Gwdiovascular
Research
EISEVIER
CardiovascularResearch32 (1996) 274-285
Infarct size-reducing effect of nicorandil is mediated by the ICAT,
channel but not by its nitrate-like properties in dogs
Tsuneo Mizumura, Kasem Nithipatikom, Garrett J. Gross *
of Pharmaco1og.v and Toxicology, Medical College of Wisconsin. 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
Received 19 October 1995;accepted14 February 1996
Abstract
Objectives: We wished to determine whether the cardioprotective effect of nicorandil to reduce infarct size is blocked by
glibenclamide, a selective KATP channel antagonist, or methylene blue, a nitric oxide (NO)/guanylate cyclase inhibitor, in dogs. The
second aim was to determine if glyceryl trinitrate produces a cardioprotective effect in the same model and to test if this effect is blocked
by methylene blue and not by glibenclamide. We also determined whether adenosine release from the ischemic-reperfused area is an
accurate index of ischemic severity in the presence of these drugs. Methods: Barbiturate-anesthetized dogs were subjected to 60 min of
left anterior descending coronary artery (LAD) occlusion followed by 3 h of reperfusion. In the first three groups, either nicorandil (100
yg/kg bolus + 10 p,g/kg/min),
glyceryl trinitrate (10 *g/kg bolus + I p,g/kg/min)
or an equivalent volume of saline was given
intravenously 15 min before LAD occlusion and continued to the time of reperfusion. In the next three groups, glibenclamide (0.3
mg/kg) was administered 15 min before drug infusion. In the final three groups, methylene blue (80 FM) was given intracoronarily 5
min before nicorandil or glyceryl trinitrate and continued until 15 min following reperfusion. Coronary venous blood samples were
collected at various times during ischemia and following reperfusion and the concentration of adenosine measured. Results: Nicorandil
produced a marked reduction in infarct size expressedas a percent of the area at risk (NC group, 12.2 f 3.2% vs. Control group,
25.7 f 4.1%, P < 0.05) and this effect was completely abolished by pretreatmentwith glibenclamide. However, intracoronary administration of methylene blue did not block the cardioprotective effect of nicorandil. On the other hand, glyceryl trinitrate also produced a
significant reduction in infarct size (GTN group, 13.0 f 3.1%) and this effect was reversedby methylene blue but not by glibenclamide.
Adenosine concentrations in coronary venous blood were significantly reduced after reperfusion in the groups with small infarctions as
comparedwith the Control group. Conclusions: These results suggestthat at equieffective cardioprotective dosesthe infarct size-reducing
effect of nicorandil in dogs is mediatedvia opening of myocardial K,,, channelsand that the cardioprotective effect of glyceryl trinitrate
is most likely to be mediated via activation of guanylate cyclase at a site yet to be determined.
Keywords:
Nicorandil; Nitroglycerin; Adenosine; Myocardial infarction; potassium channel, ATP-sensitive; Sulphonylureas; Infarct size; Dog, anesthetized: Potassiumchannel openers
1. Introduction
It is well known that nicorandil, an ATP-sensitive
potassium (K,,,) channel opener-nitrate, is a potent vasodilator and is widely used for the treatment of angina
pectoris in Japan [l]. Nicorandil has a unique pharmacological profile and has been shown to relax resistance
vessels by its KATP channel activity and to relax conductance vessels by its nitrate-like activity [2]. These dual
actions result in a reduction in preload and afterload and
these properties may be partially responsible for its antianginal efficacy [3]. In addition to these vasodilating
actions, nicorandil has also been reported to be cardioprotective in several animal models of ischemia-reperfusion
injury. Recently, we [4] reported that a non-hypotensive
dose of nicorandil produced a marked reduction in myocardial infarct size in anesthetized dogs subjected to 60 min
of ischemia and 3 h of reperfusion. Because a previous
study from our laboratory [5] has shown that the cardioprotective effect of nicorandil in stunned myocardium was
independent of its peripheral hemodynamic effects and
* Correspondingauthor. Tel.: ( + l-414) 456-8627;fax: (+ l-414) 266.
8460.
Time for primary
000%6363/96/$15X)0 Copyright 0 1996 Elsevier Science B.V. All rights reserved
PII SOOO8-6363(96)00061-2
review 35 days.
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Department
T. Mizumura et al./Cardior:ascular
2. Methods
2.1. Chemicals
Nicorandil was generously supplied by Chugai Pharmaceutical Co. Ltd., Tokyo, Japan. Glyceryl trinitrate was
purchased from Parke-Davis, Ann Arbor, MI. Methylene
blue was purchased from RBI (Research Biochemicals
International, Natick, MA), and glibenclamide from Sigma
Chemical Co., St. Louis, MO.
2.2. General preparation
of dogs
All experiments conducted in the current study were in
accordance with the “Position of the American Heart
215
Association on Research and Animal Use” adopted in
1984 by the American Heart Association, and the guidelines of the animal care committee of the Medical College
of Wisconsin. The Medical College of Wisconsin is accredited by the American Association of Laboratory Animal Care (AALAC).
Adult mongrel dogs of either sex, weighing 17.5 to 28.0
kg (mean/group = 21.4 & 0.9 to 23.8 + 1.0 kg; NS),
were fasted overnight, anesthetized with the combination
of sodium barbital (200 mg/kg) and sodium pentobarbital
(15 mg/kg), and ventilated with room air supplemented
with 100% oxygen. Atelectasis was prevented by maintaining an end-expiratory pressure of 5-7 cmH,O with a trap.
