Introduction

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Protracted myocardial ischemia induces much greater release of norepinephrine (NE) from sympathetic nerve endings than normal conditions. In this case, excessive NE release is carrier-mediated release rather than exocytosis (Schöming, 1990; Dart and Du, 1993). It is caused by reversal of the uptake 1 carrier that is responsible for reuptake of extracellular NE under normoxic condition (Schöming, 1990; Dart and Du, 1993; Kurz et al., 1995). Exaggerated NE release induced by ischemia increases oxygen demand by stimulating heart rate and contractility and decreases oxygen supply by constricting coronary vessels. This vicious cycle accelerates the progression of cell damage in ischemic myocardium and potentiates the arrhythmogenicity of NE (Braunwald and Sobel, 1988; Schöming et al., 1991; Kübler and Strasser, 1994). Therefore, the modulation of excessive NE release would be expected to protect myocardium or improve functional recovery, or limit reperfusion arrhythmias.

Antagonists of ₂-adrenoceptors, yohimbine (1 µM), idazoxan (10 µM), and rauwolscine (1 µM), were reported to attenuate not only carrier-mediated NE release from sympathetic nerve terminals but also reperfusion arrhythmias in perfused guinea pig heart model in normothermic conditions (Imamura et al., 1996).

In cardiac surgery, a surgeon cannot perform an operation without some form of cardioprotection. Hypothermia has been widely acknowledged to be the fundamental component of myocardial protection during cardiac operations (Bigelow et al., 1950a). Although hypothermia permits a long period of ischemic arrest by reduction of oxygen demand (Bigelow et al., 1950b; Reissman and van Citters, 1956) and metabolism (Lee, 1965), it is associated with a number of major disadvantages, which include its detrimental effects on enzyme function (Marttin et al., 1972), energy generation (Lyons and Raison, 1970), and cellular membrane stability (McMurchie et al., 1973). Indeed, warm heart surgery with normothermic cardioplegia improved early postoperative cardiac function (Lichtenstein et al., 1991), but inadequate distribution or interruption of normothermic cardioplegia may induce anaerobic metabolism and warm ischemic injury (Hayashida et al., 1995). Therefore, normothermic cardioplegia must be delivered continuously and homogeneously. Mild hypothermic ("tepid") cardioplegia has been reported to reduce metabolic demand but permitted immediate recovery of cardiac function (Hayashida et al., 1994, 1995). However, the influences of hypothermia on carrier-mediated NE release and the effects of pharmacological intervention on it during hypothermic ischemia were rarely reported. Therefore, we investigated the effect of presynaptic ₂-adrenoceptor blockade on NE release and reperfusion arrhythmia (ventricular fibrillation) in isolated perfused guinea pig hearts of tepid (32°C) ischemia model.

	Materials and Methods

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Ischemia/Reperfusion Experiment. Male Hartley guinea pigs (Sankyo Labo Co., Tokyo, Japan) weighing 400 to 500 g were killed by cervical dislocation under light anesthesia with CO₂ vapor. After midline thoracotomy hearts were rapidly excised and perfused through the aorta at constant pressure (30 mm Hg) with Krebs-Henseleit solution (KHS) of the following composition: 118.2 mmol/l NaCl, 4.83 mmol/l KCl, 2.5 mmol/l CaCl₂, 2.37 mmol/l MgSO₄, 1.0 mmol/l KH₂PO₄, 25 mmol/l NaHCO₃, and 11.1 mmol/l glucose. KHS was gassed with 95% O₂ and 5% CO₂. The temperature of the perfusate was adjusted to 37°C. Hearts were first perfused with gassed KHS for 30 to 45 min until heart rate, left ventricular diastolic pressure (10 mm Hg), and coronary flow reached a steady state. Heart rate was determined by electrograms recorded from the right atrium and the left ventricle. Left ventricular pressure was measured with a hand-made balloon catheter connected to a transducer (Baxter, McGaw Park, IL) and continuously recorded on a polygraph (model DS-1060; Fukuda Denshi Co., Tokyo, Japan). The epicardial temperature was regulated by covering the entire heart with a thermostatic glass chamber and continuously monitored with a temperature probe (PTI-200; Unique Medical Co., Tokyo, Japan).

NE Assay. NE was assayed in the coronary perfusate by high-performance liquid chromatography coupled to electrochemical detector (Eicom, Kyoto, Japan) and software for analysis (AD Instruments Co., Tokyo, Japan). Values were expressed in picomoles per gram of wet heart weight. Perchloric acid and EDTA were added to samples to achieve final concentrations of 0.01 N and 0.025%, respectively. After a short period of storage (within 2 weeks) at 70°C, the samples were thawed. The NE present in the effluent was absorbed on acid-washed alumina adjusted at pH 8.6 with Tris/2% EDTA buffer and then extracted into 200 µl of acetic acid. These final sample aliquots were kept frozen until set in a sampling machine (Autosampler model 33; System Instruments Co., Tokyo, Japan). The mobile phase consisted of 5 mM monochloroacetic acid, 0.5 mM Na₂EDTA, 0.5 mM sodium octylsulfate, and 1.5% acetonitrile at pH 3.0. The flow rate was kept in 0.23 ml/min by a pump (Intelligent Pump model 301; FLOM Co., Tokyo, Japan). Dihydroxybenzylamine was added to each sample as an internal standard before alumina extraction and used for recovery calculation.

Statistics. Values are expressed as mean ± S.E.M. Comparison of more than two groups were performed by ANOVA, with the Bonferroni's t test used for post hoc analysis (StatView 5.0; Abacus Concepts, Berkeley, CA). Paired Student's t test was performed for inner-group comparisons (preischemia versus postischemia). Regression analysis was performed when correlation coefficient was determined. A value of <0.05 was considered statistically significant.

Drugs. The selective ₂-adrenoceptors antagonist yohimbine, selective ₂-adrenoceptors agonist UK 14,304, desipramine (DMI), and 5-N-ethyl-N-isopropyl-amiloride (EIPA) were purchased from Sigma-Aldrich (St. Louis, MO). EIPA and UK 14,304 were dissolved in 99.8% dimethyl sulfoxide, and further dilution was made with perfusion buffer. At the concentration used (i.e., 0.1%), dimethyl sulfoxide had no effect on any preparation in these studies. Other drugs were dissolved in water.

	Results

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NE Release from Isolated Perfused Hearts Subjected to Ischemia/Reperfusion and Ventricular Fibrillation in Normothermic (37°C) and Hypothermic (32°C) Global Ischemia. The cumulative NE release during the 45-min reperfusion evoked by stop-flow normothermic (37°C) ischemia for 10, 20, and 30 min was 9 ± 1 pmol/g (n = 6), 115 ± 7 pmol/g (n = 6), and 520 ± 21 pmol/g (n = 6, i.e., control), respectively (Fig. 1A). On the other hand, NE release during the 45-min reperfusion induced by global ischemia in hypothermia (32°C) for 20, 40, and 60 min was 10 ± 1 pmol/g (n = 6), 158 ± 15 pmol/g (n = 6), and 920 ± 47 pmol/g (n = 6, i.e., control), respectively (Fig. 1B). Most of the NE was released in the first 2 min of reperfusion (Fig. 1, A and B, inset). In 20-min hypothermic ischemia models, reduction of temperature by 5°C markedly reduced NE release by 90% (10 ± 1 pmol/g, n = 6, p < 0.01 versus 37°C). Duration of ventricular fibrillation (VF) of 10-, 20-, and 30-min normothermic ischemia after reperfusion was 0 ± 0 min (n = 6), 0.7 ± 0.1 min (n = 6), and 3.0 ± 0.5 min (n = 6, i.e., control), respectively (Fig. 2A). Duration of VF induced by 20-, 40-, and 60-min hypothermic ischemia was 0 ± 0 min (n = 6), 1.2 ± 0.1 min (n = 6), and 3.9 ± 0.5 min (n = 6, i.e., control), respectively (Fig. 2B). Relationship between NE release and duration of VF is shown in Fig. 2, A and B. It was evident that the ventricular fibrillations lasted progressively longer as NE release increased.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   A, NE release from isolated perfused guinea pig hearts subjected to 10-, 20-, and 30-min global ischemia in normothermia (37°C) followed by 45-min reperfusion. Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. Inset, time course of NE release during reperfusion after ischemia. Points are mean ± S.E.M. (n = 6 in each inset) of NE release rate at the given time. B, NE release from hearts subjected to 20-, 40-, and 60-min global ischemia in hypothermia (32°C) followed by 45-min normothermic (37°C) reperfusion. Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. Inset, time course of NE release during reperfusion after ischemia. Points are mean ± S.E.M. (n = 6 in each inset) of NE release rate at the given time.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A, correlation between the magnitude of NE release and the duration of ventricular fibrillation in isolated guinea pig hearts subjected to 10-, 20-, and 30-min global ischemia in normothermia (37°C) followed by 45-min reperfusion. B, correlation between the magnitude of NE release and the duration of ventricular fibrillation in isolated guinea pig hearts subjected to 20-, 40-, and 60-min hypothermic (32°C) ischemia followed by 45-min normothermic reperfusion. Each point is the mean of six experiments (mean ± S.E.M.) in each ischemic time. The line was calculated by regression analysis; r, correlation coefficient.

