In vitro effect of air pollutants on human bronchi

A useful approach for constructing dose–response relationships and for studying the underlying mechanisms by which a xenobiotic agent enhances airway reactivity is to measure the response of an isolated airway following ex vivo exposure to a pollutant. We have in this way determined the dose–response relationship between ex vivo exposure to pollutants such as nitrogen dioxide (NO₂), the aldehyde acrolein, and ozone (O₃) and the reactivity to agonists in human isolated bronchial smooth muscle. We have also investigated the underlying alteration in the cellular mechanisms of airway smooth-muscle contraction induced by such exposure and found that it is related to alteration in calcium signaling at the site of the airway smooth-muscle cell. Finally, although there is epidemiological evidence that an increase in allergic diseases such as asthma may be linked to air pollution, there are few experimental data to address this issue. The final aim of this study was therefore to investigate the interaction between passive sensitization and exposure to pollutants in human isolated airways. We have examined (i) the effect of a pre-exposure to pollutants on the contraction of sensitized bronchi in response to a specific antigen and (ii) the effect of passive sensitization on the contraction in response to nonspecific agonists in bronchi pre-exposed to pollutants. The results indicate a combined effect of immunological sensitization and exposure to pollutants; that is, passive sensitization and exposure to pollutants act in a synergistic manner on human bronchial smooth-muscle reactivity in response to both specific antigens and nonspecific agonists. This revised version was published online in August 2006 with corrections to the Cover Date.     

Reaction of the lung to air pollutant exposure

Urban air pollutants, when administered to laboratory animals, adversely affect pulmonary structure and function. Effects include decrease in airway caliber, changes in lung elasticity, degeneration or destruction of tissue, and impaired pulmonary defense mechanisms. Urban dwellers subjected to pollutant exposures might incur similar health risks. The substances reviewed are: a) reducing pollutants — SO₂ and associated particulates and b) oxidizing (or photochemical) pollutants — NO₂ and O₃.     

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

Background

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.

Methodology/Principal Findings

By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves.

Conclusions

Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.     

Neutral endopeptidase modulates endothelin-1-induced airway smooth muscle contraction in guinea-pig trachea.

This study was designed to evaluate the role of neutral endopeptidase (NEP) in modulating the airway smooth muscle contraction induced by endothelin-1 in isolated segments of guinea-pig trachea. Endothelin-1 (10(-9)-10(-6) M) produced a concentration-dependent contraction that reached a maximum by 30 min. The NEP inhibitor leucine-thiorphan (10(-5) M) significantly increased the contractile response to endothelin-1. The addition of leucine-thiorphan to tracheal segments precontracted by 10(-9) and 10(-8) M endothelin-1 increased isometric tension by 181 +/- 65% (mean +/- 1 S.E.M.; P less than 0.05) and by 138 +/- 49% (P less than 0.05), respectively. In contrast, the kininase II inhibitor captopril and the peptidase inhibitors leupeptin and bestatin had no effect. Preincubation of endothelin-1 with 1 microgram recombinant human NEP decreased the contractile activity of endothelin-1 by 72 +/- 9%, whereas no effect was observed using heat-inactivated NEP. We conclude that NEP modulates endothelin-induced contraction of airway smooth muscle in the guinea-pig trachea.     

Combined effect of chronic hypoxia and in vitro exposure to gas pollutants on airway reactivity

