INTRODUCTION

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Acute respiratory distress syndrome (ARDS), a major cause of mortality in intensive care units, is characterized clinically by pulmonary infiltrates, hypoxemia, and the absence of an elevated pulmonary capillary wedge pressure. Lungs of patients with ARDS show increased neutrophil sequestration, augmented shunting, and intravascular coagulation as well as disruption of pulmonary capillary integrity leading to pulmonary edema. Although ARDS is observed in association with many clinical conditions, ARDS develops most commonly in patients with severe sepsis. In animals, lung injury that expresses many of the pathologic features of human ARDS can be elicited by systemic infusion of live bacteria or endotoxin of gram-negative bacteria.

Numerous mediators such as proinflammatory cytokines, prostaglandins, thromboxanes, reactive oxygen species, and nitric oxide (NO) have been implicated in the pathogenesis of sepsis-associated lung injury (1). The involvement of NO has been attributed to the induction of the inducible nitric oxide synthase (iNOS), which elicits tissue damage directly when expressed in pulmonary cells and influences lung function indirectly through the formation of peroxynitrite, a highly reactive oxidant (2). The contributions of iNOS to LPS-induced acute lung injury has recently been confirmed in mice deficient in iNOS gene. They develop a milder form of acute lung injury in response to LPS injection compared with wild type mice (2).

Recent studies have suggested that endothelins (ETs) play an important role in the pathogenesis of sepsis-induced acute lung injury and vascular failure. ETs are a family of acidic 21-amino acid peptides found in at least three distinct isoforms; endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). These peptides share sequence homology and arise through proteolytic processing of prohormones known as preproETs (3). ETs exert their biologic effects through activation of two families of G-protein coupled receptors, ET-A and ET-B. These receptors are widely distributed throughout various vascular beds and mediate numerous biologic functions of ETs. For instance, ET-1 can induce slow but sustained contraction of vascular and nonvascular smooth muscle cells (4). In isolated atrial preparations, ET-1 exerts positive inotropic and chronotropic effects (5). ET-1 has also potent effects on kidney functions such as reduction in renal blood flow and glomerular filtration rate (6). Moreover, ETs have important growth-promoting effects and potentiate the mitogenic influences of other mitogens such as platelet-derived growth factor (7). In normal lungs, ETs are synthesized by bronchial epithelial cells, endothelial cells, macrophages, vascular smooth muscles, and pulmonary neuroendocrine cells (8). Administration of exogenous ET-1 in normal mammals elicits numerous pulmonary alterations such as increased chloride secretion and mucociliary clearance in airway epithelial cells and proliferation of both airway smooth muscle cells and lung fibroblasts. ETs also modulate pulmonary vascular tone through potent stimulation of pulmonary vascular smooth muscles (8).

Recent evidence suggests that ET-1 can be considered an inflammatory cytokine because it stimulates macrophages and monocytes to release reactive oxygen species as well as cytokines such as tumor necrosis factor (TNF) and interleukins 1, 8, and 6 (9). In septic humans, plasma ET-1 concentration is elevated and correlates negatively with cardiac index (10). These results along with the observation that ET-1 concentration is elevated in the bronchoalveolar lavage fluid of septic animals suggest that ETs play an important role in sepsis-induced ARDS. However, the exact mechanisms through which ETs elicit many of the features of acute lung injury in patients with severe sepsis remain speculative.

We hypothesized that ET-1 influences lung function and blood pressure by inducing iNOS expression and enhances NO production. This hypothesis is based on the fact that elevated levels of ET-1 release many proinflammatory cytokines from immune cells and these induce iNOS expression in various pulmonary and vascular cells. We also based our hypothesis on numerous studies confirming the importance of iNOS in the development of acute lung injury and vascular failure in endotoxemic animals (2, 11). We tested our hypothesis by assessing the influence of a selective ET-A receptor antagonist on arterial pressure, pulmonary NO production, lung NOS activity, and iNOS protein expression in LPS-injected rats. The blocker was an antisense homology box-derived peptide (P1/fl) to selectively block ET-A receptors. This 22 amino-acid peptide (VLNLCALSVDRYRAVASWSRVI) is a fragment of ET-A receptor and binds to specific regions of ET-A receptor responsible for maintenance of protein shape (12). Recent in vivo and in vitro studies have confirmed that P1/fl peptide is a more potent antagonizer of the ET-A receptors than conventional ET-A antagonists such as BQ610 and BQ123 (12).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Reagents

