Introduction

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Endothelin (ET)-1 is a potent vasoconstrictor peptide that contributes to diverse biological actions through the activation of specific cell surface receptors on target tissues. To date, two different ET receptor subtypes have been identified and cloned. ET_A receptors, which mediate most of the vasoconstrictor action of ET-1, are present on vascular smooth muscle cells (Yanagisawa, 1994). ET_B receptors localized on vascular endothelium mediate the vasodilator response to ET-1 (Yanagisawa, 1994), while ET_B receptors on smooth muscle cells contribute to the vasoconstrictor effect of ET-1 (Seo et al., 1994; Teerlink et al., 1994; Haynes et al., 1995).

Congestive heart failure (CHF) is characterized by impaired cardiac function, vasoconstriction, volume retention, and activation of the sympathetic nervous system and renin-angiotensin system. In CHF, increased levels of plasma ET-1 have been demonstrated in both humans (McMurray et al., 1992; Rodeheffer et al., 1992) and experimental animal models (Cavero et al., 1990; Margulies et al., 1990). The plasma level of ET-1 has been shown to correlate closely to the severity of heart failure (Rodeheffer et al., 1992). ET-1 has also been shown to act as a potent growth factor in several cells, including fibroblasts (Takuwa et al., 1989) and cardiomyocytes (Shubeita et al., 1990), and has been found to activate several molecular markers for hypertrophy in cardiomyocytes (Shubeita et al., 1990). Thus, ET-1 may play a precipitating role in the cardiovascular system during the progression of CHF.

Increases in preproET-1 mRNA expression were reported in the left ventricle of paced dogs (Huntington et al., 1998) and the left atria of thoracic inferior vena cava constriction (TIVCC) dogs (Wei et al., 1997). The changes seen in the left atria of the TIVCC dogs were accompanied by an increase in ET-1. In addition to increases in local production of the peptide, up-regulation of ET_A receptors has been found to occur in the failing heart of rats (Sakai et al., 1996b). Recently, Luchner et al. (2000) reported that ET-1 increased in the LV of dogs with overt CHF produced by pacing over 38 days. However, the whole ET system consisting of ET-1 levels and the receptor properties of ETs in the ventricles of dogs with moderate CHF have not been reported.

Recently, several ET receptor antagonists have become available, which allows us to investigate the role of endogenous ET-1 in physiological and pathophysiological situations. Indeed, the acute administration of ET receptor antagonists has been shown to exert favorable hemodynamic responses in animal models (Shimoyama et al., 1996; Wada et al., 1997; Ohnishi et al., 1998; Onishi et al., 1999) and patients with CHF (Kiowski et al., 1995; Sütsch et al., 1998). Despite the many studies of antagonists in hypertension, there are few lines of evidence of the cardiovascular and renal effects of a balanced ET_A/ET_B receptor antagonist in dogs with CHF. Recently, we developed J-104132 [(+)-(5S,6R,7R)-2-butyl-7- [2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]-pyridine-6-carboxylic], which is a potent, orally active, balanced ET_A and ET_B receptor antagonist (Nishikibe et al., 1999). In vitro, J-104132 potently inhibited [¹²⁵I]ET-1 binding to cloned human ET_A (K_i = 0.034 ± 0.008 nM) or ET_B (K_i = 0.104 ± 0.017 nM) receptors expressed in Chinese hamster ovary cells (Nishikibe et al., 1999). The present study was designed to examine the pathophysiological roles of endogenous ET-1 in dogs with CHF after 3 weeks of rapid right ventricular pacing, which leads to the progression of CHF. To investigate the effects of changes in the entire ET system on the progression of CHF, we measured tissue immunoreactive ET-1 (irET-1) and the binding characteristics of ETs in the left and right ventricles and lung of dogs with CHF and compared the values with those in sham-operated dogs. To investigate the role of endogenous ET system in the progression of CHF, we examined the cardiovascular and renal responses to the acute intravenous administration of J-104132 in dogs with CHF. In addition, we intravenously administered J-104132 in combination with enalaprilat, an ACE inhibitor, to determine the value of an ET receptor antagonist in the treatment of CHF.

