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The Respiratory Epithelium Releases a Smooth Muscle Relaxing Factor
histamine comparable to other mast cell lines and are capable of synthesizing a variety of arachidonic acid products. Their secretory profiles vary depending on the stimulus.· Using nude mice, we have successfully propagated mastocytoma cells from four different dogs. Several of these cell lines have completed 4 passages without evidence of change in morphologic appearance, histamine content, or function. The mediators released from these cells by challenge with the calcium ionophore A23187 increased short-circuit current, and as with the lung fragments, the effect was not attributable to histamine. DISCUSSION
In this study we have used measured changes in shortcircuit current to reflect ion transport across the canine tracheal epithelium and have shown that mediators released from lung fragments by immunologic challenge stimulate ion transport in the trachea. The presence of histamine in the supernatants, although the concentration is too low to account for the short-circuit current effect, indicates that mast cell degranulation has occurred. The observation that the increase in short-circuit current can be blocked by pretreatment of the epithelial tissue with the cyclooxygenase inhibitor indomethacin suggests that the mediator effect on short-circuit current is indirect and is produced by activating the cyclooxygenase pathway in the epithelium. Leikauf et allO have observed that many of the lipoxygenase products of arachidonic acid, and bradykinin increase short-circuit current by this mechanism. Thus, these mediators or others acting by the same mechanism may be implicated in these effects of mast cell activation we have observed. It seems clear that mast cell mediators can interact with many effector systems in addition to airway smooth muscle. The role of these mediators in regulation of airway secretions is complicated, but their effect appears to be due at least in part to the activation of cyclooxygenase pathways in the airway epithelial cells. We have succeeded in developing a preparation of isolated canine mast cells and propagating these cells so that large numbers of pure, homogeneous, functionally identical mast cells are available for future studies in which we hope to define more precisely the mechanism of these mast celleffector cell interactions. REFERENCES
2
3
4 5
Shelhamer JH, Marom Z, Kaliner M. Immunologic and neuropharmacologic stimulation of mucous glycoprotein release from human airways in vitro. J Clin Invest 1980;66:1400-8 Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner M. Slow-reacting substances, leukotrienes C. and D., increase the release of mucus from human airwaysin vitro. Am Rev Respir Dis 1982; 126:449-51 MaromZ, ShelhamerJH, KalinerM. Elfectsofarachidonicacid, monohydroxyeicosatetraenoic acid and prostaglandins on the release of mucous glycoproteins from human airways in vitro. J Clin Invest 1981; 67:1695-1702 Marom Z, Shelhamer JH, Sun F, Kaliner M. Human airway monohydroxyeicosatetraenoic acid generation and mucus release. J Clin Invest 1983; 72:122-7 Phipps RJ, Denas SM, Wanner A. Antigen stimulates glycoprotein secretion and alters ion 8uxes in sheep trachea. J Appl
Physioll983; 55:1593-1602 6 Olver RE, Davis B, Marin MG, Nadel JA. Active transport of Na" and CI- across the canine tracheal epithelium in vitro. Am Rev Respir Dis 1975; 112:811-15 7 Phillips MJ, Calonico L, Gold WM. Morphological and pharmacologicalcharacterization of dog mastocytoma cells. Am Rev Respir Dis 1982; 125:63 8 Marin MG, Davis B, Nadel JA. Elfect of histamine on electrical and ion transport properties of tracheal epithelium. J Appl Physiol 1977; 42:735-8 9 Goetz! EJ, Phillips MJ, Gold WM. Stimulus specificity of the generation ofleukotrienes by dog mastocytoma cells. J Exp Med 1983; 158:731-7 10 LeikaufGD, UekilF, WiddicombeJH, NadelJA. Elfectsofleukotrienes B., C., D. and E. on electrical properties and prostaglandin release from tracheal epithelium. Fed Proc 1983; 43: 829
The Respiratory Epithelium Releases a Smooth Muscle Relaxing Factor* N. A Flaoahan, Ph.D., M.D.; and P. M. Vanhoutte, M.D.
