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Cardiovascular Research 2004 64(1):125-131; doi:10.1016/j.cardiores.2004.06.011
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Copyright © 2004, European Society of Cardiology

Extraneuronal enzymatic degradation of myocardial interstitial norepinephrine in the ischemic region

Takafumi Fujiia, Toji Yamazaki*,a, Tsuyoshi Akiyamaa, Shunji Sanob and Hidezo Moria

aDepartment of Cardiac Physiology, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan
bDepartment of Cardiovascular Surgery, Okayama University Medical School, Okayama 700-8558, Japan

* Corresponding author. Tel.: +81-6-6833-5012x2380; fax: +81-6-6872-8092. E-mail address: yamazaki{at}ri.ncvc.go.jp

Received 19 March 2004; revised 28 May 2004; accepted 14 June 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: Catechol O-methyltransferase (COMT) is believed to exert degradative action at high norepinephrine (NE) levels. Although COMT exists in cardiac tissues, the contribution of cardiac COMT activity to regional NE kinetics, particularly in ischemia-induced NE accumulation, remains unclear. We investigated the role of cardiac COMT in NE kinetics in the ischemic region. Methods: We implanted a microdialysis probe into the left ventricular myocardium of anesthetized rabbits and induced myocardial ischemia by 60-min coronary artery occlusion. We monitored myocardial interstitial levels of NE and its metabolites in the presence and absence of a COMT inhibitor. We intraperitoneally administered entacapone (10 mg/kg) 120 min before control sampling. Results: In control, entacapone increased interstitial dihydroxyphenylglycol (DHPG, intraneuronal NE metabolite by monoamine oxidase (MAO)) levels and decreased interstitial normetanephrine (NMN, extraneuronal NE metabolite by COMT) and 3-methoxy-4-hydroxyphenylglycol (MHPG, extraneuronal DHPG metabolite by COMT) levels, but did not change interstitial NE levels. Coronary occlusion increased NE levels to 165±48 nM at 45–60 min of occlusion. This increase was accompanied by increases in DHPG and NMN levels (11.3±1.1 and 9.3±1.3 nM at 45–60 min of occlusion). Entacapone augmented the ischemia-induced NE and DHPG responses (333±51 and 22.9±2.4 nM at 45–60 min of occlusion). In contrast, the ischemia-induced NMN response was suppressed by entacapone (2.0±0.4 nM at 45–60 min of occlusion). Reperfusion decreased interstitial NE levels and increased interstitial DHPG and NMN levels. Entacapone suppressed changes in NE and NMN levels, but augmented the increase in dialysate DHPG. Conclusion: Myocardial ischemia evoked increases in myocardial interstitial NE and NMN levels. COMT inhibition augmented the increase in NE (substrate of COMT) levels and suppressed the increase in NMN (metabolite by COMT) levels. In the ischemic heart, COMT contributes to the removal of accumulated NE in the myocardium.

KEYWORDS Adrenergic agonists; Autonomic nervous system; Ischemia; Reperfusion; Neurotransmitters


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
It has been reported that myocardial ischemia evokes an excessive norepinephrine (NE) accumulation in the myocardial interstitial space [1,2]. Outward NE transport through the uptake1 carrier has been proposed as an important mechanism responsible for this ischemia-induced NE accumulation [2–4]. The presence of such high NE levels in the myocardial interstitium may be involved in the progression of myocardial cell injury and a higher incidence of malignant arrhythmia [5,6].

In the non-ischemic heart, released NE is reclaimed by cardiac sympathetic nerve endings via the uptake1 carrier and repackaged or metabolized to dihydroxyphenylglycol (DHPG) by monoamine oxidase (MAO). NE, which escapes the synapses to the myocardial interstitium, spills over into the bloodstream or is taken up by extraneuronal cells via the uptake2 carrier and mainly degraded to NE metabolites by catechol O-methyltransferase (COMT) [6–9] (Fig. 1). In the ischemic heart, normal transport by the uptake1 carrier is impaired and NE spills over into the bloodstream, which is decreased due to the reduction of myocardial blood flow [1]. Therefore, extraneuronal enzymatic degradation may be the only mechanism that decreases myocardial interstitial NE. Little information, however, is available on the extraneuronal NE degradation by COMT in the ischemic region [10,11].


