Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms.

Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.     

Adrenoceptors in airway smooth muscle

This review examines the roles and functional significance of alpha and beta-adrenoceptor subtypes in airway smooth muscle, with emphasis on human airway function and the influence of asthma. Specifically, we have examined the distribution of beta-adrenoceptors in lung and the influence of age, the epithelium, respiratory viruses and inflammation associated with asthma on airway smooth muscle beta-adrenoceptor function. Sites of action, beta 2-selectivity, efficacy and tolerance are also examined in relation to the use of beta 2-agonists in man. In addition, alpha-adrenoceptor function in airway smooth muscle has been reviewed, with some emphasis on comparing observations made in airway smooth muscle with those in animal models.     

Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness.

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.     

Synergistic stimulation of airway smooth muscle cell mitogenesis

Previous studies showed that human airway smooth muscle (HASM) cells treated with lysophosphatidic acid (LPA), a pertussis toxin (PTX)-sensitive G protein-coupled (GPC) mitogen, simultaneously with epidermal growth factor (EGF), a receptor tyrosine kinase (RTK) mitogen, exhibit markedly synergistic stimulation of mitogenesis. We now show that the RTK mitogens basic fibroblast growth factor, insulin-like growth factor-1, insulin, platelet-derived growth factor-AA, and platelet-derived growth factor-BB, as well as transforming growth factor-beta, all induced synergistic stimulation of mitogenesis in the presence of LPA. The PTX-sensitive GPC mitogens carbachol and endothelin-1 and the PTX-insensitive GPC mitogens sphingosine-1-phosphate and thrombin exhibited synergistic stimulation together with EGF. Several RTK-RTK growth factor pairs and GPC-GPC mitogen pairs were also synergistic. HASM cells showed synergistic responses to serum plus EGF but not to serum plus LPA. Testing various other cell types showed that synergism between LPA and EGF occurred in other smooth muscle cells because both vascular smooth muscle cells and mesangial cells exhibited synergism. Additionally, human fetal lung fibroblasts also showed striking synergism. These results indicate that HASM cells can respond synergistically to a wide variety of mitogen combinations and that this synergism is a feature shared with other contractile cell types.     

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.     

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF-(alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.     

Antispasmodic effects of eugenol on rat airway smooth muscle

This study was undertaken to assess the effects of eugenol (EUG) on tracheal muscle (TM) and the putative mechanisms underlying these effects. Cumulatively increasing concentrations (1–1000 μm ) of EUG did not affect the resting tonus of TM. However, EUG (1–2000 μm ) reduced the contractions induced by electrical field stimulation (IC₅₀ = 842.3 ± 52.7 μm ), an effect that was unaltered by either 10 μm montelukast (IC₅₀ = 816.1 ± 70.1 μm ) or 2 μm indomethacin (IC₅₀ = 693.1 ± 170.8 μm ). EUG also completely relaxed the sustained contractile responses to 80 mM K⁺ (IC₅₀ = 597.3 ± 60.6 μm ) and 1 μm carbamoylcholine (IC₅₀ = 571.3 ± 148.8 μm ), an effect that was unaltered by indomethacin (2 μm ). Under Ca²⁺‐free conditions, EUG reduced the ACh‐induced contractions (IC₅₀ = 703.4 ± 256.1 μm ), the CaCl₂‐induced contractions in preparations pretreated with 60 μm ACh in the presence of nifedipine, and the Ba²⁺‐induced contractions in preparations depolarized with K⁺. In tracheal preparations maintained in Ca²⁺‐containing solution, EUG (300–2000 μm ) relaxed the contractile response to phorbol dibutyrate (1 μm ), an activator of protein kinase C. It is concluded that in TM, EUG induces a myogenic antispasmodic effect (not modulated by arachidonic acid derivatives) either through various mechanisms almost with the same pharmacological potency or via an action on a step common to all of them. These mechanisms seem to include blockade of voltage‐ and receptor‐operated Ca²⁺ channels, IP₃‐induced Ca²⁺ release from sarcoplasmic reticulum and reduction of the sensitivity of contractile proteins to Ca²⁺.     

