Sylvie Delaflotte

Endothelin and Ca++ agonist Bay K 8644: different vasoconstrictive properties

The mechanism of vasoconstriction induced by endothelin was investigated in rat isolated aorta in comparison with the Ca++ agonist, Bay K 8644. Endothelin (EC50 = 4 nM) induced a slow and sustained contraction in control medium whereas the one elicited by Bay K 8644 (EC50 = 14 nM) necessitating a partly K+ depolarized medium was fast with superimposed rhythmic contraction. By opposition with Bay K 8644, endothelin contraction was not inhibited by the calcium antagonists (1 microM), nifedipine, diltiazem and D 600, and substantially persisted in Ca++ free medium or after depletion of intracellular Ca++ by phenylephrine (1 microM). These data show that endothelin does not act as an activator of potential dependent Ca++ channels but probably through specific receptor(s) as suggested by its mode of vasoconstriction.

Induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in vascular smooth muscle cells

Inducible vascular nitric oxide synthase accounts for the contractile impairment observed in ondotoxemia. We provide evidence that lipoteichoic acid (LTA) from Staphylococcus aureus, a micro‐organism without endotoxin, also induces nitric oxide synthase. Our study demonstrates that on endothelium‐free rings of rat aorta. LTA‐like lipopolysaccharide induces a loss of contractility restored by Methylene blue and N G‐nitro‐l‐arginine‐methyl ester (LNAME). Moreover in cultured vascular smooth muscle cells, LTA produces a dose‐dependent increase in intracellular cyclic GMP which is antagonized by LNAME and prevented by dexamethasone.

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 α1-adrenoceptor subtypes mediate contraction in rabbit aorta and urethra

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.

Comparative effects of endothelin and phorbol 12-13 dibutyrate in rat aorta

Summary: Inducible nitric oxide (NO) synthase in vascular smooth muscle cells (SMCs) appears to play a major role for the diminished responsiveness to vasoconstrictors observed in endotoxemia. However, cardiovascular dysfunctions associated with septic shock are also observed in the absence of endotoxin (LPS). Similar hemodynamic changes are produced either by a gram‐negative bacteria (Escherichia coli) or by a gram‐positive bacteria (Staphylococcus aureus), a microorganism without LPS, suggesting a common pathway leading to cardiovascular abnormalities. In the present study, we describe the induction of NO synthase in vascular SMCs by lipoteichoic acid (LTA), a component of the membrane of gram‐positive bacteria. In cultured vascular SMCs, a 24‐h incubation with LTA produced an increase in intracellular cyclic GMP. This effect was inhibited by methylene blue (MB), an inhibitor of guanylate cyclase. Incubation with a specific inhibitor of L‐arginine, i.e., NG‐nitro‐L‐arginine methyl ester (l‐NAME), or depletion of L‐arginine attenuated the LTA‐induced cGMP production. A 5‐h incubation of endothelium‐free rings of rat aorta in the presence of LTA induced a loss of tonicity to the contractile response of phenylephrine. The contractions were restored by MB and by l‐NAME. The effect of l‐NAME was reversed by L‐arginine. These results show that LTA, like LPS, expresses NO synthase in vascular SMCs.

Endothelium independent protective effect of NG-monomethyl-L-arginine on endotoxin-induced alterations of vascular reactivity

The alpha1-adrenoceptor subtypes mediating contraction of rabbit aorta and urethra were pharmacologically characterized using an isolated organ bath technique. Although aorta was as sensitive as urethra to the contractile action of methoxamine, phenylephrine was about 10 times more potent as a contractile agonist on aorta than on urethra. In aorta, the rank order of agonist sensitivity was norepinephrine > phenylephrine > clonidine > methoxamine whereas the rank order in urethra was clonidine > methoxamine > or = phenylephrine > norepinephrine. A lack of significant correlation between the potency of different alpha1-adrenoceptor antagonists tested against the phenylephrine-induced contraction in aorta and in urethra indicated that different alpha1-adrenoceptor subtypes mediated the contractile response in the two preparations. The potency of different alpha1-adrenoceptor antagonists tested in rabbit urethra was significantly correlated with their affinity for the cloned human alpha1c-, but not alpha1a- or alpha1b-, adrenoceptor subtype. Such a clear correlation with the potency of different alpha1-adrenoceptor antagonists tested in rabbit aorta and their affinity for one subtype of cloned human alpha1-adrenoceptor was not found. Chlorethylclonidine, which produced a 10 000-fold rightward shift in the phenylephrine concentration-response curve for rat aorta, had a weak inhibitory effect in rabbit aorta and urethra as well as in other rabbit tissues (spleen, fundus, renal artery, saphenous artery). The results indicate that significant heterogeneity exists among alpha1-adrenoceptor in rabbit aorta and urethra (alpha1c-adrenoceptor). However, chlorethylclonidine does not seem to be a suitable tool for the differentiation of alpha1-adrenoceptor subtypes in the rabbit.

Verapamil as an apparent competitive antagonist of the serotonin receptor of rabbit isolated aorta.

The vasoconstrictive properties of endothelin (ET-1) and the protein kinase C activator, phorbol 12-13 dibutyrate (PDB) were comparatively investigated in isolated rat aorta. ET-1 (0.3-100 nM) and PDB (10 nM-3 microM) induced a slowly developing sustained contraction in endothelium denuded aorta. Maximal contractions induced by ET-1 and PDB were unaffected by diltiazem (10 microM). Substantial contraction to ET-1 (30 nM) and PDB (0.1 microM) remained in calcium-free medium. Contractions of ET-1 and PDB in calcium-free medium were unaffected by intracellular calcium depletion induced by phenylephrine. Following the response to ET-1 and PDB in a calcium-free medium, an additional sustained contraction was observed after calcium (2.5 mM) was added to the bath. The protein kinase C inhibitor, H7 (100 microM) was more potent in inhibiting contractions induced by phenylephrine and KCl than the ones elicited by ET-1 and PDB. The other protein kinase C inhibitors i.e. staurosporine (50 nM) and phloretin (100 microM) inhibited to a similar extent all the agonists tested. These results suggest that protein kinase C may play an important role in mediating the contraction to ET-1 in rat aorta.

The vasoconstrictor action of big endothelin-1 is phosphoramidon-sensitive in rabbit saphenous artery, but not in saphenous vein

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.

Antihypertensive profile of cicletanine, a furopyridine derivative: comparison with captopril, indapamide and prazosin.

The actions of four Ca2+-antagonists (verapamil, diltiazem, nifedipine and flunarizine) were tested on serotonin- (5-HT-) induced contraction of rabbit isolated aorta. Verapamil produced a dose-dependent, parallel shift to the right of the concentration-response curve for 5-HT (pA2 approximately equal to 7.13; Schild slope approximately equal to 1.01), indicative of competitive antagonism. The effects of diltiazem, nifedipine and flunarizine were much less pronounced and best characterized as non-competitive interactions. The effect of verapamil, which likely involves its antagonism of smooth muscle 5-HT2-receptors, might be useful in explaining its therapeutic actions and/or its side-effects.