Arterial blood pH, pCO,, and p0, were monitored at
selected intervals by an AVL automatic blood gas system
and maintained within normal physiological limits (pH
7.35 to 7.45, pC0, 30 to 35 mmHg, and p0, 85 to 100
mmHg1 by adjusting the respiration rate and oxygen flow
or by intravenous administration of 1.5% sodium bicarbonate if necessary. Body temperature was maintained at
38 + 1°C with a heating pad. Aortic blood pressure and
left ventricular pressure were monitored by inserting a
double-pressure, transducer-tipped catheter (PC 77 1, Millar) into the aorta and left ventricle via the left carotid
artery. Left ventricular dP/dt was recorded by electronic
differentiation of the left ventricular pressure pulse, and
heart rate was determined by a tachometer. The right
femoral vein and artery were cannulated for drug administration and for blood gas analysis and myocardial tissue
blood flow, respectively. A left thoracotomy was performed at the fifth intercostal space, the lung was carefully
retracted, the pericardium incised, and a catheter inserted
into the left jugular vein and gently advanced into the great
cardiac vein at its junction with the anterior interventricular vein via the right atrium and coronary sinus for the
subsequent collection of blood samples for adenosine determination. The position of the tip of the catheter in the
great cardiac vein was confirmed by visual inspection
throughout the sampling portion of the protocol. The heart
was then suspended in a pericardial cradle. A proximal
portion of the left anterior descending coronary artery
(LAD) distal to the first diagonal branch was isolated from
surrounding tissue, and a calibrated electromagnetic flow
probe (Statham SP 7515) placed around the vessel. A
flowmeter (Statham 2202) was used to measure blood
flow. A mechanical occluder was placed distal to the flow
probe such that there were no branches between the flow
probe and the occluder. The occluder was used to zero the
flow probe (LAD was occluded for 10 s 20 min before the
60 min experimental occlusion). to occlude the vessel, and
to reperfuse the myocardium. At reperfusion the occluder
was abruptly released in all groups to produce reperfusion
of the previously ischemic area. If the basal heart rate was
less than 150 beats/min, the heart was paced at that rate
with rectangular pulses of 4 ms duration and a voltage
twice threshold via bipolar electrodes clipped to the left
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
was completely blocked by glibenclamide, a selective KATP
channel antagonist, it was assumed that the infarct size-reducing effect of nicorandil is most likely to also be mediated by its myocardial K,,,
channel opening activity;
however, no studies have been performed to confirm this
hypothesis. Therefore, the first objective of the present
study was to determine if the infarct size-reducing property
of nicorandil is reversed by glibenclamide, a selective
K ATP channel antagonist.
The cardioprotective effect of glyceryl trinitrate has also
been reported by several laboratories [4,6,7], however, our
laboratory was the first to demonstrate its infarct size-reducing effect in a model of ischemia-reperfusion injury [ 11.
It has been proposed that an increased activity of soluble
guanylate cyclase in either platelets, endothelial cells, myocardium, or vascular smooth muscle may mediate these
beneficial effects of glyceryl trinitrate [8]. Thus, a second
aim of the present study was to determine if the infarct
size-reducing effect of glyceryl trinitrate is blocked by
methylene blue, a compound which has been shown to
inhibit increases in cyclic GMP resulting from NO-induced
stimulation of soluble guanylate cyclase [9] in various
tissues.
Finally, Kitakaze et al. [lo] suggested that K,,, channel openers such as cromakalim and nicorandil mimicked
the effect of ischemic preconditioning in canine hearts and
these investigators proposed that K,,
channel openers
might be exerting their beneficial effects on infarct size by
increasing the release of adenosine following reperfusion.
On the other hand, previous results from our laboratory
[4,11] have shown that coronary venous adenosine concentrations obtained during reperfusion were significantly reduced in preconditioned, nicorandil- and glyceryl-trinitrate-treated animals as compared to controls, and paralleled the reduction in infarct size. Thus, we concluded that
adenosine release from the area at risk during reperfusion
is a sensitive marker of the intensity of ischemia during
coronary occlusion. Therefore, a third objective of the
present study was to further examine this hypothesis.
Research 32 (19961274-28.5
276
T. Mizumura et al. / Cardiovascular
Jhr-Rep
/El
Fig. 1. Schematic diagram of the experimental protocol. Dogs were
randomly assigned to 9 groups. In the first three groups, nicorandil (100
kg/kg bolus and 10 p,g/kg/min infusion, NC group), glyceryl trinitrate
(10 pg/kg bolus and 1 pg/kg/min
infusion, GTN group), or an
equivalent volume of saline (Control group) were administered intravenously 15 min before a 60 min occlusion period and continued to the
time of reperfusion. In the next three groups, 0.3 mg/kg of glibenclamide
was given intravenously 15 mitt before the same amount of nicorandil
(GLB +NC group), glyceryl trinitrate (GLB +GTN), or saline (GLB
group) was administered 15 min prior to the occlusion period and
continued to the time of reperfusion. In the last three groups, intracoronary methylene blue (80 FM) was given 15 min before nicorandil
(MB + NC group), glyceryl trinitrate (MB + GTN group), or saline (MB
group) and continued to 15 min of reperfusion. In all groups, infarct size
was determined at the end of the 3 h reperfusion period. NC =
nicorandil; GTN = glyceryl trinitrate; Occ = occlusion; Rep =
reperfusion.
atria1 appendage. Pacing was not employed in the few
animals with initial rates more than 150 beats/min. Hemodynamic variables, heart rate, and coronary blood flow
were monitored and recorded by a polygraph throughout
the experiment. The left atrium was cannulated via the
appendage for radioactive microsphere injection.
2.3. Experimental design
The protocols used in this study are shown in Fig. 1.
Animals were sequentially assigned to one of 9 groups.
