Airway Epithelial Cells Drive Airway Smooth Muscle Cell Phenotype Switching to the Proliferative and Pro-inflammatory Phenotype.

The increased mass of airway smooth muscle (ASM) in the airways of asthmatic patients may contribute to the pathology of this disease by increasing the capacity for airway narrowing. Evidence for the airway epithelium as a participant in ASM remodeling is accruing. To investigate mechanisms by which airway epithelial cells induce ASM cell (ASMC) proliferation, we have employed a co-culture model to explore markers of ASMC proliferative phenotype. Co-culture with epithelial cells led to incorporation of bromodeoxyuridine into ASMCs, indicating augmented proliferation and an associated increase in mRNA of the pro-proliferative co-transcription factor Elk1. Although the mitogen heparin-binding epidermal growth factor (HB-EGF) was augmented in the co-culture supernatant, the ASMC epidermal growth factor receptor (EGFR), an effector of HB-EGF induced proliferation, did not mediate epithelial-induced proliferation. The co-culture increased the expression of ASMC mRNA for the pro-inflammatory cytokines IL-6 and IL-8 as well as the pro-proliferative microRNA miR-210. The transcriptional repressor Max-binding protein (Mnt), a putative target of miR-210, was transcriptionally repressed in co-cultured ASMCs. Together, these data indicate that the airway epithelium-induced proliferative phenotype of ASMCs is not driven by EGFR signaling, but rather may be dependent on miR210 targeting of tumor suppressor Mnt.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis.

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

Transient oscillatory force-length behavior of activated airway smooth muscle.

Airway smooth muscle (ASM) is cyclically stretched during breathing, even in the active state, yet the factors determining its dynamic force-length behavior remain incompletely understood. We developed a model of the activated ASM strip and compared its behavior to that observed in strips of rat trachealis muscle stimulated with methacholine. The model consists of a nonlinear viscoelastic element (Kelvin body) in series with a force generator obeying the Hill force-velocity relationship. Isometric force in the model is proportional to the number of bound crossbridges, the attachment of which follows first-order kinetics. Crossbridges detach at a rate proportional to the rate of change of muscle length. The model accurately accounts for the experimentally observed transient and steady-state oscillatory force-length behavior of both passive and activated ASM. However, the model does not predict the sustained decrement in isometric force seen when activated strips of ASM are subjected briefly to large stretches. We speculate that this force decrement reflects some mechanism unrelated to the cycling of crossbridges, and which may be involved in the reversal of bronchoconstriction induced by a deep inflation of the lungs in vivo.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance.

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.

Kinetics of respiratory system elastance after airway challenge in dogs.

We compared the time courses of lung mechanical changes with intravenous (iv) injection vs. aerosol administration of histamine, methacholine, and ACh in dogs. We interpret these results in terms of a spring-and-dashpot model of airway smooth muscle receiving activation via a tissue compartment when agonist is delivered by the iv route and through an additional airway wall compartment when it is delivered by the aerosol route. The model accurately accounts for the principal features of the respiratory system elastance response curves. It also accounts for the differences between iv and aerosol responses, supporting the notion that agonist delivered by aerosol has to traverse a longer pathway to the airway smooth muscle than does agonist delivered iv. The time constants representing diffusive exchange of agonist between compartments were not significantly different for the three agonists, suggesting that the three agonists shared a common principal means of clearance, which was presumably blood flow.

Helium and sulfur hexafluoride bolus washin in short-term microgravity.

