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Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α(4)β(2))-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α(7)-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α(7)) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α(7) nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α(7) nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α(7) nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α(7) nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α(7) nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.

In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited (125)I-[MePhe(7)]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K(i) = 2.2 nM and antagonized competitively NKB-induced Ca(2+) mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K(b) = 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA(2) = 8.1) and guinea pig ileal circular smooth muscles (pA(2) = 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 209 nM), and was without effect, at 1 microM, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED(50) of 0.56 mg/kg). Intraperitoneal SB 235375 (10-30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords1,2, and which has recently been cloned from man3. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (K(i)s of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC(50)s of 2, 3 and 14 nm). PL-3994 has a K(i) of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 μm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC(50)s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm-100 μm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1-1000 μg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.

The cardiopulmonary profile of three different rat strains was compared after exposure to hypoxia (9% O2) for 0, 7, or 14 days. In Sprague-Dawley (SD), Wistar (W), and high altitude-sensitive (HAS) rats, pulmonary arterial pressure (PAP) rose 30, 58, and 85% respectively, after 7 days of hypoxia, and by 108, 116, and 167%, respectively, at 14 days compared to strain- and age-matched normoxic controls. Right ventricular hypertrophy (RVH), expressed as the ratio of right free wall/left wall + septum weight, in SD, W, and HAS was increased by 24, 53, and 48%, respectively, at 7 days, and by 51, 93, and 55% at 14 days compared to normoxic littermates. Histologically, marked medial thickening and luminal stenosis of small and medium-sized arteries were observed in all hypoxic rats, being most pronounced in the HAS rats at 14 days. Treatment of HAS rats with the ET receptor antagonist SB 217242 (3.6 or 10.8 mg/day i.p. by osmotic pump) significantly inhibited the hypoxia-induced increases in PAP (70-75% decrease). RVH was inhibited by 40% at the dose of 10.8 mg/day. Histologically, the SB 217242-treated rats had almost "normal" small and medium-sized arteries, comparable to those of the normoxic HAS controls. This study demonstrates an exaggerated PAP response to chronic hypoxia in HAS compared to SD and W rats. The inhibitory influence of SB 217242 on the functional and morphologic changes induced by hypoxia provides further evidence for a role for ET and the potential utility of ET receptor antagonists in the treatment of pulmonary hypertension.

The neuropeptide tachykinins and their receptors have been implicated in the pathogenesis of lung disease, although the role of the tachykinin neurokinin-3 receptor has not been elucidated. Using confocal microscopy, we identified tachykinin neurokinin-3 receptors on human bronchial parasympathetic ganglion neurons. Electrophysiologic recordings demonstrated that activation of sensory nerve fibers, either by antidromic stimulation or capsaicin, depolarized these neurons. This response was mimicked by exogenously applied tachykinin neurokinin-3 receptor-selective agonist, senktide analogue, but not significantly by tachykinin neurokinin-1 or neurokinin-2 receptor-selective agonists. Responses to endogenous tachykinins or exogenous selective tachykinin neurokinin-3 receptor activation with senktide analogue were inhibited by the selective tachykinin neurokinin-3 receptor antagonists, SB 223412 or SB 235375. We provide the first evidence that tachykinin neurokinin-3 receptors regulate human bronchial parasympathetic ganglion neurotransmission by activation of a peripheral reflex. This pathway may play a significant role in controlling bronchomotor tone and air flow to the lung.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [ trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole; IC50 = 44 nM vs. p38α], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3–30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC50 of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

The cardiopulmonary profile of three rat strains (Sprague-Dawley, Wistar and High altitude-sensitive) was compared upon exposure to hypoxia (9% O2) for 0, 7 or 14 days. No differences were observed among the in vitro contractile (ET-1) and relaxant (carbachol) responses of pulmonary artery isolated from the three strains during normoxia. Chronic hypoxia decreased ET-1 contractile responses and diminished relaxant responses to carbachol similarly in all strains. In Sprague-Dawley, Wistar and High altitude-sensitive rats, pulmonary arterial pressure rose time-dependently and was elevated by 108%, 116% and 167%, respectively, after 14 days of hypoxia compared to normoxic controls. Right ventricular hypertrophy was increased by 51%, 93% and 55%, respectively, at 14 days. Hypoxia-induced hypertrophy and medial thickening in the pulmonary vasculature were more pronounced in High altitude-sensitive rats. Sprague-Dawley exhibited hypoxia-induced airway hyperresponsiveness to intravenous methacholine, but there were no hypoxia- or strain-related differences in in vitro tracheal contractility. Although each strain exhibited greater sensitivity for a particular hypoxia-induced parameter, pulmonary vascular functional and structural changes suggest that High altitude-sensitive rats represent a choice model of hypoxia-induced pulmonary hypertension.

The cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4, lipid products derived from arachidonic acid metabolism, have been implicated in the pathophysiology of several inflammatory diseases, in particular, asthma. This unit describes techniques and applications for the measurement of contractile responses to the CysLTs in isolated smooth muscle preparations. The contractions are assessed by standard methods for the isometric measurement of responses (contractile or relaxant) of isolated tissues to exogenous agonists, and a detailed description of the methods employed to assess CysLT-induced contractions in guinea-pig trachea is outlined. However, the same general methodology (other than parameters such as dissection for non-airway tissues) are appropriate for measuring CysLT-induced contractions in airway preparations from other animals, and in non-airways tissues (e.g., the gastrointestinal tract) from different species, and also in exploring the relaxant responses to the CysLTs that have been demonstrated in some tissues (e.g., pulmonary vein or artery).

