Department of Pharmacology and Toxicology, Medical College of
Wisconsin, Milwaukee, Wisconsin (G.J.Gross, D.A.M.), and
Department of
Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical
Research Institute, Princeton, New Jersey (P.G.S., G.J.Grover)
There has been controversy regarding whether ATP-sensitive potassium
channel activation protects hearts through adenosine A1
receptor activation or the converse. We addressed this issue by
determining the effect of the adenosine A1 receptor
antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) on the
cardioprotective activity of the ATP-sensitive potassium channel opener
bimakalim. In isolated rat hearts subjected to 25 min of global
ischemia and 30 min of reperfusion, bimakalim significantly reduced
lactate dehydrogenase release and improved postischemic recovery of
contractile function. Bimakalim increased the time to the onset of
ischemic contracture (EC25 = 1.2 µM), compared with
vehicle, and 10 µM DPCPX had no effect on this protective action
(EC25 = 1.1 µM). The 10 µM concentration of DPCPX was
sufficient to abolish the bradycardic and cardioprotective effects of
the adenosine A1 receptor agonist
(R)-(
)-N6-(2-phenylisopropyl)adenosine.
DPCPX alone had no effect on the severity of ischemia/reperfusion
damage. Glyburide completely abolished the cardioprotective effects of
bimakalim. Bimakalim (1 µg/kg, intracoronarily) given over four
periods of 5 min, interspersed with 10-min drug-free periods, before a
60-min occlusion and 3-hr reperfusion significantly reduced infarction
size in anesthetized dogs (25 ± 5 and 8 ± 2% of the left
ventricular area at risk for vehicle- and bimakalim-treated groups,
respectively). DPCPX had no effect on the infarction-sparing activity
of bimakalim (9 ± 3% of the left ventricular area at risk). The
protective effect of bimakalim was not accompanied by marked
hemodynamic changes or by changes in regional myocardial blood flow.
The results of this study suggest that the cardioprotective effects of
ATP-sensitive potassium channel openers are not dependent on adenosine
A1 receptor activation in rat or dog models of ischemia.
 |
Introduction |
KATP openers, as
a class, exert cardioprotective effects in various experimental
models of myocardial ischemia (Auchampach et al., 1991
; Ohta
et al., 1991; Grover, 1994). These protective effects are
abolished by known KATP blockers such as glyburide and
sodium 5-hydroxydecanoate (Ohta et al., 1991; Grover, 1994). This inhibition of the cardioprotective effects by KATP
blockers is uniformly observed for all KATP openers (for
review, see Grover, 1994). Glyburide abolishes not only the
cardioprotective effects of KATP openers but also their
smooth muscle relaxant activity (for review, see Edwards and Weston,
1993).
Recent studies have suggested the possibility of KATP
activation or interaction in the mechanism of preconditioning (Gross and Auchampach, 1992; Toombs et al., 1993b; Tomai et
al., 1994). Studies have also shown that KATP openers
such as bimakalim not only mimic preconditioning but also reduce the
threshold for preconditioning (Yao and Gross, 1994a). Furthermore,
several reports have suggested the potential importance of adenosine
A1 receptor activation in preconditioning (Downey et
al., 1993). Numerous studies have established a link between
adenosine A1 receptors and KATP, such that
KATP blockers can abolish the protective effects of
adenosine A1 agonists in dogs and rabbits, suggesting that
adenosine A1 receptor activation protects hearts through
KATP activation (Grover et al., 1992; Toombs
et al., 1993a; Van Winkle et al., 1994).
Conversely, several studies have suggested that the protective effects
of KATP openers are abolished by adenosine A1
receptor antagonists (Walsh et al., 1994; Armstrong et
al., 1995; Kitakaze et al., 1996). Those authors speculated that KATP openers increase adenosine release
via activation of 5
-nucleotidase or preserve adenosine
levels and therefore exert protective effects. Our hypothesis is that
KATP openers do not protect hearts via enhanced
adenosine A1 receptor activation. We addressed this by
determining the effect of adenosine A1 receptor blockade on
the cardioprotective action of bimakalim in a canine model of
infarction and an isolated rat heart model of ischemia and reperfusion.
