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(Circulation. 1996;94:742-747.)
© 1996 American Heart Association, Inc.

Articles

Endothelin-1–Induced Release of Thromboxane A₂ Increases the Vasoconstrictor Effect of Endothelin-1 in Postischemic Reperfused Rat Hearts

Christian E. Zaugg, PhD; Pius S. Hornstein, PhD; Peili Zhu, MD; David Simper, MD; Thomas F. Luscher, MD; Peter R. Allegrini, PhD; Peter T. Buser, MD

the Division of Cardiology (C.E.Z., P.S.H., P.Z., D.S., P.T.B.), University Hospital Basel; Division of Cardiology (T.F.L.), University Hospital Bern; and Biology Research Laboratory, CIBA Ltd (P.R.A.), Basel, Switzerland.

Correspondence to Christian E. Zaugg, PhD, Division of Cardiology, DIM, University Hospital, Petersgraben 4, 4031 Basel, Switzerland.

	Abstract

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Background The release and vasoconstrictor effect of endothelin-1 (ET-1) are increased after myocardial ischemia, suggesting a role for ET-1 in ischemia/reperfusion injury. However, the mechanisms of the increased vasoconstriction by ET-1 are unknown. The aim of this study was to test whether ET-1–induced release of thromboxane A₂ (TXA₂) contributes to the vasoconstrictor effect of ET-1 in nonischemic hearts and whether such release can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts.

Methods and Results ET-1–induced release of TXA₂ was assessed by measurement of the concentrations of its stable metabolite thromboxane B₂ (TXB₂) in the coronary effluent of nonischemic and reperfused isolated rat hearts before and after administration of 0.01 nmol ET-1 using an enzyme immunoassay. The contribution of ET-1–induced release of TXA₂ to the vasoconstrictor effect of ET-1 was assessed by measurement of the effects of ET-1 with and without the cyclooxygenase inhibitor indomethacin or the TXA₂/endoperoxide receptor antagonist SQ 30,741 using ³¹P magnetic resonance spectroscopy. In nonischemic hearts, ET-1 led to a small increase in TXB₂ in the coronary effluent (3.9±1.5 pg/mL; n=3), but neither indomethacin nor SQ 30,741 significantly diminished the vasoconstrictor effects of ET-1 (reduction of coronary flow, 4.0±0.4 and 4.5±0.3 mL/min, respectively, versus 4.9±0.5 mL/min for ET-1 alone; n=8, 6, and 9, respectively). In postischemic reperfused hearts, however, ET-1 led to a greater increase in TXB₂ (13.7±1.5 pg/mL; P<.05 versus nonischemic hearts; n=3), and both indomethacin and SQ 30,741 diminished the vasoconstrictor effects of ET-1 (reduction of coronary flow, 2.6±0.3 and 2.2±0.3 mL/min, respectively, versus 4.0±0.1 mL/min for ET-1 alone; n=8, 8, and 6, respectively; P<.05). Furthermore, indomethacin and SQ 30,741 prevented the detrimental effects of ET-1 on left ventricular developed pressure, intracellular pH, and phosphocreatine during reperfusion.

Conclusions ET-1–induced release of TXA₂ does not significantly contribute to the vasoconstrictor effect of ET-1 in nonischemic hearts but can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts.

Key Words: endothelin • thromboxane • ischemia • reperfusion • vasoconstriction

	Introduction

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The release of ET-1, one of the most potent vasoconstrictors, has been shown to be increased after myocardial ischemia.^{1 2 3} Additionally, the vasoconstrictor effect of ET-1 appears to be increased during postischemic reperfusion.^{1 4 5 6 7} Accordingly, ET-1 in concentrations not causing ischemia in rat hearts under nonischemic perfusion conditions caused increased vasoconstriction under postischemic reperfusion conditions, thus preventing myocardial recovery due to severe ischemia.⁸ Therefore, ET-1 has been suggested to contribute to reperfusion vascular damage¹ and to delay or even prevent postischemic recovery.⁸

