© 1997 American Heart Association, Inc.
Articles |
Expression, Function, and Regulation of E-Type Prostaglandin Receptors (EP3) in the Nonischemic and Ischemic Pig Heart
From the Institut für Pharmakologie, Heinrich-Heine-Universität Düsseldorf (Germany).
Correspondence to Dr K. Schrör, Institut für Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Moorenstr. 5, D-40225 Düsseldorf, Germany.
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Abstract The action of prostacyclin, prostaglandin E1 (PGE1), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [3H]PGE1, with a Kd of 3.7 nmol/L and a Bmax of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype–selective agonists sulprostone (EP1 and EP3) and M&B 28.767 (EP3) were active, suggesting the presence of an EP3 receptor subtype. PGE1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP3 receptor coupling to an inhibitory G protein (Gi). Additional in vivo experiments showed that intracoronary infusion of PGE1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of ß-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg IV), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-
-S–induced decrease of [3H]PGE1
binding. These data demonstrate for the first time that myocardial
receptors for PGE1 belong to the EP3 subtype.
The properties of this receptor include inhibition of adenylyl cyclase
and upregulation during regional myocardial ischemia,
suggesting an involvement in the anti-ischemic activity of E-
and I-type prostaglandins.
Key Words: E-type prostaglandin receptor • prostaglandin E1 • sarcolemma • adenylyl cyclase • myocardial ischemia
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Introduction |
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Prostaglandins exert various effects on the normal and ischemic heart. These include changes in contractility,1 energy metabolism,2 and, in the reperfused ischemic myocardium, a protective action2 3 in several experimental models, including the pig.4 5 6 In addition, a reduction by prostaglandins of arrhythmias induced by experimental ischemia has been repeatedly reported.7 8 9
The actions of prostaglandins are mediated by specific cell surface receptors that are coupled to defined postreceptor signal transduction pathways. Although the pharmacological characterization of prostaglandin receptors remains hampered by well-defined and high-affinity receptor antagonists, the knowledge about these receptors has dramatically increased after the sequencing and cloning of virtually all known prostaglandin receptors and clarification of their coupling to postreceptor signal transduction pathways.10
Surprisingly, little is known about the type, specificity, density, and postreceptor signaling of prostaglandin receptors in the heart, although ligand binding studies on bovine cardiac membranes11 have provided evidence that EP receptors are expressed in the heart and seem to be located on the sarcolemma. These receptors have been shown to inhibit myocardial adenylyl cyclase. This should be compatible with an EP3 receptor subtype but has not yet been confirmed by appropriate ligand binding studies. Subtypes of myocardial EP receptors may mediate at least some of the actions of E- and I-type prostaglandins on myocardial function in cardiac tissue under normoxic conditions and during myocardial ischemia and reperfusion.
The present work was designed to (1) characterize prostaglandin receptors located within the myocardium with respect to their agonist selectivity, affinity, and density and (2) examine the functional coupling of these receptors to postreceptor signal transduction pathways. Additional experiments were performed to (3) determine whether myocardial prostaglandin receptor activation alters myocardial contractile function. Since previous studies have shown that myocardial receptors with probable relevance for ischemic myocardial injury (eg, adrenergic receptors) may be upregulated during ischemia,12 13 14 this study was also designed to (4) evaluate myocardial prostaglandin receptor characteristics in ischemic and reperfused ischemic myocardial tissue.
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Surgical Procedures
All animal experiments were performed in accordance with the "Position of the American Heart Association on Research Animal Use" and were approved by the local governmental animal resource committee. Minipigs of either sex (Göttingen strain, 28 to 35 kg body weight) were anesthetized by intramuscular injection of 15 mg/kg ketamine and 3 mg/kg azaperon. Anesthesia was maintained by intravenous infusion of 5 mg/kg per hour metomidate, 0.8 mg/kg per hour azaperon, and 0.6 mg/kg per hour pancuronium into the left jugular vein. All animals were artificially ventilated with a mixture of room air and O2 and adjusted to an arterial PO2 between 100 and 150 mm Hg and PCO2 of 35 to 40 mm Hg. Thoracotomy was performed by midline incision of the sternum, followed by longitudinal pericardiotomy.
Fluid and electrolyte balance was maintained by intravenous infusion of Ringer's solution supplemented with 5% glucose (100 mL/h). All animals were allowed to stabilize at least 30 minutes after the surgical preparation was completed.
