Analysis of ET-A and ET-B receptors using an isolated perfused rat lung preparation

AIMS AND METHODS: The pulmonary and vascular effects of endothelin-1 receptor activation were studied in isolated perfused and ventilated lung preparations from rat. The responses to endothelin-1 (ET-1) and the endothelin B (ET(B)) receptor agonist sarafotoxin 6c (S6c) were characterized using the endothelin A (ET(A))-receptor antagonist FR 139317, the ET(B)-receptor antagonist BQ 788 and the combined ET(A)/ET(B)-receptor antagonist Bosentan. The respiratory parameter airway conductance (G(aw)) and the vascular parameter perfusion flow were analysed simultaneously. RESULTS: Concentration-response curves for ET-1 administered intra-arterially revealed that its most potent effect was on the vascular side while S6c had a more potent effect on airway conductance. ET-1, given as a bolus dose intra-arterially (100 microL of 0.2 nM), induced a strong- and long-lasting contraction of the vasculature while only a less pronounced contraction was seen in the airways. Neither of the antagonists had a significant effect per se on G(aw) or perfusion flow. FR 139317 reduced the effect of ET-1 on perfusion flow by about 50%, while airway conductance was augmented. BQ 788 enhanced the decrease in perfusion flow by ET-1 while G(aw) was not influenced. The combined ET(A)/ET(B) antagonist Bosentan powerfully prevented the ET-1-induced decrease in G(aw) but did not alter its reduction in perfusion flow. CONCLUSIONS: The potent effect of ET-1 on the vascular side of the lung is mediated mainly through ET(A) receptors, whereas both ET(A) and ET(B) receptors are involved in G(aw) in the rat lung.     

Low NaCl intake elevates renal medullary endothelin-1 and endothelin A (ETA) receptor mRNA but not the sensitivity of renal Na+ excretion to ETA receptor blockade in rats

Aims: This study was performed to investigate the effects of NaCl intake on renal mRNA expression of pre‐pro‐endothelin‐1 (ET‐1), endothelin A (ET_A) and endothelin B (ET_B) receptors as well as on renal ET‐1 content in rats. We further tested for NaCl intake‐dependent differences in the contribution of the ET system to renal sodium handling. Methods: Male Sprague–Dawley rats with telemetric devices were randomized to 0.15%, 0.60% and 1.80% NaCl diets with or without losartan. Renal sodium balance and arterial pressure were monitored. Renal blood flow and fractional sodium excretion (FENa) were measured in response to acute infusion of ET_A and ET_B blockers into the inner stripe of the outer renal medulla. Results: Medullary pre‐pro‐ET‐1, ET_A and ET_B receptor mRNA was 50%, 81% and 33% higher in rats on 0.15% vs. 1.80% NaCl. Losartan reduced medullary gene expression in rats on 0.15% NaCl. Medullary ET‐1 content was 983 ± 88 and 479 ± 42 ng mg^?1 protein in rats on 0.15% and 1.80% NaCl (P < 0.001). Chronic ET_A receptor blocker treatment reduced arterial pressure by 8–10 mmHg in rats on 0.15% vs. 1.80% NaCl without affecting renal sodium balances. Acute medullary ET_A or ET_B receptor blockade did not alter medullary blood flow and FENa in animals on either diet. Conclusion: In rats renal medullary ET‐1 content and mRNA expression of three ET system components are inversely related to NaCl intake. Higher expression levels on low NaCl intake are AT₁ receptor dependent but are not associated with increased sensitivity of renal sodium handling to ET_A receptor blockade.     

Vasoactivity mediated by endothelin ETA and ETB receptors in isolated porcine ophthalmic artery

Endothelin isopeptides and sarafotoxin S6b induced strong contractions in isolated porcine ophthalmic artery at basal tension, which were antagonized in a concentration-dependent manner by the specific ET_A antagonist FR 139317. The maximum contraction and potency of endothelin-1 and sarafotoxin S6b were similar, whereas endothelin-3 was significantly less potent and induced weaker contractions. Schild plot analysis was only obtained for endothelin-1 and sarafotoxin S6b, but indicated competitive binding at the same ET_A receptor site for these peptides. However, the slope obtained for endothelin-1 was significantly less than unity, suggesting more than one receptor. FR 139317 was a more potent antagonist of contractions induced by endothelin-3 than the other peptides. In arteries pre-contracted by prostaglandin F_2α, high endothelin-3 concentrations induced additional contraction, except in the presence of FR 139317 when a marked relaxation was observed, an ability which was otherwise masked by the strong contractile activity. The relaxation was significantly reduced in endothelin-denuded segments. Both contraction and relaxation were abolished by the ET_A/B antagonist bosentan. The results suggest the presence of ET_B as well as ET_A receptors in this artery type, though ET_B receptor activity is only demonstrated at an unusually high concentration of endothelin in this preparation in vitro.     