However, the mechanisms of this increased vasoconstriction during postischemic reperfusion are unknown. One hypothesis suggested that the loss of counteracting vasodilator mechanisms, such as prostacyclin or endothelium-derived nitric oxide, could lead to increased vasoconstriction during reperfusion.^{4 6 8} Another hypothesis suggested that the postischemically increased density of ET-1 binding sites at rat cardiac membranes⁹ could mediate increased vasoconstriction for identical concentrations of ET-1.⁸ An alternative hypothesis arises from ET-1–induced release of the cyclooxygenase product TXA₂ from rat vascular preparations.¹⁰ Such an ET-1–induced release of TXA₂ not only could contribute to the vasoconstrictor effect of ET-1 under nonischemic conditions but also could increase the vasoconstrictor effect of ET-1 under postischemic reperfusion conditions because the production of TXA₂ is increased during ischemia.¹¹

Therefore, the present study was designed to investigate whether ET-1–induced release of TXA₂ contributes to the vasoconstrictor effect of ET-1 in nonischemic hearts and whether such a release can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts. For this purpose, we tested whether ET-1 induces the release of TXA₂ in isolated rat hearts under nonischemic and postischemic reperfusion conditions. ET-1–induced release of TXA₂ was assessed by measurement of concentrations of TXB₂, the stable but inactive metabolite of TXA₂, in the coronary effluent of nonischemic and reperfused hearts before and after administration of ET-1. The contribution of ET-1–induced release of TXA₂ to the vasoconstrictor effect of ET-1 was then assessed by measurement of the effects of ET-1 with and without inhibition of cyclooxygenase activity or blockade of TXA₂ receptors in nonischemic and reperfused hearts. Vasoconstriction was assessed by measurement of the reduction of coronary flow, and the consequences of the ET-1–induced vasoconstriction were assessed by measurement of LV developed pressure, intracellular pH, phosphocreatine, ATP, and inorganic phosphate by use of ³¹P magnetic resonance spectroscopy.

	Methods

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Perfused Heart Model
Sprague-Dawley rats (CIBA Ltd, Sisseln, Switzerland), weighing 250 to 330 g, were used. After injection of heparin (1000 IU/kg IP) and sodium pentobarbital (30 mg/kg IP), hearts were excised rapidly through a midline sternotomy, and the aortas were cannulated within 30 seconds. Retrograde perfusion was performed at 37°C from a reservoir 900 mm above the aortic cannula.⁸ All hearts were perfused with a nonrecirculating Krebs-Henseleit solution containing (in mmol/L) NaCl 117.0, KCl 4.3, CaCl₂ 2.0, MgCl₂ 1.2, KH₂PO₄ 0.1, NaHCO₃ 25.0, NaEDTA 0.5, and glucose 15.0, as well as insulin 10 IU/L.^{8 12} This perfusate was saturated with a gas mixture of 95% O₂–5% CO₂ (oxygen tension, 73 to 87 kPa). A pair of platinum pacemaker wires from a pulse generator (Grass SD 5, Grass Instruments) was implanted in the RV wall and the right atrium. Throughout the experiment, hearts were paced at a constant frequency that was 120% of the spontaneous heart rate. During all experiments, the hearts were immersed in perfusate maintained between 36.8°C and 37.0°C.

Experimental Protocol
To investigate whether ET-1 induces the release of TXA₂, concentrations of TXB₂ in the coronary effluent of nonischemic and postischemic reperfused hearts were measured before and after ET-1 administration. Nonischemic hearts were first perfused under baseline conditions for 30 minutes. Subsequently, a bolus of 0.01 nmol ET-1 (Novabiochem) was administered, and TXB₂ concentrations in the coronary effluent were measured 1, 5, 10, and 30 minutes after ET-1 administration. Postischemic reperfused hearts underwent 30 minutes of baseline perfusion, 15 minutes of global isothermic ischemia, and 30 minutes of reperfusion before ET-1 administration and TXB₂ measurements.

To investigate whether ET-1–induced release of TXA₂ contributes to the vasoconstrictor effect of ET-1 under nonischemic conditions and whether such a release can increase the vasoconstrictor effect of ET-1 during postischemic reperfusion, the effects of ET-1 were measured with and without inhibition of cyclooxygenase activity or blockade of TXA₂ receptors in nonischemic and reperfused hearts. Nonischemic and reperfused hearts underwent the same protocols as described above for TXB₂ measurements. In one group of hearts, however, cyclooxygenase activity was inhibited by perfusion with 10 µmol/L indomethacin (Sigma Chemical Co) 30 minutes before ET-1 administration, ie, at the beginning of reperfusion. This concentration of indomethacin has been found to completely inhibit cyclooxygenase activity¹³ and was used previously in similar concentrations and pretreatment periods to inhibit cyclooxygenase activity in perfused rat hearts.^{4 14} In another group of hearts, TXA₂ receptors were blocked by perfusion with 0.1 µmol/L SQ 30,741 (Squibb) 30 minutes before ET-1 administration, ie, at the beginning of reperfusion. In this concentration, SQ 30,741 is a potent and selective antagonist of TXA₂ and prostaglandin endoperoxide receptors¹⁵ and was used previously in similar concentrations and pretreatment periods to block TXA₂ receptors in perfused rat hearts¹⁶ and isolated rat aorta.¹⁷ Because SQ 30,741 blocks the common receptor for TXA₂ and its precursor PGH₂, a differentiation between these two prostaglandins could not be performed, and findings of the present study concerning ET-1–induced release of TXA₂ also refer to PGH₂. After administration of a bolus of 0.01 nmol ET-1, changes of coronary flow and LV developed pressure were measured during the next 30 minutes. In reperfused hearts, intracellular pH, phosphocreatine, ATP, and inorganic phosphate were measured to assess the consequences of the ET-1–induced vasoconstriction.

To exclude instabilities of the isolated rat heart preparation during the observation time of ET-1 effects, control groups without ET-1 administration were analyzed under nonischemic and postischemic reperfusion conditions. During all experiments, endogenous ET-1 production was assumed to be inhibited by EDTA in the perfusate, a potent inhibitor of the endothelin-converting enzyme.^{18 19 20 21 22} The selected bolus of ET-1 reduced coronary flow to 50% in nonischemic perfused rat hearts but did not cause ischemic reactions under baseline conditions.⁸

Measurements of Coronary Flow and LV Pressure
To assess ET-1–induced vasoconstriction, coronary flow was measured by collection of the effluent from the RV outflow tract in graduated cylinders. LV developed pressure was measured by a fluid-filled polyethylene catheter inserted through the left atrial appendage into the left ventricle.^{1 8 12 14} The catheter was connected to a Statham P23Db pressure transducer (Gould) that was outside the magnet at the same height as the heart. LV developed pressure was defined as the difference between systolic and diastolic values of LV pressure.

TXB₂ Measurements
Measurements of TXB₂ concentrations in the coronary effluent of perfused rat hearts were performed using a commercially available enzyme immunoassay (Thromboxane B₂ EIA kit 519031, Cayman Chemical). Samples were collected from the effluent from the RV outflow tract and stored at -70°C until TXB₂ measurements were performed. Because TXB₂ concentrations of the samples were often below the TXB₂ sensitivity of this assay (20 pg/mL), samples were concentrated 10-fold by evaporation of solvent at 40°C under nitrogen dry steam. Subsequently, the concentrated samples were dissolved in UltraPure water (deionized and free of trace organic contaminants) to 1/10th of the volume before concentration. This concentration procedure did not diminish the accuracy of the TXB₂ measurements, as tested by internal standards and control measurements without TXB₂.