The experimental protocol included initial hemodynamic
measurements, arterial blood gas analysis before coronary
occlusion, and baseline arterial and venous adenosine measurements. Approximately 5 min before the 60 min LAD
occlusion period, nicorandil (NC group), 100 pg/kg bolus
followed by a 10 pg/kg/min
infusion, glyceryl trinitrate
(GTN group), 10 kg/kg
bolus followed by a 1
bg/kg/min
infusion, or an equivalent volume of saline
(Control group) were administered intravenously and continued to the time of reperfusion. The rationale for choos-
ing these doses of nicorandil and glyceryl trinitrate were
twofold: (1) we have previously shown that these doses of
nicorandil and glyceryl trinitrate produce nearly equivalent
reductions in myocardial infarct size in dogs [4] in the
absence of any systemic hemodynamic effects and (2) the
rates of drug infusion used in the present study have been
shown to yield plasma levels of both nicorandil and glyceryl trinitrate that are well within the therapeutic window
obtained in man [3,12] when these compounds are used as
antiischemic agents. In next three groups, 0.3 mg/kg of
glibenclamide was given intravenously 15 min before nicorandil (GLB + NC group), glyceryl trinitrate (GLB + GTN
group), or saline (GLB group) administration. This dose of
glibenclamide has been previously demonstrated to significantly attenuate the increase in coronary blood flow by the
potassium channel openers, EMD 56431 and nicorandil
[ 131and to block the shortening of the cardiac monophasic
action potential during ischemia in canine hearts [14]. In
addition, this dose of glibenclamide has been shown to
yield plasma levels within the normal therapeutic range
obtained in man [ 151 when glibenclamide is used to treat
diabetes. Perhaps more importantly, this dose of glibenclamide is the maximum allowable in dogs which blocks
the cardiac K,,, channel without producing an increase in
infarct size by itself [16]. In the final three groups, intracoronary methylene blue, 80 p,M, was given 15 min prior
to nicorandil (MB + NC group), glyceryl trinitrate (MB +
GTN group), or saline (MB group), and continued until 15
min into the reperfusion period. These concentrations of
methylene blue were approximated based on the infusion
rate (mg/min), the coronary artery blood flow (ml/min)
its molecular weight (319.85). This concentration of methylene blue (80 p,M) was chosen based on preliminary
studies (n = 4, each group) in which we determined a
concentration-response curve for methylene blue to block
the effect of glyceryl trinitrate to reduce infarct size and
found that 80 pM methylene blue totally abolished the
cardioprotective effect of glyceryl trinitrate (Fig. 2). In all
groups, hemodynamic measurements, blood gas analyses,
and myocardial blood flow measurements were performed
at 30 min into the 60 min occlusion period. After reperfusion, hemodynamics were measured every hour and myocardial blood flow was determined at the end of the 3-h
reperfusion period. Finally, at the end of the experiment,
the hearts were electrically fibrillated, removed and prepared for infarct size determination and regional myocardial blood flow measurement.
2.4. Infarct size determination
At the end of the 3-h reperfusion period, the LAD was
cannulated. To determine the anatomic area at risk (AAR)
and the non-ischemic area, 5 ml of Patent blue dye and 5
ml of saline were injected at equal pressure into the left
atrium and LAD, respectively. The heart was then immediately fibrillated and removed. The left ventricle was dis-
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3hr-Rep
Research 32 (1996) 274-285
T. Mizumura et al. / Cardiovascular Research32 (19961274-285
of 9.4 ml/min starting 30 s prior to the microsphere
injection and continuing for 3 min. On the following day,
the tissue slices were sectioned into subepicardium, midmyocardium, and subendocardium of non-ischemic (3
pieces) and ischemic (5 pieces) regions. Transmural pieces
were obtained from the center of several transverse sections used to determine the AAR and were at least 1 cm
from the perfusion boundaries as indicated by Patent blue
dye. All samples were counted in a gamma counter (Tracer
Analytic 1195) to determine the activity of each isotope in
each sample. The activity of each isotope was also determined in the reference blood flow samples. Myocardial
blood flow was calculated by using a preprogrammed
computer to obtain the true activity of each isotope in
individual samples and tissue blood flow was calculated
from the equation. Q, = Q, X C,/C,, where Q, is myocardial blood flow (ml/min/g
of tissue), Q,. is the rate of
withdrawal of the reference blood flow (9.4 ml/min), C,
is the activity of the reference blood flow sample
(counts/min) and C, is the activity of the tissue sample
(counts/min/g>. Transmural blood flow was calculated as
the weighted average of the three layers in each region.
2.6. Adenosine
measurements
2.6.1. Sample collection
sected and sliced into serial transverse sections 6-7 mm in
width. The non-stained ischemic area and the blue-stained
normal area were separated and both regions were incubated at 37°C for 15 min in 1% 2,3,5-triphenyltetrazolium
chloride (TTC; Sigma) in 0.1 M phosphate buffer adjusted
to pH 7.4. The TTC stains the non-infarcted myocardium a
brick-red color, indicating the presence of a formazan
precipitate that results from the reduction of TTC by
dehydrogenase enzymes in viable tissue. After storage
overnight in 10% formaldehyde, infarcted and non-infarcted tissues within the area at risk were separated and
determined gravimetrically. Infarct size was expressed as a
percent of the area at risk.
2.5. Regional
myocardial
blood fzow
Regional myocardial blood flow was measured by the
radioactive microsphere technique as previously described
in this laboratory [16]. Microspheres were administered at
30 min into the prolonged 60 min occlusion period as well
as at the end of reperfusion. Carbonized plastic microspheres (1.5 pm diameter, New England Nuclear, Boston,
MA) labeled with 14’Ce or ‘5Nb were suspended in isotonic saline with 0.01% Tween-80 added to prevent aggregation. The microspheres were ultrasonicated for 5 min
and vortexed for another 5 min before injection. One
milliliter
of the microsphere suspension (2-4 X lo6
spheres) was given via the left atria1 catheter and flushed
by 5 ml of saline. A reference blood flow sample was
withdrawn from the right femoral artery at a constant rate
and preparation
[I 71
Before LAD occlusion, arterial blood was sampled from
the right femoral artery and coronary venous blood was
withdrawn through a 8-French single-lumen (length 100
cm) catheter which was placed into the great cardiac vein.
Once the catheter was cleared of residual, stagnant blood,
1 ml of blood was aspirated over a 3-5 s period of time
into a chilled 3-ml syringe containing a stop solution
which consisted of a small amount of heparin (2 Al), 11
pM dipyridamole, and 0.6 FM erythro-9(2-hydroxy-3nonyljadenine (EHNA). Immediately after collecting the
sample, the syringe was inverted back and forth gently,
and placed in an ice bucket. Coronary venous blood was
sampled after 5, 15, and 60 min of the LAD occlusion
period, and at 5, 10, 15, and 30 min following reperfusion.
After withdrawing the last sample, all samples were centrifuged for 2 min at 30000 X g at 0°C to separate the
plasma and stop solution mixture from the cellular elements. Subsequently, 1 ml of the supernatant was removed
and transferred to a tube containing 25 pl of cold 7 M
perchloric acid to precipitate plasma proteins. After centrifugation (30000 X g, O”C, 10 min) to separate proteins,
0.6 ml of the supematant was removed and transferred to a
tube containing 25 ~1 of 5N NaOH to neutralize the
solution. Then, 250 ~1 of the solution was transferred to an
autosample vial and mixed with 20 ~1 of 2.0 M acetate
buffer, pH 4.5, and 10 1.11of chloroacetaldehyde. The vial
was capped and heated in an oven at 60°C for 4 h.
