Introduction

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The polypeptide endothelin-1 (ET-1) plays an important role in the cardiovascular system by modulating vasomotion, inotropic state, and growth processes. ET-1 exerts its different biological effects via the activation of specific receptors (ET_A and ET_B receptors) which are widely expressed on cardiac and vascular cells of both humans and other species (Rubanyi and Polokoff, 1994).

Cardiac hypertrophy is an adaptational mechanism to stresses such as neurohumoral stimulation and pressure overload (Homcy, 1998). Recent observations suggest the involvement of ET-1 in left ventricular hypertrophy, but much less is known about its role in right ventricular hypertrophy (Ito, 1997; Kirchengast and Münter, 1999). In several rat models of right ventricular hypertrophy, including exposure to normobaric hypoxia, carbon monoxide, MCT (Miyauchi et al., 1993), or volume overload (Brown et al., 1995), prepro-ET-1 mRNA transcription was increased in right atria or ventricles without consistent increase in myocardial ET-1 production. In a recent study, we found no evidence for increased release of ET-1 and big ET-1 in hypertrophic hearts and concluded that the growth process may be fueled by extracardiac ET (Brunner, 1999). Administration of ET_A antagonists (Miyauchi et al., 1993; Ichikawa et al., 1996; Prié et al., 1997; Haleen et al., 1998) or, to a lesser extent, of mixed ET_A/ET_B receptor antagonists (Chen et al., 1995; Eddahibi et al., 1995; Hess et al., 1996; Hill et al., 1997) over several weeks generally attenuated the cardiopulmonary alterations following experimental right ventricular hypertrophy. In humans with pulmonary hypertension, intravenous administration of the mixed ET_A/ET_B receptor antagonist bosentan led to potent pulmonary vasodilation (Williamson et al., 2000), an effect that may contribute to the symptomatic improvements observed after bosentan in these patients.

The present study was designed to investigate the effects of a 9-week therapy with the specific butenolide ET_A receptor antagonist PD 155080 (2-benzo[1,3]dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl-)-4-oxobut-2-enoate-sodium) (Doherty et al., 1995) on right ventricular function, ET metabolism, and intracellular calcium (Ca²⁺_i) handling in rats with right ventricular hypertrophy secondary to pulmonary hypertension. Because myocardial hypertrophy is generally associated with impaired contraction dynamics, which correlate with a prolongation of the Ca²⁺_i transient (Balke and Shorofsky, 1998; Wölkart et al., 2000), we hypothesized that ET_A antagonism might alleviate myocardial dysfunction in hypertrophy via improved Ca²⁺_i handling. Right ventricular function and the corresponding Ca²⁺_i transients were recorded on a beat-to-beat basis using the aequorin bioluminescence method. Pulmonary artery reactivity was evaluated using the organ bath setup.

	Experimental Procedures

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Animals and Experimental Design. Thirty female Sprague-Dawley rats (200-220 g) were randomized into three groups: group 1 was given a single intraperitoneal injection of MCT (50 mg/kg; MCT group, n = 12); group 2 was given the same dose of MCT, followed by daily administration over 9 weeks of the ET_A receptor antagonist PD (50 mg/kg per day, incorporated into the food; MCT + PD group, n = 9); and a third group received an intraperitoneal injection of physiological saline (same volume as MCT) and untreated chow for 9 weeks (control, n = 9). After 9 weeks, hemodynamic measurements were taken, the animals were sacrificed (urethane anesthesia), and their hearts were used in this study. PD treatment was started on the same day as MCT injection and continued until sacrifice. Previous studies have shown that PD inhibits ¹²⁵I-ET-1 binding to ET_A and ET_B receptors with IC₅₀ values of 7.8 nM and 3.5 µM, respectively, indicating ~500-fold selectivity for ET_A receptors, and that the antagonist is bioavailable after oral dosing (87%) (Doherty et al., 1995). We also reported previously that a dose of 50 mg/kg per day completely prevents ET-1-induced vasoconstriction of the hindlimb in rabbits (Rajagopalan et al., 1997). Protocols were approved by the Institutional Animal Care and Use Committee and conform to the Guiding Principles for the Use and Care of Laboratory Animals of the American Physiological Society and the National Institutes of Health.

