ETA Antagonist Research Articles

Synthesis and pharmacological activity of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acids as selective ETA antagonists

A series of 1,3,6-trisubsituted-4-oxo-1,4-dihyroquinoline-2-carboxylic acid analogs (2a–m) were designed and synthesized and their pharmacological activity determined, with the objective to better understand their SAR as potential ETA selective inhibitors. Most of the compounds displayed significant ETA antagonist activity having IC50 for inhibition of binding of the [125I]ET-1 to ETA receptor <10nM, with good selectivity for ETA antagonism over ETB receptor. Based on the in vitro results, SAR of this series of compounds requires an alkoxy substituent at the 6-position to be a straight and saturated chain up to three carbons long, since substitution of unsaturated and branched alkyloxy groups results in decrease in ETA antagonist activity. In this series, compound 2c (6-O-n-propyl analog) was found to be most potent (IC50=0.11nM) with ETB/ETA selectivity of 8303.

Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathway

Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. We measured relaxation of guinea pig internal anal sphincter strips caused by ET-related peptides and binding of (125)I-ET-1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. In the guinea pig internal anal sphincter, ET-1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or omega-conotoxin GVIA. ET-2 was as potent as ET-1. ET-3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET-1 = ET-2 > ET-3. The ET-1-induced relaxation was inhibited by BQ-123, an ET(A) antagonist, but not by BQ-788, an ET(B) antagonist. These indicate that ET(A) receptors mediate the relaxation. The relaxant response of ET-1 was attenuated by LY 83583, KT 5823, Rp-8CPT-cGMPS, tetraethyl ammonium, 4-aminopyridine and N(omega)-nitro-L-arginine, but not significantly affected by N(G)-nitro-L-arginine methyl ester, N(G)-methyl-L-arginine, charybdotoxin, apamin, KT 5720, and Rp-cAMPS. These suggest the involvement of cyclic guanosine 3',5'-cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized (125)I-ET-1 binding to the internal anal sphincter. Binding of (125)I-ET-1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ET(A) and ET(B) receptors. Taken together, these results demonstrate that ET(A) receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway.

Evidence for the use of combination targeted therapeutic approaches for the management of pulmonary arterial hypertension

The last decade has witnessed a remarkable increase in the number of effective treatment options available for the management of patients with pulmonary arterial hypertension. In this regard, agents belonging to the therapeutic classes that specifically target the prostacyclin, endothelin and nitric oxide pathways have shown the greatest efficacy in clinical studies to date. These various drug treatments have individually been shown to confer improvements in symptoms, exercise capacity, pulmonary haemodynamics and possibly survival in different patient subgroups. However, pulmonary arterial hypertension is characterised by dysregulation of a variety of pathways. In addition, disease worsening is inevitable for the majority of patients receiving monotherapy. As a consequence, there is increasing interest in the use of treatment combinations in order to target multiple targets with the aim of restoring normal pulmonary vascular function in order to improve clinical status. Indeed, use of multiple specific-treatment regimens is now part of routine clinical practice and is emphasized in recently published therapeutic guidelines. This review details the rationale for the different combination strategies and examines the clinical evidence in favour of some of the approaches that have been evaluated.

Ablation of Transient Receptor Potential Vanilloid 1 Abolishes Endothelin-Induced Increases in Afferent Renal Nerve Activity

Endothelin 1 (ET-1) and its receptors, ETA and ETB, play important roles in regulating renal function and blood pressure, and these components are expressed in sensory nerves. Activation of transient receptor potential vanilloid (TRPV) 1 channels expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity (ARNA), diuresis, and natriuresis. We tested the hypothesis that ET-1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in wild-type (WT) but not TRPV1-null mutant mice. ET-1 alone or with BQ123, an ETA antagonist, perfused into the left renal pelvis increased ipsilateral ARNA in WT but not in TRPV1-null mutant mice, and ARNA increases were greater in the latter. [Ala1, 3,11,15]-endothelin 1, an ETB agonist, increased ARNA that was greater than that induced by ET-1 in WT mice only. [Ala1, 3,11,15]-endothelin 1-induced increases in ARNA were abolished by chelerythrine, a protein kinase C inhibitor, but not by H89, a protein kinase A inhibitor. Chelerythrine, H89, and BQ788, an ETB antagonist, did not affect ARNA triggered by capsaicin in WT mice. Substance P release from the renal pelvis was increased by [Ala1, 3,11,15]-endothelin 1 in WT mice only, and the increase was abolished by chelerythrine but not by H89. Chelerythrine, H89, and BQ788 did not affect capsaicin-induced substance P release. Our data show that ET1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in WT but not in TRPV1-null mutant mice, suggesting that TRPV1 mediates ETB-dependent increases in ARNA, diuresis, and natriuresis possibly via the protein kinase C pathway.