Contribution of endothelin to the coronary vasoconstriction in the isolated rat heart induced by nitric oxide synthase inhibition

The possible involvement of endothelin-1 (ET-1) and angiotensin II in the coronary vasoconstriction induced by nitric oxide synthase (NOS) inhibition was investigated in isolated Langendorff-perfused rat hearts. Fifteen minutes of perfusion with the NOS inhibitor N ^G-nitro-L -arginine (L -NNA, 0.1 mM ) reduced coronary flow by 31%. Pre-treatment with the non-selective ET_A/ET_B receptor antagonist bosentan (1 and 10 μM ) attenuated this reduction in coronary flow to 16% (P < 0.05) and 8% (P < 0.01), respectively. The selective ET_A receptor antagonist BQ-123 (1 μM ) induced a similar inhibitory action, whereas the selective ET_B receptor antagonist BQ-788 and the angiotensin II type 1 receptor antagonist candesartan did not affect the vasoconstrictor response to L -NNA. In addition, bosentan administered after 15 min of L -NNA perfusion reversed the L -NNA-induced reduction in coronary flow in a dose-dependent manner. The high concentration of bosentan (10 μM ) increased the basal coronary flow by 17%, while the lower concentration of bosentan, BQ-123, BQ-788 and candesartan did not affect basal coronary flow. Bosentan (10 μM ) increased the level of ET-like immunoreactivity (-LI) in the coronary effluent twofold. L -NNA did not affect ET-LI level. These results indicate that ET-1 contributes to the coronary vasoconstrictor effect of L -NNA in the isolated rat heart, and that this action of ET-1 is mediated through ET_A receptor activation. Angiotensin II does not seem to contribute to L -NNA-induced vasoconstriction under the present condition.     

Endothelin(A) (ET(A)) and ET(B) receptor-mediated regulation of nitric oxide synthase 1 (NOS1) and NOS3 isoforms in the renal inner medulla

Aim: Our laboratory and others have shown that endothelin (ET)‐1 directly stimulates nitric oxide (NO) production in inner medullary collecting duct (IMCD) cells. The goal of this study was to determine which NO synthase (NOS) isoforms in IMCD are sensitive to ET‐1, and the role of ET_A and ET_B receptor activation in vivo and in vitro. Methods: NOS enzymatic activity and NOS isoform protein expression were examined in cultured IMCD‐3 cells and isolated renal inner medulla. ET_B receptor‐deficient homozygous rats (sl/sl) have elevated levels of circulating ET‐1 and lack a functional ET_B signalling pathway in kidneys, and furthermore provides a unique model to study ET_A receptor signalling in the renal inner medulla in vivo. Results: Incubation of IMCD‐3 cells with exogenous ET‐1 (50 nm ) resulted in ET_A‐dependent increased NOS1 protein expression in IMCD‐3 cells with no effect on NOS2 or NOS3 expression. ET_B receptor antagonism has no effect on NOS expression in IMCD‐3 cells. Consistent with in vitro results, cytosolic NOS1 protein expression was significantly greater in the renal inner medulla of sl/sl rats compared with heterozygous (sl/+) controls, with no alteration in NOS3 expression. In contrast to protein expression data, NOS1‐ and NOS3‐specific enzymatic activities decreased in the cytosolic fraction from the renal inner medulla of sl/sl compared with sl/+. Conclusion: These results provide evidence that both ET_A and ET_B receptors regulate NOS isoform activity in the renal inner medulla and specifically support the hypothesis that ET_A receptor activation increases NOS1 expression.     

