Cultured Airway Smooth Muscle Cells Research Articles

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.

The effects of salbutamol, beclomethasone, and dexamethasone on fibronectin expression by cultured airway smooth muscle cells.

Possible mechanisms of adverse drug effects in asthma include worsening of cellular hyperplasia and stimulation of extracellular matrix deposition. In this study, salbutamol, dexamethasone and beclomethasone were investigated to ascertain their ability to induce mitogenesis and stimulate fibronectin expression in cultured canine airway smooth muscle cells. In cells maintained in serum-free media for 72 h, salbutamol (1 nM-10 microM) caused mitogenesis. The control cells had 2.57 +/- 0.34 x 10(5) cells per ml (mean +/- SEM, N = 13), while salbutamol (1 microM) caused a maximal increase in cell number to 3.57 +/- 0.23 x 10(5) cells/ml (P < 0.01). In cells stimulated to replicate by addition of either fetal bovine serum or canine serum, no additional mitogenic effect of salbutamol was seen. Salbutamol did not have a detectable quantitative effect on fibronectin matrix expression. The glucocorticoids, beclomethasone and dexamethasone, significantly altered fibronectin expression by cultured airway smooth muscle cells. Beclomethasone increased fibronectin expression, while dexamethasone decreased expression.

Mitogen-activated signaling in cultured airway smooth muscle cells

Mitogen-activated signaling in cultured airway smooth muscle cells

ET-1 cooperates with EGF to induce mitogenesis via a PTX-sensitive pathway in airway smooth muscle cells.

We have examined the mitogenic effect of endothelin-1 (ET-1) alone or in combination with epidermal growth factor (EGF) in cultured airway smooth muscle cells (ASM) from guinea pig. ET-1 showed a weak mitogenic activity compared with the effect of EGF. However, when ET-1 and EGF were applied simultaneously, ET-1 synergistically enhanced the mitogenic activity of EGF. Neither inhibition of phospholipase C-beta nor depletion of protein kinase C affected this synergism. On the other hand, pertussis toxin (PTX), a Gi protein inhibitor, abolished the synergistic effect of ET-1 on EGF-induced mitogenesis. ET-1 induced a transient mitogen-activated protein (MAP) kinase activation peaking at 5 min. In contrast, EGF induced a stronger signal that was maintained for up to 20 min. However, concomitant stimulation of ASM with ET-1 and EGF caused an enhanced MAP kinase activation compared with EGF alone. Moreover, PTX abolished the enhanced MAP kinase activation observed in this condition. These results indicate that ET-1 can interact with an EGF-induced mitogenic axis through the Gi protein-dependent pathway, which is distinct from its direct mitogenic pathway.

Effect of interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells.

1. Increased levels of several pro-inflammatory cytokines including interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF alpha) have been found in bronchoalveolar lavage fluid from symptomatic asthmatic patients. IL-1 beta, TNF alpha and interferon-gamma (IFN gamma) are known to stimulate a number of cells to produce inflammatory mediators such as prostaglandins. Although airway smooth muscle (ASM) is known to be a rich source of prostaglandins, the regulation of cyclo-oxygenase (COX) isoforms and prostanoid production by proinflammatory cytokines have not been studied in human airway smooth muscle. 2. We studied the effects of IL-1 beta, TNF alpha and IFN gamma on the induction of two isoforms of cyclo-oxygenase and its relation to prostaglandin E2 (PGE2) release and COX activity (reflected by PGE2 synthesis from exogenous arachidonic acid) in human cultured airway smooth muscle cells. 3. IL-1 beta, but not TNF alpha or IFN gamma, caused a time- and concentration-dependent enhancement in PGE2 and other prostanoid (6-keto-PGF1 alpha, PGF2 alpha, thromboxane B2 (TXB2) and PGD2) production, with PGE2 and 6-keto-PGF1 alpha as the principal products. This stimulation was accompanied by a corresponding increase in COX activity. 4. COX-2 protein measured by Western blot analysis was not detectable in untreated cells, but was increased in a time- and concentration-dependent manner by IL-1 beta, but not TNF alpha or IFN gamma. In contrast, no variation in the expression of COX-1 protein was observed. 5. Pretreatment with the conventional non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and ibuprofen, and the selective COX-2 inhibitors, NS-398 and nimesulide, completely blocked IL-1 beta-induced PGE2 release and COX activity. The glucocorticosteroid dexamethasone and protein synthesis inhibitors, cycloheximide and actinomycin D, not only markedly inhibited IL-1 beta-stimulated PGE2 release and COX activity but also suppressed IL-1 beta-induced COX-2 induction. 6. This study demonstrates that human cultured ASM cells release prostanoids in response to IL-1 beta stimulation and that the response is mostly mediated by the induction of COX-2 rather than COX-1 isoenzyme, implying that airway smooth muscle may be an important source of prostaglandins in human airways and that COX-2 may play an important role in the regulation of the inflammatory process in asthma.

Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle cell proliferation.

Endothelin is a small peptide that is a potent bronchoconstrictor, mitogen for airway smooth muscle (ASM), and is believed to be involved in the pathogenesis of asthma. To understand how endothelin stimulates the proliferation of ASM cells in culture, we evaluated the relationship between mitogen activated protein (MAP) kinase activation and cell proliferation. Endothelin is a potent stimulator of the extracellular regulated kinase 2 (ERK2) subgroup of MAP kinases, and ERK2 activation was tightly correlated with the proliferation of rat ASM cells. PD98059, a small molecule inhibitor of MEK (MAP or ERK kinase) was used to establish the role of ERK2 activation in the endothelin-stimulated signal transduction pathway leading to cell proliferation. While PD98059 significantly inhibited the ability of endothelin to activate ERK, the drug did not appear to effect the catalytic activity of an activated MEK mutant, or ERK in vitro. The data suggest that the mechanism of PD98059 inhibition of the ERK2 pathway in ASM cells may involve inhibition of MEK activation. The endothelin signal transduction pathway that culminates in ERK2 activation was dependent on protein kinase C (PKC), since depletion of PKC significantly inhibited the ability of endothelin to activate ERK2. Taken together, the data imply that activation of ERK is a critical endpoint in the endothelin signal transduction pathway since inhibition of this kinase inhibits endothelin-induced ASM cell proliferation.

Strain Reorganizes Focal Adhesions and Cytoskeleton in Cultured Airway Smooth Muscle Cells

Abnormal mechanical stress on pulmonary structures is associated with increased airway resistance and impaired gas exchange as a result of increased airway smooth muscle (ASM) deposition. Using anin vitrosystem with cultured ASM cells, we have demonstrated that cyclic deformational strain increases ASM cellular myosin and myosin light chain kinase. To determine if these contractile protein increases were accompanied by ultrastructural changes in cells indicating phenotypic modulation, cells subjected to strain were compared to cells grown under static conditions by transmission electron microscopy (TEM) and fluorescent staining. The strained ASM cells oriented perpendicular to the strain direction were more elongated and contained more actin stress fibers than identical cells grown under physically static conditions. The stress fiber bundles were thicker and reorganized parallel to the long axis of the cell. Marked increases in the numbers and lengths of focal adhesions between the cell membrane and the substratum were found by both TEM and immunostaining for talin. Mechanical strain thus increases organization of cytoskeletal elements in cultured ASM cells. Similar effectsin vivomay serve to promote the expression of the contractile phenotype of cultured ASM cells independent of otherin vivofactors and alter cell contractility. Increased organization of cytoskeletal elements might also increase the efficiency of signal transduction from the extracellular matrix into the cell interior.

TNF-alpha inhibits isoproterenol-stimulated adenylyl cyclase activity in cultured airway smooth muscle cells.