Effects of ergometrine on airway smooth muscle contractile responses

A 26-year-old asthmatic female developed severe asthma within a few hours of receiving three oral doses of 0.4 mg ergometrine maleate for the control of postpartum haemorrhaging. This experience and two previous reports of bronchospasm in asthmatic subjects following ergometrine suggested that ergometrine altered airway smooth muscle tone. In the present investigation the effect of ergometrine was studied on canine tracheal smooth muscle strips. Ergometrine (10(-9) M-10(-4) M) induced contraction of canine tracheal smooth muscle. The concentration causing 50% of maximal contraction (EC50) was 4.73 X 10(-8) M. The acetylcholine EC50 was not altered by ergometrine (10(-9) M or 10(-8) M); however, acetylcholine (10(-4) M and 10(-3) M) induced contractions were enhanced by ergometrine (10(-8) M). The data suggest that ergometrine maleate may cause broncho-constriction in some patients with asthma.     

Maturational regulation of inositol 1,4,5-trisphosphate metabolism in rabbit airway smooth muscle.

Airway reactivity has been shown to vary with age; however, the mechanism(s) underlying this process remain unidentified. To elucidate the role of ontogenetic changes in phosphoinositide-linked signal transduction, we examined whether age-related differences in tracheal smooth muscle (TSM) contractility to carbachol (CCh) are associated with developmental changes in the production and metabolism of the second messenger, inositol 1,4,5-trisphosphate (Ins (1,4,5)P3). In TSM segments isolated from 2-wk-old and adult rabbits, both the maximal isometric contractile force and sensitivity (i.e., -logED50) to CCh (10(-10)-10(-4) M) were significantly greater in the immature vs. adult tissues (P less than 0.001). Similarly, Ins(1,4,5)P3 accumulation elicited by either receptor-coupled stimulation with CCh (10(-10)-10(-4) M) or post-receptor-mediated guanine nucleotide binding protein activation of permeabilized TSM with GTP gamma S (100 microM) was also significantly enhanced in 2-wk-old vs. adult TSM. Measurement of the activities of the degradative enzymes for Ins(1,4,5)P3 demonstrated that: (a) mean +/- SE maximal Ins(1,4,5)P3 3'-kinase activity was significantly reduced in the immature vs. adult TSM (i.e., approximately 71.7 +/- 6.0 vs. 137.8 +/- 10.0 pmol/min per mg protein, respectively; P less than 0.005); (b) by contrast, maximal Ins(1,4,5)P3 5'-phosphatase activity was significantly increased in the immature vs. adult TSM (i.e., 27.9 +/- 1.2 vs. 15.6 +/- 1.5 nmol/min per mg protein, respectively; P less than 0.001); and (c) the Km values for Ins(1,4,5)P3 5'-phosphatase were 14- and 19-fold greater than those for Ins(1,4,5)P3 3'-kinase in the 2-wk-old and adult TSM, respectively. Collectively, the findings suggest that the age-related decrease in agonist-induced rabbit TSM contractility is associated with a diminution in Ins(1,4,5)P3 accumulation which is attributed, at least in part, to ontogenetic changes in the relative activities of the degradative enzymes for Ins(1,4,5)P3.     

M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle.

Classification of muscarinic receptors in the central airways has revealed the coexistence of M2 and M3 muscarinic receptors in this tissue, with the M2 subtype being predominant. Although M3 muscarinic receptors have been linked to airway smooth muscle contraction, a functional role for the M2 subtype in this tissue has been unclear. In nonairway smooth muscle, stimulation of the M2 muscarinic receptor has been shown to be associated with inhibition of adenylyl cyclase. In the present study, characterization of muscarinic receptors in canine tracheal smooth muscle confirmed that the majority of these muscarinic receptors were of the M2 subtype (89 +/- 3%), with a minor population of M3 receptors (11 +/- 3%). In functional studies, both isoproterenol and forskolin cause a dose-dependent relaxation of precontracted airway smooth muscle. In tissues precontracted with methacholine, 11-([[2-(diethylamino)methyl]-1-piperidinyl]acetyl)5,11- dihydro-6H-pyrido[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 antagonist, shifted dose-response curves to both isoproterenol and forskolin significantly to the left. In contrast, AF-DX 116 did not alter relaxation induced by the K+ channel opener BRL 38227. Furthermore, the ability of AF-DX 116 to enhance isoproterenol-induced relaxation appears to be limited to smooth muscle precontracted with muscarinic agonists because AF-DX 116 had no effect on isoproterenol dose-response curves in muscle strips precontracted with histamine. Hexahydrosiladifenidol (HHSiD), a selective antagonist for M3 receptors, did not shift the isoproterenol dose-response curve in muscle precontracted with methacholine. This study demonstrates that stimulation of M2 muscarinic receptors in canine airway smooth muscle plays an important role in functional antagonism by reducing the relaxation caused by agents such as isoproterenol and forskolin.     

Peripheral type benzodiazepine receptor and airway smooth muscle relaxation

It has been postulated that a benzodiazepine receptor with a micromolar affinity may be associated with Ca++ channels in peripheral organs. We examined the actions of Ro5-4684 (parachlorodiazepam) and midazolam on guinea pig tracheal smooth muscle contraction. Binding studies using [3H]Ro5-4684 indicate the presence of a "peripheral" type binding site with a Kd of approximately 4 nM and maximum binding of 1 pmol/mg of protein. Midazolam did not displace radioligand. In tension studies no activity was seen for Ro5-4684 or midazolam at concentrations below 1 microM. Higher concentrations relaxed the airway smooth muscle under basal tone, the effect was augmented significantly by epithelium removal. Similar results were obtained in tissues precontracted with methacholine or KCl. Midazolam (1 or 100 microM) significantly (P less than .05) attenuated the response to Ca++ in K+-depolarized tracheal strips, the effect was greater at low Ca++ concentrations. The compounds appear to function as Ca++ antagonists in airway smooth muscle but ar not typical as shown by their ability to reduce basal tone in airway smooth muscle.     

Epithelial factors: modulation of the airway smooth muscle tone

Summary— The airway epithelium is composed of a heterogeneous population of cells. This epithelial layer is not only a physical barrier but also a target responding to a variety of inflammatory mediators. These cells can respond by releasing contracting and relaxing factors to modulate airway responsiveness. They can also metabolize some of the inflammatory mediators. Epithelial damage is a consistent feature of some respiratory conditions, but whether or not such damage contributes to airway disease is for the moment unknown. This review summarizes the literature on the known and proposed roles of the epithelium in the modulation of the airway smooth muscle tone.     

Beta-2 adrenergic responses to tulobuterol in airway smooth muscle, vascular smooth muscle and adrenergic nerves