We performed single-breath washout (SBW) tests in which He and sulfur hexafluoride (SF6) were inspired throughout the vital capacity inspirations or were inhaled as discrete boluses at different points in the inspiration. Tests were performed in normal gravity (1 G) and in up to 27 s of microgravity (microG) during parabolic flight. The phase III slope of the SBW could be accurately reconstructed from individual bolus tests when allowance for airways closure was made. Bolus tests showed that most of the SBW phase III slope results from events during inspiration at lung volumes below closing capacity and near total lung capacity, as does the SF6-He phase III slope difference. Similarly, the difference between 1 G and microG in phase III slopes for both gases was entirely accounted for by gravity-dependent events at high and low lung volumes. Phase IV height was always larger for SF6 than for He, suggesting at least some airway closure in close proximity to airways that remain open at residual volume. These results help explain previous studies in microG, which show large changes in gas mixing in vital capacity maneuvers but only small effects in tidal volume breaths.

Myosin conformational states determined by single fluorophore polarization.

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke.

Cardiogenic oscillation phase relationships during single-breath tests performed in microgravity.

We studied the phase relationships of the cardiogenic oscillations in the phase III portion of single-breath washouts (SBW) in normal gravity (1 G) and in sustained microgravity (microG). The SBW consisted of a vital capacity inspiration of 5% He-1.25% sulfurhexafluoride-balance O2, preceded at residual volume by a 150-ml Ar bolus. Pairs of gas signals, all of which still showed cardiogenic oscillations, were cross-correlated, and their phase difference was expressed as an angle. Phase relationships between inspired gases (e.g., He) and resident gas (n2) showed no change from 1 G (211 +/- 9 degrees) to microG (163 +/- 7 degrees). Ar bolus and He were unaltered between 1 G (173 +/- 15 degrees) and microG (211 +/- 25 degrees), showing that airway closure in microG remains in regions of high specific ventilation and suggesting that airway closure results from lung regions reaching low regional volume near residual volume. In contrast, CO2 reversed phase with He between 1 G (332 +/- 6 degrees) and microG (263 +/- 27 degrees), strongly suggesting that, in microG, areas of high ventilation are associated with high ventilation-perfusion ratio (VA/Q). This widening of the range of VA/Q in microG may explain previous measurements (G.K. Prisk, A.R. Elliott, H.J.B. Guy, J.M. Kosonen, and J.B. West J. Appl. Physiol. 79: 1290-1298, 1995) of an overall unaltered range of VA/Q in microG, despite more homogeneous distributions of both ventilation and perfusion.

A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap.

Two smooth muscle myosin heavy chain isoforms differ by a 7-amino-acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((-)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (approximately 10 nm) but the attachment time (t(on) for the (-)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(-insert)/Favg(+insert) = 1.1 +/- 0.2 (mean +/- SE) using the in vitro motility mixture assay, and the unitary force (F approximately 1 pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, t(on) under loaded conditions was longer for the (-)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of t(on) from d measurements to various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.

Molecular mechanics of two smooth muscle heavy meromyosin constructs that differ by an insert in the motor domain.

Phasic and tonic smooth muscles express two myosin heavy chain isoforms that differ by only a seven amino acid insert in a flexible surface loop located near the nucleotide-binding site. The inserted isoform found predominantly in phasic muscle has two times the actin-activated ATPase activity and in vitro actin filament velocity as the non-insert isoform found mainly in tonic muscle (Kelley, C.A., Takahashi, M., Yu, J.H. & Adelstein, R.S. 1993. J Biol Chem 268, 12848, Rovner, A.S., Freyzon, Y. & Trybus, K.M. 1997. J Musc Res Cell Motil 18, 103). We used a laser trap to characterize the molecular mechanics of the inserted isoform [(+)insert] and of a mutant lacking the insert [(-)insert], which is analogous to the isoform found in tonic muscles. The constructs were expressed in the baculovirus/insect cell system. Unitary displacements (Duni) were similar for both the constructs (approximately 10 nm) but the attachment time (ton) for the (-)insert was two times that of the (+)insert. These data suggest that the insert in the nucleotide-binding loop does not affect the inherent mechanics of the myosin molecule but rather the kinetics of the cross-bridge cycle.

Paradoxical helium and sulfur hexafluoride single-breath washouts in short-term vs. sustained microgravity.

During single-breath washouts in normal gravity (1 G), the phase III slope of sulfur hexafluoride (SF6) is steeper than that of helium (He). Two mechanisms can account for this: 1) the higher diffusivity of He enhances its homogeneous distribution; and 2) the lower diffusivity of SF6 results in a more peripheral location of the diffusion front, where airway asymmetry is larger. These mechanisms were thought to be gravity independent. However, we showed during the Spacelab Life Sciences-2 spaceflight that in sustained microgravity (microG) the SF6-to-He slope difference is abolished. We repeated the protocol during short periods (27 s) of microG (parabolic flights). The subjects performed a vital-capacity inspiration and expiration of a gas containing 5% He-1.25% SF6-balance O2. As in sustained microG, the phase III slopes of He and SF6 decreased. However, during short-term microG, the SF6-to-He slope difference increased from 0.17 +/- 0.03%/l in 1 G to 0.29 +/- 0.06%/l in microG, respectively. This is contrary to sustained microG, in which the SF6-to-He slope difference decreased from 0.25 +/- 0.03%/l in 1 G to -0.01 +/- 0.06%/l in microG. The increase in phase III slope difference in short-term microG was caused by a larger decrease of He phase III slope compared with that in sustained microG. This suggests that changes in peripheral gas mixing seen in sustained microG are mainly due to alterations in the diffusive-convective inhomogeneity of He that require > 27 s of microG to occur. Changes in pulmonary blood volume distribution or cardiogenic mixing may explain the differences between the results found in short-term and sustained microG.

Anomalous behavior of helium and sulfur hexafluoride during single-breath tests in sustained microgravity.

We performed single-breath wash-in tests for He and SF6 in four subjects exposed to 14 days of microgravity (microG) during the Spacelab flight Spacelab Life Sciences-2. Subjects inspired a vital capacity breath of 5% He-1.25% SF6-balance O2 and then exhaled to residual volume at 0.5l/s. The tests were also performed with a 10-s breath hold at the end of inspiration. Measurements were also made with the subjects standing and supine in 1 G. Phase III slope was measured after the dead-space washout and before the onset of airway closure. In all subjects in 1 G, whether standing or supine, phase III slope for SF6 was significantly steeper than that for He. However, in microG, the slopes became the same. Furthermore, after breath holding in microG, the SF6 slopes were significantly flatter than those for He. On return to 1 G, the changes were reversed, and there was no difference between preflight and postflight values. Because most of the phase III slope reflects events occurring in the acinar regions of the lung, the results suggest that microG causes conformational changes in the acini or changes in cardiogenic mixing in the lung periphery, but in either case the mechanism is unclear.

Time course of histamine-induced bronchoconstriction and its adrenergic and H2 modulation.

We characterized the complete time course of histamine-induced bronchoconstriction and its modulation via the release of endogenous catecholamines and by its actions on H2-receptors in anesthetized, tracheostomized, paralyzed, and artificially ventilated mongrel dogs. Respiratory resistance (R) and elastance (E) were estimated continuously with a recursive least squares estimator. Three protocols were followed in which multiple histamine bolus injections were given 1 h apart. We found that the time courses of E and R had consistent patterns (transient peak that returned to baseline within 1000 sec) even in cases of low mean arterial pressure (MAP). Indomethacin pre-treatments prevented tachyphylaxis to repeated i.v. challenges. beta-blockade produced a mild increase in baseline and a potentiation of the histamine-induced response in E and these effects were not altered with further alpha-or H2-blockade. Blockade of alpha-receptors increased the time to recovery in both E and MAP presumably by decreasing blood flow. Finally, we suggest that preventing the H2-receptor induced increase in bronchial blood flow may have increased the time to maximal E without affecting the recovery time.

On the use of the alveolar capsule technique to study bronchoconstriction.

