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Pharmacologic Preconditioning Induced by β-Adrenergic Stimulation is Mediated by Activation of Protein Kinase C

Yabe, Ken-ichi; Ishishita, Hiroyuki; Tanonaka, Kouichi; Takeo, Satoshi

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Journal of Cardiovascular Pharmacology: December 1998 - Volume 32 - Issue 6 - p 962-968
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Abstract

A brief period of ischemia and reperfusion has been shown to protect the myocardium from subsequent sustained ischemia and reperfusion (1-3). This phenomenon is known as ischemic preconditioning (I-PC), and several possible mechanisms have been suggested, including activation of protein kinase C (PKC; 4,5), attenuation of acidosis during sustained ischemia (6), and sparing of high-energy phosphates during ischemia (7,8). This protection is considered to be elicited by mediators, such as adenosine, catecholamines, and bradykinin, which are released during the I-PC period.

In a previous study, we demonstrated that pharmacologic preconditioning with a brief period of β-adrenergic stimulation (β-PC) improved cardiac function of ischemic/reperfused hearts, similar to I-PC (9). Nevertheless, it is unclear whether the cardioprotection induced by β-PC is mediated by a mechanism similar to that of I-PC.

Among the possible mechanisms, as described, there is increasing evidence that PKC activation plays a pivotal role in the mechanism of I-PC-induced cardioprotection against the development of myocardial infarction (4,10) and postischemic contractile dysfunction (5,11). If cardioprotection induced by β-PC is mediated by PKC activation, the mechanism of β-PC would be similar to that of I-PC. The purpose of this study was to determine whether the cardioprotection by β-PC is mediated by PKC activation.

MATERIALS AND METHODS

Materials

Male Sprague-Dawley rats (SLC, Shizuoka, Japan), weighing 250-300 g, were used in this study. The experimental protocol was designed according to the Guidelines of Experimental Animal Care issued by the Prime Minister's Office of Japan and approved by The University Committee of Animal Care and Welfare. The following agents were used in the study; isoproterenol hydrochloride (Iso; Nacalai Tesque Co., Ltd., Kyoto, Japan), polymyxin B sulfate (Poly B; Calbiochem, San Diego, CA, U.S.A.), timolol maleate and anti-rabbit immunoglobulin G (IgG; A-6154; Sigma Chemical Co., St. Louis, MO, U.S.A.), and anti-PKC α, δ, and ε (GIBCO BRL, Grand Island, NY, U.S.A.).

Perfusion of isolated rat heart

Rats were anesthetized with diethylether. The hearts were rapidly isolated, placed in a glass organ bath of the Langendorff apparatus, and perfused at 37°C at a constant flow rate of 9.0 ml/min with Krebs-Henseleit solution of the following composition (mM): NaCl, 120; KCl, 4.8; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; CaCl2, 1.25; and glucose, 11. The perfusion buffer was equilibrated with a gas mixture of 95% O2 + 5% CO2 (PO2 >600 mm Hg). The pH of the perfusing buffer was maintained between 7.40 and 7.42 throughout the experiment. A latex balloon with an uninflated diameter of 3.7 mm, connected to a pressure transducer (model TP-200T, Nihon Kohden, Tokyo, Japan), was inserted into the left ventricular cavity through the mitral opening and secured with a ligature that included the left atrial remnants. The ventricle was loaded with 5 mm Hg of initial left ventricular end-diastolic pressure (LVEDP). Left ventricular developed pressure (LVDP) was measured by an electronic manometer (model TP-400T; Nihon Kohden) and recorded on a thermal-pen recorder (model WT-645G, Nihon Kohden) throughout the experiment. Heart rate was triggered from the development of LVDP by a heart-rate counter (AT-601G; Nihon Kohden). Perfusion pressure was monitored through a branch of the aortic cannula by an electric manometer. Rate-pressure product (RPP) was determined by multiplying heart rate and LVDP. All hearts were preperfused for 30 min to stabilize hemodynamics before the experiment was started.

Experimental protocol

Experimental protocols are shown in Fig. 1. All hearts were subjected to 40 min of global ischemia followed by 30 min of reperfusion (standard ischemia/reperfusion insult). For prevention of hypothermia, the hearts were submerged during ischemia in the Krebs-Henseleit solution as described earlier, except for replacement of 11 mM glucose with 11 mM Tris/HCl. This solution was previously equilibrated with a gas mixture of 95% N2 + 5% CO2 (PO2, <10 mm Hg), pH 7.4, and maintained at 37°C during ischemia. The hearts were randomly assigned to the following experimental groups.