D
ysfunction of respiratory epithelial cells may contribute to the pathogenesis of bronchial hyperreactivity in asthma. U The aim of this study was to determine whether the respiratory epithelium can modulate directly the responsiveness of bronchial smooth muscle. Experiments were performed on paired rings of bronchi (length: 4--6 mm: outside diameter: 4--6 mm) taken from lower lung lobes of mongrel dogs anesthetized with pentobarbital sodium (30 rug/kg), In one ring of each pair, care was taken not to touch the epithelial surface; in the other ring, the epithelium was removed by inserting a cotton swab into- the lumen or by using forceps gently to roll the preparation over saline-loaded filter paper. The presence or absence of the epithelium was confirmed histologically. The bronchial rings were suspended between two stainless steel wires in organ chambers filled with Krebs-Ringer solution, which was gassed with 95% 0/5% COl (pH = 7.4, 3T'C). One of the wires was connected to a strain gauge for the measurement of isometric force. Concentration-effect curves to exogenous agonists were constructed by increasing organ chamber concentrations cumulatively by half-log increments. Noncumulative frequency-response curves to nerve stimulation were obtained by stimulating the preparations (9V, 2 msec, 0.5 to 16 Hz) electrically via 2 platinum electrodes which were placed parallel to the rings. Epithelium-removal did not significantly affect the lengthtension characteristics of the bronchial rings (optimal tension of rings with and without epithelium: 2.9±0.1 g and 3±0.2 g, respectively; x±SEM, n=10) or the maximal responses evoked by contractile agents, suggesting that the procedure did not affect the integrity of the bronchial smooth muscle. Removal of the epithelium increased the responsiveness of bronchial rings to acetylcholine, histamine, and 5-hydroxytryptamine, causing parallel leftward displacements in their -From the Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Reprint requests: Dr: Vanhoutte, Department of Physiology, Mayo Clinic, Rochester, Minnesota 55905 CHEST I 87 I 5 I MAY, 1985 I SuppIemenl
1898
concentration-effect curves. The magnitude of the displacement produced by epithelium-removal was similar fOr each agonist. The increased sensitivity of epithelium-denuded preparations to these agonists did not result from the removal of degradative processes. Inhibition of acetylcholinesterase (ecothiopate, 1O- 7 M) or monoamine oxidase (pargyline, lO-4M) did not prevent the increased responsiveness of the denuded rings to acetylcholine or 5-hydroxytryptamine, respectively. Neither did the increased sensitivity result from the loss of an epithelial diffusion barrier. Following contraction of the bronchial rings to acetylcholine (concentrations causing 50% of the maximum response in each ring), isoproterenol caused concentration-dependent relaxations which were significantly depressed fOllowing epithelium removal. This suggests that the epithelium releases an inhibitory factor which augments the direct inhibitory effect of p-adrenoceptor stimulation. Epithelium removal also affected the sensitivity of the bronchial rings to endogenously released acetylcholine. 'Ihmsmural nerve stimulation of canine bronchi causes activation ofparasympathetic and sympathetic nerve fibers; the parasympathetic in8uence predominates to cause contraction.3,4 The contractile responses evoked by nerve stimulation initially were not significantly different between rings with and without epithelium. However, during continued stimulation, the contractile response in unrubbed rings gradually declined, whereas in denuded preparations the response was well maintained. The epithelium-dependent decay in the neuronal response was still present following propranolol administration (5 X lO-8M). These results" indicate that the respiratory epithelium
1108