Renal endothelin system and excretory function in Wistar-Kyoto and Long-Evans rats

Aim: The role of the kidney endothelin system in the renal regulation of fluid and electrolyte excretion was investigated in Wistar–Kyoto (WKY) and Long–Evans (LE) rats in which we found previously marked differences in the renal excretory responses to endothelin A receptor blockade. Methods: The selective endothelin A and B receptor antagonists BQ‐123 (16.4 nmol kg⁻¹ min⁻¹) and BQ‐788 (25 nmol kg⁻¹ min⁻¹) were infused i.v. for 50 min in conscious chronically instrumented WKY and LE rats and their renal function and renal endothelin system were studied. Results: Without effects on glomerular filtration rate or renal blood flow, BQ‐123 and BQ‐788 decreased by more than 50% (P < 0.01) both urine flow rate and electrolyte excretion in WKY rats but only urine flow rate (P < 0.05) in LE rats. Endothelin‐1 content, preproET‐1/GPDH mRNA ratio, B_max and K_d of total endothelin receptors in renal cortex did not differ between the two strains. In contrast, plasma endothelin‐1 concentration (0.58 ± 0.04 vs. 1.05 ± 0.01 femtomol mL⁻¹; P < 0.01), renal papillary ET‐1 concentration (68 ± 5 vs. 478 ± 62 fmol mg⁻¹ protein; P < 0.01) and preproET‐1/GPDH mRNA ratio (0.65 ± 0.09 vs. 0.88 ± 0.05; P < 0.05) as well as total endothelin receptor number in renal papilla (B_max 5.3 ± 0.4 vs. and 9.0 ± 1.2 pmol mg⁻¹ protein; P < 0.05) were markedly lower in LE than in WKY rats. In vitro studies showed that in both strains ET_B receptors on renal cortical membranes amounted between 65% and 67% and on papillary membranes between 85% and 88%. Conclusion: The present data show that the selective ET_A or ET_B receptor blockade differentially affects tubular water and salt handling, which becomes apparent in conditions of low renal papillary endothelin receptor number and tissue endothelin‐1 concentration.     

Endothelin‐1‐induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin‐1 (ET‐1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca²⁺‐activated K⁺ channels. In this study, we investigated whether Ca²⁺‐activated K⁺ channels with big conductance (BK_Ca) contribute to endothelial cell proliferation induced by ET‐1. Methods: The patch‐clamp technique was used to analyse BK_Ca activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [³H]‐thymidine incorporation. Changes of intracellular Ca²⁺ levels were examined using fura‐2 fluorescence imaging. Results: Characteristic BK_Ca were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L^?1 ET‐1 caused a significant increase of BK_Ca open‐state probability (n = 14; P < 0.05; cell‐attached patches). The ET_B‐receptor antagonist (BQ‐788, 1 μmol L^?1) blocked this effect. Stimulation with Et‐1 (10 nmol L^?1) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET‐1 (10 nmol L^?1) and the highly specific BK_Ca blocker iberiotoxin (IBX; 100 nmol L^?1) did not cause a significant increase in endothelial cell growth. Ca²⁺ dependency of ET‐1‐induced proliferation was tested using the intracellular Ca²⁺‐chelator BAPTA (10 μmol L^?1). BAPTA abolished ET‐1 induced proliferation (n = 12; P < 0.01). In addition, ET‐1‐induced HUVEC growth was significantly reduced, if cells were kept in a Ca²⁺‐reduced solution (0.3 mmol L^?1), or by the application of 2 aminoethoxdiphenyl borate (100 μmol L^?1) which blocks hyperpolarization‐induced Ca²⁺ entry (n = 12; P < 0.05). Conclusion: Activation of BK_Ca by ET‐1 requires ET_B‐receptor activation and induces a capacitative Ca²⁺ influx which plays an important role in ET‐1‐mediated endothelial cell proliferation.     