FIG. 1
FIG. 1:
Experimental protocol. Hearts were preconditioned with 5-min ischemia followed by 5-min reperfusion in the absence (I-PC) and presence of 5 μM timolol (I-PC + timolol) or 50 μM polymyxin B (I-PC + Poly B). Hearts were pharmacologically preconditioned with 0.25 μM isoproterenol hydrochloride (Iso; β-PC) in the absence and presence of 50 μM polymyxin B (β-PC + Poly B). Then all hearts were subjected to 40 min of ischemia and 30 min of reperfusion.

Nonpreconditioned (Non-PC) group. The hearts were perfused without pretreatment and then subjected to the standard ischemia/reperfusion insult.

I-PC group. The hearts were subjected to 5 min of ischemia followed by 5 min of reperfusion and then subjected to the standard ischemia/reperfusion insult.

β-PC group. The hearts were perfused for 2 min with 0.25 μM Iso followed by 10 min of washout and then subjected to the standard ischemia/reperfusion insult. Iso was applied to the perfusate at an infusion rate of 0.2 ml/min from 12 to 10 min before the standard ischemia/reperfusion insult through a syringe located just proximal to the aortic cannula by using an infusion pump (Syringe pump CFV-2100; Nihon Kohden). The concentration of Iso used was chosen on the basis of the results in a preliminary study (9). That is, the hearts were treated with different concentrations of Iso ranging from 0.01 to 1.0 μM, and it was found that the peak postischemic recovery of RPP was seen in hearts treated with 0.25 μM Iso.

Poly B-treated Non-PC, I-PC, or β-PC group. The Non-PC, I-PC, or β-PC hearts were treated with the buffer containing 50 mM Poly B for the last 20 min of the preischemia. Then the hearts were subjected to the standard ischemia/reperfusion insult. During ischemia, 50 μM Poly B also was present in the submerging Krebs-Henseleit solution. This concentration of Poly B was chosen on the basis of the results in a preliminary study that 50 μM Poly B per se had no effect on the postischemic recovery of RPP of the nonpreconditioned heart. Several investigators reported that 50 μM Poly B was capable of inhibiting PKC in the preconditioning heart (4,11,12).

Timolol-treated Non-PC, I-PC, or b-PC group. The Non-PC, I-PC, or b-PC hearts were treated with 5 μM timolol for the last 15 min of the preischemia. The hearts were then subjected to the standard ischemia/reperfusion insult. This concentration of timolol completely abolished the positive inotropic effect of 0.25 μM Iso.

Assay of creatine kinase activity in effluent

During the whole period of reperfusion, the effluent from the heart was collected, and creatine kinase (CK) activity in the effluent was determined at 20°C by using the CK assay kit (Boehringer Mannheim, Mannheim, Germany). The release of CK in the effluent during preischemia was minimal (<5 nmol/min/g wet wt).

Immunoblotting

The Non-PC, I-PC, and β-PC hearts were freeze-clamped just before the standard ischemia/reperfusion and stored in liquid N2 until use. We did not examine the subcellular distribution of PKC in the Poly B-treated hearts, because it was reported that treatment with Poly B per se induces translocation of PKC (13). The myocardium was separated into membrane and cytosolic fractions according to the following technique. The myocardium was homogenized by using a Teflon homogenizer (4 times of 15-s each at 1,600 rev/min) in the solution containing (mM) Tris-HCl, 20; sucrose, 250; EDTA, 1; EGTA, 1; vanadate, 1; phenylmethylsulfonyl fluoride, 1; β-mercaptoethanol, 10; and 2 μg/ml leupeptine, a protease inhibitor, pH 7.4 at 4°C. The crude homogenate was centrifuged at 12,000 g for 30 min, and the supernatant fluid was centrifuged at 100,000 g for 1 h. The pellet and supernatant fluid and pellet thus obtained were regarded as cytosol and particulate fractions, respectively.

For Western blot analysis, 10 μg protein was applied to 7.5% (wt/vol) sodium dodecylsulfate (SDS)-polyacrylamide gels for electrophoresis and then transferred to PVDF membranes. Blots were blocked with blocking reagent (Block Ace; Snow Brand Milk Products Co., Tokyo, Japan) for 1 h at 4°C. Then they were incubated 12 h with the primary antibody (anti-PKC-δ, -α, or -ε; dilution, 1/1,000) in 10% (wt/vol) of the blocking reagent dissolved in PBS. After the incubation, the blots were exposed for 1 h at 20°C to a peroxidase-conjugated secondary antibody (dilution, 1:2000; anti-rabbit IgG). Membranes were rinsed 3 times with phosphate-buffered saline (PBS) containing 0.2% Tween 20 at intervals of each step and finally developed by the enhanced chemiluminescence Western blotting system with the aid of the ECL kit (Amersham, Buckinghamshire, U.K.).

Statistics

All values are expressed as the mean ± SEM. Functional parameters and release of CK (Fig. 3) were analyzed by a two-way analysis of variance (ANOVA). When significant differences were observed, mean values of the groups were evaluated by using post hoc Bonferroni's test. In Fig. 4, relative amounts of PKC subtypes of the cytosol and particulate groups were analyzed by using two-way ANOVA and post hoc Student's t test. Differences with a probability of ≤5% were considered significant.

FIG. 3
FIG. 3:
The recovery of rate-pressure product (A), left ventricular end-diastolic pressure at the end of reperfusion (B), and creatine kinase activity in perfusate eluted during 30 min of reperfusion (C). The hearts were nonpreconditioned (non-PC), preconditioned with 5-min ischemia followed by 5-min reperfusion (I-PC), and pharmacologically preconditioned with 0.25 μM isoproterenol hydrochloride (Iso) for 2 min followed by 10-min drug-free perfusion (β-PC) in the absence and presence of 50 μM polymyxin B (Poly B), and then subjected to 40 min ischemia and 30 min reperfusion. Values expressed as mean ± SEM of six experiments. *Significantly different from the corresponding Non-PC group (p < 0.05).
FIG. 4
FIG. 4:
The effects of preconditioning on the levels of PKC-α, δ, and ε in the cytosolic and particulate fractions of perfused rat hearts. Hearts were subjected to no drug treatment (nonpreconditioning; Non-PC), 5-min ischemia and subsequent 5-min reperfusion (I-PC), and treatment with 0.25 μM isoproterenol hydrochloride (Iso) for 2 min followed by 10-min drug-free perfusion (β-PC). Values expressed as mean ± SEM of eight experiments. *Significantly different from the corresponding Non-PC group (p < 0.05).

RESULTS

Effects of I-PC and β-PC on cardiac function

The baseline (initial) values of parameters in all groups were similar. Ischemia caused a rapid decline in RPP of the Non-PC heart. On reperfusion, RPP slightly recovered (12.3 ± 1.8% of initial, n = 6). In the preconditioned heart, RPP was decreased by 5-min ischemia and was not recovered to basal level by subsequent 5-min reperfusion (63% of initial). In the hearts treated with 0.25 μM Iso, RPP increased nearly by threefold. After Iso treatment, RPP decreased and reached 45% of the initial value before the onset of 40-min ischemia. After 40 min of ischemia, RPPs of the I-PC and β-PC heart were augmented with time after reperfusion. Both I-PC and β-PC enhanced the postischemic recovery of RPP (Fig. 2A).

FIG. 2
FIG. 2:
The time courses of changes in rate-pressure product (A), left ventricular end-diastolic pressure (B), and perfusion pressure (C) of hearts. The hearts were non-preconditioned (Non-PC), preconditioned with 5-min ischemia followed by 5-min reperfusion (I-PC), and pharmacologically preconditioned with 0.25 μM isoproterenol hydrochloride (Iso) for 2 min followed by 10-min drug-free perfusion (β-PC), and then subjected to 40 min ischemia and 30 min reperfusion. Values expressed as the mean ± SEM of six experiments.

Changes in LVEDP of the Non-PC, I-PC and β-PC hearts are shown in Fig. 2B. LVEDP of the Non-PC heart increased during ischemia and increased again immediately after the onset of reperfusion. The increase in LVEDP during reperfusion was smaller in both I-PC and β-PC hearts than in the Non-PC heart (Fig. 2B).

Changes in perfusion pressure of Non-PC, I-PC, and β-PC hearts are shown in Fig. 2C. A marked decline of perfusion pressure in the I-PC heart and a significant reduction of perfusion pressure were seen during preconditioning. During reperfusion, perfusion pressure of both I-PC and β-PC hearts was higher than that of the Non-PC heart.

