Endothelin-1 Concentration-response Curves Research Articles

MEK1/2 inhibitor U0126, but not nimodipine, reduces upregulation of cerebrovascular contractile receptors after subarachnoid haemorrhage in rats.

Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.

The role of Rho-kinase and calcium ions in constriction triggered by ET-1

Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca2+ related to activation of IP3 receptors and to Rho-kinase activity.

Functional estimation of endothelin-1 receptor antagonism by bosentan, macitentan and ambrisentan in human pulmonary and radial arteries in vitro

BackgroundEndothelin receptor antagonists are approved for pulmonary arterial hypertension. Development of selective ETA-receptor antagonists over mixed or dual receptor antagonists has depended on a range of receptor binding assays, second messenger assays and functional blood vessel assays. This study compared the 3 clinically-approved endothelin receptor antagonists in assays of human isolated pulmonary and radial arteries in vitro. MethodsHuman isolated pulmonary (i.d. 5.5mm) and human radial (i.d. 3.23mm) artery ring segments were mounted in organ baths for isometric force measurement. Single concentration-contraction curves to endothelin-1 were constructed in the absence or presence of bosentan (1–10µM), macitentan (0.03–0.3µM) or ambrisentan (0.1–1µM). ResultsAll 3 endothelin antagonists caused competitive rightward shifts in the endothelin-1 concentration-response curves in both arteries. The Clark plot and analysis gave the following pKB values: bosentan, pulmonary artery 6.28±0.13 and radial artery 6.04±0.10; macitentan, pulmonary artery 8.02±0.13 and radial artery 7.49±0.08; and ambrisentan, pulmonary artery 7.38±0.13 and radial artery 6.96±0.10. ConclusionsNoting the maximum plasma levels attained from recommended oral doses of each antagonist in volunteers, the pKB findings here show that there would be significant antagonism of endothelin-1 contraction in the pulmonary and radial arteries at therapeutic plasma levels. This functional assay confirms in human tissue that much higher plasma concentrations of endothelin-1 receptor antagonists are required to be effective than those predicted from binding or other biochemical assays.

Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion.

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

Contractile Changes in the Vasculature After Subchronic Smoking: A Comparison Between Wild Type and Surfactant Protein D Knock-Out Mice.

Cigarette smoking is a well-known risk factor for developing cardiovascular diseases, but the underlying mechanisms are largely unknown. Recent data suggest that vasocontractile receptor modulation could be an important factor. Surfactant protein D (SP-D) is important in the particle clearance in the lungs and knock-out (KO) mice for this protein develop emphysema. SP-D is also weakly expressed in the vasculature. We aimed to investigate whether SP-D was important in the cardiovascular response to cigarette smoke exposure (CSE), by utilizing SP-D KO mice and a myograph setup. Wild type (WT) and SP-D KO mice were exposed to cigarette smoke (CS) or room air for 12 weeks. The pulmonary artery, left anterior descending coronary artery, and basilar artery (BA) were isolated and mounted in wire myographs. Contractile concentration response curves to endothelin-1 and UDP were obtained. CSE caused a leftward shift in the concentration response curves for endothelin-1 in the BA for both WT and SP-D KO. UDP, acting on the purinergic P2Y6 receptor, caused reduced contraction in the left descending artery and increased contraction in the BA in the CSE WT mice. SP-D KO mice displayed no smoke induced changes, but were surprisingly similar to the CSE WT. The contractility to UDP was altered in the brain and heart vasculature of CSE mice. SP-D KO (both control and CSE) and CSE WT had similar changes in contractility compared to control WT. These results show that sub-chronic smoking induces vascular changes in the WT, mainly for the purinergic P2Y6 receptor together with minor changes for the endothelin-1 receptor. SP-D KO (both control and CSE) does not show any further changes compared to CSE WT.

Cerebrospinal fluid from patients with subarachnoid haemorrhage and vasospasm enhances endothelin contraction in rat cerebral arteries.

