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HYPOCHLOROUS ACID-INDUCED RESPONSES IN SHEEP ISOLATED PULMONARY ARTERY RINGS
Pharmacological Research, Vol. 41, No. 5, 2000 doi:10.1006rphrs.1999.0628, available online at http:rrwww.idealibrary.com on
HYPOCHLOROUS ACID-INDUCED RESPONSES IN SHEEP ISOLATED PULMONARY ARTERY RINGS U ˙ ¨ ˙ ¨ NILUFER N. TURAN, A. TUNCAY DEMIRYUREK and ˙ILKER KANZIK
Department of Pharmacology, Faculty of Pharmacy, Gazi Uni¨ ersity, Etiler, TR-06330 Ankara, Turkey Accepted 11 No¨ ember 1999
The formation of reactive oxygen species ŽROS. appears to play a significant role in many pathological states including cystic fibrosis and asthma. Although stimulated inflammatory cells represent a major source of oxygen metabolites and these cells are able to generate the potent oxidant hypochlorous acid ŽHOCl. effects of HOCl on arteries are not known. HOCl at low concentrations Ž10y7 to 10y4 M. did not affect the resting force or have an action in precontracted sheep pulmonary arteries. HOCl at 10y4 M concentration reduced histamine-induced relaxations in endothelium intact preparations. However, at high concentrations Ž10y2 to 1 M. HOCl led to constriction under resting conditions and caused vasodilation in endothelium intact and denuded serotonin Ž10 m M. precontracted arteries. These effects of HOCl were significantly reduced by pretreatment of L-arginine Ž10y3 M., sodium nitroprusside ŽSNP, 10y5 M. and N-acetyl-L-cysteine ŽNAC, 10y4 M.. The effects of SNP and NAC on HOCl-induced responses were due to direct interaction since only these compounds markedly diminished the HOCl-induced luminol chemiluminescence ŽCL.. Lack of contraction with KCl after high concentrations of HOCl showed that HOCl causes irreversible tissue damage. These results suggest that HOCl produce vasoconstriction under resting force and cause vasodilation when the pulmonary arteries precontracted. HOCl may interact with endothelium-derived mediators and contribute to tissue injury and vascular dysfunction seen in disease states. Q 2000 Academic Press KEY
WORDS:
chemiluminescence, endothelium, hypochlorous acid, nitric oxide, sheep pulmonary artery.
INTRODUCTION Under inflammatory conditions, multiple welldefined ROS are generated from phagocytic cells. Stimulated neutrophils and macrophages undergo a respiratory burst discharging large quantities of superoxide as a result of the activation of the NADPH oxidase. Superoxide dismutates to form hydrogen peroxide ŽH 2 O 2 ., and most of this H 2 O 2 is converted to the strong oxidant HOCl by the reaction of myeloperoxidase, a major enzyme from neutrophil granules w1x. Under physiological conditions, it is likely that HOCl is the major oxidant produced by neutrophils, and its reactivity with a range of biological molecules makes it a potential cause of tissue injury in inflammation w2, 3x. When formed in large amounts, HOCl is thought to contribute to tissue injury and vascular dysfuncU
Corresponding author.
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tion seen in disease states. During acute inflammatory conditions such as rheumatoid arthritis, emphysema, asthma, cystic fibrosis and acute respiratory distress syndrome ŽARDS., a dramatic increase in the number of polymorphonuclear leucocytes is observed at the site of inflammation w1x. In addition to its well-documented bactericidal action w3x, HOCl is chemically reactive and can react with several biological targets. HOCl cleaves peptide bonds w4x, oxidizes amino acids w2x, damages extracellular matrix component Žcollagen, elastin, hyalunoric acid, and proteoglycans. w5x, and reacts with Fe]S centres w6x. HOCl interacts with amines Žtaurine, lysine. and tyrosine to form N-chloramines and 3-chlorotyrosine, respectively w7]9x. Another product of the myeloperoxidase]H 2 O 2 system at plasma concentrations of Cly and amino acids is tyrosyl radical w10x and both HOCl and tyrosyl radical oxidise lipoproteins by reactions that do not require free metal ions w11x. Myeloperoxidase is thought to be a pivotal agent in the development of atherosclerotic lesions Q 2000 Academic Press
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w12x. HOCl causes rapid inactivation of a 1-antiproteinase w13x. HOCl may induce DNA alterations and cell malignant transformation through a specific inactivation of the DNA repairing enzyme poly ADPribose polymerase w14x. HOCl at low concentrations can cause an increase in endothelial permeability within minutes w15x. Under pathological conditions, HOCl may be formed at high concentrations and it is documented that myeloperoxidase is a component of human atherosclerotic lesions w12x. Despite its reactivity with such a range of biological targets, direct vascular effects of HOCl have not been well defined. Additionally, the role of endothelium in vascular reactivity of HOCl, as well as contribution of NO in HOCl-induced contraction or relaxation, has not been elucidated. Therefore, the aim of this study was to investigate the effects of HOCl on pulmonary vascular tone and on endothelial function since the lung represents a prime site for reactive oxygen metabolites.
