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NO Prevents Neutrophil-Mediated Pulmonary Vasomotor Dysfunction in Acute Lung Injury
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
63, 23–28 (1996)
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NO Prevents Neutrophil-Mediated Pulmonary Vasomotor Dysfunction in Acute Lung Injury1 RANDALL S. FRIESE, M.D., DAVID A. FULLERTON, M.D., ROBERT C. MCINTYRE, JR., M.D., THOMAS F. REHRING, M.D., JEANETTE AGRAFOJO, B.S., ANIRBAN BANERJEE, PH.D., AND ALDEN H. HARKEN, M.D. The University of Colorado Health Sciences Center, Denver, Colorado 80262 Presented at the Annual Meeting of the Association for Academic Surgery, Dearborn, Michigan, November 8–11, 1995
injury may contribute to pulmonary vasoconstriction by shifting net pulmonary vascular smooth muscle tone in favor of vasoconstriction. cGMP-mediated pulmonary vasorelaxation is impaired in the rat following endotoxin in association with increased lung neutrophil accumulation and histologic evidence of significant endothelial cell injury [5]. Another important role of endothelial-derived NO is to help prevent neutrophil adhesion to endothelial cells [6, 7]. In the coronary and mesenteric circulations, deficiency of endothelial-derived NO has been shown to exaggerate neutrophil-associated vascular injury [8, 9]. On the other hand, administration of exogenous NO (via NO donors) attenuates neutrophil-associated vascular injury [10–12]; exogenous NO may augment vascular endothelial cell defense mechanisms against neutrophils [13]. Such studies offer insight into the pathogenesis of endotoxin-induced pulmonary vasomotor dysfunction. In cultured endothelial cells, Graier and colleagues have shown that endotoxin inhibits the synthesis of NO [14]. Endotoxin also promotes the expression of neutrophil and endothelial cell adhesion molecules [15–18]. Therefore, following endotoxin, the pulmonary vascular endothelium may be particularly susceptible to neutrophil-associated injury. In the present study, we sought to augment the defense mechanisms of the pulmonary vascular endothelium by administration of exogenous NO. We hypothesized that administration of exogenous NO by a therapeutically accessible route, inhaled NO, attenuates endotoxin-induced lung neutrophil accumulation and dysfunction of endothelial-dependent cGMPmediated pulmonary vasorelaxation. The purpose of this study was to examine the effect of exogenous NO (inhaled NO) on lung neutrophil accumulation (myeloperoxidase assay, MPO) and cGMP-mediated pulmonary vasorelaxation following endotoxin injection in rats. Using isolated pulmonary arterial rings, we examined the following mechanisms of pulmonary vascular smooth muscle relaxation: (1) endothelial-dependent cGMP-mediated vasorelaxation (response to acetylcholine, ACh) and (2) endothelial-independent cGMP-me-
The purpose of this study was to examine the effect of administration of inhaled nitric oxide (NO) on lung neutrophil accumulation and pulmonary vascular endothelial cell function in endotoxin-induced acute lung injury. Mechanically ventilated rats were studied 4 hr after endotoxin (0.5 mg/kg IP). Inhaled NO (20 ppm) was administered for either the entire 4 hr after endotoxin (continuous group) or for only the first 2 of 4 hr after endotoxin (abbreviated group). Endothelialdependent (acetylcholine, ACh) and -independent cGMP-mediated relaxation (nitroprusside, SNP) pulmonary vasorelaxation were studied in isolated pulmonary arterial rings. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). Inhaled NO prevented endotoxin-induced lung neutrophil accumulation as well as pulmonary endothelial cell dysfunction. However, this protection required continuous administration of inhaled NO. We conclude that inhaled NO prevents neutrophil-mediated pulmonary vascular endothelial cell dysfunction in acute lung injury. q 1996 Academic Press, Inc.
INTRODUCTION
Net pulmonary vascular smooth muscle tone results from the mechanistic balance of vasoconstriction and vasorelaxation. In the normal lung, basal endothelial release of the endogenous vasodilator, nitric oxide (NO), contributes to the low pulmonary vascular smooth muscle tone [1]. Endothelial-derived NO lowers pulmonary vascular tone by stimulating guanylate cyclase in subjacent vascular smooth muscle cells to generate cGMP, which produces pulmonary vascular smooth muscle relaxation [2]. In acute lung injury, increased levels of circulating or local vasoconstricting agonists may contribute to increased pulmonary vascular tone [3, 4]. On the other hand, impairment of the mechanisms of pulmonary vasorelaxation in acute lung 1
Supported by NIH Grant R29HL49398.