	Materials and Methods

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CHF Model with Rapid Right Ventricular Pacing

Thirty-three adult male beagle dogs weighing 10 to 14 kg were used for the experiments. The dogs were divided into two groups. All of the dogs underwent surgery to induce chronic heart failure (n = 33). Sham-operated dogs (n = 8) were used as a control. All experimental procedures followed the guidelines of the Japanese Pharmacological Society. Before surgery, blood was taken from the cephalic vein for determination of the concentrations of ET-1, aldosterone (ALDO), atrial natriuretic peptide (ANP), norepinephrine (NE) and plasma renin activity (PRA). All surgical procedures were performed under aseptic conditions. Anesthesia was induced and maintained by isoflurane (5 and 2 vol%, respectively) with nitrous oxide (3 l/min) and oxygen (2 l/min). Artificial ventilation was performed through a tracheal tube (rate, 15 times/min; volume, 300 ml) to maintain appropriate blood gas levels. After a stabilizing period of at least 30 min, an echocardiogram was recorded for the determination of basal conditions. Thereafter, a bipolar pacemaker electrode (3272VB; Aisne Productions Cardiologiques Int., Thierry, France) was advanced through the branch of the right external jugular vein and positioned in the right ventricles. The other end of the electrode was tunneled to a subcutaneous pocket in the left lateral neck and connected to a pulse generator (SIP-501; Star Medical, Tokyo, Japan), which was buried in a subcutaneous pocket. The animals received an antibiotic for up to 3 days after implantation of the electrodes, and at 1 to 2 weeks after surgery, pacing was initiated at a rate of 240 to 250 beats/min. The dogs were periodically examined to confirm the maintenance of pacing and to monitor for incipient symptoms of CHF. The pacing was stopped when signs of CHF (ascites, decreased motor activity and appetite, and abnormal breathing) were apparent (after approximately 3 weeks of pacing). At this time, blood was withdrawn for repeat measurement of the humoral factors. Some animals with rapid right ventricular pacing were excluded in this study due to unsuccessful pacing or unsatisfactory hemodynamic measurements at baseline [less than 15 mm Hg of left ventricular end-diastolic pressure (LVEDP)].

Experimental Protocol

Experiment A. The CHF group was divided into three treatment groups. The evaluation of hemodynamic and renal function was conducted in spontaneous sinus rhythm overnight after the cessation of pacing. Baseline hemodynamic and echocardiographic parameters as well as renal function were measured before drug or vehicle treatment. Then, a bolus dose of J-104132 (1 and 3 mg/kg, n = 8, respectively) or vehicle (saline, n = 8) was administered as a 10-min infusion via the femoral vein. Hemodynamic measurements were performed at time zero (immediately after drug injection) and at 10, 20, 30, 60, 90, and 120 min after drug injection. Echocardiograms were recorded before and 30, 60, and 120 min after the injection. Urine collection was performed every 30 min to estimate renal function. Blood gases were periodically monitored to adjust the artificial ventilation.

Experiment B. Hemodynamics were evaluated as in experiment A. Enalaprilat at a dose of 0.05 mg/kg was injected via the femoral vein at a rate of 1 ml/min, and J-104132 at a dose of 1 mg/kg was injected in the same manner after 35 min of enalaprilat treatment. A higher dose of enalaprilat (0.15 mg/kg) caused severe hypotension (data not shown). Therefore, an enalaprilat dose of 0.05 mg/kg was selected in this study. Additive effects of J-104132 on cardiovascular function were evaluated as maximum response during 2 h after combination with enalaprilat, and compared with those after J-104132 alone in experiment A.

Hemodynamic Measurements

On the day of the study, each animal was anesthetized and artificially ventilated as described above. A polyethylene catheter was inserted in the left femoral artery and connected to a pressure transducer (TP-400T; Nihon Kohden Inst., Tokyo, Japan) to measure mean arterial pressure (MAP). Blood samples were taken via the arterial catheter. A polyethylene catheter was also inserted in the right and left femoral veins to inject inulin solution and drug, respectively. A 7-F Swan-Ganz pulmonary artery catheter was positioned in the pulmonary artery through the left external jugular vein, and connected to pressure transducers for the measurement of pulmonary arterial pressure (PAP), pulmonary capillary wedge pressure (PCWP), and right atrial pressure. Cardiac output (CO) was measured in triplicate by the thermodilution method using a CO computer (MTC-6210; Nihon Kohden Inst.). A 5-F high-fidelity manometer-tipped catheter (Millar Inst., Houston, TX) was positioned in the left ventricle through the left carotid artery for the measurement of left ventricle end-systolic pressure, LVEDP, and LV pressure-derived indices of contractility and relaxation. Transit doppler flow probes were attached on the right femoral artery and the left renal artery and were connected to blood flow meters (T206; Transonic System Inc., Ithaca, NY) to measure femoral blood flow and renal blood flow (RBF), respectively. Finally, a bladder catheter was used to facilitate urine collection. After completion of surgery, a priming dose of inulin (250 mg/ml) was given, followed by an infusion of saline containing inulin 2.5 mg/ml at a rate of 2 ml/min for the measurement of glomerular filtration rate (GFR). After 1 h, the infusion rate was changed to 1 ml/min at a concentration of 5 mg/ml and was kept constant throughout the subsequent experiment. Urine samples were collected every 30 min. Blood samples (1 ml) were also obtained at the end of each period for determination of inulin concentration.

A cardiotachometer triggered by limb lead II ECG provided continuous records of heart rate (HR). All pressures and HR were monitored and recorded by a polygraph system (RM-6000; Nihon Kohden Inst.).

Stroke volume was calculated as the quotient of CO and HR. Total peripheral resistance (TPR) was calculated as the quotient of MAP and CO. Pulmonary vascular resistance (PVR) was calculated as the quotient of mean PAP and CO. LV relaxation was characterized by relaxation time constant, tau, obtained according to the method of Weiss et al. (1976).

Echocardiographic Measurements

Two-dimensionally directed M-mode echocardiograms were obtained via the right parasternal approach with an ultrasound system (probe: 3.75-MHz transducer, SSA-340A; Toshiba Medical Systems, Tokyo, Japan). An LV cross-sectional view from the short-axis was taken at the chordal level. On M-mode echocardiograms, left ventricular end-diastolic inner diameter (LVEDD), left ventricular posterior wall diameter (LVPWD), and left ventricular end-systolic diameter (LVESD) were measured. Fractional shortening (FS) was calculated as 100 × (LVEDD  LVESD)/LVEDD.

Analytical Procedures

GFR was estimated from inulin clearance. Urine and plasma inulin concentrations were measured spectrofluorometrically (U-2000; Hitachi., Tokyo, Japan). Urine and plasma sodium concentrations were determined (DRI-CHEM 800; Fuji Medical System Co., Ltd., Tokyo, Japan). Plasma samples were obtained under conscious conditions before the initiation of pacing and at the end of pacing to characterize the condition of the heart failure for humoral measurement. The samples were centrifuged at 4°C and the plasma was frozen at 80°C until the assay was performed. ET-1 was estimated by enzyme immunoassay (EIA) using a kit. PRA was determined by radioimmunoassay using a kit (Dinabott Radioisotope Inst., Tokyo, Japan). ALDO was estimated by radioimmunoassay using a kit (Diagnostic Products Co., Tokyo, Japan). ANP was determined by EIA using a kit (Wako Pure Chemical Co. Ltd., Osaka, Japan). Plasma NE concentration was measured by high-performance liquid chromatography with an amperometric detector (ECD-300; Eikom, Kyoto, Japan).

Quantification of Tissue ET-1

After hemodynamic measurement, the left and right ventricular free wall and lungs from dogs with CHF (vehicle treatment in experiment A, n = 9) and from sham-operated dogs (n = 8) were removed and rapidly frozen in liquid nitrogen. The tissue samples were weighed, immediately frozen in liquid nitrogen, and stored at 80°C until measurement. Tissue was homogenized with a Polytron homogenizer for 60 s in 10 volumes of 1 M acetic acid containing 10 µg/ml pepstatin (Peptide Institute, Osaka, Japan) and immediately boiled for 10 min at 4°C. The supernatant was stored at 80°C until use. The supernatant was subjected to sandwich EIA for ET-1.

