What Makes the Airways Contract Abnormally? Is It Inflammation?

JULIAN SOLWAY

University of Chicago, Chicago, Illinois

	INTRODUCTION

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

What airway smooth muscle definitely does in asthma is contract. Because it encircles the airway, smooth muscle contraction narrows the airway lumen, thereby obstructing airflow, promoting airflow limitation and pulmonary hyperinflation, and thus producing dyspnea in a typical "asthma attack." However, little is known about airway smooth muscle function in individuals with asthma or how inflammation actually alters airway smooth muscle function. Therefore, we should consider the following as "partially known or conflicting data."

	DOES ASTHMATIC AIRWAY SMOOTH MUSCLE CONTRACT NORMALLY?

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

Does asthmatic airway smooth muscle contract normally? Yes, and perhaps no. Most studies indicate that airway smooth muscle isolated from lungs of individuals with asthma exhibits normal sensitivity to direct activators of contraction, including histamine, muscarinic agonists, cysteinyl leukotrienes, and prostanoids (1). Other studies (4, 5) instead demonstrate increased isometric force generation in response to various constrictor stimuli. Importantly, these studies used static isometric tension as the indicator of contractile response and, in doing so, this work did not fully evaluate potential abnormalities in the dynamics of asthmatic airway smooth muscle contraction. Two groups have, however, demonstrated an increase in the extent of isotonic shortening of asthmatic airway smooth muscle. Schellenberg and colleagues reported an up to threefold increase in maximal shortening of isolated smooth muscle strips from three individuals with asthma (4, 6), and Stephens and coworkers (7) found that individual myocytes dissociated from endobronchial biopsy specimens taken from five subjects with asthma shortened about 50% more than control myocytes obtained from biopsies of normal airways. Other studies in animal tissues suggest that the extent of unloaded shortening increases with the velocity of contraction (8). As such, the greater extent of shortening observed in excised asthmatic airway smooth muscle raises the possibility that asthmatic smooth muscle contracts with abnormally high velocity.

	IS VELOCITY OF SHORTENING INCREASED IN ASTHMA?

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

No published studies have directly addressed whether the velocity of shortening of airway smooth muscle is increased in asthma, although recent clinical studies are consistent with this possibility (see below). However, a series of experiments principally from Dr. Stephens' group demonstrates that passive sensitization of airway smooth muscle can alter its contraction dynamics, speeding the velocity of contraction for any given load (9), and also increasing the maximal extent of unloaded shortening (10). This phenomenon has been demonstrated for normal human airway smooth muscle (13), but has been studied more extensively in canine airway tissues (9- 12). Sensitization increases the quantity and activity of myosin light chain kinase (MLCK), an enzyme that phosphorylates the regulatory 20-kD light chain (LC20) of smooth muscle myosin, thereby increasing myosin ATPase activity (of the myosin heavy chain head) and increasing the actomyosin cycling rate. Importantly, it is this cycling rate that determines the maximal velocity of contraction, whereas it is the number of actin-myosin cross-bridges that determines contractile force; these two "features" of smooth muscle contraction act more or less independently. Thus, the principal consequence of passive sensitization of airway smooth muscle is to increase its velocity of contraction and extent of maximal shortening without increasing its force-generating capacity. Studies have identified another enzyme that phosphorylates LC20 in a calcium- calmodulin-independent manner (14), whose activity may underlie the basal actomyosin cycling activity revealed by treatment with inhibitors of myosin light chain phosphatase.

	WHAT CONTROLS CROSS-BRIDGE CYCLING RATES?

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

There are other regulatory systems that also control the actomyosin cross-bridge cycling rate (Figure 1). First, two targets of Rho family GTPases influence myosin regulatory light chain phosphorylation. Rho kinase (p160 ROCK) phosphorylates LC20 (15), as well as the myosin-binding subunit of myosin light chain phosphatase (16), thereby inhibiting its ability to dephosphorylate LC20 (17). Phosphorylation of the light chain by MLCK or other enzymes is thus more long-lived, and the smooth muscle exhibits "calcium sensitization"an augmented contractile response for any level of intracellular calcium mobilization. Complicating matters is a recent report that another Rho GTPase family member, Rnd1, antagonizes RhoA-dependent calcium sensitization (18). Rnd1 activity is induced in intestinal or arterial smooth muscle by exposure to progesterone or estrogen. Rho family GTPases also activate another signaling intermediate, designated PAK (p21-activated kinase), which phosphorylates the myosin regulatory light chain in vitro. However, the physiological significance of this process is uncertain (15) because the level of PAK activity does not correlate with the extent of LC20 phosphorylation.

View larger version (31K): [in this window] [in a new window]   Figure 1.   Potential interactions between mediators of inflammation (boxed italics) and regulators of velocity of shortening (boxed, roman). Arrows indicate positive regulation, unless accompanied by two dashes, which indicate that regulation is negative. Encircled P indicates transfer of phosphate group.