Materials for NOS activity, nitrate assay and Escherichia coli endotoxin were obtained from Sigma chemical (St. Louis, MO). Immunoblotting apparatus and reagents were obtained from Novex Inc. (San Diego, CA). Monoclonal anti-iNOS antibody was obtained from Transduction Laboratories (Lexington, KY). ECL kit was obtained from Amersham Canada Inc. (Oakville, ON, Canada). The antisense polypeptide (P1/fl) (VLNLCALSVDRYRAVASWSRVI) was synthesized and purified by HPLC at the Sheldon Molecular Biology Facility of McGill University.

Animal Preparation

The procedures were approved by the Animal Research Committee of McGill University. Pathogen-free male Sprague-Dawley rats weighing 250 to 300 g were anesthetized with sodium pentobarbital (40 mg/ kg) and were given supplemental doses as needed. The animals were tracheotomized with polyethylene tubing, and the lungs were mechanically ventilated with a volume-cycled ventilator (Harvard Rodent Ventilator Model 683; Harvard Apparatus, South Natick, MA) with a tidal volume of 2 to 3 ml, a respiratory rate of 40 to 60 breaths/min, and an inspired oxygen fraction of 1.0. To ensure complete muscle paralysis, animals were injected intravenously with pancuronium bromide (0.4 mg), and additional doses were administered as needed. An arterial catheter (22-gauge) was placed into the internal carotid artery and used for measuring blood pressure (Trantec Model 60-800; American Edwards Laboratories, Santa Ana, CA) and for sampling of arterial blood. A catheter placed into the jugular vein was used to access the venous circulation. At the end of the experimental protocol (see below), animals were killed with an overdose of sodium pentobarbital. The chest was then opened and 10 ml of phosphate-buffered saline (PBS) was injected into the right ventricle to clear the pulmonary circulation from blood cells. Lung tissues samples were then obtained from the left and right lungs and were quickly frozen in liquid nitrogen. In a few experiments (see below), the lungs were removed, weighed, and then dried in an oven at 50° C for 12 h in order to obtain pulmonary wet/dry ratios.

Plasma ET-1

Arterial blood was collected at the end of the experiment from the left ventricle and placed into chilled tubes containing EDTA. Samples were then centrifuged at 1,800 × g for 20 min to obtain plasma. The concentration of immunoreactive ET-1 in plasma was measured with a specific radioimmunoassay. In brief, the samples were extracted using C18 cartridge (SepPak; Waters, Mississauga, ON, Canada), eluted by 100% methanol, reconstituted in assay buffer, and incubated with rabbit anti-ET-1 antiserum (Peninsula Laboratories, Belmont, CA) at 4° C for 24 h. This was followed by the addition of ¹²⁵I-labeled ET-1 and a second 24-h incubation. Free and bound radioactivity was separated by the second antibody method, and gamma emission from the pelleted antibody-ET-1 complex was counted using a gamma counter.

Serum Nitrate-Nitrite

Quantification of serum nitrate (NO₃) and nitrite (NO₂) was performed using the metallic cadmium method. Cadmium fillings (0.4 to 0.7 g/filling; Fluka Chemical, Ronkonkoma, NY), one filling per sample, were washed with water, 0.1 M HCl, and then 0.1 M ammonium hydroxide. Venous serum samples were incubated with ZnSO₄ at room temperature for 15 min and then centrifuged for 5 min. The supernatants were added to the cadmium-containing microcentrifuge, incubated overnight at room temperature, and then transferred to fresh microcentrifuge tubes and centrifuged again. The supernatants were subsequently assayed for NO₂ using the Griess reagent (Sigma).

