Introduction

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It has been demonstrated that angiotensin-converting enzyme (ACE) inhibition improved indices of left ventricular (LV) function and survival in patients with congestive heart failure (CHF) (The CONSENSUS Trial Study Group, 1987; The SOLVD Investigators, 1991). More recently, angiotensin II (Ang II) type 1 (AT₁) receptor antagonists have been shown to be well tolerated in patients with CHF and may provide beneficial effects (Pitt et al., 1997). Thus, interruption of AT₁ receptor activity is an important therapeutic target for the treatment of CHF. However, although ACE inhibition, presumably through a reduction in Ang II production and AT₁ receptor activity, improved survival in patients with CHF, the rate of morbidity and mortality associated with this disease process remain significant. Thus, the development of therapeutic strategies that can operate in conjunction with interruption of AT₁ receptor activity in the setting of CHF are warranted. The progression of the CHF process is invariably associated with heightened synthesis and the release of a number of vasoactive peptides (Szatalowicz et al., 1981; Riegger and Liebau, 1982; Goldsmith et al., 1983; Benedict et al., 1993; Naitoh et al., 1994; Wei et al., 1994). For example, past clinical and experimental studies have documented that increased plasma levels of the nonapeptide vasopressin accompany the progression and/or exacerbation of CHF (Szatalowicz et al., 1981; Riegger and Liebau, 1982; Goldsmith et al., 1983; Naitoh et al., 1994). Vasopressin has been implicated as exerting effects through two distinct receptor-mediated pathways (Manning et al., 1993; Bichet, 1994; Burrell et al., 1994). The first vasopressin receptor pathway is the vasopressin V_1a receptor subtype, which is located on a number of cell types, including vascular smooth muscle. Activation of the V_1a receptor has been shown to cause peripheral vasoconstriction (Riegger and Leibau, 1982; Manning et al., 1993; Bichet, 1994, Burrell et al., 1994). The second vasopressin receptor pathway, the V₂ receptor, is located primarily in the distal tubule of the kidney and, when activated, results in water and sodium reabsorption (Manning et al., 1993; Bichet, 1994; Burrell et al., 1994). In experimental rodent models of CHF, it has been demonstrated that V₂ receptor inhibition produced beneficial hemodynamic response primarily as a result of an aquaretic effect (Nishikimi et al., 1996; Burrell et al., 1998). However, it remains unclear whether and to what degree activation of the V_1a receptor contributes to the progression and/or exacerbation of the CHF process. Accordingly, the overall goal of the present study was 3-fold: 1) to determine the direct effects of V_1a receptor blockade during the progression of CHF with respect to LV function and systemic hemodynamics, neurohormonal system activity, and contractility; 2) to compare and contrast the relative effects of V_1a receptor blockade with respect to AT₁ receptor blockade with the development of CHF; and 3) to determine the potential interaction of combined V_1a and AT₁ receptor blockade during the progression of CHF.

Chronic rapid pacing in animals has been previously demonstrated to produce changes in LV function and systemic hemodynamics, neurohormonal system activity, and contractility similar to those of the clinical spectrum of CHF (Spinale et al., 1992, 1995; Spinale, 1995; Travill et al., 1992). Specifically, pacing-induced CHF is accompanied by LV pump dysfunction and activation of several neurohormonal systems, including the renin-angiotensin pathway and vasopressin (Riegger and Liebau, 1982; Travill et al., 1992; Spinale et al., 1995, 1997a; Spinale, 1997b; Krombach et al., 1998). Moreover, the institution of ACE inhibition in this model of CHF produces beneficial effects on LV function and neurohormonal systems similar to those achieved in past clinical studies (Spinale et al., 1995, 1997a; Spinale, 1997b; Krombach et al., 1998). Thus, the pacing model may be a useful substrate to examine the effects of specific pharmacologic interruption of receptor pathways in the setting of developing CHF. Accordingly, the present study used a porcine model of pacing CHF that has been previously described, (Spinale et al., 1992, 1995, 1997a; Spinale, 1997b) to examine the effects of V_1a receptor blockade, AT₁ receptor blockade, and combined receptor blockade.