We chose these species because they both respond to KATP
openers, in terms of cardioprotection, but there are apparent differences in their mechanisms of preconditioning. Another advantage of rat hearts is that the effect of adenosine A1 receptor
blockade can be examined over the entire cardioprotective concentration range of bimakalim.
| |
Methods |
Isolated rat heart model of ischemia and reperfusion.
Male
Sprague-Dawley rats (400-500 g) were anesthetized with 100 mg/kg
sodium pentobarbital (i.p.). The trachea was intubated, and then the
jugular vein was injected with heparin (1000 U/kg). While the rats were
mechanically ventilated, their hearts were perfused in situ
via retrograde cannulation of the aorta. The hearts were
then excised and quickly moved to a Langendorff apparatus, where they
were perfused with oxygenated Krebs-Henseleit solution containing 112 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1.2 mM MgSO4, 1 mM KH2PO4, 1.2 mM CaCl2, 11.5 mM
glucose and 2 mM pyruvate, at a constant perfusion pressure (85 mm Hg).
A water-filled latex balloon attached to a metal cannula was then
inserted into the left ventricle and connected to a Statham pressure
transducer for measurement of left ventricular pressure. The hearts
were allowed to equilibrate for 15 min, at which time EDP was adjusted to 5 mm Hg; this balloon volume was maintained for the duration of the
experiment. Preischemia or predrug function, heart rate and
coronary flow (extracorporeal electromagnetic flow probe; Carolina
Medical Electronics, King, NC) were then measured. Contractile function
was calculated by subtracting EDP from left ventricular peak systolic
pressure, resulting in LVDP. Cardiac temperature was maintained
throughout the experiment by submerging the hearts in 37°C buffer,
which was allowed to accumulate in a stoppered heated chamber.
After equilibration, the hearts were subjected to one of several
treatments. Hearts were treated with vehicle (0.04% DMSO, n = 4), 0.1 to 3.0 µM bimakalim (n = 4/group) or 0.1 to 3.0 µM bimakalim plus 10 µM DPCPX
(n = 4/group). The drug treatments were given for 10 min and were included in the perfusate. At this time, the hearts were
subjected to 25 min of global ischemia and 30 min of reperfusion.
Ischemia was initiated by completely shutting off perfusate flow. At
the end of the reperfusion period, contractile function, coronary flow
and LDH release were measured. The drugs were given only before global
ischemia and were not given during reperfusion. Severity of ischemia
was determined from the time to the onset of contracture during global
ischemia, recovery of contractile function at 30 min of reperfusion and
LDH release into the reperfusate. The time to the onset of contracture
was defined as the time during global ischemia in which the first 5-mm
Hg increase in EDP was observed. Cardioprotective potency was expressed
as the concentration of bimakalim causing a 25% increase in the time
to contracture, relative to vehicle-treated hearts.
Another group of rat hearts were tested to determine whether the 10 µM concentration of DPCPX used was adequately blocking adenosine
A1 receptors. Rat hearts were isolated and prepared as
described above. They were pretreated for 10 min with vehicle (0.04%
DMSO, n = 6), 1 µM (R)-PIA
(n = 6) or 1 µM (R)-PIA plus 10 µM DPCPX
(n = 6). The hearts were rendered globally ischemic for
25 min, and this was followed by 30 min of reperfusion without drug.
Time to contracture and contractile function were measured as described
above. The effect of the KATP blocker glyburide on the
cardioprotective action of bimakalim was also determined. Rat hearts
were pretreated for 10 min with vehicle (0.04% DMSO, n = 6), 3 µM bimakalim (n = 6) or 1 µM glyburide plus
3 µM bimakalim (n = 6). The hearts were made globally
ischemic and reperfused as described above.