0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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diated vasorelaxation by stimulation of vascular smooth muscle guanylate cyclase (response to sodium nitroprusside, SNP). The results of this study demonstrate that administration of exogenous NO attenuates pulmonary vascular endothelial dysfunction following endotoxin. METHODS All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Animal housing and acclimation. Male Sprague–Dawley rats (Sasco, Inc., Omaha, NE) weighing 350–450 g were quarantined in quiet, humidified, light-cycled rooms for 2–3 weeks before use. Rats were allowed ad libitum access to food and water throughout quarantine up to the time of experimentation. Experimental protocols. After induction of anesthesia (sodium pentobarbital 50 mg/kg, IP) and tracheotomy, rats were mechanically ventilated (Pressure Controlled Respirator model RSP1002, Kent Scientific Corp.) with a fraction of inspired oxygen 0.4, respiratory rate 40 breathsrmin01, peak inspiratory pressure 9–11 cm H2O, and an inspiratory:expiratory ratio 1:2. End-tidal CO2 was continuously monitored by capnography and maintained between 35–45 mmHg. The left femoral artery was cannulated with heparinized polyethylene tubing for continuous arterial blood pressure monitoring. Arterial blood pressure was recorded at 80 Hz using a MacLab Data Interface Module (ADI Instruments, Milford, MA) and a MacIntosh computer (Apple Computer, Cupertino, CA). Anesthesia was maintained throughout the experimental protocol (sodium pentobarbital 10 mg/kg IP at 40- to 50-min intervals). Endotoxin (Salmonella typhimurium, 0.5 mg/kg IP) was freshly prepared in 0.9% saline (0.5 mg/ml). Saline-injected controls received 0.9% saline (1 cc IP). Rats were studied at either 2 or 4 hr after injection. Inhaled NO (Scott Medical Products, Plumsteadville, PA) was administered via the inspiratory arm of the ventilator circuit. The concentration of inhaled NO administered was continuously monitored by chemiluminescence (Model 42H, Thermo Environmental Instruments, Inc., Franklin, MA) and maintained at 20 ppm. Effect of exogenous NO administration. Two experimental protocols were employed to examine the effect of the timing and duration of exogenous NO administration on lung neutrophil accumulation and cGMP-mediated pulmonary vasorelaxation. Continuous NO administration. To examine the effect of NO administration from just prior to the onset of endotoxin exposure, NO was administered continuously beginning 15 min prior to endotoxin injection and for 4 hr after endotoxin injection. Rats were studied 4 hr after endotoxin injection (Fig. 1A). Abbreviated NO administration. To examine the effect of NO administration for the first 2 of 4 hr following endotoxin, inhaled NO administration was initiated beginning 15 min prior to endotoxin injection and continued for 2 hr after endotoxin injection. Inhaled NO was then stopped and rats were studied 4 hr after endotoxin injection (Fig. 1B). Isolated pulmonary arterial ring preparation. At the conclusion of the experimental protocol, median sternotomy was performed and heparin sulfate (500 USP) was injected into the right ventricular outflow tract. After removal of the heart and lungs the main pulmonary and the right and left pulmonary arteries were dissected out and placed in Earle’s balanced salts solution (EBSS) at 47C. Under dissecting microscope magnification, the surrounding tissue was dissected from the pulmonary arteries. The right and left main branch pulmonary arteries were each then cut into rings, each 3–4 mm wide; two pulmonary arterial rings were obtained from each rat. Care was taken during this process to avoid endothelial injury. EBSS is a standard physiologic salt solution and contains CaCl2 1.80 mM, MgSO4 (anhydrous) 0.83 mM, KCl 5.36 mM, NaCl 116.34 mM,
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FIG. 1. Experimental protocols used to examine the effect of inhaled nitric oxide (NO) on endotoxin-induced lung neutrophil accumulation and impairment of cGMP-mediated pulmonary vasorelaxation. (A) Continuous administration of inhaled NO; (B) abbreviated administration of inhaled NO.
NaPO4 0.40 mM (dibasic), D-glucose 5.50 mM, NaHCO3 19.04 mM, phenol red Na 0.03 mM (as pH indicator). The pulmonary artery rings were then placed on 11-mil steel wires and suspended in individual 10-cc organ chambers containing EBSS at 377C. The organ chambers were surrounded by water jackets and continually warmed (377C). Ring tension was determined by use of a force-displacement transducer (Grass FTO3, Grass Instruments Co., Quincy, MA) attached to each steel wire apparatus. Force displacement was recorded at 0.67 Hz using a MacLab Data Interface Module (ADI Instruments, Milford, MA) on a Macintosh IIci computer (Apple Computer, Cupertino, CA). Each organ chamber had continual bubbling gas flow at 40 cc/min of 21% oxygen, 5% carbon dioxide and 74% nitrogen. This produced a PO2 of 100–110 mmHg and a pH of approximately 7.4. Pulmonary vasorelaxation by cGMP-mediated mechanisms. Concentration–response curves to acetylcholine and sodium nitroprusside. Cumulative concentration–response curves were generated for ACh and SNP. In a previous study, 750 mg was determined to be the optimal resting mechanical tension (passive load) for the study of isolated pulmonary arterial rings [5]. Rings were suspended at 750 mg and allowed to reach a steady state for 1 hr, during which time the EBSS was changed every 15 min. A given ring was preconstricted with PE to achieve a PE-induced ring tension of between 200 and 400 mg. Cumulative concentration–response curves were then generated over the concentration range of 1009 –1006 M. For determination of the concentration–response curve, the ring was allowed to reach a steady state, usually requiring 2–3 min, before advancing to the next higher concentration. The ring tension remaining in the rings in response to each dose of vasorelaxing agent was expressed in milligrams of PE-induced tension. Lung harvest for myeloperoxidase assay. At the conclusion of the experimental protocol and after median sternotomy, rats were heparinized (500 USP) via injection into the right ventricular outflow tract. Lungs are perfused via the pulmonary artery for 2 min with
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TABLE 1 Lung Neutrophil Accumulation Lung MPO activity (u/g lung tissue) Saline controls Endotoxin alone NO alone Continuous NO / endotoxin Abbreviated NO / endotoxin
3.3 13.2 4.9 5.0 11.0
{ { { { {
1.0 1.0* 1.0 0.5 2.0*
Note. MPO, myeloperoxidase; NO, nitric oxide; *P õ 0.05 versus saline controls.
