Responses Of Tracheal Smooth Muscle Research Articles

Interleukin-1beta-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway.

Interleukin (IL)-1beta causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1beta. Ferrets were instilled intratracheally with IL-1beta (0.3 microg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1beta. The IL-1beta-induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1beta-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1beta. These results show that IL-1beta-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons.

Auxotonic Contractile Responses of Rat Tracheal and Bronchial Airway Smooth Muscle

The purposes of the present study were to directly compare: (1) the degree of trachealis muscle shortening and changes in tracheal dimensions and (2) ACh-mediated auxotonic contraction of trachea and intraparenchymal bronchi. The auxotonic contractile properties of tracheal and bronchial airway smooth muscle were assessed from 1–2 mm thick tracheal sections and 1 mm thick lung slices using videomicrometry in vitro at 37°C. Acetylcholine resulted in reductions in luminal area, perimeter, mean radius, length, and breadth (22.0, 10.0, 11.9, 10.7 and 12.0%, respectively). Trachealis muscle shortening reached a maximum of 39.8±4.3%. The K+channel blocker 4-aminopyridine significantly augmented the ACh-mediated reductions in tracheal luminal dimensions. In response to ACh (10−3m), reductions in bronchial dimensions were significantly greater than those of the trachea for luminal area, perimeter and mean radius (44.6 vs. 18.6, 32.0 vs. 8.0 and 28.9 vs. 9.9%, respectively). These data indicate that auxotonic contractile responses of rat tracheal smooth muscle differ from those previously reported in the dog and guinea pig, that ACh-mediated auxotonic contraction of tracheal smooth muscle is augmented by 4-aminopyridine, and that proportionate reductions in luminal dimensions in response to ACh are considerably greater for bronchial than tracheal airways.

Tumor necrosis factor-α augments contraction and cytosolic Ca 2+ sensitivity through phospholipase A 2 in bovine tracheal smooth muscle

To elucidate the effects of tumor necrosis factor-α (TNF-α) on tracheal smooth muscle contraction, we simultaneously measured isometric tension and intracellular Ca 2+ ([Ca 2+] i) in fura 2-loaded muscle strips. Smooth muscle force generation was evaluated in a high potassium (K +; 20.0–80.0 mM) solution and with acetylcholine (3 nM–10 μM ). TNF-α (1–100 ng/ml) did not directly contract muscle strips. The contractile response to acetylcholine was enhanced after application of 10 ng/ml of TNF-α for 30 min but not the response of [Ca 2+] i. The contractile response and the response of [Ca 2+] i to a high K + solution were not altered after application of TNF-α. The [Ca 2+] i–tension curve indicated that TNF-α enhanced the responsiveness of tracheal smooth muscle through the acetylcholine-mediated Ca 2+ sensitivity of intracellular contractile elements. The augmentation of the acetylcholine concentration–response curves for muscle tension in the presence of TNF-α (10 ng/ml) was inhibited in part after application of manoalide, a phospholipase A 2 inhibitor. We conclude that a low concentration of TNF-α enhances smooth muscle responsiveness to acetylcholine by agonist-mediated Ca 2+ sensitivity facilitated by phospholipase A 2.

BCG infection during pre-sensitization or even post-sensitization inhibits airway sensitivity in an animal model of allergic asthma.

The objective of this study is to investigate whether BCG infection before, during or after sensitization suppresses allergen-induced airway hyperresponsiveness and eosinophilic inflammation in allergic asthma rats, and to determine the required dose of BCG to induce such an inhibition. Eighty-seven Sprague-Dawley (SD) rats were sensitized and provoked with ovalbumin (OA). A pretreatment of 6 x 10(4) or 6 x 10(5) colony forming units (CFUs) of BCG or saline was done at four different times: 3 days before sensitization, at sensitization, 3 days before provocation, or at provocation. The assessment of tracheal smooth muscle (TSM) responsiveness to electrical field stimulation or acetylcholine (ACh) and bronchoalveolar lavage (BAL) were performed 1 day after OA provocation. Doses of 6 x 10(4) CFUs inhibited TSM sensitivity of rats infected 3 days before sensitization or at sensitization, but not 3 days before provocation or at provocation. However, doses of 6 x 10(5) CFUs significantly inhibited not only the airway eosinophilia of rats infected 3 days before sensitization or at sensitization, but also the TSM sensitivity of rats infected 3 days before provocation or at provocation. In conclusion, BCG infection suppresses the development of sensitivity of airway smooth muscle and airway eosinophilic inflammation in allergic asthma rats. Furthermore, a relatively high dose of BCG infection inhibits airway sensitivity, even after allergen sensitization.