Inflammation, increased cytokine production, and decreased responsiveness of airway smooth muscle (ASM) to beta-adrenergic agonists are characteristics of asthma. We questioned whether the cytokine tumor necrosis factor-alpha (TNF-alpha) directly impaired beta-adrenergic signal transduction in cultured canine ASM cells. Confluent ASM cells exposed to TNF-alpha (0.1-10 ng/ml) for 72 h showed lower maximal levels of adenylyl cyclase activity in response to isoproterenol (10 ng/ml; 14 +/- 4.3 vs. 7.5 +/- 1.3 pmol adenosine 3',5'-cyclic monophosphate x well(-1) x 20 min(-1), control vs. treated, respectively), despite no changes in beta-adrenergic receptor numbers (maximum number of binding sites = 4.8 +/- 0.72 vs. 4.5 +/- 0.81 fmol/mg protein, control vs. treated, respectively). Adenylyl cyclase activities in response to prostaglandin E1, NaF, or forskolin were not different in treated and untreated cells. These results demonstrate that a cytokine known to be increased during exacerbation of asthmatic symptoms directly impairs beta-adrenergic function in ASM cells and suggests a mechanism by which inflammation impairs beta-adrenergic receptor signal transduction in asthma.

The Role of Potassium Channels in the Inhibitory Effects ofβ2-adrenoceptor Agonists on DNA Synthesis in Human Cultured Airway Smooth Muscle

Opening of calcium-dependent K+channels byβ2-adrenoceptor agonists has been shown to contribute to their smooth muscle relaxant activity. In this study, the influence of K+channel activity on human cultured airway smooth muscle proliferation and on its inhibition by salbutamol have been examined. Studies of86Rb+efflux from the airway smooth muscle cell cultures confirmed that both cromakalim, an activator of ATP-dependent K+channels and salbutamol increased K+channel activity in cultured airway smooth muscle. The efflux of86Rb+was significantly attenuated by non-specific K+channel blockade. Potassium channel blockers had only small and variable effects on [3H]-thymidine incorporation in unstimulated cells and those stimulated with fetal calf serum (FCS) and no effect on FCS-induced increases in cell number. Potassium channel openers also had no substantial inhibitory effects on DNA synthesis or proliferation in cells stimulated with other mitogens including thrombin. Blockade of K+channels had no consistent effects on the inhibition of DNA synthesis and cell proliferation caused by salbutamol (0.3 nm–10μm). The inhibition of DNA synthesis caused by 8 bromoadenosine 3′5′ cyclic monophosphate (1μm–1 mm) or 8 bromoguanosine 3′5′ cyclic monophosphate (1μm–1 mm) was also unaffected by K+channel blockade. These results indicate that changes in K+channel activity have no detectable influence on DNA synthesis and proliferation of human cultured airway smooth muscle cells or on the antiproliferative effects ofβ2-agonists.

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.

Tachykinin-independent effects of capsaicin on smooth muscle in human isolated bronchi.

Contractile and relaxant responses to capsaicin and resiniferatoxin were examined in human isolated bronchus (5-12 mm o.d.). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The contractions averaged 20% of maximal contraction at 1 microM and averaged > 40% maximal contraction at 300 microM (the highest concentration studied). The capsaicin-induced contractions were mimicked by resiniferatoxin (0.1-10 microM) and inhibited by the putative capsaicin receptor antagonist, capsazepine (10 microM). The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968 (0.3 microM), whereas responses to concentrations of neurokinin A (10 nM), neurokinin B (0.1 microM), substance P (1 microM), neuropeptide gamma (10 nM), and neuropeptide K (10 nM) which produced similar-size contractions were almost abolished by 0.1 microM SR 48968. The bronchi isolated from 8 of 16 lungs also exhibited relaxations in response to capsaicin. Capsaicin-induced relaxations were not inhibited by the nitric oxide synthase inhibitor L-nitro-n-arginine (10 microM). In whole-cell patch-clamp experiments on human cultured airway smooth muscle cells, capsaicin was found to enhance outward currents due to the activation of charybdotoxin-sensitive large conductance Ca2+-activated K+ channels. Neither the capsaicin-induced contractions nor the relaxations were mimicked by angiotensin II, bombesin, or calcitonin gene-related peptide at concentrations up to 1 microM. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone, but this response does not appear to involve substance P or related neurokinins. Relaxations to capsaicin may, however, involve the activation of large conductance Ca2+-activated K+ channels.

