Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

BACKGROUND AND PURPOSE: The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. EXPERIMENTAL APPROACH: Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. KEY RESULTS: Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. CONCLUSIONS: Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells.

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1beta (0.5 ng/mL) and TNF-alpha (10 ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8 h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1 microM dexamethasone abolished enhanced release of VEGF by TNF-alpha. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.

Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

beta(2)-adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells.

1. Airway smooth muscle (ASM) is a potential source of multiple pro-inflammatory cytokines during airway inflammation. beta-Adrenoceptor agonist hyporesponsiveness is a characteristic feature of asthma, and interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha are implicated in its cause. Here, the capacity of beta-adrenoceptor agonists to prevent release of GM-CSF, RANTES, eotaxin and IL-8, elicited by IL-1 beta or TNF alpha, was examined in human ASM cells. 2. Isoprenaline (approximately EC(50) 150 nM), a non-selective beta-adrenoceptor agonist, and salbutamol ( approximately EC(50) 25 nM), a selective beta(2)-adrenoceptor agonist, attenuated release of GM-CSF, RANTES and eotaxin, but not IL-8 (EC(50) >1 microM). The maximum extent of attenuation was RANTES > or = eotaxin > GM-CSF >> IL-8, and was prevented by either propranolol (1 microM), a non-selective beta-adrenoceptor antagonist, or ICI 118511 (IC(50) 15 nM), a selective beta(2)-adrenoceptor antagonist. 3. The cyclic AMP-elevating agents, dibutyryl cyclic AMP ( approximately EC(50) 135 microM), forskolin ( approximately EC(50) 530 nM) and cholera toxin ( approximately EC(50) 575 pg ml(-1)) abolished IL-1 beta-induced release of GM-CSF, RANTES and eotaxin, but not IL-8. 4. IL-1 beta (1 ng ml(-1)) attenuated early increases (up to 1 h) in cyclic AMP formation induced by salbutamol (1 microM), but not by forskolin (10 microM). The cyclo-oxygenase inhibitor, indomethacin (1 microM) prevented later increases (3 - 12 h) in IL-1 beta-stimulated cyclic AMP content, but did not prevent the attenuation by salbutamol of IL-1 beta-induced cytokine release. 5. We conclude in human ASM cells that activation of beta(2)-adrenoceptors and generation of cyclic AMP is negatively-linked to the release, elicited by IL-1 beta or TNF alpha, of eosinophil-activating cytokines such as GM-CSF, RANTES and eotaxin, but not IL-8.

Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype.

Mature airway smooth muscle cells are characterized by a low proliferative index and expression of contractile marker proteins such as smooth muscle alpha-actin (sm-alpha-actin), calponin, and smooth muscle myosin heavy chain (sm-MHC). In the present study, defined extracellular matrix (ECM) components were examined on the proliferative and phenotypic status of mitogen-stimulated, cultured human airway smooth muscle cells. The results demonstrate that although cells adhered and spread on plates precoated with (1 to 100 microg/ml) of fibronectin (FN), collagen I (Col I), laminin (LN), or Matrigel, their subsequent proliferative response varied qualitatively. FN and Col I enhanced proliferation in response to either platelet-derived growth factor (PDGF)-BB or alpha-thrombin, compared with cells on plastic. LN, however, reduced mitogen-stimulated proliferation. A similar reduction was found in cells cultured on Matrigel. The effect of ECM substrates on contractile phenotype was determined by examining cellular expression of sm-alpha-actin, sm-MHC, and calponin using immunocytochemical and flow cytometric methods. Approximately 75% of PDGF-BB-stimulated cells, cultured on LN or Matrigel, expressed sm-alpha-actin, calponin, and sm-MHC, but only 8 to 10% stained for the Ki67 nuclear antigen proliferation marker. In contrast, more than 75% of cells cultured on FN or Col I were positive for Ki67 antigen, but only 20% were positive for contractile proteins. Flow cytometric analysis of sm-alpha-actin and DNA content confirmed the immunocytochemical findings and showed that the observed reduction in sm-alpha-actin content after culture on FN or Col I, compared with LN and Matrigel, occurred in the majority of the cell population, supporting bidirectional phenotype modulation. Overall, the data suggest that ECM substrates modulate both proliferation and phenotype of human airway smooth muscle cells in culture.

