Epithelium-derived Inhibitory Factor Research Articles

Influence of the epithelium on acetylcholine release from parasympathetic nerves of the rat trachea.

1. The present study was undertaken to investigate the influence of the airway epithelium on the release of acetylcholine (ACh) from parasympathetic nerves of the rat trachea. Epithelium-intact and epithelium-denuded preparations of rat trachea were incubated with [3H]-choline to incorporate [3H]-ACh into the cholinergic transmitter stores. Release of radiolabelled transmitter ACh was evoked by electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V). 2. Field stimulation both of epithelium-intact and epithelium-denuded radiolabelled tracheal preparations evoked an increase in the efflux of radioactivity; however, the mean stimulation-induced (S-I) efflux from epithelium-denuded preparations (2932 +/- 190 d.p.m., n = 9) was approximately 60% of that from epithelium-intact preparations (4802 +/- 820 d.p.m., n = 11). We have shown previously that, in epithelium-intact (but not epithelium-denuded) tracheal preparations, a substantial proportion of the S-I efflux is resistant to tetrodotoxin (1 microM) and to the removal of extracellular Ca2+, indicating that much of the S-I efflux is not caused by exocytotic release of neuronal [3H]-ACh. In epithelium-denuded tracheal preparations, superfused individually, phosphorylcholine (1 and 100 microM) did not alter S-I efflux. In epithelium-intact tracheal preparations, both in the absence and in the presence of atropine (1 microM), neither N(G)-nitro-L-arginine (100 microM), superoxide dismutase (100 units ml(-1)), indomethacin (10 microM), capsaicin (30 microM) nor alpha-chymotrypsin (1 unit ml(-1)) altered S-I efflux. 3. Experiments were also performed using two tracheal preparations superfused in series. When unlabelled epithelium-intact preparations were present in the upper chamber (superfused first), the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber (superfused second) did not differ significantly from radiolabelled epithelium-denuded preparations superfused individually. Moreover, there was no significant difference in the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber between experiments in which the upper chamber contained epithelium-intact or epithelium-denuded preparations. 4. Field stimulation of epithelium-intact tracheal preparations in the upper chamber with 90, 120 and 300-s periods (trains of 1 ms pulses, 5 Hz, 15 V) did not significantly alter the S-I efflux from radiolabelled epithelium-denuded tracheal preparations in the lower chamber. 5. When introduced into the upper (unlabelled epithelium-intact) and subsequently allowed to superfuse the lower (radiolabelled epithelium-denuded) tracheal preparations, the stable cholinomimetic carbachol (3 microM) markedly reduced the S-I efflux whereas ACh (0.1 and 1 microM) had no significant effect. However, in the presence of the anti-cholinesterase neostigmine (1 microM), ACh (1 microM) significantly reduced S-I efflux, indicating that ACh is subject to rapid hydrolysis by cholinesterase enzymes. When atropine (10 microM) was only exposed to radiolabelled epithelium-denuded preparations in the lower chamber, the inhibitory effects of ACh (1 microM) and carbachol (3 microM) on S-I efflux were prevented. 6. In conclusion, the findings of the present study do not support the notion that the airway epithelium exerts an inhibitory influence on ACh release from parasympathetic nerves of the rat trachea. Alternatively, if epithelium-dependent modulation of cholinergic transmission does occur in the rat trachea, then the mechanism does not appear to involve phosphorylcholine, nitric oxide, superoxide radicals, cyclo-oxygenase products of arachadonic acid, capsaicin-sensitive neuropeptides or vasoactive intestinal peptide. Moreover, the inhibitory effect of carbachol and ACh on transmitter ACh release in the rat trachea appears to be due solely to activation of prejunctional inhibitory muscarinic cholinoceptors on parasympathetic nerves and does not involve the liberation of a putative epithelium-derived inhibitory factor.

