Inhibition of Na(+)/K(+)-atpase by endothelin-1 in human nonpigmented ciliary epithelial cells

Endothelin-1 (ET-1), a potent vasoconstrictor, lowers intraocular pressure in mammals, either by enhancing the outflow of aqueous humor (AH) via the trabecular meshwork and Schlemm's canal or by reducing AH formation at the ciliary epithelium. Aqueous humor production occurs by passive diffusion of water coupled with active transport of ions, mainly involving Na(+):K(+):2Cl(-) cotransporter and Na(+)/K(+)-ATPase pump from serosal to aqueous side. Presently, we have evaluated the effects of ET-1 on Na(+):K(+):2Cl(-) cotransport and Na(+)/K(+)-ATPase activity in HNPE cells using (86)Rb(+) uptake. ET-1 (100 pM-100 nM) decreased mean (86)Rb(+) uptake by 15% during a 15-min uptake period. ET-1's effect was not prevented by BQ610, an ET(A) receptor antagonist, but was blocked by BQ788, an ET(B) receptor antagonist. ET-1's effect was mimicked by sarafotoxin, an ET(B) agonist. ET-1-induced reduction in (86)Rb(+) uptake was additive with bumetanide, a selective inhibitor of Na(+):K(+):2Cl(-) cotransporter but not with ouabain, a selective inhibitor of the Na(+)/K(+)-ATPase. ET-1 did not affect iberiotoxin-sensitive maxi K(+) channels. This suggests that ET-1-induced reduction in (86)Rb(+) uptake is mediated through the inhibition of the Na(+)/K(+)-ATPase via an ET(B)-like receptor. These findings are consistent with an ET-1 effect on active ion transport activity in HNPE cells that could explain the reduction in aqueous humor production and the lowering of intraocular pressure.     

ETA receptors mediate vasoconstriction,whereas ETB receptors clear endothelin-1 in the splanchnic and renal circulation of healthy men

The contribution of the endothelin (ET) receptors ET(A) and ET(B) to basal vascular tone and ET-1-induced vasoconstriction in the renal and splanchnic vasculature was investigated in six healthy humans. ET-1 was infused alone and in combination with the selective ET(A) receptor antagonist BQ123 or the selective ET(B) receptor antagonist BQ788 on three different occasions. BQ123 did not affect basal arterial blood pressure, splanchnic vascular resistance (SplVR) or renal vascular resistance (RVR), but inhibited the increase in vascular resistance induced by ET-1 [64+/-18 versus -1+/-7% in SplVR ( P <0.05); 36+/-6 versus 12+/-3% in RVR ( P <0.0001)]. BQ788 increased basal SplVR and RVR [38+/-16% ( P =0.01) and 21+/-5% ( P <0.0001) respectively], and potentiated the ET-1-induced vasoconstriction. Plasma ET-1 increased more after ET(B) blockade than under control conditions or after ET(A) blockade. These findings suggest that the ET(A) receptor mediates the splanchnic and renal vasoconstriction induced by ET-1 in healthy humans. The ET(B) receptor seems to function as a clearance receptor and may modulate vascular tone by altering the plasma concentration of ET-1.     

Ca2(+)-evoked [3H]dopamine release from synaptosomes is dependent on neuronal type Ca2+ channels and is not mediated by acetylcholine, glutamate or aspartate release

Elevation of potassium concentrations ([K+]) in the presence of Ca2+ is the most common method of evoking neurotransmitter release from synaptosomes. However, we have been investigating a method of releasing dopamine from synaptosomes that does not involve using elevated [K+]. In this paradigm of neurotransmitter release, dopamine is released from synaptosomes, previously exposed to micromolar or lower [Ca2+], by 1.25 mM Ca2+ in the presence of non-depolarizing [K+] (4.5 mM). The present experiments characterize the Ca2+ channel(s) involved in the Ca2(+)-evoked release of dopamine from synaptosomes, and determine whether the release is mediated by acetylcholine, glutamate or aspartate. omega-Conotoxin (10 nM), which blocks N-, L- and possibly T-type voltage-sensitive Ca2+ channels (VSCC), inhibited the Ca2(+)-evoked [3H]dopamine release from either striatal or olfactory tubercle synaptosomes to less than 50% of control. Neither 1 microM nifedipine nor 1 microM verapamil, which block L-type VSCC, affected Ca2(+)-evoked release. The N- and T-type VSCC blocker neomycin and the nonspecific Ca2+ antagonist, cobalt2+, inhibited release to a greater extent than omega-conotoxin. At 1 mM, both compounds inhibited release to approximately 30% of control. Neither the excitatory neurotransmitter glutamate nor aspartate (2mM) affected 1 microM LY-171555 (a dopamine D2 agonist) inhibition of Ca2(+)-evoked [3H]dopamine release. Also, the glutamate antagonist, glutamic acid diethyl ester, did not affect either Ca2(+)-evoked release or 1 microM LY-171555 inhibition thereof. The nicotinic antagonist hexamethonium (10 microM) and the muscarinic antagonist atropine (1 microM) were also ineffective in inhibiting Ca2(+)-evoked release or LY-171555 inhibition of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced endothelin(A) receptor-mediated calcium mobilization and contraction in organ cultured porcine coronary arteries