The effects of SB 217242, a non-peptide endothelin (ET) receptor antagonist, were investigated against hypoxia-induced cardiopulmonary changes in high altitude-sensitive rats. In isolated pulmonary artery rings, SB 217242 (30 n m ) antagonized ET-1-induced contractions with apKBof 8.0. There was no difference in the sensitivity to ET-1 or the potency of SB 217242 in pulmonary artery from normoxic rats vs. rats exposed to hypoxia (9% O2) for 14 days. However, there was a marked reduction in the maximum response to ET-1, but not to KCl or phenylephrine, in pulmonary artery from hypoxic rats; this phenomenon was inhibited by treatment of animals with SB 217242 (10.8 mg/day, ip by osmotic pump) for the 14-day hypoxic period. Furthermore, there was a significant reduction in carbachol-induced, endothelium-dependent relaxation of precontracted pulmonary artery from hypoxic animals; SB 217242 treatment during the hypoxic period did not influence this difference. Vehicle-treated rats exposed to 14-day hypoxia had 173% higher pulmonary artery pressures and 75% higher right/left+septum ventricular mass ratios compared to normoxic animals. SB 217242 (3.6 or 10.8 mg/day, ip) markedly reduced (80 and 95%, respectively) hypoxia-induced increases in pulmonary artery pressure. Right ventricular hypertrophy was inhibited by 40% at the 10.8 mg/day dose. Marked medial thickening and luminal stenosis of small and medium-sized pulmonary arteries was observed in hypoxic rats. The SB 217242-treated, hypoxia-exposed rats had comparable small and medium-sized arteries to normoxic rats. Rats treated with SB 217242 (10.8 mg/day) for the last 14 days of a 28-day hypoxic exposure had significantly lower pulmonary artery pressures than those of vehicle-treated rats. In addition, the effects of the selective ETAreceptor antagonist, SB 247083, and the selective ETBreceptor antagonist, A-192621 (3.6 or 10.8 mg/day, ip), were compared against hypoxia-induced increases in pulmonary artery pressure and plasma ET concentrations. SB 247083, but not A-192621, inhibited hypoxia-induced pulmonary hypertension, whereas A-192621, but not SB 247083, significantly exacerbated hypoxia-induced increases in ET concentrations, suggesting that hypoxia-induced pulmonary pressor responses are mediated via ETAreceptor activation, while ETBreceptor blockade may alter clearance of hypoxia-induced elevated plasma ET. The inhibitory effects of SB 217242 on the functional and remodeling changes induced by hypoxia provide further evidence that ET may play a central role in pulmonary hypertension and that ET receptor antagonists may have a utility in the treatment of this disease.

The aim of this study was to measure the effects of food restriction on antigen–induced bronchoconstriction and inflammatory cell influx in guinea pigs and to determine the role of plasma cortisol and catecholamine concentrations. Ovalbumin (OA; 0.3 mg/kg, i.v.) was administered to OA–sensitized, anesthetized guinea pigs which had been allowed free access to food or had been food restricted for 18 h prior to OA challenge. In addition to higher plasma levels of epinephrine (30% increase) and cortisol (33% increase), fasted guinea pigs had significantly lower (60% decreased) maximal bronchoconstrictor responses to OA than nonfasted, sensitized litter mates. Additionally, groups of fasted or fed animals were subdivided into two additional treatment groups: (1) saline–pretreated or (2) polyethylene glycol 400 (PEG)–pretreated (1 ml/kg, p.o., 1 h prior to antigen challenge). In saline–treated, fasted animals, bronchoconstrictor responses to antigen were significantly diminished (67% decreased) and epinephrine and cortisol levels were increased (64 and 34%, respectively) compared to the corresponding fed group. In both fasted and fed groups, the PEG–treated guinea pigs had higher plasma epinephrine and cortisol levels than animals which received saline, but no significant differences were detected within the PEG–treated group. Plasma norepinephrine concentrations were lower in all fasted groups. In a separate model in conscious guinea pigs, there were no differences in aerosol OA–induced bronchoconstriction and eosinophil influx between fasted and fed groups. However, compared to the saline pretreatment group, PEG administration reduced the antigen–induced bronchoconstriction and eosinophilia in both fed and fasted guinea pigs. We speculate that the reduced responsiveness to antigen in fasted versus fed animals may result from food–restriction–induced, stress–related release of epinephrine and cortisol from the adrenal glands, thereby suppressing mast cell degranulation or reducing responsiveness to spasmogenic and chemotactic mediators. In addition, the results suggest that oral dosing with 100% PEG may enhance this phenomenon.

Prolonged exposure to hypoxia elicits a variety of time-related morphologic and physiologic changes in the pulmonary vasculature of mammals, including humans. The study of hypoxia-induced changes in rodents generally requires a prolonged exposure to 9% oxygen for a minimum of 10 days in an airtight chamber, which has only been generally described in the literature as large (200-400 l), sealed acrylic chambers. To assist in the search for better therapies for diseases associated with chronic hypoxia using animal models, we have custom-built an airtight chamber for hypoxic exposure of rodents, and characterized the effect of chronic hypoxia on functional and morphologic changes in the pulmonary vasculature of the guineapig using this system. This chamber has been designed to alleviate any unnecessary stress related to food or water intake, cleanliness and excess illumination to the animals during the hypoxic-exposure period. Chronic exposure of the guineapig to hypoxia (0-21 days) produced time-related physiologic, morphologic, and haematologic changes. For example, after 10 days in hypoxia (9% oxygen), pulmonary artery pressure was significantly increased from 13 +/- 1 mmHg in normoxic controls (day 0, n = 6) to 26 +/- 0 mmHg (day 10, n = 4, P0.01). Right ventricular hypertrophy in hypoxic animals, presented as a ratio of right ventricle free wall weight to body weight, showed a significant increase from 0.054 +/- 0.004 (day 0) to 0.069 +/- 0.004 on day 10 (P0.05), while age-matched normoxic animals showed no changes in right ventricular weight (day 0 = 0.059, day 10 = 0.058; P0.05). Red blood cell count significantly increased over the same time period, from 5.9 +/- 0.1 (day 0) to 6.4 +/- 0.1 (day 10, P0.05), as did haematocrit, 48 +/- 0.7 (day 0) to 61 +/- 0.9 (day 10, P0.05), and haemoglobin, 16 +/- 0.2 (day 0) to 20 +/- 0.1 (day 10, P0.05). It is concluded that considerations for the well-being of the test animals (i.e. continuous water, ample food supplies, burrow-like hiding places, sanitation and protection from excess illumination) can easily be incorporated into a hypoxic chamber. The purpose of the present study was to explore modifications that may provide the animal with an optimized environment which will reduce anxiety and stress, as seen in their behaviour when inside the chambers, and to thoroughly characterize the morphologic and physiologic changes associated with chronic hypoxia which develop in a consistent time-related manner.

1. To explore potential differences between endothelin (ET) receptors in airway versus vascular smooth muscle from the same species, the ETB receptors mediating contractions produced by ET-1, ET-3 and the selective ETB ligands, sarafotoxin S6c (S6c) and BQ-3020, in rabbit bronchus and pulmonary artery were investigated by use of peptide and non-peptide ET receptor antagonists. 2. In rabbit pulmonary artery SB 209670 (10 microM), a mixed ETA/ETB receptor antagonist, was a more potent antagonist of contractions produced by S6c (pKB = 7.7; n = 9; P < 0.05), than those elicited by ET-1 (pKB = 6.7; n = 6) or ET-3 (pKB = 6.7; n = 5). BQ-788 (10 microM), an ETB receptor antagonist, inhibited responses produced by ET-3 (pKB = 5.1; n = 8), BQ-3020 (pKB = 5.2; n = 4) or S6c (pKB = 6.2; n = 9; P < 0.05 compared to potency versus ET-3- or BQ-3020-induced contractions), but was without inhibitory effect on ET-1-induced contractions (n = 5). RES-701 (10 microM), another selective ETB receptor antagonist, was without effect on contractions produced by S6c (n = 4) or ET-1 (n = 4), and potentiated ET-3- (n = 5) or BQ-3020-induced responses (n = 4). 3. The combination of BQ-788 (10 microM) and BQ-123 (10 microM), an ETA-selective receptor antagonist, antagonized contractions produced by lower concentrations of ET-1 (1 and 3 nM) in rabbit pulmonary artery, but was without effect on responses elicited by higher concentrations of ET-1 (n = 5). The combination of RES-701 (10 microM) and BQ-123 (10 microM) potentiated responses elicited by ET-1, producing a 3.7 fold shift to the left in the agonist concentration-response curve (n = 5). 4. In rabbit bronchus SB 209670 (3 microM) had similar potency for antagonism of contractions produced by ET-1 (pKB = 6.3; n = 6), ET-3 (pKB = 6.5; n = 6) or S6c (pKB = 6.1; n = 8). BQ-788 (3 microM) was without effect on responses elicited by ET-1, ET-3 or S6c (n = 6) but antagonized BQ-3020-induced contractions (pKB = 6.4; n = 4). RES-701 (3 microM) was without effect on contractions produced by S6c (n = 6) or BQ-3020 (n = 4), and potentiated rather than antagonized ET-1- or ET-3-induced responses (n = 6), reflected by a significant (about 6 fold) shift to the left in ET-1 or ET-3 concentration-response curves. The combination of BQ-788 (3 microM) and BQ-123 (3 microM) was without effect on contractions produced by ET-1 in rabbit bronchus (n = 6). The combination of RES-701 (3 microM) and BQ-123 (3 microM) potentiated responses elicited by ET-1, producing a 5.2 fold shift to the left in the agonist concentration-response curve (n = 5). 5. BQ-123 (3 or 10 microM), an ETA-selective receptor antagonist, was without effect on ET-1, ET-3 or S6c concentration-response curves (n = 3-6) in rabbit pulmonary artery or rabbit bronchus. 6. These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in rabbit pulmonary artery or rabbit bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, the qualitative and quantitative differences in the relative profiles of the various structurally diverse peptide and non-peptide antagonists examined suggests that responses produced by the ET ligands may not be mediated by a homogeneous ETB receptor population. In addition, the results suggest that differences exist in the ETB receptors mediating contraction in pulmonary vascular versus airway tissues in the same species. These receptors are not very sensitive to the standard ETB receptor antagonists, BQ-788 and RES-701. Furthermore, the results also provide further evidence that the potencies of ET receptor antagonists depend upon the ET agonist.