MATERIALS AND METHODS Lungs of freshly slaughtered sheep were obtained from a local abattoir and delivered in a cooled oxygenated physiological salt solution to the laboratory within 10 min of excision. Pulmonary arteries Ž2]5-mm outer diameter at their optimal resting force. were isolated from lungs that were mainly from the second branch of the main pulmonary artery. Arteries were cleared of fat and adhering connective tissue. Care was taken to avoid stretching and damage to the luminal surface. In some arteries the endothelium was removed by gentle rubbing of the internal surface with a cotton covered wooden stick. The removal of endothelium was confirmed by lack of relaxation to histamine w16x. The arteries were cut into rings of 4]5 mm long. Segments were suspended in a water-jacketed organ bath Ž10 ml. filled with Krebs]Henseleit solution Ž378C. of the following composition Žin mM.: NaCl 119; NaHCO3 25; KCl 4.6; MgCl 2 1.2; KH 2 PO4 1.2; CaCl 2 2.5; glucose 11. The solution was aerated with a gas mixture containing 95% O 2 ]5% CO 2 . The rings were suspended on a pair of stainless-steel hooks, one of which was fixed to an L-shaped rod inside the chamber and the other to an isometric transducer ŽMay FDT10-A, Ankara, Turkey. under optimum resting force. The stainless-steel hook was connected to the force displacement transducer. Isometric contractions were measured continuously on a polygraph ŽTumel, Izmir, Turkey. and recorded in a ¨ computer by using the Labsys computer program w17x. Arterial rings were equilibrated in Krebs] Henseleit solution for 1 h at their optimum resting
force. The optimum resting force of the pulmonary rings was determined as 3 g by comparing the tension developed by 40 mM KCl ŽEC 50 . under different resting forces. The isometric contractions were calculated as force developed per cross-sectional area w18, 19x. The cross-sectional area Ž A. of the artery was calculated by using the equation: A s blotted weight of the arteryrh = b where h s the distance Žcm. between the stainless-steel hook and rod with the artery ring under optimum resting force and b the density of the artery ring which has been shown to be 1.05 g cmy3 in the sheep carotid artery w20x. The artery rings were exposed to repeated Žgenerally three. applications of 40 mM KCl until two consecutive identical responses were observed before the start of the experimental protocol.
Experimental protocol In artery rings under resting force or precontracted with serotonin, the same protocol for both endothelium intact and denuded arteries was used. The presence of endothelium before HOCl was checked with histamine in 10 m M serotonin precontracted arteries w16x. HOCl was applied in a cumulative manner between the concentration of 10y7 to 1 M. Since exposure of high concentrations of HOCl to pulmonary arteries was found to produce marked depression in contraction, the experiments were performed in separate rings from the same lung. NO precursor ŽL-arginine, 10y3 M., donor ŽSNP, 10y5 M. and HOCl scavenger ŽNAC, 10y4 M. were incubated for 30 min. In the presence of these drugs HOCl responses were determined. At a 1 M concentration of HOCl, the responses were not maintained, therefore the readings were taken approximately 20 min after the addition of HOCl. At the end of the experiments, the presence of endothelium was confirmed by using histamine Ž10y8 to 10y4 M. in serotonin precontracted arteries. Contractility of smooth muscle was also determined by 40 mM KClinduced contractions.
Chemiluminescence Luminol CL was measured at 378C by using a chemiluminometer ŽBioOrbit, Turku, Finland. as described previously w21x. Hanks’ balanced salt solution ŽHBSS, 890 m l, pH 7.4. was mixed with luminol Ž250 m M, 100 m l. in a cuvette Žtotal volume of 1 ml.. HOCl, at 10y4 M, was injected to induce luminol CL. The CL produced was measured continuously for 5 min and recorded in a computer by using the Luminometer 1250 program Žversion 1.12, BioOrbit.. The same concentrations of L-arginine, SNP and NAC used in isolated organ bath experiments were examined in CL assay by adding before HOCl. Duplicate assays were performed in all experiments.
Drugs 5-Hydroxytryptamine creatine sulphate complex
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Žserotonin., potassium chloride, histamine dihydrochloride Žall dissolved in distilled water., L-Arginine, N-acetyl-L-cysteine, sodium nitroprusside, NaOCl Žall dissolved in PBS., luminol sodium Žprepared daily in 2 M NaOH and diluted with PBS., phosphatebuffered saline Ž10 mM KH 2 PO4 and 150 mM NaCl, pH 7.4., Hanks’ balanced salt solution Ž5.36 mM KCl, 0.441 mM KH 2 PO4 , 137 mM NaCl, 0.31 mM NaH 2 PO4 , 5.046 mM D-glucose, 1 mM CaCl 2 . were obtained from Sigma Chemical Company ŽSt. Louis, MO, USA.. HOCl was prepared as previously described by Vissers et al. w22x. NaOCl was diluted with PBS and the pH of the solution readjusted to 7.4 immediately prior to addition to the organ bath or CL cuvette. At this pH, the solution contains approximately 1:1 HOCl and OCly and is subsequently referred to as HOCl. During the studies with SNP, the organ bath was either covered with aluminium foil or situated in a dark environment in order to protect the drugs from photo-degradation.
Data analysis All results are expressed as means " SEM. n refers to the number of lungs used in the organ bath assay and the number of experiments in the CL study. Relaxations to HOCl and histamine are expressed as a percent of the level of serotonin precontracted. In the CL assay, all measurements correspond to the peak value of light emission, and the effects of drugs were expressed as a percent change from the response to the HOCl alone. Differences between the control and treated groups were analysed by using analysis of variance ŽANOVA. followed by Student] Newman]Keuls test. P values of less than 0.05 were considered to denote statistical significance of differences.