	Materials and Methods

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Rationale. The overall goal of the present study was to examine the effects of V_1a receptor blockade, AT₁ receptor blockade, and combined receptor blockade (V_1a/AT₁ receptor blockade) on LV function and myocyte contractility in a pig model of CHF. The first objective was to obtain a dosage for each receptor antagonist as well as for combined receptor blockade that would significantly blunt the appropriate receptor agonist challenge. In addition to identification of single blockade and dual blockade treatment, the second objective of the present study was to determine the effects of chronic single receptor blockade and combined receptor blockade on LV function, hemodynamics, and myocyte contractility in developing pacing CHF.

Dose Selection Studies. Nine Yorkshire pigs (25 kg, male) were chronically instrumented to measure aortic blood pressure in the conscious state as previously described (Clair et al., 1998; Krombach et al., 1998). After a recovery period of 7 to 10 days, the animal was returned to the laboratory for initial baseline hemodynamic assessment and pressor response studies. For these studies, the animals were sedated with diazepam (20 mg Valium p.o.; Hoffmann-La Roche, Nutley, NJ) and placed in a custom-designed sling that allowed the animal to rest comfortably. The vascular access port was entered using a 12-gauge Huber needle (Access Technologies, Skokie, IL) containing dual access sites, and basal, resting arterial pressure and heart rate were recorded. Pressures from the fluid-filled aortic catheter were obtained using an externally calibrated transducer (Statham P23ID; Gould, Oxnard, CA). The pressure waveforms were recorded using a multichannel recorder (Hewlett Packard, Houston, TX) as well as digitized on computer for subsequent analysis at a sampling frequency of 250 Hz (80386 processor; Zenith Data Systems, St. Joseph, MO). After these baseline measurements, a bolus infusion of Ang II (10 µg; Sigma Chemical Co., St. Louis, MO) was administered, and measurements were repeated 5 min after the Ang II infusion. This dose of Ang II was determined previously to yield a near-maximal blood pressure effect (Spinale et al., 1997a). After a 30-min recovery period, in which hemodynamics had returned to baseline levels, a bolus infusion of vasopressin (40 ng/kg; Fluka, Milwaukee, WI) was administered, and measurements were recorded as described previously. This dose of vasopressin was determined previously to yield a near-maximal blood pressure effect (Serradeil-Le Gal et al., 1993). Furthermore, previous dose-ranging experiments (0-80 ng/kg) in the conscious pig preparation showed that 40 ng/kg produced a near-maximal blood pressure response. The order of Ang II and vasopressin administration was alternated with each study.

Model of CHF and Experimental Design. Thirty-two age- and weight-matched pigs (Yorkshire, 25 kg) were anesthetized, and a left thoracotomy was performed as described earlier. In addition, a shielded stimulating electrode was sutured onto the left atrium, connected to a modified programmable pacemaker (8329; Medtronic, Inc., Minneapolis, MN), and buried in a subcutaneous pocket.

LV Function and Plasma Collection. Indices of LV pump function were obtained from simultaneously recorded pressure and echocardiographic measurements previously described at our laboratory (Tomita et al., 1991; Spinale et al., 1992, 1995). LV peak circumferential wall stress was computed using a spherical model of reference:  (grams per square centimeter) = [PD/4 h(1 + h/D)] × 1.36, where P is aortic systolic pressure, D is minor axis dimension at end-diastole, and h is wall thickness (Tomita et al., 1991). LV end-systolic wall stress was computed with the same formula but with the appropriate systolic dimensions. After these measurements, 40 ml of blood was drawn from the arterial access port into chilled tubes containing EDTA (1.5 mg/ml). A separate sample was collected into chilled tubes containing appropriate serine protease inhibitors for additional neurohormonal studies. The blood samples were immediately centrifuged (2000g, 10 min, 4°C), and the plasma was decanted into separate tubes, frozen in a dry ice/methanol bath, and stored at 80°C until the time of assay. An additional 5-ml blood sample was taken for subsequent serum osmolality and electrolyte analysis. To more carefully examine LV ejection performance, a normal control state LV fractional shortening afterload relationship was determined as described previously (Colan et al., 1984; Tomita et al., 1991). This approach provides a relatively load-independent index of ejection performance and does not require theoretical muscle models and the development of LV pressure-volume loops (Ross, 1976). Using this normal relationship, steady-state LV fractional shortening-peak wall stress values were plotted at the end of each of the treatment protocols.