Hemodynamic Measurements. Mean arterial blood pressure and heart rate were measured every 2 weeks in conscious animals, using the tail cuff method (TSE Instruments, Bad Homburg, Germany). Right ventricular pressure was measured under urethane anesthesia (1 g/kg) and ventilation with room air (60 strokes/min). The thorax was opened, and the right heart was punctured through the peritoneal space with a 23-gauge needle attached to a pressure transducer. After stabilization, systolic and diastolic pressure were recorded over 5 to 10 min. The method was highly reproducible, with a coefficient of variation of 3.1% in three consecutive measurements of systolic pressure.

Heart Perfusion. After recording right ventricular pressure, the heart was removed, arrested in ice-cold perfusion solution, and rapidly mounted on a Langendorff apparatus [Hugo Sachs Elektronik, March-Hugstetten, Germany (HSE)] as described previously (Wölkart et al., 2000). Briefly, coronary arteries were perfused at 10.0 ml/min per g of heart wet weight (fresh cardiac mass) with an oxygenated modified Krebs-Henseleit bicarbonate buffer (118 mM NaCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 4.8 mM KCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, and 11 mM glucose, pH 7.4) at 36.5-37.0°C. Isovolumic right ventricular pressure was recorded via a fluid-filled balloon made of household cellophane wrap and connected to a pressure transducer. Coronary sinus effluent was collected from the bottom of the organ chamber using a roller pump. Hearts were paced at 5 Hz. The digital signal of the right ventricular pressure tracing was further analyzed (PLUGSYS system; HSE) to obtain the following parameters: peak right ventricular systolic pressure, right ventricular end-diastolic pressure (RVEDP), right ventricular developed pressure (RVDevP), coronary perfusion pressure, time to peak systolic pressure, and time from peak systolic pressure to 90% of relaxation. The latter two parameters were obtained using a digital storage oscilloscope (DataSYS 740; Gould Instruments, Valley View, OH).

Aequorin Luminescence Measurement. Hearts were stabilized for 15 min at ambient temperature, and ~5 µl of an aequorin-containing solution (1 µg/ml) was macroinjected into the interstitium of the epicardium at the apex of the right ventricle as described previously (Kihara et al., 1989; Wölkart et al., 2000). Hearts were enclosed in a light-tight box (Blinks, 1986), and luminescence was collected onto a photomultiplier tube (Thorn EMI Electronic Tubes, Ruislip, UK) located beneath the box. The apparatus is designed to optimize collection of light emitted from the aequorin-loaded cells. Emitted light was quantified using an amplifier-discriminator (AD 6; Thorn EMI) that was connected via a voltage integrator to a storage oscilloscope. At the amplifier-discriminator, the signal was split and fed into a photon-counting module (TF830; Thurlby Thandar Instruments, Huntingdon, UK) that served to calibrate the oscilloscope and monitor dark current (light tightness of the box). Right ventricular light and pressure signals were recorded simultaneously on magnetic tape. After 25 min of perfusion at ambient temperature, the heart was immersed in perfusion solution, and perfusate temperature was gradually raised to 37°C while the aequorin light signal reached a steady state. At this point, the experimental protocol was started. Light (and pressure) signals were wave-averaged at the time of interest (generally 64 cycles), and the values for time to peak light and the 90% decline from peak light, respectively, were obtained from the averaged light signals.

Experimental Protocol. Initially, pressure-volume relations were obtained through systematically varying intraventricular balloon volume, as described previously (Wölkart et al., 2000). To determine the concentration-response relation to Ca²⁺, increasing concentrations of CaCl₂ (0.75-3 mM) were added to the perfusate, each over 10 min, and light signals and cardiac parameters were recorded as above. In these experiments, a right ventricular volume corresponding to 10 mm Hg RVEDP was chosen (Strömer et al., 1997).

Right Ventricular Wall Stress. Right ventricular wall thickness and the radius of the right ventricle were derived from the weight of the right ventricular wall and the balloon volume, respectively, as described for left ventricle (Strömer et al., 1997). Systolic and diastolic wall stress were then calculated using the relationships reported previously (Mirsky, 1979). There was no deformation of the right ventricle by the balloon at the chosen balloon volumes (35-40 µl).

Isolated Pulmonary Artery Study. Isolated vessel studies were done on the same day as heart perfusions. Hilar pulmonary arteries were dissected, with care taken not to damage the endothelial surface, and were cut into rings ~3 mm long. The rings were mounted on steel-wire hooks attached to a force displacement transducer, and changes in isometric force were recorded on a multichannel recorder (HSE). Rings were stretched to an initial tension of 2 g (maximum KCl-induced contraction of quiescent rings, as determined in separate experiments) and equilibrated for 2 h in individual organ baths containing modified Krebs-Henseleit bicarbonate buffer with the following composition: 118 mM NaCl; 4.8 mM KCl; 25 mM NaHCO₃; 1.2 mM KH₂PO₄; 1.2 mM MgCl₂; 2.5 mM CaCl₂; and 10.1 mM glucose, maintained at 37°C, and aerated with 95% O₂/5% CO₂ at a pH of 7.4. To assess the contractile activity of the vessel and functional integrity of the endothelium, respectively, increasing concentrations of KCl (10-80 mM) were added cumulatively to equilibrated tissues, followed by addition of acetylcholine (1-1000 nM). Rings were allowed to re-equilibrate in drug-free buffer until basal tension was reached again (~45 min) and contracted with 80 mM KCl, and endothelium-independent relaxation was tested with S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10 µM). Relaxation was expressed as reduction of tone induced by 80 mM KCl.

ET-1 and Big ET-1 Plasma Concentrations. Blood was collected into EDTA tubes spiked with aprotinine (final concentration, ~40 µg/ml), chilled, and centrifuged, and the plasma was stored at 20°C. ET-1 and big ET-1 were extracted from 1 ml and 0.1 ml of plasma, respectively, and determined by specific radioimmunoassay as described previously (Brunner, 1999).

Materials. PD 155080 (lot U) was synthesized at the Parke-Davis Pharmaceutical Research Laboratories (Ann Arbor, MI). MCT was obtained from Sigma (Vienna, Austria), and aequorin from Friday Harbor Laboratories (Friday Harbor, WA). All other chemicals were of the finest grade available.

Data Analysis. Group data are presented as arithmetic mean values ± S.E.M., unless otherwise specified. A one-way analysis of variance followed by the Scheffe test was performed for comparisons of cardiac functional parameters and pulmonary relaxation curves. Half-effective concentration (EC₅₀) and E_max values were obtained with the use of a five-parameter logistic function with Kaleidagraph curve-fitting software (Synergy Software, Reading, PA) on an Apple Macintosh Power PC (Cupertino, CA). Differences were considered significant at P < 0.05.

	Results

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Plasma Levels of PD after Chronic Dosing. Figure 1 shows the plasma levels of PD during 9 weeks of oral treatment with 50 mg/kg per day (n = 5 rats). Plasma concentrations were determined with a sensitive high-pressure liquid chromatography method (Doherty et al., 1995) and were 0.60 ± 0.42 µg/ml at 20 days, 1.20 ± 0.63 µg/ml at 40 days, and 1.78 ± 0.72 µg/ml at 60 days. Because rats feed mostly at night, the drug is quickly absorbed (time to maximal level after a single oral dose, ~50 min) and plasmas were drawn during the day, these levels likely reflect the middle or lower range of the steady-state concentrations.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Plasma concentrations of PD 155080 during chronic oral dosing with 50 mg/kg per day. Data are means ± S.E.M. from five animals.

Effect of PD on Survival and Right Ventricular Hypertrophy. In the MCT group, 5 of 12 animals died whereas in the MCT + PD and control groups, all animals survived. Body weight, arterial blood pressure, and heart rate were not different between experimental groups prior to, and 9 weeks after, the start of PD application (Table 1). However, right atrial and ventricular weight, right atrial weight/body weight ratio, and right ventricular weight/body weight ratio were significantly higher in MCT-injected rats, indicating substantial right ventricular hypertrophy (P < 0.05). PD substantially slowed the alkaloid-induced growth process (P < 0.05 versus MCT) so that the right heart weight was almost, albeit not completely, reduced to control level (ventricle, 1.1-fold; atrium, 1.2-fold of control; P < 0.05 versus control in both cases). The high efficacy of the drug is in line with, and corresponds to, the consistent plasma levels throughout the 9 weeks of treatment (Fig. 1). Left ventricular weight and lung weight were similar in all groups, indicating that compensatory left ventricular hypertrophy was not present in the MCT group.