Adenosine reacts with chloroacetaldehyde to form a strong
fluorescent 1,Nh-ethanoadenosine. The sample was injected directly into the HPLC from the sample vial.
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
Fig. 2. Graph showing the effects of three different concentrations of
methylene blue on infarct size expressed as a percent of the area at risk in
the presence of glycery-trinitrate-induced
cardioprotection. Methylene
blue 80 FM totally abolished the infarct size-reducing effect of glyceryl
trinitrate (MB 80 JLM + GTN) whereas lower concentrations (10 to 20
FM MB+GTN)
of methylene blue did not significantly block the
reduction in infarct size produced by GTN ( ^ P < 0.05). GTN = glyceryl
trinitrate: MB = methylene blue.
211
T. Mizumura et al. / Cardiocascular Research 32 (19961274-285
278
2.62. HPLC analysis
A newly developed HPLC method was utilized for the
determination of adenosine concentrations in plasma [17].
Briefly, 5 p,l of the samples were injected and chromatographed on a 1090 Series II Liquid Chromatograph
(Hewlett-Packard Co., Palo Alto, CA) using an autosampler and a column switching valve. A shielded hydrophobic phase column, HiSep, 250 X 2.1 mm (Supelco, Inc.,
Bellefonte PA) and an ODS-2 C,,, 250 X 2.0 mm
(Metachem Technologies, Inc., Torrance, CA) with an
isochratic mobile phase of 10% acetonitrile and 90% of 0.1
M sodium acetate and 0.002 M 1-octanesulfonic acid,
sodium salt was used for separation. The flow rate was
0.20 ml/min. The eluent from the HiSep column was
bypassed to waste 6 s after injection. After 5 min, the
eluent was switched back to the C,, column for further
separation and to the detector. The fluorescence was detected by an FS 970 LC Fluorometer (Kratos Analytical
Instruments, Ramsey, NJ) with an excitation wavelength of
274 nm and a 370 nm long-pass filter for emission. The
chromatograms were recorded and the peaks integrated on
a 3392 integrator (Hewlett-Packard). The run time was 20
Baseline
HR(bpm)
Control group (n = 7)
NC group (n = 6)
GTN group ( n = 6)
GLB group (n = 7)
GLB+NCgroup(n=7)
GLB+GTNgroup(n=6)
MB group (n = 6)
MB+NCgroup(n=6)
MB+GTNgroup(n=6)
152+2
154+3
149+_4
15s+3
156+2
156+2
157+4
155+6
156+5
MBP CrnrnHg)
Control
NC
GTN
GLB
GLB + NC
GLB + GTN
MB
MB+NC
MB+GTN
F39+9
101+5
100+ 15
99+7
85+4
78+5
87+5
91+5
90+8
PRP CmmHg/ min / 1000)
Control
NC
GTN
GLB
GLB + NC
GLB + GTN
MB
MB+NC
MB + GTN
16+2
17*1
17f3
18+1
15+1
14&l
15+1
16J11
16i2
CBF (ml / mini
Control
NC
GTN
GLB
GLB + NC
GLB + GTN
MB
MB+NC
MB+GTN
33+5
26+7
38f5
50*5*
38*4
34*5
40+7
27k5
40*4
After
blocker
After NC
or GTN
Occlusion
Reperfusion
30 min
Ih
2h
3h
-
154+2
154+3
154+ I
-
157+4
152+4
153+2
156k6
155+3
160+2
16Ok6
141 i ll*
16Ok6
156+4
152+5
155+2
151+6
157+3
157+4
157+6
154k8
160+5
155+4
152*5
152-12
156+ 1
158k3
153*4
163+5
154+9
162?5
152+5
153*5
155&l
155+2
159+4
1541-5
160&8
154*9
163*7
84,13
86+3
90+ 12
92+6
71,6
69+5
92+6
83511
78+4
87+12
93+3
97+8
87+4
78+3
72+5
90+5
95+ 10
84
97f9
97+2
99+8
93+_4
83k4
84+7
89+5
98k9
94+7
92k9
107+4
102k7
92+3
89+4
83+5
81+7
9957
95+5
15+2
14+ 1
15+2
16+2
12+ 1
1211
17+1
14+2
14* 1
16+2
16*1
17+1
15*1
13+1
11+2*
16t 1
17+2
16+1
17+ 1
17Fl
17k2
16+ 1
15f 1
14i 1
17+ 1
17+2
1752
16+1
18+_1
18+1
16+ 1
16+ 1
14+ 1
15+2
18+2
18+2
0
0
0
0
0
0
0
0
0
39+8
39*6
45+5
71 f9*$
56+13
49*6P
58+ 128
52f7’j
61+6’p
33+7
44+9
3714
60+8*9
41*4
43_+8
42+9
34*3
49+6
2918
40* 10
33+3
52*8*
39*5
36+7
31*5
3011
43+4
158+3
152+2
157+3
156+6
156k5
103*6
91&I
89+4
88*6
loo*4
97,6
18k 1
16+7
15*1
16k2
17* 1
17*1
46*4
37+3
36+4
61+10t§
5o*@I
55+7Q:
157fl
157+2
158+7
15954
158+5
88&8
9655
99+ 13
87+3
75+7
89+7
84+6
16k2
16il
17+2
15+1
1311
16+1
15+1
33*5
34+79
38+5
35+3
28+3
56*8*‘$
56+8*$
HR = heart rate; MBP = mean blood pressure; PRP = pressure-rate product; LV = left ventricle; CBP = coronary blood flow. Values are given as
meansfs.e.m. tP < 0.01, *P < 0.05 vs. corresponding time in Control group. ‘P < 0.01, “P < 0.05 vs. control value,
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
Table 1
Hemodynamics in the different groups
T. Mizumura
et al. / Cardkwascular
min with 5 min post-run-time. To study percent recovery
of adenosine, adenosine standards at 0.02, 0.20 and 1.9
PM were added to tubes containing stop solution before
withdrawing blood samples (3 sets of 8 samples). The
recoveries in both cases were greater than 90%. For each
dog, at least 2 samples were spiked with known concentrations of adenosine (0.05-2.0 FM) after treatment with
adenosine deaminase to verify the experimental procedure
and to test for possible sample influences as previously
described [17].