Neurohormones and Analytes. Plasma samples were assayed for norepinephrine, endothelin, vasopressin, Ang II, and atrial natriuretic peptide (ANP) levels. Plasma samples were also assayed for drug levels of SR49059 and the AT₁ receptor antagonist. Plasma norepinephrine was measured using HPLC and normalized to picograms per milliliter of plasma; this assay had a less than 4% coefficient of variation (Anilytics, Bethesda, MD). For the endothelin determinations, the plasma was first eluted over a cation exchange column (C18 Sep-Pak; Waters Associates, Milford, MA) and then dried by vacuum-centrifugation. The samples were reconstituted in 0.02 M borate buffer, and a high-sensitivity radioimmunoassay was performed (RPA545; Amersham, Arlington Heights, IL). The recovery from the extraction procedure was 75 ± 5% based on spiked plasma standards (4-20 fmol/ml). The interassay variation was 10% and the intra-assay variation was 9% for the endothelin radioimmunoassay procedure. For the Ang II determinations, the plasma was eluted with methanol, and a high-sensitivity radioimmunoassay was performed (NR 79980, Angiotensin II Pasteur; Sanofi Diagnostics Pasteur, Montpellier, France). For lysine-vasopressin determinations, plasma was eluted with ethanol, and a high-sensitivity radioimmunoassay for lysine-vasopressin was performed (NR 23065; DIASORIN, Stillwater, MN). ANP levels were determined from eluted plasma by radioimmunoassay. Serum Na⁺ was measured by ion selective potentiometry. Plasma levels of SR49059 and the AT₁ receptor antagonist were determined by a mass spectrophotometry method (ESI liquid chromatography-mass spectrometry/mass spectrometry) and HPLC, respectively. Serum blood urea nitrogen (BUN) and creatinine were determined by measurements of a chromogenic substrate (Vitros; Johnson & Johnson, Rochester, NY). Serum osmolality was measured by freezing-point osmometry.

Myocyte Isolation and Contractile Function Studies. After the final set of LV function measurements and plasma collection, the animals were anesthetized as described earlier, a sternotomy was performed, and the heart was quickly extirpated and placed in a phosphate-buffered ice slush. The region of the LV free wall incorporating the circumflex artery (5 × 5 cm) was excised and prepared for myocyte isolation. The region of the LV free wall composing the left anterior descending coronary artery (3 × 5 cm) was cannulated and prepared for perfusion fixation.

Data Analysis. Indices of LV function, systemic hemodynamics, and neurohormonal profiles were compared among the treatment groups by ANOVA for repeated measures. An ANOVA with a randomized-block split-plot design was used for the myocyte function studies. For these myocyte studies, each pig was a block and drug treatment was the parameter for the split-plot design. If the ANOVA revealed significant differences, pairwise tests of individual group means were compared by the use of Bonferroni's probabilities. All statistical procedures were performed with the BMDP statistical software package (BMDP Statistical Software Inc., Los Angeles, CA). Results are presented as mean ± S.E. Values of P < .05 were considered to be statistically significant.

	Results

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All of the animals entered into the individual protocols successfully completed the study.

Pressor Response Studies. The results of the pressor responses to Ang II and vasopressin in normal pigs is summarized in Fig. 1. The 60-mg/kg dose of the V_1a receptor antagonist resulted in a slight blunting of the response to Ang II compared with control levels and a greater than 50% reduction in the response to vasopressin compared with control values. The 30 mg/kg dose of the AT₁ receptor antagonist resulted in a significant blunting of the Ang II pressor response compared with control levels. However, there was a significant potentiation of the vasopressin pressor response after AT₁ receptor blockade compared with control measurements. The dual blockade treatment yielded a significant blunting of both the Ang II and the vasopressin response compared with control values.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Top, the change in mean aortic pressure after the i.v. administration of Ang II (10 µg) in the control state, after V1a receptor blockade (SR49059 60/mg/kg b.i.d. 3 days), AT1 receptor blockade (irbesartan 30 mg/kg b.i.d. 3 days), and dual treatment (irbesartan 30 mg/kg b.i.d. 3 days and SR49059 60/mg/kg b.i.d. 3 days). The response to Ang II was significantly blunted after V1a receptor blockade, AT1 receptor blockade, and dual treatment (*P < .05 versus control, n = 3 for each drug treatment). Bottom, the change in mean aortic pressure after i.v. administration of arginine vasopressin (AVP 40 ng/kg) in controls, after V1a receptor blockade, AT1 receptor blockade, and dual treatment. The response to AVP was potentiated with AT1 receptor blockade. However, the response to AVP was significantly blunted after V1a receptor blockade and dual treatment (*P < .05 versus control, n = 3 for each drug treatment).

Plasma Compound Levels. The plasma levels (drawn at the midpoint between the morning and evening doses) were determined for each receptor antagonist on the final day of the chronic CHF study. For the V_1a receptor antagonist, SR49059, plasma levels were 8.93 ± 2.69 mg/l in the monotherapy group. The plasma levels for the V_1a receptor antagonist were similar to monotherapy values in the dual blockade group (10.65 ± 0.75 mg/l, P = .56). Based on past in vitro studies, the plasma concentration of SR49059 reflects an approximate 2000-fold higher concentration than necessary to inhibit V_1a-mediated vessel contractility (Serradeil-Le Gal et al., 1993).

LV Function and Hemodynamics. Complete LV function and hemodynamic measurements at week 3 of rapid pacing are summarized in Table 1. Ambient resting heart rate was increased in all of the rapid pacing groups compared with baseline values. In the AT₁ blockade group and the dual blockade group, ambient resting heart rate was lower than that of untreated rapid pacing values. In all rapid pacing groups, mean aortic pressure was lower than baseline values and was further reduced in the dual blockade group. Moreover, in the AT₁ blockade group, mean aortic pressure was significantly reduced from baseline, pacing-only, and all other receptor blockade groups. LV wall stress patterns increased approximately 3-fold in the rapid pacing-only group and was significantly reduced in all receptor blockade groups. LV wall stress was reduced to the greatest degree in the AT₁ blockade group and the dual blockade group compared with untreated pacing values.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Isochronal LV peak wall stress versus fractional shortening points obtained at baseline in which an incremental phenylephrine infusion was performed (shaded triangle; 6 mg, n = 27). A linear relationship was observed (y = 0.05x + 46.98) and a 99% confidence interval was constructed as indicated by the dashed lines. Steady-state basal values for LV peak wall stress versus fractional shortening were then plotted for each treatment group with respect to the normal stress-shortening relationship. In the pacing-only group (), a significant downward shift to the right was observed indicative of intrinsic defects in LV myocardial performance (Ross, 1976; Colan et al., 1984; Tomita, 1991). Treatment with either V1a receptor blockade () or AT1 receptor blockade () during the pacing period resulted in an upward shift of the stress-shortening relationship. In the group undergoing dual receptor blockade (), a further shift toward normal values for this relationship was observed. However, in all pacing groups, regardless of treatment, the steady-state values fell below the 99% confidence interval constructed for the normal stress-shortening relationship.