Experiments were designed to determine the mechanism of action of the bronchodilator drug tulobuterol. Tissues were suspended in organ chambers for isometric tension recording. Tulobuterol caused concentration-dependent relaxations of guinea pig tracheae, canine saphenous veins and canine bronchi; the compound relaxed canine coronary arteries only at high concentrations and did not affect spontaneously beating guinea pig atria. A metabolite of tulobuterol, 4-hydroxytulobuterol, was more potent in relaxing guinea pig tracheae than tulobuterol, salbutamol and isoproterenol. Other metabolites (3-hydroxy-, 5-hydroxy- and 4,5-dihydroxytulobuterol) were less efficacious than 4-hydroxytulobuterol. Both tulobuterol and 4-hydroxytulobuterol acted as partial agonists. The effects of tulobuterol in the saphenous vein (but not in the coronary artery) were antagonized by the selective beta-2 adrenergic blocker ICI 118,551 but were not affected by the selective beta-1 adrenergic inhibitor metoprolol. In bronchi, removal of the epithelium reduced the relaxations caused by tulobuterol. The drug did not inhibit responses of canine bronchi to electrical stimulation of the cholinergic nerves more than those to exogenous acetylcholine. Tulobuterol caused a moderate augmentation of the evoked release of [3H]norepinephrine in canine saphenous veins previously incubated with the labeled transmitter. Thus, tulobuterol is a selective beta-2 adrenergic agonist with minimal nonselective inhibitory effect on airway and vascular smooth muscle. It also facilitates adrenergic neurotransmission, which may help to explain its bronchodilator effect in the intact organism. Tulobuterol does not activate beta-1 adrenoceptors and has no direct positive chronotropic effect. A metabolite of tulobuterol, 4-hydroxytulobuterol, is more active than the parent compound.     

The responsiveness of airway smooth muscle in vitro from dogs with airway hyper-responsiveness in vivo

To determine whether smooth muscle from airways made hyper-responsive by ozone exposure is hyper-responsive in vitro, tracheal smooth muscle strips taken from five dogs with airway hyper-responsiveness induced by ozone exposure were compared with strips from five control animals. On 1 day, airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus pulmonary resistance. On a second day, five dogs were exposed to ozone (3.0 p.p.m. for 2 h) and five were exposed to filtered air. Then airway responsiveness to acetylcholine was reassessed. All dogs were then killed, the trachea was rapidly removed and strips of smooth muscle were prepared from the central one-third of the trachea. The responsiveness of each strip to electrical field stimulation (contractions inhibitable by atropine and tetrodotoxin) and exogenous acetylcholine was assessed after 2 and 6 h of incubation and washing. Ozone caused a marked increase in responsiveness in vivo to acetylcholine with a fall in mean provocation concentration from 0.15 g % to 0.026 g % (P less than 0.001) while sham exposure had no effect. The responsiveness of muscle strips to electrical field stimulation in ozone-exposed dogs after 2 h of incubation and washing was increased when compared with 6 h of incubation and washing and with the control dogs (P less than 0.05 for EF50, the frequency of stimulation giving 50% maximum contraction). However, responsiveness to exogenous acetylcholine was similar in all strips from both ozone-exposed and control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sodium-transport inhibitors on airway smooth muscle contractility in vitro

1. To determine whether alterations in membrane sodium transport in airway smooth muscle can alter its contractility, we studied the effect of ouabain (a Na+/K(+)-adenosine triphosphatase inhibitor) and amiloride on contractile responses in bovine trachea and human bronchial rings in a series of studies. 2. Ouabain (10(-6)-10(-4) mol/l) caused concentration-related contraction of bovine trachea with a maximum effect at 30 min; the mean increases in tension with 10(-6), 10(-5) and 10(-4) mol/l ouabain were 19, 27, and 32%, respectively, of the maximum response seen with 10(-3) mol/l histamine (n = 6). In human bronchial rings, ouabain (10(-5) mol/l) caused a mean contraction which was 40% of the maximum response to methacholine (n = 8). 3. Calcium-free fluid (plus ethylenediaminetetraacetic acid) and nifedipine (10(-5) mol/l) inhibited ouabain-induced contractions, suggesting that contraction was mediated in part by calcium entry via voltage-dependent calcium channels. Phentolamine (10(-5) mol/l) was without effect. 4. Ouabain (10(-5) mol/l) did not alter histamine responsiveness in bovine trachea or methacholine responsiveness in human bronchial rings. 5. Amiloride did not affect resting tone in bovine trachea but caused a concentration-dependent relaxation of bovine tracheal strips preconstricted with carbachol, 10(-3) mol/l amiloride relaxing strips completely over 15 minutes (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)