Effects of PKC inhibition on RPP, LVEDP, and the release of CK of I-PC and β-PC hearts

In the Poly B-treated Non-PC group, a decrease in RPP was observed. RPP just before the onset of 40-min ischemia was 74.8 ± 2.5% of the initial. Treatment with Poly B did not affect the postischemic contractile function of the Non-PC hearts, but markedly abolished the I-PC- and β-PC-induced cardioprotection against ischemia/reperfusion injury (Fig. 3A). In contrast, the attenuation of the increase in LVEDP of the reperfused heart with I-PC or with β-PC was not abolished by treatment with Poly B (Fig. 3B). No appreciable effects on perfusion pressure of the ischemic/reperfused heart were seen by treatment with Poly B or timolol. Creatine kinase activity of the effluent during 30 min of reperfusion is shown in Fig. 3C. Negligible amounts of CK (<5 nmol/min/g wet wt) were released from hearts with normoxic perfusion for 70 min. The standard ischemia/reperfusion induced a marked increase in the release of CK from the reperfused heart as compared with that of the preperfused heart. I-PC and β-PC attenuated the ischemia/reperfusion-induced release of CK. The release of CK of the Poly B-treated Non-PC heart was similar to that of the Non-PC heart. Treatment with Poly B abolished the attenuation of CK release induced by I-PC or β-PC (Fig. 3C).

Effects of β-adrenoceptor blockade on I-PC and β-PC hearts

Treatment with 5 μM timolol did not affect the postischemic hemodynamics of both the Non-PC and I-PC hearts (Fig. 3A and B). Timolol did not abolish the I-PC-induced limitation of CK release (Fig. 3C). In contrast, treatment with timolol abolished β-PC-induced effects on RPP and LVEDP of the heart and the release of CK.

Subcellular distribution of PKC

Several isozymes of PKC (α, δ, and ε) in the immunoblots of cytosol and particulate fraction were detected by Western analysis and semiquantified (Fig. 4). Each of these immunoblots appeared to be a single band or unresolved doublet of the approximate size indicated: α, 80 kDa; δ, 80 kDa; ε, 90 kDa. The immunoblots for PKC-α in the particulate fraction were not observed. Effects of I-PC or β-PC on the subcellular distribution of PKC are shown in the lower panels of Fig. 4. I-PC and β-PC increased PKC-δ in the particulate fraction. Cytosolic PKC-α tended to be decreased by I-PC and β-PC, but this was not significant. Neither I-PC nor β-PC altered the distribution of PKC-ε between cytosol and particulate fraction.

DISCUSSION

In this study, we showed that treatment with Iso before sustained ischemia/reperfusion resulted in an enhanced recovery of postischemic RPP of the heart, which was similar to that of I-PC. The enhanced recovery was associated with distribution of PKC-δ from the cytosol to the particulate fraction in the heart. Because changes in distribution of PKC from cytosol to the particulate fraction are taken as a marker of activation of PKC (14), β-PC is capable of exerting preconditioning effects on ischemic/reperfused hearts through PKC activation.

We observed that both I-PC and β-PC showed similar changes in the subcellular distribution of PKC, that is, an appreciable increase in PKC-δ in the particulate fraction and an insignificant change, a trend of decrease, in PKC-δ in the corresponding cytosol of the heart. PKC protein level in the particulate fraction is estimated to be <20% of the total PKC present in the cardiac cell (15). Thus it is possible that β-PC- and I-PC-induced reduction in PKC-δ in the particulate fraction could be seen without a significant change in the PKC-δ in the cytosol.

As described in the Methods section, we did not examine changes in distribution of PKC-δ in Poly B-treated, β-PC hearts in our study. Kiss et al. (13) showed the in vitro results that although Poly B inhibits the activation of PKC, treatment with Poly B per se induces translocation of PKC in isolated cells. If so, the Poly B-treated, β-PC heart may exhibit a complicated distribution of PKC. In a preliminary study, we examined the distribution of PKC-δ of the Poly B-treated heart and found a marked translocation of PKC-δ into the particulate fraction. This may confuse the analysis of distribution of PKC in the Poly B-treated, β-PC heart. Thus we considered that it is not appropriate to examine changes in distribution of PKC subtypes in our study.

Treatment with Poly B canceled the β-PC-induced improvement of RPP and suppression of the release of CK, whereas it did not abolish the I-PC and β-PC effects on LVEDP (Fig. 3). The findings appear to indicate that there are diverse mechanisms involving preconditioning effects. Generally, an increase in LVEDP of perfused hearts is believed to depend on an increase in calcium overload (16) or depletion of myocardial high-energy phosphates or both (17). Although an increase in LVEDP of the heart occurred 10-15 min after the onset of ischemia, there was no apparent increase in tissue calcium content, at least during 35-min ischemia (18). In addition, although high-energy phosphate levels in the perfused rat heart markedly declined during ischemia and partially recovered with time after reperfusion, an increase in LVEDP of the heart was not dependent on the period of ischemia or reperfusion. Thus the causes for an increase in LVEDP of the ischemic/reperfused heart are extremely complicated, and we cannot address which possibility is plausible. We also cannot address the reason that the I-PC and β-PC effects on the increase in LVEDP were not canceled by Poly B treatment.