Neurohormones and Analytes. Plasma neurohormone values are summarized in Table 1. An approximately 5-fold increase in plasma norepinephrine was observed in the untreated pacing group compared with baseline values. Plasma norepinephrine was significantly reduced in all receptor blockade groups compared with pacing-only values. In the AT₁ blockade and dual blockade groups, plasma norepinephrine was further reduced from rapid pacing-only values. Plasma endothelin levels are also summarized in Table 1. As with plasma norepinephrine, a 5-fold increase in plasma endothelin was observed in the untreated pacing group compared with baseline values. Plasma endothelin values were reduced from the untreated pacing group in the AT₁ blockade and the dual blockade groups. Plasma Ang II levels were increased from baseline values in all pacing groups but were lower in the V_1a and dual receptor blockade groups. Plasma lysine-vasopressin was increased in the pacing-only and in the dual blockade groups. Plasma ANP was increased from baseline values in all pacing groups but was reduced from pacing-only values in both the AT₁ and dual receptor blockade groups.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Changes in serum electrolytes and osmolality from baseline values in the pacing-only group, rapid pacing with concomitant V1a receptor blockade (V1a blockade), rapid pacing with AT1 receptor blockade (AT1 blockade), and rapid pacing with concomitant dual blockade (V1a/AT1 receptor blockade; dual blockade). Serum sodium values decreased from baseline in all pacing groups; however, serum sodium levels were lower in the single blockade groups. Serum sodium levels were also reduced in the dual blockade group, but this did not reach statistical significance (P = .11). Serum osmolality decreased in the dual blockade group compared with baseline values. Serum BUN was increased from baseline values in all pacing groups; however, in the V1a blockade group, this did not reach statistical significance (P = .11). BUN levels were significantly reduced in all three treatment groups from pacing-only values. Serum creatinine levels were significantly increased from baseline values in all pacing groups regardless of treatment. The different drug treatments did not significantly affect serum creatinine levels from pacing-only values (*P < .05 versus baseline, #P < .05 versus pacing only).

Myocyte Contractility. Myocyte contractile function was examined in more than 1000 LV myocytes from all treatment groups, and this analysis is summarized in Table 2. Indices of myocyte contractile function were reduced by approximately 50% in all rapid pacing groups compared with normal control values. There was no significant improvement in basal contractile performance in the V_1a blockade or the AT₁ blockade groups. However, a significant improvement in myocyte contractile function was observed in the dual blockade group compared with rapid pacing-only values.

	Discussion

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The goal of the present study was to examine the effects of V_1a receptor blockade, AT₁ receptor blockade, and dual receptor blockade in a pacing model of CHF. Using a single or a combined dose of the V_1a and AT₁ receptor antagonists that inhibited the pharmacologic response to the appropriate agonist (vasopressin, Ang II, or both) during the development of pacing CHF, two important observations were made. First, V_1a receptor blockade, AT₁ receptor blockade, and dual receptor blockade reduced LV wall stress and plasma norepinephrine from untreated CHF values. However, only dual receptor blockade resulted in a positive effect on LV pump function. Second, at the level of the myocyte, basal contractile function was increased from untreated CHF values in the dual receptor blockade group only. Thus, the present study demonstrated a potential interaction with V_1a and AT₁ receptor blockade in a model of CHF.

Pressor Response Studies. In the present study, the doses selected for the V_1a and the AT₁ receptor antagonists provided adequate blockade of the appropriate receptor in response to agonist infusion. However, there was a significant potentiation of the vasopressin pressor response after AT₁ receptor blockade compared with control measurements. It has been shown previously in a conscious rat preparation that Ang II binding of the AT₁ receptor influences the release of vasopressin (Yamaguchi et al., 1982). Therefore, although remaining speculative, inhibition of the AT₁ receptor may have reduced local vasopressin release and increased the number of unoccupied V_1a receptors. This may have resulted in heightened activity of the V_1a receptor system after exogenous vasopressin administration and yielded a potentiated vasopressin response in the AT₁ receptor blockade group. It must be recognized that these vasopressin studies were performed in only the normal pig preparation. Interpretation of results from pressor studies performed in the setting of CHF can be problematic due to endogenous neurohormonal system activation.

