buffering
capacity. However, an increase in intracellular buffering capacity was
not evident in the preconditioned groups as measured by
Pi (Table 1). Indeed, despite less
Pi in CI-treated hearts, ischemic
acidosis remains attenuated. These results are consistent with a recent
study that also failed to find any difference in buffering capacity in
CI-PC rat hearts (1).
The NHE functions as an electroneutral, gradient-driven, facilitated
transport mediator across the lipid bilayer for
Na+ and
H+ (22, 26). Activation of the NHE
can occur through either acidification of the cytosol, which
allosterically alters its affinity for
H+, or by direct phosphorylation
of the antiporter protein (10, 22, 26). The activity of the NHE is
primarily driven by a transsarcolemmal proton gradient, and it is
therefore incapacitated if the transmembrane gradients of pH and
Na+ collapse (i.e., high
[Na+]i
with or without extracellular acidification). The NHE is exquisitely sensitive to small changes in pHi
yet is active over a relatively small range of pH. Thus, during global
ischemia in nonconditioned hearts, NHE activity has been
difficult to detect (20, 26). Conversely, during reperfusion, its role
is more prominent (26). However, direct measurements of pH regulation
by the NHE under perfused conditions are partially obscured by other
mechanisms of Na+-linked pH
regulation (notably
Na+-HCO
3
transport) (26, 33). Therefore, Avkiran and co-workers (26) have shown
(in nonconditioned rat hearts) that the role of
Na+/H+
exchange is often difficult to demonstrate in the presence of bicarbonate buffer (26). In agreement with the literature, HOE-694 or
EIPA did not alter the course of ischemic acidification in nonconditioned hearts, indicating that pH regulation thorough this
mechanism is not normally significant during global ischemia in
rat hearts. In contrast, in both CI- and PE-PC rat hearts (perfused with physiological bicarbonate-based Krebs-Henseleit buffer), the
exchanger appears to be active during global ischemia (Figs. 3
and 4). Upon inhibition of the NHE, these PC stimuli can no longer
attenuate the development of ischemic acidosis. Hence, the ischemic pH
drops into the acidic range (pH 6.5), similar to control values. We
infer that proton extrusion via the NHE may play a principal role in
the antiacidotic effects of the PC stimuli tested.
The cardiac PKC mechanism that affects
H+ extrusion during global
ischemia may not involve direct phosphorylation of the NHE. NHE-1, the predominant isoform in the heart (10, 29), is an 815-residue, 200-kDa membrane-spanning dimer with three consensus sites
for glycosylation and several putative sites for phosphorylation. These
sites do not appear to be PKC consensus sites (10). Therefore, the NHE
may not be directly activated by PKC. One possibility is PKC
phosphorylation of regulatory protein units that modify the NHE.
Alternatively, PKC may activate mitogen-activated protein kinases or
calmodulin-dependent protein kinase II, which have phosphorylation
sites on the NHE-1 (10). Berk and co-workers (29) have proposed that
ribosomal S6 kinase may be one such NHE-1 kinase that regulates the
allosteric and pH set point. At the other extreme, it is equally
possible that the NHE is not modified at all. Indeed, the PKC family
impacts on a large range of proteins (other than kinases) that could
help preserve the transmembrane driving gradients over longer ischemic
periods and thereby permit NHE function to continue during
ischemia.
We conclude that decreased H+
production during ischemia is not sufficient to explain how PC
preserves the ischemic pH. In contrast with normal rat hearts, the
contribution of the NHE to ischemic pH regulation becomes significant
after PC with either CI or the
1-adrenergic agonist PE. The PC
stimuli achieve this physiological adaptation through PKC signaling,
but the exact sites of regulation require further
examination.
Although our data show that NHE activity occurs during ischemia
in preconditioned hearts, neither CI-PC (27, 31) nor PE-PC (31) appears
to create a corresponding intracellular
Na+ overload in these hearts.
Steenbergen et al. (27) found that intracellular
Na+ levels were comparable to
those in untreated controls, whereas intracellular
Ca2+ overload was attenuated (27).
Similarly, Tosaki and co-workers (31) found that both CI-PC and PE-PC
hearts avoided excess Na+ as well
as Ca2+ accumulations. Our
estimations of the ionic contents in CI-PC hearts at end
ischemia [performed according to Tosaki et al. (30, 31)] are confirmatory (unpublished data). Ramasamy et al. (23) observed a small increase in intracellular
Na+ resonance area after CI-PC,
but only during the first 10 min of ischemia (not thereafter).
They found that CI-PC upregulated intracellular
Na+ efflux noticeably during
reperfusion and suggested that both the NHE and the
Na+-K+-2Cl
exchanger may be involved. Although some studies have suggested that
1-adrenergic receptors
stimulate NHE activity, detailed investigations show that this does not
appear to involve PKC (22). Therefore, it is possible that NHE activity
is not upregulated per se but is simply drawn along kinetically because
of Na+ extrusion by ancillary
processes.
We speculate that the PC stimulus sets up an adaptive mechanism for the
ischemic myocardium to remove its intracellular
Na+ load without incurring the
lethal increase in intracellular
Ca2+. Mechanisms that extrude
intracellular Na+ together with
counteranions (24) such as bicarbonate (22), lactate (33), and
phosphate (21) are present in heart and could be PKC targets (24). This
could allow the heart to effectively export
H+ during ischemia,
together with a dispensable counteranion, electroneutrally and without
consuming ATP. Other effects might include other synergistic actions
such as inhibition of
Na+/Ca2+
exchange or glycolysis.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants GM-08315, HL-44186, and HL-43696. J. I. Shapiro is supported by an American Heart Association Established Investigator Grant.
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FOOTNOTES |
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Address for reprint requests: A. Banerjee, Dept. of Surgery, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave. (C-320), Denver, CO 80262.
Received 17 May 1996; accepted in final form 5 May 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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(
significantly different from CI
(P < 0.05).
