Latrophilin receptors: novel bronchodilator targets in asthma.

BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.

Universal behavior of the osmotically compressed cell and its analogy to the colloidal glass transition.

Mechanical robustness of the cell under different modes of stress and deformation is essential to its survival and function. Under tension, mechanical rigidity is provided by the cytoskeletal network; with increasing stress, this network stiffens, providing increased resistance to deformation. However, a cell must also resist compression, which will inevitably occur whenever cell volume is decreased during such biologically important processes as anhydrobiosis and apoptosis. Under compression, individual filaments can buckle, thereby reducing the stiffness and weakening the cytoskeletal network. However, the intracellular space is crowded with macromolecules and organelles that can resist compression. A simple picture describing their behavior is that of colloidal particles; colloids exhibit a sharp increase in viscosity with increasing volume fraction, ultimately undergoing a glass transition and becoming a solid. We investigate the consequences of these 2 competing effects and show that as a cell is compressed by hyperosmotic stress it becomes progressively more rigid. Although this stiffening behavior depends somewhat on cell type, starting conditions, molecular motors, and cytoskeletal contributions, its dependence on solid volume fraction is exponential in every instance. This universal behavior suggests that compression-induced weakening of the network is overwhelmed by crowding-induced stiffening of the cytoplasm. We also show that compression dramatically slows intracellular relaxation processes. The increase in stiffness, combined with the slowing of relaxation processes, is reminiscent of a glass transition of colloidal suspensions, but only when comprised of deformable particles. Our work provides a means to probe the physical nature of the cytoplasm under compression, and leads to results that are universal across cell type.

MEK modulates force-fluctuation-induced relengthening of canine tracheal smooth muscle.

Tidal breathing, and especially deep breathing, is known to antagonise bronchoconstriction caused by airway smooth muscle (ASM) contraction; however, this bronchoprotective effect of breathing is impaired in asthma. Force fluctuations applied to contracted ASM in vitro cause it to relengthen, force-fluctuation-induced relengthening (FFIR). Given that breathing generates similar force fluctuations in ASM, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. Thus it is of considerable interest to understand what modulates FFIR, and how ASM might be manipulated to exploit this phenomenon. It was demonstrated previously that p38 mitogen-activated protein kinase (MAPK) signalling regulates FFIR in ASM strips. Here, it was hypothesised that the MAPK kinase (MEK) signalling pathway also modulates FFIR. In order to test this hypothesis, changes in FFIR were measured in ASM treated with the MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Increasing concentrations of U0126 caused greater FFIR. U0126 reduced extracellular signal-regulated kinase 1/2 phosphorylation without affecting isotonic shortening or 20-kDa myosin light chain and p38 MAPK phosphorylation. However, increasing concentrations of U0126 progressively blunted phosphorylation of high-molecular-weight caldesmon (h-caldesmon), a downstream target of MEK. Thus changes in FFIR exhibited significant negative correlation with h-caldesmon phosphorylation. The present data demonstrate that FFIR is regulated through MEK signalling, and suggest that the role of MEK is mediated, in part, through caldesmon.

The temperature dependence of cell mechanics measured by atomic force microscopy.

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

Effective pulmonary ventilation with small-volume oscillations at high frequency.

At high oscillation frequencies (4 to 30 hertz), effective alveolar ventilation can be achieved with tidal volumes much smaller than the anatomic dead space. An explanation of this phenomenon is given in terms of the combined effects of diffusion and convection and in terms of data consistent with the hypothesis. Theory and experimental results both show that the significant variable determining the effectiveness of gas exchange is the amplitude of the oscillatory flow rate independent of the individual values of frequency and stroke volume.

Regional alveolar pressure during periodic flow. Dual manifestations of gas inertia.

