Rho-Kinase Inhibition of Active Force and Passive Tension in Airway Smooth Muscle: A Strategy for Treating Airway Hyperresponsiveness in Asthma.

Rho-kinase inhibitors have been identified as a class of potential drugs for treating asthma because of their ability to reduce airway inflammation and active force in airway smooth muscle (ASM). Past research has revealed that, besides the effect on the ASM's force generation, rho-kinase (ROCK) also regulates actin filament formation and filament network architecture and integrity, thus affecting ASM's cytoskeletal stiffness. The present review is not a comprehensive examination of the roles played by ROCK in regulating ASM function but is specifically focused on passive tension, which is partially determined by the cytoskeletal stiffness of ASM. Understanding the molecular basis for maintaining active force and passive tension in ASM by ROCK will allow us to determine the suitability of ROCK inhibitors and its downstream enzymes as a class of drugs in treating airway hyperresponsiveness seen in asthma. Because clinical trials using ROCK inhibitors in the treatment of asthma have yet to be conducted, the present review focuses on the in vitro effects of ROCK inhibitors on ASM's mechanical properties which include active force generation, relaxation, and passive stiffness. The review provides justification for future clinical trials in the treatment of asthma using ROCK inhibitors alone and in combination with other pharmacological and mechanical interventions.

Critical roles of airway smooth muscle in mediating deep-inspiration-induced bronchodilation: a big stretch?

BACKGROUND: Deep inspiration (DI) has been shown to induce bronchodilation and bronchoprotection in bronchochallenged healthy subjects, but not in asthmatics. Strain-induced relaxation of airway smooth muscle (ASM) is considered one of the factors responsible for these effects. Other factors include the release or redistribution of pulmonary surfactant, alteration in mucus plugs, and changes in airway heterogeneity. MAIN BODY: The present review is focused on the DI effect on ASM function, based on recent findings from ex vivo sheep lung experiments showing a large change in airway diameter during a DI. The amount of stretch on the airways, when applied to isolated airway rings in vitro, caused a substantial decrease in ASM contractility that takes many minutes to recover. When challenged with a bronchoconstrictor, the increase in pulmonary resistance in the ex vivo ovine lungs is mostly due to the increase in airway resistance. CONCLUSIONS: Although non-ASM related factors cannot be excluded, the large strain on the airways associated with a DI substantially reduces ASM contractility and thus can account for most of the bronchodilatory and bronchoprotective effects of DI.

Potentiation of active force by cyclic strain in sheep carotid arterial smooth muscle.

The ability to generate force in large arteries is known to be augmented by cyclic strain that mimics the mechanically dynamic in vivo environment associated with blood pressure fluctuation experienced by these arteries. Cyclic strain does not induce a contractile response, like that observed in the myogenic response seen in small arteries, but prompts a substantial increase in the response to electrical stimulation. We coined this phenomenon "force potentiation." Because protein kinase C (PKC) and rho-kinase (ROCK) are known to play a role in increasing contractility of arterial smooth muscle by inhibition of myosin light chain phosphatase, and integrin-link kinase (ILK) is crucial in mechanotransduction, we examined how inhibition of these kinases affected force potentiation in sheep carotid artery. We found that phosphorylation of the regulatory myosin light chain was enhanced by cyclic strain, but the enhancement was observed only in activated, not in relaxed muscle. Inhibition of ROCK diminished force potentiation and active isometric force, likely due to the disinhibition of myosin light chain phosphatase. Inhibition of PKC abolished force potentiation without an effect on active force, suggesting a more exclusive role of PKC (compared with ROCK) in mediating force potentiation. Inhibition of ILK had a similar effect as PKC inhibition, suggesting that ILK may be an upstream kinase for PKC activation by mechanical stimuli. Taken together, the findings suggest that ILK, PKC, and ROCK are important kinases in the signal transduction pathway that mediate the effect of mechanical strain on force potentiation.NEW & NOTEWORTHY When subjected to mechanical strain, smooth muscle from large arteries has the ability to increase its force generation (force potentiation), which could be important in autoregulation of blood pressure. This phenomenon, however, does not involve a myogenic response, such as the one seen in small arteries and arterioles. Our work shows the involvement of ILK, PKC, and ROCK in the signal transduction pathway that mediates the force-potentiating effect of mechanical strain in large arteries.

Sustained contractile force regulated by rho-kinase and protein kinase C in sheep carotid arterial smooth muscle.

The time course of smooth muscle contraction can be divided into two phases, the initial phase is associated with force development, whereas the sustained phase is associated with force maintenance. Cumulative evidence suggests that the two phases are regulated by different signaling pathways and that ρ-kinase (ROCK) and protein kinase C (PKC) play an important role in regulating isometric force in sustained contractions. Since the maintenance of sustained force is critical to the function of vascular smooth muscle, unraveling the complex mechanism of force maintenance is crucial for understanding the cell biology of the muscle. The present study examined the effects of ROCK and PKC on the level of phosphorylation of the 20-kD myosin light chain (MLC20) and isometric force during a sustained contraction. We used partial activation and inhibition of ROCK and PKC to reduce the isometric force by 50% of the maximal isometric force in fully activated muscle, Fmax. We then examined the level of MLC20 phosphorylation in each case. We found that in partially activated muscle the level of MLC20 phosphorylation required to maintain 50% Fmax was much lower than that required in muscles where 50% reduction in Fmax was achieved by partial inhibition of ROCK and PKC. The results can be explained by a model containing a contractile apparatus and a cytoskeletal scaffold where force generated by the contractile apparatus is transmitted to the extracellular domain through the cytoskeleton. The results indicate that ROCK and PKC play an important role in force transmission through the cytoskeleton.NEW & NOTEWORTHY The study supports a model that the maintenance of sustained force during a contraction of arterial smooth muscle is dependent on the intracellular transmission of force through the cytoskeleton and that ρ-kinase and protein kinase C plays an important role in the regulation of cytoskeletal integrity and its efficiency in force transmission.

Airway and parenchymal tissue resistance and elastance in ex vivo sheep lungs: effects of bronchochallenge and deep inspiration.

Lung resistance (RL) is determined by airway and parenchymal tissue resistance, as well as the degree of heterogeneity in airway constriction. Deep inspirations (DIs) are known to reverse experimentally induced increase in RL, but the mechanism is not entirely clear. The first step toward understanding the effect of DI is to determine how each of the resistance components is affected by DI. In the present study, we measured RL and apparent airway resistance (RAW, which combines the effects of airway resistance and airway heterogeneity) simultaneously before and after a DI in acetylcholine (ACh)-challenged ex vivo sheep lungs. We found that at normal breathing frequency (0.25 Hz) ACh-challenge led to a doubling of RL, 80.3% of that increase was caused by an increase in RAW; the increase in apparent tissue resistance (RT) was insignificant. 57.7% of the increase in RAW was abolished by a single DI. After subtracting RAW from RL, the remaining RT was mostly independent of ACh-challenge and its reduction after a DI came mostly from the change in the mechanical properties of lung parenchyma. We conclude that at normal breathing frequency, RL in an unchallenged lung is mostly composed of RT, and the increase in RL due to ACh-challenge stems mostly from the increase in RAW and that both RAW and RT can be greatly reduced by a DI, likely due to a reduction in true airway resistance and heterogeneity, as well as parenchymal tissue hysteresis post DI.

Lung resistance and elastance are different in ex vivo sheep lungs ventilated by positive and negative pressures.

Lung resistance (RL) and elastance (EL) can be measured during positive or negative pressure ventilation. Whether the different modes of ventilation produce different RL and EL is still being debated. Although negative pressure ventilation (NPV) is more physiological, positive pressure ventilation (PPV) is more commonly used for treating respiratory failure. In the present study, we measured lung volume, airway diameter, and airway volume, as well as RL and EL with PPV and NPV in explanted sheep lungs. We found that lung volume under a static pressure, either positive or negative, was not different. However, RL and EL were significantly higher in NPV at high inflation pressures. Interestingly, diameters of smaller airways (diameters <3.5 mm) and total airway volume were significantly greater at high negative inflation pressures compared with those at high positive inflation pressures. This suggests that NPV is more effective in distending the peripheral airways, likely due to the fact that negative pressure is applied through the pleural membrane and reaches the central airways via the peripheral airways, whereas positive pressure is applied in the opposite direction. More distension of lung periphery could explain why RL is higher in NPV (vs. PPV), because the peripheral parenchyma is a major source of tissue resistance, which is a part of the RL that increases with pressure. This explanation is consistent with the finding that during high frequency ventilation (>1 Hz, where RL reflects airway resistance more than tissue resistance), the difference in RL between NPV and PPV disappeared.

Airway diameter at different transpulmonary pressures in ex vivo sheep lungs: implications for deep inspiration-induced bronchodilation and bronchoprotection.

Deep inspiration (DI)-induced bronchodilation is the first line of defense against bronchoconstriction in healthy subjects. A hallmark of asthma is the lack of this beneficial effect of DI. The mechanism underlying the bronchodilatory effect of DI is not clear. Understanding the mechanism will help us unravel the mystery of asthma pathophysiology. It has been postulated that straining airway smooth muscle (ASM) during a DI could lead to bronchodilation and bronchoprotection. The hypothesis is currently under debate, and a central question is whether ASM is sufficiently stretched during a DI for its contractility to be compromised. Besides bronchoconstriction, another contributor to lung resistance is airway heterogeneity. The present study examines changes in airway diameter and heterogeneity at different lung volumes. Freshly explanted sheep lungs were used in plethysmographic measurements of lung resistance and elastance at different lung volumes, whereas the airway dimensions were measured by computed tomography (CT). The change in airway diameter informed by CT measurements was applied to isolated airway ring preparations to determine the strain-induced loss of ASM contractility. We found that changing the transpulmonary pressure from 5 to 30 cmH2O led to a 51% increase in lung volume, accompanied by a 46% increase in the airway diameter with no change in airway heterogeneity. When comparable airway strains measured in the whole lung were applied to isolated airway rings in either relaxed or contracted state, a significant loss of ASM contractility was observed, suggesting that DI-induced bronchodilation and bronchoprotection can result from strain-induced loss of ASM contractility.

Filament evanescence of myosin II and smooth muscle function.

Smooth muscle is an integral part of hollow organs. Many of them are constantly subjected to mechanical forces that alter organ shape and modify the properties of smooth muscle. To understand the molecular mechanisms underlying smooth muscle function in its dynamic mechanical environment, a new paradigm has emerged that depicts evanescence of myosin filaments as a key mechanism for the muscle's adaptation to external forces in order to maintain optimal contractility. Unlike the bipolar myosin filaments of striated muscle, the side-polar filaments of smooth muscle appear to be less stable, capable of changing their lengths through polymerization and depolymerization (i.e., evanescence). In this review, we summarize accumulated knowledge on the structure and mechanism of filament formation of myosin II and on the influence of ionic strength, pH, ATP, myosin regulatory light chain phosphorylation, and mechanical perturbation on myosin filament stability. We discuss the scenario of intracellular pools of monomeric and filamentous myosin, length distribution of myosin filaments, and the regulatory mechanisms of filament lability in contraction and relaxation of smooth muscle. Based on recent findings, we suggest that filament evanescence is one of the fundamental mechanisms underlying smooth muscle's ability to adapt to the external environment and maintain optimal function. Finally, we briefly discuss how increased ROCK protein expression in asthma may lead to altered myosin filament stability, which may explain the lack of deep-inspiration-induced bronchodilation and bronchoprotection in asthma.

Mechanopharmacology of Rho-kinase antagonism in airway smooth muscle and potential new therapy for asthma.

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma.

The Huxley crossbridge model as the basic mechanism for airway smooth muscle contraction.

The cyclic interaction between myosin crossbridges and actin filaments underlies smooth muscle contraction. Phosphorylation of the 20-kDa myosin light chain (MLC20) is a crucial step in activating the crossbridge cycle. Our current understanding of smooth muscle contraction is based on observed correlations among MLC20 phosphorylation, maximal shortening velocity (Vmax), and isometric force over the time course of contraction. However, during contraction there are changes in the extent of phosphorylation of many additional proteins as well as changes in activation of enzymes associated with the signaling pathways. As a consequence, the mechanical manifestation of muscle contraction is likely to change with time. To simplify the study of these relationships, we measured the mechanical properties of airway smooth muscle at different levels of MLC20 phosphorylation at a fixed time during contraction. A simple correlation emerged when time-dependent variables were fixed. MLC20 phosphorylation was found to be directly and linearly correlated with the active stress, stiffness, and power of the muscle; the observed weak dependence of Vmax on MLC20 phosphorylation could be explained by the presence of an internal load in the muscle preparation. These results can be entirely explained by the Huxley crossbridge model. We conclude that when the influence of time-dependent events during contraction is held constant, the basic crossbridge mechanism in smooth muscle is the same as that in striated muscle.

Mechanopharmacology and Synergistic Relaxation of Airway Smooth Muscle.

Asthmatic airways are stiffer than normal. We have shown that the cytoskeletal passive stiffness of airway smooth muscle (ASM) can be regulated by intracellular signaling pathways, especially those associated with Rho kinase (ROCK). We have also shown that an oscillatory strain reduces the passive stiffness of ASM and its ability to generate force. Here, we investigated the combined effect of inhibiting the ASM contraction with ß2 agonist and decreasing the ASM cytoskeletal stiffness with ROCK inhibitor and/or force oscillation (FO) on the relaxation of contracted ASM. We hypothesize that the ASM relaxation can be synergistically enhanced by the combination of these interventions, because drug-induced softening of the cytoskeleton enhances the FO-induced relaxation and vice versa. Sheep tracheal strips were isotonically contracted to acetylcholine (3 × 10-5 M). At the plateau of shortening, ß2 agonist salbutamol (10-7 M), ROCK inhibitor H1152 (10-7 M), and FO (square wave, 1 Hz, amplitude 6% maximal active force) were applied either alone or in combination. After adjusting for nonspecific time-dependent variation, relengthening by individual interventions with low-dose salbutamol or H1152, or small amplitude FO was not significantly different from zero. However, significant relengthening was observed in all combination treatments. The relengthening was greater than the mathematical sum of relengthening caused by individual treatments thereby demonstrating synergistic relaxation. The ASM stiffness did not change with salbutamol or H1152 treatments, but was lower with FO in combination with H1152. The results suggest that the mechanopharmacological treatment can be an effective therapy for asthma.

Smooth muscle function and myosin polymerization.

Smooth muscle is able to function over a much broader length range than striated muscle. The ability to maintain contractility after a large length change is thought to be due to an adaptive process involving restructuring of the contractile apparatus to maximize overlap between the contractile filaments. The molecular mechanism for the length-adaptive behavior is largely unknown. In smooth muscle adapted to different lengths we quantified myosin monomers, basal and activation-induced myosin light chain (MLC) phosphorylation, shortening velocity, power output and active force. The muscle was able to generate a constant maximal force over a two fold length range when it was allowed to go through isometric contraction/relaxation cycles after each length change (length adaptation). In the relaxed state, myosin monomer concentration and basal MLC phosphorylation decreased linearly, while in the activated state activation-induced MLC phosphorylation and shortening velocity/power output increased linearly with muscle length. The results suggest that recruitment of myosin monomers and oligomers into the actin filament lattice (where they form force-generating filaments) occurs during muscle adaptation to longer length, with the opposite occurring during adaptation to shorter length.

Ovalbumin sensitization of guinea pig at birth prevents the ontogenetic decrease in airway smooth muscle responsiveness.

Airway smooth muscle (ASM) displays a hyperresponsive phenotype at young age and becomes less responsive in adulthood. We hypothesized that allergic sensitization, which causes ASM hyperresponsiveness and typically occurs early in life, prevents the ontogenetic loss of the ASM hyperresponsive phenotype. We therefore studied whether neonatal allergic sensitization, not followed by later allergen challenges, alters the ontogenesis of ASM properties. We neonatally sensitized guinea pigs to ovalbumin and studied them at 1 week, 3 weeks, and 3 months (adult). A Schultz-Dale response in isolated tracheal rings confirmed sensitization. The occurrence of inflammation was evaluated in the blood and in the submucosa of large airways. We assessed ASM function in tracheal strips as ability to produce force and shortening. ASM content of vimentin was also studied. A Schultz-Dale response was observed in all 3-week or older sensitized animals. A mild inflammatory process was characterized by eosinophilia in the blood and in the airway submucosa. Early life sensitization had no effect on ASM force generation, but prevented the ontogenetic decline of shortening velocity and the increase in resistance to shortening. Vimentin increased with age in control but not in sensitized animals. Allergic sensitization at birth without subsequent allergen exposures is sufficient to prevent normal ASM ontogenesis, inducing persistence to adulthood of an ASM hyperresponsive phenotype.

Models to understand contractile function in the airways.

Although the role of contractile function in the airways is controversial, there is general consensus on the importance of airway smooth muscle (ASM) as a therapeutic target for diseases characterized by airway obstruction, such as asthma or chronic obstructive pulmonary disease. Indeed, the use of bronchodilators to relax ASM is the most common and effective practice to treat airflow obstruction. Excessive pathologic bronchoconstriction may originate from primary alterations of ASM mechanical function and/or from the effects exerted on ASM function by disease processes, such as inflammation and remodeling. An in depth knowledge of the potentially multiple mechanisms that distinctively regulate primary and secondary alterations in ASM contractile function would be essential for the development of new therapeutic approaches aimed at preventing the occurrence or reducing the severity of bronchoconstriction. The present review discusses studies that have addressed the mechanisms of altered ASM contractile function in models of airway hyperresponsiveness. Although not comprehensively, in the present review, animal models of intrinsic airway hyperresponsiveness, normal ontogenesis, and allergic sensitization are analyzed in the attempt to summarize the current knowledge on regulatory mechanisms of ASM contractile function in health and disease. Studies in human ASM and the need for additional models to understand contractile function in the airways are also discussed.

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release.

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.

Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase.

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47(phox-/-) than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox-/- compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 +/- 2.4 and 9.2 +/- 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation.

Three paradigms of airway smooth muscle hyperresponsiveness in young guinea pigs.

Evidence for contributions of airway smooth muscle (ASM) to the hyperresponsiveness of newborn and juvenile airways continues to accumulate. In our laboratory, 3 novel paradigms of hyperresponsiveness of newborn and young ASM have recently emerged using a guinea pig model of maturation in 3 age groups: 1 week (newborn), 3 weeks (juvenile), and 2-3 months (adult). The first paradigm includes evidence for a natural decline after newborn and juvenile life of the velocity of ASM shortening associated with a decrease in regulatory myosin light chain phosphorylation and a parallel decline in the content of myosin light chain kinase. Associated with the decrease in ASM shortening with age is an increase in the internal resistance to shortening. Dynamic stiffness is shown to relate inversely to the expression of myosin light chain kinase. This suggests that developmental changes in shortening relate inversely to the stiffness of the ASM early in shortening, suggesting a dynamic role for the cytoskeleton in facilitating and opposing ASM shortening. This relationship can be approximated as (dP/dt)max approximately (dP/dL)passive x (dL/dt)max (the maximal rate of increase of active stress generation approximately to the passive stiffness x the maximal shortening velocity). The second paradigm demonstrates that newborn ASM, unlike that in adults, does not relax during prolonged electric field stimulation. The impaired relaxation is related to changes in prostanoid synthesis and acetylcholinesterase function. The third paradigm demonstrates that, whereas oscillatory strain serves to transiently relax adult ASM, in newborns it induces (after the initial relaxation) a sustained potentiation of active stress. This is related to developmental changes in the prostanoid release. Together, these paradigms demonstrate that ASM contributes by multiple mechanisms to the natural hyperresponsiveness of newborn and juvenile airways. Future studies will elaborate the mechanisms and extend these paradigms to ASM hyperresponsiveness following sensitization in early life.

Nox4 mediates TGF-beta1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-beta1 have not been fully elucidated. In this study, we demonstrate that TGF-beta1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-beta1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-beta1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-beta1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-beta1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-beta1-induced proliferation and hypertrophy of human airway smooth muscle.

S-nitrosoglutathione inhibits alpha1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery.

Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to G(q), we hypothesized that S-nitrosothiols modify the alpha1-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10(-8)-10(-6) M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or LY-83583} or G kinase inhibitor (KT-5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the alpha1-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of alpha1-adrenergic receptor subtype expression and receptor density by saturation binding with 125I-HEAT showed that GSNO decreased alpha1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible alpha1-adrenergic receptor inhibition by S-nitrosothiols.