2.7. Exclusion
criteria
2.8. Statistical
analysis
All values are expressed as mean f s.e.m. Differences
between groups in hemodynamics were compared by using
a two-way (for time and treatment) analysis of variance
(ANOVA) with repeated measures and Fisher’s least significant difference (LSD) test if significant F-ratios were
obtained. Differences between groups in tissue blood flows,
area at risk, and infarct size were compared by one-way
ANOVA and comparisons between groups by Fisher’s
LSD. Differences in adenosine concentrations at various
times following reperfusion between treatment groups and
the control group were compared by using an unpaired
Student t-test. ANCOVA was used to determine whether
the relationship between transmural collateral blood flow
and infarct size differed between the control and drugtreated groups. Differences between groups were considered significant if P < 0.05.
3. Results
3. I. Mortality
and exclusions
Sixty-nine dogs were initially used in the present study.
Eight dogs were excluded because transmural collateral
blood flow was more than 0.16 ml/min/g
(two in the MB
group and one each in the Control, NC, GTN, GLB,
GLB + GTN, and MB + GTN groups), three in the GLB
+ NC and the MB + NC group because more than three
32 f lU%l
274-285
219
consecutive attempts were needed to convert ventricular
fibrillation with DC pulses, and one dog in the MB + NC
group because of heartworms. Therefore, 57 dogs were
used for data analysis: 7 dogs for the Control, GLB, and
GLB + NC groups, and 6 dogs each for the rest of the
groups.
3.2. Hemodynamic
responses
The hemodynamic data are summarized in Table 1.
There were no significant differences in heart rate, mean
arterial blood pressure, pressure-rate product, or LAD
blood flow at baseline between groups except for the LAD
blood flow in the GLB group. In all groups, there were no
significant differences in systemic hemodynamics between
groups throughout the experiment except for the following
variables. Heart rate was significantly lower in the MB +
NC group at 30 min of occlusion as compared to the
control group. Pressure-rate product was reduced in the
GLB + GTN group at 1 h of reperfusion. Coronary blood
flow was significantly higher in the GLB group at baseline
and after reperfusion, and was increased after the methylene blue infusion in all MB-pretreated groups.
3.3. Myocardial
infarct size
Myocardial infarct size and area at risk data are shown
in Fig. 3. The anatomical area at risk expressed as a
percent of the left ventricle was not significantly different
between groups (Fig. 3a): Control group, 32.3 + 2.0%; NC
group, 28.7 f 1.7%; GTN group, 28.7 + 1.0%; GLB group,
33.8 + 1.5%; GLB + NC group, 30.5 + 1.7%; GLB +
GTN group, 29.7 f 1.8%; MB group, 33.2 t- 1.l%; MB +
NC group, 28.0 f 2.0%; MB + GTN group, 30.7 f 1.6%.
Myocardial infarct size expressed as a percent of the area
at risk (Fig. 3b) was significantly reduced in NC group,
12.2 + 3.2%; GTN group, 13.0 + 3.1%; GLB + GTN
group, 12.2 f 2.1%; and MB + NC group, 13.2 k 2.2% as
compared to the Control group, 25.7 k 4.1% (P < 0.05).
There were no differences in infarct size in GLB + NC
group, 26.8 f 6.0%; GLB group, 26.8 f 4.6%; MB + GTN
group, 26.0 + 3.2%; and MB group, 23.8 f 4.0% compared with controls. Fig. 4A, B and C shows the relationship between transmural collateral blood flow measured at
30 min of occlusion and infarct size expressed as a percent
of the area at risk. In all groups, there was an inverse
relationship between these two variables. Glibenclamide
(GLB) and methylene blue (MB) alone did not affect this
relationship as compared to the control group (Fig. 4A);
however, nicorandil (NC, Fig. 4B) and glyceryl trinitrate
(GTN, Fig. 4C) produced a significant (P < 0.05) downward shift in this relationship compared to the control
group. In the case of MB + NC (Fig. 4B) or GLB + GTN
(Fig. 4C) this downward shift was not blocked; however,
GLB + NC (Fig. 4B) and MB + GTN (Fig. 4C) groups
were not different from control.
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
Dogs were excluded if (1) heartworms were found after
the animals were killed, (2) transmural collateral blood
flow was greater than 0.16 ml/min/g,
(3) heart rate was
higher than 180 beats/min at the beginning of the experiment, or (4) more than three consecutive attempts were
needed to convert ventricular fibrillation with low-energy
DC pulses applied directly to the heart. Previous studies of
Longeril et al. [ 181 showed that l-3 attempts at defibrillating canine hearts did not affect infarct size, whereas
experience in our laboratory indicates that small epicardial
infarcts appear in the non-ischemic zone if more than 3
attempts are needed to convert ventricular fibrillation.
Research
280
T. Mizumura
3.4. Regional
myocardial
et al. / Cardiol,ascular
blood fzow
Research
32 (19961274-285
(A)
60
1
Transmural collateral blood flow data in the nonischemic (left circumflex coronary artery) and ischemic
(LAD) regions are summarized in Table 2. There were no
significant differences in transmural collateral blood flows
during occlusion between groups, which indicates that all
groups were subjected to similar degrees of ischemia.
V
V
0**
;‘....
0‘.
..
-.*
9
Q.,
‘.
0
....0 ....
GLB
--o-.
MB
.‘..._
-.-.....
SY.
“....v
\.
0
\
3.5. Coronary
venous adenosine concentrations
I
0.1
0
40
1
(B)
60 -
0
e
40-
60
CC)
d
40
‘\
0
l ,*\
1
A
l e
Collateral
T
I
Control
hC
GTN
I
I
CLB
GLB
N;
&
(;I.*
I
MB
MB
Yic
&N
Control
----o----
NC
--O--.
GLB+NC
-.-A---
MB+NC
+
Control
....f... GTN
I
1
+
--O-.
