Macrophages are novel sites of expression and regulation of retinol binding protein-4 (RBP4).

Obesity is linked to a low-level chronic inflammatory state that may contribute to the development of associated metabolic complications. Retinol-binding protein 4 (RBP4) is an adipokine associated with parameters of obesity including insulin resistance indices, body mass index, waist circumference, lipid profile, and recently, with circulating inflammatory factors. Due to the infiltration of adipose tissue in obesity by macrophages derived from circulating monocytes and, on the other hand, the existence of a close genetic relationship between adipocytes and macrophages, we decided to examine if RBP4 is expressed in monocytes and/or primary human macrophages. While we did not detect expression of RBP4 in undifferentiated monocytes, RBP4 expression became evident during the differentiation of monocytes into macrophages and was highest in differentiated macrophages. Once we demonstrated the expression of RBP4 in macrophages, we checked if RBP4 expression could be regulated by inflammatory stimuli such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), or the endotoxin lipopolysaccharide (LPS). We observed that while RBP4 expression was strongly inhibited by TNF-alpha and LPS, it was not affected by IL-6. Our results highlight the complexity behind the regulation of this adipokine and demonstrate that RBP4 expression in macrophages could be modulated by inflammatory stimuli.

Synergistic protective effects of antioxidant and nitric oxide synthase inhibitor in transient focal ischemia.

Both nitric oxide synthase (NOS) inhibitors and free radical scavengers have been shown to protect brain tissue in ischemia-reperfusion injury. Nitric oxide and superoxide anion act via distinct mechanisms and react together to form the highly deleterious peroxynitrite. Therefore the authors examined the effects and the interaction between the NOS inhibitor, NG nitro-L-arginine (LNA) and the antioxidant/superoxide scavenger, di-tert-butyl-hydroxybenzoic acid (DtBHB) in the rat submitted to 2 hours of middle cerebral artery occlusion. Posttreatment was initiated 4 hours after the onset of ischemia and infarct volume was measured at 48 hours. The dose-related effect of LNA resulted in a bell-shaped curve: 15, 56, 65, and 33% reduction of total infarct for 0.03, 0.1, 0.3, and 1 mg/kg (intravenously [IV]) respectively and 11% increase in infarct volume for 3 mg/kg (IV). Whereas DtBHB (20 mg/kg; intraperitoneally [IP]) was ineffective, the dose of 60 mg/kg produced 65% protection in infarct volume. The combination of a subthreshold dose of LNA (0.03 mg/kg; IV) and DtBHB (20 mg/kg; IP) resulted in significant reduction (49%) in infarct volume. These results show that LNA and DtBHB act synergistically to provide a consistent neuroprotection against ischemic injury when administered 4 hours after ischemia. This suggests that nitric oxide and free radicals are involved and interact in synergy in ischemia-reperfusion injury.

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 endotoxemia. 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 NG-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.

Competitive inhibition of 5-HT receptors in rabbit isolated aorta by the Ca(2+)-antagonist methoxyverapamil (D 600).

Experiments conducted with rabbit isolated aorta have revealed that the Ca(2+)-antagonist 3-methoxy-verapamil (D 600) is a potent competitive antagonist of the contractile action of 5-HT. Thus, the anti-hypertensive action of D 600, as well as some of its side effects, could be related to its antagonism of 5-HT receptors. D 600 might be useful in neurochemical and neurophysiological experiments that are aimed at examining 5-HT receptors, as well as in treating certain cerebrovascular disorders of man (e.g., migraine).

BN 80933 inhibits F2-isoprostane elevation in focal cerebral ischaemia and hypoxic neuronal cultures.