NE Release and Its Pharmacological Modulation: Effects of NE Neuronal Transporter Inhibitor and Na⁺-H⁺ Exchanger Inhibitor in Normothermic (37°C, 30 min) and Hypothermic (32°C, 60 min) Global Ischemia. Pretreatment with DMI (100 nM), a neuronal uptake 1 inhibitor, markedly suppressed NE release during 45-min reperfusion after 30-min normothermic ischemia by 84% (85 ± 8 pmol/g, n = 6, p < 0.01 versus control) or after 60-min hypothermic ischemia by 89% (100 ± 11 pmol/g, n = 6, p < 0.01 versus control) (Fig. 3, A and B, respectively). EIPA (10 µM), a Na⁺-H⁺ exchanger inhibitor, also decreased NE release after 30-min ischemia by 69% (163 ± 40 pmol/g, n = 6, p < 0.01 versus control) (Fig. 3A) and after hypothermic ischemia 35% (594 ± 35 pmol/g, n = 6, p < 0.01 versus control) (Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A, NE release from isolated perfused guinea pig hearts subjected to 30-min global ischemia followed by 45-min reperfusion in either the absence (control) or the presence of the NE transporter inhibitor DMI (100 nM) or the Na+-H+ exchanger inhibitor EIPA (10 µM). Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. B, NE release from hearts subjected to 60-min hypothermic (32°C) ischemia followed by 45-min normothermic (37°C) reperfusion in either the absence (control) or the presence of the NE transporter inhibitor DMI (100 nM), or the Na+-H+ exchanger inhibitor EIPA (10 µM). Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. , p < 0.01, significantly different from control by ANOVA with Bonferroni's t test used for post hoc analysis.

NE Release and Its Pharmacological Modulation: Effects of ₂-Adrenoceptor in Normothermic (37°C, 30 min) and Hypothermic (32°C, 60 min) Global Ischemia. Yohimbine (1 µM; Imamura et al., 1996), an ₂-adrenoceptor antagonist, suppressed NE release during reperfusion after 30-min normothermic ischemia by 45% (284 ± 35 pmol/g, n = 6, p < 0.01 versus control) and after 60-min hypothermic ischemia by 46% (499 ± 27 pmol/g, n = 6, p < 0.01 versus control) (Fig. 4, A and B). UK 14,304 (UK) (10 µM; Imamura et al., 1996), an ₂-adrenoceptor agonist, markedly increased NE release in normothermia to 248% (1289 ± 81 pmol/g, n = 6, p < 0.01 versus control) and in hypothermia to 240% (2216 ± 165 pmol/g, n = 6, p < 0.01 versus control) (Fig. 4, A and B). But yohimbine prevented the potentiated effect of UK in normothermia (UK + Yo: 512 ± 64 pmol/g, n = 6, p < 0.01 versus UK) and in hypothermia (UK + Yo: 535 ± 83 pmol/g, n = 6, p < 0.01 versus UK).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A, NE release from isolated perfused guinea pig hearts subjected to 30-min normothermic (37°C) ischemia followed by 45-min reperfusion in either the absence (control), the presence of the 2-adrenoreceptor antagonist Yo (1 µM), the 2-adrenoreceptor agonist UK (10 µM), or combination of UK (10 µM) and Yo (1 µM). Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. B, NE release from hearts subjected to 60-min hypothermic (32°C) ischemia followed by 45-min reperfusion in either the absence (control) or the presence of the 2-adrenoreceptor antagonist Yo (1 µM) or the 2-adrenoreceptor agonist UK (10 µM), or combination of UK and Yo. Each column (mean ± S.E.M., n = 6) represents the total NE release during 45-min reperfusion. , p < 0.01, significantly different from control; , p < 0.01, significantly different from UK by ANOVA with Bonferroni's t test used for post hoc analysis.