RESULTS
Effects of HOCl on endothelium intact and denuded pulmonary arteries under resting force HOCl did not alter the resting force at low concentrations Ž10y7 to 10y4 M. but caused concentration-dependent contractions at high concentrations Ž10y3 to 10y1 M. ŽFig. 1.. In two out of ten preparations, HOCl Ž1 M. produced further transient contractions Žfrom 6.3 to 10.2 mN mmy2 . in endothelium intact arteries. However, in five out of six preparations, HOCl Ž1 M. generated slight but transient contractions Žfrom 8.5" 1.9 to 9.7" 3.5 mN mmy2 . in endothelium denuded arteries under resting force. Since all the responses to 1 M HOCl were not maintained and declined to resting force at approximately 20 min after the HOCl administration, effects of drugs were compared at 10y1 M. The
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contractions of HOCl Ž7.6" 0.5 mN mmy2 , 10y1 M, n s 10. were significantly reduced by pretreatment of L-arginine Ž1.4" 0.3 mN mmy2 , n s 7., SNP Ž2.6 " 0.8 mN mmy2 , n s 6. and NAC Ž5.6" 0.7 mN mmy2 , n s 5. in endothelium intact arteries wFig. 2ŽA.x. At 10y1 M HOCl concentration, maximum inhibition was recorded in the presence of L-arginine. Also these drugs prevented the inhibition of contraction seen in the presence of 1 M HOCl. Similarly, pretreatment of these drugs markedly inhibited HOCl-induced contractions in endothelium denuded arteries. HOCl-induced contractions Ž10y1 M. were changed from 8.5" 1.9 mN mmy2 Ž n s 6. to 2.2" 0.3 mN mmy2 Ž n s 5., 1.4" 0.3 mN mmy2 Ž n s 5. and 3 " 0.6 mN mmy2 Ž n s 5. by L-arginine, SNP and NAC, respectively wFig. 2ŽB.x.
Effects of HOCl on endothelium intact and denuded serotonin precontracted pulmonary artery rings There was an approximately 80% inhibition in serotonin contraction in the presence of SNP Žfrom 23 " 1.9, n s 10 to 3.7" 0.8 mN mmy2 , n s 5.. However, L-arginine and NAC did not affect serotonin contractions in endothelium denuded arteries. HOCl was not effective at low concentrations, but HOCl caused vasodilation at high concentrations on 10 m M serotonin precontracted arteries. However, there was no marked difference in HOCL-induced relaxation between endothelium intact and denuded arteries. Preincubation with L-arginine Ž86.6" 2.2%, n s 5., SNP Ž140 " 3.3%, n s 6. and NAC Ž77 " 3.6%, n s 5. inhibited HOCl-induced relaxation at 1 M concentrations in endothelium intact arteries wFig. 3ŽA.x. Also in endothelium denuded arteries HOClinduced responses changed to 64 " 4% Ž n s 5., 117 " 3.5% Ž n s 6. and 71.7" 2.8% Ž n s 5. in the presence of L-arginine, SNP and NAC, respectively wFig. 3ŽB.x.
Presence of endothelium and ¨ ascular contractility
After treatment at low concentrations Ž10y7 to 10y4 M. of HOCl, histamine-induced endotheliumdependent relaxation was markedly inhibited Ž10y4 M, 30% reduction, n s 6. after HOCl treatment ŽFig. 4.. Following high concentration Ž1 M. of HOCl, KCl Ž40 mM. contractions were abolished Žfrom 19.4" 3.2, n s 8, to 0 mN mmy2 , n s 8. in endothelium intact arteries. There was also significant reduction in serotonin Ž10 m M.-induced contractions Žfrom 20.4" 2, n s 7 to 0 mN mmy2 , n s 7.. Similarly KCl Ž19.7" 3.2 mN mmy2 , n s 7. and serotonin-induced contractions Ž23.8" 2.3 mN mmy2 , n s 5. were markedly attenuated Žto 0 and 1.4" 0.1 mN mmy2 , respectively. in endothelium denuded arteries. Lack
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Fig. 1. Typical recorder tracings representing the effects of high concentrations of HOCl Ž10y2 to 1 endothelium intact Ža. and denuded Žb. sheep pulmonary arteries under resting force.
of contractions to KCl and serotonin after high concentrations of HOCl showed that HOCl caused tissue paralysis.
Chemiluminescence
HOCl at 10y4 M concentration produced 8613 " 457 mV Ž n s 5. luminol CL. This signal was not altered by L-arginine Ž9649 " 271 mV, n s 5.. However, SNP Ž170 " 9 mV, n s 5. and NAC Ž37 " 3 mV, n s 5. caused a marked inhibition in HOClinduced luminol CL ŽFig. 5..
DISCUSSION
The activation and accumulation of neutrophils at sites of tissue injury, leading to the formation of HOCl and other reactive species, is an essential feature of inflammation. Neutrophil oxidants, in particular HOCl, can cause injury to healthy tissues at sites of inflammation. In our experiment, we have
M.
in both
shown that HOCl is a vasoactive metabolite and produces both contraction under resting force or dilation in precontracted pulmonary arteries. During exposure of hearts to the oxidant HOCl, a progressive and not rapidly reversible coronary constriction was also observed, the causal mechanism remaining unclear w23x. Failure of acetylsalicylic acid and the PAF antagonist WEB 2086 to prevent the diminution in basal coronary flow excludes the cyclooxygenase product thromboxane A 2 and PAF, respectively, as possible mediators. At high concentrations, vessels were unresponsive to the contractile agonist showing that HOCl is able to cause an irreversible tissue injury to the smooth muscle of the artery. It is well known that HOCl reacts rapidly with cells to alter membrane deformability and permeability w22x. HOCl and monochloramine rapidly increased microvascular permeability in isolated perfused rat lungs w24x. HOCl can increase endothelial permeability by causing cytoskeletal shortening and cell retraction, possibly as a result of the oxidation of intracellular sulphydryls
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Fig. 2. Effects of preincubation with L-arginine Ž10y3 '., SNP Ž10y5 M, l., and NAC Ž10y4 M, v. on contractile effects of high concentrations of HOCl Ž10y3 to 1 M, B. in both endothelium-intact Ža. and denuded Žb. pulmonary arteries under resting force. U P- 0.05 indicates significantly different from the control group Ž n s 5]10..