Figure 1
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  		Fig. 1 Schema of putative factors affecting norepinephrine (NE) degradation in the cardiac sympathetic nerve ending and the extraneuronal cell. COMT, catechol O-methyltransferase; DHPG, dihydroxyphenylglycol; MAO, monoamine oxidase; NMN, normetanephrine; MHPG, 3-methoxy-4-hydroxyphenylglycol.

 
Until now, available methodology for examination of organ-specific NE degradation has been limited to the assessment of radiolabelled-catecholamine kinetics [12]. Previously, using the dialysis technique in the in vivo heart, we demonstrated that coronary occlusion evokes a marked increase of myocardial interstitial NE levels in the ischemic region [1,2,4] and that outward NE transport through the uptake1 carrier is involved in this NE efflux [2–4]. Moreover, we recently reported that the dialysis technique makes it possible to simultaneously monitor interstitial levels of NE and extraneuronal metabolites in the rabbit skeletal muscle [13]. Therefore, we consider it possible to elucidate extraneuronal NE metabolism and the role of COMT in its metabolism in the ischemic region. In the present study, we applied the dialysis technique to the heart of anesthetized rabbits and investigated myocardial interstitial levels of NE and its extraneuronal metabolites during coronary occlusion and reperfusion and examined the effect of COMT blockade on myocardial interstitial levels of NE and its metabolites.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1. Animal preparation
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult male Japanese white rabbits (2.5–3.2 kg) were anesthetized with pentobarbital sodium (30–35 mg/kg iv). The level of anesthesia was maintained with a continuous intravenous infusion of pentobarbital sodium (1–2 mg/kg/h). The rabbits were intubated and ventilated with room air mixed with oxygen. Body temperature was maintained at around 38 °C with a heating pad and lamp. Heart rate, arterial pressure, and electrocardiogram were monitored and recorded continuously. Heparin sodium (200 IU/kg) was first administered intravenously and then maintained with a continuous infusion (5–10 IU/kg/h) to prevent blood coagulation. With the animal in the lateral position, the fifth or sixth rib on the left side was partially removed to expose the heart. A small incision was made in the pericardium, and the dialysis probe was implanted in the region perfused by the left circumflex coronary artery (LCX) of the left ventricular wall. A snare was placed around the main branch of LCX to act as the occluder for later coronary occlusion. To ensure that the sampling area was in the ischemic region, we examined the color and motion of the ventricular wall during a brief occlusion and confirmed that the dialysis probe was correctly located. To avoid a preconditioning effect, the duration of occlusion was limited to within seconds.

2.2. Dialysis technique
Materials suitable for cardiac dialysis probes have been described in detail elsewhere [14]. Briefly, we designed a handmade long transverse dialysis probe. One end of a polyethylene tube (25-cm length, 0.5 mm OD, and 0.2 mm ID) was dilated with a 27-gauge needle (0.4 mm OD). Each end of the dialysis fiber (8-mm length, 0.31 mm OD, and 0.20 mm ID; PAN-1200 50 000 molecular weight cutoff, Asahi Chemical Japan) was inserted into the polyethylene tube and glued. We used a fine guiding needle (30-mm length, 0.51 mm OD, and 0.25 mm ID) for implantation of the dialysis probes. We connected a guiding needle to a dialysis probe with a stainless rod (5-mm length and 0.25 mm OD). At perfusion speed of 2 µl/min, in vitro recovery rates of NE, DHPG, normetanephrine (NMN), and 3-methoxy-4-hydroxyphenylglycol (MHPG) were 46±8%, 48±1%, 33±3%, and 46±2%, respectively (number of dialysis probes=3) [15].

