Session 16 : Pharmacologie cardiovasculaire

0

Session 16 : Pharmacologie cardiovasculaire

149

ENDOTHELIAL REGROWTH IN VIVO IS NOT MODIFIED BY ADDITION OF L-ARGININE OR L-NAME: RESULTS IN A RABBIT BALLOON INJURY MODEL

I. Six*, E. Van Belle**, R. Bordet*, D. Corseaux***, B. Dupuis*, C. Bauters**, M. E. Bertrand**

* Laboratoire de Pharrnacologie, ** Hopital Cardiologique,*** Laboratoire d’Hématologie, CHRU, 1 place de Verdun 59037 Lille Cedex France

Nitric oxide (NO) has been shown to have growth regulatory properties on cultured endothelial cells. An effect of the NO pathway on endothelial regrowth was thought to be one the mechanisms of the beneficial effect observed with the NO precursor L-arginine in balloon angioplasty and atherosclerosis models. The aim of our study was to investigate the effect of L-arginine and NO inhibitor (N

w nitro L-arginine methyl ester; L-NAME) on reendothelialization after balloon iliac injury in 49 rabbits. After angioplasty, rabbits were randomized in three groups L-arginine (22.5 g/L), L-NAME (225 mg/L) and controls. Reendothelialization was quantified 28 days after injury using Evans blue. The results were confirmed by endotllelial-specific immunostaining (CD-31, n=13). In conclusion: L-arginine, despite a significant increase in plasma L-arginine levels, and L-NAME have no effect on reendothelialization after balloon injury. This suggests that, in spite of previously demonstrated beneficial effects in balloon injury and atherosclerosis models, the NO pathway has no effect on endothelial regrowth in-vivo.

Plasma L-arginine level µmol/L

% of reendothelialization

Controls

167

± 11 *

34.64

± 4.43

L-arginine

404

± 86

46.94

± 3.56

L-NAME

143

± 10**

32.86

± 6.17

* p=0.0015 L-arg versus Controls, ** p=0.0011 L-arg versus L-NAME. Controls n=17, L-arginine n=9, L-NAME n=10

150

CHARACTERISATION OF THE PERTUSSIS TOXIN-SENSITIVE G-PROTEINS IN RAT TAIL ARTERY AND AORTIC SMOOTH MUSCLE

M.A. Petitcolin, E. Spitzbarth1, J.-L. Bueb, C. Capdeville-Atkinson1 and E.J. Tschirhart

Centre de Recherche Public de la Santé, 120 Route d’Arlon, L-1150 Luxembourg, Grand-Duché de Luxembourg and 1Laboratoire de Pharmacologie Cardiovasculaire, UHP-Nancy 1, 54000 Nancy, France.

The calcium sensitivity of agonist-induced contraction of vascular smooth muscle is lowered by pertussis toxin (PTX) suggesting that Gi/o proteins are involved in amplification of signal transduction (Spitzbarth et al., this meeting). The aim of this study was to develop methods to isolate and characterise the PTX-sensitive G-proteins in smooth muscle cells from tail artery and aorta.

The aorta (elastic artery) and tail (muscular) artery were dissected out from adult, male, Wistar rats (350-400 g). After removal of endothelium with a wire, membrane proteins were isolated by consecutive centrifugation (Kwan et al., 1983). Proteins were resuspended in a 15 mM Tris buffer with 1% cholic acid, separated on a 16.5% polyacrylamide gel and blotted. G-proteins were detected using antibodies directed against Gi

a subunits 1-2 and 3 (Bio-Rad, 3USA) and Goa subunits (Calbiochem, USA).
G-protein function was assessed by transfer via PTX (1 µg.ml-1) of ADP-ribose using [a 32P]NAD (10 µCi) (NEN, Belgium).

Both Gi and Go proteins were detected in rat tail artery smooth muscle cell membranes. Gi

a l-2 proteins were the predominant form in comparison to Gia 3 (ratio Gia 3/Gia l-2= 1/2) and Goa (traces). In rat aortic smooth muscle membranes, we also detected Gi and Go but at a lower level than in the tail artery. The ratio Gia 3/Gia l-2 was lower (1/3). PTX induced ADP-ribosylation of Ga i/o subunits in rat tail artery and aorta.

We have demonstrated the existence of Gi/o proteins and their sensitivity to PTX in rat tail artery smooth muscle cells membranes. This provides the biochemical correlate of the role of Gi/o proteins in amplification of calcium sensitivity of agonist-induced contraction in vascular smooth muscle.

Kwan C.Y. et al. (1983) Prep. Biochem. 13, 275-314; Spitzbarth E. et al. (this meeting).

151

DELAYED CORONARY ENDOTHELIAL PROTECTIVE EFFECTS OF MONOPHOSPHORYL LIPID A AFTER ISCHEMIA AND REPERFUSION IN RATS.

V Richard, E Danielou, N Kaeffer, C. Thuillez

Department of Pharmacology (VACOMED, IFRMP n°23), Rouen University Medical School, France.

We have shown previously that preconditioning with brief periods of intermittent ischemia (I) induced delayed (24 hours) protection against endothelial injury induced by prolonged ischemia and reperfusion (R), thus extending to endothelial cells the concept of delayed protection, already described for other aspects of ischemia/reperfusion injury. The search for pharmacological compounds which mimic such delayed protection may lead to the development of new anti-ischemic interventions. However, very few compounds have been shown to induce delayed protection, similar to that of preconditioning. Monophosphoryl lipid A (MLA) is an endotoxin analogue which induces delayed myocardial protection in various animal models of I/R injury, possibly through increased expression of inducible NO synthase (iNOS). However, the i potential endothelial protective effects of this drug have not been evaluated. The present study was designed to assess whether MLA exerts X delayed protective effects against R-induced coronary endothelial dysfunction in rats. Wistar rats received a single i.v injection of MLA (450 µg/kg) or solvent. Twenty four hours later, they were anesthetized and subjected to 20 min I with 60 min R, in the absence or the presence of the iNOS inhibitor aminoguanidine (300mg/kg). At the end of R. 1.5-2mm coronary segments (average diameter 250µm) were removed distal to the site of occlusion and mounted in wire myographs. Endothelium – dependent relaxations to acetylcholine were determined in arteries precontracted by serotonin. I/R induced a marked decrease in the coronary responses to acetylcholine (maximal relaxations: sham 70

± 4%, n=8; I/R: 34± 7%, n=8; p