Role of nitric oxide and guanosine 3',5'-cyclic monophosphate in mediating nonadrenergic, noncholinergic relaxation in guinea-pig pulmonary arteries.

1. Nonadrenergic, noncholinergic (NANC) nerves mediate vasodilatation in guinea-pig pulmonary artery (PA) by both endothelium-dependent and endothelium-independent mechanisms. The transmitter(s) involved in the endothelium-independent pathway have not yet been identified. We have therefore investigated the possibility that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) may mediate this neural vasodilator response in guinea-pig branch PA rings denuded of endothelium. 2. Electric field stimulation (EFS, 50 V, 0.2 ms) induced a frequency-dependent (1-24 Hz), tetrodotoxin-sensitive relaxation of the U44069-precontracted PA rings in the presence of adrenergic and cholinergic blockade. 3. The NO synthase inhibitors NG-monomethyl L-arginine (L-NMMA, 100 microM) and NG-nitro L-arginine methyl ester (L-NAME, 30 microM), and the guanylyl cyclase inhibitor methylene blue (5 microM) inhibited the EFS (16 Hz)-induced relaxation by 53 +/- 5, 74 +/- 9 and 82 +/- 9% respectively (n = 5-7, P < 0.01, compared with control rings). 4. Excess concentrations of L-, but not D-arginine (300 microM) completely reversed the inhibitory effect of L-NMMA. 5. The EFS-elicited relaxation (4 Hz) was potentiated by 1 microM zaprinast, a type V phosphodiesterase inhibitor which inhibits guanosine 3':5'-cyclic monophosphate (cyclic GMP) degradation, but was unaffected by 0.1 microM zardaverine, a type III/IV phosphodiesterase inhibitor which inhibits cyclic AMP degradation. 6. EFS (50 V, 0.2 ms, 16 Hz) induced a 3 fold increase in tissue cyclic GMP content, an action which was inhibited by L-NMMA (100 microM). 7. Pyrogallol (100microM), a superoxide anion generator, also inhibited the EFS-induced relaxation by 53 +/- 9%, and this effect was prevented by superoxide dismutase.8. Chemical sympathetic denervation with 6-hydroxydopamine had no effect on the relaxant response to EFS in the endothelium-denuded PA rings.9. In endothelium-denuded branch PA rings at resting tone, L-NMMA (100 microM) significantly augmented the adrenergic contractile response, an effect which was completely reversed by L-arginine,but not by D-arginine. In the same groups of vessel rings, L-NMMA had no significant effect on the matched contractile response to exogenous noradrenaline.10. These results suggest that NO may be released from intramural nerve endings other than adrenergic nerves (probably NANC nerves), and this leads to vasodilatation via activation of guanylyl cyclase.

Differential inhibitory effects of opioids on cigarette smoke, capsaicin and electrically-induced goblet cell secretion in guinea-pig trachea.

1. Goblet cell secretion in guinea-pig airways is under neural control. Opioids have previously been shown to inhibit neurogenic plasma exudation and bronchoconstriction in guinea-pig airways. We have now examined the effects of morphine and opioid peptides on tracheal goblet cell secretion induced by either electrical stimulation of the cervical vagus nerves, exogenous capsaicin, or acute inhalation of cigarette smoke. The degree of goblet cell secretion was determined by a morphometric method and expressed as a mucus score which is inversely related to mucus discharge. 2. Morphine, 1 mg kg-1, completely blocked goblet cell secretion induced by electrical stimulation of the vagus nerves. Morphine also inhibited the response to cigarette smoke given either at a low dose (10 breaths of 1:10 diluted in air), which principally activates cholinergic nerves, or at a high dose (20 breaths of undiluted), which activates capsaicin-sensitive sensory nerves, by 100% and 73% respectively. In contrast, morphine had no significant inhibitory effect on capsaicin-induced goblet cell secretion. The inhibitory effect of morphine was reversed by naloxone. 3. Selective mu- or delta-opioid receptor agonists, [D-Ala2, NMePhe4, Glyol5]enkephalin (DAMGO) or [D-Pen2, D-Pen5]enkephalin (DPDPE) respectively, caused a dose-related inhibition of low dose cigarette smoke-induced goblet cell discharge, with DPDPE more potent than DAMGO. A kappa-receptor agonist, trans-3,4-dichloro-N-methyl-N-(2-(1-pyrollidinyl)cyclohexyl) benzeneacetamine (U-50,488H), had no inhibitory effect. DPDPE had no inhibitory effect on goblet cell secretion induced by exogenous methacholine. 4. DAMGO dose-dependently blocked the response to high dose cigarette smoke with a maximal inhibition of 95% at 2 x 10(-7) mol kg-1. Neither DPDPE nor U-50,488H had any significant inhibitory effect. The increase in goblet cell secretion induced by exogenous substance P was not affected by DAMGO.5. We conclude that opioids inhibit neurally-mediated goblet cell secretion via actions at prejunctional delta and mu-receptors on cholinergic nerves and at mu-receptors on sensory nerve endings, and that capsaicin activation of sensory nerves is via a different mechanism from that of electrical or cigarette smoke activation.

