Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase

Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1-1 x 10(6) cells/ml), attachment of human ASMC was reduced to 12.3 +/- 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 +/- 8.1% compared with baseline of 3.1 +/- 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 +/- 0.7%; baseline 1.1 +/- 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by alpha(1)-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1-3 microg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.     

A neutrophil elastase inhibitor, sivelestat, ameliorates lung injury after hemorrhagic shock in rats

Hemorrhagic shock followed by resuscitation (HSR) causes neutrophil sequestration in the lung which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play a pivotal role in the pathogenesis of ALI. This study investigated whether sivelestat, a specific NE inhibitor, can attenuate ALI induced by HSR in rats. Male Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood so as to maintain a mean arterial blood pressure of 30+/-5 mm Hg for 60 min followed by resuscitation with the shed blood. HSR-treated animals received a bolus injection of sivelestat (10 mg/kg) intravenously at the start of resuscitation followed by continuous infusion for 60 min (10 mg/kg/h) during the resuscitation phase, or the vehicle. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, myeloperoxidase (MPO) activity, gene expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), DNA binding activity of nuclear factor (NF)-kappaB, and immunohistochemical analysis of intercellular adhesion molecule (ICAM)-1. HSR treatment induced lung injury, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, MPO activity, gene expression of TNF-alpha and iNOS, and DNA-binding activity of NF-kappaB, and enhanced expression of ICAM-1. In contrast, sivelestat treatment significantly ameliorated the HSR-induced lung injury, as judged by the marked improvement in all these indices. These results indicate that sivelestat attenuated HSR-induced lung injury at least in part through an inhibition of the inflammatory signaling pathway, in addition to the direct inhibitory effect on NE.     

Combined effects of a neutrophil elastase inhibitor (sivelestat sodium) and a free radical scavenger (edaravone) on lipopolysaccharide-induced acute lung injury in rats

Objective and design

The present study aimed to investigate the combined effects of a neutrophil elastase inhibitor, sivelestat sodium, with a free radical scavenger, edaravone, on lipolysaccharide (LPS)-induced acute lung injury (ALI).

Materials and methods

Adult male Sprague–Dawley rats were anesthetized and instilled intratracheally with 2?mg/kg LPS. Sivelestat sodium (10?mg/kg, i.p.) and/or edaravone (8?mg/kg, i.p.) were administered 1?h after LPS instillation. The severity of pulmonary injuries was evaluated 12?h after inducing acute lung injury.

Results

In lung tissues, either sivelestat or edaravone treatment alone showed significant protective effects against neutrophil infiltration and tissue injury, as demonstrated by myeloperoxidase activity and histopathological analysis. Sivelestat or edaravone treatment also attenuated the LPS-induced production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in rat lungs. However, the LPS-induced elevation of malondialdehyde levels in rat lungs was reduced only by edaravone, but not by sivelestat. In addition, combined treatment with both sivelestat and edaravone demonstrated additive protective effects on LPS-induced lung injury, compared with single treatments.

Conclusions

Combination of sivelestat and edaravone shows promise as a new treatment option for ALI/acute respiratory distress syndrome patients.     

Effect of genistein on agonist-induced airway smooth muscle contraction

Inflammation Research - .     

Adenosine potentiates neurally- and histamine-induced contraction of canine airway smooth muscle

We studied the effect of adenosine on airway reactivity of isolated canine bronchial smooth muscle under isometric conditions in vitro. Administration of adenosine and its analogs increased the contractile responses of bronchial segments to electrical field stimulation in a dose-dependent fashion, where the rank order potency was N-ethylcarboxamideadenosine greater than adenosine greater than N-cyclohexyladenosine, but had no effect on those to exogenous acetylcholine. This potentiation was more pronounced at relatively low than at high stimulus frequencies, the maximal increase from the baseline responses being 56.3 +/- 9.6% at 1 Hz (mean +/- SE, p less than 0.01). Adenosine also increased the histamine-induced contraction causing a leftward shift of the histamine dose-response curves, an effect that was abolished in the presence of atropine. These results suggest that adenosine potentiates airway responsiveness to vagal stimulation and to histamine through the activation of prejunctional A2 receptor, probably involving the accelerated release of acetylcholine from the cholinergic nerve terminals.     