[125I]-endothelin-1 ([125I]-ET-1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non-asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non-asthmatic and asthmatic lung. The use of the receptor subtype-selective ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non-asthmatic alveolar wall tissue demonstrated that 29-32% of specific [125I]-ET-1 binding was to ETA sites and 68-71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.

1. The present study was undertaken to determine the mechanism of action of endothelin-1 (ET-1)-induced contraction of the guinea-pig isolated trachea. 2. ET-1 (1 nM-0.3 microM) produces a concentration-dependent contraction of guinea-pig trachea with an EC50 of approximately 25 nM. The combination of the peptidoleukotriene receptor antagonist, SKF 104353 (10 microM) and the H1-histamine receptor antagonist, mepyramine (10 microM), which abolishes antigen-induced contraction in guinea-pig trachea, was without effect on ET-1 concentration-response curves. Furthermore, the platelet-activating factor (PAF) receptor antagonist, WEB 2086, (1 or 10 microM) did not inhibit ET-induced contraction. 3. ET-1 (0.3 microM) did not stimulate histamine or immunoreactive peptidoleukotriene release from guinea-pig isolated trachea. 4. The release of various prostanoids from guinea-pig trachea was increased significantly by ET-1 (0.3 microM); the profile of release was prostaglandin D2 (PGD2) = PGE2 = 6-keto PGF1 alpha (PGI2 metabolite)thromboxane B2 = PGF2 alpha9 alpha, 11 beta PGF2 (PGD2 metabolite). ET-1-induced release of prostaglandins, which was about 30% of that elicited by antigen in sensitized tissues, was not affected by epithelium removal and was observed in tissues from which the smooth muscle had been removed. Previous studies in our laboratory indicated that indomethacin potentiated contraction produced by high concentrations of ET-1, whereas a thromboxane receptor antagonist was without appreciable effect on ET-1 concentration-response curves. 5. Pretreatment of tissues for 1 h with capsaicin (10 microM), which depletes different sensory neurones, produced a small, but significant, inhibitory effect on ET-1 concentration-response curves in the presence but not the absence of the epithelium. The combination of the NK1 tachykinin receptor antagonist,CP-96,345 (0.1 microM), and the NK2 tachykinin receptor antagonist, SR 48968 (0.1 microM), was without effect on ET-l concentration-response curves but substantially antagonized capsaicin-induced contraction.6. The present data suggest that in guinea-pig isolated trachea, ET- 1 produces contraction predominantly via a direct mechanism: there is no significant contribution of the release of histamine,leukotrienes, PAF, or tachykinins (acting on NK1 or NK2 receptors). Although ET-1 evokes the release of an array of prostanoids from the trachea they do not appear to have a major influence on the contractile response.

1. In this study the endothelin (ET) receptor subtypes mediating contractions produced by ET-1 in human and guinea-pig pulmonary tissues were investigated. In addition the receptor responsible for ET-1-induced prostanoid release in human bronchus was determined. 2. In human bronchus and human pulmonary artery ET-1 (0.1 nM-0.3 microM) was a potent and effective contractile agent (pD2 = 7.58 +/- 0.15, n = 6, and 8.48 +/- 0.11, n = 7, respectively). BQ-123 (1-10 microM), a potent and selective ETA receptor antagonist, potently antagonized ET-1-induced contraction in human pulmonary artery (pKB = 6.8 with 1 microM BQ-123, n = 7) but had no effect in human bronchus (n = 6). 3. Sarafotoxin S6c (0.1 nM-0.1 microM), the ETB-selective agonist, did not contract human pulmonary artery (n = 5), but potently and effectively contracted human bronchus: pD2 = 8.41 +/- 0.17, maximum response = 74.4 +/- 3.1% of 10 microM carbachol; n = 5. BQ-123 (1-10 microM) did not antagonize sarafotoxin S6c-induced contraction in human bronchus (n = 5). 4. ET-1 potently contracted guinea-pig trachea, bronchus, pulmonary artery and aorta (pD2 = 8.15 +/- 0.14, 7.72 +/- 0.12, 8.52 +/- 0.12, and 8.18 +/- 0.12, respectively, n = 6-14). BQ-123 (0.1-10 microM)antagonized ET-1-induced contractions in guinea-pig pulmonary artery (pKB = 6.7 with 1 microM BQ-123,n = 6), aorta (pKB = 7.1 with 1 microM BQ-123, n = 6) and trachea (pKB = 6.2 with 1 microM BQ-123, n = 6) butwas without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nM-0.l microM) did not contract guinea-pig aorta (n = 4) or guinea-pig pulmonary artery (n = 6) but potently and effectively contracted guinea-pig bronchus: pD2= 8.55 +/- 0. 1; maximum contraction = 63.6 +/0 3.1% of 10 microM carbachol,n = 4. Sarafotoxin S6c (0.1 nM-0. 1 microM) was a much less effective agonist in guinea-pig trachea:maximum contraction = 13.9 +/- 2.5% of 10 JM carbachol, n = 4; P< 0.0001, compared to bronchus.Contractions produced by sarafotoxin S6c in guinea-pig bronchus or trachea were unaffected by BQ-123(IO microM, n=4).5. Significant differences were observed in the efficacy, relative to carbachol, but not the potency of sarafotoxin S6c in guinea-pig airways, with a much greater maximum contractile response in bronchus(69.6 +/- 2.4% of 10 microM carbachol, n = 6) or lower region of the trachea (48.5 +/- 5.9% of 10 microM carbachol,n = 6) than in the middle region of the trachea (14.4 +/- 4.0% of 10 microM carbachol, n = 6) or the upper region of the trachea (19.3 +/- 2.7% of 10 microM carbachol, n = 6). There were minimal regional differences in either ET-1-induced contraction or the potency of BQ-123 (3 microM) for inhibition of responses to ET-1 in guinea-pig airways.6. Release of various prostanoids in human bronchus induced by ET-1 (0.3 microM) was essentially abolished with 10 IM BQ-123.7. These data provide evidence that distinct ET receptors mediate ET-1-induced contraction in human pulmonary artery, guinea-pig pulmonary artery and guinea-pig aorta (ETA subtype) compared with human bronchus and guinea-pig bronchus (non-ETA, perhaps ETB subtype). Contractions to ET-1 in guinea-pig trachea appear to involve both ETA and non-ETA (ETB?) receptor subtypes. Furthermore,regional differences appear to exist in the relative distribution of ET receptor subtypes in guinea-pig airways. In human bronchus ET-1-induced prostanoid release, unlike the contractile response, appears to be mediated via ETA receptor activation.

The aim of this study was to determine whether methoxamine and barium stimulate 45 Ca 2+ uptake or efflux in the rat vas deferens in a manner that correlates with their contractile activity, and whether 45 Ca 2+ movements are inhibited by verapamil or nifedipine. Basal La 3+ -resistant (cellular) 45 Ca 2+ uptake was significantly greater in the epididymal half (791 ± 27 nmol g −1 ) than in the prostatic half (654 ± 14 nmol g −1 ) of the rat vas deferens and was unaffected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) was without effect on 45 Ca 2+ uptake in either half but BaCl 2 (1 mM) increased 45 Ca 2+ uptake by 31% in the prostatic half and by 22% in the epididymal half. The barium-induced increases in 45 Ca 2+ uptake were markedly reduced or abolished by verapamil (2 μM) or nifedipine (0.3 μM), which at these concentrations have no effect on the rhythmic contractions but abolish the initial small phasic contraction induced by barium. The basal rate of 45 Ca 2+ efflux from the intact vas deferens (into Ca 2+ containing Krebs-Henseleit solution or into Ca-free Krebs-Henseleit solution ± EGTA 0.05 mM) was not affected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) produced a marked, transient and reversible increase in 45 Ca 2+ efflux into 2.5 mM CaCl 2 Krebs-Henseleit in 50% of the intact vasa deferentia examined which was augmented by verapamil (61 μM). BaCl 2 (1 mM) produced a small increase in 45 Ca 2+ efflux into Ca 2+ -containing and Ca 2+ -free Krebs-Henseleit solutions from some intact vasa deferentia and this was not inhibited by nifedipine (14 μM). It is concluded that the BaCl 2 -induced increase in 45 Ca 2+ uptake is likely to be associated with the initial phase of the contractile response, rather than the secondary rhythmic contractions. Rhythmic contractions initiated by methoxamine and barium may be initiated by ‘trigger Ca’ entering through a population of calcium channels that have low affinity for calcium antagonist drugs.

1. The present study was undertaken to examine further the contribution of hypoxia to airway epithelium-dependent relaxation of rat aorta in the co-axial bioassay. 2. Endothelium-denuded rat aorta contracted with phenylephrine (0.05 microM) relaxed in a time-dependent manner (t1/2 = 8.3 +/- 0.4 min, n = 38) when the bathing solution was bubbled with 95% N2 and 5% CO2. In co-axial bioassays, the t1/2 for histamine (100 microM; guinea-pig trachea)- and methacholine (100 microM; rabbit bronchus)- induced relaxation was 1.9 +/- 0.2 min (n = 14) and 1.2 +/- 0.1 min (n = 26), respectively. 3. Hypoxia-induced relaxation was not associated with a rise in intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP). This contrasts with previous findings of an elevation in cyclic GMP associated with epithelium-dependent relaxation of rat aorta in co-axial bioassays. 4. Hypoxia-induced vascular relaxation was antagonized by the ATP-sensitive K+ channel blocker, glibenclamide (100 microM). In contrast, glibenclamide (100 microM) failed to inhibit histamine (100 microM; guinea-pig trachea)- and methacholine (0.1-100 microM; rabbit bronchus)-induced release of epithelium-derived inhibitory factor (EpDIF), in co-axial bioassays. Glibenclamide (100 microM) antagonized BRL 38227 (lemakalin), but not isoprenaline-induced relaxation of phenylephrine-contracted rat aorta. 5. These data strongly suggest that the airway epithelium-dependent relaxant responses observed in co-axial bioassays cannot be attributed to hypoxia.

