Epithelium-denuded Preparations Research Articles

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

BackgroundIn addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.MethodsGrowth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP1-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.ResultsEpidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP1-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.ConclusionThe results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.

Factors that determine acetylcholine responsiveness of guinea pig tracheal tubes

Acetylcholine administered to the inside of epithelium-denuded tracheal tubes did cause a potent contraction (2486±120 mg). In contrast, a response was hardly observed in tissues with an intact epithelial layer (674±81 mg), which was due to both the synthesis of nitric oxide and the activity of acetylcholinesterase, since the contractions to acetylcholine were significantly enhanced after preincubation with N ω-nitro- l-arginine methyl ester ( l-NAME) or physostigmine (1374±65 and 1120±65 mg, respectively). In addition, the suppressive effect was caused by the barrier function of the epithelial layer, since preincubation of epithelium-denuded tissues with physostigmine significantly increased the pD 2 value for acetylcholine (7.48±0.04) compared to intact tissues preincubated with physostigmine (6.32±0.10) and epithelium-denuded preparations without physostigmine (6.37±0.06). Increasing concentrations of physostigmine administered to the inside of tissues with epithelium did induce a potent spontaneous contraction (1440±350 mg) that was prevented by atropine. In contrast to what was expected, the contractile response was diminished in tracheal tubes without epithelium (665±221 mg). It is concluded that contractions of epithelium-denuded tissues are more pronounced to exogenous than to endogenous acetylcholine, and that the production and breakdown of this neurotransmitter is very rapid in intact guinea pig airways. Moreover, the release of nitric oxide and the barrier function of the epithelium did suppress the responsiveness to acetylcholine.

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.

Evidence that neuroepithelial endocrine cells control the spontaneous tone in guinea pig tracheal preparations.

The hypothesis that neuroepithelial endocrine (NEE) cells control spontaneous tone in isolated guinea pig tracheal preparations was examined. Epithelium-denuded preparations were unable to develop a normal oscillating tone in 12% oxygen (corresponding to systemic arterial oxygen levels) and, instead, developed a strong, smooth tone, similar to the "classic" tone in 94% oxygen. Inhibition of the hydrogen peroxide-producing NADPH oxidase in the NEE cells by 20 microM diphenyleneiodonium chloride transformed, in intact preparations in 94% oxygen, the tone from a strong, smooth type to an oscillating tone of considerably less force. Similar experiments in denuded preparations showed no change of tone and no oscillations. After pretreatment with the catalase inhibitor 3-amino-1,2, 4-triazole (1 mM), addition of 2 mM hydrogen peroxide to intact preparations displaying the oscillating tone caused a transformation to a strong, smooth type. These findings support the hypothesis that the spontaneous tone in this preparation is largely controlled by the oxygen-sensing NEE cells. For the first time, previous findings on isolated cells can be linked to effects in intact tissue preparations. The results also suggest that the regulation by the NEE cells involves the release of powerful relaxing and contracting factors from the epithelium.

Transition of functional innervation in the developing porcine airway from nitrergic to catecholaminergic.