Endothelin Receptor Assay

Each tissue sample was cut into small pieces and placed in solution I buffer containing 154 mM NaCl, 10 mM KCl, 0.8 mM CaCl₂, 10 mM 4-morpholinepropanesulfonic acid, and 20% w/v sucrose at 4°C and homogenized for 30 s with a Polytron homogenizer. Homogenization was repeated two times. The homogenates were centrifuged at 1000g for 10 min at 4°C. Then, the supernatants were centrifuged at 10,000g for 15 min at 4°C. The resultant supernatants were centrifuged at 100,000g for 60 min at 4°C. Then, the pellets were suspended in 5 mM HEPES buffer, pH 7.4, homogenized for 10 s with a Polytron homogenizer and centrifuged at 100,000g for 60 min at 4°C. The pellets as membrane fraction were suspended in 5 mM HEPES buffer, pH 7.4, and homogenized and stored at 80°C until use in the binding study. The protein concentration of the preparation was measured using the bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Richmond, CA) with BSA as a standard. The protein concentration in the membrane samples was adjusted to obtain 2 to 5 mg/ml.

A binding assay was performed in binding buffer (58 mM Tris/HCl, 12 µM CaCl₂, 12 µM MgCl₂, 120 µM phenylmethylsulfonyl fluoride, 1.2 µM pepstatin A, 2.3 µM leupeptin, 1.2 µM 1,10-phenanthrolin, and 0.1% BSA) with 25 µg of membrane proteins per tube in a final volume of 400 µl. Nonspecific binding was defined in the presence of 250 nM unlabeled ET-1.

Saturation binding experiments were performed by adding [¹²⁵I]ET-1 (15-20 pM) in the absence and presence of unlabeled ET-1 (100 fM-20 nM). Competition binding was performed by using [¹²⁵I]ET-1 in the presence of various concentrations of BQ-123 (200 pM-30 µM) and BQ-788 (500 pM-1 µM). The study was performed at room temperature for 4 h.

Membrane-bound [¹²⁵I]ET-1 was separated with free fraction by Whatman GF/C filters presoaked in 5 mM HEPES buffer containing 0.3% BSA. The filter was washed three times with the same buffer, and the radioactivity remaining on the filter was determined in a gamma radiation counter. All assays were conducted in triplicate.

The B_max and K_d values were calculated by the LIGAND program from the National Institutes of Health. The ratio of ET_A to ET_B receptors was calculated from the competition curve for BQ123 and BQ788 in both ventricles. In the lung, the measurement of the ratio of ET_A to ET_B receptors was calculated by displacement of [¹²⁵I]ET-1 binding with BQ123 and sarafotoxin S6c instead of BQ788. Because of completely displaced [¹²⁵I]ET-1 binding, BQ788 did not give ET_B receptor estimation.

Chemicals

The nonpeptide ET_A/B receptor antagonist J-104132, BQ123, and BQ788 were synthesized by Banyu Pharmaceutical Co. Ltd. (Ibaraki, Japan). J-104132 was dissolved in isotonic saline. The antibiotic cefamezin was purchased from Fujisawa Pharmaceutical Company (Tokyo, Japan).

Statistics

All data are represented as the mean ± S.E.M. The statistical analyses were performed using SAS software. The data obtained before and after the development of CHF were compared using Student's t test. The comparisons between the drug and vehicle groups were made with analysis of variance followed by Dunnett's test (group A). The comparisons between J-104132 alone and in combination with enalaprilat were made with analysis of variance followed by Student's t test (group B). Statistical significance was considered to be p < 0.05.