Myocardial tissue was obtained by occlusion of the superior and inferior venae cavae and the ascending aorta, followed by intracoronary infusion of 1000 mL ice-cold (4°C) St Thomas' Hospital cardioplegic solution. The hearts were then rapidly excised and frozen with clamps precooled in liquid nitrogen for further preparation of sarcolemmal membranes.
In the experiments designed to examine EP receptor properties in ischemic myocardial tissue, the LAD was carefully dissected free from the epicardium over a distance of 5 mm, halfway between its origin and apex. A silk suture (0.3-mm diameter) was placed around the vessel for later atraumatic occlusion. The animals were allowed to recover for at least 30 minutes after surgery. Then the LAD was occluded either for 15 minutes (n=6) or for 60 minutes (n=7). Thereafter, the hearts were arrested with cardioplegic solution as described above. To identify the ischemic left ventricular tissue, 20 mL of cardioplegic solution containing 20 mg of Evans blue was injected into the LAD at the site of the previous occlusion. Initial control experiments had excluded an interference of the dye with the preparation of sarcolemmal membranes and the biochemical parameters measured. Ischemic and control left ventricular myocardial tissue was separately frozen for preparation of sarcolemmal membranes.
In an additional group of pigs (n=4), regional myocardial ischemia (60 minutes) was induced as described, with the exception that the microtubule-disrupting agent colchicine (2 mg/kg IV) was administered 60 minutes before coronary occlusion. Another group of pigs (n=5) also underwent LAD occlusion for 60 minutes. Thereafter, the ligature was released, and reperfusion was allowed for 60 minutes.
Effect of Intracoronary PGE1 on
Contractile Function
Further experiments (n=5) were designed to assess a potential
effect of prostaglandin receptor activation on regional
myocardial contractile activity. PGE1 (1 nmol/min)
was infused via a 27-gauge needle into the LAD close to the origin of
the first diagonal branch, a site where coronary blood flow is
20 mL/min. The infusion of PGE1 lasted for 10 minutes.
Immediately before (control) and during the time of PGE1
administration (10 minutes), left ventricular
contractility was measured by ultrasound crystals,
which were placed in subepicardial and subendocardial positions
(Schuessler & Associates sonomicrometer). One pair of
crystals was implanted within the perfusion bed of the LAD; another was
placed within the left lateral wall, apart from the perfusion bed of
the LAD. Contractile function was quantified by the maximum
systolic change of left ventricular wall thickness.
Thirty minutes after the infusion of PGE1 was terminated,
isoprenaline was infused intravenously at a dose of 3
µg/kg per minute. The inotropic action stabilized within 10 to
20 minutes. Thereafter, PGE1 infusion was repeated, and
regional contractile function was recorded as before. Before and
during intracoronary PGE1 infusion, cardiac output
was measured by an electromagnetic blood flow probe placed around the
ascending aorta. Left ventricular pressure was
simultaneously measured by a catheter-tipped manometer
introduced into the left ventricle via the left carotid artery.
Systemic vascular resistance was calculated from peak left
ventricular pressure and cardiac output using a
Macintosh-based data analysis system (MacLab 16s, Wisstech).
Preparation of Sarcolemmal Membranes
Cardiac sarcolemma was prepared according to procedure II as
described by Jones (1992).15 About 15 g of
ischemic or nonischemic myocardial tissue was minced
with scissors, suspended in 120 mL of buffer A (10 mmol/L
histidine and 0.75 mol/L NaCl), and homogenized for
5 seconds at half-maximum speed with an Ultraturrax
homogenizer (Janke & Kunkel). The
homogenate was centrifuged at 14 000g
for 20 minutes. Then the pellet was washed twice in buffer B (5
mmol/L histidine and 10 mmol/L NaHCO3),
suspended in 5 mL of buffer C (10 mmol/L histidine and
300 mmol/L NaCl), and placed on top of a discontinuous
sucrose gradient, consisting of two layers of buffer C (7 mL each)
supplemented with of 0.25 and 0.6 mol/L sucrose, respectively.