Formation of the lipid peroxidation product 8-epi-prostaglandin2alpha (8-epi-PGF2alpha) a bioactive marker of oxidative stress, was quantified in in vitro and in vivo models of neuronal death. In culture media of primary rat cortical neurones exposed to hypoxia followed by reoxygenation, a 3.7-fold increase of 8-epi-PGF2alpha concentration was observed in comparison to control cells. In rats submitted to 2h middle cerebral artery occlusion followed by a 22h reperfusion period, a 27-fold increase of 8-epi-PGF2alpha was observed in the ischaemic hemisphere compared with the corresponding hemisphere of sham-operated rats. Treatment with the neuroprotective agent BN 80933 significantly reduced both 8-epi-PGF2alpha elevations in vitro and in vivo. These data suggest that 8-epi-PGF2alpha elevations might reflect the damaging free radical overproduction and subsequent lipid peroxidation during neuronal injury induced by hypoxia and ischaemia. Inhibition of 8-epi-PGF2alpha elevations participates to the neuroprotective effects of BN 80933.

Different alpha1-adrenoceptor subtypes mediate contraction in rabbit aorta and urethra.

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.

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

The effects of big endothelin-1 and endothelin-1 on vascular reactivity were compared in isolated rabbit saphenous artery and vein. The contractile potency of endothelin-1 was three times higher in the vein than in the artery. In contrast, big endothelin-1 was two times more effective in the artery. Phosphoramidon (100 microM), a metalloproteinase inhibitor, antagonised the contractile action of big endothelin-1 in the artery but not in the vein. These data suggest that the biosynthetic pathway leading to the formation of endothelin differs in arterial and venous vessels.

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

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.

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

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.

Differential effects of tyrosine kinase inhibitors on contraction and relaxation of the aortas of normotensive and hypertensive rats.

The contribution of tyrosine kinase activity to vasoreactivity in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats was investigated on isolated aortic preparations by the use of two tyrosine kinase inhibitors: methyl-2,5-dihydroxycinnamate (30 microM) and genistein (30 microM). The pretreatment of endothelium denuded aorta with methyl-2,5-dihydroxycinnamate reduced the sensitivity of the rings to noradrenaline to a larger extent in SHR than in WKY. The relaxing effects evoked by methyl-2,5-dihydroxycinnamate and genistein on the sustained contraction induced by endothelin-1 were also more pronounced in SHR denuded rings. Furthermore, in presence of methyl-2,5-dihydroxycinnamate, the endothelium-independent contractile responses to equipotent doses of cyclopiazonic acid were more depressed in SHR than in WKY. In WKY and SHR endothelium-intact aortas contracted with either phenylephrine or endothelin-1, carbachol and cyclopiazonic acid evoked endothelium derived relaxing factor (EDRF)/nitric oxide (NO)-dependent relaxations which were reduced by pretreatment of the rings with methyl-2,5-dihydroxycinnamate or genistein. These inhibitory effects were larger in WKY rings and more important on the cyclopiazonic acid response. In addition, sodium orthovanadate (30 microM) potentiated the noradrenaline-mediated contractions of endothelium-denuded SHR rings and reduced the cyclopiazonic acid-induced relaxation of endothelium-intact WKY rings. The present study suggests a regulatory role for tyrosine kinase in the smooth muscle contraction and the endothelium-dependent relaxation in WKY and SHR aortas and demonstrates the existence of a different relationship in the effect of tyrosine kinase inhibitors on vasoreactivity between SHR and WKY. We propose that an increase in the tyrosine kinase activity in SHR could lead to an enhanced reactivity of Ca2+-linked contractile mechanisms. In addition, our results suggest a link between the loss of tyrosine kinase activity and the altered endothelium-dependent relaxation associated with hypertension.

Effects of angiotensin I and angiotensin II in blood vessels: greater influence of converting enzyme activity in the rabbit basilar artery.

