Atelier : Utilisation de préparations d'origine humaine

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LE MUSCLE LISSE BRONCHIQUE HUMAIN—APPROCHE FONCTIONNELLE

C. Advenier*, E. Naline*, M. Molimard**, P. Devillier***

Facultés de Médecine *Paris-Ouest, **Bordeaux, ***Reims

Dans le cadre de cette présentation, nous tenterons de montrer quelle contribution l’utilisation de bronches humaines isolées a apporté à la connaissance des récepteurs des tachykinines impliqués dans la contraction du muscle lisse bronchique et au développement d’antagonistes spécifiques. Les tachykinines, substance P, neurokinine A et neurokinine B sont les médiateurs du système non adrénergique non cholinergique. Elles agissent avec des activités préférentielles sur trois types de récepteurs, dénommés respectivement NK1, NK2 et NK3. Des sous-types de récepteurs ont été identifiés en fonction de l’espèce animale, conduisant, par exemple dans le cas des récepteurs NK2 à des récepteurs NK2A (cobaye, furet, singe, chien) et NK2B (hamster) (Maggi et coll., 1993) mais aussi en fonction de ligands et ainsi, dans le cas des récepteurs NK1, ont été identifiés des récepteurs au septide (Petitet et coll., 1992). Les études fonctionnelles sur des bronches humaines isolées provenant pour la plupart de pièces d’exérèses pulmonaires de patients ex-fumeurs ont permis de démontrer que la contraction du muscle lisse bronchique est de type NK2A (Naline et coll., 1989) mais, qu’au niveau des bronches de très petit calibre elle peut impliquer aussi des récepteurs NK1 (Naline et coll., l996). Des études biochimiques associées aux études fonctionnelles permettent de montrer que la stimulation NK2 est couplée à la formation de phosphoinositides alors que celle des récepteurs NK1 l’est à celle de prostanoïdes. L’utilisation des antagonistes des tachykinines a permis de confirmer le caractère NK2A prédominant de la contraction bronchique (Advenier et coll.,1992). A l’inverse, la bronche humaine se révèle être un outil précieux pour préciser la puissance et la spécificité de nouveaux antagonistes. Enfin, les études sur bronches humaines peuvent apporter une contribution intéressante sur un plan quantitatif à la mise en place des essais cliniques ainsi qu’en atteste, dans le cas de l’antagoniste NK2, le SR 48968, le rapprochement des concentrations antagonistes qui a pu être constaté entre les expériences in vitro et les essais cliniques, réalisés chez l’asthmatique, dans des tests de provocation à la NKA (Van Schoor et coll., 1996).

Maggi et coll., 1993, ERJ, 6: 735; Petitet et coll., 1992, C.R.Acad.Sci., 314:299; Naline et coll., 1989, ARRD, 140:679; Naline et coll., 1996, Am. J. Physiol., 271 :L-763; Advenier et coll., 1992, ARRD, 146:1177; Van Schorr et coll., 1996, ERJ,9:289s).

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IN VITRO-INDUCED HUMAN AIRWAY HYPERRESPONSIVENESS

M. Molimard1, E. Naline2, V. Lagente3 and C. Advenier2

1 University Bordeaux 2, F-33076 Bordeaux, 2 Faculté de Médecine, F-75270 Paris, 3 Faculty de Pharmacie, F-35043 Rennes.

Human isolated bronchus model is interesting to study drug-receptor interactions in ” normal ” preparations. Several attempt have been done to prepare in vitro models of airway hyperresponsiveness case to the pathophysiology of asthma. In our lecture, we will present some results performed with LPS and interleukin 1ß (IL-1ß).

LPS (100 ng/ml, during 3 to 6 hours) or IL-1ß potentiated bradykinin and the tachykinin NK-1 selective receptor agonist [Sar9, Met-O2] SP -induced human isolated bronchi contraction in vitro (IL-1ß3 10-10 M, at 37°C during 1 to 3 hours for bradykinin or at 21 °C during 15 hours for [Sar9, Met-O2] SP in Krebs-Henseleit solution). As in control bronchi, the effect of bradykinin and of [Sar9, Met-O2] SP after interleukin 18 pretreatment were abolished by indomethacin (104 M), the thromboxane A2 antagonist GR 32191 suggesting that prostanoids remain involved under these experimental conditions. Although bradykinin and [Sar9, Met-O2] SP -induced contractions were mediated by thromboxane receptor stimulation, the thromboxane A2 (TxA2) mimetic U46619 induced contraction of human bronchi was not enhanced by IL-1ß pretreatment. The cycboxygenase 2 (cox-2) inhibitor CGP 28238 (10-6 M) inhibited IL-1ß-induced potentiation of [Sar9, Met-O2] SP but not of bradykinin effect. Bradykinin and [Sar9, Met-O2] SP induced a release of TxB2, the stable metabolite of TxA2, in the organ bath and this release was increased by IL-1ß pretreatment. Bradykinin- induced release of 6 keto prostaglandin F1

(the stable metabolite of prostaglandin I2) was not enhanced by IL-1ß. Taken together, our results suggest that IL-1ß is able to potentiate the effect of bradykinin or tachykinin receptor agonists on the human isolated bronchus. Several mechanisms might be involved including an increase of thromboxane synthase synthesis and/or activity in the case of bradykinin and of short term incubation (3 h, 37 °C) or an increase of synthesis and/or activity of cox-2 for tachykinin and for long term incubation (15h, 21 °C).