Relationship between NE Release and VF and Its Pharmacological Modulation in Normothermic (37°C, 30 min) or Hypothermic (32°C, 60 min) Global Ischemia. In normothermic conditions, duration of ventricular fibrillation after 30-min ischemia, under the intervention of DMI (100 nM) and EIPA (10 µM), was 0.2 ± 0.1 min (n = 6, p < 0.01 versus control) and 0 ± 0 min (n = 6, p < 0.01 versus control), respectively (Fig. 5). Duration of VF after 30-min ischemia under the intervention of yohimbine (1 µM), UK (10 µM), or combination was 0.4 ± 0.1 min (Yo: n = 6, p < 0.01 versus control), 4.3 ± 0.4 min (UK: n = 6, p < 0.01 versus control), and 1.5 ± 0.2 min (UK + Yo: n = 6, p < 0.01 versus UK), respectively (Fig. 5). In hypothermic ischemia models, DMI (100 nM) or EIPA (10 µM) decreased duration of VF to 2.2 ± 0.1 min (n = 6, p < 0.05 versus control) or to 0 ± 0 min (n = 6, p < 0.01 versus control), respectively (Fig. 6). Under the intervention of yohimbine (1 µM), UK (10 µM), or combination, duration of VF was 1.2 ± 0.5 min (Yo: n = 6, p < 0.01 versus control), 6.2 ± 0.5 min (UK: n = 6, p < 0.01 versus control), and 2.0 ± 0.4 min (UK + Yo: n = 6, p < 0.01 versus UK), respectively (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Correlation between the magnitude of NE release and the duration of ventricular fibrillation in isolated guinea pig hearts subjected to 30-min global ischemia in normothermia (37°C) followed by 45-min reperfusion. Each point is the mean of six experiments (± S.E.M.). The line was calculated by regression analysis; r, correlation coefficient. Concentrations were 100 nM DMI, 10 µM EIPA, 1 µM Yo, and 10 µM UK.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Correlation between the magnitude of NE release and the duration of ventricular fibrillation in isolated guinea pig hearts subjected to 60-min hypothermic (32°C) ischemia followed by 45-min normothermic (37°C) reperfusion. Each point is the mean of six experiments (± S.E.M.) in each ischemic time. The line was calculated by regression analysis; r, correlation coefficient. Concentrations were 100 nM DMI, 10 µM EIPA, 1 µM Yo, and 10 µM UK.

Hemodynamic States of Isolated Perfused Guinea Pig Hearts before and after Global Ischemia. Heart rate (HR), coronary flow (CF), and left ventricular developed pressure (LVDP) under diastolic left ventricular pressure adjusted to 10 mm Hg by balloon inflation are presented in Tables 1, 2, and 3, respectively. In preischemic (baseline) or postischemic period, there were no significant differences between control and drug preparation groups, but EIPA (10 µM) reduced HR, CF, and LVDP significantly, compared with another drug groups (p < 0.05). After normothermic or hypothermic ischemia, HR, CF, and LVDP were reduced in all groups, compared with respective baseline values (p < 0.05 by paired t test). In terms of ₂-adrenoceptors, hemodynamics of yohimbine (1 µM), UK (10 µM), and its combination group were similar to control after normo- or hypothermic ischemia.

	Discussion

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Our findings demonstrated that NE release induced by protracted ischemia (60 min) in tepid temperature (32°C) was due to carrier-mediated release, and the modulation of presynaptic ₂-adrenoceptors by yohimbine had the ability to attenuate carrier-mediated NE release and reperfusion arrhythmia in tepid-temperature ischemia.