modified Krebs-Hensleit solution at 377C (pH 7.35–7.45) containing 4 g/100 ml Ficoll and 10 mg/liter N-2 hydroxyethylpiperazine N1-2ethnesulfonic acid (HEPES) at 0.04 ml-g body wt01 min01 via perfusion with a peristaltic pump (Masterflex pump; Cole Parmer, Inc., Chicago, IL). Lungs were externally rinsed with saline, blotted dry, and separately weighed. Lung myeloperoxidase assay. Lung tissue was homogenized for 30 sec (Virtishear homogenizer; Virtis Co., Inc., Gardner, NY) in a 4-ml 20-mmole/liter potassium phosphate buffer, pH 7.4 and centrifuged for 30 min at 40,000 g, 47C (Sorvall RC-5B centrifuge, 5 M245 rotor, 18,000 rpm) (Dupont Instruments, Inc., Irving, TX). The pellet was resuspended in 4 ml of 50 mM potassium phosphate buffer, pH 6.0, containing 0.5 g/dl cetrimonium bromide. Resuspended pellets were frozen at 0707C until the myeloperoxidase assay was performed. Frozen samples were thawed, sonicated for 90 sec at full power (ultrasonic cell disrupter; Kontes, Vineland, NJ), incubated in a 607C water bath for 2 hr, and centrifuged for 10 min at maximum speed (Beckman Microfuge 12; Beckman Instruments, Irvine, CA). Supernatant, 0.1 ml, was added to 2.9 ml of 50 mM potassium phosphate buffed, pH 6.0, containing 0.167 mg/ml o-dianisidine and 5 1 1004% hydrogen peroxide; absorbance of 460 nm visible light was measured for 3 min (Beckman DU7 spectrophotometer; Beckman Instruments). MPO activity per gram wet lung (gwl) was calculated by Myeloperoxidase activity (units/gwl) Å
(A460 )(13.5) , Lung Weight (gm)
FIG. 2. Endothelial-dependent cGMP-mediated pulmonary vasorelaxation was significantly impaired following endotoxin. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
U/g lung tissue. Four hours following endotoxin injection, MPO activity was increased to 13.2 { 1.0 U/g lung tissue (P õ 0.05 versus control). As shown in Fig. 2, endothelial-dependent cGMPmediated pulmonary vasorelaxation was impaired following endotoxin. In saline-injected controls, pulmonary artery rings were preconstricted with PE to 292 { 16 mg tension and ACh 1006 M produced complete relaxation of isolated pulmonary artery rings (11 { 4 mg ring tension remaining). Four hours following endotoxin, ACh 1006 M produced relaxation to 186 { 21 mg tension (P õ 0.05 versus control). On the other hand, maximal ring relaxation by endothelial-independent cGMP-mediated pulmonary vasorelaxation was not impaired following endotoxin. As shown in Fig. 3, the response to SNP was different from control only at a concentration of 1007 M 4 hr following
where A460 is the change in absorbance of 460 nm light from 1 to 3 min after the initiation of the reaction. The coefficient 13.5 was empirically determined such that 1 U myeloperoxidase activity is the amount of enzyme that will reduce 1 mmole peroxide/min [19]. Reagents. Reagents were obtained from Sigma Chemical Company (St. Louis, MO). Fresh solutions were prepared daily with either deionized water or normal saline as the diluent. The concentrations are expressed as the final molar concentrations in the organ chambers. Statistical analysis. Statistical analyses were performed with a MacIntosh Computer and StatView software (Brain Power, Inc., Calabasas, CA). Data are presented as mean { one SEM. Statistical evaluation of the concentration–response curves and for determination of lung MPO utilized standard one way analysis of variance (ANOVA) with post hoc Bonferroni-Dunn test. A P value of less than 0.05 was accepted as statistically significant.
RESULTS
Effect of endotoxin alone. As shown in Table 1, lung neutrophil accumulation was significantly increased 4 hr after endotoxin. After 4 hr of mechanical ventilation following saline injection, MPO activity was 3.3 { 1.0
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FIG. 3. Endothelial-independent cGMP-mediated pulmonary vasorelaxation was not impaired following endotoxin. Maximal relaxation (efficacy) produced by sodium nitroprusside was not different from controls. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of nitroprusside.
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mained as compared to 11 { 4 mmHg tension in controls (P õ 0.05 versus control, not different from endotoxin alone). DISCUSSION
FIG. 4. Continuous administration of inhaled nitric oxide (NO) prevented endotoxin-induced impairment of endothelial-dependent cGMP-mediated pulmonary vasorelaxation. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
endotoxin. However, at a concentration of SNP 1006 M, complete ring relaxation was produced in controls as well as following endotoxin. Effect of continuous NO administration following endotoxin. Continuous administration of inhaled NO prevented the endotoxin-induced accumulation of lung neutrophils. As shown in Table 1, lung MPO activity following 4 hr of administration of inhaled NO alone was not increased over controls. Lung MPO activity determined 4 hr after endotoxin plus continuous administration of NO was likewise not different from control (5.0 { 0.5 U/g lung tissue). Continuous NO administration attenuated the dysfunction of endothelial-dependent pulmonary vasorelaxation at 4 hr following endotoxin. As shown in Fig. 4, pulmonary artery rings from rats treated with endotoxin plus 4 hr of continuous NO were preconstricted to 292 { 19 mg tension. ACh 1006 M resulted in 39 { 13 mg PE-induced tension remaining (P õ 0.05 versus endotoxin alone, not different from control). Effect of abbreviated NO administration following endotoxin. Administration of inhaled NO for only the first 2 of 4 hr after endotoxin failed to prevent endotoxin-induced lung neutrophil accumulation at 4 hr following endotoxin. As shown in Table 1, lung MPO activity was 13.2 { 1.0 U/g lung tissue 4 hr after endotoxin alone. In the abbreviated NO treatment group, lung MPO was 11.0 { 2.0 U/g lung tissue 4 hr after endotoxin (P õ 0.05 versus controls, not different from endotoxin alone). Endothelial-dependent cGMP-mediated pulmonary vasorelaxation remained significantly impaired in the abbreviated NO treatment group and was not different from endotoxin alone. As shown in Fig. 5, pulmonary artery rings from rats in the abbreviated NO treatment group were preconstricted to 270 { 16 mmHg. At ACh 1006 M, 139 { 23 mmHg PE-induced ring tension re-
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The results of this study demonstrate that endothelial-dependent cGMP-mediated pulmonary vasorelaxation is impaired following endotoxin. This vasomotor dysfunction was associated with a significant increase in lung neutrophil accumulation. Examination of endothelial-independent cGMP-mediated pulmonary vasorelaxation demonstrated that the concentration of SNP required to produce maximal pulmonary artery ring relaxation (efficacy of SNP) [20] was unchanged after endotoxin. Administration of inhaled NO beginning just prior to endotoxin exposure and for the duration of the experimental period (continuous group) was found to prevent the increase in lung neutrophil accumulation and to attenuate the impairment of endothelial-dependent vasorelaxation. Administration of inhaled NO for only the first 2 of 4 hr following endotoxin exposure (abbreviated NO group) did not prevent increased lung neutrophil accumulation at 4 hr after endotoxin, nor was dysfunction of endothelial-dependent pulmonary vasorelaxation prevented in the abbreviated NO group. Together these data suggest that administration of exogenous NO by a therapeutically available route (inhaled NO) is able to prevent endotoxin-induced lung neutrophil accumulation and attenuate dysfunction of endothelial-dependent pulmonary vasorelaxation in endotoxin-induced acute lung injury. However, these effects may require a continuous exposure to exogenous NO from prior to the onset of endotoxin exposure; cessation of inhaled NO in the setting of the inflammatory milieu created by endotoxin exposure (abbreviated group) resulted in increased lung neutro-
FIG. 5. Abbreviated administration of inhaled nitric oxide (NO) did not prevent endotoxin-induced impairment of endothelial-dependent cGMP-mediated pulmonary vasorelaxation. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
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phil accumulation and impairment of endothelial-dependent pulmonary vasorelaxation. Endotoxin produces lung injury in laboratory animals, which resembles the adult respiratory distress syndrome (ARDS) both functionally and histologically [21]. Using lung neutrophil accumulation as a parameter of lung injury, endotoxin injection produced acute lung injury in the present study. Preservation of endothelial-dependent vasorelaxation by administration of inhaled NO in the continuous delivery group was associated with a decrease in endotoxin-induced lung neutrophil accumulation. These data implicate the neutrophil in the pathogenesis of pulmonary vascular endothelial cell dysfunction in endotoxin-induced acute lung injury. An important function of endothelial-derived NO is the prevention of neutrophil/endothelial cell adherence. Using cat coronary artery rings subjected to ischemia/reperfusion, Lefer and colleagues found an association between decreased basal NO production from cat coronary artery rings and increased neutrophil adhesion to the coronary endothelium [8, 9]. These results implicate endothelial injury and the resultant loss of basal NO production in the development of pathologic vasoconstriction following vascular injury. Lefer and colleagues have also demonstrated attenuation of vascular endothelial cell dysfunction following ischemia/ reperfusion using NO donating compounds [10–12]. Kubes and colleagues reported the leukocyte adherence was increased in the feline mesenteric artery following administration of inhibitors of NO production (LNAME and L-NMMA) suggesting that endothelial-derived NO may be an important modulator of leukocyte adherence [6]. Arndt and colleagues also reported a significant increase in leukocyte adherence in rat mesenteric arteries perfused with inhibitors of NO production (L-NAME) [7]. In the lung, Addih and colleagues reported NO attenuation of ischemia/reperfusion induced lung injury in the rat [22]. Pulmonary hypertension secondary to pulmonary vasoconstriction is the major hemodynamic feature of ARDS [23]. The use of inhaled NO as a treatment modality for ARDS is based upon the ability of inhaled NO to selectively vasodilate the pulmonary circulation [24]. This pulmonary vasodilation is generally thought to occur by direct pulmonary vascular smooth muscle relaxation by the inhaled NO. However, the results of the present study suggest that inhaled NO may preserve pulmonary vascular endothelial function in acute lung injury. In this way, pulmonary vascular tone may be reduced. In summary, administration of exogenous NO (inhaled NO) prevented increased lung neutrophil accumulation and impairment of endothelial-dependent pulmonary vasorelaxation following endotoxin. We conclude that inhaled NO prevents neutrophil-mediated pulmonary vasomostor dysfunction in acute lung injury.
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REFERENCES 1. Dinh-Xuan, A. T., Higenbottam, T. W., and Clelland, C. A. et al. Impairment of endothelium-dependent pulmonary artery relaxation in chronic obstructive lung disease. N. Engl. J. Med. 324: 1539, 1991. 2. Ignarro, L. J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. Rev. Pharmacol. Toxicol. 30: 535, 1990. 3. Gwitner, G. H., Knoblauch, A., Smith, P. L., Sies, H., and Adkinson, N. F. Oxidant- and lipid-induced pulmonary vasoconstriction mediated by arachidonic acid metabolites. J. Appl. Physiol. 55: 949, 1983. 4. Tate, R. M., Vanbenthuysen, K. M., Shasby, D. M., McMurtry, I. F., Repine, J. E. Oxygen radical mediated permeability edema and vasoconstriction in isolated perfused rabbit lungs. Am. Rev. Respir. Dis. 126: 802, 1982. 5. Fullerton, D. A., McIntyre, R. C. Jr., Hahn, A. R., Agrafojo, J., Koike, K., Meng, X., Banerjee, A., and Harken, A. H. Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxininduced acute lung injury. Am. J. Phyiol. 268: L1029, 1995. 6. Kubes, P., Suzuki, M., and Granger, D. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. U.S.A. 88: 4651, 1991. 7. Arndt, H., Russell, J., Kurose, I., Kubes, P., and Granger, D. Mediators of leukocyte adhesion in rat mesenteric venules elicited by inhibition of nitric oxide synthesis. Gastroenterology 105: 675–680, 1993. Am. Rev. Respir. Dis. 126: 802, 1982. 8. Lefer, A., and Ma, X. Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler. Thromb. 13: 771, 1993. 9. Ma, X., Weyrich, A., Lefer, D., and Lefer, A. Diminished basal nitric oxide release after myocardial ishemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ. Res. 72: 403, 1993. 10. Siegfried, M. R., Erhardt, J., Rider, T., Ma, X., and Lefer, A. M. Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion. J. Pharmacol. Exp. Ther. 260: 668, 1992. 11. Siegfried, M. R., Carey, C., Ma, X., and Lefer, A. M. Beneficial effects of SPM-5185, a cysteine-containing NO donor in myocardial ischemia-reperfusion. Am. J. Physiol. 263: H771, 1992. 12. Carey, C., Siegfried, M. R., Ma, X., Weyrich, A. S., and Lefer, A. M. Antishock and endothelial protective actions of a NO donor in mesenteric ischemia and reperfusion. Circ. Shock 38: 209, 1992. 13. Lefer, A., Ma, X., Weyrich, A., and Lefer, D. Endothelial dysfunction and neutrophil adherence as critical events in the development of reperfusion injury. Agents Actions Suppl. 41: 127, 1993. 14. Grair, W. F., Myers, P. R., Rubin, L. J., Adams, R., and Parker, J. L. Exchericia coli endotoxin inhibits agonist-mediated cytosolic calicium mobilization and nitric oxide biosynthesis in cultured endothelial cells. Circ. Res. 75: 659, 1994. 15. Finn, A., Strobel, S., Levin, M., and Klein, N. Endotoxin-induced neutrophil adnerence to endothelium: relationship to CD11b/CD18 and L-selectin expression and matrix disruption. Ann. NY Acad. Sci. 725: 173, 1994. 16. Lynam, E., Simon, S., Rochon, Y., and Skiar, L. Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation. Blood 83: 3303, 1994. 17. Hess, D., Bhutwala, T., Sheppard, J., Zhao, W., and Smith, J. ICAM-1 expression on human brain microvascular endothelial cells. Neurosci. Lett. 168: 201, 1994. 18. Montgomery, K., Osborn, L., Hession, C., Tizard, R., Goff, D., Vassallo, C., Tarr, P., Bomsztyk, K., Lobb, R., and Harlan, J. Activation of endothelial-leukocyte adhesion molecule-1
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19. Barmann, T. Enzyme Handbook. New York: Springer Verlag, 1996. Pp. 234–236. 20. Ross, E. M. Pharmacodynamics: Mechanisms of drug action and the relationship between drug concentration and effect. In: A. G. Goodman, T. W. Rall, A. S. Nies, and P. Taylor (Eds.), Goodman and Gilman’s The Pharmacologic Basis of Therapeutics, 8th Edition. New York: McGraw-Hill, Inc., 1993. Pp. 33– 48. 21. Anderson, B. O., Bensard, D. D., Brown, J. M., Repine, J. E., Shanley, P. F., Leff, J. A., Terada, L. S., Banerjee, A., and Harken, A. H. FNLP injures endotoxin-primed rat lung by neu-
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trophil-dependent and -independent mechanisms. Am. J. Physiol. 260: R413, 1991. 22. Abidh, H., Kelly, C., Bouchier-Hayes, D., William, R., Watson, G., Redmond, H., and Burke, P. Nitric oxide (endothelium derived relaxing factor) attenuates revascularization induced lung injury. J. Surg. Res. 57: 39, 1994. 23. Zapol, W. M., and Snider, M. T. Pulmonary hypertension in severe acute respiratory failure. N. Engl. J. Med. 296: 476, 1977. 24. McIntyre, R. C. Jr., Moore, F. A., Moore, E. E., Piedalue, F., Haenel, J. S., and Fullerton, D. A. Inhaled nitric oxide variably improves oxygenation and pulmonary hypertension in patients with acute respiratory distress syndrome. J. Trauma 39: 418, 1995.
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NO Prevents Neutrophil-Mediated Pulmonary Vasomotor Dysfunction in Acute Lung Injury1 RANDALL S. FRIESE, M.D., DAVID A. FULLERTON, M.D., ROBERT C. MCINTYRE, JR., M.D., THOMAS F. REHRING, M.D., JEANETTE AGRAFOJO, B.S., ANIRBAN BANERJEE, PH.D., AND ALDEN H. HARKEN, M.D. The University of Colorado Health Sciences Center, Denver, Colorado 80262 Presented at the Annual Meeting of the Association for Academic Surgery, Dearborn, Michigan, November 8–11, 1995
injury may contribute to pulmonary vasoconstriction by shifting net pulmonary vascular smooth muscle tone in favor of vasoconstriction. cGMP-mediated pulmonary vasorelaxation is impaired in the rat following endotoxin in association with increased lung neutrophil accumulation and histologic evidence of significant endothelial cell injury [5]. Another important role of endothelial-derived NO is to help prevent neutrophil adhesion to endothelial cells [6, 7]. In the coronary and mesenteric circulations, deficiency of endothelial-derived NO has been shown to exaggerate neutrophil-associated vascular injury [8, 9]. On the other hand, administration of exogenous NO (via NO donors) attenuates neutrophil-associated vascular injury [10–12]; exogenous NO may augment vascular endothelial cell defense mechanisms against neutrophils [13]. Such studies offer insight into the pathogenesis of endotoxin-induced pulmonary vasomotor dysfunction. In cultured endothelial cells, Graier and colleagues have shown that endotoxin inhibits the synthesis of NO [14]. Endotoxin also promotes the expression of neutrophil and endothelial cell adhesion molecules [15–18]. Therefore, following endotoxin, the pulmonary vascular endothelium may be particularly susceptible to neutrophil-associated injury. In the present study, we sought to augment the defense mechanisms of the pulmonary vascular endothelium by administration of exogenous NO. We hypothesized that administration of exogenous NO by a therapeutically accessible route, inhaled NO, attenuates endotoxin-induced lung neutrophil accumulation and dysfunction of endothelial-dependent cGMPmediated pulmonary vasorelaxation. The purpose of this study was to examine the effect of exogenous NO (inhaled NO) on lung neutrophil accumulation (myeloperoxidase assay, MPO) and cGMP-mediated pulmonary vasorelaxation following endotoxin injection in rats. Using isolated pulmonary arterial rings, we examined the following mechanisms of pulmonary vascular smooth muscle relaxation: (1) endothelial-dependent cGMP-mediated vasorelaxation (response to acetylcholine, ACh) and (2) endothelial-independent cGMP-me-
The purpose of this study was to examine the effect of administration of inhaled nitric oxide (NO) on lung neutrophil accumulation and pulmonary vascular endothelial cell function in endotoxin-induced acute lung injury. Mechanically ventilated rats were studied 4 hr after endotoxin (0.5 mg/kg IP). Inhaled NO (20 ppm) was administered for either the entire 4 hr after endotoxin (continuous group) or for only the first 2 of 4 hr after endotoxin (abbreviated group). Endothelialdependent (acetylcholine, ACh) and -independent cGMP-mediated relaxation (nitroprusside, SNP) pulmonary vasorelaxation were studied in isolated pulmonary arterial rings. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). Inhaled NO prevented endotoxin-induced lung neutrophil accumulation as well as pulmonary endothelial cell dysfunction. However, this protection required continuous administration of inhaled NO. We conclude that inhaled NO prevents neutrophil-mediated pulmonary vascular endothelial cell dysfunction in acute lung injury. q 1996 Academic Press, Inc.