Tachykinin control of ferret airways: mucus secretion, bronchoconstriction and receptor mapping.

The effects of synthetic tachykinin receptor agonists on mucus secretion by ferret trachea was determined in vitro in Ussing chambers using 35SO4 as a mucus marker and the synthetic peptides [Sar9,Met(O2)11]substance P (SarSP), [beta Ala8]neurokinin A-(4-10) and [MePhe7] neurokinin B which are selective for NK1, NK2 and NK3 tachykinin-receptors respectively. The bronchomotor effects of the same agonists were also studied in vitro and tachykinin receptors were localized by autoradiographic mapping. SarSP was the only synthetic agonist able to elicit a concentration-dependent increase in mucus secretion and was much more potent than SP. The EC50 for SarSP was 1.7 x 10(-6) M. Moreover, the maximal increase in release of 35SO4 produced by SarSP 10(-5) M was 95% of the increase produced by methacholine 10(-4) M indicating that this concentration of SarSP induced a near maximal secretory response. There was no significant difference in the secretory action of SP administered from the luminal or the submucosal side of the tissue. Only the NK2 agonist was able to produce a concentration-dependent contractility of bronchial ring preparations and its effect was relatively weak (EC50 6.4 x 10(-6) M). Capsaicin (10(-5) M) produced only a slight increase in tracheal mucus secretion (28 +/- 5%; n = 6) and was completely ineffective in inducing bronchoconstriction. Binding sites for [125I]-Bolton Hunter SP were more evident than sites for [125I]-NKA on submucosal glands and epithelium. In contrast, only binding sites to NKA could be observed over the smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

K+ channel activator inhibition of neurogenic goblet cell secretion in guinea pig trachea.

A potassium (K+) channel activator, BRL 38227, inhibited goblet cell secretion in guinea-pig trachea induced by either electrical stimulation of the vagus nerves or acute inhalation of cigarette smoke, two stimuli which activate both cholinergic nerves and capsaicin-sensitive sensory nerves. BRL 38227 failed to inhibit methacholine- or substance P-induced goblet cell secretion which suggests that K+ channel activators inhibit neurogenic goblet cell secretion via a prejunctional effect on cholinergic and sensory nerves.

Neurogenic goblet cell secretion and bronchoconstriction in guinea pigs sensitised to trimellitic anhydride.

Trimellitic anhydride is a cause of occupational asthma in humans. We have previously found that tracheal instillation of trimellitic anhydride conjugated to guinea pig serum albumin induces acute bronchoconstriction and airway plasma exudation in sensitised animals, responses mediated primarily via histamine release. In the present study, neural mechanisms mediating bronchoconstriction and goblet cell secretion were determined in trimellitic anhydride-sensitised guinea pigs using the ganglionic blocker hexamethonium to eliminate efferent reflex mechanisms, pretreatment with capsaicin to eliminate afferent mechanisms, or cimetidine and mepyramine to eliminate histamine-mediated mechanisms. The magnitude of secretion of intracellular mucus from tracheal goblet cells was quantified morphometrically as a mucus score which is inversely related to the degree of discharge. Guinea pigs were injected intradermally either with 0.1 ml 0.3% trimellitic anhydride in corn oil or with corn oil alone as control. Fourteen to eighteen days later all sensitised animals had developed specific immunoglobulin (Ig) G1 antibodies whereas the controls had not. Tracheal instillation of conjugated trimellitic anhydride in anaesthetised animals significantly increased airway lung resistance (RL) 24-fold in sensitised guinea pigs (34.3 +/- 7.9 cm H2O.ml-1.s) compared with controls (1.4 +/- 0.1 cm H2O.ml-1.s). Mucus score was significantly reduced by 51% (indicating goblet cell secretion) in sensitised guinea pigs (183 +/- 22 mucus score units) compared with controls (372 +/- 41 mucus score units). The antihistamines significantly inhibited conjugated trimellitic anhydride-induced bronchoconstriction by 89%, but did not significantly affect goblet cell discharge. Hexamethonium alone did not significantly affect conjugated trimellitic anhydride-induced bronchoconstriction or goblet cell secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cigarette smoke-induced airway goblet cell secretion: dose-dependent differential nerve activation.