and mobilization of zinc w24x. Both direct w25x and indirect w9, 26x studies showed that HOCl is much less effective than H 2 O 2 at causing endothelial cell lysis, despite its much higher reactivity with cellular proteins and thiols w13, 27x. Paradoxically, despite its apparent low cytotoxicity, HOCl is highly reactive with the endothelium at low concentrations, and is much more effective than other oxidants at causing rapid changes in a number of endothelial functions without causing concomitant structural damage w23x. This observation suggests that HOCl was produced in substantial concentrations at points of neutrophil] endothelium contact w28x. The high concentrations of HOCl Ž50]200 m M. were found to be disruptive in SR Ca2q-ATPase and sarcolemmal Naq]KqATPase activity w29x. At low concentrations of HOCl, we observed that endothelium-dependent relaxation responses to histamine were markedly attenuated showing that there is a functional damage in endothelium. This observation is in line with the results of the other studies
showing that pretreatment of the isolated perfused guinea pig heart with intact coronary system to the HOCl was associated with a complete loss of vasodilation in response to acetylcholine, bradykinin, the poorly hydrolysable ATP-derivative b ,g-methyleneATP and adenosine in low concentration, whereas the action of SNP remained unaltered w23x. The conclusion of this study was also that the coronary endothelium was functionally disturbed w23x. Our data show that HOCl-induced contractions under resting force and relaxations in serotonin precontracted arteries were markedly diminished in the presence of exogenously generated NO by SNP, possibly through increasing vasodilator influence. SNP abolished HOCl-induced relaxation and caused further contractions only in endothelium intact precontracted arteries. Although the mechanism of this is not known, it can be speculated that SNP caused approximately 80% inhibition of the serotonin contraction and the level of precontraction in this group can influence the responses. HOCl-induced CL is
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Fig. 3. Effects of preincubation with L-arginine Ž10y3 '., SNP Ž10y5 M, l., and NAC Ž10y4 M, v. on vasodilatory effects of high concentrations of HOCl Ž10y3 to 1 M, B. in both endothelium-intact Ža. and denuded Žb. serotonin precontracted pulmonary arteries. U P- 0.05 indicates significantly different from the control group Ž n s 5]6..
Fig. 4. Histamine-induced endothelium-dependent vasodilation before ŽB. and after Ž%. treatment of low concentration of HOCl Ž10y4 M. in serotonin precontracted pulmonary arteries. U P- 0.05 indicates significantly different from the control group Ž n s 6..
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Fig. 5. HOCl Ž10y4 M.-induced luminol chemiluminescence and direct interaction between HOCl with L-arginine Ž10y3 M., SNP Ž10y5 M., and NAC Ž10y4 M.. U P- 0.05 indicates significantly different from the control group Ž n s 5..
markedly diminished by SNP and therefore it is possible that SNP andror released NO directly interact with HOCl. In fact, we observed such an interaction in CL assay in which SNP abolished HOCl response. These results are in agreement with the previous studies showing that HOCl reacts with nitric oxide and nitrite, the autoxidation product of NO in biological fluids w30x. These reactions generate species capable of nitrosating and nitrating organic substrates. HOCl is a strong two-electron oxidant w30x and would favour the conversion of nitrite to the nitryl cation or an ‘nitryl cation-like’ species. Reaction of nitrite with HOCl forms reactive intermediate species, postulated to be nitryl chloride andror chlorine nitrite, that are also capable of nitrating, chlorinating, and dimerizing phenolic substrates such as tyrosine at physiological pH w31x. The formation of nitrotyrosine with these reactions offers an additional or alternative mechanism of tyrosine nitration independent of peroxynitrite formation w31x. Additionally, since this further contraction was observed only in endothelium intact arteries, it is likely that this interaction may generate reactive intermediates, which are able to stimulate endothelium-dependent contractile factors, which are not studied in these experiments. At the concentrations tested, L-arginine produced inhibition in HOCl-induced vasoconstriction and dilation both in endothelium intact and denuded arteries suggesting that these inhibitory effects are not dependent on endogenous NO. The underlying mechanism of this inhibition is unknown but these effects were not due to direct interaction of Larginine with HOCl since there was no inhibition of HOCl responses in the CL assay. HOCl-induced vascular effects and CL response were abolished in
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the presence of NAC indicating that it is a potent scavenger of HOCl as shown previously w32x. The estimated airway concentrations of HOCl in cystic fibrosis patients are reported to be 2.65 mM on average or a maximum of 8.15 mM w33x. HOCl will diffuse from the site of its release and might cause more remote damage; concentrations as low as 10]20 m M can oxidize membrane proteins w27x. Therefore, it is very likely that the HOCl concentrations used in our experiments could occur in the pulmonary circulation of cystic fibrosis patients as well as other patients with chronic lung infection. Recent studies have demonstrated that myeloperoxidase, catalyses HOCl formation, is a component of sputum from cystic fibrosis patients w34x, as well as other inflammatory lung diseases, and of human atherosclerotic tissue w12x, underscoring the potential importance of HOCl in the pathology of each of these disease cases. On the other hand, it is known that although basal levels of free NO are normally quite low Žnanomolar., local NO concentrations have been shown to increase to levels ranging from 4 to 30 m M under pathologic conditions w35x. NO might be involved in the protective effects against HOClinduced vasoactivity and injury. However, it is very likely that L-arginine produces protective actions against HOCl via NO-independent manner. HOCl may interact with endothelium or endothelium derived mediators, and contribute to tissue injury and vascular dysfunction seen in inflammatory disease states.
