Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

RANTES release by human airway smooth muscle: effects of prostaglandin E(2) and fenoterol.

In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers.

Most of the studies linking chronic obstructive pulmonary disease (COPD) with oxidative stress are in vitro, using invasive techniques, or measuring systemic oxidative stress. The aim of this study was to quantify oxidative stress in the lungs in patients with COPD and in healthy smokers, as reflected by 8-isoprostane concentrations in breath condensate. This is a noninvasive method to collect airway secretions. 8-Isoprostane is a prostaglandin-F(2alpha) isomer that is formed in vivo by free radical-catalyzed peroxidation of arachidonic acid. We also studied the acute effect of smoking on exhaled 8-isoprostane in healthy smokers. Exhaled 8-isoprostane was measured by a specific enzyme immunoassay in 10 healthy nonsmokers and 12 smokers, 25 COPD ex-smokers, and 15 COPD current smokers. 8-Isoprostane concentrations were similar in COPD ex-smokers (40 +/- 3.1 pg/ml) and current smokers (45 +/- 3.6 pg/ ml) and were increased about 1.8-fold compared with healthy smokers (24 +/- 2.6 pg/ml, p < 0.001), who had 2.2-fold higher 8-isoprostane than healthy nonsmokers (10.8 +/- 0.8 pg/ml, p < 0.05). Smoking caused an acute increase in exhaled 8-isoprostane by about 50%. Our study shows that free radical production is increased in patients with COPD and that smoking causes an acute increase in oxidative stress.

Role of peptidase and cyclooxygenase inhibitors in the guinea-pig bronchial response to the synthetic endothelin ET(B) agonist IRL 1620 and antagonist BQ-788.

In isolated guinea-pig bronchial preparations the selective endothelin ETB agonist, IRL 1620 caused a concentration-dependent contraction. The pD2 value (7.16 +/- 0.09, n = 6) was significantly increased in the presence of peptidase inhibitors (thiorfan 1 microM, captopril 1 microM, bestatin 1 microM) (pD2 = 7.75 +/- 0.09, n = 6). Indomethacin (5 microM) did not appear to influence the ETB-agonist pD2 value (6.92 + 0.11, n = 6) but potentiated its maximal response significantly (67.23 +/- 4.81% vs. 53.37 +/- 4.80%). The concentration-response curve for the contractile response to IRL 1620 (pD2=7.83 +/- 0.01, n=16); was reproducible, although not completely, since the second curve to this selective ETB agonist was shifted significantly to the right (pD2 = 7.34 +/- 0.09, n = 16) and a decrease in the maximal response was observed (20.0 +/- 2.0%). BQ 788, a selective antagonist for ETB receptors, employed in concentrations ranging from 1.5 to 150 nM, caused a dose-dependent shift to the right of the concentration-response curve to IRL 1620, with a pIC50 value of 8.11 +/- 0.03; this action was not influenced by adding enzyme inhibitors (pIC50 = 8.17 +/- 0.29). Our data show that IRL 1620 undergoes a hydrolytic metabolism in guinea-pig bronchial preparations, which could influence the calculation of the pD2. Pretreatment of the tissue with peptidase inhibitors and indomethacin is consequently significant in the evaluation of IRL 1620 activity, while it does not influence the action of the antagonist, BQ 788.

Suc-[Glu9,Ala11,15]-endothelin-1 (8-21), IRL 1620, identifies two populations of ET(B) receptors in guinea-pig bronchus.