We measured pressure excursions at the airway opening and at the alveoli (PA) as well as measured the regional distribution of PA during forced oscillations of six excised dog lungs while frequency (f[2-32 Hz]), tidal volume (VT [5-80 ml]), and mean transpulmonary pressure (PL [25, 10, and 6 cm H2O]) were varied. PA's were measured in four alveolar capsules glued to the pleura of different lobes. The apex-to-base ratio of PA's was used as an index of the distribution of dynamic lung distension. At low f, there was slight preferential distension of the lung base which was independent of VT, but at higher f, preferential distension of the lung apex was found when VT's were small, whereas preferential distension of the lung base was found when VT's approached or exceeded dead space. These VT-related changes in distribution at high frequencies seem to depend upon the branching geometry of the central airways and the relative importance of convective momentum flux vs. unsteady inertia of gas residing therein, which, in this study, we showed to be proportional to the ratio VT/VD*, where VD* is an index of dead space. Furthermore, they imply substantial alteration in the distribution of ventilation during high frequency ventilation as f, VT, and PL vary. The data also indicate that alveolar and airway opening pressure costs per unit flow delivered at the airway opening exhibit weakly nonlinear behavior and that resonant amplification of PA's, which has been described previously for the case of very small VT's, persists but is damped as VT's approach dead space values.

Respiratory system impedance from 4 to 40 Hz in paralyzed intubated infants with respiratory disease.

To describe the mechanical characteristics of the respiratory system in intubated neonates with respiratory disease, we measured impedance and resistance in six paralyzed intubated infants with respiratory distress syndrome, three of whom also had pulmonary interstitial emphysema. We subtracted the effects of the endotracheal tube after showing that such subtraction was valid. Oscillatory flow was generated from 4 to 40 Hz by a loudspeaker, airway pressure was measured, and flow was calculated from pressure changes in an airtight enclosure mounted behind the flow source (speaker plethysmograph). After subtraction of the endotracheal tube contribution, resistance ranged from 22 to 34 cmH2O liter-1 s; compliance from 0.22 to 0.68 ml/cmH2O; and inertance from 0.0056 to 0.047 cmH2O liter-1 s2. Our results indicate that, for these intubated infants, the mechanics of the respiratory system are well described as resistance, compliance, and inertance in series. Most of the inertance, some of the resistance, and little of the compliance are due to the endotracheal tube. When the contribution of the endotracheal tube is subtracted, the results are descriptive of the subglottal respiratory system. These data characterize the neonatal respiratory system of infants with respiratory distress syndrome (with or without pulmonary interstitial emphysema) in the range of frequencies used during high frequency ventilation.

Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.

Axial dispersion in respiratory bronchioles and alveolar ducts.

The mixing of gases in the pulmonary acinus was characterized by analyzing axial gas dispersion during steady flow in models of respiratory bronchioles and alveolar ducts. An analysis (method of moments) developed for addressing dispersion in porous media was used to derive an integral expression for the axial dispersion coefficient (D*). Evaluation of D* required solving the Navier-Stokes equations for the flow field and a convection-diffusion type equation arising from the analysis. D* was strongly dependent on alveolar volume per central duct volume, the aperture size through which the alveoli communicate with the central duct, and the Péclet number (Pe). At smaller Pe (flow rate) D* was substantially smaller than the molecular diffusion coefficient, whereas at larger Pe (flow rate) D* was much greater than the Taylor-Aris result for flow-enhanced dispersion in straight tubes. Also, flow-enhanced dispersion became appreciable at smaller Pe than indicated by the Taylor-Aris result. These behaviors transcend both the lower and upper limits established previously for gas mixing in the pulmonary acinus.

On the imperfect elasticity of lung tissue.

This paper deals with a unifying hypothesis addressed at lung tissue resistance and its responses to neurohumoral and biophysical stimuli. The hypothesis holds that dissipative and elastic processes within lung tissue are coupled at the level of the stress-bearing element. Such a description leads naturally to consideration of a readily measured attribute of organ-level dissipative behavior called lung tissue hysteresivity, eta. On preliminary analysis this attribute is found to be nearly frequency independent and numerically conserved across species. To the degree that the numerical value of eta might be conserved during an intervention in which tissue dynamic elastance changes, such behavior would be consistent with the notion that elastic energy storage and dissipative energy loss reside within the very same stress-bearing element and, moreover, that those processes within the stress-bearing element bear an approximately fixed relationship. Tissue hysteresivity is closely related to the parameter K used by Bachofen and Hildebrandt (J. Appl. Physiol. 30: 493-497, 1971) to describe energy dissipation per cycle, and both lend themselves directly to interpretation based on processes ongoing at the levels of microstructure and molecule. Intraparenchymal connective tissues, surface film, and contractile elements appear to submit individually to this description and, in doing so, yield respective hysteresivities that are relatively well matched; this suggests that such hysteretic matching may be a necessary condition for synchronous expansion of the alveolar duct. The overriding simplicity with which this description organizes diverse observations implies that it may capture some unifying attribute of underlying mechanism.