Using the alveolar capsule technique, we studied the time courses of respiratory mechanical parameters at various sites on the lung surface during bronchoconstriction. Six mongrel dogs were anesthetized, tracheostomized, paralyzed and artificially ventilated (12-25 ml/kg, 19-22 breaths/min). Sternotomy was performed and alveolar capsules were glued to various parts of the lungs. Tracheal pressure and flow and alveolar pressure were measured continuously for 25 min after i.v. bolus injections of histamine (0, 0.05, 0.5, 5.0, 50.0 mg). The challenges were spaced 1 h apart. Estimates of lung tissue resistance and elastance were obtained with our recursive least-squares estimator (Lauzon and Bates, J. Appl. Physiol., 1159-1165, 1991). We found that the time courses of the parameters of most capsules were initially uniform but quickly diverged as bronchoconstriction developed. Also, we found that the differences in time course of mechanics between alveolar regions either developed randomly with step-like features presumably reflecting intermittent opening and closing of the airways leading to the various alveolar capsules, or in a progressive dose-dependent manner, possibly reflecting a gradual but structurally pre-set pattern of bronchoconstriction, or with a combination of these two patterns. We explain our results in terms of inhomogeneous mechanical properties of the lungs and examine some artifacts introduced by the alveolar pressure measurement technique.

Mechanical behaviour of the canine respiratory system at very low lung volumes.

We studied the changes in dynamic elastance and resistance of the respiratory system in 6 supine, anaesthetized, paralysed, tracheostomised and open chested dogs. Tracheal pressure (Ptr), tracheal flow (V) and 3 alveolar pressures (Palv by alveolar capsule) were measured continuously for 20 min at 5 levels of positive end expiratory pressure (PEEP) between 0.1 and 0.5 kPa. The lungs were inflated to total lung capacity (TLC) at the start of each recording period. Lung elastance (EL) and resistance (RL) were estimated by fitting the equation Ptr = RLV + ELV + K to the measured data for each breath by multiple linear regression (V = volume, K = constant). Airway resistance (Raw) was obtained from the difference between Ptr and Palv. EL increased progressively in the 20 min following lung inflations. The increase in EL over this time was about 45% of its baseline value at a PEEP of 0.1 kPa compared to an increase of only about 10% at a PEEP of 0.5 kPa. In contrast, RL changed very little over the recording period at all levels of PEEP. At low levels of PEEP Palv often bore no resemblance to Ptr indicating that significant airway obstruction or closure had occurred. These results suggest that the increase in EL at low PEEP was primarily due to the accretion of airspace closure, and that nonlinear tissue mechanical properties were responsible for the lack of change in RL.

Temporal dynamics of pulmonary response to intravenous histamine in dogs: effects of dose and lung volume.

We measured tracheal pressure (Ptr) and tracheal flow (V) in open-chest anesthetized paralyzed dogs. The lungs were maintained at a fixed volume (initial positive end-expiratory pressure 0.5 kPa) for 80 s while small-amplitude oscillations in V at 1 and 6 Hz were applied simultaneously at the tracheal opening. A bolus of histamine was given intravenously at the start of the oscillation period. The time course of lung elastic recoil pressure (Pel) was obtained by passing a running average over Ptr to smooth out its oscillations. The oscillations themselves were separated into their 1- and 6-Hz components, as were those in V. By fitting models to the 1- and 6-Hz components of Ptr and V by recursive least squares, we obtained time courses of lung resistance at 6 Hz (RL6), dynamic lung elastance at 1 Hz (EL1), and the difference between dynamic lung resistance at 1 and 6 Hz (RL1-RL6). In four dogs we studied the effects of histamine doses of 0.05, 1.0, and 20 mg. We found that Pel increased quickly and plateaued, RL6 continued to increase throughout the oscillation period, and EL1 exhibited features of both Pel and RL6. Furthermore, the ratio of RL1-RL6 to EL1 was qualitatively similar in time course to Pel. We explain these varied time courses in terms of the development of regional ventilation inhomogeneity throughout the lung as the reaction to histamine develops. In four dogs we also studied the effects of reducing the initial positive end-expiratory pressure by 0.25 kPa and found that the changes in RL6, EL1, and RL1-RL6 were greatly magnified, presumably because of the reduced forces of parenchymal interdependence.