Okadaic acid,a phosphatase inhibitor,produces a Ca2+ and calmodulin-independent contraction of smooth muscle

The effects of okadaic acid, a phosphoprotein phosphatase inhibitor, on the contractile response and on myosin light chain phosphorylation were studied in intact lamb tracheal smooth muscle. The effects of okadaic acid were compared to the response to the same fibers stimulated with 1 M methacholine, a concentration that induces 90% of maximal force. Okadaic acid (50 M) produced a slow but maximal contraction that was accompanied by an increase in phosphorylation of the 20 kDa light chain of myosin. The myosin light chain phosphorylation pattern induced by okadaic acid, however, differed from that induced by methacholine. Ca²⁺ depletion, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, blocked or attenuated methacholine-induced contractions but had no significant effect on force development or myosin light chain phosphorylation induced by okadaic acid. These results suggest that phosphorylation of the 20 kDa light chain of myosin is essential for smooth muscle contraction; they also suggest that okadaic acid either uncovers or activates an apparently Ca²⁺ and calmodulin-independent protein kinase activity that phosphorylates the 20 kDa light chain of myosin at multiple sites.Abbreviations used LC₂₀ the 20 kDa light chain of smooth muscle myosin - OA okadaic acid - DMSO dimethylsulfoxide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide - H-7 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine - TBS 150 mM NaCl, 20 mM Tris-HCl, pH 7.5     

P物质对气道平滑肌细胞增殖的调节作用

目的：观察Ｐ物质对犬气道平滑肌细胞增殖的调节作用。方法：以第四代ＡＳＭＣ为研究对象,分别以ＳＰ和「Ｓａｒ＾９,Ｍｅｔ（Ｏ２）＾１１」－ＳＰ（ＮＫ－１受兴奋剂）刺激,观察两者对细胞增殖的影响。又分别以ＧＲ ７１２５１（ＮＫ－１受体拮抗剂）,新霉素「磷脂酶Ｃ（ＰＬＣ）抑制剂」和尼群地平为干预因素,观察它们对ＳＰ作用的影响。     

Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction

An increase in the cytosolic Ca²⁺ concentration is a prerequisite in activation of contractile activity of smooth muscle. The shape of the Ca²⁺-signal is determined by spatial distribution and kinetics of Ca²⁺-binding sites in the cell. The increase in cytosolic Ca²⁺ activates myosin light chain kinase (MLCK) which in turn phosphorylates the regulatory light chains of myosin II. This Ca²⁺-dependent MLC₂₀ phosphorylation is modulated in a Ca²⁺-independent manner by inhibiting the constitutive active myosin light chain phosphatase mediated by the monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C or by increasing its activity through cGMP. Furthermore, the activity of MLCK may be decreased due to phosphorylation by CaM kinase II and perhaps p21 activated protein kinase. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades which not only show a temporal but also a spatial activation pattern.     

The effects of leptin on airway smooth muscle responses

Obesity is associated with asthma and airway hyperresponsiveness. Leptin modulates some of the proinflammatory effects observed in obesity. The objective of this study was to determine the effects of leptin on airway smooth muscle responses. The effect of leptin (0.1-100 ng/ml) on migration (toward platelet-derived growth factor [PDGF], 10 ng/ml, across collagen-coated membrane in Transwell culture plates), proliferation (by BrDU incorporation), and cytokine production (by Bioplex bead assay) of cultured human airway smooth muscle cells from nine nonasthmatic donors was assessed. Effects of leptin on the contractile responses were studied in bovine tracheal smooth muscle rings. Leptin receptor expression and activation of STAT-3, Src kinase, Suppressor of Cytokine Signaling-3 (SOCS-3), and COX were evaluated by Western blotting and PCR. PGE(2) levels in supernatant were assessed by enzyme immunoassay. Human airway smooth muscle cells express leptin receptor, which, when engaged, phosphorylated STAT-3. Leptin inhibited PDGF-induced human airway smooth muscle migration and proliferation and IL-13-induced eotaxin production. Leptin did not stimulate cytokine synthesis and did not evoke contractile responses or inhibit isoproterenol-induced relaxation of carbachol-induced contraction of bovine tracheal rings. The inhibitory effects on migration and eotaxin production are not due to activation of SOCS-3 but are partly due to increased production of PGE(2) because they were attenuated by indomethacin. In conclusion, leptin inhibited human airway smooth muscle proliferation, migration toward PDGF, and IL-13-induced eotaxin production. This is partly mediated by PGE(2) secretion from smooth muscle cells induced by leptin. The association between obesity and asthma is unlikely to be due to a direct effect of leptin on airway smooth muscle.     