Animal Models of Airway Sensitization

AbstractAirway inflammation and hyperresponsiveness (AHR) are hallmarks and distinguishing features of bronchial asthma. This unit contains protocols that employ a mouse model for the study of allergen‐induced AHR. It includes methods for sensitizing mice to allergens via the airways and assessing tracheal smooth muscle reactivity in vitro and in vivo. Protocols to measure and evaluate immunological mechanisms that contribute to the development to allergen‐induced asthma are also provided.

Mucolytics and antioxidant activity

We investigated effects of the mucolytics ambroxol and N-acetylcysteine on airways reactivity evoked by histamine in guinea pigs exposed to toluene vapors. We did not find significant changes in reactivity of tracheal smooth muscle from animals treated with mucolytics compared to the control group. However, the administration of ambroxol and N-acetylcysteine caused a significant decrease in lung tissue reactivity. The effect of ambroxol was more pronounced after intraperitoneal injection than after inhalation, while N-acetylcysteine was only effective after inhalation. The protective effects of mucolytics in the lung tissue may be due to their antioxidant activity together with other mechanisms.

Development of eosinophilic airway inflammation and airway hyperresponsiveness requires interleukin-5 but not immunoglobulin E or B lymphocytes.

We previously defined a role for B cells and allergen-specific immunoglobulins in the development of allergic sensitization, airway inflammation, and airway hyperresponsiveness (AHR), using a 10-d protocol in which allergen exposure occurred exclusively via the airways, without adjuvant. In the present protocol, normal and B-cell-deficient (microMt(-/-)) mice were sensitized intraperitoneally to ovalbumin (OVA) and challenged with OVA via the airways in order to examine the requirements for AHR with this protocol. T-cell activation (antigen-specific proliferative responses and Th2-type cytokine production) and eosinophil infiltration in the peribronchial regions of the airways, with signs of eosinophil activation and degranulation, occurred in both experimental groups. In contrast to the 10-d protocol, increased in vivo airway responsiveness to methacholine and in vitro tracheal smooth-muscle responses to electrical field stimulation were observed in both normal and B-cell-deficient mice, and these responses were inhibited by anti-interleukin (IL)-5 administration before airway challenge. These data show that IL-5, but not B cells or allergen-specific IgE, are required for eosinophil airway infiltration and the development of AHR following allergen/alum sensitization and repeated airway challenge with allergen. These results emphasize that the use of different sensitization and challenge protocols can influence the requirements for development of AHR.

Beta(2)-adrenoceptor agonist-induced upregulation of tachykinin NK(2) receptor expression and function in airway smooth muscle.