Time course of respiratory mechanics during histamine challenge in the dog.

We studied the dynamics of respiratory mechanical parameters in anesthetized tracheostomized paralyzed dogs challenged with a bolus of histamine injected either venously (venous group) or arterially (arterial group). The venous group was further divided into two groups: the first was bilaterally vagotomized and received hexamethonium bromide (denervated group), and the second also received atropine sulfate (atropine group). In the venous group, tissue resistance (Rti) and tissue elastance (Eti) increased biphasically, whereas airway resistance was monophasic and synchronized with the second rise of the tissue parameters. In the arterial group, Rti, Eti, and airway resistance increased synchronously. The denervated and atropine groups showed dynamics similar to those of the venous group. We postulate that the first phase observed in Rti and Eti in the venous group is due to constriction of the smooth muscles of the peripheral airways and blood vessels distorting the parenchyma. The second and larger phase is then due to histamine reaching the bronchial circulation and constricting the central airways, again distorting the parenchyma. The results from the arterial group support this hypothesis, whereas those from the denervated group ascertain that none of the phases observed in the venous group was due to nervous reflexes.

A nonstatistical approach to estimating confidence intervals about model parameters: application to respiratory mechanics.

Estimates of parameters obtained by fitting models to physiologic data are of little use unless accompanied by confidence intervals. The standard methods for estimating confidence intervals are statistical, and make the assumption that the fitted model accounts for all the deterministic variation in the data while the residuals between the fitted model and the data reflect only stochastic noise. In practice, this is frequently not the case, as one often finds the residuals to be systematically distributed about zero. In this paper, we develop an approach for assessing confidence in a parameter estimate when the order of the model is clearly less than that of the system being modeled. Our approach does not require a parameter to have a single value located within a region of confidence. Instead, we let the parameter value vary over the data set in such a way as to provide a good fit to the entire data set. We apply our approach to the estimation of the resistance of the respiratory system in which a simple model is fitted to measurements of tracheal pressure and flow by recursive multiple linear regression. The values of resistance required to achieve a good fit are represented as a modified histogram in which the contribution of a particular resistance value to the histogram is weighted by the amount of information used in its determination. Our approach provides parameter frequency distribution functions that convey the degree of confidence one may have in the parameter, while not being based on erroneous statistical assumptions.

Estimation of time-varying respiratory mechanical parameters by recursive least squares.

Continuous estimation of time-varying respiratory mechanical parameters is required to fully characterize the time course of bronchoconstriction. To achieve such estimation, we developed an estimator that uses the recursive linear least-squares algorithm to fit the equation Ptr = RV + EV + K to measurements of tracheal pressure (Ptr) and flow (V). The volume (V) is obtained by numerical integration of V. The estimator has a finite memory with length into the past at each point in time that varies inversely with the difference between the current measurement of Ptr and that predicted by the model, to allow the algorithm to track rapidly varying parameters (R, E, and K). V usually exhibits significant drift and must be corrected. Of the several correction methods investigated, subtraction of the recursively weighted average of V before integration to V was found to perform best. The estimator was tested on simulated noisy data where it successfully followed a fivefold increase in R and a twofold increase in E occurring over 10 s. Three dogs and two cats were anesthetized, paralyzed, tracheostomized, and challenged with a bolus of methacholine (approximately 13 mg/kg iv). Increases of 3- to 10-fold were observed in R and 2- to 3-fold in E, beginning within 10-40 s after the bolus injection. In some animals we found that the increase in E occurred more slowly than that in R, which the V signal suggested was due to dynamic hyperinflation of the lungs. These results demonstrate that our recursive estimator is able to track rapid changes in respiratory mechanical parameters during bronchoconstrictor challenge.