Isolated Airway Smooth Muscle Cells Research Articles

Cellular senescence is increased in airway smooth muscle cells of elderly persons with asthma.

Senescent cells can drive age-related tissue dysfunction partially via a senescence-associated secretory phenotype (SASP) involving proinflammatory and profibrotic factors. Cellular senescence has been associated with a structural and functional decline during normal lung aging and age-related diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Asthma in the elderly (AIE) represents a major healthcare burden. AIE is associated with bronchial airway hyperresponsiveness and remodeling, which involves increased cell proliferation and higher rates of fibrosis, and resistant to standard therapy. Airway smooth muscle (ASM) cells play a major role in asthma such as remodeling via modulation of inflammation and the extracellular matrix (ECM) environment. Whether senescent ASM cells accumulate in AIE and contribute to airway structural or functional changes is unknown. Lung tissues from elderly persons with asthma showed greater airway fibrosis compared with age-matched elderly persons with nonasthma and young age controls. Lung tissue or isolated ASM cells from elderly persons with asthma showed increased expression of multiple senescent markers including phospho-p53, p21, telomere-associated foci (TAF), as well as multiple SASP components. Senescence and SASP components were also increased with aging per se. These data highlight the presence of cellular senescence in AIE that may contribute to airway remodeling.

PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal KATP channel

Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10–275μM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

Kcnq Channels in Airway Smooth Muscle

The role of KCNQ (Kv7) channels is well established in neurons, where they play dominant roles in control of resting membrane potentials and cell excitability. Recent studies have revealed expression of KCNQ channels in different types of smooth muscle. Here we focused on airway smooth muscle (ASM), whose membrane potential is primarily, determined by BK-type K+ channels. RT-PCR and immunostaining suggest that KCNQ 1, 4 and 5 are the predominant subtypes in rodent airway. To investigate the contribution of KCNQ channels in cholinergic-induced ASM contraction, we patch-clamped freshly isolated ASM cells and isolated KCNQ current as the non-inactivating component at the end of 2s depolarizations, with BK channels blocked with 1μM paxilline. The KCNQ current was enhanced by the KCNQ channel opener, flupirtine (10μM), and abolished by XE991 (10μM). Although XE991 depolarized the resting membrane potential, it did not affect that of ASM cells pre-treated with carbachol. Similarly, XE991 did not effect carbachol-evoked contractions, whereas flupirtine induced a significant relaxation. The flupirtine effect is likely via opposing voltage-dependent Ca2+ influx, since no effect of flupertine occurred in a low K+ (1mM) bathing solution. Pre-treatment with the muscarinic M3-receptor antagonist, fumarate (5nM), to prevent Gq/11-mediated PIP2 hydrolysis did not reveal an effect of XE991 on contractions, suggesting the mechanism of KCNQ inhibition in ASM is not due to PIP2 depletion. Interestingly, an XE991-dependent increase in contractility was revealed in mice with the β1 BK channel subunit knocked out, an effect enhanced by the M3 receptor antagonist. Thus, KCNQ channels may normally play a secondary role to BK channels in control of cholinergic evoked contractions in ASM; however, given that alteration in BK β1-subunit expression is known to occur in asthma, KCNQ channels in ASM cells may be potential targets for therapeutic intervention in respiratory disease.