A final group of rat hearts were tested to determine whether DPCPX
alone can affect ischemia/reperfusion injury. The rat hearts were
pretreated with either vehicle (0.04% DMSO, n = 8) or
10 µM DPCPX (n = 8). The hearts were subjected to 25 min of global ischemia and 30 min of reperfusion (without drug). Time
to contracture, LDH release and cardiac function were measured as
described above.
Canine model of infarction.
Adult mongrel dogs of either
gender (19.5-29.3 kg) were fasted overnight, anesthetized with a
combination of sodium barbital (200 mg/kg) and sodium pentobarbital (1 mg/kg) and ventilated (by a respirator) with room air supplemented with
100% oxygen. Arterial blood gases and pH were monitored throughout the
study by an automatic blood gas system (AVL 99; AVL Scientific Corp.) and maintained within their normal ranges. This was done by modulation of the respirator or oxygen flow or administration of sodium
bicarbonate. Body temperature was maintained at 38 ± 1°C with a
heating pad. Aortic blood pressure and left ventricular pressure were
monitored by insertion of a transducer-tipped catheter (PC 771; Millar
Instruments) 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 with a tachometer. The right
femoral vein and artery were cannulated for blood gas measurement
(artery), drug administration (vein) and measurement of reference blood
flow (artery; see below). A left thoracotomy was performed at the fifth
intercostal space, the pericardium was cut and the heart was suspended
in a pericardial cradle. A proximal portion of the LAD distal to the
first diagonal branch was isolated, and an electromagnetic flow probe
(Statham SP 7515; Gould-Statham) was placed around this vessel. A
mechanical occluder was placed distal to the probe for later occlusion
of the LAD. Hemodynamics, heart rate and LAD blood flow were monitored and recorded with a polygraph (model 7; Grass Instruments) throughout the experiment. The left atrial appendage was cannulated for
radioactive microsphere injection.
Figure 1 shows the protocol used in this study. The
animals were randomly assigned to three groups. In one group, vehicle (saline) was given 70 min before 60 min of complete LAD occlusion. In
another group, vehicle (saline) was given for 10 min, followed by four
5-min periods of bimakalim infusion (1 µg/min, intracoronarily), separated by 10 min of washout. After the final 10-min washout period,
the LAD was completely occluded for 60 min, and the hearts were then
reperfused for 3 hr. In a final group of dogs, DPCPX (1 mg/kg, i.v.)
was administered 10 min before four 5-min periods of bimakalim
infusion. Bimakalim was given directly into the LAD to avoid the
hemodynamic consequences of its potent vasodilator activity. DPCPX was
given i.v. because of its innocuous hemodynamic profile.
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Fig. 1.
Diagram for the experimental design for the
isolated rat heart and dog heart models of ischemia and reperfusion.
BK, bimakalim; HR, heart rate; Rep, reperfusion; IS, infarction size.
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After 3 hr of reperfusion, the LAD was cannulated for determination of
the AAR. Patent blue dye (5 ml) and saline (5 ml) were injected at
equal pressure into the left atrium and LAD, respectively. The heart
was immediately fibrillated and removed. The left ventricle was sliced
into transverse sections (6-7-mm wide). The nonstained ischemic area
and the blue-stained area were separated, and both regions were
incubated at 37°C for 15 min with 1% 2,3,5-triphenyl tetrazolium
chloride (Sigma Chemical Co., St. Louis, MO) in 0.1 M phosphate buffer
adjusted to pH 7.4. Triphenyl tetrazolium chloride stains the
noninfarcted myocardium red. After storage overnight in 10%
formaldehyde, infarcted and noninfarcted tissues within the AAR were
separated and determined gravimetrically. Infarction size was expressed
as a percentage of AAR.
Regional myocardial blood flow during ischemia was measured using
radioactive microspheres (Mizumura et al., 1995).