ACKNOWLEDGEMENTS This study was supported by a research grant from Gazi University ŽProject No: SBE 11r95-17.. The chemiluminometer used in this study was provided by a project ŽSBAG-1243. of the Scientific and Tech¨ .. nical Research Council of Turkey ŽTUBITAK
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HYPOCHLOROUS ACID-INDUCED RESPONSES IN SHEEP ISOLATED PULMONARY ARTERY RINGS U ˙ ¨ ˙ ¨ NILUFER N. TURAN, A. TUNCAY DEMIRYUREK and ˙ILKER KANZIK
Department of Pharmacology, Faculty of Pharmacy, Gazi Uni¨ ersity, Etiler, TR-06330 Ankara, Turkey Accepted 11 No¨ ember 1999
The formation of reactive oxygen species ŽROS. appears to play a significant role in many pathological states including cystic fibrosis and asthma. Although stimulated inflammatory cells represent a major source of oxygen metabolites and these cells are able to generate the potent oxidant hypochlorous acid ŽHOCl. effects of HOCl on arteries are not known. HOCl at low concentrations Ž10y7 to 10y4 M. did not affect the resting force or have an action in precontracted sheep pulmonary arteries. HOCl at 10y4 M concentration reduced histamine-induced relaxations in endothelium intact preparations. However, at high concentrations Ž10y2 to 1 M. HOCl led to constriction under resting conditions and caused vasodilation in endothelium intact and denuded serotonin Ž10 m M. precontracted arteries. These effects of HOCl were significantly reduced by pretreatment of L-arginine Ž10y3 M., sodium nitroprusside ŽSNP, 10y5 M. and N-acetyl-L-cysteine ŽNAC, 10y4 M.. The effects of SNP and NAC on HOCl-induced responses were due to direct interaction since only these compounds markedly diminished the HOCl-induced luminol chemiluminescence ŽCL.. Lack of contraction with KCl after high concentrations of HOCl showed that HOCl causes irreversible tissue damage. These results suggest that HOCl produce vasoconstriction under resting force and cause vasodilation when the pulmonary arteries precontracted. HOCl may interact with endothelium-derived mediators and contribute to tissue injury and vascular dysfunction seen in disease states. Q 2000 Academic Press KEY
WORDS:
chemiluminescence, endothelium, hypochlorous acid, nitric oxide, sheep pulmonary artery.
INTRODUCTION Under inflammatory conditions, multiple welldefined ROS are generated from phagocytic cells. Stimulated neutrophils and macrophages undergo a respiratory burst discharging large quantities of superoxide as a result of the activation of the NADPH oxidase. Superoxide dismutates to form hydrogen peroxide ŽH 2 O 2 ., and most of this H 2 O 2 is converted to the strong oxidant HOCl by the reaction of myeloperoxidase, a major enzyme from neutrophil granules w1x. Under physiological conditions, it is likely that HOCl is the major oxidant produced by neutrophils, and its reactivity with a range of biological molecules makes it a potential cause of tissue injury in inflammation w2, 3x. When formed in large amounts, HOCl is thought to contribute to tissue injury and vascular dysfuncU
Corresponding author.
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tion seen in disease states. During acute inflammatory conditions such as rheumatoid arthritis, emphysema, asthma, cystic fibrosis and acute respiratory distress syndrome ŽARDS., a dramatic increase in the number of polymorphonuclear leucocytes is observed at the site of inflammation w1x. In addition to its well-documented bactericidal action w3x, HOCl is chemically reactive and can react with several biological targets. HOCl cleaves peptide bonds w4x, oxidizes amino acids w2x, damages extracellular matrix component Žcollagen, elastin, hyalunoric acid, and proteoglycans. w5x, and reacts with Fe]S centres w6x. HOCl interacts with amines Žtaurine, lysine. and tyrosine to form N-chloramines and 3-chlorotyrosine, respectively w7]9x. Another product of the myeloperoxidase]H 2 O 2 system at plasma concentrations of Cly and amino acids is tyrosyl radical w10x and both HOCl and tyrosyl radical oxidise lipoproteins by reactions that do not require free metal ions w11x. Myeloperoxidase is thought to be a pivotal agent in the development of atherosclerotic lesions Q 2000 Academic Press
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w12x. HOCl causes rapid inactivation of a 1-antiproteinase w13x. HOCl may induce DNA alterations and cell malignant transformation through a specific inactivation of the DNA repairing enzyme poly ADPribose polymerase w14x. HOCl at low concentrations can cause an increase in endothelial permeability within minutes w15x. Under pathological conditions, HOCl may be formed at high concentrations and it is documented that myeloperoxidase is a component of human atherosclerotic lesions w12x. Despite its reactivity with such a range of biological targets, direct vascular effects of HOCl have not been well defined. Additionally, the role of endothelium in vascular reactivity of HOCl, as well as contribution of NO in HOCl-induced contraction or relaxation, has not been elucidated. Therefore, the aim of this study was to investigate the effects of HOCl on pulmonary vascular tone and on endothelial function since the lung represents a prime site for reactive oxygen metabolites.
MATERIALS AND METHODS Lungs of freshly slaughtered sheep were obtained from a local abattoir and delivered in a cooled oxygenated physiological salt solution to the laboratory within 10 min of excision. Pulmonary arteries Ž2]5-mm outer diameter at their optimal resting force. were isolated from lungs that were mainly from the second branch of the main pulmonary artery. Arteries were cleared of fat and adhering connective tissue. Care was taken to avoid stretching and damage to the luminal surface. In some arteries the endothelium was removed by gentle rubbing of the internal surface with a cotton covered wooden stick. The removal of endothelium was confirmed by lack of relaxation to histamine w16x. The arteries were cut into rings of 4]5 mm long. Segments were suspended in a water-jacketed organ bath Ž10 ml. filled with Krebs]Henseleit solution Ž378C. of the following composition Žin mM.: NaCl 119; NaHCO3 25; KCl 4.6; MgCl 2 1.2; KH 2 PO4 1.2; CaCl 2 2.5; glucose 11. The solution was aerated with a gas mixture containing 95% O 2 ]5% CO 2 . The rings were suspended on a pair of stainless-steel hooks, one of which was fixed to an L-shaped rod inside the chamber and the other to an isometric transducer ŽMay FDT10-A, Ankara, Turkey. under optimum resting force. The stainless-steel hook was connected to the force displacement transducer. Isometric contractions were measured continuously on a polygraph ŽTumel, Izmir, Turkey. and recorded in a ¨ computer by using the Labsys computer program w17x. Arterial rings were equilibrated in Krebs] Henseleit solution for 1 h at their optimum resting
force. The optimum resting force of the pulmonary rings was determined as 3 g by comparing the tension developed by 40 mM KCl ŽEC 50 . under different resting forces. The isometric contractions were calculated as force developed per cross-sectional area w18, 19x. The cross-sectional area Ž A. of the artery was calculated by using the equation: A s blotted weight of the arteryrh = b where h s the distance Žcm. between the stainless-steel hook and rod with the artery ring under optimum resting force and b the density of the artery ring which has been shown to be 1.05 g cmy3 in the sheep carotid artery w20x. The artery rings were exposed to repeated Žgenerally three. applications of 40 mM KCl until two consecutive identical responses were observed before the start of the experimental protocol.
Experimental protocol In artery rings under resting force or precontracted with serotonin, the same protocol for both endothelium intact and denuded arteries was used. The presence of endothelium before HOCl was checked with histamine in 10 m M serotonin precontracted arteries w16x. HOCl was applied in a cumulative manner between the concentration of 10y7 to 1 M. Since exposure of high concentrations of HOCl to pulmonary arteries was found to produce marked depression in contraction, the experiments were performed in separate rings from the same lung. NO precursor ŽL-arginine, 10y3 M., donor ŽSNP, 10y5 M. and HOCl scavenger ŽNAC, 10y4 M. were incubated for 30 min. In the presence of these drugs HOCl responses were determined. At a 1 M concentration of HOCl, the responses were not maintained, therefore the readings were taken approximately 20 min after the addition of HOCl. At the end of the experiments, the presence of endothelium was confirmed by using histamine Ž10y8 to 10y4 M. in serotonin precontracted arteries. Contractility of smooth muscle was also determined by 40 mM KClinduced contractions.
Chemiluminescence Luminol CL was measured at 378C by using a chemiluminometer ŽBioOrbit, Turku, Finland. as described previously w21x. Hanks’ balanced salt solution ŽHBSS, 890 m l, pH 7.4. was mixed with luminol Ž250 m M, 100 m l. in a cuvette Žtotal volume of 1 ml.. HOCl, at 10y4 M, was injected to induce luminol CL. The CL produced was measured continuously for 5 min and recorded in a computer by using the Luminometer 1250 program Žversion 1.12, BioOrbit.. The same concentrations of L-arginine, SNP and NAC used in isolated organ bath experiments were examined in CL assay by adding before HOCl. Duplicate assays were performed in all experiments.
Drugs 5-Hydroxytryptamine creatine sulphate complex
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Žserotonin., potassium chloride, histamine dihydrochloride Žall dissolved in distilled water., L-Arginine, N-acetyl-L-cysteine, sodium nitroprusside, NaOCl Žall dissolved in PBS., luminol sodium Žprepared daily in 2 M NaOH and diluted with PBS., phosphatebuffered saline Ž10 mM KH 2 PO4 and 150 mM NaCl, pH 7.4., Hanks’ balanced salt solution Ž5.36 mM KCl, 0.441 mM KH 2 PO4 , 137 mM NaCl, 0.31 mM NaH 2 PO4 , 5.046 mM D-glucose, 1 mM CaCl 2 . were obtained from Sigma Chemical Company ŽSt. Louis, MO, USA.. HOCl was prepared as previously described by Vissers et al. w22x. NaOCl was diluted with PBS and the pH of the solution readjusted to 7.4 immediately prior to addition to the organ bath or CL cuvette. At this pH, the solution contains approximately 1:1 HOCl and OCly and is subsequently referred to as HOCl. During the studies with SNP, the organ bath was either covered with aluminium foil or situated in a dark environment in order to protect the drugs from photo-degradation.
Data analysis All results are expressed as means " SEM. n refers to the number of lungs used in the organ bath assay and the number of experiments in the CL study. Relaxations to HOCl and histamine are expressed as a percent of the level of serotonin precontracted. In the CL assay, all measurements correspond to the peak value of light emission, and the effects of drugs were expressed as a percent change from the response to the HOCl alone. Differences between the control and treated groups were analysed by using analysis of variance ŽANOVA. followed by Student] Newman]Keuls test. P values of less than 0.05 were considered to denote statistical significance of differences.