	Results

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Characterization of CHF

After 3 weeks of pacing, dogs developed heart failure characterized by ascites, decreased motor activity and appetite, and abnormal breathing. Compared with sham-operated dogs, dogs with CHF demonstrated decreases in MAP, left ventricular pressure corrected for pressure (+dp/dt/P) and CO (all p < 0.05), and increases in LVEDP, PAP, PCWP, PVR, and prolongation of tau (all p < 0.05) (Table 1). Echocardiogram showed typical eccentric hypertrophy; increases in LVEDD (+38% from 29.1 ± 0.5 mm before pacing, p < 0.05), and decreases in LVPWD (30% from 8.2 ± 0.2 mm, p < 0.05) and FS (53% from 30 ± 1%, p < 0.05) (Table 2). Accompanying the morphological and functional changes, there were remarkable increases in plasma levels of ANP (10 times the value before pacing, p < 0.05), NE (two times the value before pacing, p < 0.05), and PRA (two times the value before pacing, p < 0.05) at the end of the pacing period. However, the plasma level of ALDO remained unchanged after pacing for three weeks (Table 4). The animals with heart failure showed marked reductions in urine volume (62% versus control value of 0.26 ± 0.03 ml/min, p < 0.05), urinary excretion of sodium (72% versus control value of 59.9 ± 8.4 µl/min, p < 0.05), and fractional excretion of sodium (64% versus control value of 1.1 ± 0.2%, p < 0.05) (Table 3). GFR did not change, compared with that in sham-operated dogs (Table 3).

ET-1 Levels in Plasma and Tissues

The concentration of plasma ET-1 in dogs with CHF was approximately 2-fold higher than that of sham-operated dogs (Table 4). As shown in Fig. 1, the level of ET-1 in the left and right ventricles was significantly higher in dogs with CHF than in sham-operated dogs. In contrast, the ET-1 level in the lungs did not differ between the two groups. Plasma ET-1 levels were positively correlated with tissue ET-1 levels in both the left and the right ventricle. Tissue ET-1 levels in the left ventricle were positively correlated with LVEDP, PAP, PVR, PCWP, and LVEDD (all p < 0.05) and negatively correlated with MAP, left ventricle end-systolic pressure, +dp/dt/P, LVPWD, and FS (all p < 0.05). Tissue ET-1 levels in the right ventricle were positively correlated with right atrial pressure and tau (both p < 0.05) and negatively correlated with LVPWD and FS (both p < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   ET-1 level in left and right ventricles and lung of sham-operated does (n = 8) and CHF does (n = 8). Values are mean ± S.E.M. **p < 0.001, compared with value in sham-operated dogs.

Receptor Binding Assay

Neither the affinity nor the density of ET receptors in the ventricles and lungs was changed in dogs with CHF, compared with those in sham-operated dogs. Again, no difference was detected in the ratio of ET_A to ET_B receptors in all tested tissues (Table 5).

Cardiovascular Effects of J-104132

Experiment A. There were no significant differences among the three groups in baseline hemodynamic, echocardiographic, and renal function parameters. Intravenous injection of J-104132 at 1 and 3 mg/kg gradually decreased MAP, an effect that persisted until the end of the experiment (Fig. 2). There were no significant changes in HR among the three groups. J-104132 at both doses decreased systolic PAP and PCWP. These reductions were accompanied by significant decreases in TPR and PVR. CO was increased from 1.35 ± 0.26 and 1.39 ± 0.34 l/min at baseline to 1.54 ± 0.25 and 1.61 ± 0.33 l/min by J-104132 at doses of 1 and 3 mg/kg, respectively (Fig. 3). The increased CO levels were similar to those in dogs without heart failure. Acute administration of J-104132 had no effect on isovolumetric contraction indicated by +dp/dt/P or isovolumetric relaxation indicated by time constant (tau). J-104132 at a dose of 1 mg/kg increased FS (+38 ± 7% from baseline at 20 min), although this change was not statistically significant. The higher dose of J-104132 induced significant improvement in FS (+42 ± 6% from baseline at 30 min, p < 0.05). None of the treatments altered LVEDD.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of J-104132 (, 1 mg/kg; , 3 mg/kg) and vehicle () on systemic (MAP) and PAP, and PCWP. Values are mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the values in vehicle group at the corresponding time point.

