INTRODUCTION

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ENDOTHELIN-1 (ET-1) has been described as the most powerful vasoconstrictor yet discovered and produces its effect via stimulation of specific subtypes of the receptors ET_A and ET_B (6, 18). It has been shown that ET_A and ET_B receptors are present in vascular smooth muscle, where they mediate vasoconstriction. ET_B receptors also exist on endothelial cells, where they mediate vasodilation via release of nitric oxide (NO) and prostaglandins. ET-1 has potent action in the kidney where it can decrease glomerular filteration rate (GFR) via both ET_A and ET_B receptor-mediated vasoconstriction.

ANG-converting enzyme (ACE) is located mainly on endothelial cells, where it transforms ANG I into ANG II and degrades bradykinin. ANG II exerts its vasoconstrictor and sodium-retaining effects via the stimulation of ANG type 1 receptor (AT₁). Bradykinin activates B₂ receptors, which in turn, increase the production of NO and prostacyclin. Therefore, ACE inhibitors not only prevent the formation of a potent vasoconstrictor with proliferative properties but also increases local concentrations of bradykinin. It is possible that the increase in kinin levels associated with ACE inhibition can oppose the hypertensive effects of vasoconstrictor peptides such as ET-1 and ANG II (22, 26).

There is increasing evidence for an interaction between the endothelin and ANG systems, but there has been very little investigation into the relationship between the ET-1 and the kinin system. It has been reported that a high dose of ET-1 can decrease renal blood flow and urinary sodium excretion, and some of these effects may be due to the stimulation of the renin-ANG system (3). Infusion of ET-1 in rats also increases renin release and has been shown to stimulate ACE activity in cultured pulmonary artery cells (5, 7). Additionally, captopril, an ACE inhibitor, prevented chronic hypertension produced by infusion of ET-1 (16). Therefore, there is reasonable evidence to hypothesize that the renal and hypertensive effects of ET-1 infusion are at least in part due to the stimulation of ANG activity. Experiments were conducted to further elucidate the mechanisms of ET-1-induced increases in mean arterial pressure (MAP) and decreases in GFR by contrasting the effects of 1) the ACE inhibitor enalapril, 2) the AT-1 receptor antagonist candesartan, and 3) the combined inhibitor of ACE and neutral endopeptidase (NEP) omapatrilat in male Sprague-Dawley rats. We further explored the possibility that kinins play a role in modulating the systemic and renal response to ET-1 using a B₂ receptor-selective antagonist.

	METHODS

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All studies were conducted on male Sprague-Dawley rats (200-250 g, Harlan Laboratories) maintained at the Medical College of Georgia animal care facility for 1 wk before the study. Rats were housed in temperature-controlled conditions with a 12 h:12 h light-dark cycle. Rats were anesthetized with Inactin (100 mg/kg ip; 5-sec-butyl-5-ethyl-2-thiobarbituric acid; Sigma, St. Louis, MO) and placed on a servo-controlled heating table to maintain rectal temperature constant at 37°C. With the use of a polyethylene (PE)-205 tubing, a tracheostomy was performed to allow unobstructed breathing. A catheter (PE-50) was inserted in the right femoral artery for measuring MAP using a Maclab data acquisition system. The right jugular vein and femoral vein were cannulated (PE-50) for continuous infusion of 0.9% NaCl (10 µl/min) and 0.9% NaCl containing [³H]inulin (4 µCi · h¹ · 100 g¹; 10 µl/min), respectively. Another catheter (PE-90) was placed in the urinary bladder to allow urine collection. After a 60-min equilibration period, a 30-min baseline urine collection period was begun that included a blood sample taken at the midpoint of the period. Separate groups of rats were then given an intravenous bolus of either 1) saline (1 ml/kg), 2) the ACE inhibitor enalapril (10 mg/kg), 3) the ANG II receptor antagonist candesartan (10 mg/kg), 4) the vasopeptidase inhibitor omapatrilat (30 mg/kg), 5) the bradykinin receptor antagonist REF-000359 (1 mg/kg), or 6) REF-00059 + enalapril. This was immediately followed by ET-1 infusion (10 pmol · kg¹ · min¹). Two additional 30-min urine collection periods were obtained during ET-1 infusion with a blood sample taken at the midpoint for measurement of [³H]inulin. Urine volume was measured gravimetrically. Blood pressure changes were recorded, and GFR was determined as the clearance of [³H]inulin. In preliminary experiments, the ability of the selected doses of enalapril, candesartan, and omapatrilat to completely block the pressor response to intravenous bolus injections of ANG I (100-500 ng/kg) was confirmed (data not shown). Furthermore, the ability of the selected dose of kinin antagonist to block the decrease in MAP following an intravevous bolus injections of bradykinin (1-10 nmol) was also confirmed in separate rats (data not shown). ET-1 was obtained from American Peptide (Sunnyvale, CA). Candesartan, omapartilat, and REF-000359 were generous gifts from AstraZeneca Pharmaceuticals (Mölndal, Sweden). Enalapril was obtained from Sigma.