RESULTS
Effects of HOCl on endothelium intact and denuded pulmonary arteries under resting force HOCl did not alter the resting force at low concentrations Ž10y7 to 10y4 M. but caused concentration-dependent contractions at high concentrations Ž10y3 to 10y1 M. ŽFig. 1.. In two out of ten preparations, HOCl Ž1 M. produced further transient contractions Žfrom 6.3 to 10.2 mN mmy2 . in endothelium intact arteries. However, in five out of six preparations, HOCl Ž1 M. generated slight but transient contractions Žfrom 8.5" 1.9 to 9.7" 3.5 mN mmy2 . in endothelium denuded arteries under resting force. Since all the responses to 1 M HOCl were not maintained and declined to resting force at approximately 20 min after the HOCl administration, effects of drugs were compared at 10y1 M. The
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contractions of HOCl Ž7.6" 0.5 mN mmy2 , 10y1 M, n s 10. were significantly reduced by pretreatment of L-arginine Ž1.4" 0.3 mN mmy2 , n s 7., SNP Ž2.6 " 0.8 mN mmy2 , n s 6. and NAC Ž5.6" 0.7 mN mmy2 , n s 5. in endothelium intact arteries wFig. 2ŽA.x. At 10y1 M HOCl concentration, maximum inhibition was recorded in the presence of L-arginine. Also these drugs prevented the inhibition of contraction seen in the presence of 1 M HOCl. Similarly, pretreatment of these drugs markedly inhibited HOCl-induced contractions in endothelium denuded arteries. HOCl-induced contractions Ž10y1 M. were changed from 8.5" 1.9 mN mmy2 Ž n s 6. to 2.2" 0.3 mN mmy2 Ž n s 5., 1.4" 0.3 mN mmy2 Ž n s 5. and 3 " 0.6 mN mmy2 Ž n s 5. by L-arginine, SNP and NAC, respectively wFig. 2ŽB.x.
Effects of HOCl on endothelium intact and denuded serotonin precontracted pulmonary artery rings There was an approximately 80% inhibition in serotonin contraction in the presence of SNP Žfrom 23 " 1.9, n s 10 to 3.7" 0.8 mN mmy2 , n s 5.. However, L-arginine and NAC did not affect serotonin contractions in endothelium denuded arteries. HOCl was not effective at low concentrations, but HOCl caused vasodilation at high concentrations on 10 m M serotonin precontracted arteries. However, there was no marked difference in HOCL-induced relaxation between endothelium intact and denuded arteries. Preincubation with L-arginine Ž86.6" 2.2%, n s 5., SNP Ž140 " 3.3%, n s 6. and NAC Ž77 " 3.6%, n s 5. inhibited HOCl-induced relaxation at 1 M concentrations in endothelium intact arteries wFig. 3ŽA.x. Also in endothelium denuded arteries HOClinduced responses changed to 64 " 4% Ž n s 5., 117 " 3.5% Ž n s 6. and 71.7" 2.8% Ž n s 5. in the presence of L-arginine, SNP and NAC, respectively wFig. 3ŽB.x.
Presence of endothelium and ¨ ascular contractility
After treatment at low concentrations Ž10y7 to 10y4 M. of HOCl, histamine-induced endotheliumdependent relaxation was markedly inhibited Ž10y4 M, 30% reduction, n s 6. after HOCl treatment ŽFig. 4.. Following high concentration Ž1 M. of HOCl, KCl Ž40 mM. contractions were abolished Žfrom 19.4" 3.2, n s 8, to 0 mN mmy2 , n s 8. in endothelium intact arteries. There was also significant reduction in serotonin Ž10 m M.-induced contractions Žfrom 20.4" 2, n s 7 to 0 mN mmy2 , n s 7.. Similarly KCl Ž19.7" 3.2 mN mmy2 , n s 7. and serotonin-induced contractions Ž23.8" 2.3 mN mmy2 , n s 5. were markedly attenuated Žto 0 and 1.4" 0.1 mN mmy2 , respectively. in endothelium denuded arteries. Lack
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Fig. 1. Typical recorder tracings representing the effects of high concentrations of HOCl Ž10y2 to 1 endothelium intact Ža. and denuded Žb. sheep pulmonary arteries under resting force.
of contractions to KCl and serotonin after high concentrations of HOCl showed that HOCl caused tissue paralysis.
Chemiluminescence
HOCl at 10y4 M concentration produced 8613 " 457 mV Ž n s 5. luminol CL. This signal was not altered by L-arginine Ž9649 " 271 mV, n s 5.. However, SNP Ž170 " 9 mV, n s 5. and NAC Ž37 " 3 mV, n s 5. caused a marked inhibition in HOClinduced luminol CL ŽFig. 5..
DISCUSSION
The activation and accumulation of neutrophils at sites of tissue injury, leading to the formation of HOCl and other reactive species, is an essential feature of inflammation. Neutrophil oxidants, in particular HOCl, can cause injury to healthy tissues at sites of inflammation. In our experiment, we have
M.
in both
shown that HOCl is a vasoactive metabolite and produces both contraction under resting force or dilation in precontracted pulmonary arteries. During exposure of hearts to the oxidant HOCl, a progressive and not rapidly reversible coronary constriction was also observed, the causal mechanism remaining unclear w23x. Failure of acetylsalicylic acid and the PAF antagonist WEB 2086 to prevent the diminution in basal coronary flow excludes the cyclooxygenase product thromboxane A 2 and PAF, respectively, as possible mediators. At high concentrations, vessels were unresponsive to the contractile agonist showing that HOCl is able to cause an irreversible tissue injury to the smooth muscle of the artery. It is well known that HOCl reacts rapidly with cells to alter membrane deformability and permeability w22x. HOCl and monochloramine rapidly increased microvascular permeability in isolated perfused rat lungs w24x. HOCl can increase endothelial permeability by causing cytoskeletal shortening and cell retraction, possibly as a result of the oxidation of intracellular sulphydryls
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Fig. 2. Effects of preincubation with L-arginine Ž10y3 '., SNP Ž10y5 M, l., and NAC Ž10y4 M, v. on contractile effects of high concentrations of HOCl Ž10y3 to 1 M, B. in both endothelium-intact Ža. and denuded Žb. pulmonary arteries under resting force. U P- 0.05 indicates significantly different from the control group Ž n s 5]10..