IntroductionPrevious studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ETA and ETB receptors.MethodsIntact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ETA and ETB receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.ResultsCompared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ETA receptors and new expression of ETB receptors was apparent; 5) reduction in the enhanced response to endothelin after ETB blockade in the low range and after ETA blockade in the high range of endothelin concentrations; 6) after combined ETA and ETB blockade a complete inhibition of endothelin contraction was observed.ConclusionsOur experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ETA and ETB receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.

Diabetes enhances the intrahepatic vascular response to endothelin-1 in cirrhotic rats: association with the ETA receptor and pERK up-regulation

Cirrhosis is characterized by increased intrahepatic vascular resistance and enhanced vasocontractile responsiveness that impedes portal inflow and elevates portal pressure, in which endothelin-1 (ET-1) plays a role. Diabetes and glucose influence vasoresponsiveness but their impact on the intrahepatic vascular bed in cirrhosis is unknown. To investigate intrahepatic ET-1 vasoresponsiveness in cirrhotic rats with and without diabetes and to explore the underlying mechanisms. Spraque-Dawley rats received common bile-duct ligation (BDL) to induce cirrhosis. Streptozotocin was injected to induce diabetes in the BDL rats (BDL/STZ). In situ liver perfusion was performed to obtain the ET-1 concentration-response curves. The basic hemodynamics and hepatic protein expressions of ET-1 receptors, pERK, ERK, pAkt, Akt, iNOS, eNOS, peNOS and calmodulin were evaluated. The circulating concentrations of N-terminal pro-brain natriuretic peptide (NT-ProBNP), blood urea nitrogen (BUN) and creatinine were also determined. Body weight, mean arterial pressure, heart rate and survival rate were significantly decreased in the BDL/STZ rats. The perfusion pressure changes in response to ET-1 were higher in the BDL/STZ group for all perfusates. ETA receptor and pERK expressions were enhanced in the BDL/STZ group. The circulating concentrations of NT-ProBNP, BUN and creatinine, as well as SMA flow, were not significantly different between the BDL and BDL/STZ groups. Cirrhotic rats with diabetes showed higher intrahepatic ET-1 vasoresponsiveness than normoglycemic cirrhotic rats. This effect is not affected by changes in perfused glucose concentration and may be related, at least in part, to intrahepatic ETA R receptor and pERK over-expression.

Ethanol Consumption Increases Endothelin-1 Expression and Reactivity in the Rat Cavernosal Smooth Muscle

We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.

Sphingosine-1-phosphate augments agonist-mediated contraction in the bronchial smooth muscles of mice

The effects of sphingosine-1-phosphate (S1P) on bronchial smooth muscle (BSM) contractility were investigated in naive mice. S1P had no effect on the basal tone of the isolated BSM tissues. However, in the presence of S1P(10-6 M), the BSM contractions induced by acetylcholine (ACh) and endothelin-1 (ET-1) were significantly augmented: both the ACh and ET-1 concentration-response curves were significantly shifted to the left. In contrast, the pretreatment with S1P had no effect on the contractions induced by high K+ depolarization. It is thus possible that S1P augments BSM contraction induced by the activation of G protein-coupled receptors.