Periodic flow at airway bifurcations. I. Development of steady pressure differences.

In studies of large-amplitude periodic flows at an airway bifurcation, we found an appreciable steady-state pressure difference between the terminal units. To elucidate the fluid dynamic origins of such steady-state pressure differences, we studied single asymmetric bifurcation models with various area ratios and branching angles. The daughter ducts were identical in size and were terminated into identical elastic loads. Sinusoidal flow oscillations were applied at the parent duct so that the upstream Reynolds number ranged from 30 to 77,000 and the Womersley parameter from 2 to 30. The steady-state component (time averaged) of the pressure measured at the terminal with the smaller branching angle was found to be consistently higher than that at the other terminal. This steady-state pressure difference scaled approximately as a fixed fraction of the parent duct dynamic head. Guided by the results of flow-visualization studies, we modeled such behavior based on the temporal and spatial differences of head loss between the two branches of the bifurcation. Our results suggest that interlobar heterogeneity of mean alveolar-pressure observed in excised canine lungs during high frequency oscillation (Allen et al., J. Appl. Physiol. 62: 223-228, 1987) arises solely from fluid dynamic origins: differential head loss due to asymmetry of central airway branching structure.

Historical perspective on airway smooth muscle: the saga of a frustrated cell.

Despite the lack of a clearly defined physiological function, airway smooth muscle receives substantial attention because of its involvement in the pathogenesis of asthma. Recent investigations have turned to the ways in which the muscle is influenced by its dynamic microenvironment. Ordinarily, airway smooth muscle presents little problem, even when maximally activated, because unending mechanical perturbations provided by spontaneous tidal breathing put airway smooth muscle in a perpetual state of "limbo," keeping its contractile machinery off balance and unable to achieve its force-generating potential. The dynamic microenvironment affects airway smooth muscle in at least two ways: by acute changes associated with disruption of myosin binding and by chronic changes associated with plastic restructuring of contractile and cytoskeletal filament organization. Plastic restructuring can occur when dynamic length changes occur between sequential contractile events or within a single contractile event. Impairment of these normal responses of airway smooth muscle to its dynamic environment may be implicated in airway hyperresponsiveness in asthma.

Periodic flow at airway bifurcations. II. Flow partitioning.

The distribution of flow among parallel pathways is believed to be determined by the balance of downstream mechanical loads or time constants. We studied the influence of upstream flow conditions and airway geometry vs. downstream mechanical impedances in determining flow partitioning at airway bifurcations. Each model consisted of a single rigid bifurcation with various branching angles and area ratios but having identical pathway impedances. Sinusoidal volumetric oscillations were applied at the parent duct with various frequencies and tidal volumes. Measuring the terminal pressures continuously, we calculated the flow distribution. When flow amplitude was small, flow partitioning was homogeneous and synchronous, as expected in a system possessing homogeneous pathway impedances and time constants. But when flow amplitude was large and frequency was high, appreciable heterogeneity and asynchrony of flow partitioning arose; during midinspiration the high-velocity flow stream preferentially favored the axial pathway. This effect vanished in the absence of a net area change at the bifurcation. For a given bifurcation geometry, these observations could be organized using only two nondimensional parameters, neither of which incorporated consideration of fluid friction. The description of temporal events required, in addition, a nondimensional time. Therefore these flow-dependent phenomena and their underlying mechanisms differ fundamentally from those described in classical impedance models. The complex pattern of nonuniform interregional behaviors apparent in whole lungs when tidal volume and frequency are large (Allen et al., J. Clin. Invest. 76: 620-629, 1985) is reiterated faithfully in models consisting of only two compartments with homogeneous time constants. As such, the behaviors observed in lungs would appear to be attributable in large part to fluid dynamic factors in central airways.