Arterial injury models for coronary artery disease have demonstrated an enhanced expression and function of either the endothelin(A) or endothelin(B) (ET(A) or ET(B)) receptor subtype. We hypothesized that organ culture would enhance the physiological function of ET receptors in the porcine right coronary artery. Arteries were either cold stored (4 degrees C) or organ cultured (37 degrees C) for 4 days. After 4 days, the artery was either 1) sectioned into rings to measure the ET-1-induced isometric tension response (3 x 10(-10)-3 x 10(-7) M), or 2) enzymatically dispersed and the isolated smooth muscle cells imaged using fura-2 to measure the myoplasmic calcium (Ca(m)) response to 3 x 10(-8) M ET-1 ( approximately EC(50)). Isometric tension and Ca(m) to ET-1 were measured in the absence and presence of bosentan (nonselective ET(A) or ET(B) receptor antagonist), BQ788 (ET(B)-selective antagonist), and BQ123 (ET(A)-selective antagonist). Compared with cold storage, organ culture induced a 2-fold increase in tension development (3 x 10(-7) M ET-1) and Ca(m) (3 x 10(-8) M ET-1), which was inhibited with bosentan, thus confirming the enhanced responses to ET-1 were due to ET receptor activation. BQ123 also inhibited the enhanced contraction and Ca(m) responses to ET-1. In contrast, BQ788 failed to inhibit tension development and Ca(m) responses to ET-1 in organ culture and cold storage. Sarafotoxin 6C (ET(B) agonist) failed to elicit an increased Ca(m) response in organ culture compared with cold storage. Our results indicate the increased tension development and Ca(m) responses to ET-1 in organ culture are attributable to ET(A) receptors, and not ET(B) receptors.     

Reduction in hepatic endothelin-1 clearance in cirrhosis

Circulating endothelin-1 (ET-1) levels are increased in cirrhosis. The liver is an important site for circulating ET-1 clearance through the ET(B) receptor. We evaluated ET-1 kinetics in cirrhosis to determine if a reduced liver clearance contributes to this process. Cirrhosis was induced by carbon tetrachloride in rats. Hepatic ET-1 clearance was measured in isolated perfused livers using the single bolus multiple indicator-dilution technique. Plasma ET-1 levels doubled in cirrhosis from 0.49+/-0.04 fmol/ml (mean+/-S.E.M.) to 1.0+/-0.18 fmol/ml ( P <0.01). Liver ET-1 extraction was reduced from 81+/-1% (mean+/-S.E.M.) in controls to 50+/-6% in cirrhosis ( P <0.01). Kinetic modelling revealed a major irreversible binding site for ET-1 that is blocked by the selective ET(B) receptor antagonist BQ788 and a minor non-specific reversible binding site that cannot be blocked with BQ788 or the selective ET(A) antagonist BQ123. Reduced hepatic clearance correlated with the biochemical markers of cirrhosis, portal vein perfusion pressure ( r =-0.457; P <0.001) and the increase in ET-1 levels ( r =-0.462; P =0.002). Immunohistofluorescence with specific anti-(ET(B) receptor) antibodies revealed a preponderant distribution of ET(B) receptors on hepatic stellate cells, which was increased in cirrhosis. We conclude that cirrhosis reduces ET-1 clearance probably by capillarization of hepatic sinusoids and reduced access to ET(B) receptors. This relates to the severity of cirrhosis and may contribute to the increase in circulating ET-1 levels.     

Cytochrome P450 omega/omega-1 hydroxylase-derived eicosanoids contribute to endothelin(A) and endothelin(B) receptor-mediated vasoconstriction to endothelin-1 in the rat preglomerular arteriole

The preglomerular arteriole of the rat was used to evaluate the contribution of cytochrome P450-derived eicosanoids to the vasoconstrictor effect of endothelin (ET)-1 and to determine the receptors mediating the response. ET-1 (4 x 10(-11) to 2 x 10(-9) M) produced dose-dependent reductions in the intraluminal diameter of the renal arteriole ranging from 25 +/- 8 to 142 +/- 16 micrometer. BMS182874 [(5-dimethylamino)-N-(3, 4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide; 3 microM], an ET(A) receptor antagonist, or BQ788 (N-cis-2, 6-dimethyl-piperidino-carbonyl-L-gamma-methylleucyl-D-1-methoxy carbonyl-tryptophanyl-D-norleucine; 1 microM), an ET(B) receptor antagonist, attenuated ET-1 vasoconstriction by 59 +/- 4 and 50 +/- 10%, respectively. The combined administration of both ET receptor antagonists increased inhibition of ET-1 vasoconstriction to 75 +/- 4%. 17-Octadecynoic acid (17-ODYA, 2 microM) or 12, 12-dibromododec-enoic acid (2 microM), inhibitors of 20-hydroxyeicosatetraenoic acid (20-HETE) production, attenuated ET-1-induced vasoconstriction by 50 +/- 6 and 40 +/- 3%, respectively, as did indomethacin (10 microM), an inhibitor of cyclooxygenase. Miconazole (2 microM), the epoxygenase inhibitor, was without effect. 20-HETE (10(-8) and 2 x 10(-8) M) elicited a dose-related vasoconstriction that was inhibited by 10 microM, but not 5 microM, indomethacin. The inhibition by 17-ODYA of ET-1 vasoconstriction was not greater when combined with BMS182874 or BQ788. Moreover, vasoconstriction induced by ET-3, an ET(B)-selective agonist, was inhibited by 17-ODYA. These data indicate that both ET(A) and ET(B) receptors mediate ET-1 vasoconstriction and that 20-HETE production linked to both receptors makes a major contribution to ET-1-induced renal arteriolar vasoconstriction in the rat.     

内皮素1增高对大鼠大脑皮质神经元凋亡的作用

背景作为一种强效血管和神经活性肽,内皮素1在各种中枢神经系统病理生理情况下表达增加,可能对神经组织产生有害作用.然而,增高的内皮素1有无直接诱导神经元凋亡的作用尚不清楚.目的观察内皮素1有无直接诱导原代培养神经元凋亡的作用.设计以细胞为研究对象,完全随机对照实验研究.单位一所大学医院的神经内科和一所大学的病理学教研室、生命科学技术学院组织与移植免疫实验中心.材料实验于2001-03/2002-02在暨南大学医学院病理教研室和生命科学和技术学院组织移植与免疫研究所实验室进行.来自于新生大鼠大脑皮质的原代神经元培养,新生大鼠由中山大学医学中心动物研究所提供.干预原代大脑皮质神经元培养5 d后分别加入0.2,20 nmol/L内皮素1处理24 h,用Annexin V,Hoechst 33258染色半定量测定细胞凋亡.再用流式细胞仪分别定量检测内皮素受体A拮抗剂或内皮素受体B拮抗剂对20 nmol/L内皮素1诱导神经元凋亡的效果.对照组加入等量磷酸盐缓冲液.主要观察指标观察内皮素1直接诱导培养神经元凋亡的作用,以及其通过的ET受体亚型.结果加入0.2 nmol/L内皮素1 24 h后,Annexin-V和Hoechst 33258阳性染色细胞率分别为(23.00±9.96)%,(9.82±0.95)%,与对照组相比差异无显著性意义[(Annexin-V(13.50±3.35)%;Hoechst 33258(8.21±2.17)%].加入20 nmol/L后24 h,Annexin-V以及Hoechst33258染色阳性细胞率(50.50±10.78)%,(13.78±1.52)%显著增高,与对照组比较,差异有显著性意义.流式细胞仪检测对照组和20 nmol/L内皮素1组凋亡率分别为(0.20±0.15)%和(26.11±3.28)%;内皮素受体A拮抗剂,内皮素受体B拮抗剂分别部分阻断了内皮素1诱导培养神经元凋亡的作用,内皮素受体A拮抗剂+内皮素1组凋亡率为(13.58±4.92)%,内皮素受体B拮抗剂+内皮素1组为(9.99±3.30)%,与内皮素1组比较,差异有显著性意义.结论较高浓度的内皮素1(20 nmol/L)可直接诱导培养大鼠大脑皮质神经元凋亡,其作用可能是通过其A受体和B受体亚型共同实现的.     

Contribution of nitric oxide to the presynaptic inhibition by endothelin ETB receptor of the canine stellate ganglionic transmission.

We previously reported that endothelin (ET) 3 inhibited presynaptically the dog stellate ganglionic transmission. Here, we report the investigation of the possible involvement of nitric oxide pathway in the endothelin-induced inhibition of the ganglionic transmission. The amount of acetylcholine released by preganglionic stimulation for 10 min was concentration-dependently inhibited after exposure to ET-3 (10(-9)-10(-6) M) or IRL-1620, endothelin ET(B) receptor agonist (10(-8)-10(-5) M). The inhibition was antagonized by pretreatment with a nonselective endothelin receptors antagonist (bosentan) and an ET(B) receptor antagonist (BQ-788) or a neuronal nitric oxide synthase inhibitor, 3-bromo-7-nitroindazole, but was not inhibited by a selective ET(A) receptor antagonist, BQ-123. The reduction induced by ET-3 was also antagonized by treatment with a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one. In addition, similar reductions were also mimicked by exposure to cGMP analog, 8-bromoguanosine-3, 5-cyclic monophosphate and nitric oxide donor, S-nitroso-N-acetylpenicillamine. Exposure to ET-3 or IRL-1620 for a 30-min period increased the levels of total nitric oxide (NO), nitrite plus nitrate NO(x) concentration in the incubation medium, with the increase in NO(x) also being antagonized by BQ-788 at the same concentration. The ET-3-induced increase in NO(x) was antagonized by treatment with the same concentration of 3-bromo-7-nitroindazole or a selective inhibitor of receptor-mediated Ca(2+) entry, 1-[b-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole (10(-5) M), and with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. These results indicate that ET(B) receptor activation inhibits the sympathetic ganglionic transmission via reducing acetylcholine release from presynaptic nerve terminals, although this inhibition also seems to involve the ET(B) receptor-operated Ca(2+)-calmodulin-dependent activation of endogenous nitric oxide production.     

Characterization and distribution of endothelin receptors in the pulmonary circulation: investigation of isolated, perfused, and ventilated rabbit lungs.

The aim of the study was to investigate the distribution of 2 subtypes of endothelin-receptors, mediating the effects of endothelin-1 (ET-1) in the pulmonary circulation. Until now, it is still unclear, whether ET(A) receptors or ET(B) receptors or even both are localized in pulmonary vessels. The experiments were performed on 72 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. The arterial pressure and the lung weight gain were continuously registered. Intermittently perfusate samples were taken for determination of thromboxane A2 (TXA2) and prostacyclin (PGI2). The injection of ET-1 (10(-8) M, n = 6) resulted in a biphasic increase in pulmonary arterial pressure (PAP) that was accompanied by the generation of TXA2 and PGI2. Pretreatment with the ET(A)-receptor antagonist LU135252 (10(-6) M, n = 6) suppressed the pressure response after ET-1 application (P < 0.01 at 120 min) and reduced the generation of TXA2 (P < 0.05 at 120 min) and PGI2 (P < 0.05 at 120 min). Pretreatment with the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) also reduced the PAP increase after ET-1 injection. In contrast to this, the pulmonary vascular pressure reaction after ET-1 application was elevated, when ET(B)-receptor antagonist BQ788 (10(-6) M; n = 6) was given. Furthermore, the PGI2 to TXA2 ratio was shifted from 2.3 to 0.9, reflecting a predominance of vasoconstrictive TXA2. The simultaneous application of LU135252 and BQ788 significantly reduced the PAP increase after ET-1 application, but no beneficial effects were observed compared with the application of LU135252 solely. The injection of the ET(B)-receptor agonist sarafotoxin S6c (S6c; 10(-8) M, n = 6) also induced an increase in PAP that was not attenuated by pretreatment with the ET(B)-receptor antagonist BQ788 (10(-6) M, n = 6). LU135252 (n = 6) as well as the application of LU135252 in combination with BQ788 (n = 6) failed to suppress the pressure response after S6c, whereas the cyclooxygenase inhibitor diclofenac (10 microg/mL, n = 6) alone and in combination with LU135252 and BQ788 (n = 6) was able to prevent the PAP increase after S6c injection (P < 0.001). The results demonstrate that the ET-1-induced increase in pulmonary vascular resistance is mainly mediated via ET(A) receptors, whereas ET(B) receptors seem to mediate vasodilation, which was shown by an imbalance of TXA2 and PGI2 generation. On the other hand, the ET(B)-receptor agonist S6c induced vasoconstriction, which was only attenuated by the cyclooxygenase inhibitor diclofenac. From the current results we conclude that, apart from vasoconstrictor ET(A) receptors, at least 2 ET(B)-receptor subtypes are expressed in the pulmonary circulation, one mediating vasoconstriction, which was not blocked by BQ788, and one mediating vasodilation, which was influenced by BQ788.     