This study investigated the interaction between exposure to air pollutants and chronic hypoxia (CH). We used a hypobaric chamber (14 days at barometric pressure 380 mmHg) to produce CH in rats. Exposure to various doses of acrolein or ozone did not modify the mechanical response to cholinergic agonists. Exposure to 3 microM/min acrolein did not alter epithelium-free trachea responsiveness. In contrast, direct exposure of freshly isolated myocytes to 2 and 3 microM/min acrolein enhanced the amplitude of the first intracellular [Ca(2+)] rise in response to 0.1 microM ACh and the calcium oscillation frequency in response to 10 microM ACh. CH alone did not alter smooth muscle cross-sectional area (SMA) or epithelium-plus-submucosa thickness. CH decreased maximal contractile response (maximal force normalized to SMA) but increased sensitivity (pEC(50)) to cholinergic agonists. We conclude that unlike in normoxic rats, exposure to air pollutants does not induce airway hyperresponsiveness in CH rats, although it increased calcium signaling. These results cannot be explained by change in smooth muscle accessibility, but may be linked to the effect of CH on calcium-contraction coupling.     

Interest and limits of in vitro studies in renal vascular endocrinology

Various in vitro preparations have been utilized to study the cellular activity of vasoactive agents on renal cortical microvessels and on mesangial cells. The receptors and the transduction pathways of bradykinin and atrial natriuretic factor were characterized on cultured cortical vascular smooth muscle cells from the rabbit kidney. A preparation of afferent arterioles that had been freshly isolated from the rat kidney was used to study the NO-dependent regulation of renin release. The influence of endothelin and angiotensin II on mesangial cell proliferation was evaluated, using cocultures of human endothelial and mesangial cells. These appropriate in vitro preparations have provided new insights on renal vascular endocrinology. However, extrapolation of in vitro data to in vivo physiology must be cautious because the phenotype of vascular cells often changes in culture conditions.Abbreviations ANF atrial natriuretic factor - BK bradykinin - CNP C-type natriuretic peptide - ET-1 endothelin-1 - HUVEC human umbilical vein endothelial cells - IBMX isobutylmethylxanthine - NEP neutral endopeptidase - PKA protein kinase A - RCVSMC renal cortical vascular smoothmuscle cells     

ETB-type receptors mediate endothelin-stimulated contraction in the aortic vascular smooth muscle of the spiny dogfish shark,Squalus acanthias

Using various agonists, and the specific antagonist BQ-123, we have examined the sensitivity to endothelin of the vascular smooth muscle of the ventral aorta of the spiny dogfish shark, Squalus acanthias. Human endothelin-1 produced significant contraction of isolated vascular smooth muscle rings, with an EC₅₀ of 10 nmol·1^-1. The presence of an intact endothelium did not alter this response but the magnitude of the contraction was greater in rings with an intact endothelium. The response to 0.2 mol·1^-1 endothelin-1 was equivalent to that of 0.1 mmol·1^-1 acetylcholine, and significantly greater than that to 80 mmol·1^-1 KCl, suggesting high sensitivity even to the heterologous, mammalian peptide. The Hill plot of the contractile response was a straight line with a slope of 1.12, indicating that a single receptor was mediating the response. Endothelin-1, endothelin-3, and sarafotoxin S6c produced similar concentration-response curves, and the response to endothelin-1 was insensitive to the ET_A-specific inhibitor BQ-123. Our data are consistent with the hypothesis that the receptor involved in the contractile response to endothelin of shark aortic vascular smooth muscle is of the ET_B-rather than the ET_A-type.Abbreviations ACh acetylcholine - ANP atrial natriuretic peptide - CA celiacomesenterie artery - CRC concentration response curve - DMSO dimethylsulphoxide - ET endothelin - STX sarafotoxin - VSM vascular smooth muscle - EDCF endothelium derived contraction factor     

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca²⁺/calmodulin-dependent myosin light chain kinase, Ca²⁺-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K⁺-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.     

Neutral endopeptidase inhibitor potentiates endothelin-1-induced airway smooth muscle contraction.

To study the role of neutral endopeptidase (NEP) on endothelin-1-induced contraction of the airway smooth muscle, we examined the contractile effect of endothelin-1 in the isolated guinea pig trachea and human bronchus in the presence or absence of NEP inhibitor phosphoramidon. After incubation with phosphoramidon (10(-8) to 10(-5) M), we added endothelin-1 cumulatively from 10(-11) to 10(-7) M to the airway tissues in organ baths. Phosphoramidon significantly potentiated the endothelin-1-induced contraction in a concentration-dependent fashion in both guinea pig trachea and human bronchus, and it shifted the concentration-response curves to the left. Because NEP is known to cleave tachykinins, we next studied whether endothelin-1 contracts airway tissues by releasing endogenous tachykinins from bronchial C-fibers. After incubation with phosphoramidon (10(-5) M), we added endothelin-1 cumulatively from 10(-11) to 10(-7) M to the tissues that were treated with capsaicin to deplete the tachykinins. Phosphoramidon significantly potentiated the endothelin-1-induced contraction in the capsaicin-treated tissues, suggesting that endothelin-1 causes the contraction, at least in part, without releasing tachykinins. In contrast to the effect of phosphoramidon, captopril (an angiotensin-converting enzyme inhibitor), leupeptin (a serine protease inhibitor), and bestatin (an aminopeptidase inhibitor) did not modulate the effect of endothelin-1-induced contraction in both guinea pig trachea and human bronchus. From these results, we conclude that NEP plays an important role in regulating endothelin-1-induced contraction in the guinea pig trachea and human bronchus.     

Design,synthesis and biological evaluation of 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one derivatives as potent β2-adrenoceptor agonists

A series of β₂-adrenoceptor agonists with an 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one moiety is presented. The stimulatory effects of the compounds on human β₂-adrenoceptor and β₁-adrenoceptor were characterized by a cell-based assay. Their smooth muscle relaxant activities were tested on isolated guinea pig trachea. Most of the compounds were found to be potent and selective agonists of the β₂-adrenoceptor. One of the compounds, (R)-18c, possessed a strong β₂-adrenoceptor agonistic effect with an EC₅₀ value of 24 pM. It produced a full and potent airway smooth muscle relaxant effect same as olodaterol. Its onset of action was 3.5 min and its duration of action was more than 12 h in an in vitro guinea pig trachea model of bronchodilation. These results suggest that (R)-18c is a potential candidate for long-acting β₂-AR agonists.     

Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways.     

Comparison of Functional β-Adrenoceptor Heterogeneity in Central and Peripheral Airway Smooth Muscle of Guinea Pig and Man

Abstract

The nature of the β-adrenoceptor population(s) mediating the relaxation of guinea pig and human airway smooth muscle was investigated. On the basis of a preferential blockade by β₁ and β₂ selective antagonists of the relaxation induced by β₁ and β₂- selective agonists, guinea pig tracheal strip relaxation was found to be mediated both by β₁ - and β₂ - adrenoceptors, the relative participation of which depending on the relative affinities of the agonist towards these two receptors. With highly selective antagonists the noradrenaline(NA)-induced relaxation could be split up biphasically into a β₁- and a β₂ - component. In contrast, no such differential blockade was observed with the guinea pig lung parenchyma strip relaxation which is mediated by a homogenous β₂ -adreno-ceptor population. On comparison of the tracheal, the spirally cut main bronchus- and intrapulmonary airway smooth muscle strips it could be shown that both the sensitivity of NA for neuronal uptake and the apparent affinity of the relaxation by NA decreased in the direction of the lung periphery. Using the same techniques it was ascertained that the relaxation of human tracheal smooth muscle (autopsy, obtained within 6 hours after death), main bronchus and intrapulmonary smooth muscle (operation) are mediated by homogenous β₂ -adrenoceptor populations. In addition, neuronal and extraneuron-al uptake sites were not operative in these preparations, whether obtained from operation or from autopsy.     

Viral infection increases contractile but not secretory responses to substance P in ferret trachea.

Viral infection increases the airway smooth muscle response to substance P. This effect is due to decreased activity of neutral endopeptidase (EC 3.4.24.11), an enzyme that degrades substance P. Inhibition of neutral endopeptidase activity also potentiates substance P-induced 35SO4-labeled macromolecule secretion. Therefore we examined the in vitro effects of substance P on 35SO4-macromolecule secretion from the tracheae of influenza-infected ferrets. Despite a virus-induced loss of neutral endopeptidase activity (demonstrated in muscle bath experiments), there was no difference between control and infected tracheae in either baseline secretion [697 +/- 125 vs. 579 +/- 67 (SE) cpm/15 min; n = 15 tissues) or in the response to 10(-6) M substance P (increased by 218 +/- 63 and 195 +/- 51, respectively) or 10(-5) M substance P (increased by 416 +/- 95 and 354 +/- 54, respectively). Although phosphoramidon (10(-6) M) potentiated the secretory response to substance P, there was again no difference between control and infected tracheae. These data show that although viral infection decreases airway neutral endopeptidase activity, virus-induced hypersecretion is not due to a resulting increase in the secretory response to substance P.     

Inhibition of photosynthesis ofPopulus euramericana andHelianthus annuus by SO2, NO2 and O3

Populus euramericana cv. I-214 andHelianthus annuus L. cv. Russian Mammoth were exposed to various concentrations of O₃ SO₂ or NO₂ for 2 h in a cylindrical assimilation chamber. The threshold concentrations of air pollutants for inhibition of net photosynthesis differed between the two species and also between the pollutants tested. Furthermore, the lethal concentrations where the net photosynthetic rates were completely inhibited, also differed between species and between pollutants. For SO₂ and NO₂,P. euramericana was more tolerant photosynthetically thanH. annuus when related to the concentration of pollutants used during the experiment. However, when related to the cumulative uptake rate of each pollutant, the photosynthetic tolerance of the two species was similar. In contrast to the effects of SO₂ or NO₂, the influence of O₃ on net photosynthesis was quite different. The relative rates of net photosynthesis in both species showed the same linear relationship with O₃ concentration. However, the relationship between the relative rate of net photosynthesis and the cumulative uptake rate of O₃ differed between the two species, although it was linear in both cases.     

Neutral endopeptidase modulates neurotensin-induced airway contraction

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.     

Targeting the restricted α-subunit repertoire of airway smooth muscle GABAA receptors augments airway smooth muscle relaxation

The prevalence of asthma has taken on pandemic proportions. Since this disease predisposes patients to severe acute airway constriction, novel mechanisms capable of promoting airway smooth muscle relaxation would be clinically valuable. We have recently demonstrated that activation of endogenous airway smooth muscle GABA(A) receptors potentiates β-adrenoceptor-mediated relaxation, and molecular analysis of airway smooth muscle reveals that the α-subunit component of these GABA(A) receptors is limited to the α(4)- and α(5)-subunits. We questioned whether ligands with selective affinity for these GABA(A) receptors could promote relaxation of airway smooth muscle. RT-PCR analysis of GABA(A) receptor subunits was performed on RNA isolated by laser capture microdissection from human and guinea pig airway smooth muscle. Membrane potential and chloride-mediated current were measured in response to GABA(A) subunit-selective agonists in cultured human airway smooth muscle cells. Functional relaxation of precontracted guinea pig tracheal rings was assessed in the absence and presence of the α(4)-subunit-selective GABA(A) receptor agonists: gaboxadol, taurine, and a novel 8-methoxy imidazobenzodiazepine (CM-D-45). Only messenger RNA encoding the α(4)- and α(5)-GABA(A) receptor subunits was identified in RNA isolated by laser capture dissection from guinea pig and human airway smooth muscle tissues. Activation of airway smooth muscle GABA(A) receptors with agonists selective for these subunits resulted in appropriate membrane potential changes and chloride currents and promoted relaxation of airway smooth muscle. In conclusion, selective subunit targeting of endogenous airway smooth muscle-specific GABA(A) receptors may represent a novel therapeutic option for patients in severe bronchospasm.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