Characteristics of myogenic reactivity in isolated rat mesenteric veins

Mechanisms of mechanically induced venous tone and its interaction with the endothelium and key vasoactive neurohormones are not well established. We investigated the contribution of the endothelium, l-type voltage-operated calcium channels (L-VOCCs), and PKC and Rho kinase to myogenic reactivity in mesenteric vessels exposed to increasing transmural pressure. The interaction of myogenic reactivity with norepinephrine (NE) and endothelin-1 (ET-1) was also investigated. Pressure myography was used to study isolated, cannulated, third-order rat mesenteric small veins and arteries. NE and ET-1 concentration response curves were constructed at low, intermediate, and high transmural pressures. Myogenic reactivity was not altered by nitric oxide synthase inhibition with N(ω)-nitro-L-arginine (L-NNA; 100 μM) or endothelium removal in both vessels. L-VOCCs blockade (nifedipine, 1 μM) completely abolished arterial tone, while only partially reducing venous tone. PKC (chelerythrine, 2.5 μM) and Rho kinase (Y27632, 3 μM) inhibitors largely abolished venous and arterial myogenic reactivity. There was no significant difference in the sensitivity of NE or ET-1-induced contractions within vessels. However, veins were more sensitive to NE and ET-1 when compared with corresponding arteries at low, intermediate, and high transmural pressures, respectively. These results suggest that 1) myogenic factors are important contributors to net venous tone in mesenteric veins; 2) PKC and Rho activation are important in myogenic reactivity in both vessels, while l-VOCCs play a limited role in the veins vs. the arteries, and the endothelium does not appear to modulate myogenic reactivity in either vessel type; and 3) mesenteric veins maintain an enhanced sensitivity to NE and ET-1 compared with the arteries when studied under conditions of changing transmural distending pressure.

Increased sympathetic venoconstriction and reactivity to norepinephrine in mesenteric veins in anesthetized DOCA-salt hypertensive rats

Increased sympathetic nervous activity (SNA) elevates venomotor tone in deoxycorticosterone acetate (DOCA)-salt hypertension. We studied the mechanisms by which the SNA increases venomotor tone in DOCA-salt hypertension by making in situ intracellular recordings of venous smooth muscle cell (VSMC) membrane potential (E(m)) and measurement of outside diameter (OD) in mesenteric veins (MV) and mesenteric arteries (MA) of anesthetized rats. We also studied norepinephrine (NE)- and endothelin-1 (ET-1)-induced increases in MA or MV perfusion pressure (PP) in vitro. E(m) in DOCA-salt MV was depolarized compared with sham MV. Prazosin hyperpolarized VSMC E(m) in DOCA-salt but not in sham MV. NE concentration-response curves (CRCs) for OD decreases in MV from DOCA-salt rats were left-shifted with an increased maximum response (E(max)) compared with sham MV. NE CRCs for OD decreases in MA were right-shifted with reduced E(max) in DOCA-salt compared with sham rats. ET-1 CRCs were similar in DOCA-salt and sham MV but were right-shifted with reduced E(max) in DOCA-salt MA. NE CRCs for MAPP increases were left-shifted without a change in E(max) in DOCA-salt rats. NE did not change MVPP. MAPP and MVPP for ET-1 CRCs were similar in sham and DOCA-salt rats, but E(max) for MAPP was reduced in DOCA-salt rats. Hematoxylin staining revealed hypertrophy in DOCA-salt MA but not in MV. We conclude that there is increased reactivity to NE released from the sympathetic nervous system in DOCA-salt MV that causes VSMC depolarization and increased venomotor tone. In DOCA-salt rats, in vivo ET-1 reactivity is maintained in MV, but reduced in MA.

Inotropic response of rabbit ventricular myocytes to endothelin-1: difference from isolated papillary muscles.

Endothelin-1 (ET-1) increased cell shortening and Ca2+ transients over the concentration of 3 x 10(-11) M to 10(-9) M with EC50 of 8.3 x 10(-11) M in rabbit single ventricular myocytes. Thus ET-1 was approximately 60 times more potent in single myocytes than in papillary muscles (EC50 = 5.1 x 10(-9) M) of the same species. In single myocytes, ET-1 at 10(-8) M elicited an inhibitory response that counteracted the facilitatory response: the concentration-response curve (CRC) for ET-1 was bell shaped. The ET(A)-receptor antagonist BQ-485 shifted CRC for ET-1 to the right in parallel; however, the facilitatory response to 10(-8) M ET-1 was markedly enhanced by BQ-485 and also by the ET(B) antagonist BQ-788. The ET(A)/ET(B) antagonist TAK-044 abolished the ET-1-induced response. These findings indicate that the response to ET-1 of single myocytes is different from that of papillary muscles in concentration dependence, characteristics of the response, and susceptibility to ET-receptor antagonists. Anomalous pharmacological characteristics of ET-1-induced response in rabbit papillary muscles may be due to integrated regulatory mechanisms that may involve also various types of noncardiac cell in ventricular myocardium.