A cytosolic inhibitor of human neutrophil elastase and cathepsin G.

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is alpha 1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase or 125I-cathepsin G with neutrophil cytosol or alpha 1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of alpha 1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100 degrees C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37 degrees C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either alpha 1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.     

Comparison of substance P-induced and compound 48/80-induced neutrophil infiltrations in mouse skin.

It has recently been shown that substance P induces neutrophil infiltration in the skin, which is mediated through mast cell degranulation. Since substance P activates both skin mast cells and vascular endothelial cells, we compared the potencies of substance P and a mast cell-degranulating agent, compound 48/80, which is inactive for vascular endothelial cells, in inducing neutrophil infiltration in mouse skin. We also examined the effect of the C-terminal peptide of substance P, SP6-11, which is active for vascular endothelial cells, on compound 48/80-induced neutrophil infiltration in the skin. Subcutaneous administrations of substance P (10(-7) to 10(-5) M; 0.1 ml) and compound 48/80 (0.5-50 micrograms/ml) induced neutrophil infiltrations and mast cell degranulations in mouse skin in a concentration-dependent fashion. Moreover, substance P induced more neutrophil infiltrations than compound 48/80 in terms of the magnitude of mast cell degranulations. SP6-11 (10(-6) to 10(-4) M) induced no significant neutrophil infiltration or mast cell degranulation, but increased the vascular permeability of endothelial cells in the skin. Furthermore, SP6-11 enhanced compound 48/80-induced neutrophil infiltration without any increase in mast cell degranulation. Our results indicate that, in addition to mast cell degranulation, the activation of vascular endothelial cells is involved in substance P-induced neutrophil infiltration in the skin.     

Pentobarbital and contraction of vascular smooth muscle.

This study, with isolated rat aortic strips and portal veins, was undertaken to : 1) study the effects, if any, of pentobarbital Na (PTB) (5 x 10(-5) to 2 X 10(-3) M) on reactivity to epinephrine, serotonin, and KCl; 2) determine whether certain concentrations of PTB induce direct actions on aortic strips and portal veins; and 3) gain some insight into how these effects are brought about. The results indicate that PTB can: a) inhibit development of spontaneous mechanical activity in these vessels in anesthetic concentrations; b) dose-dependently attenuate contractions induced by epinephrine, serotonin, and KC1; c) cause a noncompetitive type displacement of the dose response curves of these vasoactive agents; d) attenuate Ca2+- induced contractions of potassium-depolarized aortic strips and portal veins concomitant with a dose-dependent displacement of these dose-response curves to the right; and e) rapidly relax drug as well as Ca2+ -induced contractions of aortas and portal veins. In addition, the data indicate that rat portal venous smooth muscle is more sensitive to the inhibitory actions of PTB than rat aortic smooth muscle. Overall, these data suggest that concentrations of PTB used to induce surgical anesthesia can exert profound depressant effects on at least two different types of vascular smooth muscle that may be related to actions on movement and/or translocation of Ca2+.     

Excitation and contraction in bovine tracheal smooth muscle.