We studied the effect of acute inhalation of middle-tar cigarette smoke on airway goblet cell secretion in anesthetized guinea pigs. Secretion induced by a low dose of smoke (10 breaths diluted 1:10 in air) was blocked by either hexamethonium or by filtering out the particulate phase of the smoke. The response was partially inhibited by atropine but was not inhibited by propranolol, phentolamine, or capsaicin pretreatment. Cutting the nerve supply to the airways did not inhibit the response to low-dose smoke. In contrast, goblet cell secretion induced by a high dose of cigarette smoke (20 breaths undiluted) was inhibited by capsaicin pretreatment but not by autonomic receptor blockade nor by filtering out the particulate phase. Secretion induced by the vapor phase of the high dose of cigarette smoke was blocked by capsaicin pretreatment but was not inhibited by hexamethonium. We conclude that in guinea pig airways the particulate phase of low doses of smoke activates cholinergic nerves via stimulation of parasympathetic ganglia, whereas the vapor phase of high doses of smoke activates capsaicin-sensitive sensory nerves.

Capsaicin and sensory neuropeptide stimulation of goblet cell secretion in guinea-pig trachea.

1. We studied the effect of capsaicin and sensory neuropeptides on tracheal goblet cell secretion in anaesthetized guinea-pigs using a semi-quantitative morphometric technique whereby the magnitude of discharge of stained intracellular mucus, expressed as a mucus score (MS), was related inversely to discharge. 2. Capsaicin (i.v.) induced goblet cell secretion: a decrease of 50% in MS below control (indicative of increased secretion) was maximal at 3.3 x 10(-9) mol/kg. 3. Capsaicin-induced secretion was unaffected either by prior vagus nerve section or by pre-treatment with atropine, propranolol and phentolamine which suggests that local axon reflexes with release of sensory neuropeptides are involved in the response. 4. Intravenous substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and calcitonin gene-related peptide (CGRP) produced dose-related increases in goblet cell secretion, with SP the most potent. Doses (mol/kg) causing a 50% decrease in MS from control were 3.5 x 10(-12) for SP; 72 x 10(-10) for NKA; 1.6 x 10(-9) for NKB; and 1.2 x 10(-8) for CGRP. The maximal increase in goblet cell secretion was 75% of control and occurred with SP at 10(-10) mol/kg. 5. SP-induced mucus discharge was not inhibited by atropine or the histamine receptor antagonists mepyramine or cimetidine. 6. We conclude that in guinea-pig trachea, goblet cell secretion is under the control of capsaicin-sensitive sensory nerves and release of neuropeptides from these nerves may induce mucus discharge via tachykinin receptors of the NK-1 subtype (indicated by an order of potency of SP greater than NKA greater than NKB).

Neural control of goblet cell secretion in guinea pig airways.

We studied the neural control of goblet cell secretion in the lower airways of anesthetized guinea pigs using a semiquantitative morphometric technique. The magnitude of discharge of intracellular mucus was determined in histological sections of the trachea and main bronchi stained for mucus glycoproteins. Bilateral electrical stimulation of the cervical vagus nerves induced goblet cell secretion. The magnitude of the effect was dependent on the frequency, voltage, and pulse width of the stimulus, and the duration of stimulation. At 10 Hz, 5 V, and 5 ms for 3 min, there was a 62% decrease in the amount of intracellular mucus below that with sham stimulation. The secretion was blocked either by atropine or by pretreatment with capsaicin but was not significantly inhibited by idazoxan, an alpha-adrenoceptor antagonist. The magnitude of goblet cell discharge in animals pretreated with propranolol was intermediate between that in controls and that with nerve stimulation, although not significant to either. These results demonstrate that goblet cell secretion is under neural control in guinea pig airways and suggest that cholinergic, nonadrenergic-noncholinergic, and possibly adrenergic neural pathways, may contribute to the secretion.

Endotoxin-induced plasma exudation in guinea-pig airways in vivo and the effect of neutrophil depletion.

The contribution of neutrophils to the action of endotoxin on plasma exudation in the airways of anaesthetized guinea-pigs was quantified by measuring the extravasation of Evans blue dye. Endotoxin (Salmonella enteritidis) caused a dose-dependent increase in microvascular leakage to Evans blue dye which was maximal after 25 min (p less than 0.05). The minimum dose tested that induced a significant rise in leakage was 1.5 mg.kg-1 for "central" intrapulmonary airways (ipa); 4.5 mg.kg-1 for trachea and main bronchi and 7.5 mg.kg-1 for nasal mucosa, larynx and "peripheral" ipa. Depletion of circulating neutrophil numbers by 97% using an antibody to guinea-pig neutrophils caused no significant diminution of the effects of endotoxin on leakage in any part of the airway. There was no significant influx of neutrophils into the airway interstitium at the time of maximum extravasation of Evans blue. We conclude that endotoxin-induced airway microvascular permeability is dependent upon mechanisms other than circulating neutrophils.

Pneumolysin induces the salient histologic features of pneumococcal infection in the rat lung in vivo.

Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement.