Temporal and spatial patterns of sea lice levels on sea trout in western Scotland in relation to fish farm production cycles.

The relationship between aquaculture and infestations of sea lice on wild sea trout (Salmo trutta) populations is controversial. Although some authors have concluded that there is a link between aquaculture and lice burdens on wild fish, others have questioned this interpretation. Lice levels have been shown to be generally higher on Atlantic salmon farms during the second years of two-year production cycles. Here we investigate whether this pattern relates to lice burdens on wild fish across broad temporal and spatial axes. Within Loch Shieldaig across five successive farm cycles from 2000 to 2009, the percentage of sea trout with lice, and those above a critical level, were significantly higher in the second year of a two-year production cycle. These patterns were mirrored in 2002-2003 across the Scottish west coast. The results suggest a link between Atlantic salmon farms and sea lice burdens on sea trout in the west of Scotland.

Polymorphisms in the endothelin-1 (EDN1) are associated with asthma in two populations.

Endothelin-1 (EDN1) has been reported to be implicated in the pathophysiology of asthma. Literature results on the genetic association of EDN1 in asthma are inconsistent. Eleven single nucleotide polymorphisms in EDN1 were genotyped in 342 and 100 families from UK and Norway, respectively. Asthma, bronchial hyperreactivity (BHR) and atopic asthma phenotypes were analyzed for the family-based association. Five single nucleotide polymorphisms (SNPs) were associated with asthma (0.0017

Endothelin and the respiratory system.

Pharmacological research involving the endothelin peptides has emphasized their activities in vascular systems, from both physiological and pathophysiological perspectives. However, the endothelins are known also to be synthesized and released from respiratory epithelial cells and to have potent effects in nonvascular components of the respiratory tract. Douglas Hay, Peter Henry and Roy Goldie summarize present understanding of the pharmacology of the endothelins in the respiratory system and assess the potential pathophysiological role in asthma.

Airway epithelium-derived inhibitory factor.

Various bronchoactive agents can induce the release from the airway epithelium of an inhibitory substance that is able to relax certain tissues including rat aorta and possibly also airway smooth muscle. This substance, whose existence has recently been confirmed using a new bioassay system, is distinct from nitric oxide (EDRF) and is also known to be non-prostanoid in nature. Roy Goldie and colleagues describe the properties of this factor, and its potential clinical significance.

Cysteinyl leukotrienes in asthma: old mediators up to new tricks.

The cysteinyl leukotrienes have long been suspected to play a role in the pathogenesis of asthma. This speculation was based largely on their release in human lung following antigen challenge as well as their potent bronchoconstrictor activity. However, there is increasing evidence that the cysteinyl leukotrienes also produce several pro-inflammatory effects and alter the activity of neuronal pathways in the airways. Douglas Hay, Theodore Torphy and Bradley Undem review these recent data and discuss the therapeutic possibilities of cysteinyl leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

Chronic obstructive pulmonary disease: emerging therapies.

Despite the high prevalence of and mortality from chronic obstructive pulmonary disease, extensive research on the underlying pathophysiology and specific therapeutics for this disease is, relatively, in its infancy. Several novel molecular targets are being investigated as potential treatments for the disease. The most exciting new class of compounds is the phosphodiesterase 4 inhibitors; Ariflo (SB 207499)-a member of this class, and the most advanced in development (Phase III)-was reported recently to have significant clinical efficacy in patients with chronic obstructive pulmonary disease. Phosphodiesterase 4 inhibitors, such as Ariflo, possibly represent the most important advance in pulmonary medicine in recent years.

Interleukin-8 receptor antagonists in pulmonary diseases.

Inflammatory cells are thought to be instrumental in the pathophysiology of pulmonary diseases, and control of their recruitment and activation in the lung would appear to be an attractive strategy for therapeutic intervention. Interleukin-8 and related CXC chemokines are involved in the function of neutrophils and T cells, and have been implicated in several lung diseases. Small-molecule antagonists of the interleukin-8 receptors have been identified, which may help elucidate the role of interleukin-8 and related chemokines in the pathophysiology of lung diseases.