Localization and function of ET-1 and ET receptors in small arteries post-myocardial infarction: upregulation of smooth muscle ET(B) receptors that modulate contraction.

Endothelin-1 (ET-1) has been implicated as a mediator of increased vascular tone during development of heart failure post-myocardial infarction (MI). In the present study, expression and pharmacology of ET-1 and its receptors were studied in small mesenteric arteries from rats at 5 and 12 weeks after coronary artery ligation for induction of MI, or sham-operation. In vessels from sham-operated and 5 week post-MI rats preproET-1mRNA, immunoreactive (ir) ET-1, ET(B) receptor mRNA and irET(B) receptor were confined to the endothelium, while ET(A) receptor mRNA was distributed throughout the media. At 12 weeks post-MI, preproET-1 and irET(A) receptor localization was similar but ET(B) receptor mRNA and immunoreactivity were detectable in the media, as well as the endothelium. The ET-1 concentration-response curve (CRC) was progressively shifted to the right in pressurized third generation mesenteric arteries from 5 and 12 week post-MI rats relative to sham-operated rats, with no change in the maximum. The ET(A) receptor antagonist BQ-123 (10(-6) M) induced a rightward shift of the ET-1 CRC in all vessels. Desensitization of ET(B) receptors, by exposure to SRTX S6c (3x10(-8) M), had no effect on the ET-1 CRC in vessels from 5 week post-MI or sham-op rats but induced a leftward shift in vessels from 12 week post-MI rats. These results identify the endothelium as the primary site of ET-1 synthesis in small arteries and the ET(A) receptor as mediating the effects of ET-1 in these vessels. However, ET(B) receptor expression increases in vascular smooth muscle post-MI and is linked to mechanisms that inhibit the contractile response to ET-1.

Novel nitric oxide donors reverse endothelin-1-mediated constriction in human blood vessels.

Cardiovascular disease is associated with elevated circulating plasma levels of endothelin-1 (ET-1). Our aim was to compare the ability of the nitric oxide donors (NO-donors) 3-morpholinylsydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) with the novel nitric oxide donors (NONOates) diethylamine NONOate (DEA/NO), and diethylenetriamine NONOate (DETA/NO) in order to physiologically antagonize ET-1-mediated constriction of human internal mammary arteries (IMA) in vitro. Both SNAP and DETA/NO caused a significant rightward shift in the ET-1 concentration-response curve. All four NO-donors were found to completely reverse an established contraction to a submaximal concentration ET-1 (decreasing order potency: SNAP >DEA/NO > SIN-1 > DETA/NO). These data suggest that the NONOates DEA/NO and DETA/NO can physiologically antagonize the effects of ET-1 in human arteries and may prove to be useful therapeutic agents in the treatment of cardiovascular disease.

Novel Nitric Oxide Donors Reverse Endothelin-1-Mediated Constriction in Human Blood Vessels

Summary: Cardiovascular disease is associated with elevated circulating plasma levels of endothelin-1 (ET-1). Our aim was to compare the ability of the nitric oxide donors (NO-donors) 3-morpholinylsydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) with the novel nitric oxide donors (NONOates) diethylamine NONOate (DEA/NO), and diethylenetriamine NONOate (DETA/NO) in order to physiologically antagonize ET-1-mediated constriction of human internal mammary arteries (IMA) in vitro. Both SNAP and DETA/NO caused a significant rightward shift in the ET-1 concentration-response curve. All four NO-donors were found to completely reverse an established contraction to a submaximal concentration of ET-1 (decreasing order of potency; SNAP > DEA/NO > SIN-1 > DETA/NO). These data suggest that the NONOates DEA/NO and DETA/NO can physiologically antagonize the effects of ET-1 in human arteries and may prove to be useful therapeutic agents in the treatment of cardiovascular disease.

