Session 7

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80

SIMULATED MICROGRAVITY INCREASES

-ADRENERGIC LIPOLYSIS IN HUMAN ADIPOSE TISSUE

P. Barbe, J. Galitzky, I. De Glisezinski**, D. Rivière**, C. Thalamas*, J.M. Senard, F. Crampes**, M. Lafontan, and M. Berlan.

Laboratoire de Pharmacologie Médicale et Clinique (INSERM Unité 317), *Centre d’Investigation Clinique and Laboratoire des Adaptations de l’Organisme à l’Exercice Musculaire**, Université Paul Sabatier, 31073 Toulouse.

The effect of a sustained decrease of sympathetic nervous activity, achieved through a S-day head down bed rest (HDBR), on the ß-adrenergic lipolytic activity of subcutaneous adipose tissue were studied in eight healthy men.

The in situ ß-adrenoceptor (AR) sensitivity was studied using the microdialysis method. Local perfusion of increasing concentrations of isoprenaline showed an increased 13-AR sensitivity on lipolysis (assessed by extracellular glycerol concentration) and on vascular tone (assessed by the ethanol clearance).

The adrenergic sensitivity of isolated adipocytes was studied in vitro. Basal lipolysis and the response to non-selective (isoprenaline) or selective (dobutamine, terbutaline, CGP 12177) ß-AR agonists were increased after HDBR as was the lipolytic effect of dibutyryl-cAMP. When data were expressed in per cent of dibutyryl-cAMP effect in order to rule out the post-receptor events, basal and lipolytic responses to ß-AR agonists where similar before and during HDBR. The

2-AR-mediated antilipolytic effects of adrenaline were not modified.

Lymphocyte ß-AR number were unchanged during HDBR.

Our results demonstrate that a sustained sympathoinhibition induces an increase of the lipolytic ß-adrenergic response in adipose tissue and suggest that this hypersensitization is linked to an increase in the post-receptor steps of the lipolytic cascade in the adipocyte rather than to changes in ß-adrenoceptors.

This work was supported by the Centre National d’Etudes Spatiales.

81

LIPOLYTIC EFFECTS OF CONVENTIONAL ß3-ADRENOCEPTOR AGONISTS AND OF CGP 12,177 IN RAT AND HUMAN FAT CELLS: PRELIMINARY PHARMACOLOGICAL EVIDENCE FOR A PUTATIVE ß4-ADRENOCEPTOR

J. Galitzky, P. Verwaerde, D. Langin, J.L. Montastruc, M. Lafontan, M. Berlan

Laboratoire de Pharmacologie Médicale et Clinique, INSERM U 317, Faculté de Médecine, Toulouse, France

The nature of rat and human fat cell ß3-adrenoceptors was investigated by studying the effects of the new ß3-adrenoceptor selective antagonist, SR 59-230A, on lipolysis induced by the conventional ß3-adrenoceptor agonists, CL 316,243 and SR 58,611A, and by the non-conventional partial ß3-adrenoceptor agonist CGP 12,177 (a potent ß1- and ß2-adrenoceptor antagonist with partial ß3-adrenoceptor agonist property).

In rat fat cells, the rank order of potency of agonists was: CL 316,243>isoprenaline>SR58,611A>CGP 12,177. The three former agents were full agonists whereras CGP 12,177 was a partial agonist (intrinsic activity of 0.70). In human fat cells, the lipolytic effect of CGP 12,177 reached 25 % of isoprenaline effect. CL 316,243 was a poor inducer of lipolysis and SR 58,611A was ineffective. In rat fat cells, lipolysis induced by CL 316,243 and SR 58,611A was competitively antagonized by SR 59,230A. Schild plots were linear with pA2 values of 6.89 and 6.37, respectively. Conversely, 0.1, 0.5 and 1 µM SR 59,230A did not modify the concentration-response curve of CGP 12,111. A rightward shift of the curve was however observed with 10 and 100 µM of SR 59,230A. The apparent pA2 value was 5.65. The non-selective ß-adrenergic antagonist, bupranolol, competitively displaced the concentration-response curve of CGP 12,177 and CL 316,243. Schild plots were linear with pA2 values of 6,70 and 7,59, respectively. CL316,243-mediated lipolytic effect was not antagonized by CGP 20,712A. In human fat cells, CGP 12,177-mediated lipolytic effect was antagonized by bupranolol and CGP 20,712A. SR 59,230A (0.1, 1 and 10 µM) did not modify the concentration-response curve of CGP 12,177. A rightward shift was however observed at 100 I1M leading to an apparent pA2 value of 4.32.

The results suggest that the non-conventional partial agonist CGP 12,177 can activate lipolysis in fat cells through the interaction with a ß-adrenoceptor pharmacologically distinct from the ß3-adrenoceptor, i.e. through a putative ß4-adrenoceptor L mediates lipolytic effect of CGP12,177 in human fat cells.

82

A STUDY OF THE CARDIOVASCULAR ACTIONS OF SR 58611 A, A BETA3-ADRENOCEPTOR AGONIST IN DOGS: CENTRAL VERSUS PERIPHERAL MECHANISMS

J.L. Montastruc, P. Verwaerde, M. Pelat, J. Galitzky, D. Langin, M. Lafontan and M. Berlan

Laboratoire de Pharmacologie Médicale et Clinique, lNSERM U 317, Faculté de Médecine, Toulouse, France

In order to investigate the putative role of beta3-adrenoceptors in central and peripheral cardiovascular regulations, the effects of intracisternal (i.c.) and intravenous (i.v.) injections of SR 58611 A (10, 50, 100 and 200 nmol kg-1), a selective beta3-adrenoceptor agonist, were investigated in chloralose-anaesthetized dogs.

In normal dogs, i.v. SR 58611 A (100 and 200 nmol kg-1) induced a dose-dependent increase in heart rate with no change in blood pressure. After i.c. injection, SR 58611 A falled to modify blood pressure and heart rate (except at the highest dose 200 nmol kg-1) which induced a positive chronotropic effect. The positive chronotropic effect of SR 58611 A (200 nmol kg-1) appeared earlier and was significantly more pronounced after i.v. than i.c. administration. In sinoaortic denervated dogs (i.e., animals deprived of baroreceptor pathways), SR 58611 A (200 nmol kg-1 i.v.) induced a marked hypotensive effect with a slight decrease in heart rate. The positive chronotropic effect of i.v. SR 58611 A (200 nmol kg-1) was reduced (but not suppressed) by pretreatment with beta-adrenoceptor antagonists (propranolol, nadolol, bupranolol or the beta3-adrenoceptor selective antagonist, SR 59230 A
(2 mg kg-1i.v.).

These experiments do not show evidence for central cardiovascular effects of SR 58611 A and suggest the lack of involvement of beta3-adrenoceptors in central control of blood pressure and heart rate. The positive chronotropic effect is mainly of peripheral origin and can be attributed to a baroceceptor-mediated reflex due to the beta3-adrenoceptor mediated vasodilation. It is also suggested that SR 58611 A also exerts its positive chronotropic action through a direct cardiac effect, the mechanism of which is unknown.

83

ACTIVATION OF ANTILIPOLYTIC

2-ADRENOCEPTORS BY ADRENALINE DURING EXERCISE IN HUMAN ADIPOSE TISSUE

V. Stich, I. De Glisezinski**, J. Galitzky*, F. Crampes**, M. Lafontan*, J. Hejnova and M. Berlan*.

Dep. of Sport Med., Charles University, Prague, Czech Republic, *Lab. de Pharmacologie Médicale et Clinique, Faculté de Médecine, (INSERM, Unité 317), Toulouse and **Lab. des Adaptations de l’Organisme à l’Exercice Musculaire, CHU Purpan, Toulouse

The sympathetic nervous system activation (SNS) induces lipomobilization during exercise through activation of ß-adrenergic receptors (AR) in adipose tissue (AT). The goal of the investigation was to study in situ the involvement of adrenaline and of its

2- adrenergic antilipolytic effects in the control of lipolysis during exercise.

For that, a differential activation of the SNS and of the adrenal medulla secretion was obtained in overnight fasting male healthy subjects (n=7; age=24.1±2.2 years) performing two repeated bouts of prolonged exercise during 60 min at 45% of their maximal aerobic power and separated by a resting period of 60 min. The in situ lipolysis was evaluated by the microdialysis method. Two probes (Carnegie Medecin, Stockholm, Sweden) of 20 x 0.5 mm and 20,000-MW cut-off were implanted in abdominal subcutaneous AT. One probe was perfused with Ringer solution alone and in the second probe with Ringer ± 0.1 mM phentolamine (

2-AR antagonist). The increase of plasma noradrenaline was similar during each exercise-bout whereas the increase of plasma adrenaline level was 4 fold higher during the second exercise-bout. The increase in plasma glycerol level was significantly higher and the decrease of plasma insulin level more prononced during the second exercise-bout. The increase in glycerol production (evaluated by the extracellular concentration) in AT was more marked during the second exercise-bout (±270%) than during the first one (±170%). The addition of phentolamine was without significant effect on glycerol production during the first exercise-bout. Oppositely, the presence of phentolamine potentiate (±403%) the glycerol production during the second exercise-bout.

These results show 1) that adrenaline activates lipomobilization during exercise and 2) that the stimulation of

2-AR by adrenaline partially inhibits lipolysis in subcutaneous AT, during exercise in human.

84

SELECTIVE ACTIVATION OF ß3-ADRENOCEPTORS BY OCTOPAMINE IN MAMMAL FAT CELLS

C. Carpéné, J. Galitzky, C. Atgié, E. Fontana, M. Lafontan and M. Berlan.

INSERM U317, Lab. de Pharmacologie Médicale et Clinique, Univ. P. Sabatier, 37 Allées Jules Guesde, 31073 Toulouse.

The ß3-adrenergic receptor (AR) is stimulated by endogenous catecholamines and by synthetic ß3-AR agonists. In mammalian fat cells, stimulation of the ß3-ARs increase lipolysis. However, no selective physiological agonist for the ß3-AR has been described.

The lipolytic effects of compounds metabolically related to catecholarnines were compared to those of noradrenaline and ß3-AR agonists (BRL 37,344 and CL 316,243) in fat cells from various species. Octopamine, but not tyramine or ß-phenylethylamine, is fully lipolytic in fat cells from species in which selective ß3-agonists act as lipolytic agents (rat, hamster, dog). Octopamine had no effect on guinea pig or human fat cells, in which the ß3-AR agonists were devoid of lipolytic action. In rat fat cells, the effect of 100 µM octopamine is inhibited by bupranolol and propranolol (IC50: 0.03 and 1.16 µM). The selective ß3-AR antagonist SR 59,230A had an IC50 value of 1.38 µM whereas CGP 20712A or ICI 118,551 (ß1- and ß2-AR antagonist, respectively) inhibited octopamine effects at concentrations > 10 µM. A similar order of potency was found when lipolysis was stimulated with ß3-AR agonists. BRL and octopamine shared a very low affinity for [3H]CGP 12,177 binding sites (IC50 > 100 µM) in dog fat cells. In fat cells from dog, and after blockade of ß1- and ß2-ARs, noradrenaline stimulated lipolysis with a pD2 of 6.54 and octopamine with a pD2 of 5.58. In rat brown fat cells, CL, isoprenaline, noradrenaline and octopamine stimulated 02 consumption (pD2: 9.0, 8.4, 7.9 and 5.8) and lipolysis (pD2: 8.4, 7.9, 6.6 and 5.1). Other biogenic amines (epinine, synephrine and phenylethanolamine) partially increased glycerol production. Their effects, also found in species which barely respond to ß3-AR-agonists, were supressed by selective ß-AR antagonists.

These data suggest that, among the biogenic amines metabolically related to catecholamines, octopamine can be considered as the endogenous ligand the most selective for the ß3-ARs present in mammals.

85

HYPERADRENERGIE ET HEMODYNAMIQUE APRES ENVENIMATION SCORPIONIQUE EXPERIMENTALE

Kh. Zeghal, S. Hammami, Z. Sahnoun, T. Rebaï.

Laboratoire de Pharmacologie de la Faculté de Médecine de Sfax -3002 SFAX- (TUNISIE).

Le mécanisme physiopathologique, ainsi que le traitement de l’envenimation scorpionique (E.S) reste encore un sujet de discussion.

Notre étude expérimentale chez le Rat a pour but d’explorer certains mécanismes physiopathologiques d’ordre hémodynamique de l'(ES) par les venins de scorpion tunisiens: buthus occitanus (BO) et androctonus australis (AA).

Méthodes

: L’étude a été réalisée chez le Rat anesthésié et ventilé. Les paramètres étudiés sont la pression artérielle (PAS et PAD) et la fréquence cardiaque (FC). Un dosage des catécholamines plasmatiques et une étude anatomopathologique ont été effectués.

Résultats

: L’administration de venin de BO ou d’AA entraîne pour toutes les doses (300-850-1250µg/kg) une variation biphasique de PAS, PAD et FC. Une élevation fugace est suivie d’une baisse progressive et durable des paramètres hémodynamiques. Ces perturbations sont doses dépendantes (p