Neurokinin A (NKA) induces bronchoconstriction mediated by tachykinin NK(2) receptors in animals and humans, and may be increased in asthma. Because beta(2)-adrenoceptor agonists are the most widely used bronchodilators in asthma, we investigated the effects of the beta(2)-adrenoceptor agonist fenoterol on NK(2) receptor messenger RNA (mRNA) and receptor density as well as the functional responses of bovine tracheal smooth muscle to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10) in vitro, using Northern blot analysis, receptor binding, and organ bath studies. Incubation with fenoterol induced a time- and concentration-dependent upregulation of NK(2) receptor mRNA (71% increase after 12 h at 10(-7) M fenoterol), which was abolished by propranolol (a nonselective beta-adrenoceptor agonist) and ICI118551 (a selective beta(2)-adrenoceptor antagonist), but not by CGP20712A (a selective beta(1)-adrenoceptor antagonist), indicating that fenoterol acts via beta(2)-adrenoceptors. These effects were mimicked by forskolin and prostaglandin E(2) (PGE(2)), both agents that increase cyclic adenosine monophosphate (cAMP), and by the cAMP analogue 8-bromo-cAMP. The upregulation was blocked by cycloheximide, indicating that it requires new protein synthesis, and was accompanied by an increase in both the stability of NK(2) receptor mRNA and the rate of NK(2) receptor gene transcription. Radioligand binding assay using the selective NK(2) receptor antagonist [(3)H]SR48968 showed a significant increase in the number of receptor binding sites after 12 h and 18 h, which was accompanied by an increased contractile responsiveness to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10). Dexamethasone completely prevented the fenoterol-induced increase in NK(2) receptor mRNA and in the contractile response. We conclude that beta(2)-adrenoceptor agonists induce upregulation of functional NK(2) receptors in airway smooth muscle by increasing cAMP, and that this can be prevented by a corticosteroid. The increased responsiveness could be relevant to asthma control and mortality.

Moxonidine acting centrally inhibits airway reflex responses.

We examined the role of I1-imidazoline (I1-IR) receptors in control of airway function, by testing the effects of systemic administration of the I1-IR agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either alpha-chloralose anesthetized or decorticate, paralyzed, and mechanically ventilated beagle dogs. Moxonidine (10-100 micrograms/kg) administered via three different routes (femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways and caused a decrease in arterial pressure and heart rate. These effects were dose dependent and were significantly reversed by efaroxan (an I1-IR and alpha 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10-100 micrograms/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1-100 micrograms/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I1-IR sites and alpha 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

Subchronic Exposure to Diisocyanates Increases Guinea Pig Tracheal Smooth Muscle Responses to Acetylcholine

Objective: In order to study the threshold concentrations of isocyanates (IC) for induction of lung disorders, constrictive responses of tracheal smooth muscles to acetylcholine (ACH) in guinea pigs with and without diisocyanate [toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI)] exposure were investigated. Methods: An IC-induced increase in smooth muscle responsiveness was studied by measuring cumulative ACH dose responses (10^–10 to 10^–4 M ACH). Basal ACH dose-response curves, measured twice in intervals of 1 h using tracheal preparations of 11 guinea pigs previously not exposed to IC, were reproducible. Results: Subchronic in vivo exposures to TDI, HDI, and MDI atmospheres of 10 and 20 parts per billion (ppb) on 5 consecutive days led to significantly (p < 0.05) increased ACH responsiveness of tracheal smooth muscle, whereas concentrations of 2.5 and 5 ppb were not effective. Exposure to HDI atmospheres of 10 ppb for 1, 2, 4, or 8 weeks resulted in a time-dependent increase in ACH responses (p < 0.05) of guinea pig tracheal smooth muscle. Increased tracheal muscle responses to ACH were transient since tracheal preparations from animals exposed to 10 and 20 ppb MDI for 4 weeks and with an exposure-free interval of 8 weeks before preparation did not show enlarged ACH responses, which were present in preparations at the end of the exposure period (p < 0.05). Exposure to low IC concentrations as present in workplaces cause increased ACH responsiveness of guinea pig tracheal smooth muscle. The increased responsiveness of the airways seems to be largely reversible, since normal responses were found after 8 weeks of IC avoidance. Conclusion: Reversibility of IC-induced airway hyperresponsiveness is of great occupational and preventive medical importance. Workers with acquired airway hyperresponsiveness might escape lung damage if the changes are detected in an early stage before alterations in lung function are in a chronic stage.

