© 1999 American Heart Association, Inc.
Vascular Biology |
Selective Activation of the Prostanoid EP3 Receptor Reduces Myocardial Infarct Size in Rodents
From the William Harvey Research Institute (K.Z., A.O., J.P., C.T.), St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK; Klinische Chemie (G.H.), Gutenberg-Universitat, Mainz, Germany; and the Minase Research Institute (K.K.), ONO Pharmaceuticals Co Ltd, Osaka, Japan.
Correspondence to Prof C. Thiemermann, The William Harvey Research Institute, Charterhouse Square, London EC1 M 6BQ, UK. E-mail C.Thiemermann{at}mds.qmw.ac.uk
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Abstract—The cardioprotective effects of E-type prostaglandins (EPs) have been attributed to vasodilatation, inhibition of platelet and neutrophil function (EP2 mediated), and an unknown "cytoprotective effect." We have hypothesized that selective activation of EP3 receptors may cause cardioprotection. The prostanoid derivative ONO-AE-248 selectively binds to murine EP3
receptors
expressed in Chinese hamster ovary (CHO) cells
(Ki, 15 nmol/L) and prevents the rise in
cAMP caused by forskolin in CHO cells (IC50 
1 nmol/L) in
which the EP3 receptor had been expressed. In
anesthetized rats subjected to regional myocardial
ischemia for 25 or 45 minutes and 2 hours of reperfusion,
infusion of ONO-AE-248 (5 µg · kg-1 ·
min-1 IV) caused a significant reduction in infarct size,
from 60±3% (n=8) to 36±6% (n=7) and from 78±2% (n=11) to 58±4%
(n=9), respectively. The reduction in infarct size caused by ONO-AE-248
in rats subjected to 25 minutes of ischemia and reperfusion was
abolished by a selective inhibitor of ATP-sensitive
potassium (KATP) channels, 5-hydroxydecanoate (n=6), and
the protein kinase C inhibitors staurosporine
(n=6) and chelerythrine (n=6). In anesthetized rabbits
subjected to coronary artery occlusion for 45 or 60 minutes and
2 hours of reperfusion, infusion of ONO-AE-248 (5 µg ·
kg-1 · min-1 IV) caused a significant
reduction in infarct size, from 61±2% (n=10) to 36±4% (n=8) and
from 63±4% (n=7) to 42±4% (n=7), respectively. The reduction in
infarct size caused by ONO-AE-248 in the rabbit was also abolished by
5-hydroxydecanoate. The cardioprotective effect of ONO-AE-248 in rats
or rabbits was not associated with any hemodynamic
effects. Selective activation of the prostanoid EP3
receptor reduces myocardial infarct size in rodents by a mechanism(s)
that may involve the activation of protein kinase C and the opening of
KATP channels.
Key Words: E-type prostaglandin–receptor agonist • myocardial injury • rodents • protein kinase C • ATP-sensitive potassium channels
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Introduction |
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The effects of E-type prostaglandins (EPs) are mediated by specific G protein–coupled receptors that activate intracellular signal transduction pathways. EP receptors have been classified into 4 subtypes, EP1, EP2, EP3, and EP4.1 In principle, EP3 receptors may be coupled to Gs, Gi, and Gq proteins.1 The G-protein coupling of EP3 receptors depends on posttranscriptional splicing. The known splice variants (eg, EP3
)
of this receptor family differ within their C-terminal amino
acid domain, which appears to determine the class of G protein that
responds to receptor stimulation.2 3 4 There is
evidence that the EP3 and the
EP3ß receptor are coupled to
Gi.5 Interestingly,
EP3 receptors are expressed on cardiac
sarcolemmal membranes and appear to inhibit adenylate
cyclase activity via Gi.6 EPs including prostaglandin E1 (PGE1) exert beneficial effects on biochemical, electrocardiographic, and functional indices of myocardial ischemia/reperfusion injury7 8 9 and reduce myocardial infarct size.10 The cardioprotective effects of these eicosanoids may be secondary to a reduction in afterload, an increase in coronary blood flow, inhibition of platelet function, and/or inhibition of the activation and extravasation of polymorphonuclear cells.11 All of these effects are secondary to the activation of EP2 receptors, which activate Gs and cause an activation of adenylate cyclase.1 In addition, the protection of isolated cells or organs by prostaglandins has been attributed to an ill-defined "cytoprotective" or "cardioprotective" effect of these agents. The mechanism(s) or the prostanoid receptor(s) mediating this effect is unknown.12
In 1995 through 1996, we discovered that the cardioprotective effects of EPs are due, at least in part, to activation of EP1 or EP3 receptors, which in turn leads to the opening of ATP-sensitive potassium (KATP) channels.10 13 This hypothesis is supported by the following findings: (1) The cardioprotective effects of PGE1 (nonselective agonist for all EP receptors) and sulprostone (selective agonist of EP1 and EP3 receptors) are abolished by inhibition of KATP channels with glibenclamide or 5-hydroxydecanoate (5-HD).10 13 (2) Sulprostone causes cardioprotection without having any hemodynamic (EP2-mediated) effects.13 (3) Activation of EP1 and EP3 receptors may result in activation of protein kinase C (PKC) and the opening of KATP channels1 14 Because EP3 receptors are expressed on cardiomyocytes and are upregulated after ischemia of the heart,6 15 we hypothesized that it is the activation of EP3, rather than of EP1, receptors that accounts for the cardioprotective and/or cytoprotective effects of EPs.
