Racemic salbutamol administration to guinea‐pigs selectively augments airway smooth muscle responsiveness to cholinoceptor agonists

1 An aim of this study was to investigate whether continuous in vivo administration of a low dose of salbutamol to guinea‐pigs alters the responsiveness of airway smooth muscle in vitro. 2 Osmotic minipumps containing a solution of racemic salbutamol were implanted subcutaneouly in guinea‐pigs. The drug was infused at a dose of 0.2 mg kg^?1 day^?1 for 10 days and, at the end of that time, the trachea was isolated and concentration–response relationships to several contractile agonists were examined. 3 This treatment resulted in significant increases in the maximum tension developed by tracheal preparations in response to cholinoceptor agonists, carbachol and methacholine. 4 Cumulative concentration–response curves for histamine, leukotriene D4, and KCl were similar in tracheal segments from saline‐control and salbutamol‐infused animals. 5 Time course experiments showed that augmented airway contractile responsiveness to cholinoceptor agonists was reversible within 3 days after cessation of the 10 day salbutamol infusion. 6 Our findings support the hypothesis that β₂‐adrenoceptor agonist drugs, administered over time in vivo, induce a transient hyperresponsiveness of airway smooth muscle to cholinergic bronchoconstrictor stimuli.     

Different immunological sensitizing regimens and airway responsiveness in vitro to contractile agonists in guinea-pigs

Groups of guinea-pigs were sensitized to ovalbumin, using different regimens to induce either IgG-like antibodies or IgE-like and IgG-like antibodies and the responsiveness to histamine and carbachol in vitro of tracheal and lung parenchymal strips was determined. EC50 values for histamine and carbachol in both tracheal and lung parenchymal strips were not significantly different for either group of sensitized guinea-pigs or their respective controls. The maximal tension developed in parenchymal strips was not significantly different for either sensitized or control groups. For tracheal strips, maximal tension was not significantly different in each group except in animals with IgG-like antibodies, which developed less maximal tension with histamine than controls. The absence of any increase in maximal tension or any change in EC50 to histamine or to carbachol in tracheal or parenchymal strips from immunized guinea-pigs indicates that immunological sensitization does not increase the responsiveness of smooth muscle to these contractile agonists.     

Ageing and epithelial integrity as modulators of airway smooth muscle responsiveness to endothelin-1

This study has examined the effects of animal age on the contractile responsiveness and inositol phosphate-generating capacities of guinea-pig (0-104 weeks) and rat (4-104 weeks) isolated tracheal smooth muscle in response to endothelin-1 (ET-1). The influence of animal age on the specific binding of [125I]ET-1 to guinea-pig and rat isolated tracheal tissue was also examined. The potency (pD2) of ET-1 was three to four times greater in tracheal tissue taken from 4-week-old rats than in similar tissue from 12- to 32-week-old animals, although maximum response (Emax) was not significantly altered. Neither pD2 nor Emax were influenced by ageing in epithelium-intact guinea-pig tracheal preparations. In contrast, removal of the airway epithelium significantly increased the contractile potency of ET-1 by two- to three-fold in tissue from animals of 6-20 weeks of age, but not in tissue from newborn animals. Significant falls in specific [125I]ET-1 grain density with ageing were demonstrated during the maturation phase in both species. In the rat, the decrease between 4 and 12 weeks was reflected in the fall in ET-1 potency at 12 weeks. However, the age-associated reduction in airway smooth muscle ET receptor number in the guinea-pig was not mirrored by significant changes in sensitivity to ET-1, suggesting the presence of a functional receptor reserve. ET-1 (1 nM) caused significant increases in intracellular inositol phosphates, with levels generally higher in rat than in guinea-pig trachea. ET-1-induced inositol phosphate accumulation decreased significantly with respect to animal age in both guinea-pig and rat isolated tracheal tissue. However, this was not correlated with changes in contractile pD2 or Emax. For example, in both rat and guinea-pig, the smallest ET-1-induced increases in intracellular inositol phosphates were measured in airway smooth muscle from the oldest animals tested, although tissue sensitivity to ET-1 was stable in both species after 12 weeks of age. These data suggest that relatively low levels of inositol phosphates were required to elicit Emax, consistent with the presence of more than one signal transduction process.     

The role of sodium pump in the inhibition of smooth muscle responsiveness to agonists during potassium restoration

1. Isometric contractions of cat splenic capsular smooth muscle in response to noradrenaline and histamine were recorded.