Bradykinin-Induced Responses in a Coaxial Bioassay System Composed of Rat Anococcygeus Muscle and Guinea Pig Trachea

1. Epithelium-dependent effects of bradykinin (BK) were investigated in a coaxial bioassay system which consisted of guinea pig trachea as donor organ and rat anococcygeus muscle as test tissue. 2. BK (10 −9 to 10 −5 M) produced concentration-dependent relaxations on the phenylephrine (3×10 −6 M)-precontracted rat anococcygeus muscle mounted alone. Relaxations decreased significantly when muscle was mounted in epithelium-intact trachea. There was also a significant difference between the relaxations obtained in the muscle within epithelium-intact and epithelium-denuded trachea (at 10 −7 to 10 −5 M concentrations). 3. Capsaicin (10 −5 M) pretreatment did not change BK (10 −9 to 10 −5 M)-induced relaxations in each preparation compared with vehicle pretreatment. Indomethacin (10 −6 M) in combination with thiorphan (10 −5 M) and atropine (10 −6 M) did not affect the BK-induced relaxations of the muscle within capsaicin-pretreated epithelium-intact or denuded trachea. 4. CGS 8515 (a specific 5-lipoxygenase inhibitor, 10 −6 M) did not change BK (10 −5 M)-induced relaxation on the muscle alone, and caused an increase of BK-induced relaxation on the muscle within epithelium-intact trachea compared with that obtained without CGS 8515. 5. Results showed that epithelial or nonepithelial factors were capable of modulating the responsiveness of rat anococcygeus muscle to BK. The decreased relaxation by BK in anococcygeus muscle did not occur by the release of cyclooxygenase products or tachykinins from tracheal epithelium, but it may have occurred by the contractile action of lipoxygenase product secreted by nonepithelial sources. In addition, BK might stimulate the secretion of an epithelium-derived inhibitory factor from the trachea.

The epithelial barrier and airway responsiveness.

Epithelial injury and bronchial hyperresponsiveness are commonly associated with airway disease, and are widely considered to occur as the result of inflammatory changes in the airway wall. Mechanistically, the airway epithelium may influence the sensitivity of the airways to provocative stimuli through its primary function as a cellular barrier between the air and the interstitium, or by liberating a variety of bronchoactive mediators, e.g., lipoxygenase and cyclooxygenase products, nitric oxide, and an epithelium-derived relaxing factor (EpDIF). Much attention has focused on the latter function of the epithelium, particularly the putative EpDIF, which has an action considered to be analogous to that of endothelium-derived relaxing factor in blood vessels. The modulation of airway calibre by the epithelium has recently been investigated in vitro using tubular preparations of bronchi, where removal of, or damage to, the epithelium increases the sensitivity to agonists by several orders of magnitude. This contrasts with the effect of removing the epithelium on strips or rings of airway wall, where the increase in sensitivity is small and rather variable, but this has been the primary observation for proposing a putative EpDIF. This review evaluates the barrier or protective function of the airway epithelium and the major role it plays in the modulation of airway responsiveness. A role of a putative EpDIF seems, at best, to be of minor functional significance.

Evidence for preservation of epithelial function in cryopreserved porcine and human bronchi

Porcine and human bronchi have been investigated in vitro without or after storage at -196 degrees C in Krebs-Henseleit solution containing 2.0 M dimethyl sulphoxide and 0.1 M sucrose as cryoprotectants. In bronchi from both species maximal post-thaw contractile responses to acetylcholine (ACh) were reduced by about 25-30% compared to unfrozen bronchi. To assess the viability of bronchi and endothelium-denuded rat aortic strips, was employed. The release of an epithelium-derived inhibitory factor (EpDIF) was induced by ACh and assessed in terms of concentration-dependent relaxation of the endothelium-denuded rat aortae. Following removal of bronchial epithelium, ACh failed to elicit any relaxation of rat aorta. With cryopreserved bronchi from both pig and human about 5 and 30 times higher concentrations of ACh, respectively, were required to elicit the same relaxant response as with unfrozen bronchi. The results suggest that after the freezing-thawing process both smooth muscle and epithelial function is largely preserved and provide support for the use of cryopreservation for storage of airway preparations for pharmacological studies.

Possible roles of airway epithelial cells in the control of airway smooth muscle tone

While the airway epithelium provides a diffusion barrier to the access of inhaled stimulants, it has recently been considered to have a more active physiological and pharmacological role in the regulation of the airway smooth muscle tone. The initial evidence for this comes from the fact that mechanical removal of the airway epithelium increases the in vitro responsiveness of airway smooth muscle to various spasmogens 3-5 times, although the nature of these regulatory mechanisms on the smooth muscle by the epithelium remains largely unresolved. One of the possible explanations is that the epithelium a generates factor(s) that inhibits the responsiveness of the underlying smooth muscle cells (epithelium-derived inhibitory or relaxing factor EpDIF/EpDRF). Furthermore, recent investigations revealed that airway epithelial cells also release a factor(s) that modulates the vascular smooth muscle tone, excitatory-neuroeffector transmission and resting membrane potential of the airway smooth muscle cells (epithelium-derived hyperpolarizing factor: EpDHF). This review will present and discuss the evidence indicating that the epithelium generates and releases a factor(s) that modulates smooth muscle tone, resting membrane potential of airway smooth muscle cells (EpDHF) and excitatory neuro-effector transmission in the airway.

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.

Evidence that epithelium-dependent relaxation of vascular smooth muscle detected by co-axial bioassays is not attributable to hypoxia.

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.

Modulation by the epithelium of the extent of bronchial narrowing produced by substances perfused through the lumen

1 Airway narrowing was determined in vitro as a measure of bronchial reactivity. A bronchial segment from pig lung was perfused with a Krebs solution and the change in flow rate to drugs and small ions perfused intraluminally was compared with that obtained by application to the serosal surface. 2 The sensitivity (EC50) to acetylchloline was 30 times greater on the serosal surface than on the luminal surface. Concentrations of histamine and carbachol which had threshold responses on flow rate when perfused intraluminally virtually stopped flow on the serosal surface. Potassium depolarizing solutions (containing either KCl or K2SO4) and vanadate (VO3-) had little or no effect intraluminally but completely stopped flow through the bronchial segment when applied to the serosal surface, i.e. they closed off the airway. 3 After removal of the epithelium the sensitivity to drugs and K+ perfused intraluminally was increased to equal that on the serosal surface. 4 No evidence for suppression of smooth muscle contraction by a putative epithelium-derived inhibitory factor (EpDIF) could be obtained: no inhibition of smooth muscle contractility was seen when the agents listed above were perfused intraluminally and their perfusion continued while they were applied to outside. 5 It was concluded that the epithelium plays a crucial role as an impermeant barrier in modulating the responsiveness of the airways smooth muscle.

The release of a non‐prostanoid inhibitory factor from rabbit bronchus detected by co‐axial bioassay

1. Methacholine relaxed phenylephrine-contracted aorta of the rat with the endothelium intact. This effect was inhibited by haemoglobin, methylene blue, gossypol, phenidone and L-NG-nitroarginine methyl ester (L-NAME). Rat aorta denuded of endothelium failed to relax in response to methacholine, histamine and the peptidoleukotrienes C4, D4 and E4. 2. Methacholine and histamine but not leukotrienes C4, D4 and E4 relaxed phenylephrine-contracted rat aorta without endothelium when surrounded by rabbit epithelium-intact bronchus. The muscarinic antagonist atropine antagonized the methacholine-induced relaxation. 3. Removal of the epithelium either mechanically or chemically, abolished methacholine-induced relaxation of rat aorta in the co-axial bioassay. These data indicate that the epithelium is responsible for the observed relaxant effect to methacholine and histamine. 4. The cyclo-oxygenase inhibitor, indomethacin, the phospholipase A2 inhibitor, mepacrine and the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), failed to inhibit methacholine-induced relaxation of rat aorta in the co-axial bioassay. This indicates that the epithelium-derived inhibitory factor (EpDIF) is not a product of the cyclo-oxygenase or lipoxygenase pathway or a product derived from activation of phospholipase A2. 5. Haemoglobin, methylene blue, phenidone, gossypol and L-NAME failed to inhibit the relaxation of rat aorta in the co-axial bioassay. These results demonstrate that EpDIF detected in the co-axial bioassay is not endothelium-derived relaxing factor (EDRF) or nitric oxide. Similarly, catalase was without effect. 6. EpDIF is unlikely to be a peptide since papain and alpha-chymotrypsin failed to alter the methacholine-induced relaxation of rat aorta in the co-axial bioassay. Furthermore, thiorphan, captopril and aprotinin were also without effect, suggesting that EpDIF is not a substrate for airway peptidases. 7. The results presented in this paper demonstrate the release of a vasoactive epithelium-derived inhibitory factor (EpDIF) from rabbit intrapulmonary bronchi by use of a co-axial bioassay preparation.