Effect of PD on Right Ventricular Pressure and Plasma Endothelins. Right ventricular overload was verified by measuring right ventricular pressures in vivo (Fig. 2). Peak systolic right ventricular pressure was significantly higher in MCT-treated than control rats (52 ± 2 versus 25 ± 2 mm Hg, P < 0.05). This was also the case for diastolic pressure (13 ± 1 versus 4 ± 1 mm Hg, P < 0.05). Because the right ventricular systolic pressure and right ventricular diastolic pressure of rats treated with MCT + PD were significantly reduced and no different from the control values (29 ± 2 and 4 ± 1 mm Hg, respectively), these data indicate that at the dose of 50 mg/kg, PD completely normalized right ventricular pressures elevated by MCT. The concentrations of ET-1 and big ET-1 in plasma were significantly elevated in MCT-treated rats (Fig. 3). ET-1 rose from 9.7 ± 0.6 to 14.9 ± 1.3 pg/ml (1.5-fold) and big ET-1 from 431 ± 21 to 910 ± 70 pg/ml (2.1-fold; P < 0.05 in each case). Chronic treatment with PD had no effect on elevated ET-1 and big ET-1 peptide levels (1.9- and 2.1-fold over control, respectively; P > 0.05 versus MCT group).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Right ventricular systolic (A) and right ventricular diastolic (B) pressure in the control group (n = 9) and in rats treated with MCT (n = 7) or with MCT + PD (n = 9). Values in the MCT group differed significantly (*, P < 0.05) from control rats; values in the MCT + PD group were not different from control. Data are expressed as means ± S.E.M.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Plasma concentration of ET-1 (A) and big ET-1 (B) in the control group and in rats treated with MCT or with MCT + PD. Values in the MCT group and MCT + PD group differed significantly (*, P < 0.05) from control rats. Data are expressed as means ± S.E.M.; n = 5.

Effect of PD on Right Ventricular Function. To assess the effects of PD on right ventricular function and intracellular Ca²⁺ handling, we increased the concentration of Ca²⁺ in the perfusate stepwise from 0.75 to 3 mM and recorded ventricular function and aequorin light transients. Hypertrophic hearts generated greater right ventricular peak systolic pressure and a higher RVDevP up to 2.5 mM extracellular Ca²⁺ (Fig. 4; P < 0.05), whereas RVEDP was not different between groups (12.2 ± 0.9 to 15.9 ± 1.2 mm Hg in controls and 10.9 ± 0.8 to 16.0 ± 1.4 mm Hg in hypertrophic hearts; P > 0.05 MCT versus control group at all Ca²⁺ concentrations). In hearts from the MCT + PD group, right ventricular peak systolic pressure tended to be lower and RVEDP to be higher than in the MCT group, resulting in a significantly lower left ventricular developed pressure curve (P < 0.05 versus MCT group). We also calculated wall stress, an estimated value of force per unit of myocardium (Fig. 5). In hearts from the MCT group, right ventricular peak systolic and right ventricular end-diastolic wall stress were ~3 to 5 kilodyne/cm² lower, not higher, than in control hearts, indicating effective compensation (P > 0.05 for systolic wall stress) or a tendency to overcompensation (P > 0.05 for end-diastolic wall stress) of the reduced compliance resulting from right ventricular hypertrophy. After treatment with PD, wall stress was indistinguishable from that of control hearts throughout the range of contractile states induced by different perfusate Ca²⁺ levels. Thus, ET_A receptor blockade with PD prevented the negative effects of hypertrophy on systolic and diastolic performance so that right ventricular pump function was no longer different from that in control hearts (Figs. 4 and 5).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of increasing extracellular Ca2+ concentrations (abscissa) on peak systolic pressure (A), end-diastolic pressure (B), and right ventricular developed pressure (C) in control rats and in rats treated with MCT or with MCT + PD. *, P < 0.05 control versus MCT group; values in the MCT + PD group were not different from control. Data are expressed as means ± S.E.M.; n = 9 (control and MCT + PD) and n = 7 (MCT), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of increasing extracellular Ca2+ concentrations (abscissa) on right ventricular peak systolic wall stress (A) and right ventricular end-diastolic wall stress (B) in control rats and in rats treated with MCT or with MCT + PD. Although values in the MCT group were not significantly different from control, PD tended to restore wall stress to control values. Data are expressed as means ± S.E.M.; n = 9 (control and MCT + PD) and n = 7 (MCT), respectively.

