Caveolin-1 and force regulation in porcine airway smooth muscle

Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca(2+)](i)) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-β-cyclodextrin (CD) or 5 μM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 μM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 μM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 μM fasudil or 10 μM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca(2+) sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.     

Neurokinin-neurotrophin interactions in airway smooth muscle

Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca(2+) concentration ([Ca(2+)](i)) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression (P < 0.05). Furthermore, [Ca(2+)](i) responses to 1 μM ACh as well as BDNF (30 min) effects on [Ca(2+)](i) regulation were enhanced by prior SP exposure, largely via increased Ca(2+) influx (P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 μM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-F(c); 1 μg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects (P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca(2+)](i) (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca(2+)](i) regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders.     

IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to na?ve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.     

Inhibition of cholinergic neurotransmission in human airways by beta 2-adrenoceptors

The purpose of the study was to determine whether catecholamines modulate cholinergic neurotransmission in isolated human airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension, and contractions were induced by either electrical field stimulation (EFS) or exogenous acetylcholine (ACh). Isoproterenol, epinephrine, and norepinephrine in that order of potency produced concentration-dependent inhibition of comparable responses to EFS and ACh. However a potency difference of 100-fold for isoproterenol (IC50 = 4.80 X 10(-8) M for EFS and 3.70 X 10(-6) M for ACh) and 10-fold for both epinephrine and norepinephrine was observed for inhibition of responses to EFS compared with responses to ACh. The inhibitory effects of isoproterenol on responses to EFS were prevented by propranolol and ICI 118551 (a beta 2-antagonist) but not by betaxolol (a beta 1-antagonist). Tyramine had no effect on contractions elicited by EFS. These experiments demonstrate that beta-agonists inhibit cholinergic nerve-induced contractions of human bronchi more potently than contractions induced by exogenous ACh, suggesting modulation of cholinergic neurotransmission by prejunctional beta 2-receptors.     

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.     

Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.     

Mechanisms of protein kinase C regulation of airway contractility

To elucidate the role of protein kinase C (PK-C) in regulating airway contractility, the effects of PK-C activation with phorbol esters, 12-deoxyphorbol 13-isobutyrate (DPB), and phorbol 12-myristate 13-acetate (PMA), and with the diacylglycerol analogue 1-oleoyl-2-acetate-rac-glycerol (OAG) were separately evaluated in isolated rabbit tracheal smooth muscle (TSM) segments. The latter agents produced dual and opposing contractile effects, with DPB being the most potent. Lower doses of DPB (less than or equal to 10(-6) M) elicited significant increases in isometric tension in both untreated TSM, as well as in TSM half-maximally precontracted with methacholine. These potentiated TSM contractions were inhibited by the Ca2+ channel blockers, nifedipine (10(-4) M) and diltiazem (10(-5) M). In contrast, higher doses of DPB (greater than or equal to 10(-6) M) induced airway relaxation, which was ablated by preinhibition of the electrogenic Na+-K+ pump with ouabain (5 x 10(-6) M) or K+-free buffer. Indeed, in separate experiments DPB (10(-7) M) was found to significantly potentiate the functional activity of the Na+-K+ pump, an effect occurring independent of inhibition of Na+-H+ exchange with amiloride (10(-4) M) or extracellular Ca2+ influx with nifedipine (10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Responsiveness of the isolated airway during simulated deep inspirations: effect of airway smooth muscle stiffness and strain.

In vivo, breathing movements, including tidal and deep inspirations (DIs), exert a number of beneficial effects on respiratory system responsiveness in healthy humans that are diminished or lost in asthma, possibly as a result of reduced distension (strain) of airway smooth muscle (ASM). We used bronchial segments from pigs to assess airway responsiveness under static conditions and during simulated tidal volume oscillations with and without DI and to determine the roles of airway stiffness and ASM strain on responsiveness. To simulate airway dilations during breathing, we cycled the luminal volume of liquid-filled segments. Volume oscillations (15 cycles/min) were set so that, in relaxed airways, they produced a transmural pressure increase of approximately 5-10 cmH(2)O for tidal maneuvers and approximately 5-30 cmH(2)O for DIs. ACh dose-response curves (10(-7)-3 x 10(-3) M) were constructed under static and dynamic conditions, and maximal response and sensitivity were determined. Airway stiffness was measured from tidal trough-to-peak pressure and volume cycles. ASM strain produced by DI was estimated from luminal volume, airway length, and inner wall area. DIs produced substantial ( approximately 40-50%) dilation, reflected by a decrease in maximal response (P < 0.001) and sensitivity (P < 0.05). However, the magnitude of bronchodilation decreased significantly in proportion to airway stiffening caused by contractile activation and an associated reduction in ASM strain. Tidal oscillations, in comparison, had little effect on responsiveness. We conclude that DI regulates airway responsiveness at the airway level, but this is limited by airway stiffness due to reduced ASM strain.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