Effect of angiotensin II and endothelin‐1 receptor blockade on the haemodynamic and hormonal changes after acute blood loss and after retransfusion in conscious dogs

Aim: This study investigates angiotensin II and endothelin‐1 mediated mechanisms involved in the haemodynamic, hormonal, and renal response towards acute hypotensive haemorrhage. Methods: Conscious dogs were pre‐treated with angiotensin II type 1 (AT₁) and/or endothelin‐A (ET_A) receptor blockers or not. Protocol 1: After a 60‐min baseline period, 25% of the dog's blood was rapidly withdrawn. The blood was retransfused 60 min later and data recorded for another hour. Protocol 2: Likewise, but preceded by AT₁ blockade with i.v. Losartan. Protocol 3: Likewise, but preceded by ET_A blockade with i.v. ABT‐627. Protocol 4: Likewise, but with combined AT₁plus ET_Ablockade. Results: In controls, haemorrhage decreased mean arterial pressure (MAP) by approximately 25%, cardiac output by approximately 40%, and urine volume by approximately 60%, increased angiotensin II (3.1‐fold), endothelin‐1 (1.13‐fold), vasopressin (116‐fold), and adrenaline concentrations (3.2‐fold). Glomerular filtration rate and noradrenaline concentrations remained unchanged. During AT₁ blockade, the MAP decrease was exaggerated (?40%) and glomerular filtration rate fell. During ET_A blockade, noradrenaline increased after haemorrhage instead of adrenaline, and the MAP recovery after retransfusion was blunted. The decrease in cardiac output was similar in all protocols. Conclusions: Angiotensin II is more important than endothelin‐1 for the short‐term regulation of MAP and glomerular filtration rate after haemorrhage, whereas endothelin‐1 seems necessary for complete MAP recovery after retransfusion. After haemorrhage, endothelin‐1 seems to facilitate adrenaline release and to blunt noradrenaline release. Haemorrhage‐induced compensatory mechanisms maintain blood flow more effectively than blood pressure, as the decrease in cardiac output – but not MAP – was similar in all protocols.     

Endothelin ETB1 receptor agonism as a new therapeutic strategy in pulmonary arterial hypertension and chronic heart failure

Pulmonary arterial hypertension and post-ischemic chronic heart failure are highly prevalent diseases with high morbidity and mortality rates due to chronic vascular injury and extensive remodeling responses at the level of the vessel walls. Endothelins play a central role in this setting, through a complex signaling system that mainly affects endothelial and vascular smooth muscle cells. ET_A and ET_B2 endothelin receptors are thought to mediate pro-ischemic responses, while ET_B1 receptor activity could account for the overall protective effect of ET_B signaling in physiology. The pharmacologic modulation of the endothelin system has mainly focused on the dual non-selective blockade of ET_A and ET_B endothelin receptors or to the selective blockade of ET_A-related pathways to date. Good clinical results were achieved in the setting of pulmonary hypertension but no advantage has been demonstrated for heart failure. Restoring and enhancing the physiological protective role of ET_B1-signaling with concomitant blockade of ET_B2 could possibly improve the efficacy of current therapies in the setting of pulmonary arterial hypertension and post-ischemic chronic heart failure.     

Involvement of endothelin-1 in habitual exercise-induced increase in arterial compliance

Aim: Habitual aerobic exercise results in a significant increase in central arterial compliance. Endothelin‐1 (ET‐1) is a potent endothelium‐derived vasoconstrictor peptide and could play a role in mediating the habitual aerobic exercise‐induced increase in central arterial compliance. The aim of the present study was to examine whether ET‐1 is involved in the mechanisms underlying the increase in central arterial compliance with aerobic exercise training. Methods: Seven apparently healthy middle‐aged and older (60 ± 3 years) adults underwent systemic endothelin‐A/B (ET_A/B)‐receptor blockade (500 mg of Tracleer^®) before and after 12 weeks of aerobic exercise training (70 ± 1% of maximal heart rate, 44 ± 2 min day^?1, 4.4 ± 0.1 days week^?1). Results: Basal carotid arterial compliance (via simultaneous B‐mode ultrasound and arterial applanation tonometry on the common carotid artery) increased significantly after exercise training. Resting plasma ET‐1 concentration decreased significantly after exercise training. Before exercise intervention, carotid arterial compliance increased significantly with the administration of the ET_A/B‐receptor blockade. After training, however, increases in carotid arterial compliance previously observed with the ET_A/B‐receptor blockade before training were abolished. Conclusions: Regular aerobic exercise training enhances central arterial compliance in middle‐aged and older humans. The increase in arterial compliance was associated with the corresponding reduction in plasma ET‐1 concentration as well as the elimination of ET‐1‐mediated vascular tone. These results suggest that reductions in ET‐1 may be an important mechanism underlying the beneficial effect of exercise training on central artery compliance.     

In vivo effects of endothelin-2, endothelin-3 and ETA receptor blockade on arterial,venous and capillary functions in cat skeletal muscle

Ekelund , U. 1994. In vivo effects of endothelin-2, endothelin-3 and ET_A receptor blockade on arterial, venous and capillary functions in cat skeletal muscle. Acta Physiol Scand 150, 47–56. Received 31 March 1993, accepted 25 May 1993. ISSN 0001–6772. Department of Physiology & Biophysics, University of Lund, Sweden. This study describes, in quantitative terms, the effects of endothelin-2 and endothelin-3 on vascular tone (resistance) in large-bore arterial resistance vessels (> 25 /μm), small arterioles (< 25 μm) and the veins, as well as on capillary pressure and fluid exchange in cat gastrocnemius muscle in vivo. Infusion of endothelin-2 or endothelin-3 (200–1600 ng kg^-1 min^-1, i.a.) elicited an initial transient dilation, followed by a dose-dependent, slowly developing constrictor response, being maintained after cessation of the infusion. At the dose of 400 ng kg^-1 min^-1 (n= 9), infused i.a. during 20 min, endothelin-2 caused an average increase in total regional vascular resistance of 80%, and endothelin-3 of 35%, and the site of constrictor action of both peptides was preferentially located to the small arterioles. Endothelin-2 also constricted the veins and, hence, evoked a pronounced capacitance response, whereas endothelin-3 was devoid of any venoconstrictor effect. This difference, via effects on the pre-/post-capillary resistance ratio, led to a more pronounced fall of capillary pressure in response to endothelin-3 than to endothelin-2. The new specific competitive ET_A receptor antagonist, FR 139317, abolished the vasoconstrictor response to both endothelin-2 and endothelin-3 in vivo, whereas the preceding vasodilator responses were unaffected. These results, taken together with those of our previous analogous study of the effects of endothelin-1, indicated that all three endothelins were approximately equally as effective in eliciting the transient dilator response in skeletal muscle in vivo, whereas the order of vasoconstrictor activity was endothelin-1 > endothelin-2 > endothelin-3. Due to an especially pronounced venoconstrictor activity of endothelin-1, this peptide, in contrast to endothelin-2 and -3, evoked a rise in capillary pressure, with a consequent net transcapillary fluid filtration and muscle tissue oedema formation. The results further indicated that the vasoconstrictor responses to all endothelins in skeletal muscle were mediated by the ET_A receptor, whereas the initial transient vasodilator responses seemed to be mediated by the ET_B receptor.     

The novel non-peptide selective endothelin A receptor antagonist LU 135 252 protects against myocardial ischaemic and reperfusion injury in the pig

The aim of the study was to investigate the efficacy of the novel non-peptide selective endothelin A (ET_A) receptor antagonist LU 135 252 to limit the extent of myocardial ischaemic and reperfusion injury. Administration of LU 135 252 (1 and 5 mg kg^–1 i.v.) to anaesthetised pigs reduced mean arterial pressure (MAP) from 91 ± 4 to 79 ± 3 mmHg (P < 0.05) and 96 ± 3–82 ± 3 mmHg (P < 0.01), respectively. Heart rate, coronary blood flow and coronary vascular resistance were not affected by LU 135 252. The infarct size induced by 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4-h reperfusion in pigs was 81 ± 5% of the area at risk in control animals given vehicle (n = 8). In pigs receiving 1 mg kg^–1 (n = 6) or 5 mg kg^–1 (n = 8) of LU 135 252 i.v. 20 min before ischaemia the infarct size was reduced to 64 ± 3% (P < 0.05) and 35 ± 4% (P < 0.001), respectively, of the area at risk. During the reperfusion period there was a non-significant trend towards a higher coronary blood flow and a lower coronary vascular resistance in the groups given LU 135 252 compared to controls. Myocardial overflow of ET-like immunoreactivity was increased during the reperfusion period but it was not affected by administration of LU 135 252. It is concluded that administration of the selective ET_A receptor antagonist LU 135 252 effectively protects the myocardium from ischaemia/reperfusion injury, indicating that the ET_A receptor subtype is involved in the development of ischaemia/reperfusion injury.     

Inhibitory effect of co‐administration of atorvastatin and endothelin‐1 receptor antagonist on the progression of atherosclerosis in rabbit

Atorvastatin, a hydroxymethylglutaryl‐CoA reductase inhibitor, and endothelin‐1 (ET‐1) receptor antagonist have been separately indicated to ameliorate disease progression in atherosclerosis. However, no study has evaluated the effect of their combination on atherosclerosis. The objective of the current study was to evaluate the direct in vivo effects of a combination regimen of atorvastatin and ET‐1 receptor antagonist on male New Zealand white rabbit models of atherosclerosis (injury‐induced). Thirty‐two atherosclerotic rabbits were divided into four experimental groups: (a) injury group – fed high‐fat diet; (b) ET‐1 receptor antagonist preventive group – fed high‐fat diet, but with intragastric administration of the ET‐1 receptor antagonist, darusentan; (c) combined preventive group – fed high‐fat diet, but with intragastric administration of both darusentan and atorvastatin; and (d) treatment group – fed high‐fat diet for the first 8 weeks, followed by normal diet and intragastric administration of both darusentan and atorvastatin up to 16 weeks. A further eight non‐atherosclerotic rabbits were fed normal diet and classified as the control group. At the end of 8 and 16 weeks, compared with the injury group, the combined preventive group had significant reduction in both the concentration of serum lipids and inflammatory factors and atherosclerosis formation, indicative of a multifaceted anti‐atherosclerotic impact. The relative area of atherosclerotic lesions in the injury group (30.84%) was significantly higher than the control group (4.62%; p < 0.05). The combined preventive group showed a significantly robust effect on lowering serum lipid, inflammatory cytokines, and maintained homeostatic balance of free radicals, and important downstream effectors like ET‐1 and matrixmetalloproteinase‐9. Our data show that atorvastatin and ET‐1 receptor antagonist co‐administration may decrease lipid levels, stabilize plaques and relieve vascular inflammation. By reducing the plaque burden, this regimen may minimize the risk of atherosclerotic plaque rupture or arterial occlusion.     

Effects of endothelin receptor antagonists on bradykinin-induced increases in macromolecular efflux

The goal of this study was to determine the effects of endothelin receptor antagonists on agonist-induced increases in macromolecular extravasation in the hamster cheek pouch in vivo. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mol wt=70 K) to examine extravasation from postcapillary venules in response to bradykinin and endothelin before and following application of inhibitors of endothelin receptors (ET_ABand ET_A). Increases in extravasation of macromolecules were quantitated by counting the number of venular leaky sites. Bradykinin (0.5 and 1.0M) and endothelin-1 (0.01 and 0.1 nM) produced a dose-related increase in the number of venular leaky sites and superfusion of PD 142893 (ET_ABantagonist), and PD 147953 and BQ-123 (ET_Aantagonists) significantly decreased bradykinin- and endothelin-induced responses. Addition of calcium to the superfusate restored bradykinin-induced increases in venular leaky sites in the presence of endothelin receptor antagonism. Thus, the findings of the present study suggest that endothelin receptor antagonists abrogate bradykinin- and endothelin-induced increases in macromolecular efflux from postcapillary venules. The mechanism for the effects of endothelin receptor antagonists appears to be related to inhibition of the ET_Areceptor which, in turn, alters the mobilization of calcium across venular endothelium.     

In-vivo effects of endothelin-1 and ETa receptor blockade on arterial,venous and capillary functions in skeletal muscle

Results from in vitro studies have indicated that endothelin-1 is a main candidate for endothelium-derived contracting factors. The aim of this in vivo study was to describe in quantitative terms the effects of endothelin-1 (ET-1), and of ET_a receptor blockade, on vascular tone (resistance) in large-bore arterial resistance vessels (> 25 μm), small arterioles (< 25 μm) and the veins, as well as on capillary pressure and fluid exchange in cat gastrocnemius muscle. Endothelin-1 (100–1600 ng kg-1 min-1, i.a.) elicited, after an initial transient dilation, a strong dose-dependent constrictor response in all three consecutive vascular sections, yet with a preferential action on the small arterioles and the veins. The vasoconstriction developed very slowly over about 1 h and was also long-lasting after cessation of the infusion. Our main quantitative analysis refers to effects elicited by 20 min long i.a. infusions of ET-1 at a dose of 400 ng kg-1 min-1. At the end of this period, the peptide caused, on average, a three-fold increase in total regional vascular resistance, in turn explained by a 70% increase in large-bore arterial resistance, a 280% increase in arteriolar resistance and a 220% increase in venous resistance. The latter effect was also manifested as a pronounced capacitance response, and as a decrease in the pre- to post-capillary resistance ratio leading regularly to a rise in capillary pressure, net transcapillary fluid filtration and oedema formation which is unusual for a vasoconstrictor. The new specific competitive ET_A receptor antagonist FR 139317 was found to be fully effective in vivo, insofar as it abolished the constrictor response to endothelin-1. ET_A receptor blockade, or administration of phosphoramidon, an inhibitor of ET-1 production, did not influence the level of basal vascular tone, indicating no significant endogenous release of ET-1 under resting conditions. This contrasts to the established pronounced endogenous release of endothelium-derived nitric oxide. Finally, vascular myogenic regulation was found not to be mediated by ET-1. The results, taken together, suggest a possible role of ET-1 in long-term, rather than short-term, regulation of vascular tone in vivo, perhaps especially during pathophysiological conditions.     

Immunohistochemical localization of endothelin-1, endothelin-3 and endothelin receptors in human pheochromocytoma and paraganglioma

Endothelln (ET) and its receptor system have been shown to exert varlous biological effects on dlfferent types of cells In addition to their well-known vasoconstrictor activity. Recently ET-1, ET-3 and the ET₃ receptor have been shown to play an Important role In the development of neural crest-derived cells and, in this context, pheochromocytomas have been reported to harbor ET-1. Endothelin-3 or ET receptor subtypes, however, have not been examined in pheochromocytoma and paraganglioma so far. In the present study the Immunohistochemical localization of ET-1/big ET-1, ET-3/big ET-3 and the ET_A and ET_B receptors were lnvestigated to clarify the biological characteristics of these two tumors using 32 pheochromocytomas and 11 extra-adrenal paragangliomas. Endothelin-lhig ET-1 was detected in 19 pheochromocytomas (59%) and eight paragangliomas (72%), while ET-3hIg ET-3 was detected in 10 pheochromocytomas (31%) and three paragangllomas (27%). The ET_A receptor was found in 21 pheochromocytomas (66%) and In eight paragangllomas (73%), whlle the ET_B receptor was found in 25 pheochromocytomas (78%) and In eight paragangllomas (73%). Normal adrenomedullary cells lacked each antigen examined. Endothelin-immunoreactive tumor cells were dlstrlbuted focally or In a manner scattered, whlle receptor-immunostained tumor cells were distributed wlth a focal pattern for the ETa receptor and wlth a focal or diffuse pattern for the ET_B receptor. Endothelln and its receptor coexlsted In the same tumor in 21 of 28 ET-posltive pheochromocytomas and in eight of 10 ET-positlve paragangliomas. In additlon, seven pheochromocytomas and two paragangllomas revealed posltivlty of the receptor(s) irrespective of the absence of ET-immunoreactlvlty. In concluslon, ET and Its receptor are frequently and concomitantly expressed in the pheochromocytoma and paraganglloma. From the highly frequent expression of this system or the receptor(s), ET-receptor-mediated slgnal transduction of these tumors concernlng growth and/or cell survival Is expected, although definite blological slgniflcance of thls llgand-receptor system in these tumors awaits further Investigation.     

Endothelin B receptor-like immunoreactivity is associated with LHRH-immunoreactive fibers in the rat hypothalamus

Antisera were raised against amino-acid residues 425–439 of the rat endothelin B (ET_B) receptor. Massive ET_B receptor immunoreactive fibers were observed in the median eminence and organum vasculosum lamina terminalis of rats. Double labeling studies with anti-luteinizing hormone-releasing hormone (LHRH) antiserum revealed that ET_B receptor-like immunoreactivity associated with LHRH-immunoreactive fibers in these areas. However, ET_B receptor-like immunoreactivity was not detected in LHRH-immunoreactive somata. This result suggests that endothelin affects the LHRH neuronal systems via ET_B receptors on the axon at the terminal field.     

Endothelin receptors as novel targets in tumor therapy

The endotelin (ET) axis, that includes ET-1, ET-2, ET-3, and the ET receptors, ET_A and ET_B, plays an important physiological role, as modulator of vasomotor tone, tissue differentiation and development, cell proliferation, and hormone production. Recently, investigations into the role of the ET axis in mitogenesis, apoptosis inhibition, invasiveness, angiogenesis and bone remodeling have provided evidence of the importance of the ET-1 axis in cancer. Data suggest that ET-1 participates in the growth and progression of a variety of tumors such as prostatic, ovarian, renal, pulmonary, colorectal, cervical, breast carcinoma, Kaposi's sarcoma, brain tumors, melanoma, and bone metastases. ET-1 receptor antagonists beside providing ideal tools for dissecting the ET axis at molecular level have demonstrated their potential in developing novel therapeutic opportunity. The major relevance of ET_A receptor in tumor development has led to an extensive search of highly selective antagonists. Atrasentan, one of such antagonists, is orally bioavailable, has suitable pharmacokinetic and toxicity profiles for clinical use. Preliminary data from clinical trials investigating atrasentan in patients with prostate cancer are encouraging. This large body of evidence demonstrates the antitumor activity of endothelin receptor antagonists and provides a rationale for the clinical evaluation of these molecules alone and in combination with cytotoxic drugs or molecular inhibitors leading to a new generation of anticancer therapies targeting endothelin receptors.     

Effects of endothelins on renin secretion from rat kidneys

Using a preparation of isolated rat kidneys perfused at constant renal artery pressure (80 mmHg) we investigated the role of endothelins in the regulation of renin release. Addition of three related endothelins (ET-1, ET-2, ET-3) at a concentration of 10 pmol L^-1 tended to enhance renin secretion rates. Higher doses (100 pmol L^-1, 1 nmol L^-1) of different ETs such as the selective ET_B, receptor agonist sarafotoxin S6c (100 pmol L^-1, 1 nmol L^-1) inhibited renin release and increased renal vascular resistance with similar potency. These effects of ETs were blunted when calcium ions were removed from the perfusate. Renin release activated by isoproterenol (10 nmol L^-1) was also significantly reduced with ET-1, -2 and -3 (1 nmol L^-1). BQ-123 (500 nmol L^-1), a selective ET_A receptor antagonist, only attenuated, whilst the nonselective ET receptor blocker bosentan (Ro 47–0203, 10 μmol L^-1) almost abolished the renal vasopressor and renin inhibitory action of ET-1 and sarafotoxin S6c. BQ-123 and bosentan alone did not affect either perfusate flow or basal renin secretion rates in isolated perfused kidneys. These findings indicate that all three ET peptides equipotently inhibit renin secretion from the kidneys. Most of the vasopressor and renin inhibitory effect of ETs is mediated by ET_b, rather than ET_A receptors involving a calcium-dependent signal transduction mechanism. Moreover, our results suggest that intrarenally released ETs do not contribute to the regulation of renin secretion from isolated perfused rat kidneys.     

Age-associated alterations in retinal arteriole reactivity to endothelin-1 differ between the sexes

Endothelin-1 (ET-1) is a vasoconstrictor implicated in age-related retinal pathologies. This study determined whether responses to ET-1 differed in retinal arterioles isolated from adult (2–3 months) and aged (>20 months) Fischer 344 rats of both sexes. Risk factors for retinal disease (retinal perfusion pressure, intraocular pressure, blood glucose) were not affected by age. However, sensitivity to ET-1 declined with age, especially in females. Vasoconstrictor responses to 50 mM KCl and Ca²⁺ release by caffeine (10 mM) were similar in all groups. Retinal ET_A and ET_B receptor expression also was similar in young and aged rats, regardless of sex. Contractions elicited by 10 nM ET-1 were inhibited by the ET_A antagonist BQ-123 (1 μM) in all groups. In contrast, the ET_B antagonist BQ-788 (1 μM) restored ET-1-induced contractions in aged female vessels, but had no effect in any other group. Removal of the endothelium also restored contractions in vessels from aged females but not males. Thus, responsiveness to ET-1 declines with age in retinal microvasculature. In males, this is likely mediated by age-related changes in the ET_A receptor signaling pathway. By contrast, effects of ET-1 on endothelial ET_B receptors attenuate vasoconstrictor responses in aged females.