releases a smooth muscle relaxing factor to inhibit the effects of bronchoconstrictor agonists and to augment the direct inhibitory effectsofrelaxing stimuli. Moreover,the in8uence of epithelium removal on the responses to nerve stimulation suggests that this inhibitory factor might be released following activation of the epithelium by these agonists. For example, the delay in the epithelium-dependent inhibitory response during nerve stimulation might represent the diffusion of nerve-released acetylcholine to the epithelial cells. Preliminary results have now been obtained using a cascade system to bioassay epithelium-derived factors. This system demonstrates that the respiratory epithelial cells continuously release a potent inhibitory substance which causes relaxation of bronchial smooth muscle. It is possible that under conditions in which the epithelium is damaged, eg, in asthma. bronchial hyperreactivity results from interruption of the production of this inhibitory substance. REFERENCES 1 Nadel JA. Bronchial reactivity. Adv Intern Med 1983; 28:207-23 2 Hogg Ie, Eggleston PA. Is asthma an epithelial disease? Am Rev Respir Dis 1984; 129:207-8 3 Russell JA. Responses of isolated canine airways to electric stimulation and acetylcholine. I Appl Physiol 1978; 45:690-8 4 Vermeire PA, Vanhoutte PM. Inhibitory effects of catecholamines in isolated canine bronchial smooth muscle. I Appl Physioll979; 46:787-91 5 Flavahan NA, Aarhus LL, Rimele TJ, Vanhoutte PM. The respiratory epithelium inhibits bronchial smooth muscle tone. I Appl Physiol (in press)
In this study we have used measured changes in shortcircuit current to reflect ion transport across the canine tracheal epithelium and have shown that mediators released from lung fragments by immunologic challenge stimulate ion transport in the trachea. The presence of histamine in the supernatants, although the concentration is too low to account for the short-circuit current effect, indicates that mast cell degranulation has occurred. The observation that the increase in short-circuit current can be blocked by pretreatment of the epithelial tissue with the cyclooxygenase inhibitor indomethacin suggests that the mediator effect on short-circuit current is indirect and is produced by activating the cyclooxygenase pathway in the epithelium. Leikauf et allO have observed that many of the lipoxygenase products of arachidonic acid, and bradykinin increase short-circuit current by this mechanism. Thus, these mediators or others acting by the same mechanism may be implicated in these effects of mast cell activation we have observed. It seems clear that mast cell mediators can interact with many effector systems in addition to airway smooth muscle. The role of these mediators in regulation of airway secretions is complicated, but their effect appears to be due at least in part to the activation of cyclooxygenase pathways in the airway epithelial cells. We have succeeded in developing a preparation of isolated canine mast cells and propagating these cells so that large numbers of pure, homogeneous, functionally identical mast cells are available for future studies in which we hope to define more precisely the mechanism of these mast celleffector cell interactions. REFERENCES
2
3
4 5
Shelhamer JH, Marom Z, Kaliner M. Immunologic and neuropharmacologic stimulation of mucous glycoprotein release from human airways in vitro. J Clin Invest 1980;66:1400-8 Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner M. Slow-reacting substances, leukotrienes C. and D., increase the release of mucus from human airwaysin vitro. Am Rev Respir Dis 1982; 126:449-51 MaromZ, ShelhamerJH, KalinerM. Elfectsofarachidonicacid, monohydroxyeicosatetraenoic acid and prostaglandins on the release of mucous glycoproteins from human airways in vitro. J Clin Invest 1981; 67:1695-1702 Marom Z, Shelhamer JH, Sun F, Kaliner M. Human airway monohydroxyeicosatetraenoic acid generation and mucus release. J Clin Invest 1983; 72:122-7 Phipps RJ, Denas SM, Wanner A. Antigen stimulates glycoprotein secretion and alters ion 8uxes in sheep trachea. J Appl
Physioll983; 55:1593-1602 6 Olver RE, Davis B, Marin MG, Nadel JA. Active transport of Na" and CI- across the canine tracheal epithelium in vitro. Am Rev Respir Dis 1975; 112:811-15 7 Phillips MJ, Calonico L, Gold WM. Morphological and pharmacologicalcharacterization of dog mastocytoma cells. Am Rev Respir Dis 1982; 125:63 8 Marin MG, Davis B, Nadel JA. Elfect of histamine on electrical and ion transport properties of tracheal epithelium. J Appl Physiol 1977; 42:735-8 9 Goetz! EJ, Phillips MJ, Gold WM. Stimulus specificity of the generation ofleukotrienes by dog mastocytoma cells. J Exp Med 1983; 158:731-7 10 LeikaufGD, UekilF, WiddicombeJH, NadelJA. Elfectsofleukotrienes B., C., D. and E. on electrical properties and prostaglandin release from tracheal epithelium. Fed Proc 1983; 43: 829
The Respiratory Epithelium Releases a Smooth Muscle Relaxing Factor* N. A Flaoahan, Ph.D., M.D.; and P. M. Vanhoutte, M.D.