The overall aim of this study was to elucidate whether selective
activation of prostanoid EP3 receptors reduces
myocardial infarct size in anesthetized rodents. We report the
discovery of a prostanoid derivative, ONO-AE-248 (Figure 1
), which selectively binds and
activates EP3 receptors in Chinese
hamster ovary (CHO) cells in which this receptor has been expressed.
Subsequently, we have investigated whether ONO-AE-248 reduces the
infarct size caused by regional myocardial ischemia and
reperfusion in anesthetized rats and rabbits. In addition, we
have investigated whether the observed reduction in infarct size caused
by ONO-AE-248 is attenuated by the KATP-channel
blocker 5-HD (in rats and rabbits) or by the PKC inhibitors
staurosporine and chelerythrine (rats).
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Receptor Binding Studies
Cloned prostanoid receptors (EP1, EP2, EP3
,
EP4, FP, TP, and IP) were expressed (separately)
in CHO cells. The transfection of the respective cDNAs into CHO cells
was performed as previously described.16 In brief, CHO
cells were cultured for 72 hours, and thereafter, membrane fractions
were prepared.16 The standard assay mixture used in the
subsequent binding studies contained the following: (1)
[3H]PGE2 or
[3H]PGF2 (2.5 nmol/L)
or [3H]iloprost or
[3H]SQ29548 (5 nmol/L); (2) 10 to 30 µg of
membranes obtained from CHO cells expressing the following prostanoid
receptors: murine EP1, EP2,
EP3, EP4, and FP as well
as human TP or IP; (3) 200 µL of either buffer A (used for the
binding studies involving
[3H]PGE2 or
[3H]PGF2 and
comprising 10 mmol/L potassium phosphate, 1 mmol/L EDTA,
10 mmol/L MgCl2, and 100 mmol/L NaCl,
pH 6.0) or buffer B (used for the binding studies involving
[3H]iloprost or
[3H]SQ29548 and comprising 10 mmol/L
Tris-HCl and 100 mmol/L NaCl, pH 7.4). After incubation for 1 hour
([3H]PGE2 or
[3H]PGF2) or 30
minutes ([3H]iloprost or
[3H]SQ29548) at room temperature, the reaction
was terminated by the addition of 2 mL of ice-cold buffer (A or B, as
appropriate), after which the mixture was rapidly filtered through a
Whatman GF/B glass filter to remove unbound label. The filter was then
washed 3 times with the same buffer, and the radioactivity was measured
by scintillation counting (ACS-II, Amersham). The specific binding was
calculated by subtracting the nonspecific binding from the total
binding. The dissociation constants (KD)
were calculated from Scatchard plots. The concentration of ONO-AE-248
that displaced 50% of the specific binding of the respective
prostanoids (IC50) was then calculated. The
respective Ki values (inhibition constant
values) were calculated by using the following equation:
Ki=IC50/(1+[C]/KD),
in which [C] equals the concentration of the respective
radioligand used.
Measurement of Forskolin-Stimulated cAMP Formation in CHO
Cells
Activation of EP3 or
EP3ß receptors with specific agonists results
in a reduction of the intracellular levels of cAMP (see introduction).
To elucidate whether ONO-AE-248 attenuates the rise in cAMP caused by
forskolin in CHO cells expressing murine EP3
receptors, these cells were seeded (105 per well)
