Contraction of human airways by oxidative stress protection by N-acetylcysteine.

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 +/- 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was approximately 100 microM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 +/- 0.83 to 26.82 +/- 1.49 nmol mg(-1) protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 +/- 2.6 to 6.9 +/- 1.9 nmol mg(-1) protein) and an increase of oxidized glutathione (from 0.09 +/- 0.03 to 0.18 +/- 0.03 nmol mg(-1) protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 +/- 1.19 nmol mg(-1) protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity. N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.

Antagonism of calcium by zinc in guinea-pig isolated taenia caeci and trachealis muscle.

1 In guinea-pig isolated taenia caeci and trachealis bathed in a K+-rich, Ca2+-free medium, CaCl2 (0.01-10 mM) produced a concentration-dependent contraction. Zn2+ (0.01-1 mM), Cd2+ (0.01-1 mM), verapamil (0.01-100 microM) and trifluoperazine (1-100 microM) were effective antagonists of CaCl2-induced responses. 2 Zn2+ and Cd2+ in concentrations from 0.01 to 1 mM were without effect on the tone of taenia and trachea in normal Tris solution. Conversely, Zn2+ and Cd2+, in concentrations of 1 mM, caused contraction of these tissues in a K+-rich, Ca2+-free medium. Zn2+ (1 mM)-induced contractions of taenia and trachea were completely inhibited by verapamil (10 microM). 3 In taenia and trachea skinned of their plasma membranes, tension development induced by Ca2+ (10 microM or 1 microM, respectively) was unaffected by verapamil (100 microM), whereas trifluoperazine (100 microM) depressed the maximal tension produced by Ca2+. Segments of skinned preparations contracted in response to low concentrations of Zn2+ (10 microM) or Cd2+ (10 microM). 4 It is concluded that Zn2+ may suppress Ca2+-induced spasm by a direct action on the binding sites of the Ca2+ channel.

Characterization of 5-HT receptors on human pulmonary artery and vein: functional and binding studies.

1. This study aimed to investigate the 5-hydroxytryptamine (5-HT) receptors mediating contraction of ring preparations isolated from human pulmonary arteries and veins. In functional studies, the responses to 5-HT, sumatriptan, ergotamine, serotonin-O-carboxymethyl-glycyl-tyrosinamide (SCMGT), alpha-methyl 5-HT (alpha-Me) and 2-methyl 5-HT (2-Me) were studied with WAY100635, GR127935, ritanserin, zacopride and SB204070 as antagonists. 2. All agonists produced concentration-dependent contractions of human pulmonary artery and vein preparations. The order of potency (-log ECS0 values) was ergotamine (6.88) > 5-HT (6.41) > or = SCMGT (6.20) = sumatriptan (6.19) > or = alpha-Me (6.04) in the artery, and ergotamine (7.84) > 5-HT (6.96) > sumatriptan (6.60) = alpha-Me (6.56) > SCMGT (6.09) in the vein. The potency of each agonist, except for SCMGT, was greater in vein than in artery preparations. Contractile responses to 5-HT were similar in intact and endothelium-denuded preparations but responses to sumatriptan were enhanced in artery rings without endothelium. 3. GR127935 (1 nM to 0.5 microM) produced an unsurmountable antagonism of the response to 5-HT, sumatriptan, ergotamine and SCMGT. Ritanserin (1 nM to 1 microM) also reduced the maximum contractile responses to 5-HT, ergotamine and alpha-Me in artery and vein preparations without affecting those to sumatriptan and SCMGT. In endothelium-denuded preparations, surmountable antagonism of sumatriptan by GR127935 (in the presence of ritanserin) and of alpha-Me by ritanserin (in the presence of GR127935) allowed for the calculation of the apparent pK(B) values of GR127935 (9.17+/-0.11 in artery and 9.11+/-0.05 in vein) and ritanserin (8.82+/-0.09 in artery and 8.98+/-0.12 in vein). 4. WAY100635 (1 nM to 1 microM), zacopride (1 nM to 1 microM), or SB204070 (1 nM) did not significantly alter the concentration-response curves for 5-HT, sumatriptan, ergotamine, SCMGT or 2-Me in human pulmonary artery or vein thus indicating that 5-HT1A, 5-HT3 and 5-HT4 receptors are presumably not involved in the contractile response to these agonists. 5. Binding studies using selective radioligands for different 5-HT receptors could not detect the presence of 5-HT1A receptor binding in human pulmonary blood vessels whereas the 5-HT(1B/1D) radioligand [3H]-5CT significantly labelled a population of specific binding sites in both vessel types. The presence of 5-HT2A receptors could also be inferred from the level of binding of [3H]-ketanserin to membranes obtained from human pulmonary vessels, although significance could not be reached for arteries. 5-HT4 specific receptor binding was scarce in veins and absent in the case of arteries. 6. These findings indicate that the human pulmonary artery and vein have a mixed functional population of 5-HT(1B/1D) and 5-HT2A receptors mediating the contractile response to 5-HT which is consistent with results of the binding studies.

The spasmogenic effects of vanadate in human isolated bronchus.

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 microM-3 mM) produced concentration-dependent, well-sustained contraction. Its -logEC50 was 3.74 +/- 0.05 (mean +/- s.e.mean) and its maximal effect was equivalent to 97.5 +/- 4.2% of the response to acetylcholine (ACh, 1 mM). 2. Vanadate (200 microM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 microM), zileuton (10 microM), a mixture of atropine, mepyramine and phentolamine (each at 1 microM), or by mast cell degranulation with compound 48/80. 3. Vanadate (200 microM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 microM) or to a Ca2+-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 microM) in Ca2+-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca2+-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca2+ stores. In such tissues cyclopiazonic acid (CPA; 10 microM) prevented Ca2+-induced recovery of vanadate-induced contraction. 4. Tissue incubation in K+-rich (80 mM) PSS, K+-free PSS, or PSS containing ouabain (10 microM) did not alter vanadate (200 microM)-induced contraction. Ouabain (10 microM) abolished the K+-induced relaxation of human bronchus bathed in K+-free PSS. This action was not shared by vanadate (200 microM). The tissue content of Na+ was increased and the tissue content of K+ was decreased by ouabain (10 microM). In contrast, vanadate (200 microM) did not alter the tissue content of these ions. Tissue incubation in a Na+-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 microM). 5. Vanadate (200 microM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 microM), staurosporine (1 microM) and calphostin C (1 microM). Genistein (100 microM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate. 6 Vanadate (0.1-3 mM) and ACh (1 microM- 3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca2+-free medium either alone or in combination with ryanodine (10 microM). 7. In human cultured tracheal smooth muscle cells, histamine (100 microM) and vanadate (200 microM) each produced a transient increase in intracellular Ca2+ concentration ([Ca2+]i). 8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 microM) in human bronchus was associated with cellular depolarization. 9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca2+ from an intracellular store. The Ca2+ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca2+]i that are close to basal.

Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes.

1. Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function. 2. Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 microM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50 approximately 100 microM). 3. Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2 approximately 4.5). Glaucine (10 microM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isoprenaline. The glaucine-induced relaxation was resistant to H-89, a protein kinase A inhibitor. Glaucine depressed the contractile responses to Ca2+ (pD'2 approximately 3.62) and reduced the sustained rise of [Ca2+]i produced by histamine in cultured human airway smooth muscle cells (-log IC50 approximately 4.3). 4. Glaucine augmented cyclic AMP levels in human polymorphonuclear leukocytes challenged with N-formyl-Met-Leu-Phe (FMLP) or isoprenaline, and inhibited FMLP-induced superoxide generation, elastase release, leukotriene B4 production, [Ca2+]i signal and platelet aggregation as well as opsonized zymosan-, phorbol myristate acetate-, and A23187-induced superoxide release. The inhibitory effect of glaucine on superoxide generation by FMLP was reduced by H-89. 5. In conclusion, Ca2+ channel antagonism by glaucine appears mainly responsible for the relaxant effect of glaucine in human isolated bronchus while PDE4 inhibition contributes to the inhibitory effects of glaucine in human granulocytes. The very low PDE4/binding site ratio found for glaucine makes this compound attractive for further structure-activity studies.

Pharmacological activity of PF-904 in guinea pig in vivo, and on human bronchus and neutrophils in vitro.

The effects of PF-904 (4-amino-1-ethyl-6-methylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide), a pyrazinothiadiazine derivative, were examined in guinea-pig airways in vivo, in human isolated bronchus and human polymorphonuclear leukocytes. PF-904 (12.5-200 mg/kg, intraduodenal) reduced bronchoconstriction in response to histamine, arachidonic acid, platelet-activating factor (PAF) and methacholine. PF-904 (50-200 mg/kg) prevented PAF-induced airways hyperreactivity and inhibited antigen-induced bronchoconstriction, airway microvascular leakage and eosinophil lung accumulation, but antigen-induced airways hyperresponsiveness was not reduced. PF-904 (1 microM-1 mM) produced complete inhibition of spontaneous (-logEC50 = 3.57+/-0.04; n = 10) and histamine-stimulated tone (-logEC50 = 3.66+/-0.07; n = 10) of human isolated bronchus. Glibenclamide (10 microM) or precontraction with KCl (80 mM) did not impede PF-904-induced bronchial relaxation. PF-904 inhibited cyclic AMP (-logIC50 = 2.83+/-0.25; n = 8) and cyclic GMP (-logIC50 = 2.90+/-0.21; n = 8) phosphodiesterase activity in human bronchus. The activity of type IV phosphodiesterase was inhibited by PF-904 (-logIC50 = 3.43+/-0.11; n= 3). PF-904 also inhibited superoxide release by N-formylmethionyl-leucyl-phenylalanine-stimulated human polymorphonuclear leukocytes, but the maximal effect was approx. 50% of that produced by rolipram (10 microM). This profile of activities of PF-904 suggests that this compound has potential therapeutic value as an anti-asthma drug.

Effects of Ca2+ channel antagonists and benzodiazepine receptor ligands in normal and skinned rat urinary bladder.

The effects of Ca2+ channel antagonists and benzodiazepine receptor ligands against concentration-dependent contractions of rat urinary bladder induced by CaCl2 (0.1-50 mM, in K(+)-depolarized tissues), KCl (1-100 mM) and acetylcholine (0.1 microM to 1 mM) were studied. Nifedipine (0.001-0.1 microM), verapamil (0.01-1 microM), diltiazem (0.01-1 microM), cinnarizine (1-100 microM), and trifluoperazine (1-100 microM) each produced a concentration-related inhibition of the log concentration-effect curve for CaCl2. The rank order of potencies of these antagonists, measured as the IC50 against Ca2+ (25 mM)-induced contraction of depolarized bladder, was nifedipine (0.01 microM) > diltiazem (0.36 microM) approximately verapamil (0.41 microM) > or = cinnarizine (2.57 microM) > trifluoperazine (17.4 microM). These antagonists depressed KCl-induced contractions with an effectiveness and potency similar to that displayed against CaCl2-induced contractions. Nifedipine, verapamil, and diltiazem but not cinnarizine and trifluoperazine had a preferential inhibitory effect on the contractions elicited by KCl when compared to those elicited by acetylcholine. Ro 5-4864, diazepam, midazolam and the non-benzodiazepine PK 11195, each at 1-100 microM, depressed CaCl2- and KCl-induced contractions (IC50 values in the micromolar range). Benzodiazepines and PK 11195, all at 100 microM, markedly depressed acetylcholine-induced contractions. Flumazenil was scarcely effective. Cinnarizine (100 microM) and trifluoperazine (100 microM), but not the other Ca2+ channel antagonists and benzodiazepine receptor ligands tested, depressed Ca2+ (20 microM)-evoked contractions of skinned bladder. It is concluded that the action of nifedipine, verapamil, and diltiazem is restricted to the plasmalemma whereas cinnarizine and trifluoperazine also act on the intracellular contractile apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of selective phosphodiesterase inhibitors on platelet-activating factor- and antigen-induced airway hyperreactivity, eosinophil accumulation, and microvascular leakage in guinea pigs.

There is currently interest in the potential use of selective inhibitors of cyclic nucleotide phosphodiesterases (PDE) in the treatment of asthma. In this study we examined the effects of three selective PDE inhibitors, milrinone (PDE III), rolipram (PDE IV) and zaprinast (PDE V), on the broncoconstriction produced by antigen and histamine, the airway hyperreactivity and microvascular leakage after aerosol exposure to platelet-activating factor (PAF) and antigen, and the antigen-induced eosinophil infiltration in guinea-pig lung. Inhaled rolipram (0.01-10 mg ml-1) inhibited dose dependently the bronchospasm produced by aerosol antigen (5 mg ml-1) an anaesthetised, ventilated guinea-pigs. Rolipram (10 mg ml-1) produced maximal inhibition of antigen-induced bronchoconstriction but only partial inhibition of the response to aerosol histamine (1 mg ml-1). Milrinone and zaprinast (each 10 mg ml-1) showed weak, or no, inhibitory effects against bronchoconstriction produced by aerosol antigen or histamine. Pretreatment with rolipram (10 mg kg-1, i.p.) prevented airway hyperreactivity to histamine which develops 24 h after exposure of conscious guinea-pigs to aerosol PAF (500 micrograms ml-1) or antigen (5 mg ml-1). The pulmonary eosinophil infiltration obtained with 24 h of antigen-exposure was inhibited by rolipram. In contrast, milrinone and zaprinast (each 10 mg kg-1, i.p.) failed to reduce either the airway hyperreactivity of the eosinophil accumulation in these animals. Rolipram (1-10 mg ml-1) reduced the extravasation of Evans blue after aerosol PAF (500 micrograms ml-1) at all airway levels while a lower dose (0.1 mg ml-1) was only effective at intrapulmonary airways. Rolipram (0.01-1 mg ml-1) markedly reduced airway extravasation produced by inhaled antigen (5 mg ml-1). Zaprinast (1-10 mg ml-1) was also effective against airway microvascular leakage produced by aerosol PAF or antigen while milrinone (10 mg ml-1) had no antiexudative effect. These data support previous suggestions that pharmacological inhibition of PDE IV results in anti-spasmogenic and anti-inflammatory effects in the airways and may be useful in the treatment of asthma.

Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.

Effects of zinc sulphate on gastric mucosal blood flow and gastric emptying of the rat.

Zinc sulphate (50 mg kg-1 p.o.) did not modify basal gastric mucosal blood flow, as measured by [3H]aniline clearance, but inhibited its reduction by noradrenaline (3.5 micrograms kg-1 min-1). Zinc sulphate also influenced gastric emptying of phenol red but its effects depended upon the dose; 30 mg kg-1 caused no variation whereas 80 mg kg-1 induced a significant delay. The nature of both actions is discussed and their implications in the development and prevention of gastric ulceration have been analysed.

Differential effects of cyclooxygenase inhibitors on the cardiovascular response to hyperosmotic mannitol.

The intravenous infusion of mannitol (1.37 mosm kg-1 min-1 during 4 min) to anesthetized, open-chest, intact dogs produced a hemodynamic response characterized by a positive inotropic effect and a decrease in total peripheral resistance. This response is attributable to a direct effect of hypertonicity (plasma osmolality raised from 309 +/- 4 to 332 +/- 5 mosm kg-1 H2O). Pretreatment with indomethacin (5 mg kg-1 i.v.) or meclofenamic acid (2 mg kg-1 i.v.) attenuated the mannitol-induced response but acetylsalycilic acid (10 mg kg-1 i.v.) was without effect. Indomethacin (5 mg kg-1 i.v.) did not change the decrease in canine hindlimb perfusion pressure produced by intra-arterial mannitol 5 mosm kg-1. Indomethacin (3 microM) did not alter the chronotropic and inotropic responses to mannitol (150 mosm above normal) in isolated rabbit atria. These results suggest that the cardiovascular response to hyperosmotic mannitol may be partly mediated in intact animals by prostaglandin release.

Effects of antisecretory drugs on mucosal erosions induced by intragastric distension in rats: a comparative study.

The antiulcer effects of two classical antisecretory agents, cimetidine and pirenzepine, were compared with those of prostaglandin E2 and carbenoxolone sodium. The acid inhibitory action of drugs was not important in our study since the ulcer model employed consisted of perfusing the stomach continuously, at a high intraluminal pressure (120 mm H2O), with a simulated gastric juice (0.1 M HCl plus 600 mg pepsin/l). Cimetidine and pirenzepine failed to inhibit gastric erosions, whereas prostaglandin E2 and carbenoxolone greatly reduced the severity of the mucosal lesions. Long-term treatment with cimetidine did not prevent gastric damage. These findings indicate that pure antisecretory agents do not directly increase the capacity of the mucosa to resist damage.

Differential effects of verapamil on various gastric lesions in rats.

Verapamil (3, 10, 20 mg/kg-1) increases the necrotizing effects of oral 25% NaCl or 100% ethanol. Damage by 0.6 N HCl was not equally affected since 1 mg/kg-1 of verapamil decreased the ulcer index whereas the higher doses augmented it. Pharmacologically induced gastric lesions were also differently affected by verapamil, ulcers produced by histamine being greatly enhanced and those of reserpine inhibited. Neither indomethacin nor compound 48/80 ulcers were modified. These results suggest that verapamil modifies the susceptibility of the gastric mucosa to damage.