LV Function and Hemodynamics. In the present study, V_1a receptor blockade did not significantly affect resting heart rate and aortic blood pressure from untreated CHF values. However, a small, but significant, reduction in the degree of LV dilation and wall stress occurred with V_1a receptor blockade, suggesting some favorable effects on LV loading conditions. Nevertheless, this was not translated into an improvement in LV pump function as determined by LV fractional shortening. In the AT₁ receptor blockade group, a significant reduction occurred in mean aortic blood pressure and LV wall stress patterns. However, AT₁ receptor blockade was not accompanied by significantly improved LV pump function. Dual receptor blockade reduced LV loading conditions (as defined by LV wall stress), and this effect was translated into an overall improvement in global LV pump performance. Interestingly, mean aortic blood pressure was higher in the dual blockade group compared with the AT₁ receptor blockade group. The increase in blood pressure in the dual blockade group was likely due to heightened plasma norepinephrine and plasma endothelin levels observed in this group compared with the AT₁ group. The reduction in resting aortic blood pressure with AT₁ receptor blockade was not apparently associated with significant hemodynamic compromise as evidenced by a reduction in resting heart rate compared with untreated pacing values. Nevertheless, the significant reduction in resting blood pressure that occurred in the AT₁ receptor blockade group makes comparisons of load-dependent indices of LV pump function difficult with respect to other treatment interventions.

Neurohormonal Systems. In the present study, V_1a receptor blockade reduced plasma norepinephrine levels from that of untreated pacing CHF values. This was likely due to a secondary effect of improved systemic hemodynamics achieved with V_1a receptor blockade. In the AT₁ receptor blockade group, the significant reduction in plasma norepinephrine levels was probably due to a favorable effect on systemic hemodynamics, as well as direct Ang II-mediated inhibition of sympathetic activation (Zimmerman et al., 1972; Brasch et al., 1993). Another neurohormonal system that is activated with the progression of CHF is the endothelin system (Wei et al., 1994; Clair et al., 1998; Krombach et al., 1998). Consistent with the clinical phenotype of CHF, pacing CHF resulted in increased plasma endothelin. In both the AT₁ and dual receptor blockade groups, plasma endothelin levels were reduced from untreated pacing values. The reduction in endothelin levels in both the AT₁ and dual receptor blockade groups was a probable contributory factor for the improvements in myocyte contractility because endothelin has been shown previously to influence contractile performance (Thomas et al., 1997). Ang II levels were reduced with V_1a receptor blockade compared with CHF values. This reduction in Ang II levels was likely due to favorable hemodynamic effects observed in the V_1a and dual receptor blockade groups. However, in the AT₁ receptor blockade group, Ang II levels were not different from pacing-only values, which was probably due to a loss of receptor feedback inhibition. In the present study, lysine-vasopressin levels were examined because this is the predominant form of vasopressin in pigs (Stebbins et al., 1994). In the AT₁ receptor blockade group, plasma vasopressin levels were lower than pacing-only values, which was likely due to favorable hemodynamic effects. In the V_1a and dual receptor blockade groups, plasma vasopressin was not different from pacing-only values. One contributory factor for this effect may be an interaction between both the V_1a and the AT₁ receptors (Yamaguchi et al., 1982). Plasma ANP was reduced in the AT₁ receptor blockade groups from pacing-only values. This was likely a result of the reduced systemic pressures and LV chamber dimensions observed in these treatment groups.

