Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation

The hemodynamic and proinflammatory effects of endothelin-1 (ET-1) in proximal (1st/2nd order) and terminal (3rd/4th order) arterioles and venules were examined in small intestine submucosa of anesthetized guinea pigs. Vessel diameter (D), red blood cell velocity, and blood flow (Q) were determined in eight proximal and eight terminal microvessels before and at 20 min of ET-1 suffusion (10(-10), 10(-9), and 10(-8) M) and then with endothelin-A (ET(A))-receptor blockade with BQ-123 (10(-5) M). This protocol was repeated with platelet-activating factor (PAF) inhibition (WEB-2086, 1.0 mg/kg iv; n = 16). The ET-1-mediated microvascular responses were also examined with endothelin-B (ET(B))-receptor blockade using BQ-788 (10(-5) M; n = 11) alone or with ET(A+B)-receptor blockade with BQ-123 + BQ-788 (n = 10). Microvascular permeability was assessed by FITC-albumin (25 mg/kg iv) extravasation in seven series: 1) buffered modified Krebs solution suffusion (n = 6), 2) histamine suffusion (HIS; 10(-3) M, n = 5), 3) ET-1 suffusion (10(-8) M, n = 5), 4) BQ-123 (10(-5) M) plus ET-1 suffusion (n = 5), 5) PAF inhibition before ET-1 suffusion (n = 5), 6) histamine-1 (H1)-receptor blockade (diphenhydramine, 20 mg/kg iv) before ET-1 suffusion (n = 5), and 7) ET(B)-receptor blockade before (BQ-788 10(-5) M; n = 3) or with ET-1 suffusion (n = 3). D and Q decreased at 10(-8) M ET-1 and returned to control values with BQ-123 and BQ-123+BQ788 but not with BQ-788 in proximal microvessels. D did not change in terminal microvessels with ET-1 (10(-8) M) but decreased with BQ-788 and increased with BQ-123. PAF inhibition did not affect the D and Q responses of proximal microvessels to ET-1 but prevented the fall in Q in terminal microvessels with ET-1. ET-1 increased vascular permeability to approximately 1/3 of that with HIS; this response was prevented with BQ-123 and WEB-2086 but not with H1-receptor blockade. This is the first evidence that submucosal terminal microvessel flow is reduced with ET-1 independent of vessel diameter changes and that this response is associated with increased microvascular permeability mediated via ET(A)-receptor stimulation and PAF activation.     

Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia

We have reported that eucapnic intermittent hypoxia (E-IH) causes systemic hypertension, elevates plasma endothelin 1 (ET-1) levels, and augments vascular reactivity to ET-1 and that a nonspecific ET-1 receptor antagonist acutely lowers blood pressure in E-IH-exposed rats. However, the effect of chronic ET-1 receptor inhibition has not been evaluated, and the ET receptor subtype mediating the vascular effects has not been established. We hypothesized that E-IH causes systemic hypertension through the increased ET-1 activation of vascular ET type A (ET(A)) receptors. We found that mean arterial pressure (MAP) increased after 14 days of 7 h/day E-IH exposure (109 +/- 2 to 137 +/- 4 mmHg; P < 0.005) but did not change in sham-exposed rats. The ET(A) receptor antagonist BQ-123 (10 to 1,000 nmol/kg iv) acutely decreased MAP dose dependently in conscious E-IH but not sham rats, and continuous infusion of BQ-123 (100 nmol.kg(-1).day(-1) sc for 14 days) prevented E-IH-induced increases in MAP. ET-1-induced constriction was augmented in small mesenteric arteries from rats exposed 14 days to E-IH compared with those from sham rats. Constriction was blocked by the ET(A) receptor antagonist BQ-123 (10 microM) but not by the ET type B (ET(B)) receptor antagonist BQ-788 (100 microM). ET(A) receptor mRNA content was greater in renal medulla and coronary arteries from E-IH rats. ET(B) receptor mRNA was not different in any tissues examined, whereas ET-1 mRNA was increased in the heart and in the renal medulla. Thus augmented ET-1-dependent vasoconstriction via vascular ET(A) receptors appears to elevate blood pressure in E-IH-exposed rats.     