The importance of biomechanical feedback in the generation of asthma

Invited EditorialThe importance of biomechanical feedback in the generation of asthmaLincoln E. FordLincoln E. FordDepartment of Environmental Health, Harvard School of Public Health, Boston, MassachusettsPublished Online:01 Oct 2011https://doi.org/10.1152/japplphysiol.01041.2011This is the final version - click for previous versionMoreSectionsPDF (27 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat those of us who pursue biological research do so mainly because of the intellectual satisfaction derived from making discoveries. But we are blessed with generous grants from our governments whose motivations are more practical and include improving the health of our populations, promoting new therapies, and enhancing the knowledge on which future practical discoveries can be made. Thus it is refreshing to read the occasional basic research paper describing a mechanism in a way that its practical importance is obvious. Such is the case with the paper by Connolly et al. (1) in this issue of the Journal of Applied Physiology.People who suffer from asthma soon learn that taking and holding a deep inspiration can provide some measure of relief. The several mechanisms contributing to this relief are likely to include 1) resetting the length of the activated smooth muscle by tearing off attached cross bridges so that if they reattach, it will be at a longer muscle length and 2) reducing the cooperative effect of multiple attached crossbridges and thus reducing the number of sites for cross bridge reattachment. On the other hand, they also learn that the relief provided by this maneuver attenuates with repeated use, so that they should use it sparingly, if at all, to avoid hyperinflated lungs.The paper discussed here examines this behavior in isolated airway smooth muscle cells grown on Silastic membranes and finds that repeated 10% stretches of these cultured cells for up to 10 days reduced resistance to stretch, in both the cyclically stretched and nonstretched cells, but that the chronically oscillated cells recover their resistance to stretch three times more rapidly than the unstretched cells. Of the possible mechanistic explanations for this behavior perhaps the most likely is an increase in myosin light chain kinase activity in the chronically oscillated cells, and this was confirmed by the authors' biochemical assay.This relatively simple finding could have enormous practical implications, some of which might be investigated with the same techniques. For example, there is belief that asthma begets more asthma. To the extent that this is so, would blocking of light-chain kinase prevent the transition from early childhood wheezing to chronic asthma. A trustworthy answer to this question could only be obtained with the clinical gold standard of a placebo-controlled, randomized, double-blind study, but substantial preliminary progress might be made with the same laboratory model used by the authors. Such laboratory experiments might address the very important practical issues of the strength and duration of treatment required to interrupt the progression to chronic asthma. Thus I view the present report as a first installment in what I hope will become a series of studies defining the pathways leading to chronic asthma and strategies for blocking these pathways.A final point to be made from these observations is that responses described have the characteristics of positive feedback, and there are two general characteristics of positive feedback that may be of particular relevance in the treatment of asthma. The first is that once started, the feedback is self-sustaining, and the second is that the output of the system increases rapidly to its maximum. These considerations suggest a mechanistic explanation for the clinical saw that the treatment of an asthma attack should be sufficiently vigorous to end all symptoms, i.e., to break the feedback loop, and that it should continue until all signs and symptoms of bronchospasm have fully abated.GRANTSThis work was supported by National Institutes of Health Grant HL-052762.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.REFERENCES1. Connolly SC , Smith PG , Fairbank NJ , Lall CA , Cole DJ , MacKinnon JD , Maksym GN. Chronic oscillatory strain induces MLCK-associated rapid recovery from acute stretch in airway smooth muscle cells. J Appl Physiol; doi:10.1152/japplphysiol.00812.2011.ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: L. E. Ford, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (e-mail: [email protected]harvard.edu). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 111Issue 4October 2011Pages 954-954 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.01041.2011PubMed21868686History Published online 1 October 2011 Published in print 1 October 2011 Metrics

Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca(2+). In this work, we found that the contraction caused by histamine depends on external Na(+), possibly involving nonselective cationic channels (NSCC) and the Na(+)/Ca(2+) exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 +/- 1% of control during external Na(+) substitution by N-methyl-D-glucamine(+), whereas substitution by Li(+) led to no significant change (91 +/- 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 +/- 5.6%), whereas preincubation with nifedipine did not (89.7 +/- 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 +/- 3%, significantly different from nifedipine alone (49 +/- 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 +/- 1% and 19 +/- 7%, respectively. Intracellular Ca(2+) measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 +/- 0.1 and 0.19 +/- 0.09 for KB-R, 0.43 +/- 0.16 and 0.47 +/- 0.18 for SKF, expressed as mean of differences). Moreover, Na(+)-free solution only inhibited the sustained response (0.54 +/- 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na(+) influx through NSCC that promotes the Ca(2+) entry mode of NCX and Ca(V)1.2 channel activation, thereby causing contraction.

Estrogen Reduces Carbachol-Induced Constriction of Asthmatic Airways by Stimulating Large-Conductance Voltage and Calcium-Dependent Potassium Channels

Both the incidence and severity of asthma in women are influenced by fluctuations in estrogen (E(2)) levels, raising the possibility that E(2)s may reduce the hyperresponsiveness that is characteristic of asthma. We examined the effect of E(2) and its downstream signaling pathways in isolated mouse bronchial and tracheal rings passively sensitized either with serum from patients with atopic asthma (ATR) or with serum from control subjects (CTR). ATR exhibited significantly higher sensitivity to carbachol than CTR. Pretreatment of ATR with E(2) shifted the carbachol concentration-response curve (CCRC) toward that of CTR. The E(2) effect was abolished by the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, the soluble guanyl cyclase inhibitor, quinoxalin-1, or the protein kinase G inhibitor, KT5823. Inhibition of the large-conductance, calcium-activated potassium (BK(Ca)) channel activity with iberiotoxin also attenuated the E(2) effect on ATR. In patch-clamp studies, E(2) increased by 50-fold the BK(Ca) channel activity in freshly isolated airway smooth muscle cells. This increase was completely blocked by KT5823. These studies suggest that, at physiologic concentrations, E(2) can prevent cholinergic-induced constriction of asthmatic tracheal rings by activating the nitric oxide-cGMP-protein kinase G pathway to increase BK(Ca) channel activity.

Commentary

commentary

Interaction of extracellular albumin and intravenous anaesthetics, etomidate and propofol, on calcium signalling in rat airway smooth muscle cells.

It has been shown in vitro that general anaesthetics modify airway responsiveness via, at least partially, a direct inhibitory effect on calcium signalling in airway smooth muscle cells. However, in vivo, these anaesthetic compounds bind serum proteins. We have investigated the effect of exposure to extracellular albumin of freshly isolated airway smooth muscle cells on the propofol- and etomidate-induced inhibitory effect on calcium signalling. [Ca2+]i was measured by microspectrofluorimetry in rat isolated tracheal smooth muscle cells using the fluorescent dye indo-1. Propofol (3 x 10(-4) M) and etomidate (10(-4) M) were the lowest 'effective' concentrations that altered the [Ca2+]i response. This alteration consisted of a decrease in both the amplitude of the [Ca2+]i peak (from 358 +/- 13 nM to 65 +/- 15 and 108 +/- 27 nM for propofol and etomidate, respectively) and the percentage of responding cells (from 80% to 37 and 25% respectively) in response to the low concentration of ACh and a decrease in the Ca2+ oscillation frequency (from 9.9 +/- 0.3 min(-1) to 4.7 +/- 0.4 and 6.9 +/- 0.4 min(-1), respectively) in response to the high concentration of ACh. Increasing the concentration of albumin reduced the inhibitory effect of etomidate and propofol on the [Ca2+]i response to ACh. When extracellular albumin concentration was kept constant (20 g/L), increasing the concentration of etomidate by one log restored its inhibitory effect on the calcium signal. This study indicates that increasing the concentration of extracellular albumin reduces the inhibitory effect of intravenous anaesthetics on calcium signalling in airway smooth muscle cells. This report suggests that, in extrapolating in vitro dose-response relationships to those from in vivo conditions, the effect of the concentration of extracellular protein can be estimated.

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.

Patch clamp techniques to study effects of anesthetics on airway smooth muscle cells

Inhalation anesthetics have a direct relaxant effect on airway smooth muscle. Recent research has done much to clarify the cellular mechanisms for contraction and relaxation in airway smooth muscles and has suggested a possible mechanism of action for inhalation anesthetics in these muscles [1,2]. As in other muscle cells, the free intracellular concentration of CaZ+([Ca2+]i ) plays a central role in regulation of airway smooth muscle tone [3]. Yamakage [1] demonstrated that relaxation of precontracted tracheal smooth muscle by halothane was associated with a decrease in [Ca2+]i . Because sustained contraction of this tissue requires the continued entry of e x t r a c e l l u l a r Ca 2+, a possible mechanism for the airway relaxant effect of inhalation anesthetics is inhibition of voltage-dependent Ca > channels (VDC) [4]. Patch clamp techniques provide a means for directly studying properties of membrane ion channels [5]. Applications of these methods to airway smooth muscle cells have demonstrated a variety of K + and Ca 2+ channels that are likely of functional importance [6,7]. One patch clamp method, whole-cell recording, is particularly useful for quantifying Ca 2+ current (Ic,) through VDC. In this report, we describe the results of a preliminary study that demonstrates inhibition by enflurane of Ica in isolated airway smooth muscle cells. Porcine tracheal smooth muscle, dissected free of epithelium and connective tissue, was minced. The tissue fragments were digested in Ca2+-free Tyrode's solution containing 0.1% collagenase, 0.05% trypsin

Effect of IgG1 and its fragments on resting membrane potential of isolated tracheal myocytes

We enzymatically isolated airway smooth muscle cells from the trachea of guinea pigs (400-600 g body wt). After removal of connective tissue, strips of trachealis muscle were cleaned under a dissecting microscope and incubated with collagenase (type I, 1 mg/ml) and elastase (type I, 15 U/ml) for 20 min. Cells were resuspended in Dulbecco's modified Eagle's medium and seeded on 1.5% gelatin-coated Petri dishes (35 mm). The viability of cells was assessed by trypan blue exclusion. Individual myocytes were impaled with glass microelectrodes (input resistance 90-100 M omega). Resting membrane potential (Em) was determined before and after administration of 1) immune serum, 2) highly purified specific immunoglobulin G1 (IgG1), and 3) enzymatically prepared fragments of IgG1-F(ab')2 and Fc. We found that 1) Em of isolated tracheal myocytes is -60.5 +/- 0.5 mV; 2) some myocytes exhibit spontaneous electrical rhythm with mean frequency of 16.9 +/- 12 min-1 and mean amplitude of 3.7 +/- 0.6 mV; 3) immune serum, IgG1, and Fc fragments induced a biphasic change in Em: the initial mean depolarization (-51.5 +/- 0.8 mV) was followed by a steady-state hyperpolarization (-68.3 +/- 0.6 mV); and 4) pretreatment of myocytes with amiloride (10(-5) M) or exposure of myocytes to a low-sodium environment prevented changes in Em induced by the passive in vitro sensitization. It is likely that airway smooth muscle cells have a low-affinity Fc receptor, the occupancy of which leads to activation of amiloride-sensitive sodium influx.

Hormonal regulation of calcium current in freshly isolated airway smooth muscle cells.

Single freshly isolated smooth muscle cells of adult bovine trachea were voltage clamped, and the calcium inward current was separated from K+ currents by blocking the large outward currents with intra- and extracellular Cs+ and extracellular tetraethylammonium chloride. Isoproterenol stimulated peak calcium current (ICa) in a dose-dependent manner through the beta-adrenergic receptor. The isoproterenol effect was not mediated or caused by the stimulation of a K+ or Na+ current, a decrease in the intracellular concentrations of Ca2+ or H+, the stimulation of the Na(+)-H+ or the Na(+)-Ca2+ exchanger. Neither basal nor isoproterenol-stimulated ICa was affected by internal dialysis of the cell with adenosine 3',5'-cyclic monophosphate (cAMP), cAMP analogues, or the catalytic subunit of cAMP-kinase. Internal dialysis of the cells with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the stimulation of isoproterenol whereas dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) induced an isoproterenol-like maximal increase of ICa. These results show that the beta-adrenergic receptor stimulates the L-type calcium current of isolated tracheal smooth muscle cells independent of cAMP and cAMP-kinase through a GTP/GDP regulated protein.