Inhibition of High-Affinity Choline Uptake and Acetylcholine Synthesis by Quinuclidinyl and Hemicholinium Derivatives

Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure-activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC-3, acetylsecohemicholinium, and HC-15 are potent inhibitors of HAChU, HC-3 being the most potent (I50 = 6.1 X 10(-8) M). In the present study, the most potent quinuclidinyl derivative was the N-methyl-3-quinuclidinone (I50 = 5.6 X 10(-7) M). This compound had approximately 100-fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3-hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N-functional group from a methyl to an allyl in the alcohol led to a 10-fold increase in activity. However, removal of the quaternizing N-methyl group yielding the tertiary amine, 3-quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2-benzylidene-3-quinuclidinone derivatives studied, only the m-chloro derivative significantly reduced HAChU.     

Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.     

Effect of cholecystokinin octapeptide and somatostatin on the motility of guinea pig and canine gallbladder

Species differences have been observed in the effect of cholecystokinin octapeptide (CCK OP) on the canine and guinea pig gallbladder smooth muscle motility. 1. CCK OP was more potent stimulant in canine than in guinea pig gallbladder smooth muscles. Its pD2 values were 10 and 9.2, respectively. 2. The acetylcholine (10(-4) M)-induced maximum contractions in canine gallbladder muscle strips were by 50% lower as compared to the CCK OP (10(-8) M) maximum responses while in guinea pig gallbladder muscle strips the acetylcholine (ACh) maximum responses were by 20% lower than the CCK OP maximum responses. 3. CCK OP increased [3H]ACh release by 27% in canine gallbladder and by 40% in guinea pig gallbladder. 4. Somatostatin (SOM) had not any direct myogenic effect in guinea pig and canine gallbladder but it decreased [3H]ACh release from gallbladder intrinsic cholinergic neurons.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

Nerve growth factor-enhanced airway responsiveness involves substance P in ferret intrinsic airway neurons

Nerve growth factor (NGF), a member of the neurotrophin family, enhances synthesis of neuropeptides in sensory and sympathetic neurons. The aim of this study was to examine the effect of NGF on airway responsiveness and determine whether these effects are mediated through synthesis and release of substance P (SP) from the intrinsic airway neurons. Ferrets were instilled intratracheally with NGF or saline. Tracheal smooth muscle contractility to methacholine and electrical field stimulation (EFS) was assessed in vitro. Contractions of isolated tracheal smooth muscle to EFS at 10 and 30 Hz were significantly increased in the NGF treatment group (10 Hz: 33.57 +/- 2.44%; 30 Hz: 40.12 +/- 2.78%) compared with the control group (10 Hz: 27.24 +/- 2.14%; 30 Hz: 33.33 +/- 2.31%). However, constrictive response to cholinergic agonist was not significantly altered between the NGF treatment group and the control group. The NGF-induced modulation of airway smooth muscle to EFS was maintained in tracheal segments cultured for 24 h, a procedure that causes a significant anatomic and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. The number of SP-containing neurons in longitudinal trunk and superficial muscular plexus and SP nerve fiber density in tracheal smooth muscle all increased significantly in cultured trachea treated with NGF. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the NGF-induced increased contraction to EFS in cultured segments but had no effect in saline controls. These results show that the NGF-enhanced airway smooth muscle contractile responses to EFS are mediated by the actions of SP released from intrinsic airway neurons.     

Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs.

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0-20 cmH(2)O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls (P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi (P < 0.01) and smooth muscle strips (P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.     

Neurochemical effects of the synthetic ACTH4-9-analog Hoe 427 (Ebiratide) in rat brain

The ACTH4-9-analog Hoe 427 systemically injected in a dose range from 0.01-10 micrograms/kg caused a fall in acetylcholine (ACh) content in different brain areas of the rat. This effect occurred 0.5 hour after a single administration and lasted up to 24 hours. The decrease in ACh content induced by Hoe 427 was more pronounced when the animals were pretreated with dexamethasone (over 7 days 1 mg/kg SC, daily). Coadministration of the choline uptake inhibitor hemicholinium-3 (HC-3) and Hoe 427 potentiated the decrease in ACh content induced by HC-3. In the same dose range Hoe 427 acutely evoked an increase of the activity of the enzyme choline acetyltransferase as well as an elevation of brain cyclic GMP content. These data indicate that Hoe 427 enhances ACh metabolism in rat brain after systemic administration.