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Neuropeptides in endobronchial biopsies from asthmatics
NEUROPEPTIDES:
193
FLJNCTION AND PHARMA COLOGY
&ys,18f4;3days,19f4;10days,5f1;4months,4 f 1). By 4 months nerves were only seen in airways. These results suggest that NO-synthase is present in puhnonary nerves and neurally derived NO may be a significant factor in the adaptation to extra-uterine life.
P18 Neuropeptides in Endobronchial Biopsies from Asthmatics D. R. Springall, P. H. Howarth*, P. Chanezt, A. Redington*, J. Bousquett, S. T. Holgate*, J. M. Polak Royal Postgraduate Medical School, London, *Southampton General Hospital, Southampton and THopital l’Aiguelongue, Montpellier, France Asthma in a clinical syndrome characterised by bronchial hyperresponsiveness and having an ir&mmatory component. Treatment with corticosteroids helps resolve the inflammation but the level of bronchial responsiveness remains within an abnormal range, suggesting another underlying mechanism. We have therefore investigated the innervation of asthmatic lung in comparison with controls. Endobronchial biopsies were obtained from carinae of the 2nd and 3rd division airways from 30 mild to severe atopic asthmatics, 10 atopic rhinitic non-asthmatic subjects and 20 non-atopic healthy controls. The biopsies were immediately fixed in Zamboni’s fixative, frozen sections cut and stained by immunofluorescence with antisera to the pan-neuronal marker PGP 9.5 and the neuropeptides, VIP, SP and CGRP. Immunofluorescent nerve density was graded in epithelium, smooth muscle, seromucous glands and blood vessels larger than arterioles and venules. There was a tendency for a higher PGP 9.5 scores in the asthmatics, but subdivision of the nerves into VIP, SP and CGRP immunoreactive failed to identify any differences between asthmatic and non-asthmatic airways. It is concluded that if neuropeptides are involved in asthma, the abnormality must be at the receptor level or due to the prolonged bronchoconsticting action of sensory peptides caused by an absence of neutral endopeptidase.
P19 ANP Binding Sites in Rat Lung are Decreased After Hypoxic Exposure D. R. Springall, M. M. Mannan, A. MoradoghliHaftvani, J. M. Polak Department of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London, W 12 ONN, UK Atria1 natriuretic peptide (ANP) has vasodilator and bronchodilator effects and rats exposed to hypoxic conditions have elevated plasma levels after 24 h which persist to at least 7 d. To determine whether ANP receptor binding sites are also affected, we studied Iungs fkom male Wistar rats exposed to normobaric hypoxia (FiO2 = 0.1) for peri-
odsmngingfrom4htolOd(n=5foreachperiod)together with normoxic controls (n = 5). Quantitative automdiography was used to estimate the density of ‘IJI-aANP~-2e binding in frozen tissue sections. ANP receptor binding levels were decreased in all blood vessels of rat lung after 7 and 10 d ofhypoxic exposure (control: 5 1. I8 amol/mmz, S.E.M. f 5.27, 7 d hypoxia: 33.51 f 2.08, 10 d hypoxia: 33.92 f 2.45; p < 0.05). No changes in binding levels were observed in airways after similar periods of hypoxia. These results suggest that hypoxia leads to a down-regulation of vascular ANP receptors, possibly as a desensitizing response to counteract the effect of elevated plasma levels. Gur fmdings may be important in unde&anding the regulation of natriuresis and pulmonary tone in hypoxia.
PZOBombesin and Bronchoconstriction E. Lath, Y. Lam&y and J.-P. Gies Laboratoire de Neuroimmunophannacologie INSERM CJF-9 105, Universite Louis Pasteur, 67401 Illkirch Cedex, France Bombesin acts predominantly as a growth factor in lung tissue. However in guinea pig in vivo, bombesin also exerts a bronchoconstrictor effect. We demonstrate that bombesin is able to pmduce a potent dose-dependent bronchoconstriction in vitro in the presence of 1es M of DL-thiorphan (endopeptidase 24.11. inhibitor). The maximal contraction observed is of 119 f 7 mg i.e. 57 f 3 % of the maximal contractile response to carbachol. Bombesin-related agonists produce a contractile response of peripheral airways with the following relative potencies: bombesin > GRP > neuromedm C > > neuromedin B. Specific GRP-preferring receptor antagonists, [DPhe6JBBS+umethyl ester and [DPhe6,Cpa14, Yl3--14]BBS~~~@-I~ inhibit bombesin-induced bronchoconstriction with high potency (PA2 of 7.1 and 7.2 respectively). The less specific antagonist [Leu14,Y 1314]BBS has a lower one (PAZ of 5.6). These results agree with the hypothesis that GRP- preferring bombesin receptors are involved in bombesin-induced bronchoconstriction. Since the contraction is unaffected by atropine, hexamethonium, propranolol, triprolidine, methysergide, Ro 19-3704 (platelet activating factor antagonist) and indomethacin or AA-86 1 (lipoxygenase inhibitor), we can suggest that the bombesin response does not occur via a mechanism involving the corresponding endogeneous agents or via the release of arachidonic acid metabolites. Moreover, the effect of bombesin is insensitive to capsaicin pretreatment, excluding the involvement of endogeneous neuropeptides. Our present results provide evidence that bombesininduced bronchoconstriction results from a direct effect of bombesin on bronchial smooth muscle GRP-preferring receptors.