1. We determined the distribution and chemical nature of the inhibitory neurotransmitter(s) to the airway smooth muscle (ASM) before and after birth. 2. Relaxation responses to electrical field stimulation (EFS) were studied in isovolumic bronchial segments from foetal (approximately 100/115 days gestation) and adult (25 kg) pigs, and in isovolumic tracheal segments from the foetus, and tracheal smooth muscle strips from the adult pig. Preparations were conditioned in low doses of atropine (10(-7) M) to reduce the effects of excitatory neurotransmission and then exposed to carbachol to produce submaximal muscle tone. Some studies were also carried out on bronchial segments from 4 week old pigs. 3. EFS (65 V, 2 ms, 5-20 Hz for 5 s) produced a TTX-sensitive relaxation in epithelium-intact and epithelium-denuded preparations. In foetal bronchial and tracheal preparations, EFS-induced relaxation was strongly inhibited by N(G)-nitro-L-arginine (L-NOARG, 10(-6) to 10(-4) M; P<0.01-0.001). However, in the adult, only relaxations of the trachea were inhibited by L-NOARG; bronchi were resistant to L-NOARG and also to N-nitro-L-arginine methyl ester (L-NAME, 10(-4) M). The inhibitory actions of L-NOARG (10(-6) to 10(-4) M) were substantially reversed by 10(-2) M L-arginine. Experiments with bronchial segments from 4 week old pigs showed partial inhibition of relaxations by L-NOARG. 4. The L-NOARG-insensitive relaxations recorded in the adult bronchus were blocked by propranolol (10(-6) M). 5. The onset of relaxation to EFS was more prompt and the rate of relaxation more rapid in foetal bronchi than in adult bronchi (P<0.0005). Maximum relaxation and recovery times were the same. 6. Foetal and adult bronchi were relaxed by sodium nitroprusside (SNP) with similar sensitivity and maximum effect. The rate of relaxation to SNP was not different in the two ages. 7. In the absence of atropine and carbachol, excitatory cholinergic responses to EFS (65 V, 2 ms, 5 Hz for 20 s) were not altered by L-NOARG (10(-4) M) or L-NAME (10(-4) M) in the adult bronchus but were modestly increased by L-NOARG in the foetal bronchus (P<0.01). 8. The tracheobronchial tree appears functionally innervated by nitrergic input to the smooth muscle before birth. However, at or after 4 weeks of age the inhibitory neural input to the bronchi is catecholaminergic, but it remains nitrergic in the trachea. There is also a weak nitrergic pre- or postsynaptic inhibition of the effects of cholinergic neurotransmission in the foetal bronchus but not in the adult.

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea.

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.

Histamine-induced contraction of human isolated bronchus is enhanced by endogenous prostaglandin F 2α and activation of TP receptors

The effect of histamine on the production of prostaglandin F 2α and the actions of prostaglandin F 2α on the responsiveness of human isolated bronchial smooth muscle were examined by organ bath techniques using bronchi from lung tissue resected from 18 patients. Following exposure to histamine, epithelium-intact bronchi generated 34.26±16.3 pg of prostaglandin F 2α/mg of tissue and epithelium-denuded preparations produced 32.62±11.83 pg/mg, suggesting that histamine-induced release of prostaglandin F 2α was from non-epithelial sources, presumably smooth muscle. The histamine H 2 receptor antagonist ranitidine did not affect the release of prostaglandin F 2α, suggesting that its generation may have resulted from histamine H 1 receptor activation. Carbachol did not influence prostaglandin F 2α generation. Contractile responses to histamine, prostaglandin F 2α and carbachol were measured in the presence and absence of the prostaglandin TP receptor antagonist SQ 29,548 ([1 S-[1α,2β(5 Z),3β,4α]]-7-[3[[2-[9-phenylamino)carbonyl]hydrazino]methyl-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) (0.4 μM). SQ 29,548 abolished responses to prostaglandin F 2α suggesting that contractions were mediated via TP receptors. Exposure to SQ 29,548 also produced a 3-fold rightward shift in the concentration-effect curve for histamine ( P=0.01) without influencing the maximum response. SQ 29,548 did not affect responses to carbachol. These results suggest that histamine selectively stimulates the generation of prostaglandin F 2α from epithelium-denuded human airway tissue (presumably from the smooth muscle), which in turn, amplifies the contractile responses of human airway smooth muscle to histamine.

