Pericyte abnormalities precede strial capillary basement membrane thickening in Alport mice.

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Sustained, delayed, and small increments in glomerular permeability to macromolecules during systemic ET-1 infusion mediated via the ETA receptor.

Emerging evidence indicates that endogenous production of endothelin (ET)-1, a 21-amino acid peptide vasoconstrictor, plays an important role in proteinuric kidney disease. Previous studies in rats have shown that chronic administration of ET-1 leads to increased glomerular albumin leakage. The underlying mechanisms are, however, currently not known. Here, we used size-exclusion chromatography to measure glomerular sieving coefficients for neutral FITC-Ficoll (molecular Stokes-Einstein radius: 15-80 Å, molecular weight: 70 kDa/400 kDa) in anesthetized male Sprague-Dawley rats (n = 12) at baseline and at 5, 15, 30, and 60 min after intravenous administration of ET-1. In separate experiments, ET-1 was given together with the selective ET type A (ETA) or ET type B (ETB) receptor antagonists JKC-301 and BQ-788, respectively. At both 15 and 30 min postadministration, the glomerular sieving coefficient for macromolecular Ficoll (70 Å) was significantly increased to 4.4 × 10-5 ± 0.7 × 10-5 (P = 0.024) and 4.5 × 10-5 ± 0.8 × 10-5 (P = 0.007), respectively, compared with baseline (2.2 × 10-5 ± 0.4 ×10-5). Decreased urine production after ET-1 prevented the use of higher doses of ET-1. Data analysis using the two-pore model indicated changes in large-pore permeability after ET-1, with no changes in the small-pore pathway. Administration of ETA blocker abrogated the permeability changes induced by ET-1 at 30 min, whereas blockade of ETB receptors was ineffective. Mean arterial pressure was only significantly increased at 60 min, being 123 ± 4 mmHg compared with 111 ± 2 mmHg at baseline (P = 0.02). We conclude that ET-1 evoked small, delayed, and sustained increases in glomerular permeability, mediated via the ETA receptor.

Afferent arteriole responsiveness to endothelin receptor activation: does sex matter?

BACKGROUND: The pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1. METHODS: Male and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10-12 to 10-8 M) and sarafotoxin 6c (S6c, ETB agonist, 10-12 to 10-8 M) using the blood-perfused juxtamedullary nephron preparation. RESULTS: Control afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 µm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10-8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10-8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10-8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10-12 to 10-9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation. CONCLUSION: These data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

Role of endothelin-1 and its receptors, ETA and ETB, in the survival of human vascular endothelial cells.

Our previous work showed the presence of endothelin-1 (ET-1) receptors, ETA and ETB, in human vascular endothelial cells (hVECs). In this study, we wanted to verify whether ET-1 plays a role in the survival of hVECs via the activation of its receptors ETA and (or) ETB (ETAR and ETBR, respectively). Our results showed that treatment of hVECs with ET-1 prevented apoptosis induced by genistein, an effect that was mimicked by treatment with ETBR-specific agonist IRL1620. Furthermore, blockade of ETBR with the selective ETBR antagonist A-192621 prevented the anti-apoptotic effect of ET-1 in hVECs. However, activation of ETA receptor alone did not seem to contribute to the anti-apoptotic effect of ET-1. In addition, the anti-apoptotic effect of ETBR was found to be associated with caspase 3 inhibition and does not depend on the density of this type of receptor. In conclusion, our results showed that ET-1 possesses an anti-apoptotic effect in hVECs and that this effect is mediated, to a great extent, via the activation of ETBR. This study revealed a new role for ETBR in the survival of hVECs.

The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive Myocardial Hypertrophy.

Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 (ET-1) system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1 , ETB2 , and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET-1 on cardiomyocytes. Indeed, ET-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET-1-dependent reduction in cell shortening and attenuated up-regulation of CaR. Down-regulation of RAMP-1 reduced CaR responsiveness. In conclusion, ET-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508-2518, 2017. © 2016 Wiley Periodicals, Inc.

TNFα-induced airway smooth muscle cell proliferation depends on endothelin receptor signaling, GM-CSF and IL-6.

UNLABELLED: Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes. HYPOTHESIS: endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.

Therapeutic Modulation of Aortic Stiffness.

Aortic stiffness (AS) is an important predictor of cardiovascular morbidity in humans. The present review discusses the possible pathophysiological mechanisms of AS and focuses on a survey of different therapeutic modalities for decreasing AS. The influence of several nonpharmacological interventions is described: decrease body weight, diet, aerobic exercise training, music, and continuous positive airway pressure therapy. The effects of different pharmacological drug classes on AS are also discussed: antihypertensive drugs-renin-angiotensin-aldosterone system drugs, beta-blockers, alpha-blockers, diuretics, and calcium channel blockers (CCBs)-advanced glycation end product cross-link breakers, statins, oral anti-diabetics, anti-inflammatory drugs, vitamin D, antioxidant vitamins, and endothelin-1 receptor antagonists. All of these have shown some effect in decreasing AS.

Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease. EXPERIMENTAL APPROACH: Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice. KEY RESULTS: In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. CONCLUSIONS AND IMPLICATIONS: These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Endothelin as a local regulating factor in the bovine oviduct.

Endothelin (EDN) is a possible regulating factor of oviductal motility, which is important for the transport of gametes and embryo. To clarify the factors that control the secretion of EDN in the bovine oviduct, the expression of EDNs, EDN-converting enzymes (ECEs) and EDN receptors (EDNRs) were investigated. All isoforms of EDN (EDN1-3), ECE (ECE1 and ECE2) and EDNR (EDNRA and EDNRB) were immunolocalised in the epithelial cells of the ampulla and the isthmus. EDNRs were also immunolocalised in smooth-muscle cells. The mRNA expression of EDN2 and ECE2 was higher in cultured ampullary oviductal epithelial cells than in isthmic cells. The expression of EDN1, EDN2 and ECE2 in the ampullary tissue was highest on the day of ovulation. Oestradiol-17ß increased EDN2 and ECE1 expression, while progesterone increased only ECE1 expression in cultured ampullary epithelial cells. These results indicate that EDNs are produced by epithelial cells and their target site is smooth-muscle and epithelial cells, and suggest that ovarian steroids are regulators of endothelin synthesis in ampullary oviductal epithelial cells.

Endothelin-1 and Endothelin-3 Regulate Endothelin Receptor Expression in Rat Coronary Arteries.

In ischaemic hearts, endothelin (ET) levels are increased, and vasoconstrictor responses to ET-1 are greatly enhanced. We previously reported that ETB receptors are up-regulated in the smooth muscle layer of coronary arteries after myocardial ischaemia-reperfusion and that the MEK-ERK1/2 signalling pathway is involved in ETB receptor up-regulation. Whether ETs are directly involved in receptor regulation has not been determined. We suggest that ET-1 and ET-3 alter the expression/activity of ET receptors in coronary vascular smooth muscle cells. Vasoconstrictor responses were studied in endothelium-denuded coronary artery segments from rats that were subjected to experimental ischaemia-reperfusion or in organ-cultured segments. Post-ischaemic and cultured coronary arteries exhibited similar increased sensitivity to ET-3. ETA receptor-mediated vasoconstriction was dominant in fresh and non-ischaemic arteries. Organ culture significantly up-regulated ETB receptors and down-regulated ETA receptor expression. Co-incubation with ET-1 (1 nM) or ET-3 (100 nM) induced further down-regulation of the ETA receptor mRNA, while the function and protein level of ETA remained unchanged. ET-3 (100 nM) further up-regulated ETB receptor mRNA and proteins but abolished ETB receptor-mediated vasoconstriction, suggesting a desensitization of ETB receptors that was not observed with ET-3 (1 nM). In conclusion, ET-1, which is the most prevalent isoform in the cardiovascular system, induces down-regulation of ETA receptor expression without changing ETA or ETB receptor function or protein levels. Intermediate concentrations of ET-3 had an effect that was similar to that of ET-1, such that high concentrations of ET-3 (100 nM) up-regulated the ETB receptor at the gene and protein levels but switched off the function of the ETB receptors via desensitization.

Activation of either the ETA or the ETB receptors is involved in the development of electrographic seizures following intrahippocampal infusion of the endothelin-1 in immature rats.

The period around birth is a risky time for stroke in infants, which is associated with two major acute and subacute processes: anatomical damage and seizures. It is unclear as to what extent each of these processes independently contributes to poor outcome. Furthermore, it is unclear whether there is an interaction between the two processes - does seizure activity cause additional brain damage beyond that produced by ischemia and/or does brain damage foster seizures? The model of focal cerebral ischemia induced by the intrahippocampal infusion of endothelin-1 (ET-1) in 12-day-old rat was used to examine the role of the endothelin receptors in the development of focal ischemia, symptomatic acute seizures and neurodegeneration. ET-1 (40pmol/µl) was infused either alone or co-administered with selective antagonists of ETA (BQ123; 70nmol/µl) or ETB receptors (BQ788; 70nmol/1µl). Effects of activation of ETB receptors were studied using selective agonist 4-Ala-ET-1 (40pmol/1µl). Regional cerebral blood flow (rCBF) and tissue oxygenation (pO2) were measured in anesthetized animals with a Doppler-flowmeter and a pO2-sensor, respectively. Seizure development was assessed with video-EEG in freely moving rats. Controls received the corresponding volume of the appropriate vehicle (10mM PBS or 0.01% DMSO-PBS solution; pH7.4). The extent of hippocampal lesion was determined using FluoroJade B staining performed 24h after ET-1 infusion. Infusion of ET-1 or ET-1+ETB receptor antagonist reduced rCBF to ~25% and pO2 to ~10% for about 1.5h, whereas selective ETB agonist, ET-1+ETA antagonist and the PBS vehicle had only negligible effect on the rCBF and pO2 levels. Reduction of rCBF was associated with the development of lesion in the injected hippocampus. In all groups, except sham operated and PBS controls, epileptiform activity was observed after activation of the ETA or the ETB receptors. The data revealed a positive correlation between the severity of morphological damage and all the measured seizure parameters (seizure frequency, average and total seizure duration) in the ET-1 group. In addition, the severity of morphological damage positively correlated with the average seizure duration in animals after infusion of ET-1+ETA receptor antagonist or after infusion of ET-1+ETB receptor antagonist. Our results indicate that the activation of ETA receptors is crucial for ischemia development, however either ETA or ETB receptors mediate the development of seizures following the application of ET-1 in immature rats. The dissociation between the ischemic-producing and seizure-producing processes suggests that damage is not necessary to induce seizures, although it may exacerbate them.

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

Interplay between endothelin and erythropoietin in astroglia: the role in protection against hypoxia.

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48-72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca²âº]i in mesenteric microvessels of pregnant rats.

BACKGROUND AND PURPOSE: Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ET(A)R), but could activate ET(B)R in the endothelium and release vasodilator substances. However, the roles of ET(B)R in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear. EXPERIMENTAL APPROACH: Pressurized mesenteric microvessels from pregnant and virgin Sprague-Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca²âº]i. KEY RESULTS: High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [Ca²âº]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca²âº]i. Pretreatment with the ET(B)R antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [Ca²âº]i, and was more potent in pregnant than in virgin rats. ET-1 + ET(A)R antagonist BQ-123, and the ET(B)R agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [Ca²âº]i, suggesting up-regulated ET(B)R-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ET(B)R in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ET(B)R. CONCLUSIONS AND IMPLICATIONS: The enhanced ET(B)R-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.

p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to G(q)-protein-coupled receptor stimuli, endothelin-1 and α(1)-adrenergic receptor agonists.

ERK1/2 (extracellular-signal-regulated kinase 1/2) and their substrates RSKs (p90 ribosomal S6 kinases) phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. In the present study we investigated the role of RSKs in the transcriptomic responses to the G(q)-protein-coupled receptor agonists endothelin-1, phenylephrine (a generic α(1)-adrenergic receptor agonist) and A61603 (α(1A)-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>A61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased the nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of the 213 RNAs up-regulated after 1 h, 51% required RSKs for their up-regulation, whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical with, endothelin-1. As with endothelin-1, PD184352 inhibited the up-regulation of most phenylephrine-responsive transcripts, but the greater variation in the effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α(1A)-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, up-regulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to G(q)-protein-coupled receptor stimulation.

Comparison of human ETA and ETB receptor signalling via G-protein and ß-arrestin pathways.

AIMS: To determine the pharmacology of ET(A)- and ET(B)-mediated ß-arrestin recruitment and compare this to established human pharmacology of these receptors to identify evidence for endothelin receptor biased signalling and pathway specific blockade by antagonists. MAIN METHODS: The ability of ET-1, ET-2, ET-3, sarafotoxin 6b and sarafotoxin 6c to activate ET(A) and ET(B)-mediated ß-arrestin recruitment was determined in CHO-K1 cells. Affinities were obtained for ET(A) selective (BQ123, sitaxentan, ambrisentan), ET(B) selective (BQ788) and mixed (bosentan) antagonists using ET-1 and compared to affinities obtained in competition experiments in human heart and by Schild analysis in human saphenous vein. Agonist dependence of affinities was compared for BQ123 and BQ788 in the ET(A) and ET(B) ß-arrestin assays respectively. KEY FINDINGS: For ß-arrestin recruitment, order of potency was as expected for the ET(A) (ET-1≥ET-2>>ET-3) and ET(B) (ET-1=ET-2=ET-3) receptors. However, at the ET(A) receptor sarafotoxin 6b and ET-3 were partial agonists. Antagonism of ET peptides by selective and mixed antagonists appeared non-competitive. BQ123, but not BQ788, exhibited agonist-dependent affinities. Bosentan was significantly more effective an inhibitor of ß-arrestin recruitment mediated by ET(A) compared to the ET(B) receptor. In the ET(A) vasoconstrictor assay, ET-1, ET-2 and S6b were equipotent, full agonists and antagonists tested behaved in a competitive manner, although affinities were lower than predicted from the competition binding experiments in left ventricle. SIGNIFICANCE: These data suggest that the pharmacology of ET(A) and ET(B) receptors linked to G-protein- and ß-arrestin mediated responses was different and bosentan appeared to show bias, preferentially blocking ET(A) mediated ß-arrestin recruitment.