We investigated the constrictor effects of Angiotensin I (Ang I) and Angiotensin II (Ang II) on rabbit peripheral (aorta, carotid artery, mesenteric artery, saphenous artery) and cerebral (basilar artery) vessels and in rat aorta in functional organ bath studies. The effect of angiotensin converting enzyme (ACE) inhibition by captopril was also assessed in these preparations. Ang II elicited concentration-dependent contractions with comparable potency in rabbit and rat endothelium-free vascular rings (pD2 about 8.5) which indicates a lack of species and regional variation in the contractile responses to Ang II. The responses to Ang II were reduced by the presence of a functional endothelium in rabbit mesenteric artery and in rat aorta. Since ACE determines the plasma and tissue conversion of Ang I to active Ang II, we calculated the ratio R (EC50 Ang I-induced contraction: EC50 Ang II-induced contraction) as an indicator of the tissue ACE effectiveness. In the aorta without endothelium, Ang I was found to be much less potent than Ang II in the rabbit (R = 44) compared with the rat (R = 3.5). This species difference in the aortic conversion of Ang I to Ang II was confirmed by the use of captopril. Captopril (10(-6) M) shifted the Ang I concentration/ response curve by 2- and 14-fold to the right in rabbit and rat respectively. In other rabbit blood vessels, the rank order of potency to Ang I in endothelium denuded rings was basilar artery > > carotid artery > or = aorta > or = saphenous artery. In addition, the R value was the lowest for the basilar artery (R = 2.5). This is in agreement with the highest rightward shift (78-fold) of the Ang I concentration/response curve by captopril for basilar artery in comparison with only 3-, 8- and 3-fold shifts observed in carotid artery, saphenous artery and aorta respectively. In conclusion, our data provide evidence for a greater influence of ACE in rabbit basilar artery than in peripheral vessels.

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

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

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.

Role of endothelium on phenylephrine-triggered contractile events in aorta of spontaneously hypertensive rats.

The ability of basal release of endothelium derived relaxing factor (EDRF) to alter contractile events in phenylephrine (PE)-triggered contraction was tested on ring segments of the thoracic aorta removed from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). In normal medium, PE (1 microM) elicited similar whole contractions in endothelium denuded arteries of SHR and WKY. The presence of endothelium only reduced the WKY response. On aorta incubated in a Ca2+ free-medium, PE (1 microM) induced an initial phasic contraction due to intracellular Ca2+ release. This was followed by a tonic contraction after Ca2+ (2.5 mM) was restored to the bath. This sustained contraction was dependent on extracellular calcium influx. Identical phasic and tonic contractions were observed in endothelium denuded rings of SHR and WKY. However, the presence of endothelium only reduced the sustained contraction of WKY arteries. When experiments were carried out in medium containing D600 (1 microM), the presence of endothelium diminished the whole contraction of both SHR and WKY rings whereas the sustained but not the phasic contractions of WKY was also inhibited. This inhibitory effect of endothelium on WKY sustained contraction was significantly higher in the presence of D600. The calcium antagonist reduced both the whole and the tonic contractions of all preparations but was ineffective on the phasic one. The D600 inhibitory action on the sustained contraction was more pronounced in denuded SHR rings than in the corresponding WKY arteries. Thus it is concluded that there is a basal influence of endothelium in both SHR and WKY. Under our conditions, the endothelial function inhibited the extracellular Ca2+ influx and especially the part of Ca2+ influx insensitive to D600. This part of Ca2+ influx is diminished in SHR and thus the efficacy of endothelium products (e.g. EDRF) is reduced in this strain.

Contractions induced by phenylephrine and noradrenaline are differently affected by endothelium-dependent relaxation in rat aorta.

