1632
Beneficial Effects of o^-Adrenoceptor Activity
on Ischemic Myocardium During Coronary
Hypoperfusion in Dogs
Masafumi Kitakaze, Masatsugu Hori, Koichi Gotoh, Hiroshi Sato, Katsuomi Iwakura,
Akira Kitabatake, Michitoshi Inoue, and Takenobu Kamada
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
We have previously reported that o^-adrenoceptor stimulation enhances adenosine-induced
coronary vasodilation. In the present study, we tested the hypothesis that o^-adrcnoceptor
activity exerts beneficial effects on myocardial ischemia through augmentation of vasodilatory
effects of released adenosine. In open-chest dogs, the left anterior descending coronary artery
was perfused through an extracorporeal bypass tube from the carotid artery. Propranolol was
infused into the bypass tube to exclude the metabolic effects of norepinephrine. When clonidine
(0.24 / u g/kg/min i.e.) was infused for 10 minutes after reduction of coronary blood flow by
partial occlusion of the bypass tube, coronary blood flow was increased by 43% from 27 ± 1
ml/100 g/min despite no changes in coronary perfusion pressure (38±5 mm Hg) and a slight
decrease in adenosine release. Both fractional shortening and lactate extraction ratio of the
perfused area were significantly improved (fractional shortening, 1.8±1.0 to 10.9±1.5%,
p<0.001; lactate extraction ratio, -57.8±6.5 to -31.9±2.4%,p<0.005). Identical results were
observed in the denervated hearts, indicating that the beneficial effect of clonidine is not
attributed to the prevention of norepinephrine release from the sympathetic nerve terminals.
The beneficial effects of clonidine were prevented by yohimbine, an a^-adrenoceptor blocking
agent. An adenosine receptor antagonist, 8-phenyltheophylline, also prevented the beneficial
effects of clonidine, indicating that these beneficial effects are mediated by effects of adenosine.
Furthermore, the extent of augmentation of coronary flow in the ischemic heart was coincided
with that of augmentation of exogenous adenosine-induced hyperemic flow (40%) by clonidine.
Production of cyclic AMP in the coronary artery during myocardial ischemia was augmented by
clonidine. In 12 other dogs, myocardial ischemia was produced by intracoronary embolization
of microspheres (15 ftm in diameter). Clonidine enhanced (39%) the hyperemic coronary flow
and improved both fractional shortening and lactate extraction ratio. Thus, we conclude that
Oi-adrenoceptor stimulation can ameliorate myocardial ischemia mainly due to enhancement of
vasodilatory effects of adenosine released from the ischemic myocardium. (Circulation Research
1989;65:1632-1645)
C
oronary circulation in the ischemic myocardium is mainly regulated by metabolic
vasodilation and sympathetic vasoconstriction.1-9 a2-Adrcnoceptor stimulation is reported to
be responsible for sympathetic coronary vasoconstriction,4-8 and Heusch and Deussen3 demonstrated that sympathetic nerve stimulation under ar
From The First Department of Medicine, Osaka University
School of Medicine, Osaka, Japan.
Supported by a grant-in-aid for Scientific Research on Priority
Areas (#62624005) from the Ministry of Education, Science, and
Culture, Japan.
Address for reprints: Masafumi Kitakaze, MD, PhD, The First
Department of Medicine, Osaka University School of Medicine,
1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan.
Received October 4, 1988; accepted June 28, 1989.
and ^-adrenoceptoT blockades (i.e., a2-adrenoceptor
stimulation) increases coronary vascular resistance
distal to severe stenosis and causes myocardial
ischemia. Furthermore, Seitelberger et al7 showed
that intracoronary infusions of a2-adrenoceptor
antagonists improve myocardial ischemia during
exercise due to attenuating exercise-induced coronary vasoconstriction. These lines of evidence suggest that diminished sympathetic vasoconstriction
by potent a2-adrenoceptor blocking agent attenuates myocardial ischemia when sympathetic tone is
activated. In contrast, we have recently provided
the evidence that moderate o^-adrenoceptor stimulation can enhance adenosine-induced coronary vasodilation, although potent a2-adrenoceptor stimulation
exerts prominent vasoconstricfion in the normoxic
Kitakaze et al Effect of o^-Adrenoceptor Activity on Ischemia
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
heart.10-12 The latter observation seems corresponding to reports of Heusch and Deussen5 and Seitelberger et al.7 However, if the former mechanism
(i.e., enhancement of adenosine-induced coronary
vasodilation) overcomes its direct vasoconstriction,
a2-adrenoceptor stimulation can increase coronary
blood flow during ischemia, because a massive
amount of adenosine is released from ischemic
myocardium.-Thus, in the present study, to test whether a2adrenoceptor stimulation has an ability to enhance
endogenous adenosine-induced coronary vasodilation and ameliorate myocardial ischemia clonidine,
an o;2-adrenoceptor agonist, was infused into the
coronary artery during hypoperfusion and measured regional contractile and metabolic functions.
Furthermore, to exclude the possibility that this
beneficial effect of clonidine does not involve the
effect of withdrawal of norepinephrine release from
the presynaptic vesicles, we measured adenosine
and norepinephrine concentrations in the arterial
and coronary venous blood and performed identical
experiments in the denervated hearts. Finally, to
observe the subcellular mechanism of this phenomenon, we measured cyclic AMP of coronary smooth
muscles with and without a2-adrenoceptor stimulation during myocardial ischemia.
Materials and Methods
Instrumentation
Fifty-nine mongrel dogs weighing 15-20 kg were
anesthetized with pentobarbital sodium (30 nag/kg
i.v.). In 10 dogs, systemic chemical sympathectomy
was performed by intravenous injection of 50 mg/kg
of 6-hydroxydopamine, 5 days before the experiment.13-14 Prevention of deleterious side effects of
6-hydroxydopamine was provided by previous
injections of propranolol (1 mg/kg) and phentolamine (1 mg/kg), and three fractional doses of
6-hydroxydopamine (10, 20, and 20 mg/kg) were
administered over a period of 24 hours.14
The trachea was intubated, and the animal was
ventilated with room air mixed with oxygen. The
chest was opened through the left fifth intercostal
space, and the heart was suspended in a pericardial
cradle. The left anterior descending coronary artery
(LAD) was cannulated and perfused with blood via
the left carotid artery through an extracorporeal
bypass tube. Coronary perfusion pressure (CPP)
was monitored at the tip of the coronary arterial
cannula, and coronary blood flow of the perfused
area (CBF) was measured with an electromagnetic
flow probe attached at the bypass tube. A small,
short collecting tube (1 mm in diameter and 7 cm in
length) was inserted into a small coronary vein near
the center of the perfused area to sample coronary
venous blood. The drained venous blood was collected in the reservoir placed at the level of the left
atrium and was returned to the jugular vein. High
fidelity left ventricular (LV) pressure was measured
1633
by a micromanometer (model P-7, Konigsberg, Pasadena, California) placed in the LV cavity through
the apex. A pair of ultrasonic crystals were placed
in the inner one third of the myocardium about 1 cm
apart to measure myocardial segment length with an
ultrasonic dimension gauge (Schuessler, 5 MHz).
Heart rate averaged 129 beats/min in the intact
heart and 96 beats/min in the denervated heart.
Heart rate was not changed during each study.
Experimental Protocols
Protocol I: Effects of clonidine on myocardial
ischemia produced by coronary hypoperfusion.
Forty-two dogs were used in this protocol. Propranolol was injected (0.3 mg/kg) and continuously
infused (10 jtg/kg/min) into the perfused area to
inhibit metabolic effects of norepinephrine. We confirmed that this treatment of propranolol prevents
the effects of isoproterenol (0.1 pig/kg i.e.) on the
fractional shortening and coronary blood flow at
least for 40 minutes. After hemodynamic stabilization, coronary arterial and venous blood were sampled for blood gas analysis and determination of
lactate, adenosine, and norepinephrine concentrations. Hemodynamic functions (i.e., LV pressure
[LVP], dP/dt, and segment length of the perfused
area) were measured. End-diastolic length (EDL)
was determined at the R wave of the electrocardiogram, and end-systolic length (ESL) was determined at the minimal dP/dt.15 Fractional shortening
(FS) was calculated by (EDL-ESL)/EDL as an
index of myocardial contractility of the perfused
area. With an occluder attached at the extracorporeal bypass tube, CPP was reduced so that CBF
decreased to one third of the control CBF. After a
low CPP was determined, the occluder was adjusted
exactly to keep CPP constant at the low level
(n=32). All hemodynamic parameters were measured 3, 5, 7, and 10 minutes after the onset of
hypoperfusion, and both coronary arterial and
venous blood for the metabolic parameters were
sampled at 10 minutes. After these measurements,
clonidine (0.24 /xg/kg/min) was infused into LAD
and all hemodynamic and metabolic parameters
were measured again. The dose of clonidine was
determined so that it maximally enhanced exogenous adenosine-induced coronary vasodilation but
did not cause direct coronary vasoconstriction.
Ten minutes later, clonidine infusion was discontinued, and hemodynamic and metabolic parameters
were obtained when hemodynamic parameters were
stabilized (n=T). Although clonidine is reported to
be a selective a2-adrenoceptor agonist, we tested
that the effects of clonidine were blunted by an
a2-adrenoceptor antagonist. Instead of withdrawal
of clonidine infusion, yohimbine (9 /ig/kg/min) was
additionally infused into LAD during coronary hypoperfusion (n=5). Furthermore, to test that the effect
of clonidine during coronary hypoperfusion is related
to adenosine's effect, clonidine was infused into
LAD during hypoperfusion under intracoronary infu-
1634
Circulation Research Vol 65, No 6, December 1989
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sion of 8-phenyltheophylline (30 /ig/kg/min i.e.), a
potent surface membrane adenosine receptor antagonist (n=5). The treatment of 8-phenyltheophylline
was initiated 10 minutes before the coronary hypoperfusion. In the preliminary study, this dose of
8-phenyltheophylline abolished the coronary
vasodilatory effect of exogenous adenosine (5 fig!
kg/min i.e.).
In five other denervated dogs, identical procedures and measurements of all variables were performed. We confirmed that the norepinephrine content in the myocardium in systcmically denervated
(n=5) and innervated (untreated) dogs (n=5) was
12±3 and 366±28 pg/mg (/><0.001), respectively.
These 10 dogs were killed immediately after anesthesia, and the myocardial tissue was sampled from
the LAD area for measurement of norepinephrine
content without any intervention.
To examine the subcellular molecular mechanisms of enhancement of coronary vasodilation by
a2-adrenoceptor stimulation, cyclic AMP in the
LAD coronary artery was measured with (n=5) and
without (n=5) clonidine treatment during hypoperfusion. Coronary hypoperfusion was continued for
20 minutes, and clonidine was infused 10 minutes
after the onset of hypoperfusion. As a control, the
content of cyclic AMP in the left circumflex (LCX)
coronary artery was measured. In these 10 dogs, to
exclude the effect of )3-adrenoceptor activity on
cyclic AMP production of both LAD and LCX
coronary arteries, propranolol (0.2 mg/kg/min) was
infused intravenously. We confirmed that this dose
of propranolol blocked effects of isoproterenol (0.1
Mg/kg i.e.).
Protocol II: Effects of aradrenoceptor activity on
myocardial ischemia induced by coronary microembolization. If a2-adrenoceptor stimulation enhances
adenosine-induced coronary vasodilation, myocardial ischemia due to microembolization can be
improved by a2-adrenoceptor stimulation because
coronary hyperemic flow induced by the embolization of microspheres is attributed to adenosine
released from the ischemic foci.6 To test this idea,
microspheres (diameter: 15±1 (xm, 5.0xl0 4 /CBF
(ml/min); 3M, St. Paul, Minnesota) were repeatedly
injected until CBF reduced less than 8 ml/min with
(n=5) and without (n=l) intracoronary infusion of
clonidine (0.24 /ig/kg/min). When hemodynamic
parameters were stabilized 5 minutes after embolization and measured, coronary arterial and venous
blood were sampled.
Protocol III: Effects of aadrenoceptor activity
on exogenous adenosine-induced coronary vasodilation. To examine the extent of the enhancement
of adenosine-induced coronary vasodilation by clonidine (0.24 /tg/kg/min i.e.) in the normoxic condition, changes in CBF during infusion of adenosine
(0.5, 1, and 2 jig/kg/min i.e.) were Observed in five
dogs with and without intracoronary infusion of
clonidine before microsphere embolization.
Since the vasodilatory effect of adenosine is
reported to be modified when myocardium is
acidotic,16 the potency of interaction between adenosine and aj-adrenoceptor activity may be changed.
To mimic the condition of myocardial acidosis
during ischemia, five dogs were ventilated with
room air mixed with 5% CO2-95% O2. Five to 10
minutes after this ventilation, pH in arterial blood
was reduced from 7.39±0.01 to 7.27±0.02 and
reached a stable state, keeping O2 in the arterial
blood at a control level (105±5 mm Hg). Vasodilatory potency of three doses of adenosine were
tested with and without clonidine under this acidotic condition.
Chemical Analysis
Coronary arteriovenous blood oxygen difference
(AVO2D) was assessed by the difference between
coronary arterial and venous oxygen contents. MVO2
(ml/100 g/min) was calculated by CBF (ml/100 g/
min)xAVO2D(ml/dl). Lactate was assessed by the
enzymatic assay, and lactate extraction ratio was
obtained by coronary arteriovenous difference in
lactate concentration multiplied by 100 and divided
by arterial lactate concentration.
Adenosine measurement. The method of adenosine measurement has been reported previously.6-9-17
One milliliter of blood was drawn into a syringe
containing 0.5 ml dipyridamole (0.01%) and 0.1 ml
MnCl2 (10 mM) to block the uptake of adenosine by
red blood cells and degradation of adenosine. After
centrifugation, the supernatant was mixed with an
equal volume of 10% trichloroacetic acid to remove
the coagulated protein. Residual trichloroacetic acid
was removed by water saturated ether from the
extraction of the supernatant, and radioimmunoassay methods for analyzing the adenosine content
were employed. Briefly, 100 fil dioxane containing
succinic acid anhydride and triethylamine succinylated adenosine in the plasma (100 /A). After a
10-minute incubation, the mixture was diluted with
800 fj\ of 0.3 M imidazole buffer (pH 6.5). The assay
mixture contained 100 /xl of the sample, 100 u\
succinyl [3H]adenosine (25,000 counts/min in an
amount of 1 pmol), and 100 (JL\ of diluted antiadenosine serum. After the mixture was kept in an
ice-cold water bath for 24 hours, a cool suspension
of dextran-coated charcoal (500 /xl) was added. The
charcoal was spun down, and 0.5 ml of the supernatant was counted for radioactivity in a liquid
scintillation counter. The amount of adenosine degradation during sampling procedure and degradation rate of adenosine were reported negligibly
small.
Norepinephrine measurement. The method of
norepinephrine measurement has been described
previously.18 Five milliliters of coronary arterial
and venous blood taken into a tube containing
EDTA was immediately placed in iced water and
centrifugated for 20 minutes. The plasma was kept
at —$ff C. Within 2 weeks, plasma norepinephrine
KUakaze et al Effect of a^-Adrenoceptor Activity on Ischemia
TABLE 1.
1635
Coronary Hemodynamic and Metabolic Parameters Before and After 0-Adrenoceptor Blockade in Intact (Innervated) Hearts
Untreated
Propranolol
(baseline)
CPP
(mm Hg)
CBF
(ml/100
g/min)
LER
(%)
AVOzD
(ml/dl)
MVO2
(ml/100
g/min)
AdR
(nmol/100
g/min)
FS
(%)
NE(A)
(pg/ml)
NE(V)
(pg/ml)
110±5
92±2
30.8±l.6
8.58±0.50
7.90±0.54
2.0+0.4
26±3
386±63
499 ±57
107±5
86±2
28.2±3.1
8.08±0.57*
6.93±0.47*
1.9±0.5
23±3
376±52
397+64
Values are mean±SEM (n=7). CPP, coronary perfusion pressure; CBF, coronary blood flow, LER, lactate extraction ratio; AVOjD,
coronary arteriovenous oxygen difference; MVO2, myocardial oxygen consumption; AdR, adenosine release; FS, fractional shortening;
NE(A) and NE(V), norepinephrine concentrations in coronary arterial and venous blood, respectively.
*p<0.05 vs. the untreated control condition.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
was adsorbed on alumina and separated by highperformance liquid chromatography (pump, LC3A; column, Zpax-SCX; Shimazu Seisakusho,
Kyoto, Japan). Plasma norepinephrine was determined spectrofluorometrically by the trihydroxyindole method (Shimazu spectrofluorophotometer RF500LCA). In this system, sensitivity of the assay is
10 pg/ml plasma and the intra-assay coefficient of
variation is 6.8%.18
To determine norepinephrine concentration in
myocardial tissue, myocardial tissue from the LAD
area was sampled within 5 seconds and was frozen
in liquid nitrogen. The frozen tissue was stored at
-80° C. Within 1 week, myocardial tissue was
homogenized with the EDTA (0.1 M), NaHSO3 (1 M),
and HCIO3 (0.05 M) solution. After centrifugation, the
norepinephrine concentration in the supernatant was
determined by the method described above.