2. Removal of potassium from the bathing medium did not change the resting tension or the responsiveness to noradrenaline. Restoration of potassium inhibited responses to noradrenaline or histamine only if the muscles were stimulated with an agonist while in the K-free medium.

3. This inhibition of responses to the agonists due to potassium was reversed rapidly by removing the ion or reversed slowly by prolonged exposure to the ion. The inhibition was also blocked by procedures or agents which block the sodium pump (ouabain, substitution of NaCl by LiCl), inhibit active processes (low ambient temperature) or prevent intracellular accumulation of sodium (substitution of choline for sodium).

4. It is proposed that under special circumstances such as when there is an increase in internal sodium concentration, the sodium pump is probably electrogenic and causes relaxation when activated by external potassium. In the normal muscle the pump is probably electrically neutral.

     

Relationship of airway responsiveness to agents causing bronchoconstriction and cough in sensitized guinea-pigs

The relationship between airway responsiveness to bronchoconstrictor- and cough-inducing stimuli has been examined in Ascaris suum-sensitized conscious guinea-pigs. Guinea-pigs were sensitized to Ascaris suum [4000 PNU and 100 mg Al(OH)₃ i.p. on days 1 and 7] and then challenged with aerosolized antigen on days 21, 28 and 35. At day 35, antigen-exposure produced an early bronchoconstrictor response (EBR) and in about 50% of the animals also a late bronchoconstrictor response (LBR) commencing 4–8 h later. The bronchial responsiveness to inhaled histamine was increased in sensitized guinea-pigs and increased further 20–24 h after acute antigen challenge. Guinea-pigs developing only EBR were equally sensitive to histamine as those having both EBR and LBR. In contrast, the cough and reflex bronchoconstriction produced by inhaled citric acid (0.40 m, acting on capsaicin-sensitive sensory neurons) and cigarette smoke (3 min exposure; exciting both capsaicin-sensitive neurons and rapidly adapting stretch receptors) were not altered by sensitization. Furthermore, acute antigen challenge did not alter the effect of citric acid as measured 24 h later. The antigen-induced airway hyperresponsiveness to histamine was not accompanied by an altered sensitivity of airway sensory nerves mediating cough (and reflex bronchoconstriction), demonstrating that bronchial- (airway obstruction) and sensory- (cough) hyperresponsiveness involve separate and independent mechanisms.     

Tachyphylaxis to beta-adrenoceptor agonists in guinea pig airway smooth muscle in vivo and in vitro.

Beta-Adrenoceptor tachyphylaxis was induced by incubating spirally cut guinea pig tracheas with isoproterenol (2.4 x 10(-7) M) for 20 min. This incubation reduced the relaxant effects of catecholamines but not of dibutyryl cyclic AMP, theophylline or sodium nitrite. Tracheas incubated with norepinephrine, phosphodiesterase inhibitors or cyclic nucleotides became tachyphylactic to isoproterenol. Pretreatment with indomethacin prevented induction of tachyphylaxis. Incubation with adenosine, methoxamine or sodium nitrite did not induce beta-adrenoceptor tachyphylaxis. When we gave isoproterenol intramuscularly to guinea pigs, airway sensitivity to aerosolized histamine was unchanged but the toxicity of parenterally administered histamine was increased. A prolonged treatment with isoproterenol reduced airway sensitivity to histamine aerosols; this reduced sensitivity was reversed by indomethacin. Thus, beta-adrenoceptor tachyphylaxis may not explain increased toxicity of parenteral histamine after isoproterenol treatment. Elevated levels of cyclic AMP and an increased synthesis of prostaglandins may result in diminished response to beta-receptor stimulation.     

Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture.