Asthma is a complex, multifactorial disease that is underpinned by airway inflammation. A variety of cytotoxic substances are released into the airway from infiltrating inflammatory cells, especially the eosinophil. These cytotoxic substances, including reactive oxygen metabolites, produce damage to the airway epithelium, a histologic feature of chronic asthma. Damage to the airway epithelium, in turn, is thought to be a major factor responsible for the development of airway hyperreactivity, a hallmark of asthma. One notable molecular target for novel antiasthmatic drugs is the cyclic AMP-specific phosphodiesterase (PDE) or PDE IV. This isozyme is the predominant form of cyclic nucleotide PDE activity in inflammatory cells. Thus, in view of the putative role of cyclic AMP as an inhibitory second messenger in these cells, PDE IV inhibitors have been shown to suppress inflammatory cell activity. The purpose of the present experiments was to examine the effect of the PDE IV inhibitor, R-rolipram, on three key functions of the guinea pig eosinophil: a) superoxide anion (O2-) production, b) adhesion to human umbilical vein endothelial cells (HUVECs), and c) infiltration into the airway. R-rolipram-elevated eosinophil cyclic AMP content (EC50 = 1.7 microM) and inhibited fMLP-induced O2- production in a concentration-dependent manner (IC50 = 0.3 microM). In contrast, neither siguazodan, a PDE III inhibitor, nor zaprinast, a PDE V inhibitor, had an appreciable effect. R-rolipram (30 microM) also reduced by 25 to 40% the adhesion of eosinophils to HUVECs stimulated with phorbol myristate acetate or tumor necrosis factor-alpha, particularly under conditions in which both cell types were simultaneously exposed to the PDE IV inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Our laboratory has been comparing the activity of a water extract of cotton bract (CBE) with the isolated trachealis smooth muscle of the dog, guinea pig, and cat. CBE induced contractions that were not mediated by 5-hydroxytryptamine (5-HT), histamine, or muscarinic receptors. The active agent(s) in CBE was dialyzable (less than 14,000 molecular weight), and substantial activity was retained after low-temperature ashing. CBE potentiated contractions of dog trachealis to histamine and 5-HT and relaxation responses to isoproterenol, whereas it had no effect on responses to methacholine and KCl. In the guinea pig trachealis, CBE reduced responsiveness to KCl, potentiated relaxations to adenosine and ATP, and did not alter the responses to the remaining agents. Responses of cat trachealis to KCl and isoproterenol were potentiated by CBE, while those to 5-HT were unaffected. Neurogenic cholinergic contractile responses were potentiated by CBE in the trachealis of the dog, but not of the guinea pig, while neurogenic relaxations were potentiated by CBE in guinea pig trachealis but not in the dog trachealis. There are thus marked species differences in the acute effects of CBE on airway smooth muscle. Due to recent interest in the possible involvement of bacterial endotoxins in the etiology of byssinosis, we examined the effects of E. coli lipopolysaccharide (LPS) in guinea pig trachealis. An initial examination revealed that LPS potentiated responses to histamine, but not those to methacholine and isoproterenol. This effect vanished upon a second appraisal with a different batch of LPS. The effect of LPS in airway smooth muscle is thus, at present, equivocal.

Local anaesthetic activity was measured on the rat phrenic nerve at 37 degrees C: Verapamil HCl had 5.1X and methoxyverapamil HCl had 3.0X the potency of lidocaine. Flunarizine was about equipotent with lidocaine, while nifedipine, diazoxide and sodium nitroprusside were without effect. It is concluded tht the smooth muscle inhibitory actions of the calcium antagonists, even when used in concentrations possessing local anaesthetic activity, are due to a reduction in Ca2+ permeability. In rat isolated vasa deferentia, lidocaine had both stimulant and inhibitory actions. On its own it induced rhythmic contractions (not blocked by phentolamine) and it also augmented the frequency of rhythmic contractions produced by methoxamine or barium. These actions may be due to block of K+ channels. Lidocaine also reduced the amplitude of methoxamine and barium induced contractions and both phases of the KCl contraction. The KCl response was restored by increasing "Ca2+]0, and it is therefore concluded that lidocaine was acting by blockade of Ca2+ channels.

Removal of the epithelium increased the responsiveness of isolated guinea-pig trachea to the contractile effects of endothelin. This phenomenon was observed in the presence of indomethacin (5 microM), captopril (10 microM), bacitracin (20 micrograms/ml) or leupeptin (50 microM), but was inhibited by phosphoramidon (10 microM). Bacitrain enhanced contraction produced by endothelin. The increased responsiveness of denuded guinea-pig trachea to endothelin may be due to removal of an epithelium-derived phosphoramidon-sensitive peptidase.

In the rat isolated vas deferens, methoxamine 8.1 microM produced an initial phasic response that declined towards baseline and was followed by rhythmic contractions that continued until wash-out. These responses were predominant in the epididymal half. BaCl2 1 mM produced a similar type of response which was not mediated by noradrenaline release or activation of alpha-adrenoceptors. The barium responses were similar in the epididymal and prostatic halves. Incubation in nominally Ca2+-free solution caused abolition or near abolition of rhythmic contractions produced by barium or methoxamine. The initial phasic response to methoxamine was abolished in Ca2+-free solution, whereas that produced by barium persisted. Rhythmic contractions produced by methoxamine or barium were inhibited by Mg2+ (2.4-20 mM) and by La3+ (1-5 mM). Mg2+ had selectivity for inhibition of the frequency of methoxamine- but not barium-induced rhythmic contractions. Despite their dependence on [Ca2+]o, barium- and methoxamine-induced rhythmic contractions were resistant to inhibition by calcium channel inhibitors. Verapamil, nifedipine and flunarazine inhibited the amplitude of rhythmic contractions more readily than the frequency (methoxamine IC50 for verapamil: amplitude = 29.8 +/- 5.40 microM, n = 6, frequency = 96.7 +/- 31.0 microM, n = 5, for nifedipine: amplitude = 2.42 +/- 0.34 microM, n = 7, frequency = 3.24 +/- 0.75 microM, n = 7, and for flunarizine: amplitude = 15.9 +/- 5.95 microM, n = 7, frequency = 153 +/- 28.6 microM, n = 7). There was no differentiation between inhibition of methoxamine and barium-induced responses. Like Mg2+, methoxyverapamil selectively inhibited the frequency of methoxamine-induced contractions (IC50: amplitude = 16.8 +/- 2.86 microM, n = 5, frequency = 2.07 +/- 0.81 microM, n = 5) but not barium-induced contractions (IC50: amplitude = 13.9 +/- 1.95 microM, n = 5, frequency = 48.5 +/- 8.98 microM, n = 5). Diazoxide (43.3-2167 microM) and nitroprusside (3.36-6712 microM) had only a small effect on barium contractions, but produced a dose-related reduction in the amplitude of methoxamine-induced responses. Diazoxide and nitroprusside caused methoxamine contractions to occur in groups, although they had no effect on their overall frequency. It is concluded that barium- and methoxamine-induced rhythmic contractions in the rat vas deferens are mediated by the entry of [Ca2+]o via membrane calcium channels that have a lower affinity (10-100 X) for calcium channel inhibitors than those mediating the KCl response. Channels activated by methoxamine are concentrated in the epididymal half, whereas those opened by barium are evenly distributed. However, although responses to methoxamine and barium are similar in form, differences in the effects of some of the drugs tested, together with the results of previous studies, indicate that they produce contractions by different mechanisms.

1 5-Hydroxytryptamine (5-HT) (5.16-1291 microM) produced a phasic contraction followed later by rhythmic contractions in the rat vas deferens, primarily in the epididymal half. 5-HT (129 microM) produced no response in Ca2+-free solution. Nifedipine (0.29 microM) or verapamil (2.04 microM) inhibited the initial phasic response to 5-HT, but inhibition of the rhythmic contractions required concentrations 5 fold (nifedipine) or 30 fold (verapamil) higher. 2 Methysergide (2.13 microM) abolished the phasic and reduced the frequency of the rhythmic contractions. Phentolamine (2.65 microM) did not affect the phasic response but reduced the amplitude of the rhythmic contractions. The combination of phentolamine (2.65 microM) and methysergide (2.13 microM) completely abolished the response to 5-HT (129 microM). 3 Desipramine (1.32 microM) had no effect on the phasic response to 5-HT (129 microM), but the rhythmic contractions were reduced in amplitude with no effect on their frequency. 4 In vasa deferentia removed from reserpine-treated or from guanethidine-denervated rats, both phasic and rhythmic components of the 5-HT (129 microM) contraction were augmented due to supersensitivity. 5 It is concluded that the phasic component of the 5-HT contraction is mediated by post-junctional 5-HT receptors, while the rhythmic component is mediated by the combination of post-junctional 5-HT receptors and noradrenaline released from neuronal stores. Assuming that nifedipine and verapamil are acting solely by inhibition of calcium channels, the phasic and rhythmic components of the 5-HT response may be mediated through separate Ca2+ channels. If this is correct, one channel might be a voltage-dependent channel and the other could be similar to, but distinct from the channel mediating the response to methoxamine.

1 Both phasic and tonic responses to KCl 160 mM were reduced by Ca2+ deprivation. After 90 min, the phasic response was abolished but 13 +/- 1.5% of the tonic response remained. This resistant component was still present if the Ca2+-free solution contained EGTA 0.1 mM. The tonic response was more resistant to deprivation in the prostatic half, while the phasic was more resistant in the epididymal half. KCl-induced contractions were completely restored 5 min after readmission of Ca2+. 2 Both the phasic and the tonic responses were reduced on lowering, and increased on raising [Ca2+]0. In 0.1 mM Ca2+, the phasic response was abolished, but 23 +/- 4% of the tonic response remained (mainly attributable to the prostatic half). These resistant contractions indicate that some of the extracellular Ca2+, especially in the prostatic half, is bound with high affinity, probably to the plasma membrane. 3 Incubation with LaCl3 (0.3-10 mM) for 15 min inhibited the phasic response more than the tonic. After incubation for 1 h, 3 mM LaCl3 abolished both phases. It is concluded that La3+ blocks Ca2+ channels most readily when they are opened during the spike. Hydralazine (0.76-5.1 mM) resembled LaCl3 in that it reduced the phasic response with little effect on the tonic. 4 MnCl2 (0.3-10mM) reduced the phasic but increased the tonic response at all concentrations. The augmenting effect may be due to release of intracellular Ca2+ or to inhibition of Ca2+ efflux. 5 The tonic response was inhibited more than the phasic response by nifedipine (0.002-0.01 microM), methoxyverapamil (0.06-2 microM), verapamil (0.2-1 microM), flunarizine (0.2-100 microM) and diazoxide (22-650 microM). With higher concentrations, only flunarizine, remained selective for the tonic response. It is concluded that flunarizine blocks Ca2+ channels most readily when opened during sustained spike-free depolarization. 6 Methoxyverapamil 48 microM and verapamil 100 microM virtually abolished both phases of the contraction to KCl 160 mM, but no more than 80% inhibition could be produced with nifedipine. It is concluded that voltage-sensitive Ca2+ channels exist in two sub-types, one of which is blocked by nifedipine, and both are blocked by verapamil, methoxyverapamil and flunarizine. Nitroprusside 17 microM had no effect on the phasic response but inhibited the nifedipine-resistant component of the tonic response. 7 Increasing [ca2+]0 reversed the effects of verapamil, methoxyverapamil, nifedipine and MnCl2, but not the effects of LaCl3. 8 Dantrolene sodium (1.25-25 microM) had no effect on KCl-induced contractions.

In the prostatic half of the rat vas deferens, responses to noradrenaline 30 microM were biphasic. Ca-deprivation reduced both phases. The initial part of the noradrenaline contraction was resistant to verapamil (IC50 74.2 microM), methoxyverapamil (IC50 approximately 100 microM) and especially nifedipine (no effect up to 14.4 microM). The late part of this response and the entire response to noradrenaline in the epididymal half were abolished by verapamil (10.2 microM), methoxyverapamil (9.6 microM) or nifedipine (1.44 microM). The first phase of the twitch to single pulse transmural stimulation was abolished by nifedipine (14.4 microM) and inhibited by methoxyverapamil (57.6-96 microM); verapamil was less potent. The second phase of the twitch was totally resistant to nifedipine (14.4 microM), but more sensitive to inhibition by verapamil or methoxyverapamil than was the first. It was concluded that the calcium channel inhibitors can distinguish between three distinct types of calcium ion channel in the rat vas deferens: (1) sensitive calcium channels mediating the noradrenaline response in the epididymal half, and the late part of the noradrenaline response in the prostatic half (2) calcium channels responsible for the first phase of the twitch, blocked by high concentrations of nifedipine (3) calcium channels mediating the second phase of the twitch and the initial response to noradrenaline in the prostatic half, unaffected by nifedipine.

The effects of KCl 160 mM on 45Ca uptake and efflux in the rat isolated vas deferens were investigated using a modification of the lanthanum method and a superfusion system respectively. In the prostatic half, basal cellular 45Ca uptake was 679 +/- 21 nmol g-1 tissue wet weight and KCl 160 mM increased this by 155%. In the epididymal half, basal cellular 45Ca uptake was 730 +/- 28 nmol 45Ca g-1 and KCl 160 mM increased this by 46%. Verapamil 2.04 microM or nifedipine 0.29 microM had no or little effect on basal 45Ca efflux or on basal 45Ca uptake. The KCl-induced increase in 45Ca uptake in both halves was inhibited by verapamil 2.04 microM or nifedipine 0.29 microM, concentrations which markedly reduce the contractile response. It is concluded that high K+ contracts the rat vas deferens by stimulation of the entry of extracellular Ca2+ to the intracellular compartment. KCl 160 mM produced a large, rapid and reversible increase in the rate of 45Ca efflux into Ca2+-containing and Ca2+-free Krebs-Henseleit solutions, which was not inhibited by verapamil 2.04 microM, nifedipine 0.29 microM or nitroprusside 1,678 microM. The relative size of the slow component of 45Ca efflux was larger in the prostatic half compared to the epididymal half of bisected tissues, suggesting that the postulated high affinity binding sites are predominant in this region. However, the rates of the fast and slow components of 45Ca efflux from prostatic and epididymal halves were identical. KCl 160 mM produced a similar increase in 45Ca efflux in prostatic and epididymal halves.

Using a novel preparation of guinea-pig trachea, direct evidence was obtained for the release of an epithelium-derived inhibitory factor modulating contractions elicited by antigen. Thus, epithelium removal produced a 6.1-fold leftward shift in ovalbumin concentration-response curves in tracheal strips from ovalbumin-sensitized guinea-pigs. This increase in sensitivity to ovalbumin was not evident when a tracheal portion containing epithelium was positioned in close proximity to the tissue from which tension was recorded.

1. In co-axial bioassays, in the presence of indomethacin, addition of histamine (100 microM) or methacholine (100 microM) to guinea-pig trachea produced an epithelium-dependent relaxation of precontracted rat aorta which was associated with an approximately 2 fold elevation in tissue levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Removal of the airway epithelium abolished the histamine-induced relaxation of rat aorta and the associated increase in intracellular cyclic GMP. 2. Epithelium-dependent relaxation was not associated with altered adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in rat aorta. Unstimulated intact or denuded guinea-pig trachea also did not affect the levels of cyclic AMP or cyclic GMP in rat aorta. 3. Methylene blue (10 microM) abolished the methacholine-induced, endothelium-derived relaxing factor (EDRF)-mediated rise in intracellular cyclic GMP in rat endothelium-intact aorta alone. In contrast, methylene blue (10 microM) did not affect the methacholine-induced epithelium-dependent rise in intracellular cyclic GMP in rat endothelium-denuded aorta in the co-axial bioassay. 4. Relaxation of the rat aorta without endothelium was associated with increased levels of cyclic GMP (but not cyclic AMP) in response to sodium nitroprusside (5 nM) and of cyclic AMP (but not cyclic GMP) in response to isoprenaline (1 microM). 5. These results provide evidence that the postulated epithelium-derived inhibitory factor (EpDIF) may produce relaxation of vascular tissue via elevation in cyclic GMP levels. Furthermore, some data suggest that EpDIF may act by stimulation of the particulate, rather than the soluble form of guanylate cyclase.