Cyclic AMP measurement. The method of cyclic
AMP measurements in tissues has been previously
described.19-21 During hypoperfusion of the LAD
coronary artery with and without treatment of clonidine, the small segments of LAD and LCX coronary arteries were removed and frozen with precooled stainless steel scissors and tongs in liquid
nitrogen and immediately stored at -80° C in liquid
nitrogen. After removal of the adventitial connective tissues in the coronary arteries (20-40 mg), the
frozen tissue was powdered, homogenized at 4° C
in 1 ml of ice-cold 6% trichloroacetic acid, and
centrifuged at 2,500g for 20 minutes. The supernatant fluid was removed and extracted three times
with 3 ml diethyl ether saturated with water and
stored in the freezer (-80° C). The cyclic AMP
concentration in the supernatant fluid was measured by the radioimmunoassay method19"21 within
7 days. Briefly, 100 ^1 dioxanetriethylamine mixture containing succinic acid anhydride succinylated cyclic AMP in the supernatant (100 /i\). After
a 10-minute incubation, the reaction mixture was
added to 800 /xl of 0.3 M imidazole buffer (pH 6.5).
One hundred microliters of succinyl cyclic AMP
tyrosine methyl ester iodinated with I (15,00020,000 counts/min in an amount less than 10" M)
was added to the assay mixture containing 100 /xl of
the supernatant and 100 pi of diluted anti-sera in the
presence of chloramine T ; the mixture was kept at
4° C for 24 hours. A cold solution of dextran-coated
charcoal (500 fil) was added to the mixture in an
ice-cold water bath. The charcoal was spun down,
and 0.5 ml of the supernatant was counted for
radioactivity in a gamma spectrometer.
Statistical Analysis
Statistical analysis was performed with paired
(Figures 2-7 and 11, Tables 1-3) and unpaired t
tests (Figures 8-10). Multiple analysis of variance
was also used in Figures 9-11 to test the tendency
of changes in hemodynamic and metabolic variables
versus extent of embolization or dose of adenosine.
All values were expressed as mean±SEM, and
p<0.05 was considered significant.
Results
Effects of arAdrenoceptor Stimulation on
Myocardial Ischemia
Intracoronary infusion of propranolol slightly but
significantly decreased both coronary arteriovenous
oxygen difference (AV02D) and myocardial oxygen
consumption (MVO2) (Table 1). However, intracoronary infusion of clonidine changed neither coronary hemodynamic nor other metabolic parameters
(Figures 9-11).
Figure 1 shows the representative tracing from
the experiment in which infusion of clonidine
increased CBF and improved fractional shortening
during coronary hypoperfusion. After abrupt reduction of CPP, CBF decreased from 36 to 12 ml/min
and fractional shortening decreased within 1 minute,
keeping a steady state for 10 minutes. Within 1
minute, once CPP was set, CPP was kept constant.
After the initiation of intracoronary infusion of
clonidine, CBF gradually increased and fractional
shortening gradually recovered. Ten minutes after
the onset of clonidine infusion, both CBF and
fractional shortening increased despite unchanged
CPP, indicating that intracoronary infusion of clonidine increases CBF and improves depressed myocardial function of ischemic myocardium. These
data are summarized in Figure 2. Although CPP was
kept constant in 36±4% of control CPP (107±4
mm Hg), intracoronary infusion of clonidine significantly (/?<0.001) increased CBF by 43±2% and
improved fractional shortening. This beneficial effect
of clonidine was demonstrated in the aspect of
myocardial metabolism. As shown in Figure 3,
lactate extraction ratio was significantly (p<0.005)
1636
Circulation Research Vol 65, No 6, December 1989
5 „
•
•
• • • • • •
• • BM •
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uotlan sf u w w y HoU IIM> -
FIGURE 1. Representative records of coronary perfusion pressure (CPP), left ventricular pressure (LVP), first
derivatives of LVP (dP/dt), coronary blood flow of perfused area (CBF), and segment length (SL) before and during
coronary hypoperfusion. The heart was pretreated with propranolol. After initiation of infusion of clonidine, CBF
gradually increased from 12 to 17 ml/min and fractional shortening was recovered. Withdrawal of clonidine infusion
returned both CBF and fractional shortening to the preclonidine levels. Reduced CPP and systemic hemodynamic
parameters were stable throughout this protocol
increased, indicating that myocardial anaerobic
metabolism was improved by clonidine infusion,
and thus MVO2 increased (p< 0.001). Myocardial
ischemia produced a significantly (/?<0.01) greater
AVO2D; however, clonidine did not change AVO2D
significantly (ischemia before clonidine, 10.9±0.9
ml/dl; ischemia with clonidine, 10.2±0.6 ml/dl; ischemia during withdrawal of clonidine, 10.5 ±0.8 ml/
dl). Coronary venous pH at the baseline condition
was 7.39±0.01 and decreased to 7.25±0.01 during
coronary hypoperfusion. However, clonidine infusion significantly (/?<0.05) improved the pH in
coronary venous blood (7.32+0.02). The pH of
coronary arterial blood did not change throughout
this study (7.40±0.01). Intracoronary infusion of
clonidine decreased adenosine release and did not
change norepinephrine concentrations in coronary
arterial and venous blood. Figure 4 shows that these
beneficial effects of clonidine are antagonized by
intracoronary infusion of yohimbine (9 ju.g/kg/min).