Enzyme immunoassays were performed in duplicate by mixing 50 µL concentrated sample with 50 µL tracer and 50 µL antiserum on microplates. After 18 hours of incubation, 200 µL Ellmans-Reagens was added to start the enzymatic reaction, and the absorption of individual vials was measured 30 minutes later at 405 nm using a photometric microplate reader (Thermo Max, Molecular Devices). Concentrations of TXB₂ in the samples were estimated from standard curves obtained by nonlinear regression to absorptions of eight known TXB₂ concentrations ranging from 7.5 to 1000 pg/mL by use of Softmax software (Molecular Devices). Measurements of TXB₂ of the duplicates by enzyme immunoassay were essentially identical and therefore were averaged and divided by 10 (correcting for previous sample concentration) to compute TXB₂ concentrations. The intra-assay and interassay coefficients of variation were <10%. The cross-reactivity of the assay was 0.44% for prostaglandin D₂, 0.22% for prostaglandin F₂, 0.2% for 11-dehydro TXB₂, 0.05% for prostaglandin F₁, and <0.01% for prostaglandin E₂ and 6-keto prostaglandin F₁.

³¹P Magnetic Resonance Spectroscopy
³¹P magnetic resonance spectroscopy of the isolated rat heart was performed on a 4.7-T horizontal 300-mm bore magnet (Bruker Spectrospin B-C 47/30). Spectra were obtained at 81.0 MHz with a spectral width of 5000 Hz. The sample was shimmed on the water signal from the heart (line width at half-height, 3 to 10 Hz). The pulse angle was 45°, and the pulse duration was 50 µs with a repetition time of 1.0 second. Three hundred transients were accumulated at 5-minute intervals. The signal-to-noise ratio was 20:1. For each spectrum, the characteristic peaks for phosphocreatine, the three phosphate groups of ATP (ß-ATP was used for quantification purposes), inorganic phosphate, and sugar monophosphates were identified. Each spectrum and the corresponding areas were numerically integrated after definition of the baseline and expressed as percentages of the baseline values. Because differentiation between the signals of sugar monophosphates and the overwhelming inorganic phosphate was difficult during ischemia and reperfusion, the sum of these two signals was used. However, most of this sum arose from inorganic phosphate and therefore was used to discuss changes of inorganic phosphate. Intracellular pH was calculated from the chemical shift of the inorganic phosphate peak relative to the phosphocreatine peak.²³

Statistical Analysis
To test for stability of the isolated rat hearts during continuous baseline perfusion (nonischemic conditions) and during postischemic reperfusion, absolute values of control hearts were recorded during periods necessary for the observation of ET-1 effects and analyzed by repeated-measures ANOVA.

To test for eventual differences between the groups before ET-1 administration, absolute values of all groups were analyzed during baseline and after 30 minutes of reperfusion by one-way ANOVA and Bonferroni's t test. Changes of variables within groups were analyzed by a paired t test.

The ET-1–induced changes of TXB₂ concentrations within groups at 1, 5, 10, and 30 minutes after ET-1 administration were analyzed by repeated-measures ANOVA and Dunnett's test. Comparison of TXB₂ concentrations between nonischemic and reperfused hearts before ET-1 administration was performed by an unpaired t test. Similarly, ET-1–induced changes of TXB₂ concentrations during the first minute after ET-1 administration were compared between nonischemic and reperfused hearts by an unpaired t test.

To compare the effects of ET-1 with and without indomethacin or SQ 30,741, absolute changes to values before ET-1 administration were calculated. Statistical analysis of these changes was performed by one-way ANOVA and Bonferroni's t test. To better express the time course of some important variables after ET-1 administration, absolute changes of coronary flow and developed pressure after ET-1 administration were plotted against time for nonischemic and reperfused hearts.

Results are expressed as mean±SEM. For all statistical analysis, the null hypothesis was rejected at the 95% level; thus, P<.05 was considered significant.

	Results

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Control Experiments and Tests for Differences Before ET-1 Administration
To test for stability of the isolated rat hearts during nonischemic and postischemic reperfusion conditions, absolute values of control hearts (without ET-1 administration) were recorded during periods necessary for the observation of ET-1 effects (Tables 1 and 2). This analysis showed no significant differences between values during continuous baseline perfusion (nonischemic hearts) or between values after 30 and 60 minutes of reperfusion. All variables remained stable during the time needed for observations after the administration of ET-1.