I 1-imidazoline receptors and cholinergic outflow to the airways

We examined the role of I 1-imidazoline receptors in the control of airway function, by testing the effects of systemic administration of the I 1-imidazoline agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either α-chloralose anaesthetized or decorticate, paralyzed and mechanically ventilated beagle dogs. Moxonidine (10–100 μg/kg) administered via three different routes (the femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways, and caused a decrease in arterial pressure and heart rate. These effects were dose-dependent, and were significantly reversed by efaroxan (an I 1-imidazoline and α 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10–100 μg/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1–100 μg/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I 1-imidazoline sites and α 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

Human respiratory syncytial virus produces prolonged alterations of neural control in airways of developing ferrets.

A dysfunction of pathways that normally cause contraction or relaxation of airways has been proposed to explain heightened levels of responsiveness produced by various insults to the airway. For example, we previously reported (4) that infection of cotton rats with the human respiratory syncytial virus (HRSV) leads to a significant decrease in an airway's nonadrenergic noncholinergic inhibitory (NANCi) response shortly after the infection. In the present study we addressed the more chronic effects of HRSV infection on airway function in young ferrets during a period of rapid somatic growth. Animals 1 wk old received HRSV or uninfected cell culture medium intranasally. In vitro studies of airway function were performed on tracheal smooth muscle (TSM) segments at 4, 8, and 24 wk of age. To evaluate neurally mediated contractile responses, frequency-response curves to electrical field stimulation (EFS) were performed with results expressed in terms of the frequency causing 50% of the maximal contractile response (ES50). In addition, contractile responses of TSM to methacholine (MCh) were also assessed with results expressed as the concentration needed to produce 50% of the maximal contractile response (EC50). To gauge NANCi responses, TSM was contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. Relaxant responses to EFS were assessed at frequencies from 5 to 30 Hz, with results expressed as mean percent relaxation. We found increased contractile responses to EFS in infected animals compared with that in the control group in both 4- and 8-wk old animals (p = 0.001 and p = 0.008, respectively). This difference had resolved by 24 wk of age. There was no difference in TSM responses to MCh between the groups at any age. Although there were no NANCi responses in 4-wk-old ferrets from either group, NANCi responses were significantly decreased in 8-wk-old ferrets previously infected with HRSV in the first week of life (p = 0.0001). A significant difference persisted (p = 0.008), albeit to a lesser degree, at 24 wk of age. These findings demonstrate that HRSV produces prolonged alterations of TSM function in ferret airways in vitro.

Differential effects of cyclosporine A after acute antigen challenge in sensitized cats in vivo and ex vivo.

1. We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (R(L)) in vivo, and tracheal smooth muscle (TSM) contraction ex vivo after antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen-induced responses of TSM in vitro. 2. Cats (n=10) were sensitized by i.m. injection of Ascaris suum antigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin-2 secretion from feline peripheral blood mononuclear cells ex vivo. 3. Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542+/-74 pg ml(-1) post AA for CsA+ and from 74+/-19 pg ml(-1) at baseline, to 970+/-180 pg ml(-1) post AA CsA- (P<0.05; P=NS vs CsA+). 4. In excised TSM, active tension elicited by exposure to AA in vitro was 107+/-38% KCl in the CsA+ group vs 144+/-56% KCl in the CsA- group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre-incubated with 1 microM CsA in vitro (8+/-8% KCl, P<0.05 vs CsA+ and CsA-). This was associated with inhibited release of 5-hydroxytryptamine into the organ bath fluid of tissues treated with CsA in vitro only. 5. We demonstrated that CsA treatment in vivo does not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell function ex vivo do not reflect lack of effects of CsA on mast cell function in vivo in this animal model of atopic asthma.