Pulmonary airway area by the two-microphone acoustic reflection method.

We noninvasively assessed airway dimensions from acoustic reflection data measured at the mouth. We recently described a two-transducer system for measurement of the nasal airway. Here we apply this approach to the measurement of the upper airway and trachea. We describe the theoretical implications of breathing on this kind of measurement and propose a new procedure that, unlike single- and dual-transducer systems used currently, does not require the use of He-O2 for inference of geometry of subglottic airways.

Proportionality between chest wall resistance and elastance.

Fredberg and Stamenovic (J. Appl. Physiol. 67: 2408-2419, 1989) demonstrated a relatively robust phenomenological relationship between resistance (R) and elastance (E) of lung tissue during external forcing. The relationship can be expressed as omega R = eta E, where omega = 2 pi times forcing frequency and eta is hysteresivity; they found eta to be remarkably invariant under a wide range of circumstances. From data gathered in previous experiments, we have tested the adequacy and utility of this phenomenological description for the chest wall (eta w) and its major compartments, the rib cage (eta rc), diaphragm-abdomen (eta d-a), and belly wall (eta bw+). For forcing frequencies and tidal volumes within the normal range of breathing, we found that eta w remained in a relatively narrow range (0.27-0.37) and that neither eta w nor the compartmental eta's changed much with frequency or tidal volume. Compared with eta w, eta rc tended to be slightly low, whereas eta d-a tended to be slightly higher than eta w. However, at higher frequencies (greater than 1 Hz) all eta's increased appreciably with frequency. During various static nonrespiratory maneuvers involving use of respiratory muscles, eta w increased up to twofold. We conclude that in the normal ranges of breathing frequency and tidal volume 1) elastic and dissipative processes within the chest wall appear to be coupled, 2) eta's of the various component parts of the chest wall are well matched, 3) respiratory muscle contraction increases the ratio of cyclic dissipative losses to energy storage, and 4) R of the relaxed chest wall can be estimated from E.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alveolated duct structure on aerosol kinetics. I. Diffusional deposition in the absence of gravity.

We examined the effects of alveolar duct structure on particle deposition in the pulmonary acinus. The low Reynolds number velocity field of carrier gas in a geometric model of the alveolated duct was solved numerically. Particle trajectories were computed from the Langevin equation. Conditional probabilities of the trajectories were calculated with an eigenfunction expansion technique in the absence of gravity. For submicron particles, Brownian motion dominated the process; the deposition rate dramatically decreased with boundary layer growth. For fine particles, fully developed boundary layer profiles determined the deposition over most of the acinar length. The assumption of a uniform radial profile results in a substantial overestimation of the local deposition rate. The deposition rate in an alveolated duct was always smaller than that in an equivalent straight tube of the same volume. Within the alveolus the deposition pattern was markedly nonuniform, with higher deposition near the alveolar entrance ring; this finding is consistent with experimental observations in animals (e.g., see Zeltner et al. J. Appl. Physiol. 70: 1137-1145, 1991). We conclude that the structure of the alveolar duct has an important influence on aerosol particle deposition in the lung acinus.

Effects of alveolated duct structure on aerosol kinetics. II. Gravitational sedimentation and inertial impaction.

We studied the effects of alveolated duct structure on deposition processes for particle diameters > or = 1 micron. For such large particles, Brownian motion is insignificant but gravity and inertial forces play an important role. A Lagrangian description of particle dynamics in an alveolated duct flow was developed, and computational analysis was performed over the physiologically relevant range. At low flow rates gravity caused deposition. Gravitational cross-streamline motion depended on the coupled effects of curvature of gas streamlines and duct orientation relative to gravity. The detailed convective flow pattern was an important factor in determining deposition. At higher flow rates, inertial impaction contributed markedly to deposition. The curved nature of streamlines again played a major role on deposition, but duct orientation had little effect. In the medium range of flow rates, both gravitational and inertial forces simultaneously influenced particle motion. Particle inertia, per se, did not cause deposition but substantially suppressed gravitational deposition. The deposition mechanism was complex; contrary to what is often assumed in past analyses, the interaction between gravitational and inertial effects could not be described in a simple additive fashion. We conclude that the structure of the alveolar duct has an important role in gravitational sedimentation and inertial impaction in the lung acinus.