In conclusion, human isolated bronchi may be useful to analyze the respective role of compounds implicated in the pathophysiology of asthma.

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ELECTROPHYSIOLOGY AND CALCIUM SIGNALLING IN HUMAN ISOLATED AIRWAY MYOCYTES

R. Marthan, J.M. Hyvelin, E. Roux and J.P. Savineau.

Laboratoire de Physiologie Cellulaire Respiratoire, INSERM (C.R.I. 9806), Université Victor Segalen Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux cedex, France.

Cells isolated from airways have been used to precisely characterise the electrophysiological properties of this smooth muscle and, more recently, to describe the changes in cytosolic calcium concentration ([Ca2+]i) occurring upon agonist stimulation. Although most studies have produced consistent results in terms of types of ion channel and pathways of calcium signalling implicated in the mechanical activity of airways, there are differences according to (i) the site along the bronchial tree (trachea vs. bronchi); (ii) the proliferating status of the cells (freshly isolated vs. cultured) and (iii) the species (human vs. animals).

Taking into account that human isolated bronchial smooth muscle is the most relevant tissue to examine the pathophysiological mechanisms implicated in asthma-induced airway narrowing, the following issues need to be discussed. With respect to the electrophysiological properties of airway smooth muscle, the contribution to [Ca2+]i rise of Ca2+ influx through L-type voltage-dependent calcium channels depends on the balance between depolarisation related to non specific cation channels and/or chloride channels activation and hyperpolarization related to activation of a variety of potassium channels. Most of the above mentioned channels appear controlled, directly or indirectly, by agonists in human bronchial smooth muscle. With respect to calcium signalling, the pattern of agonist-induced [Ca2+] i responses, the so-called [Ca2+] i oscillations, has been recently observed in freshly isolated airway smooth muscle cells. The existence, the role and the calcium sources involved in these oscillations in human bronchial smooth muscle are currently being investigated.

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ASSOCIATIONS ENTRE PROPRIETES VISCOELASTIQUES DES GROSSES ARTERES ET LES COMPOSANTS DE LA MATRICE EXTRACELLULAIRE DANS L’ANEVRYSME DE L’AORTE ABDOMINALE CHEZ L’HOMME

P. Boutouyrie, C. Glazer, Y. Bézie, S. Laurent, P. Lacolley

INSERM U337, 15 rue de l’Ecole de Médecine, Paris, France; France.

L’objectif de ce travail était de déterminer les propriétés élastiques de l’artère carotide primitive de 21 patients référés pour cure d’anévrysme de l’aorte abdominale (AAA) (age 71 ans, range 61-84), en comparaison à deux populations contrôle, constituée de 36 hypertendus (HT: 50 ans, range 24-88) et 22 sujets normotendus (NT: 44 ans, range 23-85). Le second objectif est d’établir les relations entre les propriétés fonctionnelles de l’ACP et la sévérité de l’atteinte athéromateuse, évaluée par histomorphométrie sur les pièces opératoires. Le diamètre carotidien des patients porteurs d’anévrysme est plus grand que celui des témoins NT et HT. La distensibilité de l’anévrysme est plus faible que celle de l’aorte susjacente. Après ajustement pour l’âge et la PA; le distensibilité aortique et carotidienne est plus basse chez les patients porteurs d’AAA que chez les témoins NT et HT. Les lésions pariétales de l’AAA sont très sévères, associant fibrose extensive, inflammation et lésions athéromateuses. Leur grade n’est pas associé aux altérations fonctionnelles carotidiennes et aortiques. L’AAA est caractérisé par des anomalies sévères et diffuses de la fonction d’amortissement des grosses artères, en association à des lésions histologiques avancées.

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SPECIES DIFFERENCES IN THE CONTRACTILE AND RELAXANT RESPONSES OF ISOLATED, NORMAL PULMONARY VASCULAR PREPARATIONS TO 5-HYDROXYTRYPTAMINE: ANIMAL VS. HUMAN STUDIES

E.J. Morcillo and J. Cortijo

Departament de Farmacologia. Facultat de Medicina i Odontologia. Universitat de València, València, Spain