ANTICHOLINESTERASE DRUGS STIMULATE CONTRACTILE RESPONSE OF RAT TRACHEAL RINGS

S501 Neostigmine, physostigmine and pyridostigmine stimulate, while edrophonium does not affect phosphatidylinositol (PI) response in the airway smooth muscle [1]. However, it is not fully clarified whether these drugs stimulate contractile response of airway smooth muscle. We examined the effects of these drugs on contractile response in the airway smooth muscle. METHODS: The studies were conducted under guidelines approved by the Animal Care Committee. Sixteen Male Wistar rats weighing 250-350 g were anesthetized with 50 mg/kg intraperitoneal pentobarbital and the trachea was isolated. The trachea was chopped into 3-mm-wide ring segments with McIlwain tissue chopper. The tracheal rings were suspended between stainless hooks and the resting tension was adjusted to 1.5 g. (1) Ring contraction was induced by stepwise cumulative additions of anticholinesterase (anti-ChE) drugs from 0.1 [micro sign]M to 100 [micro sign]M at final concentrations. (2) To examine whether anti-ChE drugs act directly on muscarinic receptors in airway smooth muscle, we determined the effects of tetrodotoxin (TTX) on neostigmine-induced contraction of rat tracheal rings. Statistical significance (P < 0.05) was determined using ANOVA. RESULTS: Results are displayed on Figure 1. Neostigmine caused the contraction at a dose of 1 [micro sign]M or greater, and pyridostigmine did at 10 [micro sign]M or greater, whereas edrophonium had only minimal effect on the contraction. Blockade of neural transmission by TTX had no effect on tracheal smooth muscle response to neostigmine.Figure 1: Effects of anticholinesterase drugs on the contractile response of rat tracheal rings (mean +/- SE; n = 6-7). * P < 0.05, *** P < 0.001 vs 0.CONCLUSIONS: The data suggest that anti-ChE drugs have different effects on the airway smooth muscle contractraction, and that the contraction would be mediated with the stimulation of PI response through the direct activation by anti-ChE drugs of muscarinic receptors.

Airway responsiveness to acetylcholine in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs in vivo and in vitro.

The characteristics of airway responsiveness to acetylcholine (ACh) in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were clarified in vivo and in vitro. We measured the change in ventilatory mechanics in response to ACh inhalation by means of the bodyplethysmograph and the contractile responses of isolated trachea to ACh and carbachol (CCh). Further, muscarinic receptor subtypes involved these responses were identified. The basal values for ventilatory mechanics in BHS were not significantly different from those in BHR. Respiratory resistance to ACh was progressively increased in a time- and dose-dependent manner in BHS. The contractile responses of tracheal smooth muscle to ACh in BHS were significantly greater than those in BHR, but CCh-induced responses in BHS and BHR were similar. ACh- and CCh-induced contractions were mediated via M3 receptors. These results suggested that the falling-down of BHS in response to ACh inhalation was caused by the strong constriction of the airway and the reduction in ventilation. Moreover, the airway hyperresponsiveness to ACh in BHS might be partly dependent on the change in acetylcholinesterase activity.

The role of prostanoids in ozone-induced changes in airway responsiveness: receptor activation-specific prostanoid release

We studied the effect of in vivo ozone exposure (3 ppm, 2 h) on methacholine- and histamine-induced guinea pig tracheal smooth muscle contractions in vitro and the role of cyclooxygenase products in this process. After exposure to ozone, methacholine stimulation showed a functional hyperreactivity, whereas after stimulation with histamine a hyporeactivity was observed. These effects could be explained by the release of prostanoids. In a control situation an increase in PGF 2 α , PGE 2 and PGD 2 release is observed after stimulation of the histaminergic receptor system. After ozone exposure the release of prostanoids was also enhanced (unstimulated, PGF 2 α and TxB 2; histamine, PGF 2 α , PGE 2; methacholine, PGF 2 α , TxB 2, 6-kPGF 1 α , PGE 2). This study shows that the prostanoid release is strongly dependent on the receptor system stimulated to induce smooth muscle contraction and the importance of prostanoids in ozone-induced changes in guinea pig tracheal smooth muscle reactivity.