1. The smooth muscle layer of the bovine trachea was studied in vitro with the micro-electrode and sucrose-gap techniques. The membrane potential was stable at--47-6 plus or minus 0-98 (S.E. of mean) mV, and there was no spontaneous electrical or mechanical activity. 2. The cell membrane had strong rectifying properties, making it impossible to elicit action potentials by electrical stimulation in normal Krebs Solution. The rectification was abolished by TEA (30 mmol/l), which depolarized the membrane and produced plateau-type action potentials. 3. The spontaneous repetitive action potentials produced by TEA were associated with rhythmic oscillatory contractions of the muscle strips. 4. Histamine caused an increased tone, with superimposed rhythmic fluctuations in tension. The electrical response consisted of depolarization, with rhythmic slow oscillations in potential (slow waves) which were synchronous with the fluctuations in tension. 5. Acetylcholine produced smooth, tonic contractures of tracheal muscle strips, and caused simple depolarization of the membrane. No action potentials were recorded. 6. In calcium-free solutions containing EGTA, the mechanical response to TEA was completely abolished; the response to histamine was greatly reduced; the response to acetylcholine was reduced to a lesser extent. All responses reverted to normal when normal concentrations of extracellular calcium were restored. 7. Lanthanum added to the bathing solution abolished the contraction due to TEA even though the solution contained calcium. It reduced the histamine-induced contraction to 26% of control, and reduced the acetylcholine-induced contraction to 58% of control; extracellular calcium was present throughout. 8. It is suggested that TEA produces contraction by promoting influx of calcium ions into the cytoplasm. Acetylcholine, and to a smaller extent histamine, are less dependent upon the presence of extracellular calcium, and may be capable of releasing calcium sequestered within the cell; acetylcholine appears to be more effective in releasing sequestered calcium.     

Neuronally mediated non-cholinergic contraction of guineapig bronchial smooth muscle is inhibited by a substance P antagonist

The isolated main bronchi of the guinea-pig respond to electrical field stimulation with a twitch followed by a slow contraction. Atropine blocked the slow contraction. The substance P antagonist, (d-Pro²,d-Trp^7.9)-SP, greatly reduced the atropine-resistant contraction. The results suggest the involvement of substance P in non-cholinergic neurotransmission in the guinea-pig airways.     

Reorientation of myofilaments during contraction of a vertebrate smooth muscle.

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.     

Effect of antioxidants on airway smooth muscle contraction: action of lipoic acid and some of its novel derivatives on guinea pig tracheal smooth muscle

Objectives and design

The aim of this study was to investigate the ability of (a) antioxidants, some related to α-lipoic acid (LA), (b) histone deacetylase (HDAC) inhibitors, and (c) hybrid compounds possessing both α-lipoic acid-derived antioxidant properties and HDAC inhibitory activity to inhibit guinea pig smooth muscle contraction.

Materials and methods

Guinea pig isolated tracheal rings (GPTR) were prepared and their isometric tension measured using a transducer. Histamine, carbachol and 5-hydroxytryptamine (5-HT) served as agonists. Tests with antigen (ovalbumin) used GPTR from sensitised guinea pigs or rings from non-sensitised animals that had been incubated for at least 2 h with diluted serum from sensitised animals.

Results

All antioxidants tested showed a relaxant effect on resting tension and on tension induced by histamine or carbachol, with EC₅₀(s) of 0.2–5.0 mM and a rank order of potency: LA derivatives > glutathione (GSH) > ascorbic acid (AA). However, low concentrations (<50 μM) of GSH, AA and LA potentiated histamine-induced contractions. The benzamide HDAC inhibitor MGCD0103 inhibited mast cell activation and GPTR contraction produced by antigen and certain agonists, although a 2–6 h pre-incubation was required for those effects to be apparent. Two LA–benzamide HDAC hybrid compounds, UCL M084 and UCL M109 inhibited GPTR contraction after 30 min pre-incubation; however, even after long pre-incubation (up to 6 h) those hybrid compounds showed less potent inhibition of agonist-induced contraction than did MGCD0103.