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma.

Chronic persistent asthma is characterized by poorly reversible airflow obstruction and airways inflammation and remodelling. Histopathological studies of airways removed at post mortem from patients with severe asthma reveal marked inflammatory and architectural changes associated with airway wall thickening. Increased airway smooth muscle content, occurring as a result of hyperplastic and/or hypertrophic growth, is believed to be one of the principal contributors to airway wall thickening. In recent years, significant advances have been made in elucidating the mediators and the intracellular pathways that regulate proliferation of airway smooth muscle. The contribution that smooth muscle makes to persistent airflow obstruction may not, however, be limited simply to its increased bulk within the airway wall. Interest is growing in the possibility that reversible phenotypic modulation and increased heterogeneity of airway smooth muscle function may also be a feature of the asthmatic airway. This review focuses on possible mechanisms controlling smooth muscle phenotype heterogeneity as well as on the mediators and intracellular pathways implicated in its cellular proliferation. Particular attention is paid to mechanisms involving activation of the extracellular signal regulated kinase-, protein kinase C- and phosphoinositide 3-kinase-dependent pathways, since these appear to be the major candidate second messenger pathways for G protein- and tyrosine kinase-coupled receptor-stimulated proliferation.

Airway smooth muscle as a target in asthma.

Traditionally, the contractile properties of airway smooth muscle have been regarded as its sole contribution to the pathogenesis of asthma. However, our understanding of the role that this structural cell plays in asthma is changing. Airway smooth muscle can undergo hyperplasia and/or hypertrophy leading to structural changes in the airway wall which contribute to the development of persistent airway obstruction and increased non-specific airway hyperresponsiveness in chronic severe asthma. Many studies in vitro have characterized airway smooth muscle proliferation induced by various pro-inflammatory mediators, growth factors and components of the extracellular matrix, but the mediator(s) responsible for the observed increase in airway wall smooth muscle content in vivo remain to be determined. In addition to geometric obstruction by increased airway wall thickening, proliferating airway smooth muscle cells undergo phenotypic modulation from a contractile to synthetic-proliferative state where additional functions of airway smooth muscle such as cytokine/chemokine and extracellular matrix secretion may become more apparent. This may be especially relevant in the diseased airway where the content of airway smooth muscle as a fraction of the total cells present in the airway wall is already increased. Airway smooth muscle cells may also interact by direct contact with immunocytes such as T lymphocytes through expression of cell adhesion molecules with the result that myocyte DNA synthesis is induced. As additional functions of airway smooth muscle are described, a more contemporary view is emerging that airway smooth cells may adopt an immuno-effector role in chronic asthma by proliferating, secreting cytokines, expressing adhesion molecules and by interacting with various inflammatory cells. This may involve changes in the phenotypic status of airway smooth muscle, and as a result, these cells may play an active role in perpetuating and orchestrating airway inflammation in the remodelled airway. An important phase for future airway smooth muscle research will be to determine the extent that these putative mechanisms exist in vivo in the pathogenesis of chronic severe asthma.

Tumor necrosis factor-alpha enhances mRNA expression and secretion of interleukin-6 in cultured human airway smooth muscle cells.

Airway smooth muscle (ASM) is considered to be an end-target cell for the effects of mediators released during airway wall inflammation. Several reports suggest that activated ASM may be capable of generating various proinflammatory cytokines. We investigated the effects of tumor necrosis factor (TNF)-alpha, a potent proinflammatory cytokine, on cultured human ASM cells by examining the expression and release of the cytokine interleukin (IL)-6, cell proliferation, and the expression pattern of c-fos and c-jun, two nuclear proto-oncogenes constituting the activator protein-1 transcription factor. Growth-arrested cell monolayers were stimulated with human recombinant TNF-alpha in a concentration- and time-dependent manner. TNF-alpha stimulated the expression of IL-6 messenger RNA (mRNA), which was detected after 15 min, reaching a maximum at 1 h. IL-6 protein was readily detected in ASM cell-conditioned medium after 2 h of TNF-alpha stimulation. Protein levels increased in a time- and concentration-dependent manner. Release of IL-6 elicited by TNF-alpha was significantly inhibited by dexamethasone, cycloheximide, and nordihydroguaiaretic acid (NDGA). TNF-alpha did not alter DNA biosynthesis up to 48 h or cell numbers up to 120 h. Northern blot analysis of proto-oncogene expression revealed that c-fos and c-jun mRNA levels were elevated after 30 min of TNF-alpha incubation with maximum levels at 1 h and 45 min, respectively. Expression of c-fos mRNA was downregulated by NDGA. Four hours of TNF-alpha treatment resulted in translocation of c-jun immunofluorescence from the cytoplasm to the nucleus in human ASM cells. Our results suggest that despite the lack of a mitogenic response to TNF-alpha, upregulation of primary response genes in human ASM cells may account for the induction of proinflammatory cytokines, such as IL-6, in human airways.