in 24-well plates and cultured for 48 hours. Cells were washed with
modified Eagle's medium containing 1% (wt/vol) BSA and 10 mmol/L
HEPES-NaOH buffer (pH 7.4) and subsequently preincubated (10 minutes at
37°C) in 450 µL of this buffer containing
3-isobutyl-1-methylxanthine (1 mmol/L). Modified Eagle's medium
(50 µL) containing 10 µmol/L forskolin and appropriate
concentrations of ONO-AE-248 or PGE2 were then
added and incubated for 10 minutes at 37°C. The reaction was
terminated by addition of 500 µL of 10% (wt/vol) trichloroacetic
acid solution, and the generated cAMP was determined by
radioimmunoassay. The amounts of cAMP generated by forskolin in the
presence of PGE2 or ONO-AE-248 were expressed as
a percent of the amounts of cAMP generated by forskolin alone
(control). The cDNAs of the prostanoid receptors were obtained from Dr
S. Narumiya and Dr A. Ichikawa of Kyoto University, Kyoto, Japan.
Rodent Models of Myocardial Infarction
Ninety-five male Wistar rats (240 to 350 g; Tucks,
Reyleigh, Essex, UK) were anesthetized (thiopentone sodium, 120
mg/kg IP). After tracheotomy and ventilation (70 strokes/min; tidal
volume, 8 to 10 mL/kg; inspiratory oxygen concentration, 30%; positive
end-expiratory pressure, 1 to 2 mm Hg resulting in
PCO2 values of 36 to 44 mm Hg
and PO2 values of >150 mm Hg),
the animals were instrumented for the measurement of systemic
hemodynamics. A thoracotomy was then performed and a
suture placed around the left anterior descending coronary
artery (LAD). The LAD was occluded (25 or 45 minutes) and reperfused
for 2 hours. After injection of Evans blue dye (1 mL IV) to stain the
area at risk (AR), the heart was removed and cut into 4 or 5 horizontal
slices. The AR was determined by computer-assisted planimetry (Leica,
Quantimed 500). Subsequently, the heart slices were weighed and
incubated in p-nitro blue tetrazolium (NBT, 0.5 mg/mL; 20
minutes at 37°C) to stain the areas of viable and infarcted
myocardium, which were quantified by planimetry. The AR and
infarct size were automatically calculated and expressed as a percent
of the left ventricle or of the AR, respectively.
In a separate study, 49 male New Zealand White rabbits (2.5 to 3.0 kg; Foxfield, Petersfield, Hampshire, UK) were premedicated (Hypnorm, 0.1 mL/kg IM containing 0.315 mg/mL fentanyl citrate and 10 mg/mL fluanisone) and anesthetized (pentobarbitone, 20 mg/kg IV). After tracheotomy and ventilation (36 to 40 strokes/min; tidal volume, 18 to 20 mL), a thoracotomy was performed and a suture placed around the first anterolateral branch of the left coronary artery (LAL). Hemodynamic parameters (left ventricular systolic pressure; mean arterial blood pressure, MAP; heart rate, HR; and pressure-rate index, PRI, an indicator of myocardial oxygen consumption) were measured. The LAL was occluded (45 or 60 minutes) and opened to allow reperfusion (2 hours). The AR was determined by staining of the perfused myocardium (Evans blue dye), and infarct size was determined by staining of the AR with NBT as previously described.10 17
Experimental Design (In Vivo Studies)
The first study was designed to evaluate whether ONO-AE-248
reduces myocardial infarct size in the anesthetized rat. The
following 4 experimental groups were studied: (1) LAD occlusion (45
minutes) and reperfusion (2 hours) plus infusion of vehicle (0.15%
Tween-80/0.015% ethanol in saline), starting 10 minutes before LAD
occlusion and maintained throughout the experiment (n=11); (2) LAD
occlusion (45 minutes) and reperfusion (2 hours) plus infusion of
ONO-AE-248 (5 µg · kg-1 ·
min-1, n=9), starting 10 minutes before LAD
occlusion and maintained throughout the experiment; (3) sham operation
(no LAD occlusion) and infusion of vehicle (n=3); and (4) sham
operation and infusion of ONO-AE-248 (n=3).
The second study was designed to investigate whether a larger reduction
in infarct size can be obtained with ONO-AE-248 when the
ischemic period is reduced from 45 to 25 minutes. Having
documented that ONO-AE-248 causes a substantial (40%) reduction in
myocardial infarct size under these experimental conditions, we
subsequently investigated the role of the activation of
KATP channels and/or PKC in this cardioprotective
effect. To do this, the following additional 13 experimental groups
were studied: (1) LAD occlusion (25 minutes) and reperfusion (2 hours)
plus infusion of vehicle as above (n=8); (2) LAD occlusion and
reperfusion plus infusion of ONO-AE-248 (5 µg ·
kg-1 · min-1,
n=7); (3) sham operation (no LAD occlusion) and infusion of vehicle
(n=3); (4) sham operation and infusion of ONO-AE-248 (n=3); (5) LAD
occlusion and reperfusion plus injection of 5-HD (5 mg/kg IV 10 minutes
before LAD occlusion, n=6); (6) sham operation and injection of 5-HD
(n=3); (7) LAD occlusion and reperfusion plus administration of 5-HD
(as above) and ONO-AE-248 (as above, n=6); (8) LAD occlusion and
reperfusion plus injection of staurosporine (1 µg/kg IV
10 minutes before LAD occlusion, n=6); (9) sham operation and injection
of staurosporine (n=3); (10) LAD occlusion and reperfusion
plus administration of staurosporine (as above) and
ONO-AE-248 (as above, n=6); (11) LAD occlusion and reperfusion plus
injection of chelerythrine (0.7 mg/kg IV 10 minutes before LAD
occlusion, n=6); (12) sham operation and injection of chelerythrine
(n=3); and (13) LAD occlusion and reperfusion plus administration of
chelerythrine (as above) and ONO-AE-248 (as above, n=6).
