Etienne Roux

Prolonged Moderate Hyperoxia Induces Hyperresponsiveness and Airway Inflammation in Newborn Rats

Bronchopulmonary dysplasia is the most common cause of chronic pulmonary disease in premature infants. Airway inflammation appears to play a major pathogenetic role together with barotrauma and oxygen toxicity. The aim of the present study was to determine the effect of a 15-d exposure to moderate hyperoxia (Fio2, 50%) on airway reactivity and inflammatory response in neonatal and adult rats. We studied in isolated tracheal rings the 1) isometric contraction to cumulative concentrations of carbachol (10−8 to 10−3 M);2) epithelial, submucosal, smooth muscle, and connective tissue surface area; and 3) distribution of inflammatory cells (mastocytes, granulocytes, macrophages) by using MAb. Reactivity to carbachol was significantly increased in the hyperoxic pups, in which a 13% increase in tracheal smooth muscle surface area was observed. Type-I mast cells and macrophages (submucosa and connective tissue) and granulocytes (connective tissue) were increased in the neonatal hyperoxic group. Hyperoxia did not influence functional, morphometric, or cellular data in adult rats. In conclusion, exposure of newborn rats to moderate hyperoxia induces airway hyperresponsiveness and histologic changes similar to those reported in bronchopulmonary dysplasia. Hyperresponsiveness may be ascribed to an increase in smooth muscle related to the release of yet undetermined mediators by inflammatory cells infiltrating the airways. Lung immaturity definitely plays a role because similar alterations are not observed in adult rats.

[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity

Cytosolic calcium concentration ([Ca2+]i) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle. In isolated myocytes, acetylcholine (ACh, 0.031 μm) caused a rapid and graded increase in [Ca2+]i up to a net amplitude of 492±26 nm (n=19) which gradually declined. The EC50 for ACh was 0.13 μm. This first [Ca2+]i peak was followed, when the ACh concentration increased, in approximately 5060% of the cells, by successive peaks of decreased amplitude ([Ca2+]i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca2+]i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min−1 with an ACh concentration of 100 μm. Removal of extracellular calcium (in the presence of EGTA, 0.4 mm) or addition of the voltage‐dependent Ca2+‐channel blocker verapamil (10 μm) did not alter the first [Ca2+]i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca2+‐ATPase, thapsigargin (1 μm), completely abolished the [Ca2+]i response. Thapsigargin (1 μm) also blocked the caffeine (5 mm)‐induced transient rise in [Ca2+]i. Atropine (a non‐selective muscarinic cholinoceptor antagonist) and 4‐diphenyl acetoxy N‐methyl piperidine (4‐DAMP, a selective M3 antagonist) inhibited the [Ca2+]i response to muscarinic cholinoceptor activation with an IC50 of 13 and 20 nm, respectively. Pirenzepine (a selective M1 antagonist) also totally inhibited the [Ca2+]i response to ACh but with a higher IC50 of 2 μm. Methoctramine (a selective M2 antagonist) up to a concentration of 10 μm caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration‐response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4‐DAMP at 3, 10 and 30 nm; pirenzepine 0.3, 1 and 3 μm, and methoctramine at 1, 3 and 10 μm. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA2 values were 9.2, 8.8, 6.7 and 6.3, respectively. In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca2+]i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca2+]i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca2+]i and the contractile responses are primarily dependent on activation of the M3 receptor subtype.

Muscarinic Stimulation of Airway Smooth Muscle Cells

1. Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, is released at both ganglionic synapses and postganglionic neuroeffector junctions and acts by activation of nicotinic and muscarinic cholinoceptors. This review focuses on the effects of postjunctional muscarinic stimulation of airway smooth muscle. 2. On pharmacological criteria, four distinct subtypes of muscarinic cholinoceptor, denoted M1, M2, M3 and M4 receptors, have been identified by use of selective antagonists. Cloned muscarinic cholinoceptors are members of the family of GTP-binding protein-coupled receptors, which are characterized by seven transmembrane (TM) regions connected by intra- and extracellular loops. Between the fifth and the sixth TM regions, muscarinic receptors possess a large intracytoplasmic loop that is considered to be responsible for G-protein-coupling selectivity and exhibits high divergence between the different subtypes. 3. At the site of the smooth muscle itself, both binding and Northern blot studies have demonstrated, in a variety of species, that muscarinic receptor subtypes present are M2 and M3. M2 receptors are coupled to Gi proteins and adenylyl cyclase inhibition and thus to cAMP signaling. M3 receptors are coupled to Gq/11 protein and phosphoinositide hydrolysis and thus to calcium signaling. 4. Muscarinic-induced contraction of airway smooth muscle is mediated by M3 receptors. M2-mediated inhibition of adenylyl cyclase contributes to the prevention of bronchodilation. Cross-talk between muscarinic and beta2 adrenoceptors is likely to be present in airway smooth muscle. The pathophysiological role of this cross-talk requires further investigation.

