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.

RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

Mucin secretion by goblet cells was determined by quantifying degranulation events (DE) in isolated, superficial epithelium from canine trachea. The epithelium was isolated and explanted to a novel transparent, permeable support, and the goblet cells were visualized by video microscopy. Baseline degranulation events were quantified at 0.05 DE/min. Luminal ATP (10(-4) M, n = 10) stimulated a biphasic secretory response; a burst, maximum rate = 87.9 +/- 25.3, was followed by a plateau, rate = 1.9 +/- 0.3 DE/min. Serosal ATP elicited a complex set of responses: 9 cells failed to respond, 13 exhibited a trivial response, and 31 responded vigorously but with highly variable patterns of degranulation. Nonhydrolyzable 5'-adenylylimidodiphosphate caused degranulation from both sides of the epithelium. Luminal ADP and adenosine were ineffective. Serosal ADP and adenosine elicited a range of responses that was similar in diversity and magnitude to the ATP response. Our conclusions were as follows: 1) goblet cells in the superficial epithelium of the airway can be studied at the single-cell level in explants; 2) nucleotides stimulate goblet cells to secrete mucin; and 3) the goblet cell expresses different nucleotide receptors on its apical and basolateral membranes.

The regulation of mucin secretion by airway goblet cells is poorly understood and the receptor-based regulatory mechanisms have not been described in human airways. In the present study, we report that extracellular triphosphate nucleotides regulate the rate of granule release from goblet cells in both normal and cystic fibrosis (CF) airway epithelial explants. Explants isolated from nasal and tracheobronchial tissues were mounted in perfusion chambers and the secretory activity was assessed by videomicroscopic determination of degranulation in single goblet cells and by ELISA determination of mucins secreted into the mucosal perfusate. Baseline degranulation was measured at 0.05 degranulation events (DE)/min. In normal goblet cells, mucosal ATP (10(-4) M, n = 17) induced a biphasic secretory response comprising 29.1 +/- 4.9 DE during the first 5 min, with an initial rate of 118.2 +/- 10.2 DE/min. Mucosal UTP (10(-4) M, n = 9) induced a similar response to ATP (initial rate: 89.2 +/- 23.9 DE/min, 17.9 +/- 5.1 DE in 5 min), but mucosal 2-MeSATP was not an effective agonist (initial rate: 1.5 +/- 1.4 DE/min, 2.3 +/- 0.5 DE in 5 min). Determination of mucins by ELISA confirmed that both ATP and UTP induced similar secretory responses but that 2-MeSATP was not effective. In CF explants, mucosal UTP (10(-4) M, n = 6) induced similar responses to those observed in normal tissues (initial rate: 82.5 +/- 27.5 DE/min, 18.8 +/- 4.1 DE in 5 min). We conclude that human nasal and tracheobronchial goblet cells are stimulated by mucosal nucleotides, probably via a 5'-nucleotide receptor, and that this response is unaffected by CF.

Rationale: In the normal lung, breathing and deep inspirations potently antagonize bronchoconstriction, but in the asthmatic lung this salutary effect is substantially attenuated or even reversed. To explain these findings, the prevailing hypothesis focuses on contracting airway smooth muscle and posits a nonlinear dynamic interaction between actomyosin binding and the tethering forces imposed by tidally expanding lung parenchyma. Objective: This hypothesis has never been tested directly in bronchial smooth muscle embedded within intraparenchymal airways. Our objective here is to fill that gap. Methods: We designed a novel system to image contracting intraparenchymal human airways situated within near-normal lung architecture and subjected to dynamic parenchymal expansion that simulates breathing. Measurements and Main Results: Reversal of bronchoconstriction depended on the degree to which breathing actually stretched the airway, which in turn depended negatively on severity of constriction and positively on the depth of breathing. Such behavior implies positive feedbacks that engender airway instability. Overall conclusions: These findings help to explain heterogeneity of airflow obstruction as well as why, in people with asthma, deep inspirations are less effective in reversing bronchoconstriction.Keywords: airway; smooth muscle; bronchoconstriction; stretch; asthma Among all factors known to antagonize bronchoconstriction in a healthy lung, a deep breath is among the most effective (1-5). In the asthmatic lung, however, this protective phenomenon is substantially attenuated, and during a spontaneous asthmatic attack it is sometimes even reversed (1, 6, 7). Some have suggested that the inability of a deep breath to dilate the constricted asthmatic airway might be an important cause of excessive airway narrowing (1,6,8).To explain these observations, a new conceptual framework has called attention to the role of airway smooth muscle (ASM) and the dynamic load against which it must contract (9). With each breath (10), lung parenchyma exerts a distending force on intrapulmonary airways and stretches the bands of ASM that they contain. In this conceptual framework, these tidal stretches perturb the binding of myosin to actin, causing the myosin molecule to detach from actin much sooner than it would have otherwise and thus reducing the myosin duty cycle (11-13). As a result, the contracted ASM band within a bronchoconstricted airway relengthens and thus partially relieves the bronchoconstriction. Importantly, such force fluctuation-induced muscle relengthening has molecular determinants that differ from those that determine isometric force (9, 14-17). As such, the length of contracting ASM becomes equilibrated dynamically, not statically as assumed in earlier models (18,19), and the force generated by the muscle at any instant can be dramatically less than the force predicted by the isometric force length curve (11,20).This mechanistic framework provides a plausible basis to explain how the effects of deep breath...