D
ysfunction of respiratory epithelial cells may contribute to the pathogenesis of bronchial hyperreactivity in asthma. U The aim of this study was to determine whether the respiratory epithelium can modulate directly the responsiveness of bronchial smooth muscle. Experiments were performed on paired rings of bronchi (length: 4--6 mm: outside diameter: 4--6 mm) taken from lower lung lobes of mongrel dogs anesthetized with pentobarbital sodium (30 rug/kg), In one ring of each pair, care was taken not to touch the epithelial surface; in the other ring, the epithelium was removed by inserting a cotton swab into- the lumen or by using forceps gently to roll the preparation over saline-loaded filter paper. The presence or absence of the epithelium was confirmed histologically. The bronchial rings were suspended between two stainless steel wires in organ chambers filled with Krebs-Ringer solution, which was gassed with 95% 0/5% COl (pH = 7.4, 3T'C). One of the wires was connected to a strain gauge for the measurement of isometric force. Concentration-effect curves to exogenous agonists were constructed by increasing organ chamber concentrations cumulatively by half-log increments. Noncumulative frequency-response curves to nerve stimulation were obtained by stimulating the preparations (9V, 2 msec, 0.5 to 16 Hz) electrically via 2 platinum electrodes which were placed parallel to the rings. Epithelium-removal did not significantly affect the lengthtension characteristics of the bronchial rings (optimal tension of rings with and without epithelium: 2.9±0.1 g and 3±0.2 g, respectively; x±SEM, n=10) or the maximal responses evoked by contractile agents, suggesting that the procedure did not affect the integrity of the bronchial smooth muscle. Removal of the epithelium increased the responsiveness of bronchial rings to acetylcholine, histamine, and 5-hydroxytryptamine, causing parallel leftward displacements in their -From the Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Reprint requests: Dr: Vanhoutte, Department of Physiology, Mayo Clinic, Rochester, Minnesota 55905 CHEST I 87 I 5 I MAY, 1985 I SuppIemenl
1898
concentration-effect curves. The magnitude of the displacement produced by epithelium-removal was similar fOr each agonist. The increased sensitivity of epithelium-denuded preparations to these agonists did not result from the removal of degradative processes. Inhibition of acetylcholinesterase (ecothiopate, 1O- 7 M) or monoamine oxidase (pargyline, lO-4M) did not prevent the increased responsiveness of the denuded rings to acetylcholine or 5-hydroxytryptamine, respectively. Neither did the increased sensitivity result from the loss of an epithelial diffusion barrier. Following contraction of the bronchial rings to acetylcholine (concentrations causing 50% of the maximum response in each ring), isoproterenol caused concentration-dependent relaxations which were significantly depressed fOllowing epithelium removal. This suggests that the epithelium releases an inhibitory factor which augments the direct inhibitory effect of p-adrenoceptor stimulation. Epithelium removal also affected the sensitivity of the bronchial rings to endogenously released acetylcholine. 'Ihmsmural nerve stimulation of canine bronchi causes activation ofparasympathetic and sympathetic nerve fibers; the parasympathetic in8uence predominates to cause contraction.3,4 The contractile responses evoked by nerve stimulation initially were not significantly different between rings with and without epithelium. However, during continued stimulation, the contractile response in unrubbed rings gradually declined, whereas in denuded preparations the response was well maintained. The epithelium-dependent decay in the neuronal response was still present following propranolol administration (5 X lO-8M). These results" indicate that the respiratory epithelium
1108
releases a smooth muscle relaxing factor to inhibit the effects of bronchoconstrictor agonists and to augment the direct inhibitory effectsofrelaxing stimuli. Moreover,the in8uence of epithelium removal on the responses to nerve stimulation suggests that this inhibitory factor might be released following activation of the epithelium by these agonists. For example, the delay in the epithelium-dependent inhibitory response during nerve stimulation might represent the diffusion of nerve-released acetylcholine to the epithelial cells. Preliminary results have now been obtained using a cascade system to bioassay epithelium-derived factors. This system demonstrates that the respiratory epithelial cells continuously release a potent inhibitory substance which causes relaxation of bronchial smooth muscle. It is possible that under conditions in which the epithelium is damaged, eg, in asthma. bronchial hyperreactivity results from interruption of the production of this inhibitory substance. REFERENCES 1 Nadel JA. Bronchial reactivity. Adv Intern Med 1983; 28:207-23 2 Hogg Ie, Eggleston PA. Is asthma an epithelial disease? Am Rev Respir Dis 1984; 129:207-8 3 Russell JA. Responses of isolated canine airways to electric stimulation and acetylcholine. I Appl Physiol 1978; 45:690-8 4 Vermeire PA, Vanhoutte PM. Inhibitory effects of catecholamines in isolated canine bronchial smooth muscle. I Appl Physioll979; 46:787-91 5 Flavahan NA, Aarhus LL, Rimele TJ, Vanhoutte PM. The respiratory epithelium inhibits bronchial smooth muscle tone. I Appl Physiol (in press)