INTRODUCTION
Net pulmonary vascular smooth muscle tone results from the mechanistic balance of vasoconstriction and vasorelaxation. In the normal lung, basal endothelial release of the endogenous vasodilator, nitric oxide (NO), contributes to the low pulmonary vascular smooth muscle tone [1]. Endothelial-derived NO lowers pulmonary vascular tone by stimulating guanylate cyclase in subjacent vascular smooth muscle cells to generate cGMP, which produces pulmonary vascular smooth muscle relaxation [2]. In acute lung injury, increased levels of circulating or local vasoconstricting agonists may contribute to increased pulmonary vascular tone [3, 4]. On the other hand, impairment of the mechanisms of pulmonary vasorelaxation in acute lung 1
Supported by NIH Grant R29HL49398.
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diated vasorelaxation by stimulation of vascular smooth muscle guanylate cyclase (response to sodium nitroprusside, SNP). The results of this study demonstrate that administration of exogenous NO attenuates pulmonary vascular endothelial dysfunction following endotoxin. METHODS All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Animal housing and acclimation. Male Sprague–Dawley rats (Sasco, Inc., Omaha, NE) weighing 350–450 g were quarantined in quiet, humidified, light-cycled rooms for 2–3 weeks before use. Rats were allowed ad libitum access to food and water throughout quarantine up to the time of experimentation. Experimental protocols. After induction of anesthesia (sodium pentobarbital 50 mg/kg, IP) and tracheotomy, rats were mechanically ventilated (Pressure Controlled Respirator model RSP1002, Kent Scientific Corp.) with a fraction of inspired oxygen 0.4, respiratory rate 40 breathsrmin01, peak inspiratory pressure 9–11 cm H2O, and an inspiratory:expiratory ratio 1:2. End-tidal CO2 was continuously monitored by capnography and maintained between 35–45 mmHg. The left femoral artery was cannulated with heparinized polyethylene tubing for continuous arterial blood pressure monitoring. Arterial blood pressure was recorded at 80 Hz using a MacLab Data Interface Module (ADI Instruments, Milford, MA) and a MacIntosh computer (Apple Computer, Cupertino, CA). Anesthesia was maintained throughout the experimental protocol (sodium pentobarbital 10 mg/kg IP at 40- to 50-min intervals). Endotoxin (Salmonella typhimurium, 0.5 mg/kg IP) was freshly prepared in 0.9% saline (0.5 mg/ml). Saline-injected controls received 0.9% saline (1 cc IP). Rats were studied at either 2 or 4 hr after injection. Inhaled NO (Scott Medical Products, Plumsteadville, PA) was administered via the inspiratory arm of the ventilator circuit. The concentration of inhaled NO administered was continuously monitored by chemiluminescence (Model 42H, Thermo Environmental Instruments, Inc., Franklin, MA) and maintained at 20 ppm. Effect of exogenous NO administration. Two experimental protocols were employed to examine the effect of the timing and duration of exogenous NO administration on lung neutrophil accumulation and cGMP-mediated pulmonary vasorelaxation. Continuous NO administration. To examine the effect of NO administration from just prior to the onset of endotoxin exposure, NO was administered continuously beginning 15 min prior to endotoxin injection and for 4 hr after endotoxin injection. Rats were studied 4 hr after endotoxin injection (Fig. 1A). Abbreviated NO administration. To examine the effect of NO administration for the first 2 of 4 hr following endotoxin, inhaled NO administration was initiated beginning 15 min prior to endotoxin injection and continued for 2 hr after endotoxin injection. Inhaled NO was then stopped and rats were studied 4 hr after endotoxin injection (Fig. 1B). Isolated pulmonary arterial ring preparation. At the conclusion of the experimental protocol, median sternotomy was performed and heparin sulfate (500 USP) was injected into the right ventricular outflow tract. After removal of the heart and lungs the main pulmonary and the right and left pulmonary arteries were dissected out and placed in Earle’s balanced salts solution (EBSS) at 47C. Under dissecting microscope magnification, the surrounding tissue was dissected from the pulmonary arteries. The right and left main branch pulmonary arteries were each then cut into rings, each 3–4 mm wide; two pulmonary arterial rings were obtained from each rat. Care was taken during this process to avoid endothelial injury. EBSS is a standard physiologic salt solution and contains CaCl2 1.80 mM, MgSO4 (anhydrous) 0.83 mM, KCl 5.36 mM, NaCl 116.34 mM,
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FIG. 1. Experimental protocols used to examine the effect of inhaled nitric oxide (NO) on endotoxin-induced lung neutrophil accumulation and impairment of cGMP-mediated pulmonary vasorelaxation. (A) Continuous administration of inhaled NO; (B) abbreviated administration of inhaled NO.
NaPO4 0.40 mM (dibasic), D-glucose 5.50 mM, NaHCO3 19.04 mM, phenol red Na 0.03 mM (as pH indicator). The pulmonary artery rings were then placed on 11-mil steel wires and suspended in individual 10-cc organ chambers containing EBSS at 377C. The organ chambers were surrounded by water jackets and continually warmed (377C). Ring tension was determined by use of a force-displacement transducer (Grass FTO3, Grass Instruments Co., Quincy, MA) attached to each steel wire apparatus. Force displacement was recorded at 0.67 Hz using a MacLab Data Interface Module (ADI Instruments, Milford, MA) on a Macintosh IIci computer (Apple Computer, Cupertino, CA). Each organ chamber had continual bubbling gas flow at 40 cc/min of 21% oxygen, 5% carbon dioxide and 74% nitrogen. This produced a PO2 of 100–110 mmHg and a pH of approximately 7.4. Pulmonary vasorelaxation by cGMP-mediated mechanisms. Concentration–response curves to acetylcholine and sodium nitroprusside. Cumulative concentration–response curves were generated for ACh and SNP. In a previous study, 750 mg was determined to be the optimal resting mechanical tension (passive load) for the study of isolated pulmonary arterial rings [5]. Rings were suspended at 750 mg and allowed to reach a steady state for 1 hr, during which time the EBSS was changed every 15 min. A given ring was preconstricted with PE to achieve a PE-induced ring tension of between 200 and 400 mg. Cumulative concentration–response curves were then generated over the concentration range of 1009 –1006 M. For determination of the concentration–response curve, the ring was allowed to reach a steady state, usually requiring 2–3 min, before advancing to the next higher concentration. The ring tension remaining in the rings in response to each dose of vasorelaxing agent was expressed in milligrams of PE-induced tension. Lung harvest for myeloperoxidase assay. At the conclusion of the experimental protocol and after median sternotomy, rats were heparinized (500 USP) via injection into the right ventricular outflow tract. Lungs are perfused via the pulmonary artery for 2 min with
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TABLE 1 Lung Neutrophil Accumulation Lung MPO activity (u/g lung tissue) Saline controls Endotoxin alone NO alone Continuous NO / endotoxin Abbreviated NO / endotoxin
3.3 13.2 4.9 5.0 11.0
{ { { { {
1.0 1.0* 1.0 0.5 2.0*
Note. MPO, myeloperoxidase; NO, nitric oxide; *P õ 0.05 versus saline controls.