Experiment B. The hemodynamic effects of J-104132 were examined in enalaprilat-treated dogs. In the presence of enalaprilat (0.05 mg/kg i.v.), J-104132 (1 mg/kg i.v.) caused further decreases in MAP (maximum 26.4 ± 2.0% from 84 ± 5 mm Hg with combination therapy versus maximum 18.2 ± 1.7% from 87 ± 4 mm Hg with J-104132 alone, p < 0.05), systolic PAP (28.1 ± 3.0% from 46 ± 2 mm Hg versus 20.1 ± 2.2% from 43 ± 2 mm Hg, respectively, p < 0.05), PCWP (33.8 ± 3.1% from 29 ± 1 mm Hg versus 19.6 ± 1.9% from 28 ± 1 mm Hg, respectively, p < 0.05), and TPR (39.3 ± 1.4% from 62 ± 4 mm Hg/l/min versus 28.5 ± 3.4% from 67 ± 6 mm Hg/l/min, respectively, p < 0.05) compared with J-104132 alone. Consequently, a further increase in CO (+28.2 ± 4.3% from 1.39 ± 0.12 l/min versus +20.8 ± 3.8% from 1.35 ± 0.09 l/min, respectively) was observed, although this difference did not reach statistical significance. HR was not affected by combination therapy with enalaprilat, compared with J-104132 alone.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of J-104132 (, 1 mg/kg; , 3 mg/kg) and vehicle () on CO, TPR, and PVR. Values are mean ± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, compared with values in vehicle group at the corresponding time point.

	Discussion

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Previous studies have demonstrated that plasma ET-1 levels are elevated in animal models of CHF (Cavero et al., 1990; Margulies et al., 1990) and patients with CHF (McMurray et al., 1992; Rodeheffer et al., 1992). These lines of evidence support the present findings that plasma ET-1 levels increased about 2-fold in dogs with pacing-induced CHF compared with sham-operated dogs. The underlying mechanisms for increased plasma ET-1 in CHF may be increased production of ET-1 stimulated by either hemodynamic or neurohumoral factors, and/or decreased clearance or metabolism of this peptide (Clavell and Burnett, 1995). Several studies have demonstrated increased preproET-1 mRNA in the heart of animals with experimentally induced heart failure. Sakai et al. (1996a) reported elevation of preproET-1 mRNA expression in the left ventricle of rats with myocardial infarction. Similar results were noted in the atria and lungs of dogs with CHF produced by TIVCC. Sakai et al. (1996a) also detected increased ET-1 content in these tissues. Nevertheless, there are few reports of ventricular ET-1 levels in dogs with CHF. The present study demonstrated that left and right ventricular irET-1 is increased about 2-fold in agreement with the study conducted by Luchner et al. (2000). They reported that ET-1 levels increased in the LV of dogs with overt CHF produced by pacing with a stepwise increase of stimulation frequencies over 38 days. In the present study, we performed evaluations after 3 weeks of pacing. This duration of pacing produced not only some clinical signs but also the exacerbation of hemodynamic and echocardiographic values, although none of the animals died. However, some dogs died when the duration of the pacing regimen was longer (4 weeks of pacing) (Suzuki et al., 2001). Thus, the present study indicated that tissue ET-1 levels were increased in the LV and RV even in the progression stage of CHF. We also demonstrated that there was a significant correlation between ET-1 levels in the left or right ventricle and those in plasma. These results suggest that an increase in the regional production of ET-1 in the heart is a major cause of elevated plasma ET-1 concentration.

Recently, it has been reported that patients with myocardial infarction (New York Heart Association class II-IV) showed elevation of ECE-1 levels in the left atrium, compared with control subjects (Bohnemeier et al., 1998). Thus, conversion of big ET-1 to ET-1 as well as increases in preproET-1 might be up-regulated with progression of heart failure. In our preliminary study, the expression of ECE-1 mRNA was increased in the LV and RV but not lung of the dogs with CHF, while the expression of preproET-1 mRNA did not be increased in these tissues, using reverse transcription-polymerase chain reaction (data not shown). This result suggests a possibility that increases in ET-1 level in the LV might be due to increase in conversion from big ET-1 to mature ET-1. However, more detailed experiments should be needed to determine the ECE protein expression or its activity in this model. Using a ribonuclease protection assay, Huntington et al. (1998) reported that preproET-1 mRNA expression in the LV and lungs was increased. This discrepancy may be due to differences in the severity of CHF caused by each of the experimental methods such as dog size and pacing regimen.