Intracoronary infusion of yohimbine decreased the
increased coronary blood flow, fractional shortening, and lactate extraction ratio although intracoronary infusion of clonidine was continued. Thus, the
beneficial effect of clonidine during ischemia is not
due to its nonspecific effect but attributed to the
specific a2-adrenoceptor stimulation. Figure 5 shows
that these beneficial effects of clonidine are related
to coronary vasodilatory effect of adenosine. The
intracoronary infusion of 8-phenyltheophylline did
not change coronary hemodynamic and metabolic
parameters (Table 2). After reduction of coronary
perfusion pressure to decrease coronary blood flow
to one third, fractional shortening, myocardial oxygen consumption, and lactate extraction ratio were
significantly (/xO.OOl) reduced. Ten minutes after
initiation of coronary hypoperfusion, clonidine was
initiated. However, coronary blood flow did not
increase, and improvement neither in fractional
shortening, myocardial oxygen consumption nor
lactate extraction ratio was observed. Although
adenosine release was enhanced during ischemia
compared with the condition of ischemia without
8-phenyltheophylline (Figure 3C), amounts of adenosine release before and after clonidine infusion
were not changed (Figure 5F). These results indicate that the beneficial effect of clonidine is related
to the coronary vasodilatory action of adenosine.
a2-Adrenoceptor stimulation may decrease norepinephrine release from the presynaptic vesicles
and adversely attenuate sympathetic vasoconstriction. We tested this possibility by performing a
comparable protocol in the denervated hearts (Table
3). In the denervated hearts (Figure 6), intracoronary infusion of clonidine increased CBF by 40 ±4%
and recovered fractional shortening despite no
changes in reduced CPP. Furthermore, clonidine
infusion improved lactate metabolism and increased
MV02 (Figure 7). Clonidine infusion in the denervated hearts decreased adenosine release from the
ischemic myocardium and did not alter norepinephrine concentrations in coronary arterial and venous
blood. These results indicate that clonidine increases
reduced ~CBF~ during hypoperfusion and improves
myocardial ischemia in the denervated dogs. Thus,
withdrawal «f sympathetic activities by activation
Kitakaze et al Effect of Q<,-Adrenoceptor Activity on Ischemia
1637
Hf-f-4—^H—j-^-^^—i
10
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(*)
• n*cnt±SE
n-7
• P<0.05
+P<O.0C5
++P<0.001
v».th«wlu««tl[)m«i
10
10
20
Time after the Onset of Ischemia
FIGURE 2. Changes in coronary perfusion pressure (panel A), coronary blood
flow (panel B), and fractional shortening
(panel C) during intracoronary infusion
and withdrawal of clonidine infusion in
coronary hypoperfusion. Three to 5 minutes after clonidine infusion, coronary
blood flow was gradually increased (panel
B) and fractional shortening recovered
{panel C), indicating that intracoronary
infusion of clonidine improves mechanical
myocardialfunction during ischemia. During this protocol, reduced coronary perfusion pressure was kept constant (panel A).
30
(run)
C
A
(ml/IOOg/min)
(nmol/100e/min)
p< o.ooi
p< aooi
P<OSS
P<0.05
11
FIGURE 3. Changes in myocardial oxygen consumption (MVOb panel A), lactate extraction ratio (panel Bj, adenosine
release (panel C), and norepinephrine concentrations in the coronary arterial and
venous blood (panel D) before and after
intracoronary infusions of clonidine (0.24
)jglkg/min), and after withdrawal of clonidine during myocardial ischemia. Intracoronary infusions of clonidine significantly improved lactate extraction ratio
(panel B), indicating that clonidine infusion improves metabolic function ofischemic myocardium. With this improvement of metabolic function, myocardial
oxygen consumption was also increased
(panel A).
6-
2
B
(X)
-lO-i
.a
(pg/ml)
P<0.005
P<aO5
H 600
-20
| -30-
I -40
5 400
^ -50
I
3
I 200
S -60-
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• coronary v«in
-70
ischemia
ochmca
wrth ckjoiSn.
bch«nia
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with dori&m
1638
Circulation Research
Vol 65, No 6, December 1989
(ml/IOOg/min)
«
tt
c
I 3OH
m
20-
10-1
FIGURE 4. Effects of intracoronary infusion of
yohimbine (9 (jg/kg/min) on coronary blood flow
(panel A), fractional shortening (panel B), and
lactate extraction ratio (panel C) during coronary
hypoperfusion with intracoronary infusion of clonidine. Clonidine increased coronary blood flow by
32%. Yohimbine infusion significantly (p<0.001)
decreased improved coronary blood flow, fractional shortening, and lactate extraction ratio to
the pre-clonidine level, although clonidine infusion
was continued. Coronary perfusion pressure was
kept constant during this protocol
B
10-
e
i
5-
(A
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•-703
r
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o
10
20
30
(min)
Time after the Onset of Ischemia
A
(mmHg)
D(ml/1C0e/»ln)
6-
| 100«
4-
I
I 50
2-
FIGURE 5. Changes in coronary perfusion pressure (panel A), coronary blood flow (panel B), fractional shortening (panel C), myocardial oxygen consumption (panel D), lactate extraction ratio (panel
E), and adenosine release (panel F) by intracoronary infusion of clonidine during coronary hypoperfusion. Adenosine receptors were antagonized by
intracoronary infusion of 8-phenyltheophylline.
C
(*)
F(nmol/100g/™ln)
I'
12-
8-
I
m«ans±SE
n-5
ischemia
with clonidirw
with clonidine
Kitakaze et al Effect of Oj-Adrenoceptor Activity on Ischemia
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10
*/
FIGURE 6. Changes in coronary perfusion
pressure (panel A), coronary blood flow (panel
B), and fractional shortening (panel C) during
intracoronary infusion and withdrawal of clonidine infusion in coronary hypoperfusion in
the denervated hearts. Three to 5 minutes after
clonidine infusion, coronary blood flow gradually increased (panel B) and associated with
this, fractional shortening increased (panel C).
The extents of increases in coronary blood flow
and fractional shortening are comparable with
those in the innervated hearts, indicating that
withdrawal of sympathetic activity by presynaptic aradrenoceptor activation is not involved
in the beneficial effect of clonidine in ischemic
myocardium. During this protocol, reduced
coronary perfusion pressure was kept constant
(panel A).