modified Krebs-Hensleit solution at 377C (pH 7.35–7.45) containing 4 g/100 ml Ficoll and 10 mg/liter N-2 hydroxyethylpiperazine N1-2ethnesulfonic acid (HEPES) at 0.04 ml-g body wt01 min01 via perfusion with a peristaltic pump (Masterflex pump; Cole Parmer, Inc., Chicago, IL). Lungs were externally rinsed with saline, blotted dry, and separately weighed. Lung myeloperoxidase assay. Lung tissue was homogenized for 30 sec (Virtishear homogenizer; Virtis Co., Inc., Gardner, NY) in a 4-ml 20-mmole/liter potassium phosphate buffer, pH 7.4 and centrifuged for 30 min at 40,000 g, 47C (Sorvall RC-5B centrifuge, 5 M245 rotor, 18,000 rpm) (Dupont Instruments, Inc., Irving, TX). The pellet was resuspended in 4 ml of 50 mM potassium phosphate buffer, pH 6.0, containing 0.5 g/dl cetrimonium bromide. Resuspended pellets were frozen at 0707C until the myeloperoxidase assay was performed. Frozen samples were thawed, sonicated for 90 sec at full power (ultrasonic cell disrupter; Kontes, Vineland, NJ), incubated in a 607C water bath for 2 hr, and centrifuged for 10 min at maximum speed (Beckman Microfuge 12; Beckman Instruments, Irvine, CA). Supernatant, 0.1 ml, was added to 2.9 ml of 50 mM potassium phosphate buffed, pH 6.0, containing 0.167 mg/ml o-dianisidine and 5 1 1004% hydrogen peroxide; absorbance of 460 nm visible light was measured for 3 min (Beckman DU7 spectrophotometer; Beckman Instruments). MPO activity per gram wet lung (gwl) was calculated by Myeloperoxidase activity (units/gwl) Å
(A460 )(13.5) , Lung Weight (gm)
FIG. 2. Endothelial-dependent cGMP-mediated pulmonary vasorelaxation was significantly impaired following endotoxin. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
U/g lung tissue. Four hours following endotoxin injection, MPO activity was increased to 13.2 { 1.0 U/g lung tissue (P õ 0.05 versus control). As shown in Fig. 2, endothelial-dependent cGMPmediated pulmonary vasorelaxation was impaired following endotoxin. In saline-injected controls, pulmonary artery rings were preconstricted with PE to 292 { 16 mg tension and ACh 1006 M produced complete relaxation of isolated pulmonary artery rings (11 { 4 mg ring tension remaining). Four hours following endotoxin, ACh 1006 M produced relaxation to 186 { 21 mg tension (P õ 0.05 versus control). On the other hand, maximal ring relaxation by endothelial-independent cGMP-mediated pulmonary vasorelaxation was not impaired following endotoxin. As shown in Fig. 3, the response to SNP was different from control only at a concentration of 1007 M 4 hr following
where A460 is the change in absorbance of 460 nm light from 1 to 3 min after the initiation of the reaction. The coefficient 13.5 was empirically determined such that 1 U myeloperoxidase activity is the amount of enzyme that will reduce 1 mmole peroxide/min [19]. Reagents. Reagents were obtained from Sigma Chemical Company (St. Louis, MO). Fresh solutions were prepared daily with either deionized water or normal saline as the diluent. The concentrations are expressed as the final molar concentrations in the organ chambers. Statistical analysis. Statistical analyses were performed with a MacIntosh Computer and StatView software (Brain Power, Inc., Calabasas, CA). Data are presented as mean { one SEM. Statistical evaluation of the concentration–response curves and for determination of lung MPO utilized standard one way analysis of variance (ANOVA) with post hoc Bonferroni-Dunn test. A P value of less than 0.05 was accepted as statistically significant.
RESULTS
Effect of endotoxin alone. As shown in Table 1, lung neutrophil accumulation was significantly increased 4 hr after endotoxin. After 4 hr of mechanical ventilation following saline injection, MPO activity was 3.3 { 1.0
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FIG. 3. Endothelial-independent cGMP-mediated pulmonary vasorelaxation was not impaired following endotoxin. Maximal relaxation (efficacy) produced by sodium nitroprusside was not different from controls. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of nitroprusside.