Dialysis probes were perfused with Ringer's solution at a speed of 2 µl/min using a microinjection pump (Carnegie Medicine CMA/100). Ringer's solution consisted of (in mM) 147.0 NaCl, 4.0 KCl, 2.25 CaCl2. Sampling periods were 30 min (1 sampling volume=60 µl) in control and 15 min (1 sampling volume=30 µl) during occlusion and reperfusion. Each sample was collected in a microtube containing 3 µl of 0.1 N HCl to prevent amine oxidation. Based on the results of our previous study [1,2], we commenced the protocol followed by a stabilization period of 2 h. Taking into consideration the dead space between the dialysis fiber and sample tube, we sampled the dialysate.

Dialysate NE, DHPG, NMN, and MHPG concentrations were measured as indices of myocardial interstitial NE, DHPG, NMN, and MHPG levels. Furthermore, dialysate NE and DHPG were used as indices of COMT substrate, and dialysate NMN and MHPG as indices of COMT production. We used three distinct systems of high-performance liquid chromatography (HPLC) with electrochemical detection for the highly sensitive measurements: one for NE, one for DHPG, and one for NMN and MHPG measurement [16–18]. The mobile phase consisted of 1-octane-sulfonic acid sodium salt in phosphate buffer and methanol. In each HPLC system, the concentration of each component and the reference voltage were adjusted to the optimum condition. One-third each of the dialysate sample was used for the measurement of NE, DHPG, and NMN and MHPG. Dialysate NE concentration was measured by the first HPLC after removing interfering compounds by the alumina procedure [14,16]. Dialysate DHPG concentration was measured by direct injection into the second HPLC [17]. Dialysate NMN and MHPG concentrations were measured by direct injection into the third HPLC [18]. The detection limits of NE, DHPG, NMN, and MHPG were 0.2, 0.2, 1, and 0.9 pg/injection.

2.3. Experimental protocols
After control sampling, we occluded the main branch of LCX for 60 min and then released the occluder. We continuously sampled dialysate from the ischemic region during 60 min of coronary occlusion and 15 min of reperfusion.

2.3.1. Vehicle group (n=8)
We administered saline intraperitoneally as vehicle 120 min before control sampling. After control sampling, we observed the time course of dialysate NE, DHPG, NMN, and MHPG levels from the ischemic region during 60 min of coronary occlusion and 15 min of reperfusion.

2.3.2. Entacapone group (n=8)
To elucidate role of COMT in the ischemia-induced changes in myocardial interstitial NE and its metabolites, we observed the effect of COMT inhibitor on dialysate NE, DHPG, NMN, and MHPG levels in the ischemic region. We administered intraperitoneally a COMT inhibitor entacapone (10 mg/kg; Orion Parma, Espoo, Finland) 120 min before control sampling. Entacapone was dissolved in phosphate-buffered saline, the pH of the solution was adjusted to 7.4. The route and dose of entacapone were selected to cause the full inhibition of soluble COMT in tissue [19]. After control sampling, we observed the time course of dialysate NE, DHPG, NMN, and MHPG levels with a similar protocol to that used in the vehicle group.

At the end of each experiment, the rabbits were killed with an overdose of pentobarbital sodium, and the implant regions were checked to confirm that the dialysis probes had been implanted within the cardiac muscle.