After 90 minutes of ultracentrifugation
(300 000g), the membranes at the 0.25 to 0.6 mol/L
sucrose interface were recovered, 3-fold diluted with sucrose-free
buffer C, and sedimented (300 000g, 30 minutes). The
sarcolemmal vesicles were then suspended in 200 µL aliquots (5 mg
each) of 10 mmol/L Tris-HCl (pH 6.4) and stored frozen
(-40°C) until use. All preparation steps were performed at 4°C in
the presence of 0.1 mmol/L phenylmethylsulfonyl
fluoride.
Ligand Binding Studies
Binding experiments were performed in 200 µL of binding buffer
containing 100 mmol/L NaCl, 25 mmol/L HEPES (pH
6.5), and 25 to 50 µg membrane protein. Scatchard analysis
was performed by the cold saturation technique in the presence of 3
nmol/L [3H]PGE1. Nonlabeled
PGE1 was added in concentrations between 0.3 nmol/L
and 1 µmol/L. Nonspecific binding was determined in the
presence of 10 µmol/L PGE1. Equilibrium of
binding was achieved after 60 minutes of incubation at 37°C. At this
time, bound was separated from free activity by rapid filtration with a
24-well filtration device (Brandel) and by washing three times with 5
mL of ice-cold binding buffer. Radioactivity on the filters was counted
with standard liquid scintillation techniques. The binding and
displacement data were evaluated by computer-based nonlinear fitting
analysis (Enzfitter, Biosoft). The best fit of the data was
achieved when assuming a single class of binding sites. The binding
data were referred to membrane protein, determined according to the
method of Bradford.16
The kinetics of [3H]PGE1 binding to sarcolemmal membranes were studied according to the method described by Jaschonek et al.17 Kobs of the ligand-receptor complex was determined with [3H]PGE1 at a concentration of 5 nmol/L at 20°C. K-1 was measured as time course of [3H]PGE1 displacement after addition of 10 µmol/L PGE1 at 20°C. K+1 was calculated from the following equation: K+1=(Kobs-K-1)/ligand
Receptor-subtype specificity of binding was determined by competition of [3H]PGE1 binding with PGE0, PGE1, PGE2, sulprostone, butaprost, M&B 28.767, iloprost, cicaprost, PGD2, and SQ 29.548 (each 0.3 nmol/L to 10 µmol/L). The processing was identical to that for cold saturation with PGE1 (above).
Effect of GTP--S on [3H]PGE1
Receptor–G-protein coupling was assessed by determination of
the effect of GTP--S on the specific binding of
[3H]PGE1. Sarcolemmal membranes were
incubated in binding buffer for 60 minutes at 37°C with or without
0.1 mmol/L GTP--S, a concentration that can be expected
to achieve complete saturation of guanine nucleotide
binding sites of G-protein 
subunits. Then saturation binding
studies were carried out using [3H]PGE1 (0 to
20 nmol/L) as ligand. The effect of myocardial ischemia
(15 or 60 minutes) and reperfusion (60 minutes) on G-protein coupling
was assessed by determining the changes of specific
[3H]PGE1 (3 nmol/L) binding to
sarcolemmal membranes caused by a range of different GTP--S
concentrations (1 to 100 µmol/L).
Measurement of Na+,K+-ATPase
Activity
Sarcolemmal Na+,K+-ATPase activity was
determined by the K+-stimulated conversion of
4-nitrophenylphosphate to 4-nitrophenol at 400 nm using a double-beam
spectrophotometer (UV-160, Shimadzu) by the procedure described by
Danilenko et al.18
Measurement of GTPase Activity
Sarcolemmal GTP hydrolysis was measured as hydrolysis of
[-32P]GTP, using the procedure of Fleming and Watanabe
(1988).19
PGE1-Induced Inhibition of Sarcolemmal Adenylyl
Cyclase
Sarcolemmal membranes (50 µg protein) were suspended in 100
µL of 50 mmol/L HEPES buffer (pH 7.4) containing
(mmol/L) sodium EDTA 1, MgCl2 10, dithiothreitol 2,
methylisobutylxanthine 2, ATP 2, creatine phosphate 4, and GTP 0.01.
Creatine kinase was added at a concentration of 6.8 U/mL. Adenylyl
cyclase was stimulated with forskolin (10 µmol/L).
PGE1 was added at concentrations from 0.1 nmol/L to
1 µmol/L. Separate measurements showed that adenylyl
cyclase inhibition was maximal at 1 µmol/L. The reaction
was allowed to proceed for 10 minutes at 37°C and stopped by heat
inactivation (90°C for 5 minutes). Membrane protein was sedimented at
10 000g for 10 minutes, and cAMP was determined by
radioimmunoassay.