Several investigations indicated that the reduction of massive NE release, or the blockade of its effects, could attenuate postischemic cardiac dysfunction and associated arrhythmias (Imamura et al., 1996; Hatta et al., 1999; Levi and Smith, 2000). There are the two major mechanisms of NE release from sympathetic nerve endings (Kurz et al., 1995). One is Ca²⁺-dependent exocytosis and the other is Ca²⁺-independent carrier-mediated release. The exocytotic NE releases are evoked by electrical stimulation during normoxia or by short period ischemia (less than 10 min), which is strongly dependent on Ca²⁺ influx (Kurz et al., 1995; Hatta et al., 1999). On the other hand, NE release induced by protracted ischemia is characterized by Ca²⁺-independent carrier-mediated mechanism, and the amount of carrier-mediated NE release was much greater than that of exocytosis (Hatta et al., 1999). In protracted myocardial ischemia, the accumulation of Na⁺, due to compensatory activation of Na⁺-H⁺ exchanger, and free NE in adrenergic terminals trigger a massive release of free axoplasmic NE through a reversal of NE transporter (Levi and Smith, 2000). In our 60-min hypothermic (32°C) ischemia model, EIPA and DMI markedly attenuated NE release from sympathetic nerve endings. The most NE overflow was observed during the first 2 min of reperfusion (Fig. 2, A and B). This rapid washout of NE in the early phase suggests that carrier-mediated NE release had already occurred during protracted ischemia in prejunctional extracellular space.

NE overflow induced by protracted myocardial ischemia directly correlates with the severity of reperfusion arrhythmias (Imamura et al., 1996; Hatta et al., 1999; Levi and Smith, 2000). Changes in intracellular ion homeostasis, particularly Ca²⁺, are thought to play an important role in reperfusion arrhythmias (Tani and Neely, 1989). Indeed, a manifestation of reperfusion injury is ventricular arrhythmia that is associated with an increased intracellular Ca²⁺ density in postsynaptic myocytes, pacemaker cells, and conducting tissue (Tani and Neely, 1989). Stimulation of postsynaptic ₂- or -adrenoceptors by released NE results in an increased intracellular Ca²⁺ concentration via rapid and transient increase of inositol-1,4,5-trisphosphate (InsP₃) mediated by phospholipase C (Anderson et al., 1995; Woodcock et al., 2001) or by increased open probability of voltage-dependent transmembrane Ca²⁺ channel activity (Levi and Smith, 2000), respectively. Accordingly, this reperfusion-induced InsP₃ formation or Ca²⁺ channel activity depends on NE release from the cardiac sympathetic nerve terminals. In postsynaptic myocytes, myocardial ischemia causes the intracellular acidification that activates Na⁺-H⁺ exchanger (Avkiran, 1999). This also results in increased cytosolic Na⁺ and then accumulation of Na⁺ is removed via Na⁺-Ca²⁺ exchanger, operating in reverse mode. Thus, Ca²⁺ overload is developed in postsynaptic myocytes during the early phase of reperfusion (Anderson et al., 1995; Woodcock et al., 2001). Therefore, there are thought to be two major means for attenuation of reperfusion arrhythmias, one is presynaptic reduction of massive NE release and the other is postsynaptic regulation of Ca²⁺ overload.

In our study, more emphasis was paid to presynaptic regulation of NE release than to postsynaptic modulation. We found that the severity of reperfusion arrhythmias was associated with an increase in NE overflow (Figs. 2, 5, and 6). Moreover, it was shown that DMI, a presynaptic modulator of NE release, inhibits not only the severity of reperfusion arrhythmias but also the reperfusion-induced InsP₃ formation in postsynaptic myocardial cells (Du et al., 1998). Therefore, presynaptic reduction of excessive NE release seems to be one of the most important factors controlling reperfusion arrhythmias.

Yohimbine, an ₂-adrenoceptor antagonist, has many pharmacological actions, including modulation of catecholamine release in central and peripheral tissues. It also blocks or reverses catecholamine- or sympathomimetic drug-induced contraction of vas deferens and gastrointestinal tract, platelet aggregation, lipolysis in adipose tissue, and suppression of insulin release (Goldberg and Robinson, 1980). Some investigators show that yohimbine can improve cardiac function and metabolism of ischemic myocardium (Takeo et al., 1991; Sargent et al., 1994) or can reduce regional ischemia (Seitelberger et al., 1988).