Although we observed similar results in hearts subjected to I-PC and β-PC in terms of RPP, CK release, and LVEDP, there were apparent differences in the effects on these variables between timolol-treated, I-PC, and β-PC hearts. That is, I-PC, unlike β-PC, enhanced the recovery of RPP, suppressed the increase in LVEDP, and attenuated the release of CK in the timolol-treated hearts as well. The findings suggest the following conclusions: (a) β-PC is definitely mediated by β-receptor-stimulating action; (b) there are different mechanisms for preconditioning effects in I-PC and β-PC hearts; (c) probably β-PC may be exerted through mechanisms distinct from I-PC, although they may share certain intracellular signals.

Myocardial ischemia enhances the release of catecholamines from nerve terminals and increases their levels in the synaptic cleft, which may induce activation of α- or β-adrenoceptors or both. Contribution of α-receptor-mediated action to the mechanism of β-PC was ruled out by the findings that the α1-receptor antagonist bunazosine failed to abolish the cardioprotective effects of β-PC under the current experimental conditions (9). This finding is in good agreement with those of Asimakis et al. (19) that pretreatment with phenylephrine did not improve the postischemic contractile function in the perfused rat heart. Furthermore, we observed that β-PC mimicked I-PC in terms of enhancement of the postischemic recovery of RPP and reduction of CK release. Treatment with timolol, however, did not abolish the cardioprotective effects of I-PC. These findings further confirmed that the cardioprotective effects of β-PC somewhat differed from those of I-PC. It is considered that PKC is activated by I-PC-induced mediators such as adenosine, catecholamine, and bradykinin. Goto et al. (20) proposed that several mediators stimulate PKC during the I-PC period, and that cardioprotection is triggered when PKC stimulation exceeds the threshold level. Considering these hypotheses and findings, β-adrenoceptor is one of possible receptors that can be stimulated by I-PC.

Treatment with 0.25 μM Iso elicited a large increase in RPP during 2 min of administration and 5 min after administration, followed by a steep decline in RPP in the following 5 min. The peak increase in RPP (LVDP × heart rate) was caused by the increases of 240% LVDP and 120% heart rate, indicating a major contribution of the development of LVDP to this increase. It is possible that β-adrenoceptor stimulation may cause the excessive consumption of the myocardial energy store, and thus energy production cannot meet increased cardiac muscle contraction, particularly in hearts perfused under a constant flow rate. In fact, we observed in a previous study that myocardial creatine phosphate in the in vivo rat was decreased and lactate content was increased in the first 1 and 2 min after infusion of 2 μg/kg Iso, i.p. (21). The results suggest that catecholamine at a relatively high dose is capable of using a large amount of high-energy phosphates rapidly to meet the incidence of positive inotropic and chronotropic actions and thereby inducing metabolic imbalance between energy consumption and production in the myocardium. Thus it is probable that treatment with Iso in our study induced relative hypoxia in cardiac muscles. Such a hypoxic state in the myocardium might lead to a physiologic state similar to I-PC. It is recognized that a hypoxic state in the heart results in a reduction of tissue glycogen levels, and the partial depletion of tissue glycogen is considered to be a possible mechanism for I-PC (22). Thus treatment with Iso, like I-PC, might result in an induction of hypoxia with low levels of high-energy phosphates, which is similar to I-PC, although the degree of the β-PC-induced hypoxic state is considerably less than that of I-PC.

It is well recognized that β-stimulation can increase the intracellular concentration of Ca2+ via a cyclic adenosine monophosphate (cAMP)-dependent pathway. Furthermore, mild calcium paradox-induced increase in the intracellular Ca2+ concentration was shown to induce preconditioning as well as PKC activation (23). The increase in the intracellular concentration of Ca2+ also stimulates phospholipase C, which may lead to activation of PKC via formation of diacylglycerol. Taken together, it is likely that β-PC, which exerts cardioprotective effects against ischemia/reperfusion injury, induces the activation of PKC via an increase in the intracellular concentration of Ca2+, as with I-PC.

In conclusion, our study showed that the cardioprotection induced by β-PC is mediated by PKC activation and associated with changes in distribution of PKC-δ from cytosol to particulate fraction, as similar to I-PC. The results suggest that PKC activation plays an important role in both β-PC- and I-PC-induced cardioprotection against postischemic contractile dysfunction in perfused rat hearts.

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Keywords:

β-Adrenergic stimulation; Preconditioning; Protein kinase C; Rat hearts

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