Northern Blot Analysis of Cardiac EP3
Receptor Message
Three additional pigs underwent 60 minutes of LAD occlusion.
Total RNA was isolated from ischemic and control left
ventricular tissue by the Trizol RNA isolation procedure
(GIBCO-BRL). Poly(A+) RNA was isolated from total RNA by
paramagnetic particle separation (PolyATract, Promega) and
analyzed by electrophoresis on a 1% agarose/formaldehyde gel.
Poly(A+) RNA (20 µg) was loaded on each lane. After
transfer to Hybond N membrane (Amersham), RNA was fixed by baking at
80°C for 2 hours. The blot was hybridized overnight at
high-stringency conditions (65°C, 5x SSPE, 5x Denhardt's solution,
0.5% SDS, and 500 µg salmon sperm DNA) with a 32P random
prime–labeled fragment of the porcine EP3 receptor (0.9-kb
cDNA, EcoRI-BamHI; J. Meyer and K. Schrör,
unpublished data, 1997) and a 1.3-kb human GAPDH20
EcoRI fragment. After hybridization, the membranes were
washed twice at 37°C with 2x SSPE and 0.1% SDS and twice at 65°C
with 0.2x SSPE and 0.1% SDS. The hybridization signals were detected
on Kodak X-Omat film with exposition for 3 hours (GAPDH) or overnight
(EP3 receptor). Densitometric analysis was
performed on a PC-based gel evaluation system (Scan Pack,
Biometra).
Drugs and Chemicals
PGE0, PGE1, and PGE2 were
gifts from Dr P. Ney, Schwarz Pharma (Mannheim, Germany); sulprostone,
iloprost and cicaprost, from Dr F. MacDonald, Schering (Berlin,
Germany); butaprost, from Dr P. Gardiner, Bayer (Middlesex, UK); M&B
28.767, from Dr L. Caton, Rhone-Poulenc Rorer (Vitry sur Seine,
France); and SQ 29.548, from Dr M. Ogletree, Squibb (Princeton,
NJ). [3H]PGE1 and [-32P]GTP
were purchased from DuPont de Nemours and Amersham, respectively. All
other chemicals were from Sigma and Merck.
Statistical Analysis
All data are mean±SEM. Different groups were compared by
two-tailed t test for paired or unpaired samples, as
required. More than two groups were compared by ANOVA (repeated
measures) with Bonferroni's correction. Differences between groups
were considered significant at values of P<.05.
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Characteristics of Sarcolemmal [3H]PGE1 Binding Sites
Saturation experiments with PGE1 indicated a Kd of 3.7±0.5 nmol/L and a density of binding sites (Bmax) of 92±15 fmol/mg protein (Fig 1
). At 3 nmol/L
[3H]PGE1, nonspecific binding was 20% of
total binding. Since the Scatchard transformation of the saturation
binding data was best fit by linear regression, a single class of
binding sites can be assumed. Hill transformation of the competition
between labeled and unlabeled PGE1 revealed a slope of
-0.95. This value is close to -1, indicating competition for
independent binding sites.
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Additional kinetic experiments evaluated the time course of association and dissociation of [3H]PGE1 on porcine sarcolemma. Kobs was 1.67x10-3 · s-1, and K-1 was 0.30x10-3 · s-1, resulting in K+1 of 2.74x105 mol-1 · s-1. The calculated Kd value was 1.1 nmol/L.
Specificity of PGE1 Binding to Porcine
Sarcolemma
PGE2 displaced
[3H]PGE1 (3 nmol/L) from the
sarcolemmal binding sites with a potency similar to PGE1.
(Fig 2, Table 1). The calculated pD2 values
for PGE1 and PGE2 were almost identical. The
PGI2 mimetics iloprost and cicaprost were at least one
order of magnitude less active in displacing
[3H]PGE1 but reached the same maximum as
PGE1 and PGE2. In addition, PGE0
was equieffective to PGE1 and PGE2.
PGD2 displaced [3H]PGE1 at
micromolar concentrations. The thromboxane receptor
antagonist SQ 29.548 did not displace
[3H]PGE1 (not shown). These data collectively
indicate specificity of the sarcolemmal binding site for EPs.