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mained as compared to 11 { 4 mmHg tension in controls (P õ 0.05 versus control, not different from endotoxin alone). DISCUSSION
FIG. 4. Continuous administration of inhaled nitric oxide (NO) prevented endotoxin-induced impairment of endothelial-dependent cGMP-mediated pulmonary vasorelaxation. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
endotoxin. However, at a concentration of SNP 1006 M, complete ring relaxation was produced in controls as well as following endotoxin. Effect of continuous NO administration following endotoxin. Continuous administration of inhaled NO prevented the endotoxin-induced accumulation of lung neutrophils. As shown in Table 1, lung MPO activity following 4 hr of administration of inhaled NO alone was not increased over controls. Lung MPO activity determined 4 hr after endotoxin plus continuous administration of NO was likewise not different from control (5.0 { 0.5 U/g lung tissue). Continuous NO administration attenuated the dysfunction of endothelial-dependent pulmonary vasorelaxation at 4 hr following endotoxin. As shown in Fig. 4, pulmonary artery rings from rats treated with endotoxin plus 4 hr of continuous NO were preconstricted to 292 { 19 mg tension. ACh 1006 M resulted in 39 { 13 mg PE-induced tension remaining (P õ 0.05 versus endotoxin alone, not different from control). Effect of abbreviated NO administration following endotoxin. Administration of inhaled NO for only the first 2 of 4 hr after endotoxin failed to prevent endotoxin-induced lung neutrophil accumulation at 4 hr following endotoxin. As shown in Table 1, lung MPO activity was 13.2 { 1.0 U/g lung tissue 4 hr after endotoxin alone. In the abbreviated NO treatment group, lung MPO was 11.0 { 2.0 U/g lung tissue 4 hr after endotoxin (P õ 0.05 versus controls, not different from endotoxin alone). Endothelial-dependent cGMP-mediated pulmonary vasorelaxation remained significantly impaired in the abbreviated NO treatment group and was not different from endotoxin alone. As shown in Fig. 5, pulmonary artery rings from rats in the abbreviated NO treatment group were preconstricted to 270 { 16 mmHg. At ACh 1006 M, 139 { 23 mmHg PE-induced ring tension re-
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The results of this study demonstrate that endothelial-dependent cGMP-mediated pulmonary vasorelaxation is impaired following endotoxin. This vasomotor dysfunction was associated with a significant increase in lung neutrophil accumulation. Examination of endothelial-independent cGMP-mediated pulmonary vasorelaxation demonstrated that the concentration of SNP required to produce maximal pulmonary artery ring relaxation (efficacy of SNP) [20] was unchanged after endotoxin. Administration of inhaled NO beginning just prior to endotoxin exposure and for the duration of the experimental period (continuous group) was found to prevent the increase in lung neutrophil accumulation and to attenuate the impairment of endothelial-dependent vasorelaxation. Administration of inhaled NO for only the first 2 of 4 hr following endotoxin exposure (abbreviated NO group) did not prevent increased lung neutrophil accumulation at 4 hr after endotoxin, nor was dysfunction of endothelial-dependent pulmonary vasorelaxation prevented in the abbreviated NO group. Together these data suggest that administration of exogenous NO by a therapeutically available route (inhaled NO) is able to prevent endotoxin-induced lung neutrophil accumulation and attenuate dysfunction of endothelial-dependent pulmonary vasorelaxation in endotoxin-induced acute lung injury. However, these effects may require a continuous exposure to exogenous NO from prior to the onset of endotoxin exposure; cessation of inhaled NO in the setting of the inflammatory milieu created by endotoxin exposure (abbreviated group) resulted in increased lung neutro-
FIG. 5. Abbreviated administration of inhaled nitric oxide (NO) did not prevent endotoxin-induced impairment of endothelial-dependent cGMP-mediated pulmonary vasorelaxation. N Å 5 rats/10 pulmonary artery rings in each group. *P õ 0.05 versus control at same concentration of acetylcholine.
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phil accumulation and impairment of endothelial-dependent pulmonary vasorelaxation. Endotoxin produces lung injury in laboratory animals, which resembles the adult respiratory distress syndrome (ARDS) both functionally and histologically [21]. Using lung neutrophil accumulation as a parameter of lung injury, endotoxin injection produced acute lung injury in the present study. Preservation of endothelial-dependent vasorelaxation by administration of inhaled NO in the continuous delivery group was associated with a decrease in endotoxin-induced lung neutrophil accumulation. These data implicate the neutrophil in the pathogenesis of pulmonary vascular endothelial cell dysfunction in endotoxin-induced acute lung injury. An important function of endothelial-derived NO is the prevention of neutrophil/endothelial cell adherence. Using cat coronary artery rings subjected to ischemia/reperfusion, Lefer and colleagues found an association between decreased basal NO production from cat coronary artery rings and increased neutrophil adhesion to the coronary endothelium [8, 9]. These results implicate endothelial injury and the resultant loss of basal NO production in the development of pathologic vasoconstriction following vascular injury. Lefer and colleagues have also demonstrated attenuation of vascular endothelial cell dysfunction following ischemia/ reperfusion using NO donating compounds [10–12]. Kubes and colleagues reported the leukocyte adherence was increased in the feline mesenteric artery following administration of inhibitors of NO production (LNAME and L-NMMA) suggesting that endothelial-derived NO may be an important modulator of leukocyte adherence [6]. Arndt and colleagues also reported a significant increase in leukocyte adherence in rat mesenteric arteries perfused with inhibitors of NO production (L-NAME) [7]. In the lung, Addih and colleagues reported NO attenuation of ischemia/reperfusion induced lung injury in the rat [22]. Pulmonary hypertension secondary to pulmonary vasoconstriction is the major hemodynamic feature of ARDS [23]. The use of inhaled NO as a treatment modality for ARDS is based upon the ability of inhaled NO to selectively vasodilate the pulmonary circulation [24]. This pulmonary vasodilation is generally thought to occur by direct pulmonary vascular smooth muscle relaxation by the inhaled NO. However, the results of the present study suggest that inhaled NO may preserve pulmonary vascular endothelial function in acute lung injury. In this way, pulmonary vascular tone may be reduced. In summary, administration of exogenous NO (inhaled NO) prevented increased lung neutrophil accumulation and impairment of endothelial-dependent pulmonary vasorelaxation following endotoxin. We conclude that inhaled NO prevents neutrophil-mediated pulmonary vasomostor dysfunction in acute lung injury.
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trophil-dependent and -independent mechanisms. Am. J. Physiol. 260: R413, 1991. 22. Abidh, H., Kelly, C., Bouchier-Hayes, D., William, R., Watson, G., Redmond, H., and Burke, P. Nitric oxide (endothelium derived relaxing factor) attenuates revascularization induced lung injury. J. Surg. Res. 57: 39, 1994. 23. Zapol, W. M., and Snider, M. T. Pulmonary hypertension in severe acute respiratory failure. N. Engl. J. Med. 296: 476, 1977. 24. McIntyre, R. C. Jr., Moore, F. A., Moore, E. E., Piedalue, F., Haenel, J. S., and Fullerton, D. A. Inhaled nitric oxide variably improves oxygenation and pulmonary hypertension in patients with acute respiratory distress syndrome. J. Trauma 39: 418, 1995.
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