Abstract 5655: Autoantibodies Against Endothelin-1 Receptors Are Involved in the Pathogenesis of Pulmonary Arterial Hypertension

Background In patients with pulmonary arterial hypertension (PAH) production of endothelin-1 (ET-1) is increased and elevated ET-1 plasma levels correlate with PAH severity. After repeated detection of agonistic antibodies against the ET-1 A receptor (ETA) in PAH patients’ sera we assessed their prevalence and properties. Methods Using spontaneously beating rat neonatal cardiomyocytes as bioassay we analyzed sera of PAH patients for the presence of autoantibodies (AABs) against ETA receptors. AABs were purified by affinity chromatography. Results At the time of AAB testing, the pulmonary vascular resistance (PVR) in the 58 evaluated PAH patients varied between 500 and 1920 dyn • sec • cm −5 (median 1088 dyn • sec • cm −5 ) and 45 (77.6%) were in functional NYHA/WHO class ≥ III. Of these 58 patients 44 (78.7%) tested positive for AABs against ETA receptors. ET-1 and the AABs against ETA exerted agonistic negative chronotropic effects on the neonatal cardiomyocytes (decrease in pulsation rate), which were blocked by the ETA antagonist BQ610, but not by the ET-1 B receptor blocker BQ788. The AABs induced permanent stimulation without desensitization of the receptor mediated signal cascade and the effect increases with higher AAB concentrations. First attempts to eliminate by immunoadsorption these AABs (which appeared to belong to the IgG2 subclass) in 5 PAH patients showed encouraging results. After AAB removal, which was well tolerated by all patients, at the first controls performed 3 and 6 weeks later all 5 patients showed reduction in right ventricular diameters, lower pulmonary arterial pressure, higher cardiac index and improved exercise tolerance (increase in both VO 2 max and 6 minute walk distance). In one patient the AABs reappeared after 2 months and this AAB reappearance was associated with clinical worsening and PVR increase. Conclusions The majority of patients with advanced PAH tested positive for functional serum AABs against ETA These AABs activate the receptors, like ET-1, but prevent the desensitization of the receptor-mediated signal cascade normally seen with ongoing receptor stimulation. Our results suggest that these AABs could be involved in PAH pathogenesis. AAB removal by immunoadsorption might be a possible new therapeutic approach.

Differential Endothelin Receptor Expression and Function in Rat Myometrial Cells and Leiomyoma ELT3 Cells

Uterine leiomyoma are the most common benign tumors of the myometrium. We previously identified endothelin (ET)-1 as a proliferative and antiapoptotic factor in Eker rat-derived leiomyoma (ELT3) cells. A major role of ETB receptor in the prosurvival effect was revealed. Here we investigated, in ELT3 and myometrial cells, the respective contribution of ETA and ETB in the proliferative effect of ET-1. In myometrial cells, binding experiments show that ETA is almost exclusively expressed and stimulates phospholipase C (PLC) activity and ERK1/2 phosphorylation and proliferation. In ELT3 cells, ETB is expressed at about the same level as ETA, and the two receptors are differently coupled to Gi protein. The ETB agonist, sarafotoxin S6c, stimulates PLC activity 60% less than ET-1 but is as potent as ET-1 to increase ERK1/2 phosphorylation and induce proliferation. However, the ability of ETA to activate ERK1/2 is observed after ETB desensitization. Although ETA and ETB antagonists partially reduce ET-1 stimulated PLC activity, they are without effect on ET-1-induced ERK1/2 phosphorylation and proliferation. Only the simultaneous use of ETA and ETB antagonists reduces ET-1-triggered ERK1/2 activation. These unconventional properties of ETRs may reveal the existence of functional ETA-ETB heterodimers. Finally, treatment of ELT3 cells with ETB but not ETA-directed small interfering RNA reduces the proliferative effect of ET-1. All the data obtained in ELT3 cells strengthen the relation between ETB overexpression, which decreases the ETA to ETB ratio, and the ability of leiomyoma cells to highly proliferate and resist apoptosis.