In Vitro Aortic Contractility

In five rats, the effectiveness of P1/fl peptide in blocking ET-1-induced smooth muscle contraction (ET-A receptor activation) was assessed by using the in vitro aortic contractility preparations. To test the specificity of P1/fl for ET receptors, this preparation was also used to measure in 10 additional rats the influence of P1/fl on the phenylephrine- and KCl-induced contractions. Rats were killed by decapitation and the aortas were excised, cleaned of adherent tissues, cut into rings (4 mm in length), and suspended in 25-ml organ baths filled with a warmed modified Krebs-Ringer bicarbonate solution. The arterial rings were suspended by means of two stainless steel stirrups. One of the stirrups was anchored to the bottom of the organ chamber and the other was connected to a force transducer (Grass FT03; Grass Instrument Co., Quincy, MA) to record changes in isometric force. The rings were stretched over 60 min to a baseline tension of 2 g. At the end of the experimental protocol, the vessels were dried so that tension could be expressed in grams per milligrams dry vessel weight.

Exhaled NO Measurements

Exhaled air of mechanically ventilated animals was collected over a 15-min period by connecting the expiratory port of the ventilator to a 5-L nondiffusing gas collection bag (Hans Rudolph, Kansas City, MO). The NO in exhaled air thus collected was measured by a chemiluminescence analyzer (Model 270B; Sievers, Boulder, CO). The electrical signals were amplified with a Gould Amplifier Model 6600 (Gould Inc., Instruments Division, Cleveland, OH) and were integrated to measure the area under the curve. NO concentration in exhaled air was expressed as parts per billion (ppb).

L-Citrulline Assay

Frozen lung samples were homogenized in HEPES-based homogenization (contains various protease inhibitors), centrifuged at 10,000 rpm for 15 min at 4° C and the supernatants (50 µl) were then added to 10-ml prewarmed (37° C) tubes containing 100 µl of reaction buffer containing KH₂PO₄, valine, NOS cofactors, and L-[2,3³H]arginine. The samples were incubated for 30 min at 37° C and the reaction was terminated by the addition of cold (4° C) stop buffer. Dowex 50w resin (8% cross-linked, Na⁺ form) was then added and the supernatant was assayed for L-[³H]citrulline by using liquid scintillation counting. The procedure was repeated in the presence of EGTA, EDTA, and N^G- nitro-L-arginine methyl ester (NOS inhibitor) in order to calculate Ca²⁺/calmodulin-dependent and independent NOS activities (2).

Immunoblotting

Crude homogenates (80 µg) were loaded and separated on TRIS-glycine SDS-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes, blocked overnight with nonfat dry milk, and subsequently incubated with primary monoclonal anti-iNOS (1:500) antibody. Specific proteins were detected using horseradish peroxidase-conjugated antimouse secondary antibody and chemiluminescence reagents provided with the Enhanced Chemiluminescence Kit (Amersham Canada). The blots were scanned with an imaging densitometer, and optical densities of protein bands were quantified with software (SigmaGel; Jandel Scientific, San Rafael, CA).

Experimental Protocols

Four sets of experiments were performed.

Experiment 1. We first assessed the ability of P1/fl peptide to block ET-A receptor-mediated vasoconstriction of rat aortic smooth muscles. A total of 30 aortic rings obtained from 15 normal rats were placed in organ baths (see METHODS above) and stretched to a baseline tension of 2 g. After 60 min of equilibrium, the rings were contracted with 10 nM ET-1, 10 µM phenylephrine, or 60 mM KCl to elicit a tension equivalent to 80 to 100% of maximum tension. The effectiveness of P1/fl peptide in antagonizing aortic contractions was then assessed by adding increasing concentrations of P1/fl peptide to the organ bath. Papaverine (100 µM) was added at the end of the experiment to ensure that aortic contractions could be reversed completely.

Experiment 2. This experiment was designed to assess the ability of P1/fl peptide to antagonize the in vivo vascular responses elicited by bolus injections of ET-1. Two groups (n = 5 in each group) of anesthetized and instrumented rats were examined for a 5-h period. Rats in Group 1 (saline) were pretreated with a single (0.3 ml) injection of normal saline followed 15 min later by an intravenous injection of 0.03 nM/kg of porcine ET-1 (Peninsula Laboratories). A second intravenous bolus injection of 0.03 nM/kg ET-1 was administered 5 h later and the animals were then killed. An identical protocol was used in Group 2 except that the animals were pretreated with P1/fl peptide (20 µg/kg dissolved in 0.3 ml of PBS) instead of saline. The initial vasodilatory (endothelial ET-B receptor activation) and the late sustained pressor (smooth muscle ET-A receptor activation) responses elicited by the two ET-1 injections were measured in the two groups of animals.