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Since distinct subtypes of the EP receptor are known, it was of
interest to identify the particular subtype located on porcine
sarcolemma. This was studied by competition binding experiments with
the EP receptor subtype–selective compounds sulprostone
(EP1 and EP3), butaprost (EP2), and
M&B 28.767 (EP3). In fact, sulprostone and M&B 28.767
displaced [3H]PGE1 at a potency similar to
PGE1, whereas butaprost was inactive (Fig 3, Table 1
). This indicates that the
EP3 receptor subtype is present on porcine
sarcolemma.
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G-Protein Coupling of Sarcolemmal EP3
Receptors
[3H]PGE1 binding to sarcolemmal
membranes was evaluated in the absence and presence of the stable GTP
analogue GTP--S (0.1 mmol/L) in order to assess coupling
of sarcolemmal EP3 receptors to a G protein. As shown in
Fig 4, GTP--S caused a distinct shift
of the saturation binding curve to the right and a decreased negative
slope in the Scatchard plot. The calculated Kd
values were 2.3±0.1 and 5.7±0.5 nmol/L without and with
GTP--S, respectively, indicating that the GTP analogue decreased
ligand affinity. This suggests that sarcolemmal EP3
receptors are coupled to a G protein. Obviously, GTP--S did not
alter the number of binding sites (Bmax), since the
x-axis intercept of the Scatchard-transformed data was the
same in saturation experiments with or without the addition of
GTP--S.
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) and absence (
, control) of 0.1 mmol/L
GTP-
Further evidence in support of a G protein–coupled EP receptor was
obtained from the effect of PGE1 on sarcolemmal hydrolysis
of [-32P]GTP. PGE1 (1 nmol/L to
1 µmol/L) significantly increased GTPase activity. The
EC50 for this effect was 4.7±0.3 nmol/L (n=4), and
GTPase activity was stimulated to a maximum of 1 pmol/mg per
minute at 100 nmol/L PGE1 (Fig 5).
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Since adenylyl cyclase inhibitory G proteins
(Gi) represent an important, though not the only,
class of EP receptor–coupled G proteins, it was of interest to
determine the effect of PGE1 on sarcolemmal adenylyl
cyclase activity. It was found that PGE1 inhibited
forskolin (10 µmol/L)–stimulated activity of sarcolemmal
adenylyl cyclase in a concentration-dependent manner (Table 2). The effect was statistically
significant at 10 nmol/L and higher. PGE1 did not
alter basal adenylyl cyclase activity, determined in the absence of
forskolin.
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Functional Response to Myocardial EP3 Receptor
Activation
Intracoronary infusion of PGE1 (1
nmol/min) did not alter regional contractile function, as
determined by sonomicrometric measurement of left
ventricular wall thickness in the perfusion bed of the LAD,
distal to the site of PGE1 infusion (Fig 6). However, in the presence of
ß-adrenergic stimulation by isoprenaline (3 µg/kg per minute
IV), PGE1 caused a marked and significant delay of left
ventricular systolic wall thickening. This effect
was constant for the duration of intracoronary PGE1
infusion (10 minutes) and completely reversible within 30 minutes after
the PGE1 infusion was stopped (not shown).
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The negative inotropic action of PGE1 in the presence of ß-adrenergic stimulation was not due to a change in peripheral vascular resistance (ie, systemic vasodilation), which was comparable before and during PGE1 infusion (3180±250 dyne · s · cm-5 before versus 3110±270 dyne · s · cm-5 after 2 minutes of intracoronary PGE1 infusion, P>.05). In contrast to isoprenaline, the cAMP-independent positive inotropic action of ouabain was not altered by PGE1 (not shown).
The present data collectively indicate that normoxic myocardium contains a homogeneous class of EP3 receptors coupled to an adenylyl cyclase inhibitory G protein, allowing for a functionally relevant attenuation of the inotropic response to ß-adrenergic stimulation. This may contribute or even explain the pharmacologically important reduction of ischemic myocardial injury by PGE1 and other EP (or combined EP and IP) receptor agonists. Further experiments were therefore conducted to evaluate the regulation and G-protein coupling of EP3 receptors at short-term (15-minute) and long-term (60-minute) myocardial ischemia and reperfusion (60 minutes).