2.4. Statistical analysis
Hemodynamic and dialysate NE, DHPG, MHPG, and NMN responses to coronary occlusion in the presence and absence of COMT inhibitor were statistically analyzed by two-way analysis of variance with repeated measures on one factor [20]. When a statistically significant effect of coronary occlusion was detected as a whole, the Newman–Keuls test was applied to determine which mean values differed significantly from each other. When statistically significant effect of the COMT inhibitor was detected, the Newman–Keuls test was applied to determine which periods differed significantly between the vehicle and entacapone groups. Statistical significance was defined as P<0.05. Values are presented as means±SE.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1. Time course of heart rate and arterial pressure
The time course of heart rate and mean arterial pressure is shown in Table 1. In the vehicle group, heart rate decreased after 15 min of occlusion, whereas in the entacapone group, heart rate increased after 30 min of occlusion. There was, however, no significant difference in heart rate between groups.


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  		Table 1 Time course of heart rate and mean arterial pressure during coronary occlusion and reperfusion

 
Coronary occlusion significantly decreased mean arterial pressure in both groups. In the entacapone group, mean arterial pressure was higher than those in the vehicle group at each sampling point, while changes in mean arterial pressure were similar to those in the vehicle group.

3.2. Dialysate NE levels in the ischemic region
Coronary occlusion significantly altered dialysate NE levels (Fig. 2). In the vehicle group, dialysate NE levels were 0.39±0.07 nM in the control and increased after coronary occlusion. During 60 min of coronary occlusion, dialysate NE levels markedly increased and reached 165±48 nM at 45–60 min of occlusion. After reperfusion, dialysate NE levels decreased to 62±40 nM, although their levels were higher than those in the control. In the presence of entacapone, dialysate NE levels also markedly increased and reached 333±51 nM at 45–60 min of occlusion. These increases in dialysate NE levels after 30 min of coronary occlusion were significantly enhanced by entacapone whereas entacapone did not change dialysate NE levels in the control (0.26±0.07 nM). After reperfusion, dialysate NE levels decreased but remained higher than those in the vehicle group.


Figure 2
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  		Fig. 2 Dialysate norepinephrine (NE) levels in the ischemic region. Values are means±SE. *P<0.05 vs. control value, §P<0.05 vs. value at 45–60 min of occlusion, {dagger}P<0.05 vs. concurrent value of vehicle group.

 
3.3. Dialysate DHPG levels in the ischemic region
Coronary occlusion significantly altered dialysate DHPG levels (Fig. 3). In the vehicle group, dialysate DHPG levels were 6.5±0.5 nM in the control and did not change within 30 min of coronary occlusion. After 30 min of occlusion, dialysate DHPG levels gradually increased and reached 11.3±1.1 nM at 45–60 min of occlusion. After reperfusion, dialysate DHPG levels further increased to 29.5±2.6 nM. In the presence of entacapone, dialysate DHPG levels gradually increased during the ischemia and reached 22.9±2.4 nM at 45–60 min of occlusion. After reperfusion, dialysate DHPG levels further increased to 52.6±8.4 nM. In the presence of entacapone, dialysate DHPG levels in the control (9.9±0.8 nM) and after reperfusion were higher than those in the vehicle group.


Figure 3
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  		Fig. 3 Dialysate dihydroxyphenylglycol (DHPG) levels in the ischemic region. Values are means±SE. *P<0.05 vs. control value, §P<0.05 vs. value at 45–60 min of occlusion, {dagger}P<0.05 vs. concurrent value of vehicle group.

 
3.4. Dialysate NMN levels in the ischemic region
Coronary occlusion significantly altered dialysate NMN levels (Fig. 4). In the vehicle group, dialysate NMN levels were 2.9±0.4 nM in the control and increased after 30 min of occlusion and reached 9.3±1.3 nM at 45–60 min of occlusion. After reperfusion, dialysate NMN levels further increased (11.9±2.0 nM). Entacapone decreased dialysate NMN levels in the control to undetectable levels. Then dialysate NMN levels increased after 30 min of occlusion and reached 2.0±0.4 nM at 45–60 min of occlusion. After reperfusion, dialysate NMN levels further increased to 4.1±0.8 nM. Their NMN levels were lower than those in the vehicle group at each sampling point before, during, and after coronary occlusion.