View this table: [in this window] [in a new window]   Table 1. Effects of Indomethacin and SQ 30,741 on ET-1–Induced Effects in Nonischemic Rat Hearts

To test for eventual differences between groups before ET-1 administration, absolute values were analyzed at baseline and in reperfused hearts also after 30 minutes of reperfusion. This analysis showed no significant differences between baseline values (Table 1). Therefore, absolute changes in the values immediately before ET-1 administration were calculated to compare and display ET-1–induced effects in nonischemic hearts. In addition, the analysis showed no significant differences between values after 30 minutes of reperfusion (Table 2). Neither indomethacin nor SQ 30,741, which were both administered at the beginning of reperfusion, significantly improved postischemic recovery of the measured variables. Thus, all hearts were considered equally recovered after 30 minutes of reperfusion. Therefore, changes relative to the values after 30 minutes of reperfusion (immediately before ET-1 administration) were calculated to compare and display ET-1–induced effects.

View this table: [in this window] [in a new window]   Table 2. Effects of Indomethacin and SQ 30,741 on ET-1–Induced Effects in Postischemic Reperfused Rat Hearts

Effects of ET-1 on TXB₂ Concentrations in the Coronary Effluent
In nonischemic hearts, ET-1 caused a small but statistically significant increase in TXB₂ concentrations in the coronary effluent (Fig 1). Specifically, the administration of 0.01 nmol ET-1 increased TXB₂ concentrations by 3.9±1.5 pg/mL within the first minute of administration of ET-1. Subsequently, TXB₂ concentrations remained significantly elevated for the next 9 minutes before approaching baseline levels 30 minutes after ET-1 administration.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of 0.01 nmol ET-1 on TXB2 concentrations in the coronary effluent of perfused rat hearts under nonischemic ([symbol:Typographic Ext/34/12]) and postischemic reperfusion ([symbol:Typographic Ext/33/12]) conditions. Values are mean±SEM for three hearts in each group. *P<.05 vs corresponding value before ET-1 administration; #P<.05 vs nonischemic conditions.

In postischemic reperfused hearts, however, TXB₂ concentrations were higher and ET-1 caused a greater increase in TXB₂ concentrations than in nonischemic hearts (Fig 1). Specifically, the administration of 0.01 nmol ET-1 increased TXB₂ concentrations by 13.7±1.5 pg/mL within the first minute of administration of ET-1 (P<.05 versus the increase under nonischemic conditions). Subsequently, TXB₂ concentrations remained significantly elevated for the next 4 minutes before approaching levels before ET-1 administration.

Influence of Indomethacin and SQ 30,741 on ET-1–Induced Effects
In nonischemic hearts, neither cyclooxygenase inhibition by indomethacin nor blockade of TXA₂ receptors by SQ 30,741 significantly affected the effects of ET-1. Specifically, neither 10 µmol/L indomethacin nor 0.1 µmol/L SQ 30,741 affected the reduction of coronary flow by 0.01 nmol ET-1 (Table 1 and Fig 2A). LV developed pressure did not change significantly after ET-1 administration (Table 1 and Fig 2C). Similarly, ET-1–induced effects on LV developed pressure were not different with or without indomethacin or SQ 30,741.

View larger version (27K): [in this window] [in a new window]   Figure 2. Changes of coronary flow (A and B) and LV developed pressure (C and D) after ET-1 alone ([symbol:Typographic Ext/33/12]) and after ET-1 with indomethacin ([symbol:Typographic Ext/35/12]) or with SQ 30,741 ([symbol:Typographic Ext/58/12]) in nonischemic (A and C) and in postischemic reperfused (B and D) rat hearts. Values are mean±SEM; see Tables 1 and 2 for n.