EPITHELIUM-DEPENDENT INHIBITION OF CHOLINERGIC TRANSMISSION IN RAT ISOLATED TRACHEA BY POTASSIUM CHANNEL OPENERS

We have investigated the effects of the potassium channel openers, cromakalim and pinacidil, on cholinergic transmission in rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [ 3H]-choline to incorporate [ 3H]-acetylcholine into the cholinergic transmitter stores. In radiolabelled, epithelium-intact preparations, electrical field stimulation (60s trains of 1ms pulses, 5Hz, 15V) evoked an efflux of radioactivity that was unaffected by the removal of extracellular Ca 2+and, a large proportion of which was resistant to tetrodoxin (1μ M). In contrast, in epithelium-denuded preparations, both tetrodotoxin and Ca 2+withdrawal virtually abolished the stimulation-induced (S-I) efflux. Thus, with epithelium-denuded but not with epithelium-intact tracheal preparations, the S-I efflux reflects the release of [ 3H]-acetylcholine from cholinergic nerves. Atropine (1μ M) markedly enhanced the S-I efflux in both epithelium-intact and epithelium-denuded preparations. In epithelium-intact preparations, the combination of atropine (1μ M) and tetrodotoxin (1μ M) reduced the S-I efflux to about the same level as did tetrodotoxin alone. Thus, in epithelium-intact tracheal preparations, when prejunctional muscarinic cholinoceptors subserving autoinhibition of transmitter release are blocked, S-I efflux may be taken as an index of transmitter acetylcholine release. Cromakalim (1μ M) had no effect on the S-I efflux from either epithelium-intact or epithelium-denuded tracheal preparations. However, in epithelium-intact preparations, when atropine (1μ M) was present, cromakalim (1 and 10μ M) and pinacidil (100μ M) significantly inhibited the S-I efflux. In epithelium-denuded preparations, in the presence of atropine (1μ M), cromakalim (1μ M) and pinacidil (100μ M) were without effect on S-I efflux. The inhibition of S-I efflux produced by cromakalim (1μ M) and pinacidil (100μ M) in epithelium-intact tracheal preparations (in the presence of atropine) was prevented by the ATP-sensitive potassium channel blocking drug glibenclamide (1μ M). Glibenclamide (1μ M) alone enhanced S-I efflux from epithelium-intact preparations in the absence but not in the presence of atropine (1μ M). Glibenclamide (1μ M) was without effect on S-I efflux in epithelium-denuded preparations both in the absence or presence of atropine (1μ M). In conclusion, the present study has provided additional evidence of an inhibitory action of the potassium channel openers, cromakalim and pinacidil, on the release of acetylcholine from parasympathetic nerves of the rat trachea which is dependent upon the functional integrity of the airway epithelium. The findings suggest that cromakalim and pinacidil may inhibit transmitter acetylcholine release by opening ATP-sensitive potassium channels, presumably on epithelial cells. In addition, the enhancement of S-I efflux from epithelium-intact tracheal preparations by glibenclamide may indicate that ATP-sensitive potassium channels on epithelial cells play a functional role in the modulation of transmitter acetylcholine release from parasympathetic cholinergic nerves of the airways.

Poly-l-arginine-mediated release of acetylcholine from parasympathetic nerves in rat and guinea-pig airways

1. The synthetic cationic polypeptide, poly-L-arginine (0.03-1 mg ml-1) induced concentration-dependent contraction of guinea-pig and rat isolated trachea. In guinea-pig isolated trachea, this response was attenuated in the presence of the muscarinic cholinoceptor antagonist, atropine (0.1 microM) and augmented by the acetylcholinesterase inhibitor, ecothiophate (0.1 microM). The neuronal sodium channel blocker, tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine and acetylcholine. 2. The contractile response to poly-L-arginine in rat isolated trachea was inhibited in the presence of atropine (0.1 microM) and the 5-hydroxytryptamine (5-HT) receptor antagonist, methysergide (1 microM). Treatment of rat tracheal preparations with capsaicin (100 microM) or tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine. In contrast, ecothiophate (0.1 microM) augmented the contractile response to poly-L-arginine in rat isolated trachea. 3. Electrical field stimulation (5 Hz, 2 min) of epithelium-denuded guinea-pig tracheal preparations preloaded with [3H]-choline resulted in a contractile response and the simultaneous efflux of radioactivity into the superfusate. Both these responses were abolished in the presence of tetrodotoxin (1.5 microM). Poly-L-arginine (1 mg ml-1) also increased the efflux of total radioactivity from epithelium-denuded guinea-pig isolated tracheal preparations preloaded with [3H]-choline, but this response was tetrodotoxin-insensitive. The negatively charged polyanion, heparin (1 mg ml-1) failed to increase significantly the efflux of radioactivity from epithelium-denuded preparations. 4.In conclusion, the synthetic cationic polypeptide, poly-L-arginine, caused contraction of guinea-pig isolated tracheal preparations via the release of acetylcholine from parasympathetic nerves. Similarly,poly-L-arginine-induced contraction of rat isolated trachea is secondary to the release of acetylcholine from parasympathetic nerves and/or the release of mast cell-derived 5-HT.