Experiment 3. We assessed in this experiment the effects of pretreatment with P1/fl peptide on mean arterial pressure, exhaled NO concentration, lung NOS activity and lung iNOS protein expression. Four groups of animals (n = 7 in each group) were examined for a period of 5 h. Group 1 (saline) served as a control group and the animal received a single bolus (0.3 ml) injection of saline. In Group 2 (saline + P1/fl), P1/fl peptide (20 µg/kg) was administered 15 min prior to saline injection. In Group 3 (LPS), a bolus injection of E. coli LPS (20 mg/kg, serotype 055:B5) was injected instead of saline. Similarly, in Group 4 (LPS + P1/fl), P1/fl peptide (20 µg/kg) was administered 15 min prior to LPS or saline injection. Baseline values of arterial pressure and exhaled NO were measured 5 min prior to LPS or saline injections. Both parameters were measured throughout the experimental period (5 h). At the end of the experiment, lungs were excised and were subsequently processed for iNOS protein expression and NOS activity assay (see METHODS above).

Experiment 4. The influence of pretreatment with P1/fl peptide on plasma ET-1 concentration, serum nitrite-nitrate concentration and lung wet/dry ratio was assessed in this experiment. Four groups of animals were examined (n = 7 in each group) for a period of 5 h as in Experiment 3. Baseline arterial blood samples were obtained for the determination of nitrite-nitrate concentration and blood gases. Animals received either saline or LPS and were pretreated with or without P1/fl peptide (20 µg/kg). Serum nitrite-nitrate concentration was repeated every 60 min throughout the observation period. At the end of the experiment, arterial blood samples for the measurement of plasma ET-1 levels were obtained and the lungs were then excised and lung wet/dry ratios were measured.

Data Analysis

Data are shown as means ± standard errors of the means. The two-way analysis of variance test was employed in order to calculate statistical heterogeneity for arterial blood gases, plasma ET-1 level, serum nitrite-nitrate concentrations, exhaled NO concentration, mean arterial pressure, NOS activity, and wet/dry ratios both between and within treatment groups at different time points after saline and LPS injections. Statistical differences between mean values were detected using Student's t test.

	RESULTS

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Experiment 1 (in vitro blockade of ET-1 induced vasoconstriction). The influence of P1/fl peptide on aortic contractions induced by either ET-1, phenylephrine, or KCl is illustrated in Figure 1. ET-1-induced contraction was reversed by increasing concentrations of P1/fl peptide, with complete reversal obtained at a P1/fl peptide concentration of 10 µM (Figure 1). By comparison, P1/fl had no influence on phenylephrine- or KCl-induced contractions (Figure 1).

View larger version (21K): [in this window] [in a new window]   Figure 1.   The influence of P1/fl peptide on aortic contractions induced by ET-1, phenylephrine (PE), or KCl. Rat aortic rings were isolated in vitro and stimulated with 10 nM ET-1, 10 µM PE, or 60 mM KCl. Note that increasing concentrations of P1/fl peptide reversed ET-1-induced contractions but had no effects on PE- or KCl-induced contractions.

Experiment 2 (in vivo blockade of ET-1-induced pressure changes). In the saline group, the first injection of 0.03 nM/kg of ET-1 elicited a transient and early (within 30 s) decline in mean arterial pressure, which was followed by a sustained rise in pressure lasting for more than 15 min. The second (5 h later) injection of ET-1 elicited qualitatively and quantitatively similar vasodilatory and pressor responses to those observed after the first injection (Figure 2). Pretreatment with P1/fl peptide eliminated the sustained pressure response elicited by the first and second ET-1 injections (Figure 2).

View larger version (31K): [in this window] [in a new window]   Figure 2.   The effects of pretreatment with saline (top panel ) or P1/fl peptide (20 µg/kg) (bottom panel ) on the vasodilator (hatched columns) and the pressor (crossed columns) responses to two bolus injections of 0.03 nM/kg of ET-1 in anesthetized rats. The two injections were separated by 5 h (300 min). Open columns refer to baseline arterial pressure measured prior to ET-1 injection. *p < 0.05 compared with baseline values. Notice that pretreatment with P1/fl eliminated the pressor responses elicited by the first and second ET-1 injections.