1 beta 2-Adrenoceptor agonists may exacerbate asthma by reducing the release of the anti-proliferative and anti-inflammatory molecule, heparin from mast cells in the airway. In this study, the direct effects of the clinically used bronchodilator, salbutamol, on the proliferation of airway smooth muscle cells grown in culture and stimulated with a range of mitogens have been examined. 2 In mitogen-stimulated cells, salbutamol (0.1-100 nM) inhibited [3H]-thymidine incorporation in a concentration-dependent manner. Salbutamol (100 nM) pretreatment reduced the mitogenic responses to thrombin (0.3 u ml-1), epidermal growth factor (EGF) (300 pM) and U46619 (100 nM) by 61.7 +/- 6.1%, 46.9 +/- 13.9% and 57.6 +/- 12.7%, respectively. However, salbutamol pretreatment did not appear to reduce the small mitogenic response to endothelin-1. 3 Increases in [3H]-leucine incorporation in thrombin (0.3 u ml-1)-stimulated cells were reduced by salbutamol (100 nM) by 27.7 +/- 2.8%. Similarly, thrombin (0.3 u ml-1)-stimulated increases in cell number were also inhibited by salbutamol (100 nM) pretreatment. Thus, the effect of salbutamol in decreasing thrombin-induced [3H]-leucine incorporation may, at least in part, be explained by inhibition of cell proliferation. 4 The inhibition of cell proliferation by salbutamol was prevented by pretreatment with either the non-selective beta-adrenoceptor antagonist, propranolol (0.3 microM) or the selective beta 2-adrenoceptor antagonist, ICI 118551 (50 nM). 5 These results indicate that salbutamol, through activation of a beta 2-adrenoceptor, has a direct inhibitory effect on proliferation elicited by the mitogens thrombin, EGF, and U46619.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased response to GABA-B agonists in longitudinal smooth muscle-myenteric plexus preparations from morphine-tolerant guinea-pigs

1) Responsiveness of guinea-pig ileal longitudinal smooth muscle-myenteric plexus preparations to drugs activating GABA-B receptors was studied in morphine-tolerant animals. For this purpose morphine pellets (75 mg each) were implanted subcutaneously in guinea-pigs and experiments were performed three days later in electrically-stimulated ileal strips. 2) Activation of GABA-B receptors with GABA (10(-6) -10(-3) M) or (-)-baclofen (10(-6)-10(-3) M) caused a dose-related inhibition of twitch response that was about 80% lower in preparations from morphine-tolerant animals than in controls. This was found both in preparations maintained in the presence of morphine (10(-6) M) and in morphine-free Krebs. The effect was evident also in ileal preparations from morphine-tolerant animals in which a withdrawal syndrome was induced by the administration of naloxone before sacrifice. 3) The phenomenon was specific since the dose-response curve of the adenosine-inhibitory effect was comparable in preparations from tolerant animals and controls. 4) The hyporesponsiveness to GABA-B receptor activation began 12 h after pellet implantation and was maximal on the third day. 5) It is concluded that during tolerance to and withdrawal from morphine there is a hyporesponsiveness of GABA-B receptors in "in vitro" guinea-pig ileal longitudinal muscle-myenteric plexus preparations.     

Effects of adrenomedullin and calcitonin gene-related peptide on airway and pulmonary vascular smooth muscle in guinea-pigs.

1. The airway and pulmonary vascular effects of adrenomedullin were studied in the guinea-pig isolated trachea, main bronchi and pulmonary artery in vitro and compared to the effects of calcitonin gene-related peptide (CGRP). 2. In tracheal rings, CGRP (1 nM to 1 microM) potentiated the cholinergic contractions induced by electrical field stimulation (EFS) at 5 Hz in a concentration-dependent manner. At a concentration of 1 microM, CGRP slightly decreased the responses to log EFS frequency, producing 50% of the maximum contraction from a control value of 0.77 +/- 0.10 Hz to 0.54 +/- 0.05 Hz without a significant effect on the concentration-response curves to acetylcholine (ACh). In contrast, adrenomedullin (1 nM to 1 microM) did not alter either EFS-induced cholinergic or ACh-induced contractions. 3. In bronchial strips, CGRP (1 nM to 1 microM) slightly reduced both the non-adrenergic non-cholinergic (NANC) contraction induced by EFS at 10 Hz and the substance P (1 microM)-induced contraction in a concentration-dependent manner, whereas adrenomedullin (1 nM to 1 microM) was without effect. 4. Neither CGRP (1 microM) nor adrenomedullin (1 microM) altered NANC relaxation induced by EFS at 5 Hz in tracheal rings precontracted with histamine (10 microM). 5. Adrenomedullin (1 nM to 1 microM) and CGRP (1 nM to 1 microM) induced a concentration-dependent relaxation of the histamine (10 microM)- and prostaglandin F2 alpha (10 microM)-precontracted pulmonary arterial rings with intact endothelium with a similar potency. 6. Neither removal of the endothelium nor NG-nitro-L-arginine methyl ester (100 microM) altered the vasorelaxant effects of adrenomedullin (1 nM to 1 microM) and CGRP (1 nM to 1 microM). 7. The putative CGRP receptor antagonist, CGRP8-37 (1 microM to 10 microM) concentration-dependently attenuated the CGRP (3 nM to 30 nM)-induced vasorelaxant actions, whereas it had no effect on the relaxation of vessel rings induced by adrenomedullin (3 nM to 30 nM). 8. These results suggest that adrenomedullin is a potent vasodilator of the pulmonary artery without any bronchomotor effect in the guinea-pig lung, and that the vasorelaxant actions of adrenomedullin are not mediated via the activation of CGRP1 receptors.     