Antigen-induced Release of Airway Epithelium-derived Inhibitory Factor

The ability of guinea pig trachea to produce a vasoactive epithelium-derived inhibitory factor (EpDIF) in response to mast cell-derived mediators was assessed in a coaxial bioassay system. The mast cell degranulating agent compound 48/80 (10 micrograms/ml) and histamine caused reductions in phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tube tissue. Relaxation responses to histamine and to compound 48/80 (10 micrograms/ml) were markedly reduced in the presence of mepyramine (50 microM) or when the epithelium was removed from coaxially mounted guinea pig trachea, indicating that they were mediated via the release of EpDIF. Coaxial bioassay assemblies were also prepared using EpDIF donor tracheal tissue obtained from guinea pigs actively sensitized to ovalbumin. Subsequent challenge with ovalbumin (10(-7) to 10(-1) mg/ml) produced concentration-dependent relaxation mediated by EpDIF. Ovalbumin-induced relaxation responses were not inhibited in the presence of either mepyramine (20 and 100 microM) or SKF 104353-Z2 (10 microM) alone but were significantly reduced when both mepyramine (20 microM) and SKF 104353-Z2 (10 microM) were present. Antigen-induced relaxation was apparently mediated by EpDIF in response to mast cell-derived histamine and leukotrienes. Rat tracheal airway smooth muscle did not relax in response to EpDIF, suggesting selectivity of action on vascular smooth muscle. Vasoactive EpDIF may play a role in protecting against antigen-induced bronchoconstriction by regulating bronchial circulation flow.

Epithelium-derived inhibitory factor in human brochus

The potencies of histamine and methacholine were significantly increased by approximately 2- and 5-fold respectively in human non-diseased isolated bronchi on removal of the epithelium. In contrast, no increases in spasmogen potency were observed following epithelium removal in bronchi obtained from a sample of asthmatic human lung. The failure of epithelium removal to increase asthmatic brochial sensitivity to histamine may have been due to a reduction in the release of an epithelium-derived inhibitory factor (EpDIF) resulting from disease-induced epithal damage. A co-axial bioassay system in which endothelium-denuded rat aorta was used as the assay tissue was used to detect the release of a vasorelaxant EpDIF from human bronchial tissue. Histamine (100 μM) and methacholine (25 μM), in the presence of indomethacin (5 μM), reduced phenylephrine-induced tone in endothelium-denuded rat aorta in co-axial assemblies by 75 ± 11 and 67 ± 9% respectively. Removal of the bronchial epithelium abolished these responses, indicating that they were mediated by an EpDIF. It is possible that human airway smooth muscle is sensitive to this vasorelaxant EpDIF and that the absence of the source of this factor following epithelium removal caused the increase in sensitivity to spasmogens. Alternatively, the human bronchial epithelium may also release an EpDIF selective for airway smooth muscle.

Pharmacological evaluation of a guinea‐pig tracheal epithelium‐derived inhibitory factor (EpDIF)

1. An epithelium-derived inhibitory factor (EpDIF) released by guinea-pig tracheal epithelium was evaluated in a co-axial bioassay system consisting of an epithelium-intact guinea-pig tracheal tube surrounding endothelium-denuded rat aortic strip. 2. Histamine and several muscarinic agonists induced concentration-dependent relaxation of phenylephrine-contracted rat aorta via the release of EpDIF. However, several other agonists did not induce the release of EpDIF from guinea-pig trachea. These included the nicotinic cholinoceptor agonists nicotine (25 microM), 1,1-dimethyl-4-phenylpiperazinium (DMPP) (25 microM), calcium ionophore A23187 (0.5 microM), bradykinin (0.05-0.5 microM), substance P (5 microM), platelet activating factor (PAF, 1-100 nM), the leukotrienes (LT) LTC4, LTD4 and LTE4 (0.1-10 nM) as well as hyperosmotic stimuli. 3. Prostaglandin E2 (PGE2) induced concentration-dependent contraction of endothelium-denuded rat aortic preparations, indicating that this prostanoid could not be EpDIF. Furthermore, relaxation to histamine and methacholine, mediated via EpDIF, was not significantly altered in the presence of phenidone (50 microM) the cyclo-oxygenase/lipoxygenase inhibitor with radical scavenging properties or the cytochrome P-450 inhibitors metyrapone (1 mM) and SKF 525A (25 microM). This suggests that EpDIF is neither a prostanoid nor a cytochrome P-450 metabolite of arachidonic acid. 4. The soluble guanylate cyclase inhibitor, methylene blue (50 microM), caused small but significant increases in the potencies of both histamine and methacholine in co-axial assemblies, indicating that EpDIF did not activate this enzyme and therefore was not NO or a related substance. The beta-adrenoceptor antagonist, (-)-propranolol (1 microM), and the PAF-receptor antagonist, WEB 2086 (50 microM), also failed to alter significantly EpDIF-modulated relaxations. These data suggest that EpDIF is neither a stimulant of fiadrenoceptors nor of PAF receptors. 5. The present study provides some evidence that this vascular smooth muscle-sensitive EpDIF may not be related to the putative EpDIF previously hypothesized to modulate directly spasmogen-induced airway smooth muscle tone.

Receptors in Asthmatic Airways

Airway tissue obtained postmortem from nondiseased and severely asthmatic human lung was used in functional, radioligand binding and autoradiographic studies to investigate aspects of various receptor systems for putative mediators of asthma. Results indicate that asthma does not involve an intrinsic abnormality of smooth muscle contractility to spasmogens. Nor was there any evidence for up-regulation of histamine H1-receptor, muscarinic cholinoceptor, or alpha 1-adrenoceptor function. Conversely, severe asthma involving intense airway inflammation resulted in significant beta-adrenoceptor dysfunction, probably caused by receptor uncoupling from adenylate cyclase. Evidence was also obtained for histamine and methacholine-induced release of a nonprostanoid, airway epithelium-derived inhibitory factor (EpDIF), which may have a significant dilator effect in the adjacent bronchial circulation. The activity of this potentially protective inhibitory autacoid system would be expected to be reduced in asthma, which commonly involves epithelium damage. The autoradiographic distribution of specific binding sites for 125I-labeled substance P (I-SP) was also determined in bronchi from healthy humans and asthmatics. In sharp contrast to guinea pig airways where high levels of binding were detected over smooth muscle, specific I-SP binding was sparse over human airway smooth muscle from both sources, while dense binding was associated with structures immediately beneath the epithelium and with deep submucosal glands. These data suggest a more significant role for SP in secretory processes than in spasmogenic processes in human bronchi and highlight the potential for drawing invalid conclusions concerning human airway function from studies using animal models.