Mitogen-activated signaling in cultured airway smooth muscle cells

Airway hyperresponsiveness and excess smooth muscle mass coexist in patients with asthma and bronchopulmonary dysplasia. This increase in airway smooth muscle mass, which in part relates to smooth muscle proliferation, may increase bronchoconstrictor-induced airway narrowing, even in the absence of excessive force generation. Thus, there is need for a precise understanding of the events involved in airway smooth muscle mitogenesis. This review examines the inflammatory substances and growth factors that induce airway smooth muscle proliferation, and the signaling pathways that may be involved in the transduction of these extracellular signals to the cell nucleus. Also discussed are various antimitogenic substances and potential mechanisms underlying the inhibition of cell proliferation. Central to the discussion are the extracellular signal regulated kinases (ERKs), serine/threonine kinases of the mitogen-activated protein kinase (MAP kinase) superfamily, which upon activation, translocate from the cytoplasm to the nucleus after mitogenic stimulation. Insight gained from studies of cultured airway smooth muscle growth and mitogen-activated signaling may shed light on parallel mechanisms that may operate in asthma and in bronchopulmonary dysplasia, and may lead to therapeutic interventions against airway remodeling.

Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle.

1. The regulation of histamine-induced [3H]-inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied. 2. Histamine induced concentration-dependent [3H]-inositol phosphate formation (EC50 4 microM). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2-selective antagonist, tiotidone or the H3 receptor-selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells. 3. Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration-dependent inhibition of histamine-induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 microM). Preincubation for 22 h with 1 microM dexamethasone produced inhibition of the inositol phosphate response to histamine to all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1-20 mM) was unaltered by preincubation with dexamethasone. 4. Preincubation of human airway smooth muscle cells with 1 microM dexamethasone for time periods of < 6 h failed to inhibit histamine-induced inositol phosphate formation in human airway smooth muscle cells. 5. Histamine also induced concentration-dependent elevation of intracellular calcium levels in Fura 2-loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 microM dexamethasone. 6. We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen-induced contractile responses in the airways.

Markers of airway smooth muscle cell phenotype.

Airway smooth muscle plays a principal role in the pathogenesis of asthma. Primary cultures are being used to investigate airway myocyte proliferation and cellular pathways regulating contraction. Airway smooth muscle cells (SMC) modulate from a contractile to a noncontractile phenotype in culture, but no systematic study of the concomitant changes in expression of cytocontractile and cytoskeletal proteins has been reported. We measured temporal changes in protein marker expression of canine tracheal SMC in primary culture, using specific antibodies and cDNA probes. Immunoblot analysis revealed that when cells became proliferative after 5 days of culture, the content of smooth muscle myosin heavy chain (sm-MHC), calponin, sm-alpha-actin, and desmin diminished by > 75%; myosin light chain kinase, h-caldesmon, and beta-tropomyosin had also decreased significantly (P < 0.05). Northern blots revealed that mRNA levels for sm-MHC and sm-alpha-actin were also significantly reduced in proliferative SMC. Conversely, immunoblotting demonstrated the content of non-muscle myosin heavy chain, l-caldesmon, vimentin, alpha/beta-protein kinase C (PKC), and CD44 homing cellular adhesion molecule (HCAM) increased one- to sixfold as cells became proliferative. The content of sm-MHC and sm-alpha-actin protein increased after confluence, suggesting that cultured airway SMC are capable of phenotypic plasticity. Marker protein contents were also compared, by immunoblot assay, between SMC dissociated from trachealis or pulmonary arterial media. Cytocontractile protein content was higher in the trachea, which shortens faster than the pulmonary artery. The identification of these markers provides tools for assessing the phenotype of airway SMC in culture and the airways of asthmatic patients.

Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: Relationship to elevated cAMP levels

The link between increased usage of β-adrenoceptor agonists and worsening of asthma symptoms has raised interest in the effects of agents such as salbutamol on airway wall remodelling, and particularly airway smooth muscle proliferation. In the present study we have investigated the role of increases in intracellular cAMP in the inhibitory effect of salbutamol on airway smooth muscle proliferation. The inhibitory effects of a combination of submaximally effective concentrations of salbutamol (10 nM) and the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 100 μM) on thrombin (0.3 U/mL)-induced mitogenesis in human cultured airway smooth muscle cells was greater than that for either agent alone. In addition, agents known to increase cAMP-dependent protein kinase activity including forskolin (10 μM), 8-bromoadenosine-3′,5′-cyclic monophosphate (100 μM), and prostaglandin E 2 (1 μM) have an inhibitory effect on thrombin (0.3 U/mL)-induced proliferation. Furthermore, the cAMP antagonist, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (300 μM) significantly reduced the inhibitory effect of salbutamol (10 nM) on thrombin (0.3 U/mL)-induced DNA synthesis. In IBMX (100 μM)-pretreated cells, salbutamol (100 nM) increased intracellular cAMP levels via stimulation of a β 2-adrenoceptor. Salbutamol (10 μM), at concentrations supramaximally effective for inhibition of mitogenesis, had no effect on thrombin (0.3 U/mL)-induced increases in intracellular calcium levels. Therefore, our results suggest that the previously reported inhibition of mitogen-induced proliferation in human cultured airway smooth muscle cells by the β 2-adrenoceptor agonist, salbutamol (100 nM), is at least partly due to elevation of intracellular cAMP, while there is no effect of salbutamol on initial mitogen-induced increases in intracellular calcium.

Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells

Smooth muscle hypertrophy is often found in tissue subjected to abnormal physical stress. To determine if physical stress (strain) per se could increase the contractile potential of airway smooth muscle (ASM), we compared cultured ASM cells subjected to strain to control cells (no strain) for rates of 1) myosin light chain kinase (MLCK)-mediated myosin light chain (LC20) phosphorylation, 2) actin-activated myosin ATPase, and 3) myosin light chain phosphatase-mediated myosin dephosphorylation. Lysates from strained cells showed increases in both LC20 phosphorylation activity and actomyosin ATPase activity but decreased rates of phosphatase-dependent myosin dephosphorylation. The increased LC20 phosphorylation activity and ATPase activity of the strained cells were accompanied by increases in cellular content of MLCK and myosin, respectively, compared with control. Because the cultured ASM cells exposed to strain expressed higher MLCK activity and actomyosin ATPase activity but lower myosin light chain phosphatase activity, these data suggest that physical stress in part determines ASM potential for contractile state.

Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis

Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.

Production of PGE2 by bovine cultured airway smooth muscle cells: regulation by cAMP.

Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. Previous studies have shown that it is produced by bovine cultured airway smooth muscle (ASM) cells in large quantities, but its regulation by second messengers has not been studied in this tissue. To determine whether PGE2 production by ASM might be an important action of beta-adrenoceptor agonists in asthma, the regulation of PGE2 production by adenosine 3',5'-cyclic monophosphate (cAMP) was assessed using dibutyryl cAMP (DBcAMP), forskolin, and albuterol. DBcAMP increased PGE2 production over a 24-h time course. Forskolin and albuterol both increased PGE2 production over control cells to similar levels after 24 h. Incubation of albuterol-treated cells with propranolol significantly (70%) reduced the stimulatory effect of albuterol on PGE2 production. Incubation of forskolin-treated cells with Rp-cAMP, a cAMP antagonist, inhibited the PGE2 response evoked by forskolin by 80%. Ro-20-1724, a selective inhibitor of type IV phosphodiesterase, stimulated PGE2 production (P = 0.02). Cycloheximide, a protein-synthesis inhibitor, did not inhibit the response to DBcAMP. The effects of DBcAMP were additive with the effects of bradykinin, a proinflammatory mediator known to increase PGE2 production (P < 0.05). These studies suggest that cAMP may play an important regulatory role in stimulating PGE2 production by ASM. This may be a novel beneficial action of beta-adrenoceptor agonists in asthma.

Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase.

Severe chronic asthma is associated with structural changes in the airway wall including airway smooth muscle (ASM) hyperplasia. We have used cultured ASM cells isolated from rabbit trachealis as a model with which to investigate possible mechanisms of accelerated ASM growth to mitogenic stimuli. To elucidate the role that protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways play in the control of ASM mitogenesis, we have investigated the effect of reportedly selective inhibitors of PKC (3-[1-[3-(amidinothio)propyl]-3-indolyl]-4-(1-methyl-3-indolyl)-1H - pyrrole-2,5-dionemethanesulfonate [Ro31-8220] and 3-[1-(aminopropyl)indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione acetate [Ro31-7549]) and PTK (alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide [ST638]) on partially purified PKC, fetal calf serum (FCS)-stimulated protein phosphotyrosine content and on FCS-induced proliferation. Anion-exchange chromatography of lysed ASM cells resolved two peaks of Ca(2+)-activated, phospholipid-dependent PKC activity and one peak of Ca(2+)- and phospholipid-independent PKC activity. The selective PKC inhibitors, Ro31-8220 and Ro31-7549, abolished the main peak of PKC activity and the Ca(2+)- and phospholipid-independent peak that co-eluted with the main peak. The inhibition was dependent on the concentration of ATP in the reaction cocktail (IC50: 10 microM ATP: Ro31-8220 0.026 microM, Ro31-7549 0.073 microM; 100 microM ATP: Ro31-8220 0.065 microM, Ro31-7549 0.271 microM), consistent with these compounds inhibiting PKC at the ATP-binding site. Ro31-8220 was more potent (2- to 3-fold) than Ro31-7549. Concentrations of each inhibitor that produced maximal inhibition of the pooled kinase activity also abolished the second peak of Ca(2+)-dependent activity. The PTK inhibitor, ST638, had no effect on the kinase activity associated with any of the Ca(2+)-dependent or -independent peaks that eluted from the column. ST638, however, maximally inhibited FCS-stimulated PTK activity (IC50 25 microM). FCS-stimulated PTK was also inhibited by Ro31-8220 (IC50 0.15 microM), but only by 60%, revealing an Ro31-8220-insensitive component to the response. The ability of each protein kinase inhibitor to inhibit proliferation was also studied using four independent indices of ASM cell growth and division: 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) dye conversion, Coomassie blue protein determination, hemacytometer cell counts, and DNA synthesis. Ro31-8220 and Ro31-7549 produced concentration-dependent inhibition of FCS-stimulated proliferation of growth-arrested ASM cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of cultured airway myocytes for study of airway smooth muscle

Cultured airway smooth muscle cells provide a convenient model system for studying the regulation of a wide range of airway responses at the cellular level. This review describes the characteristics of cultured airway smooth muscle cells and differences that exist between cultured cells and acutely dissociated cells or muscle strips. Receptor and ion-channel expression and control of coupling in cultured airway smooth muscle are reviewed. The methodology for airway smooth muscle culture is discussed. The main advantage of using cultured airway smooth muscle cells is that studies can be performed to examine long-term control of cell responses. Studies of the regulation of receptor expression and coupling, desensitization of receptor or channel-mediated responses, or regulation of the expression of important enzymes or muscle proteins can be readily performed in cell culture. In addition, cultured airway myocytes provide a useful secondary screening system for the development of novel therapeutic agents targeted at airway receptors that are expressed upon these cells.