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.

On the terminology for describing the length-force relationship and its changes in airway smooth muscle. J Appl Physiol 97: 2029 -2034, 2004; doi:10.1152/japplphysiol.00884.2004.-The observation that the length-force relationship in airway smooth muscle can be shifted along the length axis by accommodating the muscle at different lengths has stimulated great interest. In light of the recent understanding of the dynamic nature of length-force relationship, many of our concepts regarding smooth muscle mechanical properties, including the notion that the muscle possesses a unique optimal length that correlates to maximal force generation, are likely to be incorrect. To facilitate accurate and efficient communication among scientists interested in the function of airway smooth muscle, a revised and collectively accepted nomenclature describing the adaptive and dynamic nature of the lengthforce relationship will be invaluable. Setting aside the issue of underlying mechanism, the purpose of this article is to define terminology that will aid investigators in describing observed phenomena. In particular, we recommend that the term "optimal length" (or any other term implying a unique length that correlates with maximal force generation) for airway smooth muscle be avoided. Instead, the in situ length or an arbitrary but clearly defined reference length should be used. We propose the usage of "length adaptation" to describe the phenomenon whereby the length-force curve of a muscle shifts along the length axis due to accommodation of the muscle at different lengths. We also discuss frequently used terms that do not have commonly accepted definitions that should be used cautiously.smooth muscle contraction; adaptation; plasticity; cytoskeleton; contractile apparatus THE CAPACITIES OF AIRWAY SMOOTH MUSCLE to generate force and to shorten are not a unique function of muscle length. Instead, they change appreciably depending on the histories of muscle loading, length, and activation. These changes can occur over the course of days, hours, and even seconds (9, 11-14, 24, 35, 41, 44, 46). As a result, the length-force relationship of airway smooth muscle is highly mutable, and its characterization is meaningful only when the histories on which the relationship is derived are included. Length-dependent force generation in other smooth muscles is also known to be influenced by various factors (18,29,34,36,39), with the extent of influence varying from one type of smooth muscle to another. The following description of phenomena and terminology is based on and intended for airway smooth muscle, and it may or may not apply to other smooth muscle types. Current terminology that describes the length-force characteristic in airway smooth muscle is borrowed from the physiology of striated muscle but is inadequate, and in some cases ill-suited, to depict the mutable relationship in airway smooth muscle. Thus there is a need to seek a consensual agreement among scientists working in the field of airway smooth muscle biomechanics concern...

Abstract. During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front.The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca 2+ signal may trigger the cytoskeletal reorganiTation requlred for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca 2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca 2+ signals. To identify these messengers, Ca 2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]t increased rapidly from ~100 nM to >.~400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of '~ 200 nM.The initial Ca 2+ spike was insensitive acutely to removal of extracellular Ca 2+ but was abolished by treatments expected to deplete internal Ca 2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lametlipod activity and suppressed the Ca 2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca 2+ spike on stimulation. Although cell flattening and larneUipod activity were induced by chemoattractant when the normal Ca 2+ response was blocked, cells failed to polarize and crawl, indicating that Ca 2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca 2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca 2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.D RECTIONAL persistence during locomotion in a stimulus gradient and rapid reorientation when the direction of the stimulus gradient changes are virtually certain to involve a complex web of highly interactive intracellular processes that stabilize directionality in a way that can be immediately and effectively overridden. Although components of the signal transduction pathways that control cell orientation have been identified in many types of cells, little is known about the mechanisms involved in translating the spatio-temporal patterns of the stimulus into appropriate changes in intracellular organization. Ca2+-sensitivity of a large number of cytoskeletal proteins suggests that Ca 2+ could play an important role. Thus considerable effort has been devoted to characteri...

Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose inf luence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.Several methods have been used in the past to probe the role of a protein in cell function, each with advantages as well as limitations. Organic compounds are available that can modulate the activity of proteins, but interpretation of effects often is complicated by their relatively slow onset and low selectivity for a specific protein. Impressive progress toward single protein specificity has been made with antisense (1) and homologous recombination (2) methods, which disrupt the expression, and thus the function of a specific protein. Interpretation of effects, or lack thereof, on a cellular function may be complicated, however, by compensatory pathways enhanced by the absence of the targeted protein. Peptides that bind to proteins with high affinity and high selectivity provide a means to rapidly and potently inhibit the activity of selected proteins, but peptides must be microinjected into cells, and microinjection itself can at least transiently alter cell function. It thus would be desirable to have a way to make a peptide that is initially inactive or ''caged'' because of a strategically placed photolabile moiety (3, 4). Such a peptide could be injected into a cell, and time allowed for it to distribute evenly and for normal cell function to be verified. The peptide's biological activity then could be unmasked by light-directed removal of the photolabile group. Each cell would be its own control, thereby diminishing effects of cell to cell variability, and active peptides could be produced rapidly (within milliseconds) and with good spatial resolution.We describe here the preparation and use of photoactivatable caged peptides targeted against calmodulin and myosin light chain kinase (MLCK). Because these two proteins are known to be essential in the control of smooth muscle contractility, the efficacy of the caged...

Birefringence and force produced by pig tracheal smooth muscles were recorded every 100 ms during electrically stimulated tetani at muscle lengths that varied 1.5-fold and at the peak of acetylcholine contractures at the same lengths. Isometric force was nearly the same at all lengths. Resting birefringence at the longest length was 30% greater than that at the shortest length. During tetani, birefringence increased with approximately the same time course as force, rising by 20% at the shortest length and 9% at the longest length, and continued to increase by an additional 0.5-1.5% of the resting value for 2-8 s after stimulation ended and force began to fall. This late increase was greatest and more sustained at longer lengths. During contractures, birefringence increased by 25 and 18% at the shortest and longest lengths, respectively. Comparison of these results with our published thick-filament densities suggests that thick-filament density increased by about 80, 72 and 50% during contractures at the short, intermediate and long lengths, and that ∼35% of birefringence in the resting muscle at the longest length was not due to thick filaments. These findings support the hypotheses that tracheal smooth muscle adapts to longer lengths by increasing thick-filament mass and that myosin thick filaments are evanescent, dissociating partially during relaxation and reforming upon activation. The results further suggest that thick-filament formation is sufficiently rapid to account for the velocity slowing and some of the force increase observed during the rise of activation of tracheal smooth muscle.

A B S T R A C T IsolatedRana pipiens sartorius muscles at 0°C were stimulated via their nerves and small stretches or releases applied during the plateau of the isometric tetanus at to. Extra heat above the isometric maintenance heat was produced during the drop in tension caused by release and, for very small releases (A-0.5% to), was completely reabsorbed during tension recovery. The extra heat was always directly proportional to the tension change. Heat absorption proportional to the tension change was also observed during the increase in tension produced by small stretches in the range 0.5% t0 -< At ~ 3.0% to. The mean heat:tension ratioR in seven experiments was -0.0084, which is within the range of values reported previously by Woledge. In addition, it was found that during tension recovery after small releases of 1.0% to -< At <_ 3.0% t0 the "contractile" component seems able to shorten about 1% #0 without producing shortening heat.

Tension and heat production were measured in frog sartorius muscles in response to small shortening ramps (releases) at high and moderate speed. Transient tension responses to fast releases (0.1 to 0.4 mm in 1 or 4 ms) were similar to the tension transients length-clamped single fibers. Tension time courses during releases at 25 mm/s were like fiber responses calculated from the first two phases of the step responses (Ford et al., 1977). We conclude that similar crossbridge transitions produce tension transients observed in whole muscles and single fibers. Heat was absorbed during rapid tension recovery after fast releases and during the later part of releases at 25 mm/s. Variation of heat absorption with release size was compared with that of crossbridge movement predicted by the Huxley-Simmons hypothesis of force generation (Huxley and Simmons, 1971). Agreement between the two supports the conclusion that heat is absorbed by the crossbridge transitions responsible for rapid tension recovery after release. The results indicate that the entropy change of these transitions is positive.

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentration-dependent. Small calcium injections affected specifically actin-containing motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates IP3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular cAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture.