No abstract

A sthma is a chronic inflammatory disease which is accompanied by extensive changes in normal airway tissue architecture, termed remodeling (1, 2). Airway remodeling in asthma comprises epithelial dysfunction, hypertrophy of the mucus glands, subepithelial vascularization, and changes in extracellular matrix composition (2). In addition, airway smooth muscle (ASM) from people suffering with asthma exhibits enhanced proliferative (3) and migratory responses (4, 5), as well as increased secretion of a myriad of pro-inflammatory cytokines/ chemokines and growth factors (6). The mechanisms that underly the exaggerated function of ASM in asthma are unknown.Smooth muscle responses to diverse stimuli are controlled by changes in the concentration of free cytosolic Ca 2ϩ ([Ca 2ϩ ] i ). Elevation of [Ca 2ϩ ] i results from increased Ca 2ϩ influx across the plasma membrane following activation of Ca 2ϩ -permeable ion channels and the Na ϩ -Ca 2ϩ -exchanger (NCX, 3Na ϩ :1Ca 2ϩ ), and by release of stored Ca 2ϩ from the sarcoplasmic reticulum (SR), in turn triggered by inositol 1,4,5-triphosphate (IP 3 ) or ryanodine receptor (RyR) channels (7). Termination of the cytosolic Ca 2ϩ signal occurs by extracellular removal of cytosolic Ca 2ϩ by the NCX and by its rapid sequestration into SR stores by the sarco/endoplasmic reticulum Ca 2ϩ (SERCA) pump (7). Impaired replenishment of SR stores arising from reduced activity of the SERCA pump could impact on a wide range of Ca 2ϩ -dependent smooth muscle functions (8) and abnormal Ca 2ϩ handling by ASM has previously been proposed to be an important determinant of the airway hyperresponsiveness that is characteristically present in asthma (9, 10).There are 3 tissue-specific members of the mammalian SERCA family, SERCA1, SERCA2 and SERCA3, each encoded by a separate gene (ATP2A1, ATP2A2, and ATP2A3) (11), with SERCA2 being the most highly expressed in smooth muscle (12, 13). The function of the different isoforms of SERCA2 is similar (14). We have investigated if the secretory and hyperproliferative phenotype of ASM in asthma is associated with impaired SERCA isoform expression. Results SERCA2Expression. SERCA2 mRNA expression was reduced in ASM cells cultured from patients with moderate, but not mild asthma compared with cells derived from healthy subjects (P ϭ 0.04, Fig. 1A). Western immunoblot showed a single band for SERCA2 at the expected size (Ϸ110 kDa) in ASM lysates (Fig. 1). SERCA2 protein expression was correspondingly reduced in ASM cells from patients with moderate asthma (P ϭ 0.015, Fig. 1B). In contrast, IP 3 R1 mRNA and protein expression did not differ between asthmatics and controls ( Fig. 1 A and B), suggesting the change in SERCA2 was not the result of a reduction in total SR. Transcripts for SERCA1, and SERCA3 were not detected in ASM. Further experiments using SERCA2A, SERCA2B, and SERCA2C specific primers demonstrated that predominant isoform in ASM is SERCA2B with the other isoforms expressed at very low levels around the limit of detection. The pattern of ...

(OA) or saline exposure of sensitized Brown Norway rats was examined on agonist reactivity, airway smooth muscle (ASM) content, and contractile protein expression in small bronchioles at 24 h, 7 days, and 35 days after challenge. OA increased ASM content (P Ͻ 0.05 vs. saline) at 24 h, which resolved by 7 days. Maximum developed tension (Tmax) to carbachol, KCl, and 4-␤-phorbol 12,13-dibutyrate was increased (P Ͻ 0.05) by OA in bronchioles at 24 h but was abrogated after correction for ASM. Differences in Tmax were not present at 7 days. In contrast, at 35 days, T max was increased (P Ͻ 0.05) after correction for ASM. Smooth muscle (sm)-␣-actin, sm-myosin heavy chain (MHC) isoform 1, calponin, smoothelin-A, and sm-myosin light chain kinase expression were reduced (P Ͻ 0.05) by OA at 24 h in bronchioles but not in trachealis. Consistent with contraction findings, no difference in expression of these proteins was detected at 7 days. At 35 days, however, with the exception of sm-␣-actin, their abundance was again reduced (P Ͻ 0.05) by OA. Nonmuscle MHC and ␤-actin were unchanged throughout by OA. These findings indicate persistent changes in contractile protein content, consistent with ASM phenotypic modulation in vivo, which occur in response to repeated OA inhalation. Thus, OA exposure induces structural changes in bronchiole ASM content and in agonist responsiveness ex vivo that resemble remodeling in asthma. asthma; inflammation; airway wall structural remodeling ASTHMA IS A DISORDER that affects the tracheobronchial tree from large to small airways (22). One of the basic characteristics of asthma is airway hyperresponsiveness (AHR) that is demonstrated by increased responses to inhaled bronchoconstrictors such as methacholine and that can be observed following allergen exposure. Induction and perpetuation of AHR may result from repeated inflammatory events involving a complex and coordinated response of multiple inflammatory and structural cells, mediators, connective tissue elements, and cytokines whose actions lead ultimately to persistent changes in airway wall structure (5). This remodeling includes epithelial cell damage and mucus gland hypertrophy, reticular basement membrane thickening, alterations in connective tissue composition, and an increase in the content of smooth muscle in the airway wall as a result of hyperplasia and hypertrophy (5).Differentiation of smooth muscle in the developing lung is characterized, as in other tissues, by the progressive replacement of nonmuscle cytoskeletal and contractile proteins with smooth muscle-specific isoforms, leading to a range of distinctive smooth muscle cell phenotype subpopulations (25,27,37). In intact, healthy mature blood vessels and airways, the majority of smooth muscle cells exist in a quiescent and fully differentiated contractile phenotype. Little attention has so far been paid to modulation of the contractile state in airway disease, although parallels in airway and pulmonary vascular remodeling are increasingly being sought (4,16,18,37). In vi...