The pharmacological properties of endothelin receptors (ETR) were investigated in guinea-pig bronchus by comparing binding and functional results. In binding assays, both the ET(B) agonists, endothelin-3 (ET-3) and N-suc-[Glu9,Ala11,15]ET-1(8-21) (IRL 1620), and the antagonist, N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D- 1-methoxycarbonyltryptophanyl-D-norleucine (BQ 788), showed biphasic inhibition curves of [125I]-endothelin-1 (ET-1) binding to bronchus membranes prepared from intact or epithelium-deprived tissue. IRL 1620 did not completely displace specifically [125I]-ET-1 bound to these tissue preparations. In the presence of the ET(A)-selective antagonist, cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu) (BQ 123, 1 microM), IRL 1620 displacement curves were shallow but a complete inhibition was reached at a concentration of 1 microM. Both curves were better represented by two-site models. In addition, BQ 788 competition curves became monophasic when binding experiments were performed in the presence of 1 microM BQ 123. The non-selective agonist, ET-1, and BQ 123 inhibited [125I]-ET binding to bronchus membranes in dose-dependent fashions with monophasic curves. The contracting activity of IRL 1620 (0.55 nM- 1.6 microM) was tested on multiple-ring bronchial preparations pretreated with peptidase and cyclo-oxygenase inhibitors. BQ 788 shifted IRL1620 concentration-response curves to the right while BQ 123 did not influence bronchial responsiveness. In addition, a potentiation of the maximal response to the agonist was observed in BQ 788 treated bronchial rings. This effect was abolished by tissue pretreatment with Nomega-nitro-L-argininemethylester (L-NAME) or epithelium removal but not by pretreatment with atropine or iberiotoxin. Our results demonstrate that guinea-pig bronchus contains two populations of ET(B) receptors with different affinities for the ET(B)-selective agonist, IRL 1620. One ET(B) receptor population appears to activate bronchial muscle contraction while another on epithelial cells causes muscle relaxation through the release of nitric oxide (NO).

Different bronchial responsiveness to Ach between normal and OA-sensitized guinea pigs after acoustic stress: a role for adenosine.

Noise-exposure makes non-sensitized guinea pigs hyporesponsive to Acetylcholine (Ach), while in Ovalbumin (OA)-sensitized guinea pigs the responsiveness to the cholinergic mediator is not modified by acoustic stress (Nieri et al., 1996). The occurrence of bronchial hyporesponsiveness after acoustic stress in non-sensitized guinea pigs was verified also with histamine, obtaining a result similar to that observed with Ach. Moreover, the role of adenosine as modulator of the bronchial responsiveness to Ach after noise-exposure was assessed both in normal and in sensitized guinea pigs. In non-sensitized noise-exposed guinea pigs, the hyporesponsiveness to Ach was abolished by pretreatment of the animals with the peripheral A1/A2 antagonist 8-p-(sulfophenyl)theophylline (8-pSPT, 3 mg/kg i.v.) or with the A2-selective blocker 3,7-dimethyl-1-propargylxanthine (DMPX, 80 microg/kg i.v.) but not with the A1-selective antagonist Xanthine Amine Congener (XAC, 0.1 mg/kg i.v.). In sensitized guinea pigs, pretreatment with theophylline (25 mg/kg i.v.) makes noise-exposed animals again hyporesponsive to Ach, while no effect was obtained with the selective A1 and A2 antagonists employed. Also enprofylline (10 mg/kg i.v.), a phosphodiesterase inhibitor more potent than theophylline, does not modify the responsiveness to Ach in sensitized noise-exposed guinea pigs. The overall data presented suggest the involvement of the peripheral purinergic system in the regulation of airway reactivity after the stressful condition and indicate an altered functionality of this system as a consequence of sensitization. Furthermore, noise-exposure makes it possible to reveal in guinea pigs an opposite influence by theophylline on airway responsiveness to Ach, in sensitized, with respect to normal, animals.

Synthesis and beta-adrenergic properties of (Z)-N-[3-(alkylamino)-2-hydroxypropylidene](aryl-methyloxy)amines: effects of the configuration around the methyloxyiminomethyl (MOIM) double bond on the biopharmacological properties of MOIM-type beta-blocking agents.

The N-isopropyl- (3a-g) and N-tert-butyl-substituted (4a-g) (Z)-N-(3-(amino)-2-hydroxypropylidene)-(arylmethyloxy)amines were synthesized in order to compare their beta 1- and beta 2-adrenergic properties with those of their previously studied corresponding analogues with the E configuration (1a-g and 2a-g). Compounds 3 and 4 were tested for their affinity for beta 1-a and beta 2-adrenoceptors by radioligand binding experiments, and the compounds with the highest affinity were also assayed for their activity towards the same types of beta-adrenoceptors by functional tests on isolated preparations. The Z-methyloxyiminomethyl (Z-MOIM) compounds 3 and 4 proved to possess, on the whole, affinity (Ki) and activity (PIC50) indices similar to those of the E isomers 1 and 2, thus indicating that for the MOIM-type beta-adrenergic antagonists 1-4, the type of configuration around the MOIM double bond does not have any appreciable effect either on the affinity or on the activity towards beta-adrenoceptors. These results are rationalized on the basis of the steric and electronic analogies existing between the MOIM groups of 1-4 in the two types of configurations (E and Z).

Histaminic bronchospasm potentiated by adenosine: investigation of the mechanisms.

In anaesthetized guinea pigs, adenosine enhances the histamine-induced bronchospasm by means of a mechanism partly involving non-adrenergic-non-cholinergic (NANC) nerves, not related to capsaicin-sensitive neurons (Breschi et al., 1994). In the present paper, we excluded any interference by adenosine with the mediators known to be present in the airway inhibitory NANC system, VIP (vasoactive intestinal polypeptide) and NO (nitric oxide). The use of alpha-chymotrypsin or L-N(G)-nitro-arginine methyl ester (L-NAME) failed to modify the potentiation under study. The effects of adenosine were further investigated by studying whether an increased release of excitatory mediators from non-neural cells, in particular 5-HT (5-hydroxytryptamine, serotonin) and arachidonic products, was involved. In this connection, methysergide did not significantly affect the modulatory action of adenosine, revealing that the release of 5-HT was also not involved. Inhibition was obtained with hydrocortisone and with nordihydroguaiaretic acid, but not with indomethacin or with the mastocyte membrane stabilizer, sodium cromoglycate. This evidence suggests that lipooxygenase products, not derived from mastocytes, probably participate in the potentiating effect of adenosine.

Effects of noise stress on EFS-mediated cholinergic and inhibitory NANC responses in tracheae from normal and sensitized guinea-pigs.

1 The aim of the present research was to study the cholinergic and inhibitory non-adrenergic-non-cholinergic (NANC) responses obtained with electrical field stimulation (EFS) of tracheal tissues from sham- and noise-exposed guinea-pigs. A comparison was also made between normal and ovalbumin (OA)-sensitized animals. 2 In proximal tracheae pretreated with indomethacin (3 microM), propranolol (1 microM), alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), frequency-dependent responses to EFS (0.1 ms width; 20 V, 0.1-100 Hz, 15 s train duration) were obtained, both contractile and relaxing in nature. The contractile responses were abolished by atropine (1 microM), and did not vary significantly between sham- and noise-exposed guinea-pigs, or between normal and sensitized animals. The NANC relaxing responses, present in spite of the pre-treatment of the tissues with L-NAME and alpha-chymotrypsin, and almost completely abolished by tetrodotoxin (TTX) treatment (10 microM), appeared to be enhanced in noise-exposed guinea-pigs, with respect to sham-exposed animals, but only when the animals were not OA-sensitized. 3 In distal tracheae contracted with histamine (10 microM), the study of the whole inhibitory NANC response (pre-treatment with propranolol, but not with alpha-chymotrypsin and L-NAME), which was mainly TTX-sensitive, revealed a statistically non-significant difference between sham- and noise-exposed guinea-pigs, both normal and OA-sensitized. When distal tracheae were preincubated with alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), in addition to propranolol, a significant residual inhibitory NANC response to EFS was observed. Surprisingly, in this case, similarly to the evidence obtained in proximal tracheae, a significantly enhanced response was revealed in noise-exposed guinea-pigs with respect to sham-exposed animals. 4 The noise-induced enhancement of the relaxant response disappeared when the tissues were pretreated with the A2 purinergic antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 1 microM), while it persisted in the presence of the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM). 5 The above data indicate that, while not modifying the cholinergic and the whole inhibitory NANC response to EFS, noise stress selectively influences an inhibitory component of the NANC system in guinea-pig trachea with a mechanism probably involving an enhanced neurally mediated release of adenosine, which relaxes the smooth muscle via A2 receptors. This effect appears to be lacking or masked in sensitized guinea-pigs.

Effects of adenosine on NANC bronchoconstriction in anaesthetized guinea-pigs.