Effects of Acute Myocardial Ischemia on Sarcolemmal
EP3 Receptor Density
In the experiments with 15 minutes of LAD occlusion, sarcolemmal
EP receptor density (Bmax) was not significantly different
between control myocardial tissue (74±13 fmol/mg protein) and
the ischemic area (62±3 fmol/mg). Furthermore, receptor
affinity (Kd) was comparable (3.0±1.6 and
2.6±1.4 nmol/L, respectively).
In contrast, LAD occlusion for 60 minutes caused a significant increase
of Bmax from 83±8 (control myocardium distant
from the ischemic area) to 126±21 fmol/mg protein,
equivalent to an increase by 52±13% (P<.005, Fig 7). This elevation of receptor density
appeared to be specific for EP3 receptors, because the
activity of K+-stimulated
Na+,K+-ATPase, a marker enzyme of sarcolemmal
membranes, was identical in membranes prepared from nonischemic
and ischemic myocardium (Fig 7). The
Kd values remained unchanged in the control and
ischemic areas (2.7±0.9 versus 2.9±1.8 nmol/L,
respectively; P>.05). Thus, 60 minutes of regional
ischemia increased the density without changing EP3
receptor affinity.
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Another group of pigs also underwent a period of ischemia (60
minutes) but was pretreated with the microtubule-disrupting agent
colchicine (2 mg/kg IV) 60 minutes before the LAD was occluded.
Colchicine did not alter blood pressure, dP/dt, heart rate, or left
ventricular end-diastolic pressure (not shown)
but completely prevented the increase in receptor density (Fig 7). In
these experiments, receptor affinity was 4.9±1.0 nmol/L in
control tissue and 4.1±1.9 nmol/L in ischemic tissue,
which was not significantly different (P>.05).
When coronary occlusion (60 minutes) was followed by 60 minutes of reperfusion, sarcolemmal EP3 receptor density was 68±6 fmol/mg in nonischemic and 46±9 fmol/mg in reperfused ischemic myocardium. This decrease did not reach statistical significance (P>.05). This was accompanied by a similar decrease of myocardial K+-stimulated Na+,K+-ATPase activity. Reperfusion did not change sarcolemmal EP3 receptor affinity, as shown by identical Kd values in reperfused ischemic and nonischemic control myocardium (2.7±0.9 versus 2.7±0.5 nmol/L, respectively).
Expression of EP3 Receptor mRNA
In three experiments, poly(A+) RNA was prepared from
left ventricular myocardial control tissue (left lateral
wall) and from ischemic myocardium (left anterior
wall) after LAD occlusion, separated on an agarose gel, and hybridized
with a 32P-labeled probe of the porcine myocardial
EP3 receptor. In all of these samples, distinct transcripts
appeared at a size of 2.2 kb (Fig 8).
GAPDH was equal in control and ischemic myocardium.
The densitometric analysis showed a comparable expression of
EP3 receptor mRNA in the control and ischemic areas
of the left ventricle. This suggests that the increased sarcolemmal
EP3 receptor density during regional myocardial
ischemia is not due to a transcriptional upregulation of
EP3 receptor message.
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G-Protein Coupling of Sarcolemmal EP3 Receptors in
Ischemic Myocardium
Regional myocardial ischemia did not impair the
GTP--S–induced decrease of [3H]PGE1 (5
nmol/L) binding to sarcolemmal membranes, indicating intact
receptor G-protein coupling during ischemia. This is shown by a
similar GTP--S (100 µmol/L)–induced decrease of
[3H]PGE1 binding in membranes from tissue
subjected to 60 minutes of LAD occlusion compared with membranes from
nonischemic control tissue (Fig 9). Similar results were obtained after
15 minutes of ischemia (not shown). Interestingly, GTP--S at
low concentration (1 µmol/L) caused a significant
(P<.05) increase of bound activity in membranes from
ischemic, but not from control, tissue.
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Reperfusion (60 minutes) subsequent to regional ischemia (60
minutes) was also associated with a decrease in
[3H]PGE1 binding after the addition of
100 µmol/L GTP--S (Fig 9). This indicates that
G-protein coupling of EP3 receptors also remained intact in
the reperfused ischemic myocardium. Similar to
ischemia alone, a lower concentration of GTP--S (1
µmol/L) increased [3H]PGE1 binding.