Polarized effects of amiloride and 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid on ATP-induced contraction of trachea

Polarity in the effects of amiloride and 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS) in the guinea-pig isolated, perfused trachea was investigated to evaluate the roles of epithelial and airway smooth muscle Na + and Cl − channels in the development of contractile responses to ATP. The blockers were applied to the mucosal (intraluminal) perfusing solution or to the serosal (extraluminal) bath before the second of two challenges with ATP (10 −4 M), which was added to the same bath as the blocker, or to the abluminal bath. In epithelium-intact tracheas, amiloride (10 −4 M) added to the extraluminal or intraluminal bath rapidly (1 min) and extensively inhibited contractions to extraluminally applied ATP (10 −4 M). In contrast, contractions to intraluminally applied ATP (10 −4 M) were relatively resistant to extraluminal and intraluminal amiloride (10 −4 M), in terms of the degree and onset of the inhibition. DIDS (10 −4 M) present in the extraluminal or intraluminal baths caused a slowly developing elevation of baseline tone. After a 30 min incubation, extraluminal DIDS potentiated responses to extraluminally added ATP, but intraluminal DIDS inhibited contractions to ATP added to the extraluminal and intraluminal baths. In contrast to the intact tracheas where there was no difference, the second response of epithelium-denuded preparations to intraluminally administered ATP was diminished. In rubbed tracheas the response to intraluminally added ATP was inhibited further by intraluminal amiloride but was potentiated by intraluminal DIDS. The results suggest that the effects of amiloride and DIDS were polarized across the tracheal wall and involved epithelial and smooth muscle ion channels.

Role of acetylcholinesterase in airway epithelium-mediated inhibition of acetylcholine-induced contraction of guinea-pig isolated trachea

To seek evidence for the involvement of acetylcholinesterase activity in the modulatory influence of the airway epithelium, we examined responses to acctylcholine (ACh), hcthancchol, histamine or KCI in isolated epithelium-intact and epithelium-denuded guinea-pig trachcalis preparations. The concentration-response curves to ACh were shifted 26-fold to the left by epithelial denudation hut the contractile response to KCl was not altered. The response to histamine in epithelium-denuded preparations increased 4-fold with no attenuation in the presence of physostigmine (30 nM). Physostigmine (30 nM) potentiated the response to ACh in epithelium-intact tissues more (about 26-fold) than in epithelium-denuded tissues (about 3.5-fold). Thus, in the presence of physostigmine removing the epithelium had only a slight effect (not statistically significant) on the potency of ACh to contract the trachea. Removing the epithelium had no effect on the potency of bethanechol, a muscarinic receptor agonist that is not a substrate for cholincsicrases. Physostigmine itself contracted the trachealis muscle but the pD 2 values and maximum responses in epithelium-intact and denuded preparations were not significantly different. The frequency-response curves to electrical field-stimulated cholinergic contractions were unaffected by removing the epithelium. In conclusion, the principal mechanism by which the epithelium inhibis contraction of guinea-pig trachea to exogenously applied ACh is via epithelium-derived acetylcholinesterase activity.

Histamine tachyphylaxis in human airway smooth muscle. The role of H2-receptors and the bronchial epithelium.