The present work assesses the effects of the acute administration of adenosine on tachykinergic bronchoconstriction induced in different ways (exogenously administered capsaicin or substance P and vagal electrical stimulation) in anaesthetized and curarized guinea-pigs. Adenosine (30-3000 micrograms kg-1, i.v.) enhanced significantly and dose-relatedly the airway narrowing induced by a single dose of capsaicin (0.5-2 micrograms kg-1, i.v.), both in normal and in vagotomized animals. A smaller and less dose-dependent enhancement by the nucleoside of the pulmonary resistance increase induced by substance P (5-15 micrograms kg-1, i.v.) was observed. This effect was almost completely prevented by the H1 antagonist diphenhydramine (1 mg kg-1, i.v.), which also unmasked an inhibitory action of adenosine at the highest doses. Diphenhydramine, on the contrary, did not significantly modify the potentiation by adenosine of capsaicin-mediated bronchoconstriction. Finally, the nucleoside dose-dependently inhibited the atropine-resistant bronchospasm following vagal electrical stimulation. The use of the selective adenosinic agonists R-N6-[2-phenylisopropyl]adenosine (1-100 micrograms kg-1, i.v.) and 5'-N-methylcarboxamidoadenosine (1-100 micrograms kg-1, i.v.) before the administration of capsaicin, revealed the ability of the first to reproduce the enhancement induced by adenosine, while the second had an inhibitory effect. It is concluded that adenosine has both excitatory and inhibitory modulatory effects on airway responsiveness to excitatory non-adrenergic non-cholinergic (e-NANC) stimuli. The excitatory effects, revealed with substance P and capsaicin, support the hypothesis that adenosine may play a role as an asthma mediator.

Acetylcholine-induced bronchoconstriction modified by noise exposure in normal but not in sensitized guinea-pigs.

1. The acute (6h) exposure of guinea-pigs to white noise (110 dB) as a stress stimulus, reduced bronchial reactivity to acetylcholine (Ach) (3-1000 micrograms kg-1 i.v.) in anaesthetized animals. 2. The hyporesponsiveness to Ach in stressed animals was not confirmed in vitro on tracheal preparations (Ach 1 x 10(-9)-1 x 10(-4) g ml-1) and disappeared in vivo when the animals were sensitized with ovalbumin (OA, 100 mg kg-1 i.p. + 100 mg kg-1 s.c.). The hyporesponsiveness was also absent in ovalbumin sensitized guinea-pigs exposed to an aerosol of ovalbumin 60 min before testing with Ach. 3. In non-sensitized guinea-pigs, pretreatment with butoxamine (1 mg kg-1 i.v.) or with theophylline (25 mg kg-1 i.v.), completely abolished the effect of noise-exposure. In contrast, pretreatment with L-NG-nitro-arginine methyl ester (L-NAME, 10 mg kg-1 i.v.), alpha-chymotrypsin (2 U kg-1 i.v.) or with enprofylline (10 mg kg-1 i.v.), did not affect it. 4. In conclusion, our experiments reveal inhibitory mechanisms upon Ach-induced bronchoconstriction activated by a stress stimulus and this is absent in sensitized animals. These mechanisms seem to be linked to the adrenergic beta 2-receptors and a role for the purinergic system (via A-receptors) may also be present.

Adenosine enhances the bronchocontractile response to histamine in anaesthetized and curarized guinea pigs through a mechanism partly blocked by hexamethonium.

The ability of adenosine to potentiate the airway narrowing induced by histamine in anaesthetized and curarized guinea pigs has been investigated in order to establish whether it could be ascribed to a modulatory activity by the nucleoside at the neuronal level. Bilateral vagotomy, atropine (2 mg/kg i.v.), and pretreatment with capsaicin (52 mg/kg s.c. 6 days before the experiment) did not result in any significant protection against the enhancement provoked by the nucleoside of the bronchocontractile effect of histamine. On the contrary, the latter was significantly reduced by the ganglionic blocking agent, hexamethonium (10 mg/kg i.v.). Moreover, the effect of adenosine on airway responsiveness to histamine was not modified in animals treated with propranolol (1 mg/kg i.v.) or guanethidine (20 mg/kg s.c. over a period of 2 days). In conclusion, current data suggest that the purine is able, in our experimental model, to potentiate the bronchospasm induced by histamine by means of a mechanism mediated, at least partly, by non-adrenergic-non-cholinergic nerves not related to capsaicin-sensitive afferent neurons.