Experiment 3. The changes in mean arterial pressure in the four groups of animals are illustrated in Figure 3. Although mean arterial pressure remained unchanged in the saline and saline + P1/fl groups, it declined progressively in the LPS group. Arterial pressure in the LPS + P1/fl group remained similar to that of the saline and saline + P1/fl groups, indicating that pretreatment with P1/fl prevented LPS-induced hypotension (Figure 3).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Changes in mean arterial pressure in the four groups of animals. LPS injection induced a progressive decline in mean arterial pressure in the LPS group. At 30 min onward, arterial pressure in the LPS group was significantly lower than that measured in the remaining three groups of animals. Note that pretreatment with P1/fl peptide eliminated LPS-induced hypotension.

The changes in arterial blood gases in the four groups of animals are shown in Figure 4. There was a significant decline in arterial pH in the LPS group, whereas no significant changes in arterial blood gases after 5 h of experimental period in the saline and saline + P1/fl groups. Pretreatment with P1/fl attenuated LPS-induced decline in arterial pH (Figure 4).

View larger version (29K): [in this window] [in a new window]   Figure 4.   Changes in arterial blood gases in the four groups of animals. *p < 0.05 compared with baseline values; +p < 0.05 compared with the LPS + P1/fl group.

Exhaled NO concentrations remained unchanged in the saline and the saline + P1/fl groups from baseline values of 6 to 7 ppb (Figure 5). LPS injection elicited about a 4-fold rise in exhaled NO concentration, which reached a plateau after 200 min of LPS injection. Pretreatment with P1/fl peptide resulted in a smaller rise (2-fold) in exhaled NO concentrations compared with those in the LPS group (Figure 5). However, exhaled NO concentrations measured at 120 min onward in the LPS + P1/fl group were significantly higher than those of the saline and saline + P1/fl groups (Figure 4).

View larger version (22K): [in this window] [in a new window]   Figure 5.   Exhaled NO concentration (expressed in parts per billion, ppb) in the four groups of animals. Note that LPS injection elicited a progressive increase in exhaled NO concentration that reached statistical significance after 120 min of LPS injection compared with baseline values (0 time). Pretreatment with P1/fl attenuated the rise in exhaled NO after LPS injection. At 150 min onward, exhaled NO concentration in the LPS group was significantly (p < 0.05) higher than those measured in the remaining three groups of animals.

Injection of LPS elicited a substantial rise in lung Ca²⁺/ calmodulin-dependent and independent NOS activities (p < 0.01 and < 0.05 compared with the saline and saline + P1/fl groups) (Figure 6). Pretreatment with P1/fl peptide eliminated LPS-induced augmentation of pulmonary NOS activity (Figure 6). An immunoblot of lung samples obtained after 5 h of experimental period in the four groups of animals is shown in Figure 7. We detected a weak 130 kD iNOS protein band in the lungs of saline and saline + P1/fl groups. Injection of LPS elicited a significant upregulation of iNOS protein expression (OD of 200% of the saline group). Pretreatment with P1/fl peptide eliminated this rise in iNOS protein expression observed after LPS injection (OD of 60% of the saline group) (Figure 7). Similar changes in lung iNOS expression were observed in immunoblotting experiments performed on four independent animals in each group.

View larger version (28K): [in this window] [in a new window]   Figure 6.   Changes in lung NO activity in various groups of rats. *p < 0.05 and **p < 0.01 compared with the saline group, respectively; +p < 0.05 and ++p < 0.01 compared with the LPS + P1/fl group, respectively. Notice that LPS injection evoked a significant rise in lung NOS activity, whereas lung NOS activity in the LPS + P1/fl group was similar to that of the saline and saline + P1/fl groups.

View larger version (31K): [in this window] [in a new window]   Figure 7.   A representative immunoblot of lung samples for iNOS expression in the four groups of animals. Lung samples were probed with a monoclonal anti-iNOS antibody. Note that significant iNOS protein expression was detected after LPS injection, whereas weak iNOS protein expression was detected in the remaining three groups of rats.