Cultured human airway smooth muscle cells stimulated by interleukin-1beta enhance eosinophil survival.

Airway smooth muscle may be an important cellular source of proinflammatory mediators and cytokines and may participate directly in airway inflammation. In this study we have examined whether airway smooth muscle cells could contribute to mechanisms of eosinophil accumulation by prolonging their survival. To investigate this possibility, conditioned medium from human airway smooth muscle cells stimulated with interleukin (IL)-1beta was examined on the in vitro survival of highly purified human peripheral blood eosinophils. After 7 d, when cultured in control medium, less than 1 +/- 0.2% of the initial eosinophil population remained viable. In contrast, culture in medium conditioned for 96 h by human airway smooth muscle cells stimulated with IL-1beta (1 pg-100 ng/ml) resulted in a concentration-dependent increase in eosinophil survival. (The concentration that produced 50% of this effect was 0.03 ng/ml IL-1beta.) Maximum eosinophil survival occurred at 1 to 3 ng/ml IL-1beta. This effect was also time-dependent and was readily detected in airway smooth muscle cell-conditioned medium after just 3 h of stimulation with IL-1beta (1 ng/ml). It continued to increase before reaching a plateau around 24 h, with no decrease in activity for up to 120 h of stimulation. Conditioned medium from unstimulated airway smooth muscle cells did not enhance eosinophil survival. The survival-enhancing activity was completely inhibited (the concentration that inhibited 50% [IC50] was 6.9 microg/ml) by a polyclonal goat antihuman antibody to granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-100 microg/ml), but antibodies (10-100 microg/ml) to IL-3 and IL-5, and a normal goat immunoglobulin G control had no effect on the eosinophil survival-enhancing activity. GM-CSF levels in culture medium from smooth muscle cells were markedly increased by IL-1beta and were maximum at 30 ng/ml (0.037 ng/ml/10(6) cells versus 3.561 ng/ml/10(6) cells, unstimulated versus 30 ng/ml IL-1beta). The IL-1 receptor antagonist inhibited both the production of GM-CSF (IC50 19. 1 ng/ml) and the eosinophil survival-enhancing (IC50 53.7 ng/ml) activity stimulated by IL-1beta. Release of GM-CSF elicited by IL-1beta was inhibited by dexamethasone but not by indomethacin. These data indicate that cultured human airway smooth muscle cells stimulated with IL-1beta support eosinophil survival through production of GM-CSF and thus may contribute to the local control of inflammatory cell accumulation in the airways.

Airway smooth muscle as a target of glucocorticoid action in the treatment of asthma.