Endothelin plays a role in contractions of isolated pig pulmonary vessels induced by diaspirin cross-linked hemoglobin

The current studies were undertaken to investigate the role of endothelin-1 (ET-1) and its receptors in contractions of isolated pulmonary vessels of the pig induced by diaspirin cross-linked hemoglobin (DCLHb). Second-order pulmonary arteries (PAs) and veins (PVs) were isolated from pigs, cut into rings (4 to 5 mm), and mounted at optimal passive tension in 37 degrees C Krebs-filled tissue baths bubbled with 95% O2/5% CO2. Isometric tension was recorded continuously. In paired rings, concentration responses to ET-1 (10(-10) to 10(-7) mol/L), DCLHb (10(-9) to 3x10(-6) mol/L), and N-nitro-L-arginine (LNA) (10(-6) to 5x10(-5) mol/L) in the presence and absence of the ET(A) receptor antagonist BQ123 (3x10(-5) mol/L) were determined. PVs and PAs with intact endothelium and rings from which the endothelium was removed (denuded) were pretreated with the ET(B) receptor antagonist BQ788 to determine the contribution of ET(B) receptors to ET-1, DCLHb, and LNA responses. ET-1, DCLHb, and LNA caused concentration-dependent increases in tension in all vessels. In the presence of BQ123, the 50% effective concentration (EC50) of ET-1 was significantly increased (by 5-fold to 10-fold) in all vessels. DCLHb concentration responses were significantly attenuated-in the PVs by 45% and in the PAs by 79%-during treatment with BQ123. BQ123 attenuated LNA responses in PVs by 35% and in PAs by 87%. Treatment with BQ788 had no effect on endothelium-intact PVs or PAs but significantly increased ET-1 EC50 (log of the molar concentration) from -9.0+/-0.22 to -7.8+/-0.05 in denuded PAs. The ET-1 EC50 was significantly decreased in denuded PAs (-9.0+/-0.22) as compared with responses in endothelium-intact PAs (-8.1+/-0.18). DCLHb concentration responses were attenuated by 71% and LNA responses by 80% during antagonism with BQ788 in the intact PAs only. These data demonstrate that ET-1 plays a role in DCLHb-induced contractions in the PA and PV. The contributions of ET are mediated by both ET(A) and ET(B) receptors in the PA but only by ET(A) receptors in the PV. These results suggest that the vasoconstrictor actions of DCLHb, which have previously been shown to depend on its interference with endothelium-generated NO, may also involve ET. This may reflect the importance of the interaction of these two endothelium-generated physiologic antagonists in the pulmonary circulation.     

Contractile and arrhythmic effects of endothelin receptor agonists in human heart in vitro: blockade with SB 209670

It is known that binding sites with endothelin(A) (ET)(A) and ET(B) receptor characteristics coexist in human heart but little is known about the receptors that mediate cardiostimulant effects of ET receptor agonists or their consequences. Functional studies were performed on isolated human cardiac tissues. The maximal positive inotropic effects of ET-1 were right atrium > left atrium = right ventricle. The rank order of potencies of agonists in right atrium was sarafotoxin S6c > ET-1 = ET-2 > or = ET-3. The ET(A) receptor-selective compounds BQ123 (10 microM) and A-127722 (1 microM) only slightly blocked (<0.5 log-unit shift) the effects of lower concentrations of ET-1, and the ET(B) receptor antagonist Ro46-8443 (10 microM) did not cause blockade. SB 209670 caused concentration-dependent rightward shifts of ET-1 and sarafotoxin S6c concentration-effect curves with Schild slopes not different from one and affinities (-logM K(B)) of 7.0 and 7.9, respectively. ET-1 caused arrhythmic contractions in right atrial trabeculae that were prevented by 10 microM SB 209670 but not 10 microM BQ123 or 1 microM A-127722, precluding ET(A) receptors. ET-1 caused a higher incidence of arrhythmic contractions in tissues taken from patients treated with beta-blockers before surgery than in tissues from non-beta blocker-treated patients. Sarafotoxin S6c produced arrhythmias that were prevented by SB 209670. The positive inotropic effects of ET-1 in human right atrial myocardium are mediated mostly by a non-ET(A), non-ET(B) receptor. Ventricular inotropic ET receptors differ from atrial inotropic ET receptors. ET-1 induced arrhythmic contractions in human atria do not appear to be mediated by an ET(A) receptor.     

Endothelin (ET)-3 stimulates cyclic guanosine 3',5'-monophosphate production via ETB receptor by producing nitric oxide in isolated rat glomerulus, and in cultured rat mesangial cells.

We investigated the effects of endothelins on receptor-mediated cyclic nucleotide metabolism in rat glomerulus, inner medullary collecting duct (IMCD), and also in cultured rat glomerular mesangial cells. Endothelin (ET)-3 dose-dependently stimulated cGMP accumulation in glomerulus, which was higher than that of ET-1 or ET-2. ETB receptor agonist IRL 1620 produced cGMP in a dose-dependent manner, mimicking the effect of ET-3. ETA receptor antagonist BQ123-Na did not inhibit ET-3- or IRL 1620-stimulated cGMP generation. NG-monomethyl-L-arginine (L-NMMA) significantly inhibited ET-3- or IRL 1620-induced cGMP production, suggesting that ET-3- or IRL 1620-stimulated cGMP generation was mediated through nitric oxide (NO). Intracellular Ca chelator BAPTA/AM and calmodulin antagonist W-7, but not Ca channel blocker nicardipine, significantly inhibited ET-3- or IRL 1620-induced cGMP generation. In cultured rat mesangial cells, ET-3 stimulated cGMP generation through NO in the presence of fetal calf serum, which was not inhibited by addition of BQ123-Na. In IMCD, ET-3 had no stimulative effect on cGMP generation. We conclude that ET-3 stimulates NO-induced cGMP generation through ETB receptor in glomerulus. This effect seems to be mediated through intracellular Ca/calmodulin, but not through Ca influx via L-type Ca channel. Mesangial cells can be a source of NO coupled to ETB receptor activation in glomerulus. From these results, mesangial ETB receptor may work to counteract the vasoconstrictive effect of endothelin caused via ETA receptor in glomerulus.     

Ethanol consumption enhances endothelin-1-induced contraction in the isolated rat carotid

We investigated the mechanisms involved in the enhancement of endothelin (ET)-1 vascular reactivity induced by ethanol consumption. Ethanol intake for 2, 6, and 10 weeks enhanced the ET-1-induced contractile response of endothelium-intact but not endothelium-denuded rat carotid rings independently of the treatment duration. Conversely, phenylephrine-induced contraction was not affected by ethanol intake. The contraction induced by IRL1620 [succinyl-(Glu(9),Ala(11,15))-ET-1-(8-21)], a selective ET(B) agonist, was increased after treatment with ethanol in endothelium-intact but not in endothelium-denuded carotid rings. Moreover, ET-1- and IRL1620-induced relaxation was reduced in endothelium-intact phenylephrine-precontracted rings from ethanol-treated rats. Acetylcholine-induced relaxation was not affected by ethanol treatment. N(G)-Nitro-l-arginine methyl ester, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin, and tetraethylammonium reduced the relaxation induced by IRL1620 in carotid glands from control but not ethanol-treated rats. The mRNA levels for ET(A) and ET(B) receptors were not altered by ethanol consumption. However, ethanol treatment reduced the protein expression of ET(B) receptors. Furthermore, immunohistochemical assays showed reduced immunostaining for endothelial ET(B) receptors after treatment with ethanol. We conclude that ethanol consumption enhances ET-1-induced contraction in the rat carotid and that this response is not different among the three periods of treatment used in this study. Finally, the potentiation of ET-1-induced vascular reactivity is probably caused by reduced expression of relaxing endothelial ET(B) receptors.     

Interleukin-18 induces mechanical hypernociception in rats via endothelin acting on ETB receptors in a morphine-sensitive manner

Interleukin (IL)-18 has an important role in the pathogenesis of arthritis, which is accompanied by movement limitation secondary to inflammatory articular nociception. Therefore, we investigated the possible mechanical hypernociceptive effect of IL-18 in rats using the paw constant pressure and the electronic pressure-meter tests. In both tests, intraplantar administration of IL-18 (20-60 ng paw(-1)) caused a dose- and time-dependent mechanical hypernociception, which peaked 3 h and reached control levels 24 h after injection. Pretreatments with indomethacin (2.5 mg kg(-1)), atenolol (1 mg kg(-1)), or 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2;2-dimethylpropanoic acid; Na (MK886) (5-lipoxygenase-activating protein inhibitor; 1 mg kg(-1)) did not inhibit IL-18-evoked hypernociception (40 ng paw(-1)), whereas dexamethasone (2 mg kg(-1)) inhibited the process. IL-18-evoked hypernociception was not inhibited by pretreatment with antiserum to rat tumor necrosis factor-alpha (50 microl paw(-1)) or IL-1 receptor antagonist (300 pg paw(-1)). Pretreatment with N-cys-2,6 dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-1-methoxycarboyl-d-norleucine (BQ788) (ET(B) receptor antagonist; 3-30 nmol paw(-1)), but not with cyclo[(D)Trp-(D)Asp-Pro-(D)Val-Leu] (BQ123) (ET(A) receptor antagonist; 30 nmol paw(-1)), dose dependently inhibited the IL-18-induced hypernociception. Pretreatment with morphine (3-12 microg paw(-1)) also dose-dependently inhibited the IL-18-induced hypernociception. Moreover, endothelin-1-induced mechanical hypernociception also was inhibited by BQ788, but not by BQ123, indomethacin, or atenolol. In conclusion, we demonstrated for the first time that IL-18 is a prohypernociceptive cytokine that induces mechanical hypernociception mediated by endothelin, via ET(B) receptor. Therefore, inhibition of the endothelin ET(B) receptor could be beneficial on controlling inflammatory hypernociception of diseases in which IL-18 plays a role in their pathogenesis.     

Pharmacological profile of ZD1611, a novel, orally active endothelin ETA receptor antagonist.

The endothelins (ETs), potent vasoconstrictor peptides, have been implicated in the pathogenesis of various cardiovascular disorders. In the present study, we describe the novel, potent, orally active, selective ET(A) receptor antagonist ZD1611 [3-(4-[3-(3-methoxy-5-methylpyrazin-2-ylsulfamoyl)-2-pyridyl ]phenyl)- 2,2-dimethylpropanoic acid]. ZD1611 competitively inhibited (125)I-labeled ET-1 binding at human cloned ET(A) and ET(B) receptors with pIC(50) values of 8.6 +/- 0.1 and 5.6 +/- 0.1, respectively, showing 1000-fold selectivity for the ET(A) receptor. ZD1611 caused a parallel rightward shift of the concentration response curve to ET-1 in the rat isolated aorta yielding a concentration of antagonist that caused a 2-fold rightward shift in the ET-1-response curve (pA(2)) of 7.5 +/- 0.3. When administered i. v. to anesthetized rats and dogs, ZD1611 caused dose-related rightward shifts of partial dose-response curves to the precursor of ET-1, big ET-1. Threshold doses for significant antagonist activity were determined as 0.1 mg/kg and 0.3 mg/kg in the rat and dog, respectively. Importantly, ZD1611 was able to reverse an established big ET-1-induced pressor response in pithed rats in the presence of continuous big ET-1 infusion. Failure of ZD1611 to inhibit the BQ3020 (ET(B)-selective)-induced depressor response in pithed rats indicated a lack of activity at the endothelial ET(B) receptor. ZD1611 was orally active in the rat at 0.3 mg/kg and had a duration of action of more than 7 h, and, in the dog, a dose of 0.6 mg/kg p.o. was active for at least 6 h. In conclusion, these data demonstrate that ZD1611 is a potent and orally active, selective ET(A) receptor antagonist with a long duration of action which may be of therapeutic use.     

Investigation into the potential anti-inflammatory effects of endothelin antagonists in a murine model of experimental monosodium urate peritonitis

Endothelin (ET)-1 has been detected in many inflammatory pathologies, including rheumatoid arthritic patients, asthma, and ischemic-reperfusion injury. In this study, we have investigated the effect of a panel of different ET-1 antagonists displaying different selectivities for the receptors in a murine model of experimental inflammatory peritonitis. Systemic treatment of mice with the ETA antagonist C33H44N6O5, N-[N-[-N(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-1-methyl-D-tryptophyl]-3-(2-pyridinyl)-D-alanine (FR139317) inhibited neutrophil accumulation. However, a greater degree of inhibition was observed with the ETB antagonist C34H51N5O7, N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH (BQ-788) and the ET(A and B) antagonist C52H65N7O10, N-acetyl-alpha-[10,11-dihydro-5H-dibenzo-[a,d]cycloheptadien-5-yl]-D-Gly-Leu-Asp-lle-lle-Trp (PD145065); all these effects occurred without altering peripheral blood cell counts. Release of the CXC chemokine KC was significantly reduced by the FR139317 and PD145065 but not by BQ-788. Evaluation of the therapeutic potential of these antagonists showed that PD145065 inhibited neutrophil migration and KC release, whereas the others caused a nonsignificant reduction in these parameters. Parameters of endothelial cell activation showed that urate-stimulated interleukin-1beta release was inhibited by BQ-788 and PD145065 but not by FR139317, whereas ET-1 was only inhibited by the mixed antagonist. A different scenario was observed with respect to release of the CXC chemokine KC with FR139317 and PD145065 being effective, whereas with a marker of polymorphonuclear activation the ETA and mixed antagonist inhibited adhesion molecule expression. These data show that ET-1 antagonists elicit different mechanisms of actions in the way they display their antimigratory effects in a murine model of monosodium urate crystal peritonitis.     

Endothelin-1 regulates cardiac L-type calcium channels via NAD(P)H oxidase-derived superoxide

It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ET(A) receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 microM)] but was not altered in the presence of an ET(B) receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 microM)]. Pretreatment of cells with the ROS scavenger tempol (100 microM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH(3)]) (5 microM) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca(2+) channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.     

Positive inotropic responses mediated by endothelin ET(A) and ET(B) receptors in human myocardial trabeculae.

The aim of the present study was to determine possible inotropic effects mediated by endothelin ET(A) and ET(B) receptors in human myocardial trabeculae from the right atrium and the left ventricle. Isolated trabeculae from human hearts were paced at 1.0 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Endothelin-1 (ET-1) and ET-3 had a strong positive inotropic effect in all trabeculae. ET-1 was significantly more potent than ET-3 in both atrial (P < 0.001) and ventricular (P < 0.05) trabeculae. Preincubation with the ET(A) receptor antagonist FR139317 (1 microM) decreased significantly (P < 0.005) the potency of ET- I in both atrial and ventricular trabeculae, without any significant changes in Emax (maximum effect obtained with an agonist). The ET(B) receptor agonist IRL 1620 had a positive inotropic effect only in some trabeculae, and the ET(B) receptor antagonist BQ 788 (1 microM) almost completely blocked this effect. These results suggest that both ET(A) and ET(B) receptors mediate positive inotropic responses at both the atrial and ventricular level in the human heart.     

L-glutamate and gamma-aminobutyric acid efflux from rat cerebrocortical synaptosomes: modulation by kappa- and mu- but not delta- and opioid receptor like-1 receptors

The modulation by delta-, kappa-, mu-, and opioid receptor like-1 (ORL(1)) agonists and antagonists of L-glutamate (L-Glu) and gamma-aminobutyric acid (GABA) efflux from superfused rat cerebrocortical synaptosomes was studied. Tetrodotoxin (0.5 microM) inhibited the spontaneous efflux of both transmitters by 20%. Ca(2+) omission decreased GABA and facilitated L-Glu efflux. The neurotransmitter overflow evoked by K(+) concentrations in the 7.5- to 10-mM range was largely Ca(2+) dependent and tetrodotoxin sensitive. Neither the delta-receptor agonist deltorphin (up to 0.3 microM) nor the ORL(1) receptor agonist nociceptin (up to 1 microM) significantly affected either spontaneous or K(+)-evoked neurotransmitter efflux. Conversely, the ORL(1) ligand [Phe(1)(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) (0.3 microM) caused a naloxone-sensitive inhibition of both L-Glu- and GABA-stimulated overflow. The kappa-receptor agonist (-)-U50,488 failed to modulate spontaneous L-Glu and GABA efflux. However, it similarly inhibited the K(+)-evoked overflow of both neurotransmitters (EC(50) approximately 100 nM; E(max) approximately 25-30% inhibition) in a norbinaltorphimine-sensitive manner. The selective mu-receptor agonist endomorphin 1 inhibited both spontaneous (EC(50) approximately 50 nM) and K(+)-evoked (EC(50) approximately 10 nM; E(max) approximately 50% inhibition) L-Glu efflux in a naloxone-sensitive manner. Conversely, it significantly inhibited only K(+)-evoked GABA efflux (EC(50) approximately 10 nM), although with a lower maximal effect (E(max) approximately 25-30% inhibition). It is concluded that, in the rat cerebral cortex, L-Glu and GABA efflux from nerve terminals is under the direct inhibitory control of kappa- and mu- (but not delta- or ORL(1)) receptors. Because glutamatergic terminals emerged as a preferential target of mu-receptor agonists, the activation of this receptor may advocate both relevant analgesic and neuroprotective effects.     

Endothelin-1 inhibits endothelium-dependent vasodilatation in the human forearm: reversal by ETA receptor blockade in patients with atherosclerosis

Several cardiovascular disorders, including atherosclerosis, are associated with endothelial dysfunction and enhanced expression of endothelin-1 (ET-1). The role of ET-1 in the development of endothelial dysfunction in vivo remains unclear. The objective of the present study was to investigate the effect of elevated circulating levels of ET-1 on endothelium-dependent vasodilatation (EDV), and to test the hypothesis that ET(A) receptor antagonism improves EDV in patients with atherosclerosis. EDV and endothelium-independent vasodilatation were determined by brachial artery infusion of acetylcholine and sodium nitroprusside respectively during measurement of forearm blood flow (FBF) with venous occlusion plethysmography. A 60 min intra-arterial infusion of ET-1 (n=10) significantly blunted EDV in young healthy males (33 +/- 13% compared with 271 +/- 74% increase in FBF induced by 10 mug/min acetylcholine; P<0.01). Noradrenaline, which evoked a similar degree of vasoconstriction, did not attenuate EDV. In a separate set of experiments, a 60 min intra-arterial infusion of the selective ET(A) receptor antagonist BQ123 evoked a significant increase in EDV in patients with atherosclerosis (n=10; 109 +/- 45% compared with 255 +/- 101% increase in FBF induced by 10 microg/min acetylcholine; P<0.01), whereas no significant change was observed in healthy age-matched controls (n=9). Endothelium-independent vasodilatation was not affected by ET-1 or BQ123. These observations demonstrate that elevated levels of ET-1 impair EDV in healthy control subjects. Furthermore, ET(A) receptor blockade improves EDV in patients with atherosclerosis, indicating that ET-1 attenuates EDV via an ET(A)-receptor-mediated mechanism.