Modulation of ET-1-induced contraction of human bronchi by airway epithelium-dependent nitric oxide release via ET(A) receptor activation.

1. The purpose of this work was to investigate whether endothelin-1 (ET-1) was able to induce the release of an inhibitory factor from the airway epithelium in isolated human bronchi and to identify this mediator as well as the endothelin receptor involved in this phenomenon. 2. In intact bronchi, ET-1 induced a concentration-dependent contraction (-logEC50 = 7.92+/-0.09, n = 18) which was potentiated by epithelium removal (-logEC50 = 8.65+/-0.11, n = 17). BQ-123 , an ET(A) receptor antagonist, induced a significant leftward shift of the ET-1 concentration-response curve (CRC). This leftward shift was abolished after epithelium removal. 3. L-NAME (3 x 10(-3) M), an inhibitor of nitric oxide (NO) synthase, induced a significant leftward shift of the ET-1 CRC, and abolished the potentiation by BQ-123 (10(-8) M) of ET-1-induced contraction. 4. In intact preparations, the ET(B) receptor antagonist BQ-788 induced only at 10(-5) M a slight rightward shift of the ET-1 CRC. In contrast, in epithelium-denuded bronchi or in intact preparations in the presence of L-NAME, BQ-788 displayed a non-competitive antagonism toward ET-1-induced contraction. 5. IRL 1620, a selective ET(B) receptor agonist, induced a contraction of the isolated bronchus (-logEC50=7.94+/-0.11, n= 19). This effect was not modified by epithelium removal or by BQ-123. BQ-788 exerted a competitive antagonism against IRL 1620 which was similar in the presence or absence of epithelium. 6. These results show that ET-1 exerts two opposite effects on the human airway smooth muscle. One is contractile via ETB-receptor activation, the other is inhibitory and responsible of NO release which counteracts via ETA-receptor activation the contraction.

Involvement of ET(A) and ET(B) receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes.

This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [32P]phosphatidylbutanol in [32P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET(B)-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET(A) receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2+/-3.5% of the ET-1 response) was defined at low (up to 1 microM) concentrations of BQ-123, yielding an estimated Ki value for BQ-123 of 21.3+/-2.5 nM. In addition, the presence of 1 microM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD2 value. Increasing concentrations of the ET(B) selective antagonist BQ-788 inhibited the S6c response with a Ki of 17.8+/-0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing Ki values of 20.9+/-5.1 nM and 439+/-110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 microM BQ-788, also revealing two components. Only one of them, corresponding to 69.8+/-4.4% of the response, was sensitive to BQ-788 which showed a Ki value of 28.8+/-8.9 nM. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET(A) and ET(B) receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET(A) and ET(B) receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET(B) receptors were not blocked, and 30-50% and 50-70%, respectively, when ET(B) receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

Endothelin-1 activates phospholipases and channels at similar concentrations in porcine coronary arteries.

Sensitivity of endothelin-1 (ET-1)-ion channel interactions has been proposed to exceed that of ET-1-phospholipase activation in vascular smooth muscle. We wanted to determine whether short-circuiting ion channels with staphylococcal alpha-toxin pores would shift the ET-1-force relation to the right as predicted from the above proposal. Medium size porcine coronary arteries (outer diameter 0.7-1.5 mm) were mounted on isometric force transducers. ET-1 concentration response curves were compared between intact rings and those subjected to alpha-toxin treatment with Ca buffered at 0.1 microM. The EC50 for treated rings (1.5 +/- 1.0 nM, n = 5 pigs) was similar to that for intact rings (1.9 +/- 0.4 nM). The Ca sensitivity of the alpha-toxin-treated rings (EC50 = 0.43 +/- 0.08 microM) was similar to that reported by other laboratories for intact and alpha-toxin-treated arteries and was shifted eightfold to the left by a high concentration of ET-1 (10 nM). Measurements of [32P]phosphatidic acid ([32P]PA) levels were used to evaluate phospholipase activity in intact arteries. The time courses for [32P]PA production and contraction were similar in response to high (100 nM) and to low (1 nM) ET-1. Significant increases in both steady-state contraction and [32P]PA occurred over a wide range of ET-1 concentrations tested (0.3-100 nM). Our findings support the concept that ET-1-phospholipase coupling is operative over the whole concentration range that induces contractile responses. It is suggested that both Ca entry and Ca sensitization processes are activated by ET-1 at low concentrations (<EC50) and that both processes contribute significantly to the integrated response.