The role of airway epithelium and H2-receptors in the development of histamine tachyphylaxis was studied using human isolated bronchial smooth muscle strips obtained from 18 patients undergoing thoracotomy. In epithelium-intact strips, a 38% reduction in the maximal contractile response (Emax) (p less than 0.002) and a 2.14-fold increase in the EC50 (p less than 0.02; n = 18) was observed after three separate histamine cumulative concentration effect curves (CCEC). In contrast, significant differences were not seen for either Emax (p greater than 0.4; n = 10) or EC50 (p greater than 0.26; n = 10) in epithelium-denuded strips. In separate experiments, both intact and denuded muscle strips were treated with the H2-receptor antagonist ranitidine (60 microM), either 30 min prior to the first or 30 min prior to the second histamine CCEC. In epithelium-intact strips, pretreatment with ranitidine caused a 1.8-fold increase in Emax in the initial CCEC (p less than 0.02), and both ranitidine schedules prevented tachyphylaxis (n = 8). In epithelium-denuded preparations, ranitidine did not enhance the responsiveness to histamine beyond that seen in untreated epithelium-denuded strips (n = 6). These data suggest that histamine-induced tachyphylaxis occurs in human airway smooth muscle and is mediated, at least in part, via H2-receptors resident on airway epithelium. In vivo, this may function as a protective mechanism, but damage to the epithelium and loss of H2-receptors may be significant in the development of histamine bronchial hyperreactivity as seen in asthma.

The modulatory effects of prostaglandins on both excitatory and inhibitory non-adrenergic non-cholinergic neurotransmission in guinea-pig airways.

Guinea-pig trachea and bronchi were used to investigate the effects of indomethacin and prostaglandin E2 (PGE2) on non-adrenergic non-cholinergic excitatory (e-NANC) and inhibitory (i-NANC) neurotransmission evoked by electrical field stimulation. Indomethacin potentiated e-NANC responses in bronchi with intact epithelium but had no effect on epithelium-denuded preparations. Inhibitory NANC responses were increased by indomethacin independent of the epithelium. Both i-NANC and e-NANC neurotransmission were suppressed by PGE2 in a dose-dependent manner. These results indicate that endogenous prostaglandins (e.g. PGE2) generated from the epithelium have an inhibitory effect on i-NANC and e-NANC nerve responses in airways. The epithelium is presumably not the only source for generation of prostaglandins that are involved in i-NANC neurotransmission.

An ubiquitous modulating function of rabbit tracheal epithelium: degradation of tachykinins.

1. To examine the role of epithelium in the responsiveness of tracheal smooth muscle in rabbit, we measured the contractile responses to acetylcholine (ACh), KCl, 5-hydroxytryptamine (5-HT), histamine, substance P (SP), neurokinin A (NKA), and electrical field stimulation EFS) in intact and epithelium-denuded preparations. 2. Removal of epithelium did not alter the contractile response to any agonist examined, except SP. 3. Removal of epithelium enhances the contractile response to SP. In the presence of phosphoramidon, the contractile response to SP was not significantly different in either group. The results suggest that the effect of epithelium is largely due to degradation of SP by enzymes in the epithelium. 4. Arachidonic acid metabolites did not seem to be related to the responses induced by contractile agonists or EFS. 5. In the presence of SP, the contractile responses and [3H]-choline outflow evoked by EFS were dose-dependently enhanced. Contractile responses to EFS and [3H]-choline outflow evoked by EFS were enhanced by SP significantly more than by NKA. Both were abolished by atropine or tetrodotoxin. 6. These results suggest that rabbit tracheal epithelium may modulate SP-induced contractions, both direct and indirect, by inactivation of SP. This phenomenon is widespread in mammals. The rabbit may be a useful model to examine airway cholinergic functions.