5-Hydroxytryptamine (5-HT, serotonin) is released from pulmonary neuroendocrine cells and platelets and it has been implicated in primary and secondary pulmonary hypertension, hypoxic pulmonary vasoconstriction, and pulmonary side-effects of various drugs. 5-HT is a potent (pEC50 ~6-7) constrictor of bovine, ovine, canine, caprine, feline and rodent (rabbit, guinea-pig and rat) isolated pulmonary arteries. Regional differences in reactivity (conduit vs. resistance pulmonary arteries) may exist. The receptor involved in vasoconstriction of pulmonary arteries is the 5-HT2A but additional presence of different 5-HT1 receptors (precontracted bovine pulmonary artery; rat) has been reported. 5-HT relaxes the porcine isolated pulmonary artery through activation of endothelial 5-HT2B receptors. Pharmacological responses of the pulmonary veins to 5-HT have been less studied and comprise both contraction (bovine[5-HT2], canine, feline, rabbit) and relaxation (ovine [5-HT4], caprine). Human isolated intrapulmonary arteries and veins (2-5 mm i.d.; isometric recording) contracted (endothelium-independent) in response to 5-HT (pEC50 ~6.5 in artery and ~7 in vein). Functional and radioligand binding studies have demonstrated that the human pulmonary artery and vein contain a mixed population of 5-HT1B/1D and 5-HT2A receptors mediating vasoconstriction but no evidence of involvement of 5-HT1A, 5-HT3 and 5-HT4 receptors. Therefore, remarkable differences in the in vitro pulmonary vasoreactivity to 5-HT -and to other agonists like sumatriptan- in humans compared to other mammals advise the use of human tissues in research addressed to study pathophysiological responses of pulmonary circulation that may have clinical relevance, including the potential therapeutic use of selective 5-HT1B/1D antagonists in pulmonary hypertension.

(Supported by SAF96-200 and SAF97-0047 from CICYT and Generalitat Valenciana).

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CHOLINERGIC CONTROL OF HUMAN AND ANIMAL PULMONARY VASCULAR TONE

X. Norel, L. Walch, B. Leconte, J.P. Gascard and C. Brink

CNRS ERS-566, Centre Chirurgical Marie Lannelongue, 133 av. de la Résistance, 92350 Le Plessis-Robinson, France.

The regulation of pulmonary vascular tone by acetylcholine (ACh) involves the activation of different subtypes of muscarinic receptors as well as cholinesterases (ChE) which are responsible for ACh degradation. Most of the studies on the cholinergic control of the pulmonary vascular tone have been performed in vessels derived from animals. The endothelium-dependent relaxation induced by ACh has been reported in isolated pulmonary vessels from different animals (1, 2) including man (3). However, the muscarinic receptors involved in the ACh induced vasodilatation of rat pulmonary artery are of the M3 subtype (4) while those characterised in human pulmonary artery are of the M3 and M1 subtypes (5). The ability of ACh to induce pulmonary vasoconstriction is also species dependent. In vessels derived from sheep lung, ACh induced contractions in veins but not in arteries (6) whereas in human pulmonary vessels the reverse is observed (7). The ChE are implicated in the vasoconstriction induced by ACh in human and rabbit pulmonary arteries (7, 8). However, in these studies while acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) activities were detected in human vessels only acetylcholinesterase activity was found in rabbit vessels. Together these results concerning the cholinergic control of the pulmonary vascular tone indicate that extrapolation of the data obtained in animal models to human vessels requires some caution.

(1) Liu et al., 1992; (2) Gruetter & Lemke, 1986; (3) Greenberg et al., 1987; (4) McCormak et al., 1988; (5) Norel et al., 1996;
(6) Toga et al. 1996, (7) Walch et al. 1997; (8) Altiere et al. 1994.

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SELECTIVE DIFFERENTIATION AND PROLIFERATION OF HUMAN EOSINOPHILS FROM UMBILICAL CORD BLOOD-DERIVED PROGENITORS: PHARMACOLOGY OF CALCIUM FLUXES AND SUPEROXIDE RELEASE.

D.M. Zardini

Neuroimmunologie & Inflammation, Centre de Recherche Public-Santé, 120 Route d’Arlon. L-1150 Luxembourg.

Eosinophils, a minority constituent of the peripheral blood, represent major effector cells in allergic inflammatory responses. Once activated, they are thought to mediate tissue damage through the release of reactive oxygen species (O2) and of a number of cytotoxic proteins stored in their cytoplasmic granules. Because direct isolation of sufficient number of purified eosinophils from peripheral blood is difficult, we developed a modified method to generate large number of eosinophils in vitro. Human umbilical cord blood mononuclear cells cultured in the presence of IL-3 and IL-5 were selectively differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. Studies were thereafter performed on these in vitro differentiated eosinophils to assess their ability to respond to a variety of stimuli by generating active oxygen species such as the superoxide anion O2 and we investigated the role of Ca2+ in the in vitro activation of these cells. Umbilical cord-derived eosinophils responded to fMLP and PAF in terms of O2 production and [Ca2+] variations. Pharmacological manipulation of extra- and intracellular calcium concentrations as well as modifications of membrane calcium fluxes, allowed a determination of essential Ca2+ requirements for their biological response in terms of O2 production. The cytological and pharmacological results obtained with this methodology suggest that human umbilical cord-derived eosinophils behave as adult human peripheral blood eosinophils do after fMLP and PAF activation, and thus may serve as a model to study eosinophil biological activities.