大黄素增强平滑肌细胞钙离子依赖的氯离子电流

本文利用全细胞膜片钳技术观察大黄素对大鼠近端结肠ClCa电流的直接作用,并探讨该作用的信号传导途径.     

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.     

Role of phosphoinositide metabolism in functional antagonism of airway smooth muscle contraction by beta-adrenoceptor agonists

Histamine and the muscarinic agonists, methacholine, oxotremorine, and McN-A-343, were used to contract guinea-pig tracheal smooth muscle preparations. Cumulative dose-relaxation curves with isoprenaline were performed subsequently. In addition, the concentration-dependent induction of phosphoinositide metabolism by the contractile agonists was measured in bovine tracheal smooth muscle. All agonists were found to induce a decrease of the apparent affinity of isoprenaline and a loss of relaxation, depending on the concentration and type of contractile agonist used. The differential effects of the contractile agonists, especially at higher and supramaximal concentrations, on these beta-adrenergic parameters could be explained by differences in phosphoinositide metabolism.     

The smooth muscle and airway hyperresponsiveness

Airway hyperresponsiveness, excessive airway narrowing caused by stimuli that normally elicit limited or no response, is one of the cardinal features of asthma. The length-dependence of smooth muscle contractility has been recognized for decades, and it forms an essential foundation for many aspects of the physiological regulation of airway contractility in vivo. This review summarizes the structural and functional alterations of airway smooth muscle in asthma and chronic obstructive pulmonary disease, that underlie pathophysiological conditions of airway hyperresponsiveness.     

Insulin induces airway smooth muscle contraction

BACKGROUND AND PURPOSE: Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. EXPERIMENTAL APPROACH: We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations. KEY RESULTS: It was found that insulin (1 nM-1 microM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 microM), Rho-kinase (Y-27632, 1 microM) and p42/44 MAP kinase (PD-98059, 30 microM and U-0126, 3 microM), but not of PI-3-kinase (LY-294002,10 microM). In addition, insulin significantly increased PGF(2alpha)-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 microM) and the EP(1)-receptor antagonist AH-6809 (10 microM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins. CONCLUSIONS AND IMPLICATIONS: Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.     

Pharmacology of airway smooth muscle proliferation

Airway smooth muscle thickening is a pathological feature that contributes significantly to airflow limitation and airway hyperresponsiveness in asthma. Ongoing research efforts aimed at identifying the mechanisms responsible for the increased airway smooth muscle mass have indicated that hyperplasia of airway smooth muscle, due in part to airway myocyte proliferation, is likely a major factor. Airway smooth muscle proliferation has been studied extensively in culture and in animal models of asthma, and these studies have revealed that a variety of receptors and mediators contributes to this response. This review aims to provide an overview of the receptors and mediators that control airway smooth muscle cell proliferation, with emphasis on the intracellular signalling mechanisms involved.     

Age-related changes in the reactivity of the rat jejunum to cholinoceptor agonists

The contractile effects of barium, potassium, acetylcholine and acetyl-beta-methylcholine were studied in jejunum of 4, 12 and 20 month-old rats. The effects of age on the sensitivity and responsiveness of the jejunum to the agonists were examined using cumulative dose administration and measuring the pD2. The maximum contraction produced by the agonists and the sensitivity to barium and potassium were not modified by aging. In the middle-aged animals, the jejunum sensitivity to acetylcholine decreased while the sensitivity to acetyl-beta-methylcholine was not statistically different from that of 4 month-old rats. Since at this age (12 months) the acetylcholinesterase activity increased, the reduction in the sensitivity to acetylcholine could have been due to the increase in the activity of the metabolizing enzyme. Otherwise, in the jejunum from the older animals the sensitivity for both cholinoceptor agonists, acetylcholine and acetyl-beta-methylcholine, increased 5-fold when compared with the middle-aged animals. However, compared with the jejunum of 4 month-old rats the sensitivity to acetylcholine, to 20 month-old animals, was not altered while the sensitivity to acetyl-beta-methylcholine was increased. At this age (20 months), the jejunum acetylcholinesterase activity was increased when compared with that of 4 month-old rats and was not modified when compared with that of 12 month-old rats. The change in supersensitivity to cholinoceptor agonists could not have been due to an alteration in muscarinic receptor affinity, since the pA2 for atropine was not modified with increasing age. These results suggest that after an increase in acetylcholinesterase activity around month 12, the jejunum develops an adaptative supersensitivity to cholinoceptor agonists.     

Evidence for a direct relationship between phosphoinositide metabolism and airway smooth muscle contraction induced by muscarinic agonists

The relationship between bovine tracheal muscle contraction and phosphoinositide metabolism was studied with the muscarinic agonists, methacholine, oxotremorine, and McN-A-343. Analysis of the dose-response curves for contraction and inositol phosphates accumulation with these agonists demonstrated a direct relationship between the two parameters, with a considerable reserve of inositol phosphate production for the full contractile agonists, methacholine and oxotremorine, and no reserve for the partial agonist, McN-A-343.     

beta(2)-adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells

1. Airway smooth muscle (ASM) is a potential source of multiple pro-inflammatory cytokines during airway inflammation. beta-Adrenoceptor agonist hyporesponsiveness is a characteristic feature of asthma, and interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha are implicated in its cause. Here, the capacity of beta-adrenoceptor agonists to prevent release of GM-CSF, RANTES, eotaxin and IL-8, elicited by IL-1 beta or TNF alpha, was examined in human ASM cells. 2. Isoprenaline (approximately EC(50) 150 nM), a non-selective beta-adrenoceptor agonist, and salbutamol ( approximately EC(50) 25 nM), a selective beta(2)-adrenoceptor agonist, attenuated release of GM-CSF, RANTES and eotaxin, but not IL-8 (EC(50) >1 microM). The maximum extent of attenuation was RANTES > or = eotaxin > GM-CSF > IL-8, and was prevented by either propranolol (1 microM), a non-selective beta-adrenoceptor antagonist, or ICI 118511 (IC(50) 15 nM), a selective beta(2)-adrenoceptor antagonist. 3. The cyclic AMP-elevating agents, dibutyryl cyclic AMP ( approximately EC(50) 135 microM), forskolin ( approximately EC(50) 530 nM) and cholera toxin ( approximately EC(50) 575 pg ml(-1)) abolished IL-1 beta-induced release of GM-CSF, RANTES and eotaxin, but not IL-8. 4. IL-1 beta (1 ng ml(-1)) attenuated early increases (up to 1 h) in cyclic AMP formation induced by salbutamol (1 microM), but not by forskolin (10 microM). The cyclo-oxygenase inhibitor, indomethacin (1 microM) prevented later increases (3 - 12 h) in IL-1 beta-stimulated cyclic AMP content, but did not prevent the attenuation by salbutamol of IL-1 beta-induced cytokine release. 5. We conclude in human ASM cells that activation of beta(2)-adrenoceptors and generation of cyclic AMP is negatively-linked to the release, elicited by IL-1 beta or TNF alpha, of eosinophil-activating cytokines such as GM-CSF, RANTES and eotaxin, but not IL-8.     

Kavain inhibits murine airway smooth muscle contraction

This study examined the effect of kavain, the principle biologically active component of kava, on murine airway smooth muscle. In isolated isometrically contracted tracheal ring preparations, kavain was noted to diminish the maximal contractile response to both muscarinic receptor activation and voltage-operated calcium channel activation. The IC50 for kavain in rings precontracted with carbachol was found to be 177 microM +/- 53.1, and, in rings precontracted with KCl, it was found to be 59.6 microM +/- 10.1. In addition, pretreatment with kavain attenuated airway smooth muscle contraction evoked with either carbachol or KCl. The EC50 for KCl was not affected by kavain pretreatment. However, the EC50 for carbachol was significantly affected by a high kavain pretreatment dose. Nitric oxide mediated relaxation was not observed to play a role in kavain's smooth muscle relaxing properties. Similarly, prostaglandin pathways are not likely involved in these effects since pretreatment of tracheal rings with indomethacin before carbachol contraction did not reduce the relaxant effect of kavain. The mechanism of kavain-induced relaxation and inhibition of contraction is likely due to a mechanism common to both contractile agonists that were employed in our study.