Endothelin-mediated vasoconstriction in postischemic newborn intestine

We previously suggested that the profound, sustained vasoconstriction noted in 3-day-old swine intestine after a moderate episode of ischemia-reperfusion (I/R) reflects the unmasking of underlying constrictor tone consequent to a loss of endothelium-derived nitric oxide (NO). In this study, we sought to determine whether endothelin-1 (ET-1) was the unmasked constrictor and whether selective loss of endothelial ET(B) receptors, which mediate NO-based vasodilation, participated in the hemodynamic consequences of I/R in newborn intestine. Studies were performed in innervated, autoperfused intestinal loops in 3- and 35-day-old swine. Selective blockade of ET(A) receptors with BQ-610 had no effect on hemodynamics under control conditions; however, when administered before and during I/R, BQ-610 significantly attenuated the post-I/R vasoconstriction and reduction in arteriovenous O(2) difference in the younger group. In 3-day-old intestine, reduction of intestinal O(2) uptake to a level similar to that noted after I/R by lowering tissue temperature had no effect on the response to BQ-610 or ET-1, indicating that the change in response to BQ-610 noted after I/R was not simply consequent to the reduction in tissue O(2) demand. In studies in mesenteric artery rings suspended in myographs, we observed a leftward shift in the dose-response curve for ET-1 after selective blockade of ET(B) receptors with BQ-788 in 3- but not 35-day-old swine. Rings exposed to I/R in vivo behaved in a manner similar to control rings treated with BQ-788 or endothelium-denuded non-I/R rings.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Endothelin ET(A) but not ET(B) receptors mediate contraction of common bile duct

Endothelin (ET) causes contraction of the gallbladder. To investigate effects of ET in the common bile duct, we measured contraction of longitudinal muscle strips from guinea pig common bile ducts induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the common bile duct. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 caused tetrodotoxin and atropine-insensitive contraction. In terms of maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy. However, sarafotoxin S6c, a selective ET(B) receptor agonist, caused only a negligible contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. The ET-1-induced contraction was inhibited by BQ-123, an ET(A)-receptor-selective antagonist, but not by BQ-788, an ET(B)-receptor-selective antagonist. In addition, the combination of both antagonists, BQ-123 and BQ-788, inhibited ET-1 induced contraction but did not potentiate the inhibition caused by BQ-123 alone. These indicate that ET(A) but not ET(B) receptors mediate the contraction. Autoradiography localized 125I-ET-1 binding to the smooth muscle layer. Binding of 125I-ET-1 to the smooth muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves indicated the presence of ET(A) and ET(B) receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ET(A) receptor mediates the contraction.     

Mechanism of hypoxic pulmonary vasoconstriction involves ET(A) receptor-mediated inhibition of K(ATP) channel

There is controversy on the role of endothelin (ET)-1 in the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV is inhibited by ET-1 subtype A (ET(A))-receptor antagonists in animals, it has been reported that ET(A)-receptor blockade does not affect HPV in isolated lungs. Thus we reassessed the role of ET-1 in HPV in both rats and isolated blood- and physiological salt solution (PSS)-perfused rat lungs. In rats, the ET(A)-receptor antagonist BQ-123 and the nonselective ET(A)- and ET(B)-receptor antagonist PD-145065, but not the ET(B)-receptor antagonist BQ-788, inhibited HPV. Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123 and BQ-788 in PSS-perfused lungs was prevented by costimulation with angiotensin II. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide also prevented inhibition of HPV by BQ-123 in both lungs and rats. These results suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ET(A) receptor-mediated suppression of K(ATP)-channel activity.     

Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and alpha1-adrenoceptor-mediated vasoconstriction in Type 2 diabetes

Regulation of vascular tone and blood flow involves interactions between numerous local and systemic vascular control signals, many of which are altered by Type 2 diabetes (T2D). Vascular responses to endothelin-1 (ET-1) are mediated by endothelin type A (ET(A)) and type B (ET(B)) receptors that have been implicated in cross talk with alpha(1)-adrenoceptors (alpha(1)-AR). ET(A) and ET(B) receptor expression and plasma ET-1 levels are elevated in T2D; however, whether this influences coronary alpha(1)-AR function has not been examined. Therefore, we examined the effect of ET(A) and ET(B) receptor inhibition on coronary vasoconstriction to ET-1 and alpha(1)-AR activation in a mouse model of T2D. Coronary vascular responses were examined in isolated mouse hearts from control and diet-induced T2D C57BL/6J mice. Responses to ET-1 and the selective alpha(1)-AR agonist phenylephrine (PE) were examined alone and in the presence of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) alone or in combination with selective ET(A) or ET(B) receptor inhibitors BQ-123 and BQ-788, respectively. Vasoconstriction to ET-1 was enhanced, whereas ET(B), but not ET(A), receptor blockade reduced basal coronary tone in T2D hearts. In the presence of l-NAME, ET(A) receptor inhibition attenuated ET-1 vasoconstriction in both groups, whereas ET(B) inhibition abolished this response only in control hearts. In addition, ET(A) inhibition enhanced alpha(1)-AR-mediated vasoconstriction in T2D, but not control, hearts following l-NAME treatment. Therefore, in this model, enhanced coronary ET-1 responsiveness is mediated primarily through smooth muscle ET(B) receptors, whereas the interaction with alpha(1)-ARs is mediated solely through the ET(A) receptor subtype.     

The role of ET(A) and ET(B) receptor antagonists in acute and allergic inflammation in mice

In this study, we investigated the effects of the selective ET(A) (BQ-123) and ET(B) (BQ-788) receptor antagonists for endothelin-1 (ET-1) against several flogistic agent-induced paw edema formation and ovalbumin-induced allergic lung inflammation in mice. The intraplantar injection of BQ-123, but not BQ-788, significantly inhibited carrageenan-, PAF-, ET-1- and bradykinin-induced paw edema formation. The obtained inhibitions (1h after the inflammatory stimulus) were 79+/-5%, 55+/-4%, 55+/-6% and 74+/-4%, respectively. In carrageenan-induced paw edema, the mean ID(50) value for BQ-123 was 0.77 (0.27-2.23)nmol/paw. The neutrophil influx induced by carrageenan or PAF was reduced by BQ-123, with inhibitions of 55+/-2% and 72+/-4%, respectively. BQ-123 also inhibited the indirect macrophage influx induced by carrageenan (55+/-6%). However, BQ-788 failed to block the cell influx caused by either of these flogistic agents. When assessed in the bronchoalveolar lavage fluid in a murine model of asthma, both BQ-123 and BQ-788 significantly inhibited ovalbumin-induced eosinophil recruitment (78+/-6% and 71+/-8%), respectively. Neither neutrophil nor mononuclear cell counts were significantly affected by these drugs. Our findings indicate that ET(A), but not ET(B), selective ET-1 antagonists are capable of preventing the acute inflammatory responses induced by carrageenan, PAF, BK and ET-1. However, both ET(A) and ET(B) receptor antagonists were found to be effective in inhibiting the allergic response in a murine model of asthma.     

Endothelin-1 and pancreatic islet vasculature: studies in vivo and on isolated, vascularly perfused pancreatic islets

Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor, which also stimulates insulin release. The aim of the present study was to evaluate whether exogenously administered ET-1 affected pancreatic islet blood flow in vivo in rats and the islet arteriolar reactivity in vitro in mice. Furthermore, we aimed to determine the ET-receptor subtype that was involved in such responses. When applying a microsphere technique for measurements of islet blood perfusion in vivo, we found that ET-1 (5 nmol/kg) consistently and markedly decreased total pancreatic and especially islet blood flow, despite having only minor effects on blood pressure. Neither endothelin A (ET(A)) receptor (BQ-123) nor endothelin-B (ET(B)) receptor (BQ-788) antagonists, alone or in combination, could prevent this reduction in blood flow. To avoid confounding interactions in vivo, we also examined the arteriolar vascular reactivity in isolated, perfused mouse islets. In the latter preparation, we demonstrated a dose-dependent constriction in response to ET-1. Administration of BQ-123 prevented this, whereas BQ-788 induced a right shift in the response. In conclusion, the pancreatic islet vasculature is highly sensitive to exogenous ET-1, which mediates its effect mainly through ET(A) receptors.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.     

Role of NO and cytochrome P-450-derived eicosanoids in ET-1-induced changes in intrarenal hemodynamics in rats

Endothelin-1 (ET-1) produces potent renal effects that we have previously shown to be dependent on cytochrome P-450 (CYP450) metabolites of aracidonic acid (24) This study evaluated the role of these metabolites in the effects produced by ET-1 on renal blood flow (RBF), cortical blood flow (CBF), medullary blood flow (MBF), and mean arterial blood pressure (MBP). ET-1 (20-200 pmol/kg) increased MBP, renal vascular resistance (RVR), and MBF but reduced CBF and RBF in a dose-dependent manner. The decreases in CBF and RBF, and increases in MBP and RVR were blunted by BMS-182874, an ET(A) receptor antagonist or BQ-788, an ET(B) receptor antagonist. Similarly, indomethacin, an inhibitor of cyclooxygenase activity, or 12,12-dibromododecenoic acid (DBDD), a CYP450-dependent inhibitor of production of 20-hydroxyeicosatetraenoic acid (20-HETE), blunted these effects. ET-3 elicited dose-related reduction in CBF and increase in MBF. Indomethacin accentuated the reduction in CBF and attenuated the increase in MBF, as did DBDD. ET-1-induced increase in MBF was attenuated by BQ-788, N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, indomethacin, or DBDD. DBDD inhibited the hemodynamic effects of L-NAME. Miconazole, the inhibitor of CYP450-dependent epoxygenase activity, was without effect. These results indicate that hemodynamic changes produced by ET-1 are mediated by vasoconstrictor prostanoids and/or prostanoid-like substances, possibly, 20-HETE via activation of ET(A) and ET(B) receptors. However, the increase in MBF is mediated by vasodilator prostanoids or by NO via ET(B) receptor activation.     

Nitric oxide and endothelin in oxygen-dependent regulation of vascular tone of human umbilical vein

We investigated the possible contribution of nitric oxide (NO) and endothelin (ET) to oxygen-dependent regulation of human umbilical vein vascular tone by simultaneous registration of intracellular membrane potential and isometric tension of vessel strips with and without NO synthase inhibition [10-4 M N omega-nitro-L-arginine methyl ester (L-NAME)], ETA receptor blockade (10(-5) M BQ-123), or ETB receptor blockade (10(-7) M BQ-788) at Po2 values in the bath solution between 5 and 104 mmHg. Increasing PO2 above the physiological intrauterine range resulted in depolarization and an increase of isometric tension, whereas lowering PO2 resulted in hyperpolarization and a decrease in isometric tension. Removal of the endothelium reversed these effects. At PO2 values below 39 mmHg, intact preparations treated with either L-NAME, BQ-788, or BQ-123 were more depolarized than controls. In the case of treatment with L-NAME or BQ-123, this was accompanied by an increase in isometric tension. We conclude that it is NO that mediates the hypoxic hyperpolarization and vasodilatation of the human umbilical vein and that ET, via activation of ETB1 receptors on endothelial cells, contributes to this effect.     

The endothelin/sarafotoxin-induced increase of the proliferation of undifferentiated and DMSO-differentiated GEM-81 goldfish erythrophoroma cells is mediated by ETB receptors

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET(B)-mediated effects on vertebrate pigment cells. GEM-81 cell line, a red pigment cell-derived cutaneous tumor of the teleost Carassius auratus, expresses ET(B) receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO-differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using (125)I ET-1 and BQ-485 (an ET(A) antagonist) or BQ-788 (an ET(B) antagonist). BQ-788, but not BQ-485, significantly reduced (125)I-ET-1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ-788. The results demonstrated that, with the exception of ET-1 (biphasic effect) and ET-3 (no significant effect) in undifferentiated GEM-81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO-differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ-788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