Instead, it appears that PAK regulates smooth muscle contraction through phosphorylation of the actin-binding protein caldesmon (15). A number of actin-binding proteins that might regulate contraction have been identified in smooth muscle, including caldesmon and h1-calponin. Each of these species has been shown to slow actin-myosin sliding in the in vitro motility assay (19), by binding to actin in a way that reduces actin-myosin ATPase activity through mechanisms now under study. Phosphorylation of either caldesmon or calponin on key residues reduces its affinity for actin and also impairs its ability to slow actin velocity in the in vitro motility assay. Caldesmon can be phosphorylated by PAK (15), p38 mitogen-activated protein kinase (23), extracellular signal-regulated kinases 1 and 2 (ERK 1/2) (21, 22), and by the epsilon isoform of protein kinase C (PKC-) (24). Calponin is also known to be phosphorylated by PKC- (24). Apparently, contraction can be induced independent of increased calcium mobilization or changes in LC20 phosphorylation simply by removing the inhibitory influence of either caldesmon or calponin on actin- myosin cross-bridge cycling. Another abundant smooth muscle protein, SM22 (also called transgelin), shares homology with calponin and binds to actin [(25); Y. Fu and J. Solway, unpublished observations], but its potential role in regulating smooth muscle contraction remains uncertain.

Above, we noted that Rho kinase can inhibit myosin light chain phosphatase. Myosin light chain phosphatases can also be inhibited by PKC [apparently acting in some circumstances through an intermediate inhibitor protein (26)]. Thus, activation of PKCs might potentiate the actomyosin cycling rate by reducing inhibition by caldesmon and calponin, and by inhibiting the dephosphorylation of LC20.

	DOES AIRWAY INFLAMMATION INFLUENCE ACTIN-MYOSIN CYCLING?

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

Does airway inflammation modulate the expression or activity of LC20 kinases or phosphatases, or of actin-binding proteins that influence actomyosin cycling? Little is known, but inferences might be drawn from studies in other systems. First, Katsumata and colleagues (27) found that pig coronary arterial inflammation induced by repeated applications of interleukin 1 (IL-1) led to enhanced LC20 diphosphorylation upon serotonin stimulation. Although this effect was inhibited by fasudil, which blocks both PKC and MLCK, the precise mechanism that led to the increased LC20 phosphorylation was not identified. Second, transforming growth factor  (TGF-), which can be released by eosinophils and resident lung cells, can transiently increase h1-calponin mRNA in cultured smooth muscle cells (28), and TGF-, but not tumor necrosis factor  (TNF), induced h1-calponin mRNA and protein in cultured Ito cells from human liver (29). TGF- exposure also appears to activate MLCK, as suggested by its MLCK-dependent disruption of endothelial adherens junctions (30). Next, a variety of stimuli that could appear in asthmatic airways, including smooth muscle mitogens, lysophosphatidic acid (from serum), and TNF can lead to the activation of Rho family GTPases and/or MAP kinases, which could enhance the actomyosin cycling rate through the mechanisms discussed above. TNF also potentiates carbachol-induced intracellular calcium elevation in airway smooth muscle (31), and thrombin (from serum) directly stimulates intracellular calcium elevation (32).

A number of studies have begun to reveal that cytokine exposure can alter airway smooth muscle isometric contractile function. For example, Hakonarson and coworkers (33) studied the influence of helper T cell type 1 (Th1)- and Th2-type cytokines on contractions of airway smooth muscle segments, some of which were presensitized by exposure to atopic asthmatic serum. Exposure of sensitized strips to the Th1 cytokines IL-2 and interferon  (IFN-) ablated their augmented constrictor responses to muscarinic stimulation, and exposure of naive strips to the Th2 cytokines IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) augmented their muscarinic responsiveness and impaired their relaxation to isoproterenol. The intracellular signaling mechanisms by which cytokine-induced alteration in contractile activation occurs are not yet established.

A study by Fan and colleagues (34) demonstrated that tracheal smooth muscle strips from antigen-sensitized SLJ mice exhibited increased velocity of shortening and greater extent of maximal shortening compared with strips from nonsensitized SLJ mice, or with strips from sensitized or control ASW mice. Interestingly, splenocytes from SLJ mice produced more IL-4 than did spleen cells from ASW mice, suggesting (but not proving) a potential causal relationship between the airway muscular effects of sensitization and IL-4 generation. IL-4 has been shown to reduce the proliferative responses of cultured airway smooth muscle cells to platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and thrombin through a cAMP-independent mechanism (35), but its potential direct effects on contraction are unknown.

While these data do not specifically demonstrate that velocity of shortening is increased in asthmatic airway smooth muscle, they suggest that airway smooth muscle subjected to an inflammatory environment might behave in this fashion. Could that lead to asthmatic airway dysfunction?

Two important consequences of increased velocity of smooth muscle shortening follow. First, as noted above, experiments demonstrate that sensitized tissues with increased velocity of shortening exhibit a greater extent of total shortening in response to contractile stimulation. Scant data also suggest that asthmatic airway smooth muscle may exhibit an abnormally increased total extent of unloaded shortening (4). Second, theory (which has not yet been proved experimentally) suggests that a faster velocity of shortening should impart contractile hyperresponsiveness, by minimizing the bronchodilating effect of a deep inhalation (36). According to this hypothesis, slowly (i.e., normally) contracting smooth muscle would have insufficient time to contract between (bronchodilating) breaths in a breathing individual, whereas rapidly contracting smooth muscle would quickly reestablish airway narrowing upon release of each tidal or deep inspiration. Interestingly, experiments have already demonstrated that individuals with asthma exhibit diminished or absent bronchodilation after a deep breath (compared with unaffected individuals) (37), even though inhalation does dilate the airways transiently (38). To date, no direct measurements of unloaded velocity of shortening of asthmatic airway smooth muscle have been reported, but a report from Jackson and colleagues (39) suggests that the rate of reestablishment of methacholine-induced bronchoconstriction after transient deep breath-induced bronchodilation may be faster in individuals with asthma (time constant, 0.2 min) than in healthy subjects (time constant, 0.7 min). Other preliminary reports link genetic differences in in vivo airway responsiveness to ex vivo velocity of airway smooth muscle shortening (7, 40).

Beyond its potential contractile dysfunction, most reports suggest that smooth muscle from asthmatic airways does not relax normally in response to -adrenergic agonists (41). This stems from uncoupling of -receptor signaling, rather than from any deficiency in -receptor abundance (44). Studies using cultured human airway myocytes show that exposure to exogenous IL-1 induces cyclooxygenase 2 expression (see below), thereby promoting prostaglandin E₂ (PGE₂) release from treated cells, and PGE₂ impairs the relaxation normally induced by -adrenergic stimulation by uncoupling intracellular signaling (45, 46). Interestingly, sensitization promotes autocrine secretion of IL-1 by airway smooth muscle (47), suggesting a mechanism by which atopy infection might contribute to the abnormal relaxation of asthmatic airway smooth muscle. Infection with rhinovirus also impairs -adrenergic agonist- induced relaxation, apparently through upregulation of Gi3 (48).

	WHAT IS THE CONTRIBUTION OF AIRWAY SMOOTH MUSCLE TO INFLAMMATION?

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

Recently, it has been recognized that airway smooth muscle may participate in the dysregulated inflammation characteristic of asthmatic airways. Cultured human airway smooth muscle stimulated with IL-1 releases GM-CSF (which prolongs eosinophil survival), through a mechanism that is inhibited by glucocorticoids but not by indomethacin (49). Upon stimulation with TNF, cultured human airway myocytes also release RANTES, an 8-kD C-C chemokine with chemoattractant activity for eosinophils, lymphocytes, and monocytes (50). This effect is potentiated by coincubation with interferon  (which does not induce RANTES secretion itself), and can be inhibited partially by exposure to the Th2 cytokines IL-4, IL-10, and IL-13 and by glucocorticoids. A cytomix incorporating IL-1, TNF, and IFN- stimulated accumulation of cyclooxygenase 2 (COX-2) mRNA and protein, and accumulation in culture supernatant of PGE₂ and 6-keto-PGF₁ (51). Furthermore, one study demonstrates that treatment of airway smooth muscle with serum from individuals with atopic asthma induces the sequential elaboration of Th2 and then Th1 type cytokines (33). Together, these data suggest that chemokines, cytokines, and prostanoids, which could enhance asthmatic airway inflammation, are produced by airway smooth muscle cells exposed to an inflammation milieu. However, it remains unknown whether this potential role in sustaining airway inflammation is quantitatively important in asthmatic airways.

	IMPORTANT QUESTIONS

TOP INTRODUCTION DOES ASTHMATIC AIRWAY SMOOTH... IS VELOCITY OF SHORTENING... WHAT CONTROLS CROSS-BRIDGE... DOES AIRWAY INFLAMMATION... WHAT IS THE CONTRIBUTION... IMPORTANT QUESTIONS REFERENCES

• Is the velocity of shortening of airway smooth muscle abnormally increased in subjects with asthma, or in individuals genetically predisposed to acquiring asthma?
• If so, what is the underlying dysregulation of contraction dynamics that accounts for the increased velocity of shortening?
• What role, if any, do inflammatory mediators play in the generation of these abnormalities?
• In model animal systems (e.g., transgenic mice overexpressing constitutively active MLCK in airway smooth muscle), does a primary increase in velocity of shortening lead to secondary airway constrictor hyperresponsiveness in the intact lung?
• Could reduction of constrictor hyperresponsiveness be accomplished by slowing the velocity of airway smooth muscle shortening as a novel asthma therapy?
• Does airway smooth muscle make an important contribution to the inflammatory environment of asthmatic airways?

	Footnotes

Correspondence and requests for reprints should be addressed to J. Solway, M.D., University of Chicago MC6026, 5841 S. Maryland Avenue, Chicago, IL 60637.

	References

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