and mobilization of zinc w24x. Both direct w25x and indirect w9, 26x studies showed that HOCl is much less effective than H 2 O 2 at causing endothelial cell lysis, despite its much higher reactivity with cellular proteins and thiols w13, 27x. Paradoxically, despite its apparent low cytotoxicity, HOCl is highly reactive with the endothelium at low concentrations, and is much more effective than other oxidants at causing rapid changes in a number of endothelial functions without causing concomitant structural damage w23x. This observation suggests that HOCl was produced in substantial concentrations at points of neutrophil] endothelium contact w28x. The high concentrations of HOCl Ž50]200 m M. were found to be disruptive in SR Ca2q-ATPase and sarcolemmal Naq]KqATPase activity w29x. At low concentrations of HOCl, we observed that endothelium-dependent relaxation responses to histamine were markedly attenuated showing that there is a functional damage in endothelium. This observation is in line with the results of the other studies
showing that pretreatment of the isolated perfused guinea pig heart with intact coronary system to the HOCl was associated with a complete loss of vasodilation in response to acetylcholine, bradykinin, the poorly hydrolysable ATP-derivative b ,g-methyleneATP and adenosine in low concentration, whereas the action of SNP remained unaltered w23x. The conclusion of this study was also that the coronary endothelium was functionally disturbed w23x. Our data show that HOCl-induced contractions under resting force and relaxations in serotonin precontracted arteries were markedly diminished in the presence of exogenously generated NO by SNP, possibly through increasing vasodilator influence. SNP abolished HOCl-induced relaxation and caused further contractions only in endothelium intact precontracted arteries. Although the mechanism of this is not known, it can be speculated that SNP caused approximately 80% inhibition of the serotonin contraction and the level of precontraction in this group can influence the responses. HOCl-induced CL is
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Fig. 3. Effects of preincubation with L-arginine Ž10y3 '., SNP Ž10y5 M, l., and NAC Ž10y4 M, v. on vasodilatory effects of high concentrations of HOCl Ž10y3 to 1 M, B. in both endothelium-intact Ža. and denuded Žb. serotonin precontracted pulmonary arteries. U P- 0.05 indicates significantly different from the control group Ž n s 5]6..
Fig. 4. Histamine-induced endothelium-dependent vasodilation before ŽB. and after Ž%. treatment of low concentration of HOCl Ž10y4 M. in serotonin precontracted pulmonary arteries. U P- 0.05 indicates significantly different from the control group Ž n s 6..
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Fig. 5. HOCl Ž10y4 M.-induced luminol chemiluminescence and direct interaction between HOCl with L-arginine Ž10y3 M., SNP Ž10y5 M., and NAC Ž10y4 M.. U P- 0.05 indicates significantly different from the control group Ž n s 5..
markedly diminished by SNP and therefore it is possible that SNP andror released NO directly interact with HOCl. In fact, we observed such an interaction in CL assay in which SNP abolished HOCl response. These results are in agreement with the previous studies showing that HOCl reacts with nitric oxide and nitrite, the autoxidation product of NO in biological fluids w30x. These reactions generate species capable of nitrosating and nitrating organic substrates. HOCl is a strong two-electron oxidant w30x and would favour the conversion of nitrite to the nitryl cation or an ‘nitryl cation-like’ species. Reaction of nitrite with HOCl forms reactive intermediate species, postulated to be nitryl chloride andror chlorine nitrite, that are also capable of nitrating, chlorinating, and dimerizing phenolic substrates such as tyrosine at physiological pH w31x. The formation of nitrotyrosine with these reactions offers an additional or alternative mechanism of tyrosine nitration independent of peroxynitrite formation w31x. Additionally, since this further contraction was observed only in endothelium intact arteries, it is likely that this interaction may generate reactive intermediates, which are able to stimulate endothelium-dependent contractile factors, which are not studied in these experiments. At the concentrations tested, L-arginine produced inhibition in HOCl-induced vasoconstriction and dilation both in endothelium intact and denuded arteries suggesting that these inhibitory effects are not dependent on endogenous NO. The underlying mechanism of this inhibition is unknown but these effects were not due to direct interaction of Larginine with HOCl since there was no inhibition of HOCl responses in the CL assay. HOCl-induced vascular effects and CL response were abolished in
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the presence of NAC indicating that it is a potent scavenger of HOCl as shown previously w32x. The estimated airway concentrations of HOCl in cystic fibrosis patients are reported to be 2.65 mM on average or a maximum of 8.15 mM w33x. HOCl will diffuse from the site of its release and might cause more remote damage; concentrations as low as 10]20 m M can oxidize membrane proteins w27x. Therefore, it is very likely that the HOCl concentrations used in our experiments could occur in the pulmonary circulation of cystic fibrosis patients as well as other patients with chronic lung infection. Recent studies have demonstrated that myeloperoxidase, catalyses HOCl formation, is a component of sputum from cystic fibrosis patients w34x, as well as other inflammatory lung diseases, and of human atherosclerotic tissue w12x, underscoring the potential importance of HOCl in the pathology of each of these disease cases. On the other hand, it is known that although basal levels of free NO are normally quite low Žnanomolar., local NO concentrations have been shown to increase to levels ranging from 4 to 30 m M under pathologic conditions w35x. NO might be involved in the protective effects against HOClinduced vasoactivity and injury. However, it is very likely that L-arginine produces protective actions against HOCl via NO-independent manner. HOCl may interact with endothelium or endothelium derived mediators, and contribute to tissue injury and vascular dysfunction seen in inflammatory disease states.
ACKNOWLEDGEMENTS This study was supported by a research grant from Gazi University ŽProject No: SBE 11r95-17.. The chemiluminometer used in this study was provided by a project ŽSBAG-1243. of the Scientific and Tech¨ .. nical Research Council of Turkey ŽTUBITAK
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