Figure 4
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  		Fig. 4 Dialysate normetanephrine (NMN) levels in the ischemic region. Values are means±SE. *P<0.05 vs. control value, §P<0.05 vs. value at 45–60 min of occlusion, {dagger}P<0.05 vs. concurrent value of vehicle group.

 
3.5. Dialysate MHPG levels in the ischemic region
Coronary occlusion significantly altered dialysate MHPG levels (Fig. 5). In the vehicle group, dialysate MHPG levels were 3.9±0.3 nM in the control. Dialysate MHPG levels transiently decreased 15–30 min after occlusion (3.6±0.4 nM), but recovered after 30 min of occlusion. After reperfusion, dialysate MHPG levels increased to 5.5±0.3 nM. Entacapone substantially decreased dialysate MHPG levels in the control (1.5±0.4 nM). In the presence of entacapone, dialysate MHPG levels further decreased during occlusion and reached 0.6±0.2 nM at 30–45 min of occlusion. After reperfusion, dialysate MHPG levels increased to 2.3±0.5 nM. Their MHPG levels were lower than those in the vehicle group at each sampling point before, during, and after coronary occlusion.


Figure 5
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  		Fig. 5 Dialysate 3-methoxy-4-hydroxyphenylglycol (MHPG) levels in the ischemic region. Values are means±SE. *P<0.05 vs. control value, §P<0.05 vs. value at 45–60 min of occlusion, {dagger}P<0.05 vs. concurrent value of vehicle group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
Using the dialysis technique in the in vivo rabbit heart, we observed myocardial interstitial levels of NE and its neuronal and extraneuronal metabolites in the ischemic region and examined the contribution of extraneuronal NE degradation by COMT to myocardial interstitial NE levels. Our data demonstrate that COMT plays an important role in NE metabolism during 60 min of coronary occlusion and reperfusion.

4.1. Myocardial interstitial NE and its metabolites under control conditions
The administration of entacapone did not alter myocardial interstitial NE levels in the control. Degradation of NE by COMT may play a minor role in the changes in myocardial NE levels [21]. In general, NE that is taken up by cardiac sympathetic nerve endings is repackaged or metabolized to DHPG by MAO. On the other hand, NE that is taken up via extraneuronal NE transport systems by extraneuronal cells is metabolized to NMN or MHPG by COMT [7–10,22,23] (Fig. 1). In the present study, entacapone increased DHPG in the myocardial interstitium but did not alter myocardial interstitial NE levels in the control. Myocardial interstitial levels of DHPG were about 16-fold higher than those of NE and about 2- to 3-fold higher than those of NMN in the control. Therefore, under physiological conditions, released NE could be largely taken up by cardiac sympathetic nerve endings via the uptake1 carrier and transferred into stored vesicle or metabolized to DHPG by MAO. A smaller percentage of released NE, which escapes the synapses, is taken up by extraneuronal cells via the uptake2 carrier and is metabolized to NMN by COMT. Compared with MAO, COMT could play a minor role on the degradation of released NE in the control.

After entacapone administration, increases in myocardial interstitial DHPG accompanied decreases in myocardial interstitial MHPG levels in the control. These metabolites of NE penetrate the cell membrane by diffusion [24]. Their values serve as indices of the neuronal and extraneuronal NE metabolism. COMT blockade suppressed the degradation of DHPG to MHPG. Therefore, the decrease in MHPG levels and increase in interstitial DHPG levels could be ascribed to inhibition of COMT by entacapone. In rabbit heart, we confirmed the existence of COMT activity with the main substrate of COMT being DHPG rather than NE.

4.2. Myocardial interstitial NE and its metabolites during coronary occlusion
Myocardial interstitial NE levels markedly increased after 15 min of occlusion. In this phase, outward transport of NE via the uptake1 carrier takes place from cardiac sympathetic nerve endings [2,3]. The marked increase in myocardial interstitial NE could be due to this non-exocytotic NE release and inhibition of neuronal reuptake via the uptake1 carrier [2–4].

Entacapone augmented increases in myocardial interstitial NE levels after 30 min of coronary occlusion. This result indicates that COMT contributes to the degradation of myocardial interstitial NE in this phase. Neuronal reuptake via the normal mode of uptake1 is dependent on the sodium gradient between the intra- and extraneuronal space [25]. Neuronal degradation of released NE via neuronal uptake cannot be expected in this phase because of a reduced sodium gradient [3,25]. Moreover, NE spillover into the bloodstream is decreased due to reduced myocardial blood flow [1]. On the other hand, extraneuronal NE uptake operates independently of the sodium gradient [26]. Burgdorf et al. [27] demonstrated that the extraneuronal monoamine transporter is activated during metabolic distress such as low flow ischemia. Previous investigations with similar preparations suggested that ketamine augments ischemia-induced NE accumulation by inhibition of extraneuronal uptake [28,29]. We consider that substantial NE in the myocardial interstitium is taken up alternatively by extraneuronal cells via the uptake2 carrier and is metabolized to NMN by COMT in this phase. Thus, COMT activity plays an important physiological role in NE degradation in the ischemic period.

Myocardial interstitial NMN levels increased after 30 min of occlusion. Entacapone decreased basal myocardial interstitial NMN levels and suppressed the ischemia-induced increase in myocardial interstitial NMN levels. This suppression is consistent with the finding that COMT contributes to the degradation of myocardial interstitial NE via extraneuronal NE uptake. Furthermore, even in the presence of entacapone, myocardial interstitial NMN levels increased after 30 min of occlusion. An increase in interstitial NE levels may overcome the inhibition of COMT by entacapone. Although COMT could play a minor role in the degradation of released NE in the control, a substantial increase in myocardial NE may cause the high affinity of the extraneuronal COMT system. In the control period, an extraneuronal COMT system may contribute to DHPG degradation, whereas in the ischemic period, both neuronal NE uptake and MAO activities may be suppressed by ischemia and alternatively the extraneuronal COMT system may promote NE degradation based on the fact that the myocardial interstitial NE levels in the ischemic period were 10-fold higher than those of DHPG. Therefore, we consider that increases in myocardial interstitial NE levels shift the main substrate of COMT from DHPG to NE.

The relationship between DHPG and MHPG supports our interpretation. In the control, entacapone increased myocardial interstitial DHPG levels and decreased myocardial interstitial MHPG levels. In contrast, in the ischemic period, increases in myocardial interstitial DHPG levels were not associated with increases in myocardial MHPG levels, but increases in myocardial NE levels accompanied increases in myocardial NMN. Thus, both DHPG and NE are metabolized by the extraneuronal COMT system, but the amount of NMN and MHPG may be dependent on the concentration of their substrate. Thus, there was a clear difference in the main metabolite by COMT between the non-ischemic and ischemic periods. The main metabolite by COMT was NMN rather than MHPG in the latter [30]. These results are limited to ischemia within 60 min because prolonged ischemia accompanies the structural membrane defects, and other mechanisms for NE release and degradation may be involved [31].

4.3. Myocardial interstitial NE and its metabolites after reperfusion
Myocardial interstitial NE levels decreased after reperfusion although they were higher than those in control. On the other hand, myocardial interstitial DHPG levels increased after reperfusion. The uptake1 carrier resumes normal transport function after reperfusion [2,4]. The inward NE transport via the uptake1 carrier could contribute to the decrease in myocardial interstitial NE levels. The increase in axoplasmic NE by uptake and the recovery of MAO activity could increase myocardial interstitial DHPG levels [2]. Thus, neuronal degradation by MAO contributes to decreasing myocardial interstitial NE after reperfusion.