Glucocorticoids are highly effective in the control of asthma and suppression of airway inflammation. The cellular and molecular mechanisms involved in the anti-inflammatory actions of glucocorticoids are becoming clearer. Although it is apparent that glucocorticoids have effects on many aspects of inflammation, it is not certain which actions on which cell types are the most critical in controlling asthma. Airway smooth muscle cells represent a significant proportion of all cells present in the airways and might therefore be expected to be a prominent cellular target for inhaled steroids. Despite this, little is known of the action of glucocorticoids on airway smooth muscle. It is becoming clear that in addition to its contractile properties, airway smooth muscle can potentially contribute to the pathogenesis of asthma by increased proliferation and by expression and secretion of pro-inflammatory cytokines and mediators, which in turn may lead to the activation and recruitment of key inflammatory cells in the airways. This review examines the action of glucocorticoids on some of the diverse functions of airway smooth muscle that are implicated in remodeling of the airways in asthma. Glucocorticoids either directly or indirectly modulate contraction of airway smooth muscle by suppressing agonist-induced increases in intracellular calcium levels or by downregulating or uncoupling receptors linked to contraction (e.g., muscarinic M2 or M3, histamine H1 receptors). In addition, glucocorticoids may augment relaxation of airway smooth muscle by increasing activation of either cyclic AMP-dependent (e.g., increased expression of beta2-adrenoceptors, reduced homologous desensitization of beta2-adrenoceptors) or AMP-independent mechanisms (e.g., increased Na+/K+ electrogenic pump activity). In addition to their effects on contraction, glucocorticoids are also effective antiproliferative agents in airway smooth muscle, but under some circumstances may also contribute to proliferation by inhibiting the antiproliferative effect of high concentrations of tumor necrosis factor alpha in these cells. Glucocorticoids also suppress induction of cyclooxygenase-2 in human airway smooth muscle cells and the subsequent synthesis and release of arachidonic acid metabolites, particularly prostaglandin E2. The potential of airway smooth muscle to recruit and activate pro-inflammatory cells such as the eosinophil may also be reduced by glucocorticoids, as they are effective in preventing the release of several cytokines (e.g., RANTES, interleukin-8, and granulocyte macrophage colony-stimulating factor). The possibility exists that as we begin to understand and speculate more about the likely role of airway smooth muscle in the pathogenesis of asthma, it may be necessary to reconsider airway smooth muscle as an important cellular target for the action of glucocorticoids in the treatment of asthma.

Potassium channels in human fetal airway smooth muscle cells.

Ion channels underlying the resting membrane potential were examined in human fetal airway smooth muscle (ASM). Tissue was obtained from the Medical Research Council Tissue Bank, London, UK. ASM cells were enzymatically dispersed, and ion currents were examined using a patch clamp. Although all cells were of similar size and stained intensely for vimentin, only approximately 50% stained intensely for smooth muscle alpha-actin or myosin heavy chain. Depolarization induced a tetraethylammonium (TEA)- and charybdotoxin (ChTX)-sensitive outward current that varied widely among cells (<50 to >2000 pA at +100 mV), and a smaller nonselective cation current that was similar in all cells (approximately 20 pA at +100 mV). The TEA-sensitive current was associated with three types of large conductance, ChTX-sensitive K+ channel: a 200-pS channel, which was active at negative potentials and low [Ca2+], as described for freshly isolated adult ASM, and two other K+ channels of 100 and 150 pS, previously observed only in adult ASM proliferating in culture. ChTX, but not 4-aminopyridine, caused a substantial depolarization in the current clamp mode, suggesting that, in contrast to ASM from other species or vascular smooth muscle, large conductance K+ channels rather than a delayed rectifier are the major determinant of membrane potential in this tissue. Our results show a distinct similarity between fetal ASM and adult ASM proliferating in culture. We suggest that the heterogeneity in current density and staining reflect different degrees of differentiation, rather than different cell types, and that the 100- and 150-pS K+ channels are specifically associated with a proliferative phenotype in human ASM.

Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type.

1. Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H2 (PGH2) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined. 2. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE2, thromboxane B2 or 6-ketoPGF1 alpha by radioimmunoassay. 3. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) each at 10 ng ml-1) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 microM; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 microM) and cyclohexamide (10 micrograms ml-1; added 30 min before the cytokines). 4. Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1 beta, TNF alpha, IFN gamma each at 10 ng ml-1 for 24 h) caused the accumulation of PGE2 and 6-keto-PGF1 alpha. 5. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1 beta in combination with TNF alpha caused a similar release of PGE2 to that when the three cytokines were given in combination. 6. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 microM for 15 min) after which PGE2 was measured. IL-1 beta and TNF alpha increased COX-2 activity and an additional small increase was produced by the three cytokines in combination. 7. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids.