Endothelin-1 decreases CD36 protein expression in vascular smooth muscle cells

Recent studies have shown that CD36 plays important roles as a major scavenger receptor for oxidized low-density lipoproteins and as a crucial transporter for long-chain fatty acids. CD36 deficiency might be associated with insulin resistance and abnormal dynamics of long-chain fatty acids. Endothelin-1 (ET-1), which is synthesized and secreted by vascular endothelial cells, is the most potent endogenous vasoconstrictor known and also stimulates the proliferation of vascular smooth muscle cells (VSMCs) and thus is believed to play an important role in the development of various circulatory disorders, including hypertension and atherosclerosis. The aim of the present study was to investigate the regulatory effect of ET-1 on CD36 expression in cultured VSMCs. VSMCs were treated for different times (0-24 h) with a fixed concentration (100 nM) of ET-1 or with different concentrations (0-100 nM) for a fixed time (24 h); then CD36 expression was determined using Western blots. CD36 expression was significantly decreased by ET in a time- and dose-dependent manner. This inhibitory effect was prevented by the ET(A) receptor antagonist BQ-610 (10 microM) but not the ET(B) receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the tyrosine kinase-mediated and MAPK-mediated pathways. The inhibitory effect of ET-1 on CD36 protein expression was blocked by inhibition of tyrosine kinase activation by use of genistein (100 microM) and by the ERK inhibitor PD-98059 (75 microM) but not by the p38 MAPK inhibitor SB-203580 (20 microM). In conclusion, we have demonstrated that ET-1, acting via the ET(A) receptor, suppresses CD36 protein expression in VSMCs by activation of the tyrosine kinase and ERK pathways.     

Endothelin blockade augments pulmonary vasodilation in the ovine fetus

Ivy, D. Dunbar, John P. Kinsella, and Steven H. Abman.Endothelin blockade augments pulmonary vasodilation in the ovine fetus. J. Appl. Physiol. 81(6):2481-2487, 1996.The physiological role of endothelin-1 (ET-1) inregulation of vascular tone in the perinatal lung is controversial.Recent studies suggest that ET-1 contributes to high basal pulmonaryvascular resistance in the normal fetus, but its role in the modulationof pulmonary vascular tone remains uncertain. We hypothesized that highET-1 activity opposes the vasodilator response to some physiological stimuli such as increased pressure. To test the hypothesis that ET-1modulates fetal pulmonary vascular responses to acute and prolongedphysiological stimuli, we performed a series of experiments in thelate-gestation ovine fetus. We studied the hemodynamic effects of twoET-1 antagonists, BQ-123 (a selectiveET_A-receptor antagonist) andphosphoramidon (a nonselective ET-1-converting enzyme inhibitor) duringmechanical increases in pressure due to partial ductus arteriosuscompression in chronically prepared late-gestation fetal lambs. Incontrol studies, partial ductus arteriosus compression decreased theratio of pulmonary arterial pressure to pulmonary artery flow in theleft lung 34 ± 6% from baseline. Intrapulmonary infusions ofBQ-123 (0.5 µg/min for 10 min; 0.025 µg/min for 2 h) orphosphoramidon (1.0 mg/min for 10 min) augmented the peak vasodilatorresponse during ductus arteriosus compression (52 ± 3 and 49 ± 6% from baseline, respectively, P < 0.05 vs. control). In addition, unlike the transient vasodilator response to ductus arteriosus compression in control studies, ET-1blockade with BQ-123 or phosphoramidon prolonged the increase in flowcaused by ductus arteriosus compression. In summary,ET_A-receptor blockade andET-1-converting enzyme inhibition augment and prolong fetal pulmonaryvasodilation during partial compression of the ductus arteriosus. Weconclude that ET-1 activity modulates acute and prolonged responses ofthe fetal pulmonary circulation to changes in vascular pressure. Wespeculate that ET-1 contributes to regulation and maintenance of highpulmonary vascular resistance in the normal ovine fetal lung.