Effect of PD on Ca²⁺_i Transients. The aequorin light signals corresponding to the different extracellular Ca²⁺ concentrations had a similar shape in control and hypertrophied hearts, i.e., they consisted of a single component that rose to the peak and declined to the baseline before the corresponding pressure response. A quantitative analysis of the isovolumic pressure and aequorin light signals is presented in Fig. 6. The time to peak light was similar in controls (18 ± 2 to 25 ± 3 ms) and MCT hearts (21 ± 2 to 27 ± 4 ms) as was time to peak pressure (58 ± 3 to 69 ± 3 ms and 53 ± 2 to 63 ± 2 ms, respectively; P > 0.05; Fig. 6, A and B). However, the time that elapsed for the decline of the pressure and aequorin light signals was significantly longer in hypertrophic than control hearts, i.e., 90% decline from peak light was 35 ± 3 to 50 ± 3 ms in control and 61 ± 3 to 73 ± 4 ms in hypertrophic hearts (P < 0.05; Fig. 6C). Likewise, 90% decline from peak pressure was 76 ± 2 to 89 ± 4 ms in control and 88 ± 3 to 104 ± 2 ms in hypertrophic hearts (P < 0.05; Fig. 6D). In hearts from animals that had been treated with PD, the slowed relaxation from peak pressure and peak light was completely abolished at all perfusate Ca²⁺ concentrations (Fig. 6, C and D). PD had no effect on the contractile phase of the heart cycle (Fig. 6, A and B).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effect of increasing extracellular Ca2+ concentrations (abscissa) on time to peak light (TPL; A), time to peak pressure (TPP; B), time to 90% decline of peak light (T90L; C) and time to 90% decline of peak pressure (T90P; D) in control hearts and in hearts from rats treated with MCT or with MCT + PD. *, P < 0.05 control versus MCT group; values in the MCT + PD group were not different from control. Data are expressed as means ± S.E.M.; n = 6 (control) and 7 (MCT and MCT + PD), respectively.

Effect of PD on Pulmonary Endothelium-Dependent Vasodilation. Acetylcholine (10⁸ to 10⁶ M) induced concentration-dependent vasodilations in isolated pulmonary artery preconstricted with excess KCl (Fig. 7). The curve-fitted E_max value for control relaxations was 0.57 ± 0.12 g (22% of KCl-induced tone), and the half-maximal response (EC₅₀) value was 6.9 (5.6-8.5). 10⁸ M (geometric mean with 95% confidence interval). Vessels from MCT-treated rats were unresponsive to acetylcholine up to 10⁶ M. However, in vessels from the MCT + PD group, the E_max value was 0.25 ± 0.02 mg and the EC₅₀ value was 5.0 (2.1-10.2) · 10⁸ M (P < 0.05 versus control). Thus, PD treatment significantly restored endothelium-mediated pulmonary artery dilation. The response to SNAP (10 µM) did not differ among the three groups (Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Relaxation induced by acetylcholine (108 to 106 M) or SNAP (10 µM) in rings of pulmonary artery from control rats (8 rings from 4 animals), rats treated with monocrotaline (12 rings from 5 animals), or rats treated with MCT + PD (14 rings from 6 animals). PD treatment partially restored endothelial function (*, P < 0.05 versus MCT group; analysis of variance). The effect of SNAP was not different between groups. Tissues were preconstricted with 80 mM KCl, which gave a mean tension of 2.6, 3.1, and 2.4 g, respectively, in the three groups. Data are means ± S.E.M. of the decrease in preconstricted tone.

	Discussion

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In this study, we investigated the role of endogenous ET-1 on the development of changes in cardiac function and intracellular Ca²⁺ handling associated with right ventricular myocardial hypertrophy. This was studied by oral administration of an antagonist specific for the ET_A receptor during the development of right ventricular pressure overload resulting from MCT-induced pulmonary hypertension. The data clearly indicate substantial antihypertrophic and pulmonary artery protective effects of long-term ET_A receptor antagonism in association with improved intracellular Ca²⁺ modulation, implicating a deleterious role for endogenous ET-1 in the cardiopulmonary changes at the level of intracellular Ca²⁺ homeostasis.

Long-Term ET_A Blockade and Cardioprotection. In the present study, adult animals were injected with MCT, which resulted in a lower mortality (42%) than when young rats were used (60%) (Brunner, 1999). As expected, MCT alone produced right ventricular hypertrophy associated with elevated pulmonary artery pressures. The isolated hypertrophic hearts generated right ventricular systolic and diastolic pressures and peak systolic and end-diastolic wall stress that were similar to those in control hearts, indicating that the hypertrophied hearts were in a compensatory (adaptive) stage of right ventricular hypertrophy, with preserved contractile reserve (Brown et al., 1998). Administration of the receptor antagonist PD increased survival to 100% and completely blocked the pulmonary arterial pressure response (in vivo and in vitro), whereas the hypertrophic response was reduced close to control level (for ventricle, 10% above control). This result is in marked contrast to most previous long-term studies with ET_A receptor antagonists, which only partly (~30-50%) prevented the rise in pulmonary artery pressure and right ventricular hypertrophy (Miyauchi et al., 1993; Ichikawa et al., 1996; Prié et al., 1997). However, this might be related to daily dose and duration of treatment. Similarly, we recently showed that sodium-2-benzo[1,3]dioxol-5-yl-4-(4-methoxyphenyl)-4-oxo-3-(3,4,5-trimethoxy-benzyl)-but-2-enoate, formerly known as PD 156707, another butenolide ET_A antagonist with structural similarity to PD (Doherty et al., 1995), can reverse existing pulmonary hypertension and prevent progression of right ventricular hypertrophy in rats exposed to chronic hypoxia (Haleen et al., 1998). Taken together, these data obtained with the MCT model and other models of right ventricular hypertrophy and employing different ET receptor antagonists selective for the ET_A subtype, provide evidence that ET-1 is involved in the pathophysiology of the right ventricular hypertrophic response. We reported recently that exogenous ET-1 had no effect on the decline of the pressure and light parameters in MCT hypertrophic hearts (Wölkart et al., 2000), casting doubt on the functional significance of the ET system in this model. A possible explanation for the discrepancy could be that an acutely added agonist will not substantially affect a functional parameter (in this case, diastolic relaxation) if that function is already maximally stimulated, whereas in the present study ET receptors were blocked chronically, which prevented the alterations from the outset.

ET_A Blockade and Normalization of Ca²⁺_i Handling. To determine whether the ET_A receptor antagonist could alter the impaired Ca²⁺_i handling known to be associated with the myocardial response to pressure overload, we evaluated averaged Ca²⁺_i transients using the Ca²⁺-sensitive bioluminescence indicator, aequorin. Previous aequorin studies have documented prolongation of twitch duration and the corresponding Ca²⁺ transient in trabecular and papillary muscles isolated from several animal models of cardiac hypertrophy (Gwathmey and Morgan, 1985; Gwathmey et al., 1995) and in cells from failing human hearts (Meuse et al., 1992). In the present study, we have avoided the limitations imposed by isolated muscle techniques by simultaneous recording on a beat-to-beat basis of both right ventricular pressure and Ca²⁺_i transients derived from the apex of the right ventricle. Hypertrophic hearts exhibited abnormalities in the time course of Ca²⁺ handling and ventricular relaxation, both of which were slightly, but significantly, prolonged. However, therapy with PD completely normalized diastolic relaxation and Ca²⁺ transport kinetics, strongly suggesting that the cardioprotection of this ET_A antagonist is related to improved diastolic function. A prolongation of the light signal was previously observed in hypertrophic ventricular preparations from several species, including humans, and has been attributed to a reduced reuptake of Ca²⁺ by the sarcoplasmic reticulum during diastole (Balke and Shorofsky, 1998), possibly due to lower transcription of mRNA for sarcoplasmic reticulum Ca²⁺-ATPase 2 (SERCA-2) (LekanneDeprez et al., 1998). Changes in phospholamban expression or function could also contribute to altered Ca²⁺ transport. This protein is often up-regulated in left ventricular hypertrophy or failure, but in pulmonary pressure-induced right ventricular hypertrophy of rats (LekanneDeprez et al., 1998) and ET-1-induced hypertrophy of isolated myocytes (Van Heugten et al., 1998), the expression of this protein was substantially down-regulated. Further experiments will show whether ET_A antagonists prevent alterations in sarcoplasmic reticulum function observed in myocardial hypertrophy or failure.

ET_A Blockade and Pulmonary Artery Function. We found that preconstricted isolated pulmonary artery rings from MCT-treated rats were unresponsive to acetylcholine, an endothelium-dependent agonist, and that relaxation was restored to ~60% of controls in rings from rats chronically treated with PD. In previous investigations, it was shown that MCT injection in rats results in progressive endothelial injury, impairs nitric oxide (NO)-mediated vasodilation, and produces pulmonary hypertension (Mathew et al., 1995; Frash et al., 1999). As was recently reported for this model, some or all of these effects are probably related to a reduced release of NO, due to a reduced sensitivity of endothelial NO synthase to Ca²⁺, or a reduced intracellular availability of the cation (Nakazawa et al., 1999). The mechanism by which therapy with ET_A antagonists could improve endothelium-dependent dilation is not known and requires further experiments. These drugs could also improve Ca²⁺ handling of endothelial cells, as shown in this report for the myocardium. Such a mechanism is in line with the findings of Nakazawa et al. (1999) mentioned above and might also partly explain the antihypertensive response of PD in the angiotensin II model of hypertension (Rajagopalan et al., 1997).