Experiment 4. Injection of LPS resulted in a significant increase in lung wet/dry ratio compared with that measured in the saline and saline + P1/fl groups (Figure 8). Pretreatment with P1/fl peptide prevented the LPS-induced rise in pulmonary wet/dry ratio (Figure 8). In the saline and saline + P1/fl groups, serum nitrite-nitrate concentration remained similar to baseline (0 time) values (Figure 9), whereas it rose significantly and progressively in the LPS and LPS-P1/fl groups. However, at 240 min onward, serum nitrite-nitrate concentration was significantly higher in the LPS group than in the LPS-P1/fl group (Figure 9).

View larger version (15K): [in this window] [in a new window]   Figure 8.   Changes in lung wet/dry ratios after saline or LPS injection in various groups of animals. Statistical symbols are identical to those shown in Figure 5.

View larger version (20K): [in this window] [in a new window]   Figure 9.   Serum nitrite-nitrate concentration in the four groups of animals. *p < 0.05 and **p < 0.01 compared with corresponding values measured in the saline group; +p < 0.05 compared with corresponding values obtained in the LPS + P1/fl group. Note the progressive rise in serum nitrite-nitrate level in the LPS group. Pretreatment with P1/fl attenuated the late rise in serum nitrite- nitrate concentration observed after LPS injection.

The changes in plasma ET-1 concentrations in the four groups of animals are illustrated in Figure 10. Plasma ET-1 concentrations rose by about 2- to 3-fold in the LPS and LPS + P1/fl groups compared with those measured in the saline and saline + P1/fl groups (p < 0.01).

View larger version (16K): [in this window] [in a new window]   Figure 10.   Changes in plasma endothelin concentration in response to saline and LPS injections. **p < 0.01 compared with the saline group. Pretreatment with P1/fl did not prevent the increase in plasma ET-1 concentration induced by LPS injection.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The main findings of this study are as follows: (1) Injection of E. coli LPS was associated with a progressive decline in arterial pressure and a significant rise in serum nitrite-nitrate levels, pulmonary NOS activity, exhaled NO concentration, and upregulation of pulmonary iNOS protein expression. (2) Pretreatment with P1/fl peptide eliminated the decline in arterial pressure, the rise in pulmonary wet/dry ratio, lung NOS activity, and iNOS expression and attenuated the increase in exhaled NO concentrations. (3) LPS-induced rise in serum nitrite-nitrate, but not the increase in plasma ET-1 concentration, was attenuated by pretreatment with P1/fl peptide.

Consideration of Methods

Numerous selective and nonselective ET-receptors antagonists have been used to evaluate the role of ETs in various pathologic processes. In this study, we injected P1/fl peptide intravenously to selectively block ET-A receptors in various vascular beds of saline and LPS-treated rats. This peptide has been designed according to the Molecular Recognition Theory, which is based on the idea that binary (hydropathy) amino acid sequences code in the antisense direction for peptides, which interact with the sense protein through shapes or structures resulting from their exactly inverted hydropathy (12). Baranyi and colleagues (12) described these regions (antihomology boxes) as 15 amino acids in length are separated by 15 amino acids. It has been postulated that these boxes may be involved in protein folding, chaperoning, and oligomer formation of proteins. Baranyi and colleagues designed several ET-A receptor antisense peptides, the most effective among which is P1/fl peptide. The rationale for using P1/fl peptide in our study was based on the following. (1) Unlike conventional ET-receptor antagonists of various chemical compositions, antisense peptides may actually exist in nature as endogenous antagonist (12). (2) Baranyi and colleagues indicated that pretreatment of septic rats with P1/fl improved lung gas exchange function, suggesting that this peptide may attenuate sepsis- induced acute lung injury. (3) Recent studies have indicated that P1/fl peptide is a more powerful antagonist of biologic actions of ETs in in vivo settings than the most commonly used ET-A receptor antagonists such as BQ610 and BQ123 (13, 14).