1
[
\
5
.(OJWVlArWvic)
30
10
20
Tim* afUr th« Onset of Ischsmia
A
(on)
c
(ml/IOOg/tr
P<(UB
P<O05
(ml/IOOg/tr
10-
p<ao5
p<ao5
6
42-
B
(K)
0
(PS/ml)
P<0J)1
P<0JB
E —10
600
400
4o
2
m«ans±SE.n«5
0!200
-40
O coronary artery
• coronary v«in
ach.nml
ach.nml
octant
•ithdrml
dckndht
•tcrwnu
hchtait
iuMn
FIGURE 7. Changes in myocardial oxygen consumption (panel A), lactate extraction ratio
(panel B), adenosine release (panel C), and
norepinephrine concentrations in the coronary
arterial and venous blood (panel D), before
and after intracoronary infusion of clonidine
(0.24 fjg/kg/min), and after withdrawal of clonidine during myocardial ischemia. The hearts
were chemicalfy denervated. In the denervated
dogs, systemic norepinephrine concentration
was higher (panel D) than that in the innervated myocardium (Figure 3D). This may be
because of compensatory mechanisms of depletion of norepinephrine in the heart.23 Intracoronary infusion of clonidine significantly
improved lactate extraction ratio (panel B),
indicating that clonidine infusion improves metabolic function of ischemic myocardium. This
improvement is related with neither the withdrawal ofpresynaptic sympathetic activity nor
the increase in adenosine release from the
ischemic myocardium (panel C). According to
this improvement of metabolic function, myocardial oxygen consumption (MVOJ was also
increased (panel A).
1640
TABLE 2.
Circulation Research Vol 65, No 6, December 1989
Coronary Hemodynamic anil1 Metabolic Parameters Before and After Treatments With Propranolol and 8-Phenyltbeophylline
CPP
(mm Hg)
CBF
(ml/100
g/min)
Untreated
Propranolol
(baseline)
105+7
93±3
104±8
89±5
8-Phenyltheophylline
103±8
89±8
NfVO,
LER
(%)
28.9±2.5
AVOjD
(ml/dl)
8.69±0.41
(ml/100
g/min)
AdR
(nmol/100
g/min)
FS
(%)
8.10±0.43
1.5±0.7
23±1
30.2+3.3
23.4±4.4
7.92±0.62
7.93±0.44
7.01 ±0.55
7.07±0.71
1.1 ±0.6*
2.6±0.8
23±3
20±l
Values are roean + SEM (n=5). CPP, coronary perfusion pressure; CBF, coronary blood flow; LER, lactate extraction ratio; AVO 2 D,
coronary arteriovenous oxygen difference; MV0 2 , myocardial oxygen consumption; AdR, adenosine release; FS, fractional shortening.
*p<0.05 vs. the untreated control condition.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
of presynaptic a2-adrcnoccptors is not involved in
the mechansim of this beneficial effect of clonidine
infusion.
Figure 8 represents the subcellular molecular
basis of the interaction between a2-adrenoceptor
stimulation and adenosine. Without treatment of
clonidine, myocardial ischemia (coronary perfusion
pressure, 94±6 to 32±5 mm Hg; coronary blood
flow, 82±3 to 27±2 miyiOO g/min) increased cyclic
AMP content of the coronary artery to 290±40
pmol/g (p<0.001). However, treatment of clonidine
during myocardial ischemia further increased
(/><0.05) cyclic AMP content of the involved coronary artery to 480±70 pmol/g.
Effects of arAdrenoceptor Stimulation on
Exogenous Adenosine-Induced
Coronary Vasodilation
Figure 11 shows the responses of CBF to exogenous adenosine with and without clonidine under
the condition that pH in the coronary venous blood
is 7.38±0.02. Adenosine-induced coronary vasodilation (1 ^ig/kg/min i.e.) was enhanced by 28±5%
with infusion of clonidine (panel A). Figure 11B
shows the responses of CBF to adenosine with and
without clonidine under the condition that pH in the
coronary venous blood is 7.27±0.03. Treatment of
clonidine (1 ^ig/kg/min i.e.) enhanced adenosineinduced coronary vasodilation by 4O±3%.
Effects of arAdrenoceptor Stimulation on
Microsphere-Induced Myocardial Ischemia
The doses of microspheres at the maximal embolization with and without clonidine infusion were
5.2±0.2xl0 5 and 5.4±O.3xlO5/g myocardium,
respectively (NS). Figure 9 demonstrates the changes
in CBF and adenosine release during repetitive
embolizations of microspheres. Intracoronary infusion of clonidine significantly (/><0.05) enhanced the
peak hyperemic CBF by 39% despite no significant
changes in adenosine release. Although coronary
micToembolization produces almost linear decreases
in fractional shortening and lactate extraction ratio
(Figure 10), these two parameters with clonidine
were significantly (p<0.05) improved compared with
those without clonidine, indicating that clonidine
improves the microsphere embolization-induced myocardial ischemia due to the enhancement of adenosine's vasodilatory effect by clonidine.
Discussion
Although there is consensus that potent a2adrenoceptor stimulation causes coronary vasoconstriction,5-7-8-24 we have previously reported that
adenosine-induced coronary vasodilation is enhanced
by moderate a2-adrenoceptor stimulation.10-12 The
present study provides evidence that a2-adrenoceptor
stimulation can increase coronary blood flow of ischemic myocardium and improve both mechanical and
metabolic functions. This increased flow is nutritional
to ischemic myocardium.
We showed in the present study that a2adrenoceptor stimulation by clonidine (0.24 fig/
kg/min i.e.) increases coronary blood flow in the
ischemic heart by 32-43% (Figures 2B, 4A, 6B, and
9A). Our previous studies10"12 showed that moderate a2-adrenoceptor stimulation enhances exogenous adenosine-induced vasodilation, and the present study indicated that acidotic condition enhances
the sensitivity of a2-adrenoceptor stimulation on aden-
TABLE 3.