In a third study, we investigated whether ONO-AE-248 reduces myocardial infarct size in the anesthetized rabbit. To do this, the following 3 experimental groups were studied: (1) LAL occlusion (45 minutes) and reperfusion (2 hours) plus infusion of vehicle (as above, n=10); (2) LAL occlusion and reperfusion plus infusion of ONO-AE-248 (5 µg · kg-1 · min-1, n=8); and (3) sham operation (no LAL occlusion) and infusion of ONO-AE-248 (n=3).
The fourth study was designed to elucidate whether the reduction in infarct size afforded by ONO-AE-248 in the rabbit is attenuated by pretreatment of the animals with the KATP-channel blocker 5-HD. The following 4 experimental groups were studied: (1) LAL occlusion (60 minutes) and reperfusion (2 hours) plus infusion of vehicle (as above, n=7); (2) LAL occlusion and reperfusion plus infusion of ONO-AE-248 (as above, n=7); (3) LAL occlusion and reperfusion plus injection of 5-HD (5 mg/kg IV, n=7); and (4) LAL occlusion and reperfusion plus injection of 5-HD and ONO-AE-248 (n=7).
Measurement of the Plasma Levels of Troponin T (TnT) in the
Rat
At the end of the experiment (study 2, groups 1 [n=8], 2
[n=7], and 3 [n=3]), a 1-mL blood sample was obtained from the
carotid cannula and centrifuged to obtain plasma. The
concentration of TnT was determined by the short turn-around-time assay
(STAT provided by Boehringer Mannheim) using an Elecixs system
2010.
Drugs
Chelerythrine and staurosporine were dissolved in
dimethyl sulfoxide (final concentration in vivo <0.2% for dimethyl
sulfoxide), ONO-AE-248 (prostanoic acid derivative, molecular weight of
380.5) was dissolved in ethanol, Tween-80, and saline (final
concentration in vivo, <0.1% for ethanol and 0.1% for Tween-80).
5-HD was dissolved in saline. Unless otherwise stated, all compounds
were obtained from Sigma Chemical Co. Thiopentone sodium (Intraval) was
obtained from May & Baker Ltd. Chelerythrine and
staurosporine were from Calbiochem.
Statistical Analysis
All values in the text, figures, and tables are expressed as the
mean±SEM of n observations. Statistical analysis was performed
by 1-way ANOVA followed, if appropriate, by Bonferroni's test for
multiple comparisons. A value of P<0.05 was considered
statistically significant.
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Results |
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Specific Binding of ONO-AE-248 to Membranes of CHO Cells Expressing EP, FP, TP, or IP Receptors
In CHO cells expressing the murine EP3
receptor, ONO-AE-248 caused a concentration-related displacement of
[3H]PGE2 with a
Ki value of 15 nmol/L (Table 1). In contrast, the
Ki values for the displacement of
[3H]PGE2 from murine
EP1, EP2, or
EP4 receptors ranged from 3.7
(EP2) to >10 µmol/L (Table 1). In
contrast to sulprostone, which potently binds to murine
EP1 receptors (Ki of
42 nmol/L), ONO-AE-248 caused only a small displacement of
[3H]PGE2 from the murine
EP1 receptor (15% at 10 µmol/L). At
concentrations of 10 µmol/L, ONO-AE-248 caused only (1) a 1%
displacement of
[3H]PGF2 from the
murine FP receptor, (2) a 9% displacement of
[3H]SQ29548 from the human TP receptor, and (3)
a 4% displacement of [3H]iloprost from the
human IP receptor (Table 1). Thus, ONO-AE-248 binds selectively
at concentrations <1 µmol/L to EP3
prostanoid receptors.
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ONO-AE-248 Attenuates the Forskolin-Induced Rise in Intracellular
cAMP in CHO Cells Transfected With the Murine EP3
Receptor
To elucidate whether binding of ONO-AE-248 to the
EP3 receptor activates signal
transduction events (ie, reduction in intracellular cAMP), whole CHO
cells in which the EP3 receptor had been
expressed were challenged with forskolin, which increases the
intracellular levels of cAMP, in the absence (control) or presence of
PGE2, a nonselective agonist of all EP receptors;
sulprostone, an EP1- and
EP3-receptor agonist; or ONO-AE-248.
PGE2 (IC50 0.1 nmol/L),
sulprostone (IC50 0.1 nmol/L), or
ONO-AE-248 (IC50 1 nmol/L) caused a
concentration-dependent inhibition of the rise in intracellular cAMP
caused by forskolin. At a concentration of 10 nmol/L, the rise in cAMP
caused by forskolin was abolished by sulprostone, reduced to 12±2% of
control by PGE2, and reduced to 42±2% of
control by ONO-AE-248 (n=3). Thus, activation of
EP3 receptors with ONO-AE-248, sulprostone, or
PGE2 attenuates the rise in cAMP caused by
forskolin. Thus, ONO-AE-248 is an agonist at the
EP3 receptor.
ONO-AE-248 Reduces Myocardial Infarct Size in Rats
Study 1 in Rats: 45 Minutes of LAD Occlusion
Values for MAP, HR, and PRI measured during the course of the
experiment (study 1) are given in Table 2. Baseline hemodynamic
data were similar (P>0.05) in all groups studied. In rats
subjected to sham operation or LAD occlusion and reperfusion,
ONO-AE-248 did not cause any significant hemodynamic
effects. The AR was similar in rats subjected to LAD occlusion and then
treated with either vehicle (control, 54±4%, n=11) or ONO-AE-248
(57±2%, n=9, P>0.05). When compared with vehicle-treated
control animals, infusion of ONO-AE-248 caused a significant reduction
in infarct size, from 78±2% (control, n=11) to 58±4%
(P<0.05, n=9) of the AR.
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Study 2 in Rats: 25 Minutes of LAD Occlusion
MAP, HR, and PRI were measured during the course of the experiment
(study 2, data not shown). Baseline hemodynamic data
were similar (P>0.05) in all groups studied. In rats
subjected to LAD occlusion for 25 minutes plus 2 hours of reperfusion,
ONO-AE-248 did not cause any significant hemodynamic
effects. Moreover, none of the other interventions studied (5-HD,
staurosporine, or chelerythrine) caused any significant
hemodynamic effects when given to either sham-operated
rats or rats subjected to LAD occlusion and reperfusion, in either the
absence or presence of ONO-AE-248.
In the 13 experimental groups studied, the AR ranged from 45±2% to
56±2% and was not different between groups (P>0.05). When
compared with vehicle-treated control animals, infusion of ONO-AE-248
caused a significant reduction in infarct size, from 60±3% (control,
n=8) to 36±6% (P<0.05, n=7) of the AR (Figure 2a). The reduction in infarct size
afforded by ONO-AE-248 was abolished by pretreatment of rats with 5-HD
(Figure 2a). Interestingly, 5-HD alone did not affect the
infarct size in rats subjected to regional myocardial
ischemia/reperfusion (Figure 2a). Similarly, the
reduction in infarct size afforded by ONO-AE-248 was abolished by
pretreatment of rats with the PKC inhibitors
staurosporine (Figure 2b) and chelerythrine (Figure 2c). Neither staurosporine nor chelerythrine,
however, affected the infarct size caused by regional myocardial
ischemia and reperfusion in the rat (Figure 2b and 2c).
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To exclude the possibility that ONO-AE-248 interferes with the NBT staining procedure, 3 rats were subjected to regional myocardial ischemia (25 minutes) and reperfusion (2 hours). The heart was removed, and the AR was incubated with NBT (as above) in the presence of 10 µmol/L ONO-AE-248. The infarct size in these experiments was 62±5% (n=3) and hence, not different from control. Thus, ONO-AE-248 does not interfere with the staining procedure.
ONO-AE-248 Reduces Myocardial Ischemia–Mediated TnT
Increases in the Rat
In rats subjected to sham operation (surgical procedure only), the
plasma levels of TnT were below the detection limit of the assay
(<0.01 µg/L, n=3). In contrast, LAD occlusion (25 minutes) and
reperfusion (2 hours) resulted in a significant increase in the plasma
levels of TnT, to 65±14 µg/L (n=8). Treatment of rats subjected to
regional myocardial ischemia and reperfusion with ONO-AE-248
attenuated this rise in the plasma levels of TnT to 21±6 µg/L
(P<0.05, n=7).
ONO-AE-248 Reduces Myocardial Infarct Size in Rabbits
Study 3 in Rabbits: 45 Minutes of LAL Occlusion
Values for MAP, HR, and PRI measured during the course of the
experiment (study 3) are given in Table 2
. Baseline
hemodynamic data were similar (P>0.05) in
all groups studied. In rabbits subjected to LAL occlusion for 45
minutes plus 2 hours of reperfusion, ONO-AE-248 did not cause any
significant hemodynamic effects. The AR was similar in
rabbits subjected to LAL occlusion for 45 minutes and treated with
either vehicle (control, 40±2%, n=10) or ONO-AE-248 (48±3%, n=8).
When compared with vehicle-treated control animals, infusion of
ONO-AE-248 caused a significant reduction in infarct size, from 61±2%
(control, n=10) to 36±4% (P<0.05, n=8) of the AR.
Study 4 in Rabbits: 60 Minutes of LAL Occlusion
MAP, HR, and PRI were measured during the course of the experiment
(study 4, data not shown). Baseline hemodynamic data
were similar (P>0.05) in all groups studied. In rabbits
subjected to LAL occlusion for 60 minutes plus 2 hours of reperfusion,
ONO-AE-248 did not cause any significant hemodynamic
effects. In the 4 experimental groups studied, the AR ranged from
38±4% to 47±2% and was not different between groups
(P>0.05). When compared with control animals, infusion of
ONO-AE-248 caused a significant reduction in infarct size, from 63±4%
(control, n=7) to 42±4% (P<0.05, n=7) of the AR (Figure 3). Pretreatment with 5-HD abolished the
reduction in infarct size afforded by ONO-AE-248 (Figure 3).
However, 5-HD alone did not affect the infarct size when compared with
vehicle control (Figure 3).
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Discussion |
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This study demonstrates that the prostanoid derivative ONO-AE-248 selectively binds to EP3
receptors expressed
in CHO cells. In these cells, ONO-AE-248 also attenuates the rise in
cAMP caused by forskolin, suggesting that ONO-AE-248 activates
Gi in these cells. Thus, ONO-AE-248 is a
selective agonist of the EP3 receptor. We also
report that ONO-AE-248 reduces the myocardial infarct size in rats and
rabbits subjected to coronary artery occlusion and reperfusion.
The reduction in infarct size caused by ONO-AE-248 was determined by
staining of the viable myocardium within the AR with the
formazan dye NBT. The reduction in infarct size caused by ONO-AE-248 as
determined by this staining procedure is indeed due to a reduction in
myocardial tissue injury, as ONO-AE-248 also attenuated the increase in
the plasma levels of TnT caused by regional myocardial ischemia
in the rat. There is good evidence that a rise in the plasma levels of
cardiac TnT are specific for myocardial tissue injury.18
Unlike plasma levels of creatine phosphokinase or lactic dehydrogenase,
which are elevated in open-chest models of myocardial infarction due to
the surgical procedure (K.Z. and C.T., unpublished observations, 1998),
the thoracotomy employed here did not result in any detectable rise in
the plasma levels of TnT. Because ONO-AE-248 also did not interfere
with the NBT staining procedure used, our data strongly support the
conclusion that ONO-AE-248 does indeed cause a significant reduction in
myocardial infarct size. What, then, is the mechanism(s) by which ONO-AE-248 causes a significant reduction in the degree of necrosis caused by myocardial ischemia and reperfusion? Clearly, a reduction in blood pressure and hence, myocardial oxygen consumption does not account for the cardioprotective effects of ONO-AE-248 reported here, because this EP3-receptor agonist did not cause any hemodynamic effects in either rats or rabbits. This also supports the notion that at the doses used in vivo, ONO-AE-248 is a selective EP3-receptor agonist and does not activate EP2 receptors, which mediate the hypotension caused by EPs. This finding is important, because it demonstrates that the cardioprotective effects of ONO-AE-248 are, unlike those of prostacyclin, iloprost, or (at higher doses) PGE1, not limited by hemodynamic side effects.
We suggest that the observed reduction in infarct size by ONO-AE-248 is due to the activation of EP3 receptors in vivo. It is unlikely that the dose of ONO-AE-248 used in this study is sufficient to activate other EP receptors for the following reasons. We demonstrated that the Ki value of ONO-AE-248 at the EP3 receptor is 0.015 µmol/L, whereas those for the EP2 and the EP4 receptors are in the range of 3 to 4 µmol/L, and those for the EP1, FP, TP, and IP receptors are in excess of 10 µmol/L. Because ONO-AE-248 did not cause a significant fall in blood pressure, it is extremely unlikely that the dose of ONO-AE-248 chosen here was sufficient to activate EP2 receptors. Thus, we propose that it is most likely that the observed effects of ONO-AE-248 are due to the activation of EP3 receptors. Hohlfeld and colleagues19 have reported in abstract form that the EP3-receptor agonist M&B28767 also causes a substantial (50%) reduction in infarct size in pigs subjected to regional myocardial ischemia. Taken together, these findings support the view that activation of EP3 receptors does indeed cause a reduction in infarct size.
What, then, is the mechanism(s) by which activation of EP3 receptors reduces infarct size? The reduction in infarct size caused by either PGE1 or the EP1/EP3-receptor agonist sulprostone is at least in part due to the activation and opening of KATP channels.10 13 Here we report that the cardioprotective effect of the selective EP3-receptor agonist ONO-AE-248 is, in rats and rabbits, abolished by pretreatment of the animals with 5-HD, a selective blocker of KATP channels. These findings suggest that activation of EP3 receptors leads to opening of KATP channels, which in turn results in cardioprotection. The signal transduction events involved in the cardioprotective effects of ONO-AE-248 are reminiscent of the ones that mediate the potent anti-ischemic effects of "ischemic preconditioning."20 Preconditioning of the heart and other organs or tissues with ischemia results in the release of adenosine and other mediators (eg, bradykinin, endothelin-1, angiotensin II, and catecholamines), which activate G protein–coupled receptors resulting in (1) translocation of PKC from the cytosol to the cell membrane, (2) activation of PKC, (3) opening of KATP channels, and (4) ultimately, cardioprotection.20 21 The reduction in infarct size afforded by ischemic preconditioning in the rabbit model of myocardial ischemia and reperfusion used here is also abolished by 5-HD.13 Thus, we have hypothesised that activation of EP3 receptors by ONO-AE-248, like ischemic preconditioning, may lead to activation of PKC, opening of KATP channels, and ultimately, cardioprotection. To support this hypothesis, we have demonstrated that the reduction in infarct size caused by ONO-AE-248 in the rat is abolished by 2 chemically distinct inhibitors of PKC, namely, staurosporine, a nonselective PKC inhibitor, and chelerythrine, a selective PKC inhibitor. The doses of staurosporine and chelerythrine employed here have been reported to specifically inhibit the activation of PKC in vivo and ex vivo22 23 24 and also abolish the cardioprotective effects of ischemic preconditioning in rodents.22 25
The cytoprotective properties of prostaglandins were first described as the ability of these agents to protect the gastric mucosa against noxious stimuli (eg, ethanol, acid, and hot water).26 Although the mechanism of these cytoprotective effects of prostaglandins has been elusive, these cytoprotective effects have been suggested to contribute to or account for the protection of certain organs or tissues, including the heart, by prostaglandins against a variety of different noxious stimuli.12 26 Thus, cytoprotection is the protection of a tissue by prostaglandins in the absence of alterations in blood flow, inhibition of platelet function, or inhibition of polymorphonuclear cell activation mediated by EP2 receptors. Activation of EP3 receptors leads to protection of the myocardium against ischemia, without causing alterations in systemic hemodynamics (vide infra) and presumably, inhibition of the function of blood-borne cells. Thus, activation of these receptors may contribute to or even account for the protection of cardiomyocytes caused by EPs.
In conclusion, this study demonstrates for the first time that (1)
ONO-AE-248 selectively binds to EP3 receptors,
(2) ONO-AE-248 causes a reduction in intracellular cAMP in CHO
cells, in which the EP3 receptor had been
expressed, and challenged with forskolin, (3) ONO-AE-248 reduces
infarct size in rats and rabbits subjected to regional myocardial
ischemia and reperfusion, (4) the cardioprotective effects of
ONO-AE-248 are abolished by the KATP-channel
blocker 5-HD (rats and rabbits), and (5) the cardioprotective effects
of ONO-AE-248 are abolished by the PKC inhibitors
staurosporine and chelerythrine (rats). We speculate that
the cytoprotective or cardioprotective effects of EPs are at least in
part due to activation by these agents of EP3
receptors. Our discovery of a selective agonist of the
EP3 receptor provides an important
pharmacological tool to elucidate the role of the prostanoid
EP3 receptors in physiology and pathophysiology.
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Acknowledgments |
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K.Z. is supported by the Deutsche Gesellschaft für Kardiologie-Herz-und-Kreislaufforschung. A.O. is supported by the Deutsche Forschungsgemeinschaft (OL 109/1-1). C.T. is a Senior Fellow of the British Heart Foundation (FS 96/018). We thank the ONO Pharmaceutical Company for the generous supply of ONO-AE-248.
Received October 6, 1998; accepted January 28, 1999.
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|---|
1. Coleman RA, Kennedy I, Humphrey PPA, Brunce K, Lumley P. In: Hansch C, Samnes PG, Taylor JB, Emmet JC, eds. Comprehensive Medicinal Chemistry. Oxford, England: Pergamon Press; 1990:643–651.
2. Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, Narumiya S. Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. [see comments]. Nature. 1993;365:166–170.[Medline] [Order article via Infotrieve]
3.
Sugimoto Y, Negishi M, Hayashi Y, Namba T, Honda A,
Watabe A, Hirata M, Narumiya S, Ichikawa A. Two isoforms of the EP3
receptor with different carboxyl-terminal domains: identical ligand
binding properties and different coupling properties with Gi proteins.
J Biol Chem. 1993;268:2712–2718.
4. Kotani M, Tanaka I, Ogawa Y, Usui T, Mori K, Ichikawa A, Narumiya S, Yoshimi T, Nakao K. Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions. Mol Pharmacol. 1995;48:869–879.[Abstract]
5. Irie A, Sugimoto Y, Namba T, Harazono A, Honda A, Watabe A, Negishi M, Narumiya S, Ichikawa A. Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase. Eur J Biochem. 1993;217:313–318.[Medline] [Order article via Infotrieve]
6.