One may argue that the observed effects of P1/fl peptide are due to the influence of this peptide on both ET-A and ET-B receptors. The following observations indicate, however, that P1/fl peptide is an effective antagonist of ET-A rather than ET-B receptors. Firstly, the vasoconstrictor effect of ET-1 on isolated rat aorta (mediated mainly by activation of vascular smooth muscle ET-A receptors) was completely reversed by P1/fl peptide (Figure 1). Secondly, previous studies indicate that selective ET-A receptor antagonists potently inhibit the in vivo pressor response to ET-1 injection, whereas the vasodilator response to that injection is eliminated by pretreatment with selective ET-B receptor antagonists (15). In our study, the in vivo vasodilator response to ET-1 injection remained intact, whereas the sustained vasoconstriction elicited by ET-1 was prevented by P1/fl peptide (Figure 2). Thirdly, blockade of ET-B receptors causes a significant rise in plasma ET-1 concentration because these receptors are involved in clearance of ET-1. However, we found no significant alterations in plasma ET-1 levels with pretreatment with P1/fl peptide in saline or LPS-injected rats (Figure 10).

ETs and Vascular Failure of Sepsis

We found that plasma ET-1 concentration increased by more than 2-fold in the LPS group. This observation is in accordance with previous findings in endotoxemic and septic animals and humans (16). The increase in plasma ET-1 levels in septic animals has been attributed to both upregulation of ET-1 synthesis as well as impaired renal and pulmonary clearance of ETs. In addition, local tissue hypoxia and changes in shear stress could be operative during septic shock and lead to significant elevation in ET-1 concentration.

The role of endogenous ETs in hypotension of sepsis and endotoxemia remains controversial. Conflicting results have been obtained regarding the effectiveness of various ET receptor antagonists in reversing LPS-induced hypotension. For instance, pre-treatment of septic rats with the nonselective ET-receptor antagonist SB209670 resulted in worsening of hypotension, hepatocellular injury, and vascular reactivity to norepinephrine infusion (17). By comparison, the nonselective ET-receptor antagonist, bosentan, improves survival but had no influence on LPS-induced hypotension in pigs (18). In another study, Ruetten and Thiemermann (19) reported that BQ788 (selective ET-B receptor antagonist) attenuated delayed hypotension, improved vascular reactivity to norepinephrine, and prevented liver injury in endotoxemic rats, whereas BQ485 (selective ET-A receptor antagonist) had no effects on these parameters. The reasons behind these contradictory results are not clear. We speculate, however, that many factors are involved in determining the influence of ET-receptor antagonists on LPS-induced hypotension such as the hemodynamic profiles of various septic shock models, the degrees to which endogenous ET levels are elevated in these models and in in vivo effectiveness and selectivity of various ET-receptor antagonists.

Our results indicate that pretreatment with P1/fl peptide completely prevented the decline in systemic arterial pressure elicited by LPS injection. We attributed this influence of P1/fl peptide to antagonism of ET-A receptors rather than to a direct influence of P1/fl peptide on vascular smooth muscle contractility because this peptide in saline-treated animals had no effect on systemic arterial pressure. The prevention of LPS- induced hypotension by P1/fl peptide suggests that activation of ET-A receptors by ETs in general and ET-1 in particular promotes the development of hypotension in this animal model. We speculate that the contribution of ET-1 to the decline in arterial pressure in LPS-injected animals is mediated through the following mechanisms. First, although systemic injections of ET-1 causes sustained elevation in arterial pressure as a result of significant elevation of systemic vascular resistance, an increase in local ET-1 concentration in septic animals is likely to cause local severe venous constriction and pooling of blood in large capacitance vascular beds, resulting eventually in a reduction in cardiac output and low arterial pressure. Hepatic, renal, and splanchnic vascular beds are likely targets of local influence of ET-1 in sepsis. Indeed, Ruetten and Thiemermann (19) proposed that prevention of liver dysfunction in endotoxemic rats by ET receptor antagonists may be due to alleviation of severe hepatic vasoconstriction elicited by endogenous ET-1 release. Secondly, there is growing evidence that ET-1 stimulates the release of proinflammatory cytokines such as TNF, platelet-activating factor, interleukin-1, interleukin-6, interleukin-8, and arachidonic acid metabolites (for review, see Reference 20). The participation of these cytokines in LPS-induced hypotension and vascular failure has been well established. Thirdly, ET-1 interacts with the NO system in the vasculature by augmenting the vasodilator and the cytotoxic effects of NO on vascular smooth muscle cells (21) and by promoting iNOS induction in the endothelial and vascular smooth muscle cells. Many investigators have established the important role of enhanced NO release by iNOS protein in delayed vascular failure elicited by LPS injection in various animal models (11). The observation that pretreatment with P1/fl peptide attenuated the rise in serum nitrite-nitrate, particularly after several hours of LPS injection, lend support to the argument that endogenous ET-1 release may promote vascular NO synthesis during the course of septic shock. The cellular pathways through which ET-1 leads to iNOS induction will be discussed below.