In postischemic reperfused hearts, however, both cyclooxygenase inhibition by indomethacin and blockade of TXA₂ receptors by SQ 30,741 diminished the effects of ET-1. Specifically, both 10 µmol/L indomethacin and 0.1 µmol/L SQ 30,741 diminished the reduction of coronary flow by 0.01 nmol ET-1 (Table 2 and Fig 2B). Consequently, both indomethacin and SQ 30,741 prevented the decrease of LV developed pressure by ET-1 (Table 2 and Fig 2D). Furthermore, indomethacin and SQ 30,741 prevented the decrease of intracellular pH, the decrease of phosphocreatine, and the increase of inorganic phosphate by ET-1 (Table 2). ß-ATP did not change significantly after ET-1 administration (Table 2). Similarly, ET-1–induced effects on ß-ATP were not different with or without indomethacin or SQ 30,741.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study in isolated perfused rat hearts demonstrated that ET-1–induced release of TXA₂ does not significantly contribute to the vasoconstrictor effect of ET-1 in nonischemic hearts but can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts. Specifically, ET-1 led to a small increase in TXB₂, the stable but inactive metabolite of TXA₂, in the coronary effluent of nonischemic hearts. This increase suggests that ET-1 induced the release of TXA₂ under nonischemic conditions. However, this release does not significantly contribute to the vasoconstrictor effect of ET-1 because both cyclooxygenase inhibition by indomethacin and blockade of TXA₂ receptors by SQ 30,741 did not diminish the effects of ET-1 in nonischemic hearts. In postischemic reperfused hearts, however, ET-1 led to a greater rise of TXB₂ in the coronary effluent than in nonischemic hearts, reflecting greater ET-1–induced release of TXA₂. In contrast to nonischemic conditions, ET-1–induced release of TXA₂ can increase the vasoconstrictor effect of ET-1 in reperfused hearts because both indomethacin and SQ 30,741 diminished the effects of ET-1 in reperfused hearts. Specifically, indomethacin and SQ 30,741 diminished the reduction of coronary flow by ET-1 and prevented the detrimental effects of ET-1 during reperfusion. Accordingly, both indomethacin and SQ 30,741 prevented the decrease of LV developed pressure by ET-1. Furthermore, they prevented the intracellular acidosis, the decrease in phosphocreatine, and the increase in inorganic phosphate by ET-1. The prevention of acidosis and of accumulating inorganic phosphate might have contributed to the beneficial effects of indomethacin and SQ 30,741 on LV developed pressure because acidosis inhibits the interaction between actin and myosin and because large amounts of inorganic phosphate reduce the calcium sensitivity of the contractile proteins.²⁴ ß-ATP did not change significantly in any group after ET-1 administration, suggesting a sufficient anaerobic ATP production or replenishment of ATP from phosphocreatine stores or both. Because SQ 30,741 blocks the common receptor for TXA₂ and its precursor PGH₂, it should be noted that the findings of the present study concerning ET-1–induced release of TXA₂ also refer to PGH₂. Recent observations suggest, however, that PGH₂ does not contribute to the vasoconstrictor effect of ET-1 in postischemic reperfused hearts.²⁵

The different contribution of ET-1–induced release of TXA₂ to the vasoconstrictor effects of ET-1 in nonischemic and reperfused hearts may shed light on the mechanism of the increased vasoconstriction by ET-1 during reperfusion.^{1 4 5 6 7 8} In nonischemic hearts, ET-1–induced release of TXA₂ does not significantly contribute to the vasoconstrictor effect of ET-1, suggesting that this effect arises primarily from acting on vascular smooth muscle cells (Fig 3, left). In postischemic reperfused hearts, however, ET-1–induced release of TXA₂ can significantly increase the vasoconstrictor effect of ET-1 (Fig 3, right). Similarly, TXA₂ has been shown to contribute to contractions evoked by ET-1 in the aortas of spontaneously hypertensive rats but not in those of normotensive rats.²⁶ Considering that TXA₂ was endothelium derived in these hypertensive rats²⁶ and that endothelial cells are an important source of TXA₂,²⁷ it is reasonable to speculate that ET-1 induced the release of endothelium-derived TXA₂ in the present study (Fig 3). Alternatively or additionally, ET-1 may have induced the release of TXA₂ from smooth muscle cells.¹⁰

View larger version (27K): [in this window] [in a new window]   Figure 3. Suggested mechanism of the contribution of TXA2 to the vasoconstrictor effects of ET-1 in nonischemic (left) and postischemic reperfused (right) rat hearts. Besides acting on smooth muscle cells, ET-1 induces the release of vasoconstricting TXA2 and/or PGH2 from endothelial cells. Under nonischemic conditions, the contribution of TXA2/PGH2 to the vasoconstrictor effect of ET-1 is insignificant. Under postischemic reperfusion conditions, however, this contribution increases the vasoconstrictor effect of ET-1.

In accordance with the suggested mechanism, indomethacin and SQ 30,741 did not significantly improve coronary flow or any other variable before administration of ET-1 because endogenous ET-1 production was inhibited by EDTA during all experiments.^{18 19 20 21 22} Consequently, the release of TXA₂ could not be induced before administration of exogenous ET-1, and both indomethacin and SQ 30,741 could not improve postischemic recovery. Furthermore, postischemically reduced production of the counteracting vasodilator prostacyclin probably did not contribute to the increased vasoconstriction by ET-1 because indomethacin, which inhibited the production of both TXA₂ and prostacyclin, did not cause further increased vasoconstriction. However, the higher density of ET-1 binding sites during reperfusion might still contribute to increased ET-1 effects during reperfusion.

The finding that ET-1–induced release of TXA₂ can increase the vasoconstrictor effect of ET-1 during postischemic reperfusion might be a crucial mechanism underlying delayed or incomplete postischemic recovery of the myocardium. Accordingly, ET-1 in concentrations not causing ischemia in rat hearts under nonischemic perfusion conditions caused severe ischemia under postischemic reperfusion conditions, preventing myocardial recovery in the present study and in a previous study.⁸ Ischemic myocardial tissue has long been noted to release TXA₂,^{11 28} which can participate in the progression of ischemic injury.²⁹ The present study suggests that part of this TXA₂ release might be dependent on release of ET-1 and therefore could be an interesting target of therapeutic strategies during postischemic reperfusion. Under in vivo conditions, however, such ET-1–induced release of endothelium-derived TXA₂ is likely to be masked by TXA₂ production of platelets and therefore might not be reflected by TXA₂ plasma concentrations. Nevertheless, local ET-1–induced release of TXA₂ from endothelial cells directed toward adjacent smooth muscle cells might contribute critically to the vasoconstrictor effect of ET-1 independent of other TXA₂ sources. Yet it remains to be determined whether the vasoconstrictor effect of endogenous ET-1 can be increased by the release of TXA₂ during reperfusion and how the degree of ischemia/reperfusion injury affects such a release.

In conclusion, this study in isolated perfused rat hearts demonstrated that ET-1–induced release of TXA₂ does not significantly contribute to the vasoconstrictor effect of ET-1 in nonischemic hearts but can increase the vasoconstrictor effect of ET-1 in postischemic reperfused hearts. This ET-1–induced release of TXA₂ might be a crucial mechanism underlying delayed or incomplete postischemic recovery of the myocardium.

	Acknowledgments

 
Drs Zaugg and Hornstein were supported by grants from the Swiss National Science Foundation. Dr Zhu was supported by the Swiss Foundation for Cardiology. Dr Buser was supported by a career development grant (SCORE No. 32-29340.90) from the Swiss National Science Foundation.

	Selected Abbreviations and Acronyms

 

ET-1 = endothelin-1 LV = left ventricular PGH2 = prostaglandin H2 RV = right ventricular TXA2 = thromboxane A2 TXB2 = thromboxane B2

Received November 21, 1995; revision received February 14, 1996; accepted February 16, 1996.

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