Leukotriene B4 potentiates airway muscle responsiveness and

We studied the effects of leukotriene B₄ (LTB₄) on guinea pig airway muscle responsiveness and . Responsiveness in vivo was assessed by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in 5 unanesthetized, spontaneously breathing guinea pigs. We found that aerosolized LTB₄, in a concentration that itself had no effect on baseline SRaw, caused a substantial increase in bronchial reactivity to i.v. ACh within 8 min of its administration. Responsiveness was assessed by measuring isometric contraction of the guinea pig trachealis upon stimulation by either chemical or electrical field stimuli. These studies showed that a concentration of LTB₄ that itself did not cause contraction, potentiated airway muscle contraction to ACh and KCl, but not to norepinephrine. This effect of LTB₄ was substantially reduced by nifedinpine. Our data suggests that amounts of LTB₄ that are themselves non-contractile or in , may directly potentiate the responsiveness of airway smooth muscle to other bronchoconstrictors.     

Effect of Parenchymal Stiffness on Canine Airway Size with Lung Inflation

Although airway patency is partially maintained by parenchymal tethering, this structural support is often ignored in many discussions of asthma. However, agonists that induce smooth muscle contraction also stiffen the parenchyma, so such parenchymal stiffening may serve as a defense mechanism to prevent airway narrowing or closure. To quantify this effect, specifically how changes in parenchymal stiffness alter airway size at different levels of lung inflation, in the present study, we devised a method to separate the effect of parenchymal stiffening from that of direct airway narrowing. Six anesthetized dogs were studied under four conditions: baseline, after whole lung aerosol histamine challenge, after local airway histamine challenge, and after complete relaxation of the airways. In each of these conditions, we used High resolution Computed Tomography to measure airway size and lung volume at five different airway pressures (0, 12, 25, 32, and 45 cm H₂O). Parenchymal stiffening had a protective effect on airway narrowing, a fact that may be important in the airway response to deep inspiration in asthma. When the parenchyma was stiffened by whole lung aerosol histamine challenge, at every lung volume above FRC, the airways were larger than when they were directly challenged with histamine to the same initial constriction. These results show for the first time that a stiff parenchyma per se minimizes the airway narrowing that occurs with histamine challenge at any lung volume. Thus in clinical asthma, it is not simply increased airway smooth muscle contraction, but perhaps a lack of homogeneous parenchymal stiffening that contributes to the symptomatic airway hyperresponsiveness.     

Tracheal smooth muscle responses to substance P and neurokinin A in the piglet.

The tachykinins substance P (SP) and neurokinin A (NKA) have been shown to induce airway smooth muscle contraction in mature animals, and the enzyme neutral endopeptidase (NEP) modulates this effect. We evaluated maturation of SP- and NKA-induced tracheal smooth muscle contraction and modulation of their effects by NEP in anesthetized, paralyzed, and artificially ventilated piglets less than 4 days, 2-3 wk, and 10 wk of age. Tracheal smooth muscle tension was measured in vivo from an open tracheal segment by use of a force transducer. Intravenous SP caused a dose-dependent increase in tracheal tension in all three age groups; however, the response in less than 4-day-old piglets was significantly weaker than in 2- to 3- and 10-wk-old piglets. NKA caused a dose-dependent increase in tracheal tension only in 2- to 3- and 10-wk-old piglets. The response of tracheal tension to NKA was weaker than the response to SP in all age groups. Atropine (2 mg/kg) significantly diminished the responses of tracheal tension to SP and NKA, indicating a cholinergic contribution to these responses at all ages. Intravenous thiorphan, a known NEP inhibitor, potentiated the effects of SP only in 2- to 3- and 10-wk-old piglets and did not affect the response of tracheal tension to NKA at any age. Biochemical analyses demonstrated a significant increase in tracheal NEP activity in comparably aged piglets over the first 10 wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)