ETs and Pulmonary NO Production

It has become evident in the past few years that elevated levels of NO in the lungs, by acting directly on various pulmonary cells and indirectly through the formation of peroxynitrite, play an important role in the development of acute lung injury in septic animals. Many investigators attributed the increase in pulmonary NO synthesis in these animals to the induction of iNOS expression, particularly in airway epithelial cells and macrophages. Indirect confirmation of the role of iNOS in LPS-induced acute lung injury has been provided by observing significant attenuation of various indices of acute lung injury and prevention of microvascular leakage by selective iNOS inhibitors (22). In addition, we have recently confirmed the critical role of iNOS by demonstrating that iNOS knockout mice develop significantly milder acute lung injury after LPS injection than do wild type mice (2).

The notion that ETs may be involved in acute lung injury is based on the observation that ET-1 increases capillary fluid leakage and potentiates leukotoxin-induced lung edema (23). Furthermore, many investigators have documented that ET-1 concentration is elevated in the lungs of endotoxemic animals (24). In humans, Langleben and colleagues (25) described for the first time that not only plasma ET-1 is elevated in patients with ARDS but pulmonary ET-1 clearance is reduced in these patients. These investigators proposed that ET-1 plays an important role in the pathogenesis of pulmonary hypertension in patients with ARDS.

We found that blockade of ET-A receptors by P1/fl ameliorated the influence of LPS injection on lung wet/dry ratio and pulmonary NO synthesis and iNOS expression. These results confirm our hypothesis that ETs contribute to the development of acute lung injury in sepsis through enhanced NO synthesis. We propose that ET-1 acts in autocrine, paracrine, and endocrine fashions to stimulate pulmonary NO synthesis through the induction of iNOS expression. Enhanced ET production by pulmonary endothelial cells and airway epithelial cells in response to LPS injection can activate ET-A receptors located on neighboring endothelial and epithelial cells and lead to an increase intracellular cAMP synthesis and to activation of the protein kinase C pathway. Both of these effects are known to cause transcriptional upregulation of iNOS expression (26). Our proposal that the iNOS isoform may be induced by ET-1 has recently been confirmed in cultured endothelial cells and monocytes (27). Enhanced ET synthesis, which has recently been documented in the lungs of septic animals (24), can also be produced by pulmonary and alveolar macrophages. This proposal is based on the observation that these cells are capable of synthesizing both ET-1 and ET-3 (28). On the basis of this finding and on our previous observation documenting the contribution of macrophage iNOS activity to exhaled NO production (29), we speculate that attenuation of LPS-induced pulmonary NO production in animals pretreated with P1/fl peptide is due to blockade of ET-A receptors located on pulmonary and alveolar macrophages, which results in attenuation of iNOS expression in these cells.

Another pathway through which ETs may interact with pulmonary NO production and iNOS expression is through the release of proinflammatory cytokines such as TNF and IL-1 from macrophages and monocytes. These particular cytokines are well-known activators of iNOS transcription in in vivo and in vitro experiments. In addition, ET-1 stimulates superoxide production by alveolar macrophages (30). Reaction of NO with superoxide anions produces peroxynitrite, which is a highly oxidative species and is capable of nitrating tyrosine residues of numerous proteins leading to the formation of nitrotyrosine. High levels of nitrotyrosine formation detected by specific antibody has been shown to develop in human acute lung injury (31).

It should be emphasized that there exists non-nitric oxide pathways through with ET-1 promotes lung injury and pulmonary edema in septic animals. These pathways include increasing pulmonary microvascular pressure and elevation of airway vascular permeability.

In summary, pretreatment with P1/fl peptide prevented the decline in arterial pressure, the rise in lung wet/dry ratio, lung NOS activity, and pulmonary iNOS expression and attenuated the increased in exhaled NO production and serum nitrite- nitrate concentration in animals injected with LPS. These results indicate that activation of ET-A receptors by ETs during the course of septic shock promotes the development of both vascular failure and acute lung injury by stimulating the induction of iNOS and increased NO synthesis.