Effect of epithelium on mucus secretion from feline tracheal submucosal glands

We studied the effect of airway epithelium on mucus secretion by use of an isolated tracheal submucosal gland preparation reported previously (J. Appl. Physiol. 60: 1237-1247, 1986). Mucus glycoconjugate release from submucosal glands of feline trachea was examined using [3H]glucosamine as a mucus precursor. Isolated glands showed significantly higher secretory responses to cholinergic, alpha-, and beta-adrenergic agonists and dibutyryladenosine 3',5'-cyclic monophosphate (average 400% of control) than the conventional tracheal mucosal explants, which contained epithelium and submucosal tissues in addition to submucosal glands (average 160% of control). The addition of isolated epithelium depressed the secretory response of isolated glands to the same level as that of tracheal explants. However, the supernatant from isolated epithelium failed to inhibit secretory responses to methacholine in isolated glands, suggesting that the epithelium-derived inhibitory factor to secretion may be short-lived. Leukotriene D4 antagonist (FPL 55712), cyclooxygenase and/or lipoxygenase inhibitors (indomethacin or BW 755C) caused no significant change in the inhibitory action of epithelium, suggesting that the inhibition is not due to arachidonic acid metabolites. The newly found secretory inhibitory action of epithelium is of particular interest in the pathogenesis of hypersecretion associated with epithelial damage.

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD 4, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE 2α and PGI 2 (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD 4, methacholine and histamine. LTD 4 (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE 2 and PGF 2α but not PGI 2, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI 2 and is unlikely to be PGF 2α or PGE 2. However, the possibility remains that the basal release of PGE 2 and/or PGF 2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE 2 and PGF 2α which may be involved in the maintenance of baseline tone.

Differential effects of epithelium removal on the responsiveness of guinea-pig tracheal smooth muscle to bronchoconstrictors.

1 The influence of the epithelium on contractions produced by the peptidoleukotrienes, 5-hydroxytryptamine (5-HT) and the thromboxane mimetic, U-44069, was examined in trachea from control and ovalbumin-sensitized guinea-pigs. 2 In control tissues removal of the epithelium produced an approximately 2 to 4 fold leftward shift in leukotriene C4 (LTC4) and LTD4 concentration-response curves, but no effect on LTE4-induced contractions. Similar results were obtained in preparations from ovalbumin-sensitized animals. 3 Responses produced by 5-HT or U-44069 were similar in the presence and absence of the epithelium in control guinea-pigs. 4 Indomethacin produced contrasting effects on leukotriene-induced contractions in control guinea-pigs: an increase in sensitivity to LTC4 in the presence but not absence of the epithelium, no effect on LTD4-induced contractions and a decrease in sensitivity to LTE4 in both epithelium-containing and epithelium-free preparations. 5 These results indicate that there is selectivity in the effects of epithelium removal on agonist-induced contractions of the guinea-pig trachea. This provides further evidence for the modulatory influence of the epithelium on the reactivity of mammalian airway smooth muscle and supports the postulated existence of an epithelium-derived inhibitory factor. The observation that in intact trachea indomethacin mimics the effects of epithelium removal on LTC4-induced responses, suggests the involvement of a prostanoid(s) in this phenomenon.

Regional differences in reactivity and in the influence of the epithelium on canine intrapulmonary broncial smooth muscle responsiveness

Differences in the reactivity and in the influence of the epithelium on responsiveness of canine 2nd and 3rd generation airway smooth muscle were examined. Epithelium-containing 3rd generation airways produced a greater maximum contraction and were more sensitive to methacholine and histamine, but not to KCl, than corresponding 2nd generation airways. Mechanical removal of the epithelium increased the sensitivity to methacholine and histamine in 2nd generation airways; there was also an increase in the maximum response elicited by histamine, but not by methacholine, in epithelium-free preparations. In contrast, there was no significant difference in the sensitivity to or the maximum response elicited by histamine or methacholine in epithelium-containing and epithelium-free 3rd generation airways. Epithelium removal had no effect on KCl-induced responses in either airway region. The inhibitory effects of verapamil (1 μM) against KCl- and methacholine-induced responses were identical in preparations containing and lacking the epithelium. The results support the postulate of an epithelium-derived inhibitory factor modulating airway smooth muscle reactivity. Furthermore, the influence of the epithelium exhibits regional differences, being greater in larger airways.

Demonstration of the release of an epithelium-derived inhibitory factor from a novel preparation of guinea-pig trachea

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.