Reperfusion caused a decrease in myocardial interstitial NE levels and an increase in myocardial NMN levels, both of which changes were suppressed by administration of entacapone. During the early reperfusion period, COMT activity promotes the degradation of NE. Myocardial interstitial NE levels were higher than those in the control. Therefore, during the ischemic and reperfusion periods, these higher NE levels in myocardial interstitium may serve as an effective substrate of COMT for NE degradation via extraneuronal NE uptake.

During the reperfusion period, increases in myocardial interstitial DHPG accompanied increases in myocardial interstitial MHPG levels. Furthermore, administration of entacapone suppressed both of these changes. Myocardial interstitial DHPG levels were similar to myocardial interstitial NE levels. These data suggest that COMT activity promotes the degradation of DHPG. Alternatively, higher NE level in myocardial interstitium may produce MHPG via COMT activity. Recently, we demonstrated in rabbit skeletal muscle that local administration of higher NE increased dialysate NMN but not MHPG levels, whereas local administration of higher DHPG increased dialysate MHPG levels [19]. Therefore, higher NE and DHPG levels serve as the substrate of COMT and independently yield NMN and MHPG during the reperfusion period. Thus, COMT activity plays an important physiological role in the reperfusion period.

4.4. Methodological considerations
In the presence of a high concentration of entacapone, mean arterial pressure was higher than that in the vehicle group at each sampling point before, during, and after coronary occlusion, but changes in mean arterial pressure were similar to those in the vehicle group. In the previous and present studies, intraperitoneal administration of entacapone did not alter control dialysate NE levels from skeletal muscle and myocardium [19]. In humans, entacapone did not alter plasma catecholamine levels or hemodynamics at rest or during exercise [32]. The influence of entacapone on pressure-regulating peptides remains unclear. An increase in mean arterial blood pressure might decrease dialysate NE levels through a baroreflex mechanism. Furthermore, baroreflex-independent and non-exocytotic NE efflux leads to high NE levels in the myocardial interstitium of ischemic regions, making it unlikely that hemodynamic change contributes to the removal of accumulated NE during myocardial ischemia.

Two major classes of COMT have been defined on the basis of their location: a soluble, cytosolic form and a membrane-bound form [33]. Entacapone inhibits both classes of COMT. The soluble, cytosolic form is generally assumed to be the predominant form of the enzyme. The membrane-bound form has been suggested to be responsible for O-methylation at low and physiologically relevant concentrations of the catecholamine neurotransmitters, whereas the soluble, cytosolic form predominates under conditions that lead to saturation of the membrane-bound form [33]. In the present study, myocardial interstitial norepinephrine levels reached 100–1000 times the normal plasma concentrations after 15 min of occlusion. Thus, a soluble, cytosolic form could contribute to the observed decrease in myocardial interstitial NE levels in the ischemic region.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
Under physiological condition, extraneuronal enzymatic degradation by COMT plays a minor role on the inactivation of myocardial interstitial NE. Under ischemic conditions, however, myocardial interstitial NE levels are markedly increased by ischemia. Normal transport by the uptake1 carrier is impaired and NE spillover into the bloodstream is decreased due to the reduction of myocardial blood flow, but extraneuronal enzymatic degradation by COMT contributes to the decrease in myocardial interstitial NE levels in the ischemic region.


   Acknowledgements
 
This work was supported by Grants-in-Aid for scientific research (15590787) from the Ministry of Education, Culture, Sports, Science and Technology; the Research Grants for Cardiovascular Disease (H13C-1) from the Ministry of Health, Labor and Welfare. The authors thank Orion-Pharma (Espoo, Finland) for the supply of entacapone.


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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Y. Kuroko, T. Yamazaki, N. Tokunaga, T. Akiyama, H. Kitagawa, K. Ishino, S. Sano, and H. Mori
Cardiac epinephrine synthesis and ischemia-induced myocardial epinephrine release
Cardiovasc Res, June 1, 2007; 74(3): 438 - 444.
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