Effects of Intravenous Anesthetics on Normal and Passively Sensitized Human Isolated Airway Smooth Muscle

Background General anesthetics may modify airway responsiveness. The authors investigated the effect of thiopental, propofol, and etomidate on airway smooth muscle. Methods Contraction experiments were done in human airway rings that were either normal or passively sensitized with asthmatic serum. The effect of propofol and etomidate was also studied on both [Ca sup +] sub i increase measured by microspectrofluorimetry in isolated myocytes and isometric contraction in the rat trachea. Results In human bronchi, thiopental (10 sup ‐7 to 10 sup ‐4 M) induced a concentration‐dependent contraction. Neither propofol nor etomidate altered baseline tone, but both anesthetics reduced histamine‐induced contraction. In human immunologically sensitized isolated bronchi, propofol (3 x 10 sup ‐4 M) reduced histamine reactivity (Delta Fmax in %) to a greater degree than in nonsensitized tissues (64.4 +/‐ 15.7% and 16.4 +/‐ 8.5%, respectively; n = 6, P < 0.05), whereas the effect of etomidate (10 sup ‐4 M) was similar in both types of tissue (24.1 +/‐ 6% and 22.3 +/‐ 15%, respectively, n = 6). In rat isolated tracheal myocytes, propofol (3 x 10 sup ‐4 M) and etomidate (10 sup ‐4 M) altered the [Ca2+]i signal in response to the depolarizing agent potassium chloride and the muscarinic agonist acetylcholine. Accordingly, the two anesthetics also reduced the mechanical response of rat tracheal rings to these agonists. Conclusions Whereas thiopental contracts human isolated bronchi, propofol and etomidate reduce histamine‐induced contraction in human isolated airway smooth muscle that were either not sensitized or passively sensitized with asthmatic serum. This effect involves inhibition of both electro‐ and pharmacomechanical coupling.

Role of Sarcoplasmic Reticulum and Mitochondria in Ca2+ Removal in Airway Myocytes

The aim of this study was to use both a theoretical and experimental approach to determine the influence of the sarco-endoplasmic Ca2+-ATPase (SERCA) activity and mitochondria Ca2+ uptake on Ca2+ homeostasis in airway myocytes. Experimental studies were performed on myocytes freshly isolated from rat trachea. [Ca2+]i was measured by microspectrofluorimetry using indo-1. Stimulation by caffeine for 30 s induced a concentration-graded response characterized by a transient peak followed by a progressive decay to a plateau phase. The decay phase was accelerated for 1-s stimulation, indicating ryanodine receptor closure. In Na2+-Ca2+-free medium containing 0.5 mM La3+, the [Ca2+]i response pattern was not modified, indicating no involvement of transplasmalemmal Ca2+ fluxes. The mathematical model describing the mechanism of Ca2+ handling upon RyR stimulation predicts that after Ca2+ release from the sarcoplasmic reticulum, the Ca2+ is first sequestrated by cytosolic proteins and mitochondria, and pumped back into the sarcoplasmic reticulum after a time delay. Experimentally, we showed that the [Ca2+]i decay after Ca2+ increase was not altered by the SERCA inhibitor cyclopiazonic acid, but was slightly but significantly modified by the mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. The experimental and theoretical results indicate that, although Ca2+ pumping back by SERCA is active, it is not primarily involved in [Ca2+]i decrease that is due, in part, to mitochondrial Ca2+ uptake.

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

BackgroundExtracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca2+]i signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.MethodsIsometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca2+]i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Students t tests for quantitative variables and χ2 tests for qualitative variables. Results were considered significant at P < 0.05.ResultsIn rat airways, extracellular ATP (10-6–10-3 M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca2+]i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca2+, external Na+ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.ConclusionExtracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca2+ influx in addition with, in IPB, P2Y receptors stimulation and Ca2+ release from intracellular Ca2+ stores. Extracellular Ca2+ influx occurs through L-type voltage-dependent channels activated by external Na+ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.

Contribution of Rho kinase to the early phase of the calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the role of Rho kinase (RhoK) in Ca2+–contraction coupling in rat airways. Isometric contraction was measured on tracheal, extrapulmonary and intrapulmonary bronchial rings. Intracellular [Ca2+] was recorded in freshly isolated tracheal myocytes. Stimulation by carbachol (0.3 and 10 μm) and 50 mm external KCl induced a short‐time, Hill‐shaped contraction obtained within 90 s, followed by a sustained or an additional delayed contraction. Responses of [Ca2+]i to acetylcholine consisted in a fast peak followed by a plateau and, in 42% of the cells, superimposed Ca2+ oscillations. The RhoK inhibitor Y27632 (10 μm) did not alter the [Ca2+]i response. Whatever the agonist, Y27632 did not modify the basal tension but decreased the amplitude of the short‐duration response, without altering the additional delayed contraction. The Myosin Light Chain Phosphatase (MLCP) inhibitor calyculin A increased the basal tension and abolished the effect of RhoK. KN93 (Ca2+–calmodulin‐dependent protein kinase II inhibitor) and DIDS (inhibitor of Ca2+‐activated Cl− channels) had no influence on the RhoK effect. We built a theoretical model of Ca2+‐dependent active/inactive RhoK ratio and subsequent RhoK‐dependent MLCP inactivation, which was further coupled with a four‐state model of the contractile apparatus and Ca2+‐dependent MLCK activation. The model explains the time course of the short‐duration contraction and the role of RhoK by Ca2+‐dependent activation of MLCK and RhoK, which inactivates MLCP. Oscillatory and non‐oscillatory [Ca2+]i responses result in a non‐oscillatory contraction, the amplitude of which is encoded by the plateau value and oscillation frequency. In conclusion, Ca2+‐dependent but CaMK II‐independent RhoK activation contributes to the early phase of the contractile response via MLCP inhibition.

Theoretical and experimental investigation of calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the Ca2+-contraction coupling in rat tracheal smooth muscle. [Ca2+]i, isometric contraction and myosin light chain (MLC) phosphorylation were measured in response to 1 mM carbachol. Theoretical modeling consisted in coupling a model of Ca2+-dependent MLC kinase (MLCK) activation with a four-state model of smooth muscle contractile apparatus. Stimulation resulted in a short-time contraction obtained within 1 min, followed by a long-time contraction up to the maximal force obtained in 30 min. ML-7 and Wortmannin (MLCK inhibitors) abolished the contraction. Chelerythrine (PKC inhibitor) did not change the short-time, but reduced the long-time contraction. [Ca2+i responses of isolated myocytes recorded during the first 90 s consisted in a fast peak, followed by a plateau phase and, in 28% of the cells, superimposed Ca2+ oscillations. MLC phosphorylation was maximal at 5 s and then decreased whereas isometric contraction followed a Hill-shaped curve. The model properly predicts the time course of MLC phosphorylation and force of the short-time response. With oscillating Ca2+ signal, the predicted force does not oscillate. According to the model, the amplitude of the plateau and the frequency of oscillations encode for the amplitude of force, whereas the peak encodes for force velocity. The long-time phase of the contraction, associated with a second increase in MLC phosphorylation, may be explained, at least partially, by MLC phosphatase (MLCP) inhibition, possibly via PKC inhibition.

Effect of engineered nanoparticles on vasomotor responses in rat intrapulmonary artery.

Pulmonary circulation could be one of the primary vascular targets of finest particles that can deeply penetrate into the lungs after inhalation. We investigated the effects of engineered nanoparticles on vasomotor responses of small intrapulmonary arteries using isometric tension measurements. Acute in vitro exposure to carbon nanoparticles (CNP) decreased, and in some case abolished, the vasomotor responses induced by several vasoactive agents, whereas acute exposure to titanium dioxide nanoparticles (TiO(2)NP) did not. This could be attributed to a decrease in the activity of those vasoactive agents (including PGF(2)(alpha), serotonin, endothelin-1 and acetylcholine), as suggested when they were exposed to CNP before being applied to arteries. Also, CNP decreased the contraction induced by 30 mM KCl, without decreasing its activity. After endoplasmic reticulum calcium stores depletion (by caffeine and thapsigargin), CaCl(2) addition induced a contraction, dependent on Store-Operated Calcium Channels that was not modified by acute CNP exposure. Further addition of 30 mM KCl elicited a contraction, originating from activation of Voltage-Operated Calcium Channels that was diminished by CNP. Contractile responses to PGF(2)(alpha) or KCl, and relaxation to acetylcholine were modified neither in pulmonary arteries exposed in vitro for prolonged time to CNP or TiO(2)NP, nor in those removed from rats intratracheally instilled with CNP or TiO(2)NP. In conclusion, prolonged in vitro or in vivo exposure to CNP or TiO(2)NP does not affect vasomotor responses of pulmonary arteries. However, acute exposure to CNP decreases contraction mediated by activation of Voltage-Operated, but not Store-Operated, Calcium Channels. Moreover, interaction of some vasoactive agents with CNP decreases their biological activity that might lead to misinterpretation of experimental data.

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

Abstract— 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 × 10−4M) and etomidate (10−4M) 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−1to 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.