10th Annual Congress of the German Association of Clinical Pharmacology

10th Annual Congress of the German Association of Clinical Pharmacology

Evidence for interaction between endothelin receptors in equine airways

Endothelin‐1 (ET), an inflammatory mediator, causes potent contraction of airway smooth muscles leading to oxidative stress (OS) by binding to ETA and ETB receptors. We investigated the interaction between the ETA and ETB receptors in contractions and OS on healthy equine bronchial rings. Rings were prepared from right diaphragmatic lobe. Rings for response studies were set in the tissue chambers containing oxygenated Tyrode's solution at 37oC and 2‐gm tension was applied. Concentration‐response relationships were determined with graded concentrations of ET‐1 on control rings, rings incubated with ETA and ETB antagonists, separately and combined. For OS studies, each ring was incubated for 30 min separately with ET, ET+ETA antagonist, ET+ ETB antagonist and ET+ ETA+ETB antagonists. Total reactive oxygen species (ROS), superoxide production and peroxynitrate were determined using Brucker EMX ESR spectrophotometer. ET caused dose‐dependent contraction which was enhanced by ETA blockade and was decreased by ETB blockade. Combined blockade significantly inhibited the response by ET. ET‐induced total ROS was decreased in all treatments. Superoxide production was slightly decreased by antagonists individually but increased when combined. Peroxynitrate production was increased by ETA antagonist, but decreased in other treatments. The study suggested that an interaction between ET‐receptors exists both in contractile response and OS. Supported by USDA Formula Funds (# LA 1433) and EHSP of LSU‐SVM.

Augmented vascular reactivity induced by ET‐1 is associated with increased O‐GlcNAcylation

O-GlcNAcylation, the modification of proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc), modulates many functions including vascular reactivity. We hypothesized that ET-1-induced changes in vascular responses are associated with increased levels of O-GlcNAc-proteins. Incubation of rat aortas with ET-1 (0.1 ?M) produced a time-dependent increase in vascular O-GlcNAc levels (at 24h, arbitrary units = 2.2±0.1 vs. 1.0±0.05 control; n=4) and decreased expression of OGT and OGA, key enzymes in the O-GlcNAcylation process. Overnight incubation with ET-1 increased phenylephrine (PE) reactivity [Emax (mN) = 19±5 vs. 11±2 control; n=4]. The effects of ET-1 we not observed when vessels were previously transfected with antibodies against-OGT or after incubation with atrasentan (ETA antagonist, 1μM, Fig.1). Aorta and resistance arteries from rats chronically treated with low doses of ET-1 (2pmol/min/Kg, for 14 day) exhibit increased O-GlcNAc-proteins (Fig.1) and displayed changes in reactivity similar to those induced by PugNAc, an inhibitor of OGA. PugNAc treatment increased O-GlcNAc levels and enhanced PE responses. Our data show that ET-1 augments O-GlcNAc levels and that this modification contributes to the vascular changes induced by this peptide. We speculate that reactive oxygen species may provide a mechanism for the augmented vascular O-GlcNAc content induced by ET-1. Figure 1Open in figure viewerPowerPoint

Involvement of NO and MEK/ERK pathway in enhancement of endothelin-1-induced mesenteric artery contraction in later-stage type 2 diabetic Goto-Kakizaki rat

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. However, no abnormalities in the vascular responsiveness to ET-1 have been identified in the chronic stage of type 2 diabetes. Our goal was to look for abnormalities in the roles played by ET receptors (ET(A) and ET(B)) in the mesenteric artery of the type 2 diabetic Goto-Kakizaki (GK) rat and to identify the molecular mechanisms involved. Using mesenteric arteries from later-stage (32-38 wk old) individuals, we compared the ET-1-induced contraction and the relaxation induced by the selective ET(B) receptor agonist IRL1620 between GK rats and control Wistar rats. Mesenteric artery ERK activity and the protein expressions for ET receptors and MEK were also measured. In GK rats (vs. age-matched Wistar rats), we found as follows. 1) The ET-1-induced contraction was greater and was attenuated by BQ-123 (ET(A) antagonist) but not by BQ-788 (ET(B) antagonist). In the controls, BQ-788 augmented this contraction. 2) Both the relaxation and nitric oxide (NO) production induced by IRL1620 were reduced. 3) ET-1-induced contraction was enhanced by N(G)-nitro-l-arginine (l-NNA; NO synthase inhibitor) but suppressed by sodium nitroprusside (NO donor). 4) The enhanced ET-1-induced contraction was reduced by MEK/ERK pathway inhibitors (PD-98059 or U0126). 5) ET-1-stimulated ERK activation was increased, as were the ET(A) and MEK1/2 protein expressions. 6) Mesenteric ET-1 content was increased. These results suggest that upregulation of ET(A), a defect in ET(B)-mediated NO signaling, and activation of the MEK/ERK pathway together represent a likely mechanism mediating the hyperreactivity to ET-1 examined in this study.

Endothelial endothelin-1 over-expression using receptor tyrosine kinase tie-1 promoter leads to more severe vascular permeability and blood brain barrier breakdown after transient middle cerebral artery occlusion

Endothelin-1 (ET-1) is up-regulated in the endothelial cells and astrocytes under ischemia. Transgenic mice with astrocytic ET-1 over-expression (GET-1) showed more severe neurological deficit and larger infarct after transient middle cerebral artery occlusion (MCAO). Here, the significance of endothelial ET-1 in ischemic brain injury was investigated using transgenic mice with the endothelial ET-1 over-expression (TET-1). Increased ET-1 level was observed in the TET-1 brain infarct core after transient MCAO. ETA receptor expression was induced in the penumbra and ETA antagonist (A-147627) partially normalized the infarct volume and neurological deficit. In the infarct core of TET-1 brain, superoxide, nitrotyrosine, and gp91phox levels were increased. TET-1 brain displayed increased matrix metalloproteinase-2 expression, water content, immunoglobulin leakage and decreased occludin level in the ipsilateral hemisphere indicative of BBB breakdown and hemispheric edema. Interestingly, AQP-4 expression was increased in the penumbra of TET-1 brain following transient MCAO leading to the water accumulation. Taken together, endothelial ET-1 over-expression and ETA receptor activation contributes to the increased oxidative stress, water accumulation and BBB breakdown after transient MCAO leading to more severe neurological deficit and increased infarct.

Drug Discovery for Overcoming Chronic Kidney Disease (CKD): The Endothelin ETB Receptor / Nitric Oxide System Functions as a Protective Factor in CKD

Accelerated cardiovascular disease (CVD) is a frequent complication of renal disease. Chronic kidney disease (CKD) develops hypertension and dyslipidemia, which in turn can contribute to the progression of renal failure. There is general agreement that endothelin-1 (ET-1), which acts through the two subtypes of receptor ETA and ETB, plays important physiological roles in the regulation of normal cardiovascular function and that excessive ET-1 production is linked to CVD and CKD. Although selective ETA or nonselective ETA/ETB receptor antagonisms have been recognized as a potential strategy for treatment of several cardiovascular disease, it remains unclear which of the antagonisms is suitable for the individuals with CKD because upregulation of the nitric oxide (NO) system via ETB receptor is responsible for renal function such as natriuresis, diuresis, and glomerular hemodynamics. Our findings clearly indicate that the blockade of ET receptors, in particular ETA-receptor antagonism, not only produces a potential renoprotective effect in CKD but also reduces the risk of CVD. In contrast, pharmacological blockade or genetic deficiency of ETB receptor seems to aggravate CKD and CVD in several experimental models of rats. Moreover, preliminary evidence in patients with CKD also suggests that both selective ETA- and nonselective ETA/ETB-receptor blockade decreases blood pressure but that selective ETA blockade has additional desirable effects on renal hemodynamics. Thus, at least in CKD, these findings support the notion that ETB receptor– mediated actions produce a renoprotective effect and that nonselective ETA/ETB-receptors blockade seem to offer no advantage over selective ETA antagonism, and if anything may potentially reduce the benefits.

Chronic ethanol intake modulates vascular levels of endothelin‐1 receptor and enhances the pressor response to endothelin‐1 in anaesthetized rats

The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg(-1), i.v.) or to the selective ETB agonist, IRL1620 (0.001-1.0 nmol kg(-1), i.v.), with or without ETA or ETB antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg(-1) and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methylleucyl1-D-1methoxycarbonyltryptophanyl-D-norleucine) at 0.25 mg kg(-1), respectively) were tested in anaesthetized rats, after 2 weeks' chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg(-1) reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg(-1)) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg(-1) were enhanced following ETB receptor blockade. Finally, we observed higher levels of ETA receptor in the heart and mesenteric artery and a reduction of ETB receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. Increased vascular reactivity to ET-1 and altered protein levels of ETA and ETB receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.

DOCA-salt treatment enhances responses to endothelin-1 in murine corpus cavernosumThis article is one of a selection of papers published in the special issue (part 1 of 2) on Forefronts in Endothelin.

The penis is kept in the flaccid state mainly via a tonic activity of norepinephrine and endothelins (ETs). ET-1 is important in salt-sensitive forms of hypertension. We hypothesized that cavernosal responses to ET-1 are enhanced in deoxycorticosterone acetate (DOCA)-salt mice and that blockade of ETA receptors prevents abnormal responses of the corpus cavernosum in DOCA-salt hypertension. Male C57BL/6 mice were unilaterally nephrectomized and treated for 5 weeks with both DOCA and water containing 1% NaCl and 0.2% KCl. Control mice were uninephrectomized and received tap water with no added salt. Animals received either the ETA antagonist atrasentan (5 mg x day(-1) x kg(-1) body weight) or vehicle. DOCA-salt mice displayed increased systolic blood pressure (SBP), and treatment with atrasentan decreased SBP in DOCA-salt mice. Contractile responses in cavernosal strips from DOCA-salt mice were enhanced by ET-1, phenylephrine, and electrical field stimulation (EFS) of adrenergic nerves, whereas relaxations were not altered by IRL-1620 (an ETB agonist), acetylcholine, sodium nitroprusside, and EFS of nonadrenergic noncholinergic nerves. PD59089 (an ERK1/2 inhibitor), but not Y-27632 (a Rho-kinase inhibitor), abolished enhanced contractions to ET-1 in cavernosum from DOCA-salt mice. Treatment of DOCA-salt mice with atrasentan did not normalize cavernosal responses. In summary, DOCA-salt treatment in mice enhances cavernosal reactivity to contractile, but not to relaxant, stimuli, via ET-1/ETA receptor-independent mechanisms.

Effect of chronic and selective endothelin receptor antagonism on microvascular function in Type 2 diabetes

Vascular dysfunction, which presents either as an increased response to vasoconstrictors or an impaired relaxation to dilator agents, results in worsened cardiovascular outcomes in diabetes. We have established that the mesenteric circulation in Type 2 diabetes is hyperreactive to the potent vasoconstrictor endothelin-1 (ET-1) and displays increased nitric oxide-dependent vasodilation. The current study examined the individual and/or the relative roles of the ET receptors governing vascular function in the Goto-Kakizaki rat, a mildly hyperglycemic, normotensive, and nonobese model of Type 2 diabetes. Diabetic and control rats received an antagonist to either the ET type A (ETA; atrasentan; 5 mg x kg(-1) x day(-1)) or type B (ET(B); A-192621; 15 or 30 mg x kg(-1) x day(-1)) receptors for 4 wk. Third-order mesenteric arteries were isolated, and vascular function was assessed with a wire myograph. Maximum response to ET-1 was increased in diabetes and attenuated by ETA antagonism. ETB blockade with 15 mg/kg A-192621 augmented vasoconstriction in controls, whereas it had no further effect on ET-1 hyperreactivity in diabetes. The higher dose of A-192621 showed an ETA-like effect and decreased vasoconstriction in diabetes. Maximum relaxation to acetylcholine (ACh) was similar across groups and treatments. ETB antagonism at either dose had no effect on vasorelaxation in control rats, whereas in diabetes the dose-response curve to ACh was shifted to the right, indicating a decreased relaxation at 15 mg/kg A-192621. These results suggest that ETA receptor blockade attenuates vascular dysfunction and that ETB receptor antagonism exhibits differential effects depending on the dose of the antagonists and the disease state.

Abstract 2301: Agonistic Autoantibodies Against the Endothelin 1 ETA - and α1-Adrenergic- Receptor in the Sera of Patients with Idiopathic Pulmonary Arterial Hypertension.

Background: Idiopathic pulmonary arterial hypertension (IPAH) is a progressive fatal disease of unknown cause. This disease is accompanied by an massive increase of pulmonary vascular resistance (remodelling and increase of vascular tonus) inducing right heart failure. It was shown that the synthesis of vasoconstrictors (endothelin 1, thromboxane A2, serotonine) is elevated in patients with IPAH. Methods: We analyzed sera of patients with PH with regards to functional autoantibodies (AABs) against G-protein coupled receptors using spontaneously beating rat cardiomyocytes as bioassay. Moreover, we investigated the effect of the AAB on the translocation of the transcription factors NFκB and AP-1. Results: The sera of patients with IPAH contain functional AABs against the α1-adrenergic (α1-AR) and the endothelin1 ETA receptor. We purified the AABs by affinity chromatography. AABs against the α1-AR exert a positive chronotropic effect. AABs against the ETA receptor induce like endothelin 1 a negative chronotropic effect. The agonistic effect of the AABs was dose-dependent and blocked by prazosine (α1-AR blocker) and BQ610 (ETA antagonist), respectively. Moreover, the agonistic effect of the AABs was neutralized by peptides corresponding to the second extracellular loop of both receptors. Therefore, the epitopes of the AABs are localized on the second extracellular loops of the receptors. The α1-AR AABs as well as the ETA-receptor AABs induce a permanent stimulation without desensitization of the receptor mediated signal cascade. The α1-adrenergic agonist phenylephrine and endothelin1 as ET receptor agonist cause a translocation of the transcription factor NFκB and AP-1 from the cytosol into the nucleus in cultured neonatal rat cardiomyocytes. The same translocation was observed using the AABs against the α1-AR and ETA receptor. Conclusion: The agonist-like AABs against the α1-AR and the ETA receptor influence in vitro the signalling of cultured cells. Moreover, the AABs prevent the desensitization of the receptor mediated signal cascade normally seen by ongoing receptor stimulation. Therefore, we assumed that the functional AABs against the α1-AR and the ETA receptor may be involved in the pathogenesis of IPAH.

IDENTIFICATION AND CHARACTERIZATION OF A DYSFUNCTIONAL CARDIAC MYOCYTE PHENOTYPE: ROLE OF BACTERIA, TOLL-LIKE RECEPTORS, AND ENDOTHELIN

Cardiac myocyte dysfunction is clearly identified as underlying the acute heart failure associated with bacterial infection, as well as the chronic syndrome following cardiac damage, but the mechanisms leading to dysfunction in each case are not fully established. It is thought that local hormones such as endothelin 1 (ET-1) can increase the risk of heart failure in acute or chronic conditions. In the current study, we characterize myocytes as populations and identify a novel phenotype of the ventricular cardiac myocyte that does not contract appropriately on electrical stimulation. The noncontractile cardiac myocytes were viable and had normal calcium transients. The proportion of noncontractile cardiac myocytes was increased by bacteria (gram-positive Staphylococcus aureus or gram-negative Escherichia coli). Using selective ligands or myocytes from genetically modified mice, we established that the effects of S. aureus were mediated by Toll-like receptor 2/6 and of E. coli by Toll-like receptor 4. The transition to the noncontractile phenotype was strongly inhibited by ETA antagonism but unaffected by inhibition of NOS, suggesting that ET-1 and not NO mediates this phenomenon. These results are the first to describe the characteristics of this noncontractile phenotype and the mechanisms of its induction by bacteria. Description of the myocyte population, instead of effects only on individual cells, will be more relevant to the prediction of the depression of cardiac function.

Evidence for Vasculoprotective Effects of ETB Receptors in Resistance Artery Remodeling in Diabetes

Vascular remodeling, characterized by extracellular matrix deposition and increased media-to-lumen (M/l) ratio, contributes to the development of microvascular complications in diabetes. Matrix metalloproteinases (MMPs) play an important role in the regulation of extracellular matrix (ECM) turnover and vascular remodeling. Vasoactive factor endothelin (ET)-1 not only causes potent vasoconstriction but also exerts profibrotic and proliferative effects that change vessel architecture, which makes it a likely candidate for a key role in vascular complications of diabetes. Thus, this study investigated the regulation of MMP activity of resistance arteries under mild-to-moderate diabetes conditions, as seen in type 2 diabetes, and the relative role of ET receptors in this process. Vessel structure, MMP activity, and ECM proteins were assessed in control Wistar and diabetic Goto-Kakizaki (GK) rats treated with vehicle, ET(A) receptor antagonist atrasentan (5 mg x kg(-1) x day(-1)), or ET(B) receptor antagonist A-192621 (15 mg x kg(-1) x day(-1)) for 4 weeks. M/l ratio was increased in diabetes. Atrasentan prevented this increase, whereas A-192621 caused further thickening of the medial layer. Increased MMP-2 activity in diabetes was prevented by atrasentan treatment. Collagenase activity was significantly decreased in diabetes, and while ET(A) antagonism improved enzyme activity, ET(B) blockade further reduced collagenase levels. Accordingly, collagen deposition was augmented in GK rats, which was reversed by atrasentan but exacerbated with A-192621. ET-1 contributes to the remodeling of mesenteric resistance arteries in diabetes via activation of ET(A) receptors, and ET(B) receptors provide vasculoprotective effects.