Endothelin-1 induces itch and pain in the mouse cheek model.

AIMS: To date, suggestions that endothelin-1 (ET-1) causes nociception and pruritus are based on results in preclinical models in which responses to pruritic and nociceptive stimuli cannot be distinguished. This study reexamines these sensory effects of ET-1 in the new mouse cheek model, in which pruritogens and algogens evoke distinct behavioral responses. MAIN METHODS: Mice received intradermal (i.d.) injections of test substances into the left cheek and bouts of hind limb scratches or forepaw wipes, directed to the injection site, were considered indicative of pruritus and nociception, respectively. KEY FINDINGS: Histamine and capsaicin selectively evoked scratching and wipes, respectively, whereas ET-1 (3-60 pmol) promoted dose-dependent bouts of both behaviors. While scratching and wipe responses to ET-1 (30 pmol) were potentiated by BQ-788 (an ET(B) receptor antagonist) and reduced by co-injection of BQ-788 plus BQ-123 (an ET(A) receptor antagonist), BQ-123 alone inhibited scratching responses only. CTOP (µ-opioid receptor selective antagonist) only augmented scratching responses to ET-1, whereas DAMGO (µ-opioid receptor selective agonist) reduced both behaviors. Loratadine (histamine H(1) receptor antagonist) marginally reduced scratching, but markedly suppressed wipes. SIGNIFICANCE: These results demonstrate that ET-1 evokes pruritic and nociceptive behaviors in the mouse cheek model. Both responses to ET-1 appear to be mediated via ET(A) receptors and subjected to limitation by simultaneous ET(B) receptor activation. Local endogenous opioids acting on µ-opioid receptors selectively modulate the pruritic response to ET-1, whereas histamine, possibly derived from mast cells and acting on H(1) receptors, contributes importantly to the nociceptive effect of ET-1 in this model.

Endothelin-1 and -2: two amino acids matter.

AIMS: Endothelin-1 (ET-1) and endothelin-2 (ET-2; Trp(6)Leu(7)ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelin(A) (ET(A))-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect. MAIN METHODS: We used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ET(A)-receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp(6)ET-1 and Leu(7)ET-1). KEY FINDINGS: ET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ET(A)-antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pK(B): 7.1 ± 0.2 versus 5.6 ± 0.4). However, 1 µM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu(7)ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256 nM Trp(6)ET-1 did not cause contraction and did not antagonize arterial responses to ET-1. SIGNIFICANCE: Arterial smooth muscle ET(A)-receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists.

Agonist-dependent modulation of arterial endothelinA receptor function.

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) causes long-lasting vasoconstrictions. These can be prevented by ET(A) receptor antagonists but are only poorly reversed by these drugs. We tested the hypothesis that endothelin ET(A) receptors are susceptible to allosteric modulation by endogenous agonists and exogenous ligands. EXPERIMENTAL APPROACH Rat isolated mesenteric resistance arteries were pretreated with capsaicin and studied in wire myographs, in the presence of L-NAME and indomethacin to concentrate on arterial smooth muscle responses. KEY RESULTS Endothelins caused contractions with equal maximum but differing potency (ET-1 = ET-2 > ET-3). ET-1(1-15) neither mimicked nor antagonized these effects in the absence and presence of ET(16-21). 4(Ala) ET-1 (ET(B) agonist) and BQ788 (ET(B) antagonist) were without effects. BQ123 (peptide ET(A) antagonist) reduced the sensitivity and relaxed the contractile responses to endothelins. Both effects depended on the agonist (pK(B): ET-3 = ET-1 > ET-2; % relaxation: ET-3 = ET-2 > ET-1). Also, with PD156707 (non-peptide ET(A) antagonist) agonist-dependence and a discrepancy between preventive and inhibitory effects were observed. The latter was even more marked with bulky analogues of BQ123 and PD156707. CONCLUSIONS AND IMPLICATIONS These findings indicate allosteric modulation of arterial smooth muscle ET(A) receptor function by endogenous agonists and by exogenous endothelin receptor antagonists. This may have consequences for the diagnosis and pharmacotherapy of diseases involving endothelins.