The role of excitatory amino acids in airway reflex responses in anesthetized dogs

In these studies we examined the role of excitatory amino acids (EAAs) neurotransmission in communicating sensory inputs to the airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPA/kainate receptor blockade on reflex and chemical responses of tracheal smooth muscle. Experiments were performed in chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs ( n=18), under hyperoxic, normocapnic, and normohydric conditions. Topical application or microinjection of NMDA receptor blockers, into the region of the ventrolateral medulla where airway-related vagal preganglionic neurons are located, insignificantly decreased the reflex changes in tracheal tone. However, topical application or microinjection of AMPA/kainate subtype of glutamate receptor selective antagonists markedly reduced reflex increase in tracheal tone induced by (1) lung deflation, (2) stimulation of laryngeal cold receptors, and (3) activation of peripheral or central chemoreceptors. These effects were potentiated by prior NMDA receptor blockade. Findings indicate that an increase in central cholinergic outflow to the airways by a variety of excitatory afferent inputs is mediated via activation of EAA receptors, mainly AMPA/kainate subtype of glutamate receptors.

Nitric oxide inhibits ACh-induced intracellular calcium oscillations in porcine tracheal smooth muscle.

With real-time confocal microscopy, the effect of three nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and diethylamine NO adduct, on the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh) was examined. ACh initiated propagating [Ca2+]i oscillations in TSM cells, which were inhibited by NO donors. 8-Bromoguanosine 3',5'-cyclic monophosphate slowed the frequency of [Ca2+]i oscillations but did not completely inhibit oscillations, suggesting that the effects of NO donors are only partially mediated via guanosine 3',5'-cyclic monophosphate-dependent mechanisms. After preexposure to NO donors, ACh induced a small biphasic [Ca2+]i response that was blocked by nifedipine, suggesting a lack of effect on Ca2+ influx through voltage-gated channels. In addition, NO donors did not inhibit Ca2+ influx induced by BAY K 8644. The [Ca2+]i response to caffeine was inhibited by NO donors, indicating inhibition of sarcoplasmic reticulum (SR) Ca2+ release. When Ca2+ influx and SR Ca2+ reuptake were blocked, basal [Ca2+]i increased, and this was inhibited by NO donors, suggesting enhanced Ca2+ efflux. These results indicate that NO donors inhibit [Ca2+]i oscillations by blocking SR Ca2+ release and enhancing Ca2+ extrusion.

Effect of aspiration of milk on mechanisms of neural control in the airways of developing rabbits.

We studied the effects of recurrent aspiration of milk on neural control of airways in young developing rabbits. Beginning at 1 week of age, rabbits received 0.5 ml/kg of whole milk or sterile physiologic saline intranasally while under light methoxyflourane anesthesia 5 days a week for a period of 3 weeks. At 4 and 8 weeks of age, in vitro studies of contractile and relaxant responses of tracheal smooth muscle (TSM) segments were evaluated. To assess the neurally mediated contractile responses, frequency response curves to electrical field stimulation (EFS) were performed with results expressed in terms of frequency of EFS causing 50% of the maximal contractile response (ES50) values. In addition, the contractile responsiveness of TSM to methacholine (MCh) as reflected by the concentration causing 50% of the maximal contractile response (EC50) values was also determined to evaluate the underlying cholinergic reactivity of this segment of airway. To assess nonadrenergic noncholinergic inhibitory (NANCi) responses, experiments were performed on TSM contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. EFS was delivered to the contracted tissue at stimulation frequencies ranging from 5 to 30 Hz with results expressed as mean percent relaxation. Recurrent aspiration of milk but not saline increased EFS-induced contractile responses, as shown by significantly lower ES50 values compared with the control group: P = 0.02 and P = 0.001 at 4 and 8 weeks of age, respectively. TSM responsiveness to MCh was no different between the two groups, suggesting that alterations in prejunctional mechanisms of neural control were most likely responsible for the increased contractile response to EFS. The NANCi responses were significantly decreased by milk aspiration at both 4 and 8 weeks of age, with the abnormalities less pronounced at the later time point. These findings demonstrate that repeated aspiration of milk leads to abnormal mechanisms of neural control within airways of developing rabbits. While aspiration of milk altered both contractile and relaxant responses to EFS, the former abnormalities became more pronounced with time while the latter appeared to be resolving. These observations suggest that injury to an airway early in development does not necessarily resolve with time but may persist, with functional abnormalities becoming more pronounced even after the airway insult has ceased.

Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells.

Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.