Statistical analysis. Student's t-test for paired data was used to determine the significant differences between control versus experimental periods (Statview; Abacus Concepts, Berkeley, CA). ANOVA with a Scheffé's post hoc test was used to determine the statistically significant differences between groups for ET-1-induced changes in MAP and GFR. Values are reported as means ± SE with P < 0.05 being considered significant; n = 5-8 rats in all groups.

	RESULTS

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Table 1 illustrates the MAP, GFR, urine flow rate, and hematocrit before and after 1 h of ET-1 infusion after treatment with enalapril, omapatrilat, candesartan, REF-00359, REF-00359 plus enalapril, or vehicle. As expected, MAP significantly increased in ET-1-infused rats (Fig. 1). Enalapril significantly attenuated the rise in MAP produced by ET-1 infusion, although neither omapatrilat nor candesartan had any effect. Consistent with renal vasoconstriction, infusion of ET-1 for 1 h significantly decreased GFR (Fig. 2). None of the inhibitors significantly influenced the effect of ET-1 to reduce GFR. ET-1 also significantly decreased urine flow compared with the baseline value; none of the inhibitors had any effect on ET-1-induced decreases in urine flow rate.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Percent change in mean arterial pressure (MAP) due to acute endothelin (ET)-1 infusion after pretreatment of bolus doses of vehicle, enalapril (ena, 10 mg/kg), omapartilat (omp, 30 mg/kg), candesartan (can, 10 mg/kg), REF-000359 (1 mg/kg), or REF-000359 + enalapril. Data are means ± SE; n = 8 rats in each group. *P < 0.05 vs. ET-1 alone.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Percent change in glomerular filtration rate (GFR) due to acute ET-1 infusion after pretreatment of bolus doses of vehicle, enalapril (10 mg/kg), omapartilat (30 mg/kg), candesartan (10 mg/kg), REF-000359 (1 mg/kg), or REF-000359 + enalapril. Data are means ± SE; n = 5-8 rats in each group.

To determine whether increased kinin activity could account for the effect of enalapril on ET-1-induced changes in MAP, another series of experiments were conducted using REF-000359, a B₂ kinin receptor antagonist. Blockade of B₂ receptors had no effect on ET-1-induced changes in MAP or GFR (Table 1 and Figs. 1 and 2). However, coadministration of B₂ receptor antagonist with enalapril restored ET-1-induced increases in MAP that were not observed with enalapril alone (Fig. 1). Decreases in GFR were the same among groups (Fig. 2). The kinin antagonist, with or without enalapril, had no effect in the decreases in urine flow rate produced by ET-1 infusion. There were no significant differences in hematocrit among these groups, although there was a tendency for ET-1 to increase hematocrit in rats given the B₂ antagonist.

	DISCUSSION

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The current study extends previous studies showing that ACE inhibition inhibits the response to ET-1. Acute infusion of ET-1 for 1 h at a rate of 10 pmol · kg¹ · min¹ significantly increased MAP and decreased GFR and urine flow rate. We observed that the ACE inhibitor enalapril significantly attenuated the short-term pressor response to ET-1 infusion, whereas the AT-1 receptor antagonist candesartan failed to do so. These results suggest that the inhibitory effect of enalapril is related to its kininase activity and not to the generation of ANG II. Therefore, we determined the effect of B₂ receptor blockade and observed that it restored the pressor response to ET-1 when it was coadministered with enalapril. These results support the hypothesis that increased kinin survival accounts for the effect of ACE inhibition on the response to ET-1.

In contrast to our results, Cao and Banks (3) previously reported that the ACE inhibitor captopril inhibited the decrease GFR, but not the increase in MAP, produced by a higher dose of ET-1. This same laboratory also reported that ANG II receptor antagonists had no effect on either the systemic or renal action of the peptide (2). The reason for the contrasting observations may be due to the use of different ACE inhibitors or the different doses of ET-1. Consistent with a role for kinins in mediating the effects of ACE inhibitors on the ET-1 response, both laboratories reported that AT₁ receptor blockade had no effect. The dose of enalapril used in the current study has been shown to inhibit ANG II formation by other investigators and was confirmed in our preliminary experiments showing complete blockade of the response to acute ANG I infusion. Additionally, we observed that the selected doses of omapatrilat and candesartan have similar abilities as enalapril to prevent ANG I-induced change in MAP.