RANTES is a basic 8-kDa polypeptide of the C-C chemokine subfamily with strong chemotactic activity for eosinophils, lymphocytes, and monocytes. We determined the regulation of RANTES production by human airway smooth muscle cells in culture. While TNF-alpha, but not IFN-gamma, increased RANTES mRNA expression and protein release, the combination of TNF-alpha and IFN-gamma caused a greater degree of expression and release in a time- and dose-dependent manner. Sequential treatment of airway smooth muscle cells with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized the cells to the stimulatory effect of TNF-alpha. Using a modified Boyden chamber technique, RANTES separated by reverse-phase liquid chromatography from cell culture supernatants of airway smooth muscle cells stimulated by TNF-alpha and IFN-gamma showed a strong chemoattractant effect on human eosinophils, an effect inhibited by an anti-RANTES Ab. RANTES production induced by TNF-alpha and IFN-gamma was inhibited partly by the Th2-derived cytokines, IL-4, IL-10, and IL-13, as well as by dexamethasone. Our studies indicate that, in addition to contractile responses and mitogenesis, airway smooth muscle cells have synthetic and secretory potential with the release of RANTES. They may participate in chronic airway inflammation by interacting with both Th1- and Th2-derived cytokines to modulate chemoattractant activity for eosinophils, activated T lymphocytes, and monocytes/macrophages.

Ion channels in freshly isolated and cultured human bronchial smooth muscle cells.

Voltage-gated ion currents were studied in human bronchial airway smooth muscle (ASM) cells. Proliferating or growth-arrested cells in culture were compared with freshly isolated cells. Three types of charybdotoxin (ChTX)-sensitive K+ channel were observed in all cell types, with conductances in symmetrical 140 mM KCl solutions ([Ca2+]i < 0.1 nM) of 206 +/- 14 pS (n = 32), 144 +/- 11 pS (n = 27) and 109 +/- 5 pS (n = 25). The relative proportion of each channel type differed substantially between the three groups of cells. In freshly isolated ASM cells large conductance K+ channels were represented almost entirely by a conductance of 206 pS, which was found in all twenty-three patches studied. In contrast, in most patches from proliferating cells the majority of channels had conductances of either 144 pS (14 of 21 patches) or 109 pS (8 of 21 patches). Cultured cells that had been growth arrested by serum depletion revealed the same set of channels as the proliferating cells, but the occurrence of the 109 pS channel was much more frequent (16 of 19 patches). As has been shown previously, 206 pS channels were active at a physiological membrane potential (-60 to -20 mV) even at a very low free [Ca2+]. The 144 pS channels could be recorded only at depolarized potentials (+80 to +100 mV), whereas 109 pS channels were active over a wide range of potentials (-60 to +100 mV), but only in the presence of GTP. In a proportion of cultured cells a tetrodotoxin-sensitive Na+ current and a hyperpolarization-induced inwardly rectifying K+ current were also observed (15 and 21%, respectively, of all cells examined). Neither of these currents were observed in freshly isolated cells. Whole-cell outward current in all groups was sensitive to tetraethylammonium, ChTX, and iberiotoxin, but not to 4-aminopyridine. In summary, it is clear that during proliferation there are considerable changes in the expression of ionic channels in ASM that have profound functional significance. In particular, these changes would tend to make the tissue more excitable, and may be of relevance to the proliferative process itself.

Localisation and expression of beta-adrenoceptor subtype mRNAs in human lung.

The cellular localisation and distribution of mRNAs encoding beta-adrenoceptor subtypes in human lung were studied by in situ hybridisation and Northern blot analysis. The 851-bp SmaI/PvuII fragment of human beta 1-adrenoceptor cDNA, the 439-bp SmaI fragment of human beta 2-adrenoceptor cDNA and the 975-bp SmaI fragment of human beta 3-adrenoceptor cDNA bound to single mRNA species of approximately 3.2 kb, 2.2 kb and 2.3 kb in size, respectively. Human lung and heart and rabbit lung expressed both beta 1- and beta 2-adrenoceptor mRNAs with no detectable level of beta 3-adrenoceptor mRNA, while rabbit perirenal adipose tissue expressed beta 1-, beta 2- and beta 3-adrenoceptor mRNAs. Cultured human airway epithelial cells and airway smooth muscle cells expressed only beta 2-adrenoceptor mRNA. In situ hybridisation in human lung, using 35S-labelled antisense RNA probes revealed a high level of expression of beta 1- and beta 2-adrenoceptor mRNAs in the pulmonary blood vessels, high level of expression of beta 2-adrenoceptor mRNA in the alveolar walls with less expression of beta 1-adrenoceptor mRNA. There was a moderate expression of beta 2-adrenoceptor but not beta 1-adrenoceptor mRNA in airway epithelium and smooth muscle of peripheral airways and no detectable beta 3-adrenoceptor mRNA in any lung structures.

Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma.

Chronic persistent asthma is characterized by poorly reversible airway obstruction. Histopathological studies of airways removed postmortem from patients with severe asthma reveal marked inflammatory and architectural changes associated with airway wall thickening. Increased airway smooth muscle content, occurring as a result of hyperplastic and/or hypertrophic growth, is believed to be one of the principal contributors to airway wall thickening. Intense interest is building to discover the mechanisms responsible for these long-term structural changes. In vitro cell culture offers a powerful and exacting approach to cellular and molecular studies of the long-term regulation of airway smooth muscle function. This review discusses the methodologies for establishing and maintaining cell cultures of airway smooth muscle. It also describes the characteristics of these cells in culture and addresses the potential importance of phenotype plasticity and its possible relationship to altered smooth muscle function in vivo. Drawing on parallels from vascular studies, this review focuses, in particular, on the synthetic nature of the airway smooth muscle cell, emphasizing its potential to alter the composition of the extracellular matrix environment and orchestrate key events in the process of chronic airway remodelling.

PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells.

The effect of recombinant platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB and -AB) on mitogenesis of human cultured airway smooth muscle (ASM) was examined using the MTT-reduction assay and [3H]thymidine incorporation. Results were correlated with expression of PDGF receptor (PDGFR)-alpha and -beta subunits in the absence and presence of fetal calf serum (FCS). When FCS was absent PDGF-AB and -BB were potent mitogens, whereas PDGF-AA was weakly mitogenic, evoking <20% of the maximum response induced by the B-chain isoforms. When FCS (2.5%) was present, all PDGF isoforms stimulated marked ASM proliferation with similar efficacy and potency. Cross-competition binding analysis in FCS-deprived cells revealed that ASM cells in culture express mainly PDGFR-beta. Preincubation with PDGF-AA or PDGFR-alpha neutralizing antiserum abolished PDGF-AA binding and decreased total receptor number by approximately 15%. The ratio of PDGFR-alpha to beta subunits was approximately 1:8, supported by intense immunofluorescence staining for PDGFR-beta and weak staining for PDGFR-alpha. In parallel studies, uptake of [3H]thymidine stimulated by PDGF-AA, but not PDGF-AB or -BB, was inhibited by PDGFR-alpha immobilization. Western immunoblot analysis confirmed expression of mature PDGFR-alpha and -beta subunits in ASM cells. FCS did not cause any detectable increase in PDGFR-alpha expression or in PDGF-AA binding. These data support a role for PDGFR-beta mediating ASM mitogenesis during FCS-free conditions, but in the presence of FCS, both PDGFR-alpha and -beta subunits are linked to mitogenesis. The enhanced mitogenicity of PDGF-AA in the presence of FCS was independent of any detectable upregulation of PDGFR-alpha, suggesting that the inability of PDGF-AA to promote mitogenesis in the absence of FCS is not simply due to relative numbers of PDGFR-alpha and PDGFR-beta.

Potassium currents in human freshly isolated bronchial smooth muscle cells.

1. K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch-clamp technique. 2. In whole-cell recordings a depolarization-induced, 4-aminopyridine (4-AP)-sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA. 3. In the majority of cells depolarization to -40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4-AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin-perforated patch technique. 4. Single channels with a conductance of about 210 pS were recorded in cell-attached patch, inside-out patch, outside-out patch and whole-cell recording configurations. Channel open state probability in inside-out patches was 0.5 at a membrane potential of 4 +/- 14 mV (mean +/- s.d., n = 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX. 5. In current-clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration-dependent depolarization. 6. These results suggest that in human bronchial smooth muscle cells, in marked contrast to other species, the majority of the outward current induced by depolarization is not due to a delayed rectifier,but to the activity of a large conductance, ChTX-sensitive K+ channel. The Ca2+- and voltage-dependency of this channel may well allow a sufficiently high open state probability for it to play a partin the regulation of the resting membrane potential.