Calcium uptake into guinea-pig trachealis: the effect of epithelium removal.

Removal of the epithelium from preparations of guinea-pig airways in vitro increases the responsiveness of the smooth muscle of normal and ovalbumin-sensitized animals to a number of contractile agents. To determine if epithelium removal results in an increase in Ca2+ entry into the smooth muscle, the effect of removing the epithelium on Ca2+ uptake into the trachealis smooth muscle was studied using a modified La3+-technique. KCl increased Ca2+ uptake in the presence and absence of the epithelium in control and sensitized animals. Methacholine did not promote Ca2+ uptake, whether or not the epithelium was present, in either control or sensitized animals. Ovalbumin did not stimulate Ca2+ uptake into the trachealis of sensitized animals. These results indicate that the increase in responsiveness of airway smooth muscle seen on epithelium removal is not a consequence of a facilitation of Ca2+ entry into the muscle. The increased responsiveness to methacholine in control animals, and to ovalbumin in preparations in tension studies in epithelium-free tissues from sensitized animals, cannot be explained by an increased availability of extracellular Ca2+ into the muscle, but, rather may reflect some other effect of the epithelium-derived modulatory factor.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 1. Identification of the 4-quinolinecarboxamide framework.

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 2. Identification of (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB 223412).

Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Pharmacological evidence for distinct endothelin receptors in guinea-pig bronchus and aorta.

The ETA receptor antagonist, BQ-123 (1 microM) potently antagonized endothelin-1 (ET-1) concentration-response curves in guinea-pig aorta (pKB = 7.1). However, 10 microM BQ-123 was without effect on ET-1-induced contractions in guinea-pig bronchus. The ETB-selective agonist, sarafotoxin S6c did not contract the aorta but was a potent and effective contractile agonist in the bronchus. BQ-123 (10 microM) was without effect on sarafotoxin S6c-induced contractions in the bronchus. These data provide evidence for distinct endothelin receptors in guinea-pig aorta and bronchus, which appear to be predominantly of the ETA and non-ETA, perhaps ETB, subtypes, respectively.

Mechanism of endothelin-induced contraction in guinea-pig trachea: comparison with rat aorta.

1. Endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of guinea-pig epithelium-containing (intact) trachea (EC50 = 30.9 nM). Endothelin was a less potent agonist than leukotriene D4 (LTD4; EC50 = 0.77 nM), but was more potent than carbachol (EC50 = 0.15 microM) or substance P (EC50 = 1.4 microM). Endothelin was a more potent contractile agent in rat endothelium-denuded aorta (EC50 = 2.1 nM) than in guinea-pig trachea. 2. Endothelin-induced contraction in guinea-pig trachea was unaffected by mepyramine (10 microM), atropine (1 microM), SK&F 104353 (10 microM), a leukotriene receptor antagonist, or SQ 29,548 (1 microM), a thromboxane receptor antagonist. The contraction produced by 0.3 microM endothelin was potentiated by cyclo-oxygenase inhibition with 5 microM indomethacin. 3. Nicardipine (0.01 or 0.1 microM) or incubation in calcium-free medium +0.1 mM EGTA for 30 min had a relatively minor or no effect on endothelin concentration-response curves in guinea-pig intact trachea, but markedly inhibited responses produced by endothelin in endothelium-denuded aorta of the rat. Increasing the EGTA concentration in calcium-free medium to 1 mM abolished endothelin-induced contraction in guinea-pig trachea. 4. In guinea-pig trachea, ryanodine (10 microM) produced a 2.1 fold shift to the right of endothelin concentration-response curves and reduced the maximum response elicited by 0.3 microM endothelin. 5. Staurosporine (0.01 microM and 0.1 microM), a protein kinase C inhibitor, was without effect on endothelin- or carbachol-induced contraction in guinea-pig trachea, but markedly inhibited the response produced by endothelin in rat aorta. 6. Endothelin (3 nM-0.3 microM) produced a concentration-dependent stimulation of phosphatidylinositol (PI) turnover in guinea-pig intact trachea, with an EC50 value of 45.9 nM. 7. Removal of the epithlium markedly potentiated endothelin-induced contraction in guinea-pig trachea, producing a 4.7 fold leftward shift in endothelin concentration-response curves and an increase in the contractile response elicited by 0.3 microM endothelin. 8. These data indicate that endothelin is a potent agonist in guinea-pig trachea whose response is markedly enhanced by removal of the airway epithelium. Endothelin-induced contraction is not mediated to a marked extent by calcium influx via dihydropyridine-sensitive calcium channels and does not involve the release of histamine, acetylcholine, leukotrienes or thromboxane. Rather, endothelin appears to produce contraction of guinea-pig trachea via a direct action which involves stimulation of PI turnover and utilization of calcium from intracellular stores and, also, calcium influx via a pathway that is not sensitive to dihydropyridine calcium channel inhibitors. Endothelin-induced contraction of rat aorta was more sensitive to the effects of incubation in Ca2 +-free medium, nicardipine or staurosporine, suggesting that differences exist in the relative mechanisms whereby endothelin produces contraction in different tissues.

The effects of calcium channel inhibitors and other procedures affecting calcium translocation on drug-induced rhythmic contractions in the rat vas deferens.

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.

Effects of KCl on 45Ca uptake and efflux in the rat vas deferens.

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.

The contractile effects of 5-hydroxytryptamine on the rat isolated vas deferens.

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.

Effects of some organic calcium antagonists and other procedures affecting Ca2+ Translocation on KCl-induced contractions in the rat vas deferens.

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.

Human urotensin-II is a potent spasmogen of primate airway smooth muscle.

The contractile profile of human urotensin-II (hU-II) was examined in primate airway and pulmonary vascular tissues. hU-II contracted tissues from different airway regions with similar potencies (pD(2)s from 8.6 to 9.2). However, there were regional differences in the efficacy of hU-II, with a progressive increase in the maximum contraction from trachea to smaller airway regions (from 9 to 41% of the contraction to 10 microM carbachol). hU-II potently contracted pulmonary artery tissues from different regions with similar potencies and efficacies: pD(2)s=8.7 to 9.3 and maximal contractions=79 to 86% of 60 mM KCl. hU-II potently contracted pulmonary vein preparations taken proximal to the atria, but had no effect in tissues from distal to the atria. This is the first report describing the contractile activity of hU-II in airways and suggests that the potential pathophysiological role of this peptide in lung diseases warrants investigation.

The effects of epithelium removal on the sensitivity of guinea-pig isolated trachealis to bronchodilator drugs.

Mechanical removal of the epithelium increased the sensitivity of tracheal strips to isoprenaline, sodium nitroprusside, and to adenosine (only in the presence of inhibitors of its uptake and metabolism). Epithelium removal was without effect on sensitivity to salbutamol or papaverine. Preincubation of tracheal strips with an inhibitor of extraneuronal uptake, corticosterone (50 microM), had no effect on tissue sensitivity to either salbutamol or papaverine. However, the steroid both increased sensitivity to isoprenaline, and abolished the effect of epithelium removal on sensitivity to this catecholamine. These results suggest that in the guinea-pig, the tracheal epithelium is a major source of extraneuronal uptake for catecholamines. Furthermore, the increase in trachealis sensitivity to isoprenaline following epithelium removal is probably due to loss of these sites of extraneuronal uptake. The fact that sensitivity to salbutamol, papaverine and adenosine (in the absence of metabolic inhibitors) was not increased by denuding the epithelium indicates that loss of a diffusion barrier to drugs is not the mechanism of increased sensitivity. Adenosine (and possibly nitroprusside) may cause the epithelium to release a smooth muscle excitatory factor. Thus, removal of the epithelium attenuates this excitatory influence and enhances smooth muscle responsiveness to adenosine. These results provide further evidence that the epithelium has an important role in modulating the sensitivity of guinea-pig trachealis to drugs.

Correlation between airway epithelium-induced relaxation of rat aorta in the co-axial bioassay and cyclic nucleotide levels.

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.