Conclusions

The results showed that GSH more potently inhibited contraction induced by histamine than that induced by 5-HT or carbachol, whereas LA, and especially UCL M084 and UCL M109, more potently blocked contraction induced by carbachol and 5-HT than that induced by histamine. For GSH, and possibly also for LA-type compounds, the inhibition of agonist-induced tracheal smooth muscle contractions may be due to NO formation. This study did not detect a synergistic relaxant effect in two compounds incorporating the structural union of a benzamide HDAC inhibitor terminus with a LA-derived antioxidant moiety.

     

Long-term effects of intracellular calcium and growth factors on excitation and contraction in smooth muscle

Modulation of vascular smooth muscle cells from a contractile to a synthetic phenotype is thought to be important in the development of the atherosclerotic lesion. Such modulation depends on growth factors and is influenced by cell—cell and cell—matrix interactions. Whereas smooth muscle cells in the vessel wall are contractile, dispersed cells in culture rapidly modulate to synthetic phenotype, which complicates long-term in vitro studies. In contrast, vascular segments or smooth muscle strips in organ culture can maintain contractility for at least a week, sufficient for studies involving altered metabolism or protein expression. Examples are effects of endogenous polyamines on membrane ion channels and excitation—contraction coupling. While smooth muscle tissue is well preserved in serum-free culture, growth stimulation with fetal calf serum (FCS) causes multiple effects, including decreased contractility, ultrastructural changes, decreased expression of l -type Ca²⁺ channels, and increased SR release of Ca²⁺ via ryanodine receptors. These are all consequences of increased basal [Ca²⁺]_i caused by FCS, as they are reversed by culture with verapamil in a concentration (1 μm ) that does not inhibit stimulation of DNA and protein synthesis by FCS. The effects of FCS on contractility and Ca²⁺ channel expression are mimicked in serum-free culture with increased [Ca²⁺]_i. Contractile protein patterns, including myosin isoform composition, are unaffected by FCS, suggesting that reversal to synthetic phenotype is limited and not the immediate cause of decreased contractility.     

Effect of antioxidants on airway smooth muscle contraction: action of lipoic acid and some of its novel derivatives on guinea pig tracheal smooth muscle

Objectives and design

The aim of this study was to investigate the ability of (a) antioxidants, some related to α-lipoic acid (LA), (b) histone deacetylase (HDAC) inhibitors, and (c) hybrid compounds possessing both α-lipoic acid-derived antioxidant properties and HDAC inhibitory activity to inhibit guinea pig smooth muscle contraction.

Materials and methods

Guinea pig isolated tracheal rings (GPTR) were prepared and their isometric tension measured using a transducer. Histamine, carbachol and 5-hydroxytryptamine (5-HT) served as agonists. Tests with antigen (ovalbumin) used GPTR from sensitised guinea pigs or rings from non-sensitised animals that had been incubated for at least 2 h with diluted serum from sensitised animals.

Results

All antioxidants tested showed a relaxant effect on resting tension and on tension induced by histamine or carbachol, with EC₅₀(s) of 0.2–5.0 mM and a rank order of potency: LA derivatives > glutathione (GSH) > ascorbic acid (AA). However, low concentrations (<50 μM) of GSH, AA and LA potentiated histamine-induced contractions. The benzamide HDAC inhibitor MGCD0103 inhibited mast cell activation and GPTR contraction produced by antigen and certain agonists, although a 2–6 h pre-incubation was required for those effects to be apparent. Two LA–benzamide HDAC hybrid compounds, UCL M084 and UCL M109 inhibited GPTR contraction after 30 min pre-incubation; however, even after long pre-incubation (up to 6 h) those hybrid compounds showed less potent inhibition of agonist-induced contraction than did MGCD0103.

Conclusions

The results showed that GSH more potently inhibited contraction induced by histamine than that induced by 5-HT or carbachol, whereas LA, and especially UCL M084 and UCL M109, more potently blocked contraction induced by carbachol and 5-HT than that induced by histamine. For GSH, and possibly also for LA-type compounds, the inhibition of agonist-induced tracheal smooth muscle contractions may be due to NO formation. This study did not detect a synergistic relaxant effect in two compounds incorporating the structural union of a benzamide HDAC inhibitor terminus with a LA-derived antioxidant moiety.