Kinins activate B₂ receptors in vascular endothelial cells, which in turn can oppose the ET-1 hypertensive effect via the increase in NO release. In addition, Momose et al. (14) showed that captopril inhibits ET-1 secretion from endothelial cells through a bradykinin-dependent mechanism. To further explore the possibility that increased kinin activity could explain how enalapril attenuates the pressor response to ET-1, we examined the effect of B₂ receptor blockade on the ability of enalapril to inhibit ET-1 responses. At a dose that completely blocks the vasodilator response to exogenous bradykinin, we observed that prior administration of a kinin antagonist plus enalapril prevented enalapril from inhibiting ET-1-induced hypertension. On the other hand, the kinin antagonist alone before ET-1 infusion had no significant effect on the response to ET-1 but showed some tendency to potentiate hypertensive and renal response to acute ET-1 infusion. This could be attributed to the decrease in NO production due to the blockage of B₂ receptors, which in turn will potentiate the ET-1 vasoconstrictor effect. Similarly, although not statistically significant, there was a tendency for ET-1 to produce a greater decrease in GFR and increase in hematocrit during B₂ receptor blockade. We suggest that further studies are warranted to discern the precise role of kinins in modulating these other responses to ET-1.

Work from Dr. Erdos's (11, 12) laboratory has shown that ACE inhibition results in B₂ receptor activation independent of kininase activity. Therefore, it is possible that direct B₂ receptor activation by ACE inhibitors may account for their ability to inhibit ET-1-induced increases in arterial pressure. We also cannot exclude the possibility that the infusion of ET-1 may be accompanied by activation of B₂ receptors through a direct communication between ET_A or ET_B receptors and the B₂ receptor.

Vasopeptidase inhibitors are a new class of antihypertensive drugs that act through the combined inhibition of ACE and NEP enzymes and are thought to be effective in low and high renin models of hypertension (4, 19). Therefore, we also examined whether vasopeptidase inhibition would have a similar effect as enalapril in attenuating the response to ET-1. In contrast to enalapril, we observed that the combined ACE-NEP inhibitor omapatrilat failed to block the response to acute ET-1 infusion. Because omapatrilat and enalapril were given at doses that maximally inhibit ACE-kininase activity, we must conclude that the inability of omapatrilat to inhibit the response to ET-1 is related to inhibition of NEP. Therefore, one can reason that the increase in kinin activity produced by ACE inhibition may be offset by a similar decreased degradation of ET-1. In support of this conclusion, Yamaguchi et al. (25) previously reported that NEP inhibition increases ET-1-induced airway smooth muscle contraction in vitro.

There may also be other vasodilatory mechanisms by which ACE inhibitors can oppose ET-1-induced hypertension. Other investigators (20, 21) have shown that ACE inhibitors may increase release of vasodilator prostaglandins. In addition, Nagase et al. (17) demonstrated that ET-1 enhances the release of oxygen-derived free radicals in vivo and in vitro, which can also increase MAP. Although it remains to be investigated, it is possible that ACE inhibitors antagonize ET-1-induced hypertension through scavenging superoxide anion produced by chronic ET-1 infusion, which in turn will increase NO availability.

There has been considerable uncertainly about the specific mechanisms responsible for the interaction between ET-1 and the renin-angiotensin system. Short-term infusion of ET-1 into intact animals has shown to increase plasma renin activity (5, 10). Additionally, Kawaguchi et al. (7) has demonstrated the potential stimulation of ACE in cultured bovine pulmonary artery endothelial cells treated with ET-1. Moroi et al. (15) also observed a twofold increase in ACE activity when treating rat aortic smooth muscle cells with ET-1, where this stimulatory effect on ACE activity in vascular smooth muscle cells was completely inhibited by 10⁷ M captopril. Contrary to the previous findings, renin secretion from juxtaglomerular cells was generally decreased by ET-1 in both in vivo and in vitro studies (1, 8, 9, 13).

Mortensen and Fink (16) previously showed that captopril prevented hypertension produced by chronic administration of ET-1 without a significant elevation in plasma ANG II level. This observation suggested that if ANG II is involved in ET-1 hypertension, it most likely occurs at a local tissue level. Furthermore, it has been shown that a chronic elevation in circulating ET-1 did not significantly change plasma renin or plasma aldosterone concentration, suggesting that ET-1 does not stimulate the renin-angiotensin system (23, 24). These chronic studies are consistent with our findings with short-term ET-1 infusion, although it is yet to be determined whether the effect of ACE inhibition during chronic ET-1-induced hypertension is related to increased kinin survival.

In summary, our results indicate that the ACE inhibitor enalapril attenuated the hypertensive and renal response to acute ET-1 infusion, at least in a part, via an increase in kinin activity. Acute ET-1 infusion does not appear to stimulate the renin-angiotensin system in vivo. Vasopeptidase inhibition is not as effective as ACE inhibition alone in antagonizing the pressor effects of ET-1.