Microspheres were administered 30 min into the prolonged 60-min
occlusion periods. Carbonized plastic microspheres (15 µm; New
England Nuclear) labeled with 141Ce or 95Nb
were suspended in isotonic saline with 0.01% Tween 80 added to prevent
aggregation. They were ultrasonicated for 5 min and vortex-mixed for
another 5 min before injection. The microspheres (2-4 × 106 spheres) were given through the left atrial catheter,
which was flushed with saline. A reference blood flow sample was drawn
from the right femoral artery, at a constant rate of 9.4 ml/min,
starting 30 sec before microsphere injection and continuing for 3 min. The tissue slices were sectioned into subepicardium, midmyocardium and
subendocardium of the nonischemic and ischemic regions. All samples
were counted in a gamma counter (Tracor Analytic 1195) to determine the
activity of each isotope in each sample as well as in the reference
blood sample. Myocardial blood flow was then calculated and expressed
as milliliters per minute per 100 g. Dogs were excluded if
transmural collateral blood flow was >20 ml/min/100 g or if more than
three consecutive attempts were needed to convert ventricular
fibrillation with low-energy d.c. pulses applied directly to the heart.
Chemicals.
(R)-PIA and DPCPX were purchased from
Research Biochemicals (Natick, MA). Glyburide was purchased from Sigma
Chemical Co. Bimakalim was provided as a gift from E. Merck or
was synthesized in the Department of Chemistry at Bristol-Myers Squibb.
Statistics.
All values are expressed as mean ± S.E.M.
Differences between groups in hemodynamics were compared by using
two-way ANOVA with repeated measures and the Fisher least significant
difference post hoc test. Differences between groups in
blood flow, AAR and infarction size were compared by using one-way
ANOVA. ANCOVA was used to determine whether the relationship between
transmural collateral blood flow and infarction size differed between
control and drug-treated groups. For the isolated heart studies,
repeated-measures ANOVA was used with the Newman-Keuls post
hoc test. Differences in regression lines plotting infarction size
as a percentage of the AAR vs. transmural collateral blood
flow were compared by ANCOVA. Statistical significance was set at
P < .05.
| |
Results |
Isolated rat heart studies.
The effect of increasing
concentrations of bimakalim on cardiac function and coronary flow is
shown in table 1. Before ischemia, bimakalim had a
slight cardiodepressant effect, without any effect on heart rate.
Bimakalim significantly increased preischemic coronary flow, although
this did not occur in a clearly concentration-dependent manner. During
reperfusion, LVDP was significantly reduced in vehicle-treated hearts,
indicating severe ischemia/reperfusion damage. Bimakalim significantly
improved reperfusion function in a concentrationdependent manner.
The effects of bimakalim on reperfusion contracture formation during
global ischemia are shown in figure 2. The time to onset
of contracture was significantly increased, in a
concentration-dependent manner. The EC25 for increasing time to contracture for bimakalim was 1.2 µM. Also shown in figure 2
are the cumulative LDH release data. LDH release was significantly reduced by bimakalim. The reperfusion double product (another index
of reperfusion cardiac work) was increased in a concentration-dependent manner by bimakalim. Reperfusion EDP (fig. 2) was significantly reduced
by bimakalim. The incidence of reperfusion fibrillation was not
affected by bimakalim.
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TABLE 1
Effect of bimakalim on pre- and postischemic cardiac function and
coronary flow in isolated rat hearts
All values are mean ± S.E.
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Fig. 2.
Effect of 0.1 to 3.0 µM bimakalim alone or in the
presence of 10 µM DPCPX on LDH release during reperfusion and percent
change in the time to onset of contracture during global ischemia in rat hearts. Bimakalim significantly reduced LDH release and EDP during
reperfusion and increased the time to contracture during global
ischemia. These effects were not abolished by DPCPX. *, significant
difference from vehicle for both bimakalim and bimakalim plus DPCPX
groups (P < .05).
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The effect of DPCPX on the cardioprotective activity of bimakalim is
shown in table 2 and figure 2. DPCPX had no effect on the preischemic cardiodepressant or coronary dilator activities of
bimakalim. DPCPX had no significant effect on the time to contracture curve for bimakalim, such that the EC25 (1.1 µM) for
bimakalim plus DPCPX was nearly identical to that for bimakalim alone.
