Mechanism of H2O2-induced modulation of airway smooth muscle

The effect of H 2 O 2 on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P i release were measured by single-turnover kinetics. H 2 O 2 treatment caused a decrease in HMM regulation from 800-to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, ϳ50% of the molecules lost the ability to bind to ATP. H 2 O 2 treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H 2 O 2 -inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H 2 O 2 -inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 ؉ 2.1 mM ATP, H 2 O 2 caused a ϳ50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H 2 O 2 directly affects the force generating complex. Dithiothreitol treatment reversed the H 2 O 2 inhibition of the maximal force by ϳ50%. These data, when compared with the in vitro kinetic data, are consistent with a H 2 O 2 -induced loss of functional myosin heads in the muscle.Contraction of smooth muscle is controlled largely by phosphorylation of the 20-kDa myosin regulatory light chain (RLC), 2 resulting in the cyclic attachment and detachment of myosin to actin (cross-bridge cycling) and the hydrolysis of ATP by actin-activated myosin ATPase (1). The level of RLC phosphorylation depends on the balance between the activities of MLCK and SMM phosphatase. MLCK is primarily activated (through Ca 2ϩ -calmodulin) by an increase in [Ca 2ϩ ] i from intracellular stores or influx of extra-cellular Ca 2ϩ , whereas inhibition of myosin phosphatase, which contributes to the amount of force at a given [Ca 2ϩ ] i (Ca 2ϩ sensitivity), occurs through a receptor-G protein-catalyzed signal transduction cascade (2-5).Reactive oxygen species, such as H 2 O 2 , modulate smooth muscle contractility (6) and contribute to the severity of numerous diseases, including acute lung injury, asthma, pulmonary hypertension, ischemia-reperfusion, and arthritis (7). H 2 O 2 reversibly inhibits receptor agonist-induced contraction of both vascular (8 -10) and ASM (11-15).The mechanisms for H 2 O 2 inhibition of smooth muscle contraction are not well understood but have been proposed to involve effects on kinase-mediated signaling cascades, cytoskeletal effects, ionotropic effects, lipid oxidation, direct inhibitory effect on smooth muscle contractile proteins, and potentially confounding effects on epithelial cells (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In general, healthy smooth muscle is quite resis...

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“…H 2 O 2 reversibly inhibits receptor agonist-induced contraction of both vascular (8 -10) and ASM (11)(12)(13)(14)(15).…”

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confidence: 99%

H2O2-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin

Penheiter

Bogoger

Ellison

et al. 2007

Journal of Biological Chemistry

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“…Rhoden and Barnes (16) on the epithelium denuded and Gao and Vanhoutte (20) on the intact and denuded guinea pig trachea observed attenuation of H 2 O 2 -induced contraction in the presence of indomethacin. In contrast, Gupta and Prasad (17) found significant attenuation of H 2 O 2 -induced relaxation, and Prasad and Gupta (26) described a reversal of the relaxant effect of hydroxyl radical to contraction on the rabbit trachea after indomethacin pretreatment. Our results are supporting those of Prasad and Gupta (26), because in intact tissue, indomethacin pretreatment changed the biphasic character of tracheal responses to H 2 O 2 and hydroxyl radical into a monophasic prolonged and increased contraction.…”

Section: Discussionmentioning

confidence: 90%

“…Under such a condition, the increased tone was persistent and the relaxant component of the response was absent. Removal of the epithelium was found to cause partial loss of relaxant factors following H2O2 administration also in other animal species (17,18). Deepithelisation of the trachea is presumably also removing superoxide anion-eliminating mechanisms of the epithelium, thus unmasking direct actions of superoxide anion on the smooth muscle.…”

Section: Discussionmentioning

confidence: 91%

“…Later existence of species differences was demonstrated. H2O2 was shown to relax the rabbit (17) and dog (18) trachea, contract the cat trachea (19) and cause biphasic effect on the guinea pig trachea (20). The effects of H2O2 are generally considered to be modulated by cyclooxygenase inhibitors or epithelium removal (16, 21 -25).…”

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confidence: 99%

“…Although there are many studies focusing on the effect of H2O2 on isolated trachea or bronchi of various animal species: rat (22), cow (21), ferret (28), rabbit (17), guinea pig (16,17,20,29), human (25), less were done on the effects of ozone (guinea pig trachea (30), feline bronchi (31)), hypochlorous acid (guinea pig trachea (32,33)), hydroxyl radical (rabbit trachea (26)) and xanthine /xanthine oxidase (guinea pig airways in vivo (34) and cat bronchi in vitro (35)). The lack of comprehensive studies that would compare the effects of long-lasting contact of systems generating hydroxyl radical and superoxide anion on the tone of the guinea pig trachea in the presence or absence of epithelium with that of H 2 O 2 on the same tissue of a single animal species prompted us to compare them in guinea pig trachea.…”

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confidence: 99%

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Effects of Various Reactive Oxygen Species on the Guinea Pig Trachea and Its Epithelium

Matyas

Pucovský

Bauer

2002

Japanese Journal of Pharmacology

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ABSTRACT-Reactive oxygen species (ROS) are key factors playing important roles in tissue damage of airways under different pathological conditions. Effects of ROS (superoxide anion, H2O2 and hydroxyl radical) were recorded on isometric tension of intact and epithelium denuded, not precontracted guinea pig trachea. Superoxide anion was produced by xanthine /xanthine oxidase and hydroxyl radical either by FeSO4/H2O2 or FeSO4/ascorbic acid. In intact preparations, the muscle tension was unaffected by superoxide anion, while H 2 O 2 and hydroxyl radical produced a biphasic response, contraction followed by relaxation. Both the amplitude and duration of contractions evoked by H 2 O 2 were larger than those caused by hydroxyl radical producing systems. On denuded tracheal strips, superoxide anion elicited also a biphasic response, and the H 2 O 2 and hydroxyl radical produced contractions were of higher amplitude and of longer duration than in intact tissues. Indomethacin pretreatment enhanced or slightly reduced the amplitude of contractions evoked by both H 2 O 2 and hydroxyl radical on the intact and denuded preparations, respectively. Moreover, the duration of contractions of the trachea induced by oxidative systems was prolonged. Indomethacin did not affect the action of superoxide anion on the intact tissues and reduced the amplitude of the biphasic response on denuded ones. Nordihydroguaiaretic acid pretreatment did not alter the responses elicited by ROS in intact preparations and reduced their action on the denuded ones. Our results suggest that a) various ROS contract tracheal smooth muscle with simultaneous release of epithelium derived relaxing factors, b) epithelium possesses superoxide anion scavenging capacity which is high enough to protect smooth muscle from its actions, and c) cyclooxygenase products participate in relaxation and lipoxygenase products in contraction caused by ROS in the guinea pig trachea.

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