GLB+GTN
---A---
MB+GTN
Blood Flow (mllminlg)
Fig. 4. Plots of the relationship between transmural collateral blood flow
(ml/mitt/g)
and infarct size expressed as a percent of the area at risk
(IS/AAR). In all groups, there was an inverse relation between transmural collateral blood flow and IS/AAR. Panel A represents data points
obtained from the Control, GLB and MB groups. Panel B shows data
points obtained from the Control, NC, GLB+NC and MB +NC, GTN,
and GLB + GTN groups. Panel C shows points from the Control, GTN,
GLB + GTN and MB + GTN groups. The regression lines for NC, MB +
NC, GTN and GLB + GTN were all shifted downward as compared to the
control group (Panels B,C) by analysis of covariance (P < 0.05).
YR
Fig. 3. Graphs illustrating the effects of nicorandil (NC) and glyceryl
trinitrate (GTN) with or without glibenclamide (GLB) and methylene
blue (MB) on the area at risk expressed as a percent of the left ventricle
(AAR/LV)
and infarct size expressed as a percent of the area at risk
(IS/AAR). (a) There were no significant differences in AAR/LV
between groups. (b) NC, GTN, GLB + GTN, and MB + NC resulted in a
marked reduction in IS/AAR. GLB and MB totally abolished the cardioprotective effect of NC and GTN, respectively. Neither GLB or MB had
any significant effect on IS/AAR when administered alone. * P < 0.05
vs. controls.
concentrations at 15 min of reperfusion in the MB + NC
group (P < O.OS>,and at 5, 10 (P < 0.05) and 15 min
(P < 0.01) of reperfusion in the GLB + GTN group. On
the other hand, in the groups which showed no significant
difference in infarct size as compared to control animals,
there were no significant differences in adenosine concentration during reperfusion except for the one at 15 min of
reperfusion in the GLB + NC group (P < 0.05). There
were no differences in adenosine concentrations in all
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
The concentrations of adenosine in coronary venous
blood samples obtained at various times during coronary
occlusion and reperfusion are shown in Fig. 5. Each group
in which myocardial infarct size was reduced showed a
decrease in adenosine concentration at 5, 10 and 15 min of
reperfusion. Pretreatment with nicorandil resulted in a
significant reduction in adenosine concentration at 5 (P <
0.01 vs. controls), 10 and 1.5 min (P < 0.05) of reperfusion. Glyceryl trinitrate also produced a significant reduction in adenosine concentration at 5 min of reperfusion
(P < 0.05). There were significant reductions in adenosine
I
0.2
T. Mizumura et al./Cardiowzscular
groups before and during LAD occlusion as compared to
the Control group.
Table 2
Transmural myocardial blood flow
Group
Control (n = 7)
NC(n=6)
GTN (n = 6)
GLB (n = 7)
GLB+NC (n= 7)
GLB+GTN(n=6)
MB(n=6)
MB+NC(n=6)
MB+GTN(n=6)
281
Research 32 (IY96) 274-285
Non-ischemic region
Ischemic region
30 min Occ 3 h Rep
30 min Occ 3 h rep
1.05+0.18
0.86kO.89
1.14+0.12
1.22zbO.11
1.06&0.16
0.78*0.05
1.20+0.16
1.15kO.21
1.06*0.12
0.06+0.02
0.06~0.01
0.07iO.01
0.05+0.02
0.06+0.01
0.07+0.02
0.04+0.01
0.04+0.01
0.04*0.01
1.09CO.16
0.79kO.79
0.96&0.14
1.47+0.31
1.12&0.09
1.10&0.25
1.04+0.21
1.17+0.21
1.28f0.20
in ml/min/g.
Occ
=
4. Discussion
The major finding obtained from this study indicates
that nicorandil, a K,,, channel opener-nitrate, when administered 15 min prior to a 60-min period of ischemia
followed by 3 h of reperfusion, produces a marked reduction in infarct size and this beneficial effect is totally
abolished by glibenclamide, a selective K,,, channel antagonist. These results are the first to demonstrate that the
infarct size-reducing effect of nicorandil is blocked by a
(‘ml
3 .(I
5
IS ho
s
IO
IS
30
(h)
Time (min)
Fig. 5. Graphs showing venous adenosine concentrations from the ischemic-reperfused area at various times during occlusion and following reperfusion. In
ah groups, there were no significant differences in adenosine concentration before and during the occlusion period between groups. After reperfusion,
however, adenosine concentrations from the area at risk at 5, 10 and 15 min of reperfusion were significantly lower in the NC group as compared to the
corresponding values in the Control group (a). Similarly, adenosine concentrations at the same time of reperfusion were significantly decreased in the CTN
(b), MB +NC (cl, and GLB +GTN groups (d) except for the following values: 10 and 15 min of reperfusion in the GTN group, 5 and IO min in the
MB + NC group. In contrast, there was no significant reduction in adenosine concentration during reperfusion between the following groups: GLB + NC
(e), MB +GTN (f), MB (g), and GLB (h) except for the value at 15 min of reperfusion in the GLB +NC group. * P < 0.05. * * P < 0.01 vs. controls.
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
All values are expressed as meanfs.e.m.
occlusion; Rep = reperfusion.
0.81~0.15
0.81 +0.19
0.68*0.10
1.13f0.15
0.99+0.12
0.80+0.18
0.54+0.10
0.80+0.14
0.92iO.12
282
T. Mizumura
et al. / Cardiovascular
4. I. Nicorandil as a KATP channel opener
In a previous study, we [4] showed that nicorandil
produced a marked reduction in infarct size and adenosine
release from the ischemic area during reperfusion in dogs
subjected to 1 h of LAD occlusion followed by 3 h of
reperfusion. In that study, glyceryl trinitrate, used for
purposes of comparison, also reduced infarct size and
adenosine release. Since other KATp channel openers such
as aprikalim [19] and bimakalim [II] have been shown to
reduce infarct size in dogs, and we [l I] demonstrated that
bimakalim reduced infarct size and adenosine release
equivalent to that previously shown with nicorandil, one
could speculate that the beneficial effect of nicorandil on
ischemic myocardium is mainly mediated via K,,, channel activation. However, since glyceryl trinitrate produced
similar effects on infarct size and adenosine release as
nicorandil, it was not clear as to which property of nicorandil is responsible for its infarct size-reducing effectK *rP channel opening or nitrate action. In the present
study, we demonstrated that glibenclamide, a selective
K *rP channel antagonist, blocks the infarct size-reducing
effect of nicorandil in canine hearts. However, it is still
32 (1996)
2746285
possible that if higher doses of nicorandil were used, the
nitrate-like effect might be sufficient to overcome the
blockade of the KATP channel produced by glibenclamide
and result in a reduction in infarct size. On the other hand,
higher doses of nicorandil, exceeding the therapeutic window, may produce marked peripheral vasodilator effects
accompanied by a reflex tachycardia which may overcome
any direct beneficial effect of nicorandil to reduce infarct
size and make data interpretation difficult. Thus, we feel
confident that at doses used clinically, the cardioprotective
effect of nicorandil is primarily the result of its myocardial
K ATP channel-opening properties and not its nitrate-like
effects. This is not to disregard the important property of
nicorandil to dilate large epicardial coronary arteries at
therapeutic concentrations which has been shown to be a
result of its nitrate-like actions and may be responsible for
increasing blood flow to the ischemic myocardium [3].