Lung tissue resistance and hysteretic moduli of lung parenchyma.

Lung tissue resistance (Rti) represents a large and labile component of total pulmonary resistance, but the mechanism is unknown. One hypothesis that has received some support in the literature is that on exposure to contractile agonists airway smooth muscle shortens and then, by the agency of elastic interdependence, induces distortion in surrounding parenchyma. Parenchymal distortion induced in the vicinity of a constricted airway is a pure shear deformation, but currently there are no data available for shear hysteresivity. Guided by a microstructural model, we have assigned stiffness and hysteresivity to microstructural elements and then computed how those properties are expressed at the macroscale in bulk hysteresivities for both shear and volumetric expansion. Hysteresivity for volumetric expansion is shown to be a stiffness-weighted average of hysteresivities of all microstructural components. But as the hysteresivity of microstructural elements increases, that for shear deformation increases to some degree but eventually attains a plateau. Blunted hysteretic response in shear seems to be an intrinsic property of pressure-supported structures, like the lung, that require an inflating pressure to ensure mechanical stability. The analysis indicates that that part of Rti attributable to parenchymal distortion can be at most a small fraction of that attributable to volumetric expansion. These results are purely theoretical in nature, and this suggests that caution is necessary in their interpretation. However, the mechanical basis of the results is sufficiently general to conclude that the hypothesis that parenchymal distortion secondary to bronchoconstriction can account for Rti and its changes seems to be implausible.

Tissue resistance in the guinea pig at baseline and during methacholine constriction.

Total lung resistance (RL), airway resistance (Raw), and tissue resistance (Rti) were measured in unconstricted and methacholine (MCh)-constricted guinea pigs while tidal volume, lung volume, and breathing frequency were varied. Measurements were made in tracheostomized ventilated guinea pigs with use of alveolar capsules. Relationships between Raw and Rti at different breathing frequencies, lung volumes, tidal volumes, and levels of constriction were compared with previously reported values in other species. Our results demonstrate that, at fixed tidal volume, Rti was inversely related to breathing frequency (Rti approximately f-0.64, where f is breathing frequency in Hz) and increased with increasing lung volume. Rti was a significantly greater percentage of RL after MCh administration (40-50%) than at baseline (15-35%), indicating a greater tissue than airway constrictor response. Rti was also 0.5 log dose more responsive to intravenous MCh than Raw on the basis of the dose required to produce 100% increase in resistance from baseline (PD100). These data show that, in the guinea pig, Rti changes with lung volume, breathing frequency, and constrictor tone in a manner similar to other species previously reported and that Rti can be an important determinant of lung dysfunction during constriction, even in species for which it is small in relation to Raw at baseline.

Toward a kinetic theory of connective tissue micromechanics.

The aim of this study is to develop unifying concepts at the microstructural level to account for macroscopic connective tissue dynamics. We establish the hypothesis that rate-dependent and rate-independent dissipative stresses arise in the interaction among fibers in the connective tissue matrix. A quantitative theoretical analysis is specified in terms of geometry and material properties of connective tissue fibers and surrounding constituents. The analysis leads to the notion of slip and diffusion boundary layers, which become unifying concepts in understanding mechanisms that underlie connective tissue elasticity and energy dissipation during various types of loading. The complex three-dimensional fiber network is simplified to the interaction of two ideally elastic fibers that dissipate energy on slipping interface surfaces. The effects of such interactions are assumed to be expressed in the aggregate matrix. Special solutions of the field equations are obtained analytically, whereas the general solution of the model field equations is obtained numerically. The solutions lead to predictions of tissue behavior that are qualitatively, if not quantitatively, consistent with reports of a variety of dynamic moduli, their dependencies on the rate and amplitude of load application, and some features associated with preconditioning.