In rings of rat aorta precontracted with phenylephrine (10 microM) or noradrenaline (10 microM), addition of carbachol (10 microM) produced an endothelium-dependent relaxation. However, regardless of the concentration of agonist tested, both the intensity and duration of the relaxation were significantly less when noradrenaline, rather than phenylephrine, was used as the precontracting agent. The different responses observed do not appear to be related to destruction of endothelium-derived relaxing factor by autoxidation of noradrenaline since neither EDTA (30 microM) nor superoxide dismutase (30 units mL-1) improved the relaxation to carbachol. In addition, in endothelium-free rings, the noradrenaline (1 microM)-induced contraction was less sensitive than the phenylephrine (1 microM)-induced contraction to sodium nitroprusside (0.1 microM) or to 8-Br-cGMP (300 microM). With phenylephrine-, but not noradrenaline-, induced contraction, the relaxation triggered by carbachol was significantly reduced by pretreatment of the aortic rings with chloroethylclonidine (50 microM), which inactivates a subpopulation of alpha 1-adrenoceptors. Thus, the results confirm that both alkylation sensitive and resistant alpha 1-adrenoceptors exist in rat aorta and indicate that EDRF may discriminate between these two alpha 1-adrenoceptor subtypes which are differently affected by phenylephrine and noradrenaline.

Increased influence of endothelium in obese Zucker rat aorta.

The ability of endothelium to alter contractile events in phenylephrine (PE)-triggered contraction has been tested on ring segments of the thoracic aorta removed from obese Zucker rats (plasma cholesterol 3.63 mM; n = 8) and from age matched lean rats (plasma cholesterol 2.38 mM; n = 8). In normal medium, PE (1 microM) elicited similar contractions in endothelium-denuded arteries of both strains. However, the presence of endothelium reduced these contractile events and the endothelium-dependent relaxation induced by carbachol (10 microM) was higher in obese rats. In rings incubated in Ca2+ free medium containing EGTA (1 mM), PE (1 microM) induced a phasic contraction and a sustained contraction following addition of Ca2+ (2.5 mM) to the medium. The phasic contraction was due to intracellular Ca2+ release, whereas the sustained response was dependent on extracellular Ca2+ influx. In endothelium-free preparations, the size of both the phasic and sustained contraction was similar for the two strains. The Ca2+ antagonist gallopamil (1 microM) reduced the sustained contraction of lean (24%) and obese (34%) rats without affecting the phasic contraction. In preparations possessing endothelium, the sustained, but not the phasic contraction, of both strains was inhibited. This inhibitory effect of endothelium on the sustained contraction was significantly higher in obese than in lean rats. Thus, it can be concluded that phenylephrine elicited quantitatively and qualitatively similar contractions in obese and lean rats. In both strains, the endothelium diminished the contraction induced by PE, however, this effect was more pronounced in obese rats than in lean ones. These results may explain, in part, the described absence of atherosclerotic lesions in the obese strain.

Effects of BN 52063 and other agents inhibiting platelet-activating factor-induced contractile responses in rat portal vein.

Platelet-activating factor (PAF-acether) is a potent agonist (EC50: 3.2 x 10(-8) M) of isolated rat portal vein. BN 52063 (composed of BN 52020, BN 52021 and BN 52022; molar ratio 2:2:1) specifically inhibits PAF-acether (10(-7) M) induced tone (IC50: 3.9 x 10(-5) M). Salbutamol (IC50: 3.1 x 10(-7) M), forskolin (IC50: 3.1 x 10(-6) M) and theophylline (IC50: 2.25 x 10(-4) M) are also effective in inhibiting PAF-acether-induced contractile responses and all excepting forskolin, show a certain specificity in this action. The basal myogenic activity of the rat portal vein is dose-dependently decreased by salbutamol (IC50: 1.2 x 10(-7) M), forskolin (IC50: 2.6 x 10(-6) M) and theophylline (IC50: 2.3 x 10(-4) M) whereas BN 52063 has no effect. The data suggest that rat portal veins possess specific PAF-acether receptors sensitive to BN 52063 and that PAF-acether effects could be inhibited by compounds which can bypass these putative receptors and modulate cAMP levels.

In vivo and in vitro effects of cicletanine in spontaneously hypertensive rats.