(625): Role of peripheral endothelin receptors in an animal model of CRPS-type I

Chronic post-ischemic pain (CPIP) is an animal model of CRPS-I developed using an ischemia-reperfusion injury of the rodent hindpaw. CPIP mice exhibit robust mechanical and cold allodynia for at least four weeks. Presently, the contribution of endothelin to nociception has been evaluated in CPIP mice. CPIP is induced by placing a tourniquet (tight fitting O-ring) on one hindlimb of an anesthetized mouse just proximal to the ankle joint for 3 h, with reperfusion prior to termination of anesthesia. We assessed sustained nociceptive behaviors (SNBs) exhibited following intraplantar (i.pl.) injection of 200 pmol of endothelin-1, -2 or -3 (ET-1, ET-2, ET-3) in day 2 CPIP mice. The effects of i.pl. ET-2 were also compared between mice with CPIP (+2 or +7 days), neuropathic (CCI +14 days and SNI +7 days) or inflammatory (CFA +1 day) pain. The effects of an ETA antagonist (BQ-123, 10 nmol, i.pl.) or an ETB antagonist (BQ-788, 30 nmol, i.pl.) or agonist (IRL-1620, 50 pmol, i.pl.) were also assessed on mechanical and cold allodynia and ET-2-induced SNBs in CPIP mice. Compared to sham, CPIP mice exhibited prolonged SNBs after injection of ET-2, but not ET-1 or ET-3. However, ET-2 did not produce significant SNBs in mice with neuropathic or inflammatory pain. The ETB agonist, but not ETA or ETB antagonists, reduced both ET-2-induced SNBs and mechanical allodynia in CPIP mice. In conclusion, CPIP mice appear to have increased nociceptive responses to ET-2, yet also are relieved of both mechanical allodynia and ET-2-induced nociception by treatment with an ETB agonist. CPIP mice may have an upregulation of ETB receptors, which produce antinociceptive effects, yet also exhibit an unexplained nociceptive response to ET-2. Further study of the differential involvement of the potential ETB subtypes in these two distinct observations is needed.

Postnatal changes in pulmonary vein responses to endothelin-1 in the normal and chronically hypoxic lung

The response of pulmonary arteries to endothelin-1 (ET-1) changes with age in normal pigs and is abnormal in pulmonary hypertension. The purpose of this study was to determine if the same is true of the pulmonary veins. We studied the wall structure and functional response to ET-1 in pulmonary veins from normal pigs from fetal life to adulthood and from pigs subjected to chronic hypobaric hypoxia either from birth for 3 days or from 3 to 6 days of age. In isolated normal veins, the contractile response decreased by 40% between late fetal life and 14 days of age with a concomitant twofold increase in endothelium-dependent relaxant response. The ET(A) antagonist BQ-123 reduced the contractile response significantly more in newborn than older animals, whereas the ET-B antagonist BQ-788 had no effect in fetal animals and maximally increased contraction at 14 days of age. Hypoxic exposure significantly increased pulmonary vein smooth muscle area and contractile response to ET-1. The relaxation response was impaired following hypoxic exposure from birth but not from 3 to 6 days of age. The ET(A) antagonist BQ-123 decreased contractile and increased dilator responses significantly more than in age-matched controls. Thus pulmonary veins show age-related changes similar to those seen in the pulmonary arteries with a decrease in ET(A)-mediated contractile and increase in ET-B-mediated relaxant response with age. Contractile response was also increased in hypoxia as in the arteries. This study suggests that pulmonary veins are involved in postnatal adaptation and the pathogenesis of pulmonary hypertension.