In CHF, excessive activation of the sympathetic nervous system is well documented, and there is evidence that the density of -adrenoceptors decreases progressively (Spinale et al., 1994). It is thought that excessive activation of the sympathetic nervous system causes down-regulation of -receptors. Sakai et al. (1996b) have reported that ET-1 binding sites were increased despite elevated ET-1 levels in the left ventricle of rats with ischemic heart failure. These results indicate that the function of the ET system was activated during CHF in rats. No previous studies have determined the changes in ET receptor kinetics in dogs. In the present study, we found no changes in the affinity and density of ET receptors or in the ratio of ET_A to ET_B receptors in the failing heart. These results demonstrated that the affinity and density of ET receptors were not altered despite high levels of circulating and local ET-1.

There is little information on the cardiovascular effects of balanced ET_A/ET_B receptor antagonists in dogs with CHF. Therefore, we used J-104132, which was recently developed as a potent, orally active balanced antagonist of both ET_A and ET_B receptors, to examine the functional roles of endogenous ET-1 in CHF. Our results demonstrated that ET_A/ET_B receptor blockade with J-104132 (at doses of 1 and 3 mg/kg) produced a marked reduction of TPR and PVR in dogs with CHF, which indicated vasoconstriction of the systemic and pulmonary vascular vessels by elevated ET-1 during CHF. These vasodilative effects of J-104132 reached a plateau 30 to 60 min after administration and persisted for 120 min. A reduction of PCWP after the administration of J-104132 at 1 and 3 mg/kg was also observed. As a result of decreases in both pre- and after-load, CO was improved to the level of sham-operated dogs. Increased CO was not due to the direct effects of J-104132 on contractile and relaxation performance of the myocardium, since there were no changes in +dp/dt/P or tau after the administration of J-104132. These results demonstrated that acute blockade of ET_A/ET_B receptors with J-104132 decreased pre- and after-load, and consequently increased CO in a canine CHF model. In addition, it is important to note that J-104132 administered after treatment with the ACE inhibitor enalaprilat caused further reductions in pre- and after-load and consequently further increases in CO in dogs compared with values obtained with J-104132 alone, since angiotensin-converting enzyme inhibitors are now considered as standard therapy in patients with heart failure.

ETs are known to have various renal effects. In the kidney, ET_A receptors were expressed in the glomerulus, vasa recta, and arcuate arteries (Terada et al., 1992), and were thought to be involved in mediating the vasoconstrictor actions of ET-1. Brooks et al. (1994) noted that ET-1-induced renal vasoconstriction is mediated by ET_A receptors in dogs. In addition to ET_A-mediated vasoconstriction, activation of ET_B receptors was probably involved in the renal vasoconstrictor response of ET-1 in anesthetized rats (Pollock and Opgenorth, 1993). In the present study, the blockade of endogenous ET with J-104132 produced marked increases in RBF with no alteration in GFR, thereby indicating the potent vasoconstrictive profile of endogenous ETs in the renal vascular bed of CHF dogs. In contrast, ET_B receptors mainly localize on the inner medullary collecting ducts and glomeruli (Terada et al., 1992). Several studies have demonstrated that ET-1 inhibits arginine vasopressin-induced reduction of water permeability and cAMP accumulation in rat IMCD (Tomota et al., 1990). Garcia and Garvin (1994) demonstrated that ET-1 had a unique biphasic action on sodium transport in the proximal tubules. In anesthetized dogs, the intrarenal administration of sarafotoxin S6c resulted in a diuretic response with no change in sodium excretion (Matsuo et al., 1997). When J-104132 was intravenously administered, there were no apparent effects on urine volume or the urinary and fractional excretion of sodium. These results suggested that the effects of endogenous ET-1 on urine formation were likely to be negligible in CHF.

In summary, the present study demonstrated that pacing-induced CHF caused irET-1 in plasma and the left and right ventricles to increase without altering levels in the lungs, compared with those in sham-operated dogs. There were no significant changes in the density and affinity of total ET receptors, and in the ratio of ET_A to ET_B receptors in the left and right ventricles and the lungs of dogs with CHF, compared with those of sham-operated dogs. Blockade of ET receptors with J-104132 significantly decreased the MAP and PAP associated with increases in CO, which resulted in reduction of both systemic and pulmonary vascular resistance in dogs with CHF. The beneficial effects of J-104132 were additive to those of enalaprilat. These results indicate that the endogenous ET system is exaggerated in moderate CHF, and has a detrimental effect on cardiac function. Thus, J-104132 is expected to be useful for the treatment of CHF in humans.