Jean-Michel Guillon

Angiotensin II and phorbol-esters potently down-regulate endothelin (ET-1) binding sites in vascular smooth muscle cells.

[125I]ET-1 binding to vascular smooth muscle cells showed an apparent single class of high affinity recognition sites with a Kd of 2.12 +/- 0.46 nM and a Bmax of 81.2 +/- 5.2 fmol/10(6) cells. The specific binding was equally and totally displaced by ET-1 and ET-2 whereas ET-3 presented a different pattern. We investigated heterologous regulation of ET-1 binding sites by preincubating the cells with angiotensin II (AII), Arg-vasopressin, bradykinin, enkephalins, serotonin, norepinephrine and carbachol, for 18 h at 37 degrees C. Only AII pretreatment resulted in an important and dose-dependent decrease of ET-1 binding capacity. Sar1-Ile8-AII inhibited the regulatory effect of AII. Furthermore, preexposure of the cells with phorbol-12,13 dibutyrate but not with phorbol-12,13 didecanoate also resulted in a concentration-dependent diminution of ET-1 binding sites. These findings suggest that AII may selectively down-regulate ET-1 binding sites in vascular smooth muscle cells by a mechanism involving protein kinase C.

Vascular mechanism of action of endothelin-1: Effect of Ca2+ antagonists

Summary The vasoconstrictive properties of the endothelium-derived peptide, endothelin-1 (ET-1), were investigated on rat isolated aorta and on cultured rat aortic smooth muscle cells. In rat isolated aorta, endothelin-1 induced a slow and sustained contraction in a Ca2+-free medium; after calcium readmission, an additional sustained contraction was elicited. In vascular smooth muscle cells, endothelin-1 provoked a dose-dependent Ca2+ influx that was not inhibited by calcium entry blockers (nifedipine, D 600, or diltiazem). In these cells, [125I]-endothelin-l bound to a specific, saturable, and high affinity recognition site (Kd about 10–9 M and Bmax = 52

Different regulation of vascular tone by angiotensin II and endothelin-1 in rat aorta

2 fmol/106 cells). The binding was not reversible and not affected by calcium antagonists. These data do not support the hypothesis that endothelin-1 acts as an endogenous agonist of the voltage-dependent Ca2+ channels. The action of endothelin-1 can be separated into two components: one dependent on Ca2+ influx but insensitive to calcium antagonists and another independent of extracellular Ca2+. The irreversible binding of endothelin-1 may reflect an internalization of the ligand inside the cell membrane, leading to multiple contractile events.

Lack of effect of atrial natriuretic factor on the tone induced in rat portal vein by platelet-activating factor

The effects of NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide (NO) synthesis was tested on the endotoxin-induced alterations of alpha-adrenoceptor function. In isolated aorta, there was no significant difference in the tension induced by phenylephrine (PE, 10 microM) on rings removed from control and endotoxin injected rats (10 mg/kg, ip). However, a lack of tonicity of the contraction was observed in rings of shocked rats (8 +/- 2.9 and 86 +/- 4.6% relaxation at 105 min for sham and shocked rings respectively). The gradual tension decrease to PE was more potent in rings possessing endothelial cells. However, in both preparations, the loss of tonicity was significantly inhibited by NMMA (30 microM). In endothelium-free rings, L-arginine (100 microM) potentiated the loss of tonicity to PE and reversed the inhibitory effect of NMMA. NMMA, like methylene blue, was also able to restore the PE-contraction. The results indicate that the endotoxin-induced alterations of vascular reactivity may be due, in part, to NO formation from L-arginine independent of the endothelium.

Cross-desensitization between angiotensin II and endothelin-1 in the pithed rat

The effects of moderate cooling and of phenylarsine oxide on the contraction induced by two vasoactive peptides, angiotensin II (AII) and endothelin (ET-1), were investigated on endothelium-free rings of rat aortas. At 37 degrees C, the contraction induced by AII (0.1 microM) was transient. This decline in tension is unlikely to be due to rapid degradation of AII. In contrast, ET-1 (10 nM) induced a slowly developing and sustained contraction similar to the one observed with phorbol 12-13 dibutyrate (PDB, 22 nM). Moderate cooling (25 degrees C) significantly potentiated and prolonged the effect of AII but reduced the velocity of the ET-1 and PDB contraction, although the rate of the phenylephrine (1 microM) response remained unchanged. Phenylarsine oxide (100 microM) reduced the decline in tension in response to AII but inhibited the contraction elicited by ET-1 and PDB. In rings incubated in calcium-free medium (37 degrees C), AII induced a phasic contraction. This was followed by a second phasic contraction after calcium (2.5 mM) had been restored to the bath. The intensity of this second contraction decreased as the time between AII and calcium injection increased. This method, using regression analysis, permitted us to determine the time taken to reduce the contraction by half (4.8 min; r: 0.96), which may reflect the half-time of receptor sequestration. In calcium-free medium, the contractions induced by ET-1 and PDB were slow and sustained. Thus, rapid AII-receptor internalization leads to a short-term regulation of vascular tone whereas activation of protein kinase C by ET-1 may induce a long-term regulation.

Blocking agents of edrf effect or formation for the treatment of shock

The spontaneous myogenic activity of the isolated rat portal vein was inhibited by atrial natriuretic factor or by sodium nitroprusside. These compounds were not effective on the tone induced by PAF-acether or carbachol. 8-Bromo cyclic GMP and dibutyryl cyclic AMP inhibited myogenic activity and reduced the agonist-induced contractions. Only dibutyryl-cyclic AMP significantly inhibited the PAF-acether-induced contractile responses. These results indicate that the tone induced by PAF-acether can be used to discriminate between drugs which selectively increase cyclic nucleotide levels.

PAF Antagonists as Potential Therapeutic Agents in Cardiac Anaphylaxis and Myocardial Ischemia

It has been shown that in vitro angiotensin II (Ang II) potently downregulates endothelin-1 (ET-1) binding sites. In this study, we investigated in vivo the interactions between the renin-angiotensin system and ET-1. Sprague-Dawley rats were pithed, ventilated, and diastolic blood pressure (DBP) was recorded. ET-1 (1 nmol/kg) induced a biphasic response: a transient hypotension followed by a sustained increase of DBP. Captopril (5 mg/kg, i.v.) or Sar1-Ile8-Ang II (10 ng/kg/min) did not affect ET-1 responses. In other experiments, DBP was increased by infusion of methoxamine (MTX: 10, 20, 25, 32.5 micrograms/kg/min) or Ang II (50, 100, 150, 200 ng/kg/min). ET-1-induced hypotension correlated with the level of DBP (r = 0.94) for both agonists. The elevation of DBP in response to ET-1 was also related to the vascular tone but was dose-dependently attenuated by Ang II infusion when compared with MTX. Conversely, infusion of ET-1 (0.1 nmol/kg/min) blunted the pressor response to Ang II (0.1 micrograms/kg) but not to MTX (50 micrograms/kg). These doses induced the same increase of DBP in pithed control rats. Similarly, increased plasma renin activity induced by chronic salt depletion (0% NaCl) in pithed rats provoked a shift to the right of the dose-response curves to Ang II and ET-1 but not to MTX. These results suggest an in vivo cross-desensitization between ET-1 and Ang II.