In contrast to ischemia alone, this was also seen in the
nonischemic control myocardium.
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The present study demonstrates specific myocardial binding sites for EPs, extending earlier data11 by the identification of a G protein–coupled EP3 receptor subtype, located on cardiac sarcolemmal membranes. Scatchard analysis of [3H]PGE1 binding revealed a Kd of 3.7 nmol/L, which agrees with the Kd values previously reported for recombinant EP receptors.21 This Kd is also consistent with the observed EC50 value for PGE1-mediated sarcolemmal GTPase activation. Although the present work shows a single homogeneous class of binding sites, bovine hearts have previously been reported to display a high-affinity and a low-affinity site for EPs.11 Based on similar affinity, the binding site found in pig sarcolemma more likely corresponds to the low-affinity site of bovine sarcolemma, since saturation requires nanomolar concentrations of PGE1. The rank order of potency for displacement of [3H]PGE1 showed highest activity for M&B 28.767, which identifies an EP3 receptor subtype. Other than M&B 28.767 and sulprostone, PGE1 has some cross affinity toward IP receptors. However, the maximum of displacement was similar for the three ligands. A contribution of IP receptors to [3H]PGE1 binding is, therefore, unlikely.
The sarcolemmal membrane preparation used in the present study may be contaminated with membranes derived from other cellular compartments and different types of tissue. Since antibodies directed against EP3 receptors are not available, this receptor cannot be localized by immunohistochemistry. Several arguments, however, suggest that the EP3 receptors described in the present study are essentially located on cardiomyocytes. First, we have used a well-characterized technique for preparation of cardiac sarcolemmal membranes.15 Second, the functional measurements showed a marked inhibitory effect of intracoronary PGE1 on the ß-adrenergic inotropic response to isoprenaline. This is supported by a recent study performed with neonatal cardiomyocytes, which showed that PGE1 in nanomolar concentration inhibits ß-adrenergic arrhythmogenic effects.22 Finally, a number of studies have demonstrated that prostaglandins such as PGE1, PGI2, and their mimetics1 2 3 4 5 6 7 8 9 reduce ischemic myocardial injury.
In general, EP3 receptors may activate
Gs, Gi, and Gp
proteins.10 The present study demonstrates G-protein
coupling of sarcolemmal EP3 receptors by a
PGE1-induced increase in sarcolemmal GTPase activity and a
GTP--S–induced shift from one single class of high-affinity binding
sites toward a single class of binding sites with decreased affinity.
This, however, does not identify the type of G protein coupled to
EP3 receptors.
It has been shown that G-protein coupling of EP3 receptors
depends on posttranscriptional splicing. The known splice variants of
this receptor mainly differ within their C-terminal amino acid
sequences,21 23 24 which appear to determine the class of
G proteins that responds to receptor stimulation.21 24 A
GTP--S–induced shift of ligand binding, similar to the one observed
in the present study, has been reported for transfected
EP3 receptors, which are coupled to inhibition of adenylyl
cyclase.24 Evidence that the G protein, which couples to
myocardial EP3 receptors, belongs to the class of
inhibitory G proteins (Gi) is provided by the
inhibition of sarcolemmal adenylyl cyclase by PGE1, which
is in agreement with a previous study by Lopaschuk et
al.11 This is also supported by the finding that
PGE1 decreases the inotropic response to ß-adrenergic
stimulation by isoprenaline, without altering basal contractile
function. Further, pertussis toxin prevents the GTP--S–induced
shift of [3H]PGE1 binding (T. Hohlfeld,
unpublished data, 1996).
Whereas PGE1 completely inhibited the isoproterenol-induced inotropic effect, adenylyl cyclase was inhibited only partially (60%). One possible explanation is that the in vitro determination of adenylyl cyclase activity does not ideally reflect the cAMP formation in vivo. Since EP3 receptors and adenylyl cyclase require access from different sides of the sarcolemma, an incomplete penetration of PGE1 and/or ATP across sarcolemmal vesicle membrane may impair the in vitro inhibition by PGE1 as well as overall cAMP formation.
A number of studies have shown that PGE1, PGI2, and their mimetics improve postischemic myocardial function and reduce myocardial infarct size (for review, see References 2 and 32 3 ). In addition, Hide et al25 also demonstrated that PGE1 and PGE0 cause ischemic preconditioning. Therefore, it was of particular interest to evaluate whether myocardial EP3 receptors remain intact and coupled to G proteins in ischemic myocardial tissue.