Nitric oxide synthase inhibition reverses arteriolar hyporesponsiveness to endothelin-1 in septic rats.

Persistent vasodilation refractory to vasopressor agents is the hemodynamic abnormality characteristic of septic shock. Induction of nitric oxide synthase (NOS) by sepsis-induced cytokines has been hypothesized to play a pathogenetic role in this refractory vasodilation. To evaluate the mechanism of vasodilation in sepsis, we used in vivo videomicroscopy to measure responses of resistance arterioles (15-20 microm) to topical suffusion of the potent vasoconstrictor, endothelin-1 (ET-1), in rat cremaster muscle. Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. Responses to topically suffused ET-1 were assessed in septic and control rats before and after superfusion of the muscle with the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Sepsis produced a decrease in ET-1-induced vasoconstriction; the ET-1 concentration-response curve was shifted to the right in septic rats (P < 0.05). Contractions at ET-1 concentrations of 1, 10, and 100 nM were 20, 28, and 32%, respectively, of sham controls. Superfusion of the muscle with L-NMMA restored arteriolar responsiveness to ET-1 in the septic rats, significantly increasing arteriolar constriction at 1 and 10 nM. This effect was reversed with superfusion of excess L-arginine (1 mM). This study demonstrates that impaired vasoconstriction in response to ET-1 in resistance arterioles of septic rats in vivo is reversed by NOS inhibition. Taken together with previous studies showing sepsis-induced impairment of vasoconstriction with norepinephrine, a vasopressor with a mechanism of action different from ET-1, these findings suggest a generalized abnormality in the responsiveness of resistance arterioles in sepsis. Reversal of hyporesponsiveness to both of these vasopressor agents by NOS inhibition suggests an important role for nitric oxide as a mediator of refractory vasodilation in sepsis.

Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ETA and ETB receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ETA receptor antagonist, BQ-123; the ETB receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ETB receptor selective antagonist, BQ-788; and the ETA/ETB antagonist, TAK-044. 2. In 3rd generation mesenteric arteries, ET-1 (10(-13)-10(-7) M) produced concentration-dependent contractions (pD2 9.86). SRTX S6c (10(-12)-10(-7) M) also induced concentration-dependent contractions in 53% of arteries studied, although the Emax was much less than that obtained with ET-1 (10.7 +/- 2.9% vs 101.9 +/- 2.6% of the 60 mM KCl-induced contraction). 3. Neither ETB receptor desensitization, by a supra-maximal concentration of SRTX S6c (10(-7) M), nor incubation with BQ-788 (3 x 10(-8) M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1. 4. In the presence of BQ-123 (10(-6) M), responses to low concentrations of ET-1 (up to 10(-10) M) were unaffected but responses to concentrations of ET-1 above 10(-10) M were significantly inhibited. 5. SRTX S6c desensitization followed by incubation with BQ-123 (10(-6) M) or co-incubation with BQ-788 (3 x 10(-8) M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10(-8) M and 3 x 10(-7) M). 6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ETA and ETB receptors. The relative role of ETB receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ETB receptor desensitization or BQ-788. The effect of ETB receptor desensitization or blockade is only revealed in the presence of ETA receptor blockade, suggesting the presence of a 'crosstalk' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.