Sulfonylureas such as glibenclamide have been used for
a long time in the treatment of non-insulin-dependent
diabetes mellitus and have also been shown to be specific
antagonists of K,,, channels in insulin-secreting cells,
coronary arteries and cardiac cells [20]. Among sulfonylureas, glibenclamide is considered to be the most potent
K ATP channel blocker [21] and has been used in a large
number of in vitro and in vivo studies. Gross [ 131 showed
that increasing concentrations of glibenclamide produced a
parallel shift to the right of the dose-response curves for
increases in coronary blood flow to intracoronary injections of nicorandil and EMD 5643 1, two potassium channel openers, while not affecting the dose-response curve
to glyceryl trinitrate in dogs. We have also shown that the
dose of glibenclamide used in this study blocks myocardial
K ATP channels in the canine heart without increasing
infarct size by itself [19,22] In agreement, Sargent et al.
[23] have demonstrated that the K ATP channel opener,
cromakalim, protected ischemic rat hearts and its effect
was reversed by glibenclamide, while the anti-ischemic
effects of mechanistically different agents such as calcium
antagonists, sodium channel blockers and calmodulin antagonists were not blocked by glibenclamide. These data
indicate that glibenclamide is a potent, selective KATP
channel blocker in the myocardium. Therefore, the finding
that the infarct size-reducing effect of nicorandil was
completely abolished by glibenclamide and not affected by
methylene blue strongly supports our hypothesis that nicorandil reduces infarct size via myocardial K,,, channel
activation.
4.2. Glyceryl trinitrate and methylene blue
Glyceryl trinitrate, a classical nitrate, has been widely
used in the treatment of angina pectoris for many years.
The beneficial effects of glyceryl trinitrate on ischemic
myocardium has been thought to be related to its vasodilating action: increased diameter of epicardial coronary artery,
cardiac unloading, or recruiting collateral blood flow into
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
K ATP channel antagonist. The results also showed that
glyceryl trinitrate reduced myocardial infarct size in the
same model and that methylene blue, a nitric oxide
(NO)/guanylate cyclase inhibitor, reversed the cardioprotective effect of glyceryl trinitrate. The infarct size-reducing effect of nicorandil was not affected by methylene blue
and that of glyceryl trinitrate was not attenuated by glibenclamide, which indicates the selectivity of these two antagonists for specific mechanisms. Neither glibenclamide nor
methylene blue affected infarct size when they were administered alone. Another important finding of the present
study was that adenosine concentrations obtained from the
ischemic-reperfused area were significantly lower in the
‘protected’ groups (NC, GTN, MB + NC, and GLB + GTN
groups), although in the GLB + NC group, there was still
a tendency for a decreased release of adenosine as compared to the control group in spite of equivalent infarct
sizes in the two groups (Fig. 5). There were no differences
in adenosine concentration between groups during ischemia. These results are in agreement with a recent study
from our laboratory [ 1I] in which we also showed that the
cardioprotective effect of bimakalim, a selective K,,,
channel opener, and ischemic preconditioning both resulted in a decreased release of adenosine following reperfusion in a similar canine model to that used in the present
study. These results also indicate that the infarct size-reducing effect of nicorandil or glyceryl trinitrate is not
mediated by an increase in adenosine released from the
area at risk during ischemia or reperfusion in this model
and also confirm our previous findings which clearly
suggest that adenosine is a sensitive marker of ischemic
injury during coronary artery occlusion [4,11].
Research
T. Mizumura et al. /Cardiovascular
283
greater than those used in in-vitro studies [27,29,30]. These
authors suggested that the binding of methylene blue to red
blood cells and its rapid reduction in the blood [32] could
explain the much higher blood concentration required to
inhibit guanylate cyclase in vivo. In agreement, the concentration of methylene blue we used in the present study
was approximately 80 p,M and in our preliminary experiments (Fig. 2) approximately 20 p.M of methylene blue
failed to block the infarct size-reducing effect of glyceryl
trinitrate. Whether these concentrations of methylene blue
block the effects of NO released by glyceryl trinitrate in
cardiac myocytes similar to smooth muscle cells is unknown and more studies are needed to address this possibility.
Another interesting finding concerning methylene blue
in the present study was an increase in coronary blood
flow during methylene blue infusion. As shown in Table 1,
there were significant increases in coronary blood flow
after methylene blue infusion in the MB, MB + NC and
MB + GTN groups. A similar effect of methylene blue
was observed by Sobey et al. 1311who found that methylene blue significantly increased femoral artery blood flow
while not affecting femoral artery diameter or arterial
blood pressure in dogs. The exact mechanism by which
methylene blue increases coronary blood flow is not clear;
however, methylene blue may dilate resistance vessels in
both hearts and hindlimbs.
4.3. Adenosine concentration and infarct size
Recently, Kitakaze et al. [lo] showed that nicorandil
mimicked the effect of ischemic preconditioning in the
canine heart and suggested that this beneficial effect was
the result of an increase in adenosine release from the
ischemic area during reperfusion. Walsh et al [33] also
found that pinacidil produced a significant reduction in
infarct size in rabbits and that this effect could be reversed
by 8-p-sulfophenyl theophylline, an adenosine receptor
antagonist. They suggested that K,,, channel openers may
be protecting the ischemic myocardium by increasing
adenosine release upon reperfusion. However, since we
observed that treatment with either nicorandil or glyceryl
trinitrate resulted in a reduction in adenosine release from
the ischemic myocardium during reperfusion as compared
to control animals and that there was no difference in
adenosine release after reperfusion between the GLB + NC,
MB + GTN, GLB, MB, and Control groups in the present
study, it is unlikely that an increase of adenosine during
ischemia and/or reperfusion contributes to the infarct
size-reducing effect of nicorandil and glyceryl trinitrate. In
agreement, Sawmiller et al. [34] showed that pinacidil did
not increase venous adenosine concentrations in isolated
guinea pig hearts. More recently, Silva et al. [35] demonstrated that the increased interstitial adenosine concentration produced by pentostatin, an adenosine deaminase
inhibitor, during 60 min of ischemia did not result in a
Downloaded from http://cardiovascres.oxfordjournals.org/ at Pennsylvania State University on March 6, 2014
the ischemic myocardium [3]. Several investigators have
shown an infarct size-reducing effect of glyceryl trinitrate
in dogs [7,24], and humans [6]. Although Jugdutt et al. 1241
reported that collateral blood flow significantly increased
in glyceryl-trinitrate-treated animals, the exact mechanism
by which glyceryl trinitrate reduced infarct size was not
clear in any of those studies. Since, in the present study,
we showed that a non-hypotensive dose of glyceryl trinitrate produced a significant reduction in infarct size in
dogs subjected to 1 h of coronary artery occlusion and 3 h
of reperfusion independent of area at risk, changes in
systemic hemodynamics, or collateral blood flow, and that
this beneficial effect was completely reversed by methylene blue, a soluble nitric oxide (NO)/guanylate cyclase
inhibitor, it is likely that the infarct size-reducing effect of
glyceryl trinitrate may be mediated by increased soluble
guanylate cyclase activity, although the present results do
not suggest the site of action responsible for the protective
effect of glyceryl trinitrate (i.e., myocardium, vascular
smooth muscle, endothelial cells, platelets or neutrophils).
Since glyceryl trinitrate has been previously shown not to
activate myocardial guanylate cyclase 1251 or affect neutrophil function in the ischemic-reperfused myocardium [4]
and produces its cardioprotective effect at a non-hypotensive dose, this suggest that an action on platelet function
and aggregation might be responsible for its beneficial
effect in this model. That glyceryl trinitrate has recently
been shown to reduce the formation of primary platelet
thrombi at non-hypotensive doses in dogs similar to those
used in the present study [26] supports this hypothesis.
Methylene blue. methylthionine chloride, is known to
be an inhibitor of soluble guanylate cyclase in vascular
smooth muscle cells [27] and has also been shown to
inhibit the actions of NO by generating superoxide anion
in cultured pulmonary artery smooth muscle cells [28].
Superoxide anion would combine with NO to form peroxynitrite, thereby decreasing effective NO concentrations.
Therefore, the results of these studies suggest two mechanisms by which methylene blue might inhibit the effect of
glyceryl trinitrate, one direct and one indirect, but both
related to inhibiting the effect of NO to activate guanylate
cyclase. In this regard, in a number of in vitro studies,
lo-50 FM of methylene blue has been shown to inhibit
the stimulation of soluble guanylate cyclase induced by
nitric oxide or nitrovasodilators in various systems such as
bovine coronary arteries [27,29] and rabbit aortas [30]. In
intact animals, Sobey et al. [3] demonstrated that continuous intraarterial infusion of methylene blue (10 mg/min)
attenuated the increase in femoral artery diameter and
femoral blood flow produced by glyceryl trinitrate in dog
hindlimbs. Interestingly, the plasma concentration of methylene blue infused into the femoral artery in their study
was approximately 600 FM at the initial femoral blood
flow while a lesser concentration of the drug (approximately 120 p,M) had no effect on the response to glyceryl
trinitrate. These concentrations of methylene blue are much
Research 32 (19961274-285
284
T. Mizumura et al./ Curdiorasculur
ischemic myocardium while glyceryl trinitrate did not
show any attenuation in neutrophil infiltration regardless
of the time it was administered in our previous study [4],
this feature may be another advantage of nicorandil over
nitrates in the treatment of patients with acute myocardial
infarction, especially during thrombolytic therapy or primary PTCA. Alternatively, the marked reduction in infarct
size observed in the presence of glyceryl trinitrate also
suggests that, in some instances, this agent may have
advantages over nicorandil, particularly in diabetic patients
taking chronic sulfonylurea antidiabetic drugs which are
known antagonists of the K,,, channel and may be less
responsive to nicorandil.
Acknowledgements
The authors would like to thank Ms. Anna Hsu and Ms.
Jeannine Moore for their excellent technical assistance and
Ms. Carol Knapp for outstanding secretarial assistance.
This study was supported by funds from NIH grant HL
083 11 and Chugai Pharmaceutical Company, Tokyo, Japan.
4.4. Clinical implications
References
From a clinical standpoint, the results of the present
study indicate that both nicorandil and glyceryl trinitrate
may be effective in reducing myocardial ischemia and
minimizing the extent of myocardial necrosis during treatment of patients with acute myocardial infarction, or coronary artery interventions such as percutaneous transluminal
coronary angioplasty (PTCA) and coronary bypass graft
surgery. One major advantage of nicorandil over glyceryl
trinitrate is the lack of development of hemodynamic
tolerance, which has limited the chronic use of organic
nitrates in clinical practice. Tsutamoto et al. [38] measured
pulmonary capillary wedge pressure in patients with
chronic heart failure during nicorandil or glyceryl trinitrate
infusions and showed that in the nicorandil-treated group
the pressure continued to decrease for 24 h, whereas in the
glyceryl-trinitrate-treated
group it returned to baseline
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GMP in the myocardium, it would be intriguing to determine if this beneficial effect of glyceryl trinitrate disappears after hemodynamic tolerance has developed. In addition, several studies have shown that K,, channel openers inhibit neutrophil function in humans and dogs
[4,1 1,19,40]. Since nicorandil, when given immediately
before and during reperfusion, still produced a significant
reduction in infarct size and neutrophil infiltration into the
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ischemia is indeed a reflection of ischemic severity during
coronary artery occlusion.
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T. Mizumura
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