Oral treatment for 2 weeks with cicletanine [1,3-dihydro-6-methyl-7-hydroxy-3-(4-chloro-phenyl)furo(3,4-c)pyridine] at 30 mg/kg/day delayed the onset of hypertension in spontaneously hypertensive rats (SHR) (15.7 mmHg, p less than 0.001). This antihypertensive effect was increased when the animals were maintained on a high-salt diet (40.8 mmHg, p less than 0.001). The ability of cicletanine to alter calcium movements in phenylephrine (PE)- and angiotensin II (ANGIO)-triggered contraction was tested on isolated SHR aorta. PE (1 microM) and ANGIO (0.1 microM) induced a phasic contraction in calcium-free medium due to intracellular calcium release. Upon addition of calcium (2.5 mM) a second sustained (PE) or biphasis (ANGIO) contraction was observed. This second contraction was dependent on extracellular calcium influx. Cicletanine (0.1-0.3 mM) both reduced the phasic (77%, p less than 0.001 for PE; 68%, p less than 0.05 for ANGIO with cicletanine 0.3 mM) and the second contraction elicited by the two agonists (27%, p less than 0.001 for PE; 84%, p less than 0.001 for ANGIO with cicletanine 0.3 mM). These results suggest that in addition to its stimulatory effect on prostaglandin, a direct vascular action of cicletanine could also be involved in the antihypertensive properties of the drug.

In vitro cardiovascular antihistamine properties of cicletanine in comparison with diphenhydramine.

Cicletanine (CIC) (pA2: 7.0) was found to be as potent as diphenhydramine (DIPH) (pA2: 7.2) in competitively inhibiting histamine-induced contraction of isolated rabbit aorta. Both CIC (pA2: 7.3) and DIPH (pA2: 7.5) similarly shifted histamine-induced endothelium-dependent relaxation of isolated rat aorta to the right. The similarity of results (in terms of pA2 values) indicates that in spite of their opposite responses on vascular tone, the H1 receptors on rabbit and rat aortae may be similar. DIPH was found to be as potent as cocaine in potentiating the chronotropic action of noradrenaline on isolated right atria, whereas CIC was without effect. The ability of DIPH to inhibit the noradrenaline uptake at the neuronal membrane seems to be independent of its H1-receptor antagonism potency. Finally, the furopyridine framework of CIC does not elicit the cocaine-like activity of the oxyethylamine residue of DIPH.

[Interaction of atrial natriuretic factor and cicletanine in relation to contractions induced by endothelin and phenylephrine on the isolated rat aorta]. / Interaction entre le facteur natriurétique atrial et le ciclétanine vis-à-vis de la contraction induite par l'endothéline et la phényléphrine sur l'aorte isolée de rat.

The interaction between cicletanine (CIC) and atrial natriuretic factor (ANF) on vessels was studied using the isolated rat aorta as model. Contractions induced by endothelin (ET1), a vasoconstrictor peptide from vascular endothelial cells, and by phenylephrine (PE), an alpha 1-adrenoceptor agonist, were recorded isometrically on aortic rings the endothelium of which had deliberately been damaged. Both agonists produced a concentration-dependent contraction (ET1:EC50 1.8 nM, Emax 2.2 g; PE:EC50 30 nM, Emax 2.4 g) which was antagonized by a 30 min pretreatment with either CIC (100 microM) or ANF (1 nM). However, PE was more sensitive than ET1 to the inhibitory action of CIC or ANF. Moreover, during inhibition by ANF spasmodic contractions were observed with PE but not with ET1; this was not observed when the antagonist was CIC. The inhibitory effect produced by the combination of CIC and ANF was additive with ET1 whereas a potentiation was observed with PE. Thus, on the isolated rat aorta model the contraction induced by PE was more sensitive to the action of ANF than that induced by ET1, which indicates that the two antagonists have a different mechanism of action. A direct action of CIC on the alpha-adrenoceptor might account for the potentiation observed between ANF and CIC against PE.