Hohlfeld T, Zucker TP, Meyer J, Schror K. Expression,
function, and regulation of E-type prostaglandin receptors
(EP3) in the nonischemic and ischemic pig heart.
Circ Res. 1997;81:765–773.
7.
Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC.
Dissimilar effects of prostacyclin, prostaglandin E1, and
prostaglandin E2 on myocardial infarct size after
coronary occlusion in conscious dogs. Circ Res. 1981;49:685–700.
8. Schror K, Thiemermann C, Ney P. Protection of the ischemic myocardium from reperfusion injury by prostaglandin E1 inhibition of ischemia-induced neutrophil activation. Naunyn Schmiedebergs Arch Pharmacol. 1988;338:268–274.[Medline] [Order article via Infotrieve]
9.
Simpson PJ, Mickelson J, Fantone JC, Gallagher KP,
Lucchesi BR. Reduction of experimental canine myocardial infarct size
with prostaglandin E1: inhibition of neutrophil migration
and activation. J Pharmacol Exp Ther. 1988;244:619–624.
10. Hide EJ, Ney P, Piper J, Thiemermann C, Vane JR. Reduction by prostaglandin E1 or prostaglandin E0 of myocardial infarct size in the rabbit by activation of ATP-sensitive potassium channels. Br J Pharmacol. 1995;116:2435–2440.[Medline] [Order article via Infotrieve]
11. Lucchesi BR, Mullane KM. Leukocytes and ischemia-induced myocardial injury. Annu Rev Pharmacol Toxicol. 1986;26:201–224.[Medline] [Order article via Infotrieve]
12. Schror K. Eicosanoids and myocardial ischaemia. Basic Res Cardiol. 1987;82:235–243.
13. Hide EJ, Thiemermann C. Sulprostone-induced reduction of myocardial infarct size in the rabbit by activation of ATP-sensitive potassium channels. Br J Pharmacol. 1996;118:1409–1414.[Medline] [Order article via Infotrieve]
14. Coleman RA, Smith WL, Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994;46:205–229.[Medline] [Order article via Infotrieve]
15. Hohlfeld T. Regulation of prostaglandin receptors in myocardial ischemia. Agents Actions Suppl. 1995;45:33–37.[Medline] [Order article via Infotrieve]
16.
Sugimoto Y, Namba T, Honda A, Hayashi Y, Negishi M,
Ichikawa A, Narumiya S. Cloning and expression of a cDNA for mouse
prostaglandin E receptor EP3 subtype. J Biol
Chem. 1992;267:6463–6466.
17. Thiemermann C, Thomas GR, Vane JR. Defibrotide reduces infarct size in a rabbit model of experimental myocardial ischaemia and reperfusion. Br J Pharmacol. 1989;97:401–408.[Medline] [Order article via Infotrieve]
18.
Adams JE, Abendschein DR, Jaffe AS. Biochemical markers
of myocardial injury: is MB creatine kinase the choice for the 1990s?
Circulation. 1993;88:750–763.
19. Hohlfeld T, Meyer J, Schror K. Myocardial EP3 receptors are externalized in ischemic porcine hearts and mediate reduction of ischemic myocardial injury. Naunyn Schmiedebergs Arch Pharmacol. 1998;357:R103. Abstract.
20. Downey JM, Cohen MV. Signal transduction in ischemic preconditioning. Z Kardiol. 1995;84:77–86.
21. Millar CG, Baxter GF, Thiemermann C. Protection of the myocardium by ischaemic preconditioning: mechanisms and therapeutic implications. Pharmacol Ther. 1996;69:143–151.[Medline] [Order article via Infotrieve]
22.
Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase
C: its role in ischemic preconditioning in the rat. Circ
Res. 1994;75:586–590.
23.
Kaye AD, Nossaman BD, Ibrahim IN, Feng CJ, Kadowitz
PJ. Influence of protein kinase C inhibitors on
vasoconstrictor responses in the pulmonary vascular bed of cat
and rat. Am J Physiol. 1995;268:L532–L538.
24.
Kozak W, Klir JJ, Conn CA, Kluger MJ. Attenuation of
lipopolysaccharide fever in rats by protein kinase C
inhibitors. Am J Physiol. 1997;273:R873–R879.
25.
Yoshida K, Kawamura S, Mizukami Y, Kitakaze M.
Implication of protein kinase C-, 
, and 
isoforms in
ischemic preconditioning in perfused rat hearts. J
Biochem (Tokyo). 1997;122:506–511.
26. Robert A. On the mechanism of cytoprotection by prostaglandins. Ann Clin Res. 1984;16:335–338.[Medline] [Order article via Infotrieve]
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