Autoinhibitory ₂-adrenoceptors are effective modulators of depolarization-evoked NE release in the normoxic condition (Langer, 1977). Previous study demonstrates that sympathetic nerve ₂-adrenoceptors are coupled to voltage-dependent N-type Ca²⁺ channels through the G_i family of proteins to inhibit neurotransmitter release (Lipscombe et al., 1989). Therefore, stimulation of the receptor by endogenous agonists results in inhibition of NE release and antagonism of the receptor unmasks activation by endogenous NE (Dart et al., 1984; Münch et al., 1996). Antagonists of peripheral presynaptic ₂-adrenoceptors increase exocytotic NE release evoked by electrical field stimulation (Imamura et al., 1994; Kurz et al., 1995).

In protracted myocardial ischemia, in contrast, a blockade of ₂-adrenoceptors with yohimbine inhibited carrier-mediated NE release in our investigations. Indeed, ₂-adrenoceptor activation by UK 14,304 profoundly enhanced NE overflow. Moreover, yohimbine prevented the potentiated effect of UK. ₂-Receptor activation has been reported to be associated with Na⁺-H⁺ exchanger (Ruffolo et al., 1991). Accordingly, subthreshold concentrations of yohimbine (30 nM) or EIPA (3 µM) could not attenuate NE release themselves, but the combination of these inhibited NE overflow (Imamura et al., 1996). This synergism between yohimbine and EIPA suggests that ₂-adrenoceptors play a significant role in the promotion of carrier-mediated NE release. Therefore, we speculate that the presynaptic blockade of ₂-adrenoceptor with yohimbine can attenuate NE overflow by means of its inhibitory action to Na⁺-H⁺ exchanger in isolated guinea pig heart model.

The essential role of myocardial hypothermia in preventing tissue damage during cardiac surgery has been documented (Buckberg, 1987). The protection provided by lowing temperature has been attributed predominantly to the reduction of energy-consuming processes that would delay the depletion of high-energy phosphates (Kirklin et al., 1979).

In our study, NE release became progressively greater with longer periods of global ischemia either in normothermia or hypothermia (Fig. 1, A and B). After more than 40-min period of ischemia under normothermia, further increased NE release would have been observed, which could not be modulated by blockade of the uptake 1 carrier (Schöming et al., 1984). This fact implies that a progressive injury of membrane structures (cell lysis) occurred during prolonged period of ischemia in normothermia. Our experiments showed that NE overflow induced by 20-min ischemia in tepid temperature was significantly smaller than that of normothermia (Fig. 1). Moreover, NE releases induced by 60-min global ischemia in tepid temperature, which might have not been attenuated under 60-min normothermic ischemia, were markedly reduced either by DMI or EIPA. The fact that NE release induced by prolonged ischemia could be attenuated with pharmacological agents represents a part of cardioprotective effect of hypothermia against ischemia.

The effect of hypothermia on carrier-mediated NE release was reported in a recent investigation (Gerber et al., 1999). This study demonstrated that hypothermia reduced NE release induced by 30-min global ischemia in a temperature-dependent manner (Gerber et al., 1999). But this investigation did not emphasize the pharmacological regulation of NE release in hypothermia. Therefore, it was the aim in our present study to determine whether presynaptic pharmacological regulations in tepid ischemia can reduce carrier-mediated NE release. NE release induced by 60-min global ischemia in tepid temperature was decreased to 10% by DMI and to 60% by EIPA, compared with the overflow without drugs. Moreover, blockade of presynaptic ₂-adrenoceptor with yohimbine also attenuated NE release 50%. These results indicate that presynaptic modulations of carrier-mediated NE release via ₂-adrenoceptor in hypothermic ischemia are as effective as in normothermic ischemia.

In conclusion, this study showed that accumulation of NE induced by protracted myocardial ischemia plays an important role in genesis of reperfusion arrhythmias (ventricular fibrillation) during the early phase of reperfusion and that the amount of NE release was associated with the severity of reperfusion arrhythmias. In mild hypothermic protracted ischemia (60 min), massive NE release was due to the carrier-mediated mechanism. Moreover, presynaptic modulations of ₂-adrenoceptors with yohimbine, or neuronal uptake 1 inhibitor and Na⁺-H⁺ exchanger inhibitor were able to attenuate NE release and reperfusion arrhythmia induced as effectively as in normothermic conditions. Our findings highlight the importance of carrier-mediated NE release in the generation of reperfusion arrhythmias and may contribute to the effective myocardial protection in cardiac surgery.