LV Myocyte Function and Inotropic Response. To more carefully examine inherent myocyte contractile performance in the absence of external loading conditions and neurohormonal system activity, isolated myocyte contractility studies were performed. Consistent with past reports from this laboratory, the development of CHF resulted in inherent defects in LV myocyte contractile function (Spinale et al., 1992, 1995; Spinale, 1997b). V_1a receptor blockade alone or AT₁ receptor blockade alone did not change basal myocyte contractile function from CHF values. Dual receptor blockade significantly increased indices of myocyte contractile function compared with untreated pacing CHF values. Therefore, the improvement in indices of LV ejection performance observed in the dual receptor blockade group was likely due to an inherent improvement in myocyte contractile function.

Vasopressin and CHF. Past clinical and experimental studies have documented that increased plasma levels of vasopressin accompany the progression and/or exacerbation of CHF (Szatalowicz et al., 1981; Riegger and Liebau, 1982; Goldsmith et al., 1983; Naitoh et al., 1994). Increased vasopressin results in the activation of not only the V_1a receptor, causing increased vascular resistance, but also the V₂ receptor, resulting in increased sodium and fluid retention at the level of the kidney (Manning et al., 1993; Bichet, 1994; Burrell et al., 1994, 1998; Nishikimi et al., 1996). It must be recognized that in the present study, we used V_1a receptor blockade. Moreover, V₂ receptor blockade, both acute and chronically, has been demonstrated to have a significant aquaretic effect as demonstrated by a reduction in organ weight accumulation and a decrease in urine osmolality (Nishikimi et al., 1996; Burrell et al., 1998). In addition, both acute and chronic administration of nonselective vasopressin receptor blockade has been demonstrated to provide beneficial hemodynamic and aquaretic effects in experimental models of CHF as demonstrated by a reduction in systemic blood pressure and increased urinary output (Mulinari et al., 1990; Naitoh et al., 1994; Wang et al., 1991). Thus, future studies in which combined vasopressin receptor blockade is chronically administered in this model of pacing CHF are warranted. The present study is the first to examine the potential interrelationship between the V_1a receptor and the AT₁ receptor with respect to LV pump function and myocyte contractile performance. The signal transduction pathways for both the AT₁ receptor and V_1a receptor involve the activation of phospholipase C with subsequent increases in intracellular Ca²⁺ and protein kinase C activation (Burrell et al., 1994). The present study provides additional evidence to suggest that an interrelationship exists between activation of the V_1a receptor and the AT₁ receptor in the progression of a CHF process.

Study Limitations and Future Directions. In the present study, we used a model of chronic rapid pacing that produces a phenotype consistent with clinical CHF. However, only one dose of each inhibitor was used, and these doses were predicated on initial dose-ranging studies for both V_1a receptor blockade and AT₁ receptor blockade in normal animal preparations. Therefore, dose-response relationships with V_1a receptor blockade in the setting of CHF remain to be established. In the animal preparation used in the present study, higher doses of the V_1a receptor antagonist resulted in tachycardia and unstable hypotension. These results suggest that a narrow therapeutic window may exist for SR49059. Based on past in vitro studies (Serradeil Le-Gal et al., 1993), the plasma concentration of SR49059 reflects an approximate 2000-fold higher concentration than necessary to inhibit V_1a-mediated vessel contractility. Thus, the plasma levels achieved in the CHF preparation significantly exceed that which is necessary to inhibit V_1a receptor activity based on in vitro computations. It must be recognized that the plasma levels of the V_1a receptor antagonist achieved in the present study have been reported to bind to the receptor (Serradeil Le-Gal et al., 1993). Therefore, the possibility that the dose of SR49059 used in the study might have also inhibited V₂ activity must be considered. Thus, the present study should be considered an initial study, and future studies are necessary to more carefully define the role of the V_1a receptor in developing CHF. Furthermore, although the present study evaluated V_1a receptor blockade in combination with AT₁ receptor blockade, a more appropriate comparison may have been with that of an ACE inhibitor; past studies have compared acute administration of V_1a receptor blockade with that of an ACE inhibitor (Arnolda et al., 1991). Thus, future studies that evaluate V_1a receptor blockade in the background of ACE inhibition in developing CHF are warranted.