DPCPX had no effect on the protective action of bimakalim on
reperfusion LDH release or recovery of contractile function. The
protective effect of bimakalim on reperfusion EDP was not
affected by DPCPX. In a separate experiment, the KATP
blocker glyburide (1 µM) completely abolished the cardioprotective
effects of 3 µM bimakalim (data not shown). Glyburide also completely
abolished the preischemic coronary vasodilator action of bimakalim.
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TABLE 2
Effect of bimakalim plus 10 µM DPCPX on pre- and postischemic cardiac
function and coronary flow in isolated rat hearts
All values are mean ± S.E.
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To ensure that the concentration of DPCPX was sufficient to effectively
block adenosine receptors, we determined its effect on the
cardioprotective activity of (R)-PIA (adenosine receptor agonist). These data are shown in table 3 and figure
3. (R)-PIA caused significant preischemic
bradycardia, which was abolished by 10 µM DPCPX. (R)-PIA
significantly enhanced postischemic recovery of function, reduced LDH
release, increased the time to the onset of contracture and attenuated
reperfusion contracture. These protective effects were completely
abolished by DPCPX. (R)-PIA had no effect on coronary flow,
as would be expected (data not shown). In a separate study, DPCPX alone
had no effect on the severity of ischemia/reperfusion injury, with
DPCPX causing a time to contracture of 17.5 ± 1.1 min and a LDH
release of 24 ± 2 U/g, which were not different from the
respective vehicle-treated group values (17.1 ± 0.7 min and
25 ± 2 U/g).
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TABLE 3
Effect of (R)-PIA, with or without DPCPX, on pre- and
postischemic cardiac function and coronary flow in isolated rat hearts
All values are mean ± S.E.
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Fig. 3.
Effect of 1 µM (R)-PIA alone or in
the presence of 10 µM DPCPX on reperfusion LDH release and EDP after
global ischemia and the time to onset of contracture during global
ischemia in rat hearts. (R)-PIA significantly reduced
LDH release and enhanced the recovery of contractile function. These
effects were completely abolished by DPCPX. *, significant difference
from vehicle group (P < .05).
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Canine infarction size studies.
The effect of bimakalim on
myocardial infarction size in dogs is shown in figure 4.
Vehicle-treated hearts had infarctions that were approximately 25% of
the AAR. Bimakalim significantly reduced infarction size, such that it
was approximately 8% of the AAR. DPCPX had no effect on the
cardioprotective effects of bimakalim.
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Fig. 4.
Effect of bimakalim alone or with DPCPX on
infarction size in dogs. Infarction size is expressed as percentage of
the AAR. The AAR was similar for all groups. Bimakalim significantly
reduced infarction size (*P < .05), compared with vehicle-treated
hearts, and DPCPX had no effect on this cardioprotective action. LV,
left ventricle.
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Linear regression analysis demonstrated an inverse relationship between
transmural collateral blood flow at 30 min of ischemia and infarction
size/AAR for all groups (fig. 5). By ANCOVA, it was
observed that there was a significant downward shift in the regression
lines in the two bimakalim-treated groups, compared with the control
group.
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Fig. 5.
Plot of transmural collateral blood flow to the
ischemic area at 30 min of occlusion vs. infarction size
expressed as a percentage of the AAR (IS/AAR). The regression lines for
the bimakalim (BK) and bimakalim plus DPCPX groups were significantly
(P < .05) shifted downward, compared with control (by ANCOVA).
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Regional myocardial blood flow was measured at 30 min into ischemia in
both the LAD-perfused region (ischemic) and the nonischemic region
(table 4). Myocardial blood flow in the nonischemic
region was similar for all groups. In the ischemic region, marked
blood flow reductions were observed for all groups and were most severe in the subendocardial region. No differences between groups in collateral blood flow were observed.
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TABLE 4
Regional myocardial blood flow in ischemic and nonischemic zones during
ischemia
All values were measured at 30 min into the 60-min ischemic period and
are expressed as mean ± S.E.M. No differences between drug
treatments were observed.
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Hemodynamic data are shown in table 5. Base-line values
for heart rate, arterial blood pressure and coronary blood flow were similar for all groups. Heart rate did not deviate from base-line levels throughout the protocol for any group. Mean arterial blood pressure was slightly reduced in vehicle-treated hearts during ischemia, and blood pressure was better maintained in the
bimakalim-treated group. No other differences were observed for
arterial blood pressure. Coronary blood flow (electromagnetic flow
probe) was similar under base-line conditions for all groups and was
unchanged during reperfusion in all groups. Preischemic blood flow was
significantly increased by bimakalim, and this was not blocked by
DPCPX. There was no coronary blood flow in the LAD during ischemia.
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TABLE 5
Effect of bimakalim, with or without DPCPX, on hemodynamic parameters
in anesthetized dogs
All values are mean ± S.E.M. Occl, 30 min into ischemia; Rep,
reperfusion; Drug, postdrug.
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| |
Discussion |
Specific KATP openers have been shown by numerous
laboratories to protect ischemic myocardium (Auchampach et
al., 1991; Ohta et al., 1991; Grover, 1994). The
cardioprotective effects of this class of agents are independent of
vasodilator activity, as well as action potential shortening (Atwal
et al., 1993; Yao and Gross, 1994b). KATP
openers appear to protect via a direct effect on the
myocardium, because they have been shown to have cardioprotective effects in isolated myocytes (Armstrong et al., 1995).
KATP openers conserve ATP in ischemic myocardium while
having minimal effects on cardiac function (Grover et al.,
1991).
KATP openers may mimic an endogenous protective mechanism,
because KATP blockers abolish preconditioning in several
species, including humans (Gross and Auchampach, 1992; Toombs et
al., 1993b; Tomai et al., 1994). The profile of
cardioprotection for preconditioning is consistent with that for
KATP openers. This has been demonstrated by studies showing
that the threshold for preconditioning is reduced by bimakalim (Yao and
Gross, 1994a). In that study, the combination of a subthreshold dose of
bimakalim with a subthreshold preconditioning stimulus resulted in
significant cardioprotection. Rat hearts can also be preconditioned,
although the linkage of this protection to KATP is not as
clear. Studies in isolated rat hearts showed that glyburide did not
abolish preconditioning (Fralix et al., 1993). Recent
studies by Gross and Schultz (1996) showed that preconditioning in an
in vivo model of ischemia and reperfusion in rats was
abolished by prolonged treatment with glyburide, so this issue remains
to be resolved.
Although most studies show KATP to be involved with
preconditioning, it is still unknown how these channels fit into the
cascade of events during preconditioning. Early studies suggested a
link between adenosine A1 receptors and KATP
(Kirsch et al., 1990; Toombs et al., 1993b; Van
Winkle et al., 1994). Because antagonists of both
KATP and adenosine A1 receptors abolish
preconditioning in most species, it is possible that these two systems
are linked in series. Studies by Kirsch et al. (1990)
suggested that adenosine A1 receptor activation can trigger
KATP activation, although this was done in normoxic rat
neonatal cardiac myocytes. We examined this in dogs and found the
cardioprotective effects of (R)-PIA to be abolished by
glyburide (Grover et al., 1991). Similar results were found
for the protective effects of adenosine using the KATP blockers glyburide and sodium 5-hydroxydecanoate in pigs and rabbits (Toombs et al., 1993a; Van Winkle et al.,
1994). Although those studies seemed fairly conclusive, several studies
contradicted those results (Walsh et al., 1994; Armstrong
et al., 1995; Kitakaze et al., 1996).
The contradictory results prompted us to evaluate, in a detailed
manner, the effect of an adenosine A1 receptor antagonist on the cardioprotective effects of a selective KATP opener
in dogs and rats. We chose to study rat hearts, in addition to dogs, because we can perform detailed doseresponse studies and therefore can properly evaluate the effects of adenosine receptor antagonists. The profiles of cardioprotection for KATP openers are
similar in rats and dogs (for review, see Grover, 1994). In the present study, bimakalim exerted clear cardioprotective effects in dogs and
rats. In rat hearts, the cardioprotective effects occurred in a
concentration-dependent manner and were completely abolished by
glyburide. DPCPX had no effect on the cardioprotective activity of
bimakalim in either species. In rat hearts, we determined the effect of
DPCPX over the cardioprotective concentration range, to investigate
whether there was a shift in this curve, and there was no shift. At 10 µM, DPCPX was found to completely block the bradycardic and
cardioprotective actions of (R)-PIA. The 1 µM concentration of (R)-PIA was found to be a maximally
cardioprotective concentration; because 10 µM DPCPX completely
abolished this action, adenosine A1 receptors were
effectively blocked (Grover et al., 1996). This
concentration of DPCPX was previously shown to be without effect on the
severity of ischemia/reperfusion injury in isolated rat hearts (Grover
et al., 1996). The concentration of glyburide used in the
rat hearts was previously shown to be without effect on the severity of
ischemia (Grover, 1994; Grover et al., 1996). Although
glyburide does affect systems other than KATP (Al-Aqati,
1995), its ability to inhibit cardioprotection is specific to
KATP openers (Sargent et al., 1991). The dose of DPCPX used in the dog study was previously shown to have no effect on
infarction size and no effect on adenosine A2
receptor-mediated increases in coronary blood flow (Auchampach and
Gross, 1993).
It is still difficult to reconcile our results with those of the
investigators finding adenosine antagonists to block KATP opener-induced protection. Because we determined the effect of DPCPX in
rats over the entire cardioprotective concentration range, we feel
confident that DPCPX has no effect on the cardioprotective activity of
bimakalim. All of the studies showing that adenosine A1
receptor blockade abolished cardioprotection used pinacidil, and
pinacidil is thought to have other activities not related to
KATP opening, although those activities have not been
thoroughly characterized (Meisheri et al., 1991). Our use of
rat hearts can be criticized because their mechanism of preconditioning
is thought to be different from that of other species, but DPCPX was
without effect on dogs as well. It is difficult to believe that
KATP openers protect dog and rat hearts via
different mechanisms, and therefore the data on both species are
relevant. It should also be pointed out that DPCPX is specific for
adenosine A1 receptors and the role of adenosine
A3 receptors in mediating KATP opener-induced cardioprotection remains unknown (Armstrong and Ganote, 1995). Previous
studies from our laboratory showed that 10 µM DPCPX had no effect on
the coronary dilator activity of adenosine, suggesting that DPCPX is
devoid of adenosine A2 receptor-blocking activity (Grover
and Sleph, 1994).
We still do not know the exact mechanism of cardioprotection for
KATP openers, but we feel confident that it is not related to adenosine A1 receptor activation. The protective effects
of KATP openers are not dependent on reduced cardiac work,
nor are they dependent on increases in coronary blood flow (for review, see Grover, 1994; Grover and Sleph, 1995). Consistent with this, we
showed no change in ischemic regional collateral blood flow with
bimakalim. Bimakalim did increase preischemic flow in the isolated
hearts, but total global ischemia was used and thus there was no
ischemic flow; therefore, bimakalim could not have improved oxygen
delivery. The cardioprotective effects of KATP openers are
not correlated with enhanced sarcolemmal potassium currents (Yao and
Gross, 1994b; Grover, et al., 1995) and have been
hypothesized to involve an intracellular mechanism. KATP
are expressed in mitochondria, and KATP openers activate
this channel within their cardioprotective concentration range (Garlid
et al., 1996). It is possible that adenosine might influence
this intracellular channel, although the nature of such an interaction
is not now clear.
AAR, anatomic area at risk;
ANCOVA, analysis of
covariance;
ANOVA, analysis of variance;
DMSO, dimethylsulfoxide;
DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
EDP, end diastolic pressure;
KATP, ATP-sensitive potassium channels;
LAD, left anterior
descending coronary artery;
LDH, lactate dehydrogenase;
LVDP, left
ventricular developed pressure;
(R)-PIA, (R)-()-N6-(2-phenylisopropyl)adenosine.
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Abstract
Introduction