Effect of capsaicin on PAF‐induced bronchial hyperresponsiveness and pulmonary cell accumulation in the rabbit

1 Platelet activating factor (PAF), but not the carrier molecule bovine serum albumin (BSA) induced bronchoconstriction in the anaesthetized rabbit. This bronchoconstriction was not altered by prior treatment with capsaicin. 2 Rabbits demonstrated increased airways responsiveness to histamine 24h after exposure to PAF but not to BSA. PAF failed to increase airways responsiveness to histamine in animals pretreated with capsaicin (80 mg kg-1). 3 A significant increase in inflammatory cells was obtained in bronchoalveolar lavage (BAL) 24h after PAF exposure in vehicle-treated rabitts. This was associated with an increase in the numbers of neutrophils and eosinophils. Capsaicin treatment inhibited the PAF-induced influx of inflammatory cells found in BAL, although this was not associated with an inhibition of PAF-induced pulmonary eosinophilia. 4 Capsaicin-induced motor effects were modest in epithelium-intact rabbit bronchial preparations, but were significantly enhanced in epithelium-denuded preparations in the presence of thiorphan. The contractile response to capsaicin was significantly inhibited in tissues exposed to a consecutive dose of capsaicin. Furthermore, ruthenium red abolished capsaicin-induced contraction in epithelium-denuded preparations. 5 Tissue content of calcitonin gene-related peptide-like immunoreactivity and substance P-like immunoreactivity was not reduced in bronchus and iris obtained from capsaicin-treated rabbits, although capsaicin-induced contractile responses in rabbit bronchus obtained from animals previously treated with capsaicin were significantly reduced. Furthermore, airway responses to histamine, methacholine and electrical field stimulation in vitro, were not altered by pretreatment of rabbits in vivo for 3 days with capsaicin. 6. In conclusion, PAF-induced airways responsiveness and pulmonary cell accumulation is inhibited by in vivo capsaicin pretreatment in the rabbit, via a mechanism that may not involve depletion of sensory neuropeptides.

Tetrodotoxin Does Not Block the Epithelium-dependent Release of Prostaglandin E2 Induced by Electrical Field Stimulation in Isolated Ferret Trachea

Electrical field stimulation (EFS) has previously been shown to induce the release of prostaglandin (PG) E2 from ferret tracheal epithelium. We have now conducted a study to see whether this effect of EFS is due to the activation of nerves or whether it is a non-neural effect. The release of PGE2 and 6-keto-PGF1 alpha into the bath fluid was assayed in isolated ferret tracheas with (E+) or without (E-) epithelium, stimulated by either EFS or direct vagal nerve stimulation (DNS) repeatedly for 120 min. EFS-stimulated E+ preparations showed a gradual decline in the contractile responses (30 +/- 1% of baseline) and an increase in PGE2 to 296 +/- 38 pg/ml. In EFS-stimulated, epithelium-denuded (E-) preparations, the decline was significantly lower (11 +/- 5%), as well as the final concentration of PGE2 (107 +/- 21 pg/ml). In DNS-stimulated E+ preparations, the contraction decline was 8 +/- 1% and the final concentration of PGE2 was less than 6 pg/ml. Although tetrodotoxin (TTX) abolished the contractile response in EFS-stimulated E+ preparations, it did not significantly reduce the release of PGE2 (260 +/- 6 pg/ml), whereas atropine partly counteracted the release. The bath concentration of 6-keto-PGF1 alpha increased, independently of the electrical stimulation, contractile response, or presence of the epithelium. We conclude that EFS activates the epithelium-dependent release of PGE2 by a TTX-resistant mechanism. This may be due to an activation of TTX-resistant nerves, or possibly to a non-neural effect, such as a direct effect on the epithelial cells. The results indicate that the airway epithelium has the ability to respond to certain stimuli with a pronounced release of PGE2, thereby counteracting bronchoconstriction.

Role of epithelium in agonist‐induced contractile responses of guinea‐pig trachealis: influence of the surface through which drug enters the tissue

1. A method has been used in guinea-pig isolated tracheal rings to achieve selective drug entry from the adventitial or mucosal surface. A study has been made of the effects of epithelium removal on responses to spasmogens entering the tissue solely from the adventitial or the mucosal surface. 2. Cumulative concentration-response curves for KCl (1 to 100 mM), acetylcholine (0.1 microM to 10 mM) and histamine (1 microM to 1 mM) were constructed in intact and epithelium-denuded tracheal rings in circumstances where drug entry was unrestricted or restricted to the adventitial or mucosal surface. 3. Epithelium removal did not alter the responsiveness or sensitivity of tracheal rings to KCl either when drug entry was unrestricted or when drug entry was restricted to the adventitial or mucosal surface. 4. When acetylcholine entered from the mucosal or adventitial surfaces of intact tracheal rings its concentration-response curve was displaced to the right with respect to that obtained for unrestricted drug entry. A greater rightward shift was observed for mucosal drug entry than for adventitial drug entry. Epithelium removal potentiated acetylcholine entering from the mucosal surface to a greater extent (27.5 fold) than it potentiated acetylcholine entering from both surfaces (4 fold). Epithelium removal did not potentiate effects of acetylcholine entering from the adventitial surface alone. 5. In intact tracheal segments, concentration-response curves for histamine entering from the mucosal surface were displaced to the right compared with those for histamine entering in an unrestricted fashion or from the adventitial surface alone. This displacement was absent in epithelium-denuded preparations. Epithelium removal potentiated (2-3 fold) histamine entering from the mucosal surface or entering in an unrestricted way. It did not potentiate histamine entering from the adventitial surface alone. 6. Our findings suggest that the epithelium does not modulate tracheal responses to KC1. Its ability to modulate responses to acetylcholine and histamine is observed when these spasmogens enter the tissue from the mucosal surface but not when they enter from the adventitial surface. The mechanism by which epithelium removal preferentially potentiates acetylcholine and histamine entering from the mucosal rather than the adventitial surface remains to be determined.

Effects of epithelium removal on relaxation of airway smooth muscle induced by vasoactive intestinal peptide and electrical field stimulation

1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP.

Epithelium-derived PGE 2 inhibits the contractile response to cholinergic stimulation in isolated ferret trachea

We have previously reported that epithelium-dependent inhibitory factors, both prostanoids and nonprostanoids, can be activated by electrical field stimulation (EFS) and direct nerve stimulation (DNS) in an in vitro nerve-muscle preparation of ferret trachea. In this study we set out to compare the release of the inhibitory prostanoids, PGE 2 and PGI 2, in preparations with intact and with removed epithelium. Ferret tracheae were mounted in organ baths and phasic contractions were induced by EFS and DNS. The bath concentrations of PGE 2 and the PGI 2-metabolite 6-keto-PGF 1α were measured using radioimmuno assays. The gradual decrease in contractile response to DNS, 2 Hz for 120 min, was 95 ± 2% of baseline (mean ± SEM) in preparations with intact epithelium compared with 29 ± 8% in epithelium-denuded preparations (p<0.001). The bath-levels of PGE 2 showed a slight but significant (p < 0.01) increase in denuded preparations with a final bath-concentration at 120 min of 13 ± 3 pg/ml. The release of PGE 2 was more pronounced in preparations with intact epithelium which resulted in a final bath-concentrations of PGE 2 of 84 ± 38 pg/ml which was significantly higher compared with epithelium-denuded preparations and also compared with time-matched controls with the same pattern of contraction induced by DNS alone. The concentrations of 6-keto-PGF 1α showed a slight increase which was of comparable magnitude (ns) in intact and denuded preparations. In conclusion this study demonstrates that the cyclooxygenase-dependent component of the epithelium-dependent inhibition of the contractile response to cholinergic nerve stimulation in ferret trachea is mediated by PGE 2 but not by PGI 2.

Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists.

The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration-effect curves in guinea-pig isolated tracheal ring preparations with and without an intact epithelium. Estimates of Emax were not significantly different in epithelium-denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium-stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. The autoradiographic localisation of binding sites for the non-selective beta-adrenoceptor ligand [125I]-iodocyanopindolol (I-CYP) showed that the epithelium of the guinea-pig trachea had a 75 +/- 16% greater density of beta-adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I-CYP binding. It was concluded that the reduced functional response of guinea-pig trachea to isoprenaline was probably not due to smooth muscle beta-adrenoceptor dysfunction. Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea-pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.