193
FLJNCTION AND PHARMA COLOGY
&ys,18f4;3days,19f4;10days,5f1;4months,4 f 1). By 4 months nerves were only seen in airways. These results suggest that NO-synthase is present in puhnonary nerves and neurally derived NO may be a significant factor in the adaptation to extra-uterine life.
P18 Neuropeptides in Endobronchial Biopsies from Asthmatics D. R. Springall, P. H. Howarth*, P. Chanezt, A. Redington*, J. Bousquett, S. T. Holgate*, J. M. Polak Royal Postgraduate Medical School, London, *Southampton General Hospital, Southampton and THopital l’Aiguelongue, Montpellier, France Asthma in a clinical syndrome characterised by bronchial hyperresponsiveness and having an ir&mmatory component. Treatment with corticosteroids helps resolve the inflammation but the level of bronchial responsiveness remains within an abnormal range, suggesting another underlying mechanism. We have therefore investigated the innervation of asthmatic lung in comparison with controls. Endobronchial biopsies were obtained from carinae of the 2nd and 3rd division airways from 30 mild to severe atopic asthmatics, 10 atopic rhinitic non-asthmatic subjects and 20 non-atopic healthy controls. The biopsies were immediately fixed in Zamboni’s fixative, frozen sections cut and stained by immunofluorescence with antisera to the pan-neuronal marker PGP 9.5 and the neuropeptides, VIP, SP and CGRP. Immunofluorescent nerve density was graded in epithelium, smooth muscle, seromucous glands and blood vessels larger than arterioles and venules. There was a tendency for a higher PGP 9.5 scores in the asthmatics, but subdivision of the nerves into VIP, SP and CGRP immunoreactive failed to identify any differences between asthmatic and non-asthmatic airways. It is concluded that if neuropeptides are involved in asthma, the abnormality must be at the receptor level or due to the prolonged bronchoconsticting action of sensory peptides caused by an absence of neutral endopeptidase.
P19 ANP Binding Sites in Rat Lung are Decreased After Hypoxic Exposure D. R. Springall, M. M. Mannan, A. MoradoghliHaftvani, J. M. Polak Department of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London, W 12 ONN, UK Atria1 natriuretic peptide (ANP) has vasodilator and bronchodilator effects and rats exposed to hypoxic conditions have elevated plasma levels after 24 h which persist to at least 7 d. To determine whether ANP receptor binding sites are also affected, we studied Iungs fkom male Wistar rats exposed to normobaric hypoxia (FiO2 = 0.1) for peri-
odsmngingfrom4htolOd(n=5foreachperiod)together with normoxic controls (n = 5). Quantitative automdiography was used to estimate the density of ‘IJI-aANP~-2e binding in frozen tissue sections. ANP receptor binding levels were decreased in all blood vessels of rat lung after 7 and 10 d ofhypoxic exposure (control: 5 1. I8 amol/mmz, S.E.M. f 5.27, 7 d hypoxia: 33.51 f 2.08, 10 d hypoxia: 33.92 f 2.45; p < 0.05). No changes in binding levels were observed in airways after similar periods of hypoxia. These results suggest that hypoxia leads to a down-regulation of vascular ANP receptors, possibly as a desensitizing response to counteract the effect of elevated plasma levels. Gur fmdings may be important in unde&anding the regulation of natriuresis and pulmonary tone in hypoxia.
PZOBombesin and Bronchoconstriction E. Lath, Y. Lam&y and J.-P. Gies Laboratoire de Neuroimmunophannacologie INSERM CJF-9 105, Universite Louis Pasteur, 67401 Illkirch Cedex, France Bombesin acts predominantly as a growth factor in lung tissue. However in guinea pig in vivo, bombesin also exerts a bronchoconstrictor effect. We demonstrate that bombesin is able to pmduce a potent dose-dependent bronchoconstriction in vitro in the presence of 1es M of DL-thiorphan (endopeptidase 24.11. inhibitor). The maximal contraction observed is of 119 f 7 mg i.e. 57 f 3 % of the maximal contractile response to carbachol. Bombesin-related agonists produce a contractile response of peripheral airways with the following relative potencies: bombesin > GRP > neuromedm C > > neuromedin B. Specific GRP-preferring receptor antagonists, [DPhe6JBBS+umethyl ester and [DPhe6,Cpa14, Yl3--14]BBS~~~@-I~ inhibit bombesin-induced bronchoconstriction with high potency (PA2 of 7.1 and 7.2 respectively). The less specific antagonist [Leu14,Y 1314]BBS has a lower one (PAZ of 5.6). These results agree with the hypothesis that GRP- preferring bombesin receptors are involved in bombesin-induced bronchoconstriction. Since the contraction is unaffected by atropine, hexamethonium, propranolol, triprolidine, methysergide, Ro 19-3704 (platelet activating factor antagonist) and indomethacin or AA-86 1 (lipoxygenase inhibitor), we can suggest that the bombesin response does not occur via a mechanism involving the corresponding endogeneous agents or via the release of arachidonic acid metabolites. Moreover, the effect of bombesin is insensitive to capsaicin pretreatment, excluding the involvement of endogeneous neuropeptides. Our present results provide evidence that bombesininduced bronchoconstriction results from a direct effect of bombesin on bronchial smooth muscle GRP-preferring receptors.