Coronary Hemodynamic and Metabolic Parameters Before and After 0-Adrenoceptor Blockade in Denervated Hearts
CPP
(mm Hg)
CBF
(ml/100
g/min)
LER
(%)
AVO 2 D
(ml/dl)
MVOj
(ml/100
g/min)
AdR
(nmol/100
g/min)
FS
(%)
NE(A)
(pg/ml)
NE(V)
(Pg/ml)
Untreated
Propranolol
90±4
84±2
21.3±2.4
7.90±1.19
6.70±1.01
2.2±0.7
31±3
516±47
476 ±57
(baseline)
84 ± 3
74±2*
22.8±1.6
8.10±1.12
5.97±0.74
1.8±0.7
29±3
500+4S
488±56
Values are mean±SEM (n=5). CPP, coronary perfuston pressure; CBF, coronary blood flow; LER, lactate extraction ratio; AVO 2 D,
coronary arteriovenous oxygen difference; MVO2, myocardial oxygen consumption; AdR, adenosine release; FS, fractional shortening;
NE(A) and NE(V), norepinephrine concentrations in coronary arterial and venous blood, respectively.
'p<0.05 vs. the untreated control condition.
Kitakaze et al
(pmol/g)
P<0.05
J^L without clonidint
j i L w i t h clomdin*
m««ns±SE,
400-
200-
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
control
rtchtmia
FIGURE 8. Changes in cyclic AMP content in the coronary arteries with and without clonidine during myocardial ischemia. Treatment with clonidine significantly
(p<0.05) increased cyclic AMP contents of the coronary
artery in the ischemia area compared with treatment
without clonidine. Hearts were treated with propranolol.
osine's effect by 40% (Figure 11B). This value corresponds well to the increment of coronary blood flow
by oz-adrenoceptor stimulation in the ischemic myocardium. Furthermore, a potent antagonist of adenosine receptors, 8-phenyltheophylline, completely abolished the effects of a2-adrenoceptor stimulation on
coronary blood flow, indicating that improvement of
myocardial ischemia with increased coronary blood
flow is due to enhancement of vasodilatory effects of
adenosine released from the ischemic myocardium by
a2-adrenoceptor stimulation.
Nevertheless, we should consider other possibilities. a2-Adrenoceptor stimulation may increase
adenosine release from the ischemic myocardium
and increase coronary blood flow. However, this is
not the case because the present study showed no
increases in adenosine release (Figures 3C, 7C, and
9B), and our previous study-showed that adenosine
release is not attributed to the a2-adrenoceptor
activity.9 a:-Adrenoceptor activation is reported to
increase in the endothelium-derived relaxing factor
and may cause coronary vasodilation.25 However,
intracoronary infusion of clonidine in the normoxic
condition did not change baseline CBF (Figure 9A),
indicating that this dose of clonidine does not facilitate release of endothelium-derived relaxing factor.
In the skeletal muscle, a2-adrenoceptor stimulation
produces vasodilation resulted from histamine
release. Histamine is reported to increase coronary blood flow through its inotropic and chronotropic effects.- However, this mechanism is not
likely because 1) intracoronary infusion of clonidine
changed neither MVO2 nor baseline CBF and 2)
positive inotropic effect of histamine may result in
improvement of contractile function but may not
Effect of aj-Adrenoceptor Activity on Ischemia
1641
improve metabolic function during myocardial ischemia (Figures 3B and 7B). In addition to this,
several lines of evidence29'30 suggest that aadrenoceptor activity itself has a positive inotropic
effect, which may enhance the contractility of ischemic region and increase coronary blood flow.
However, this mechanism is unlikely because
increased contractility neither improves the metabolic function (lactate extraction ratio and pH in
coronary venous blood) nor decreases adenosine
release. a2-Adrenoceptor stimulation can cause withdrawal of tonic sympathetic activity and exert coronary vasodilation, since activation of presynaptic
a2-adrenoceptors may decrease the release of norepinephrine from the presynaptic vesicles.31 However, in our experiment there is no evidence that
clonidine during coronary hypoperfusion decreases
norepinephrine concentration in the coronary venous
blood (see Figures 3D and 7D), and furthermore,
clonidine increases coronary blood flow even in the
denervated hearts (Figure 6B). There is also the
possibility that intracoronary infusion of clonidine
opens functional collateral vessels to the ischemic
area, which may reduce the severity of ischemia.
However, there is evidence that collateral flow is
not regulated by a-adrenoceptor activities.33 Nathan
and Feigl reported that a-adrenoceptor stimulation during coronary hypoperfusion favorably affects
intramyocardial flow distribution by causing subepicardial vasoconstriction. During hypoperfusion, a
decrease in subendocardial flow is more prominent
than in subepicardial flow, although oxygen demand
in the subendocardial region is higher than in the
subepicardial region. If subepicardial flow dominantly bypasses the subendocardial region, subendocardial ischemia may be improved. Since coronary resistance vessels in the nonischemic condition
are predominantly regulated by cr8242-adrenoceptor
activity,- this mechanism may be possible. However, BufBngton and Feigl showed that transmural
inhomogeneous flow distribution mediated by aadrenoceptor coronary vasoconstriction is evident
under moderate coronary hypoperfusion (coronary
perfusion pressures70 mm Hg). This beneficial flow
distribution is diminished by the potent metabolic
vasodilatory effect when the heart is progressively
underperfused (coronary perfusion pressure, 38
mm Hg; lactate extraction ratio, -49%). In our
experiment, because coronary perfusion pressure
during hypoperfusion was 38±5 mm Hg (Figure
2A), the beneficial effect observed by Nathan and
Feigl is not related to our present observation.
Heusch and Deussen5 demonstrated that postsynaptic a2-adrenoceptor stimulation mediates coronary vasoconstriction produced by sympathetic
nerve stimulation during critical reduction of coronary perfusion pressure and concluded that a2adrenoceptor stimulation is deleterious in myocardial ischemia. Seitelberger et al7 also demonstrated
that a2-adrenoceptor blockade attenuates the severity of exercise-induced myocardial ischemia. These
1642
Circulation Research
(ml/1002/ min)
2M-1
Vol 65, No 6, December 1989
P<0.01
FK0.05
|
I without clomdma (n =
FIGURE 9. Serial changes in
coronary blood flow (panel A)
and adenosine release (panel B)
under repetitive embolization of
microspheres into coronary
artery with and without intracoronary infusion of clonidine
(0.24 figlkglmin). Extent of
emboUzation indicates cumulative amount of injected microspheres normalized by amount
of maximal embolization. The
doses of microspheres at maximal embolization with and without clonidine infusion were
5.2±0.2xl01 and 5.4±0.3xl0sfg
myocardium, respectively (NS).
a2-Adrenoceptor
stimulation
with clonidine further enhanced
microembolization-induced coronary hyperemia (panel A)
despite no significant changes
in adenosine release from ischemic foci (panel B).
with clonidk-10 (n = 5)
am±SE
B
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
-2
6-
1
X
1
4c
X
41
2
0
(cofltrol)
20
40
40
60
100
(X)
Extant of Embolization
p<0.025
30
without clonidina (n = 7)
with donidint (nM5)
m.«m±SE
p<0.025
20-1
T
p<0.05
P<0.0l
-10
B
-20
0
(control)
20
40
60
E x t e n t of Embolization
40
FIGURE 10. Serial changes in
fractional shortening (panel A)
and lactate extraction ratio
(panel B) under repetitive embolization of microspheres into coronary artery with and without
intracoronary infusion of clonidine. Without clonidine, fractional shortening and lactate
extraction ratio were significantly (p<0.05) decreased compared with those of the clonidinetreated group, indicating that
intracoronary infusion of clonidine significantly
improves
mechanical and metabolic functions during
microsphereinduced ischemia.
Kitakaze et al
A
normal condition
Effect of Oj-Adrenoceptor Activity on Ischemia
B
acidotic condition
1643
p<0.00l
(ml/IOOt/min)
(ml/100»/"nn)
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
control
Doses of Adenosine
Doses of Adenosine
11. Effects of intracoronary infusion of clonidine on adenosine-induced coronary vasodilation under arterial
blood pH of 7.39±0.01 (panel A) and 7.27±0.01 (panel B). The vasodilatory effect of adenosine is augmented (p<0.01)
by clonidine infusion by 28±5% at 1 figlkglmin of adenosine infusion (panel A); however, in the acidotic condition (panel
B), intracoronary infusion of clonidine further enhanced (p<0.05) adenosine-induced coronary vasodilation (40±3%).
FIGURE
two reports seem contradictory to ours. Both exercise and sympathetic nerve stimulation elevate the
systemic norepinephrine level to more than 1,000
pg/ml,18-35-37 which is two to three times higher
concentrations than that of our results (Figure 3D).
If tt2-adrenoceptor stimulation is potent, direct coronary vasoconstriction is exerted and may overcome the beneficial effect of a2-adrenoceptor stimulation on adenosine-induced coronary vasodilation.
Our previous studies10-12 showed that intracoronary
infusion of norepinephrine of 0.24 /tg/kg/min
enhances adenosine-induced maximal coronary vasodilation under a,- and j8-adrenoceptor blockades;
however, 0.60 pig/kg/min of norepinephrine
adversely decreases maximal coronary blood flow,
indicating that two to three times more potent
stimulation of a2-adrenoceptor activity can reverse
its effect on coronary blood flow. Thus a2adrenoceptor stimulation increases coronary blood
flow and improves myocardial ischemia under low
norepinephrine levels in systemic arterial blood, but
further activation of aradrenoceptors under high
concentration of norepinephrine may decrease coronary blood flow and produce severe ischemia
during coronary hypoperfusion. It is noteworthy
that a2-adrenoceptor stimulation by norepinephrine
plays a protective role in ischemic myocardium,
because several lines of evidence suggest only the
deleterious effects of catecholamine,' and the phys-
iological role of a-adrenoceptor activity during ischemia has not been clarified.
Activations of the adenosine receptor (A2) and
/3-adrenoceptor in coronary arteries increased cyclic
AMP through the activation of adenylate cyclase
activity. However, interference of /3-adrenoceptor
stimulation during ischemia was minimal because
the experiments were done under propranolol infusions, and thus the increase in cyclic AMP level by
ischemia was mainly attributed to the stimulation of
adenosine receptor in the coronary arteries. Our
data (Figure 8) also showed that myocardial ischemia increases cyclic AMP content of the coronary
artery. a2-Adrenoceptor stimulation during myocardial ischemia enhanced this production of cyclic
AMP in the coronary artery; however, stimulation of
a2-adrenoceptors alone is not responsible for increases
in intracellular cyclic AMP concentrations.31 This
result indicates that our observation (i.e., the interaction between a2-adrenoceptors and adenosine receptors) is tightly coupled to the changes in cyclic AMP
levels in the involved coronary arteries. Indeed,
several investigators reported that an increase in
cyclic AMP level by stimulation of adenosine receptors is amplified by the a382-adrenoceptor stimulation
in the cerebellum and islet cells.
Although we did not elucidate the regulatory
mechanisms of amplification of cyclic AMP level by
a2-adrenoceptor stimulation, we provided the novel
1644
Circulation Research
Vol 65, No 6, December 1989
evidence that a2-adrenoceptor stimulation can be
beneficial in acute myocardial ischemia through the
enhancements of adenosine-induced cyclic AMP
production and coronary vasodilation.
Acknowledgments
The authors gratefully acknowledge the helpful
discussion and generous assistance of Drs. T. Matsuyama, H. Ozaki, and K. Iwai in the catecholamine measurements.
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1645
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KEY WORDS • a2-adrenoceptor activity • clonidine
myocardial ischemia • adenosine • catecholamine
•
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Beneficial effects of alpha 2-adrenoceptor activity on ischemic myocardium during coronary
hypoperfusion in dogs.
M Kitakaze, M Hori, K Gotoh, H Sato, K Iwakura, A Kitabatake, M Inoue and T Kamada
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Circ Res. 1989;65:1632-1645
doi: 10.1161/01.RES.65.6.1632
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1989 American Heart Association, Inc. All rights reserved.
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