Ligand binding clearly showed that receptor affinity was unchanged at
short (15-minute) and prolonged (60-minute) durations of
ischemia. Receptor density remained unchanged during short-term
ischemia but was significantly increased during long-term
ischemia in the presence of intact G-protein coupling, as
indicated by a similar decrease of [3H]PGE1
binding by the GTP analogue GTP--S. The increase in receptor density
cannot be attributed to an enhanced yield of sarcolemmal membranes in
ischemic tissue, since the activity of
Na+,K+-ATPase, a marker of sarcolemmal
membranes, was not altered.
The observed increase in receptor density during coronary occlusion might be caused by de novo receptor synthesis. However, EP3 receptor mRNA expression was similar in control and ischemic myocardium, arguing against a transcriptional increase of receptor synthesis. Alternatively, an intracellular receptor pool may have been externalized during ischemia, as has been shown for the ischemia-induced upregulation of myocardial ß-adrenergic receptors.12 13 14 26 27 Notably, the time course and extent of upregulation appear to be similar for ß-adrenergic12 and EP3 receptors.
A pool of intracellular ("light") membranes is a source of myocardial ß-adrenergic receptor upregulation.12 Several studies have shown that microtubule-disrupting agents, such as colchicine, interfere with the translocation of adrenergic receptors between cell surface and an intracellular compartment.28 29 It may be hypothesized, therefore, that EP3 receptors are upregulated by a similar mechanism. In fact, the present study shows that colchicine prevents EP3 receptor upregulation, suggesting that microtubule-dependent externalization of EP3 receptor protein is the explanation for EP3 receptor upregulation during ischemia. It should be noted in this context that intracellular eicosanoid receptors (ie, thromboxane A2/PGH2 receptors) have been described in platelets30 and that platelet thromboxane A2/PGH2 receptors are upregulated in patients with acute myocardial infarction.31
The upregulation of myocardial EP3 receptors suggests a role in prostaglandin-mediated protection against tissue injury in reperfused ischemic myocardium. This is supported by several arguments. First, EP3 receptor–mediated inhibition of adenylyl cyclase antagonizes the detrimental effects of catecholamines released during acute myocardial ischemia,32 eg, by reducing the deleterious effects of Ca2+ overflow. Second, inhibitory G proteins may open inwardly rectifying33 34 and ATP-sensitive35 K+ channels. There seems to be a consensus that glibenclamide-sensitive K+ channels are involved in myocardial protection by ischemic preconditioning.36 Third, G proteins that belong to the Gi species are known to activate sarcolemmal Na+-Ca2+ exchange37 and thus may contribute to the control of intracellular calcium concentration.
All types of prostaglandins are formed within the heart. These include PGE238 and PGI2,6 which are the most likely candidates for endogenous EP3 receptor agonists. However, under which circumstances and by which extent PGE2 and PGI2 achieve concentrations sufficiently high to interact with EP3 receptors is not entirely clear at present. Since we6 and others39 have found that cyclooxygenase inhibition adversely affects ischemic myocardial injury, it is likely that in reperfused ischemic myocardium prostaglandin concentrations are high enough to activate cardiac EP3 receptors.
In summary, we have shown that myocardial tissue expresses EP receptors that belong to the EP3 subtype. EP3 receptor activation causes in vitro inhibition of adenylyl cyclase and decreases the inotropic response to ß-adrenergic activation. In addition, this receptor subtype is subject to upregulation during acute myocardial ischemia, whereas G-protein coupling remains intact. These data offer a rational basis for understanding the cardioprotective actions of EPs. In addition, they may stimulate the further development of therapeutic strategies to reduce or prevent ischemic myocardial injury by prostaglandin mimetics, since new and highly selective agonists of EP3 receptors become increasingly available.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB 242). The authors thank Kirsten Bartkowski and Elke Vasilescu for skillful technical assistance and Erika Lohmann for competent secretarial support. We thank Bayer UK (butaprost), Rhone-Poulenc Rorer (M&B 28.767), Schering AG (iloprost, sulprostone), Schwarz Pharma (PGE1), and Squibb (SQ 29.548) for their generous supply of compounds.
Received November 25, 1996; accepted August 7, 1997.
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References |
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