ETB Receptor Agonist Research Articles

529: SOVATELTIDE (TYCAMZZI) IN PATIENTS WITH CEREBRAL ISCHEMIC STROKE: A RANDOMIZED MULTICENTER STUDY

Introduction: Sovateltide (Tycamzzi™) is a novel first-in-class ETB receptor agonist and a synthetic analog of endothelin-1, which promotes neuroprotection, neuroregeneration, and neurovascular remodeling following CNS damage. Methods: A phase III, prospective, multicentric, randomized study was conducted in acute cerebral ischemic stroke (ACIS) patients 18 through 78 years of age. Following radiologic confirmation of ischemic stroke, patients could be enrolled with NIHSS score greater than 5. Excluded were patients with intracranial hemorrhage and those receiving endovascular therapy. Sovateltide was administered in three doses, each dose of 0.3 μg/kg, as an intravenous bolus at an interval of 3 hours ± 1 hour on day 1, day 3, and day 6 (total dose/day: 0.9 µg/kg). The primary objectives were to determine the neurological outcome based on the modified Rankin Scale (mRS) score, National Institute of Health Stroke Scale (NIHSS) score, and Barthel Index (BI) scale score from day 1 through day 90. Results: A significantly (p=0.004) greater number of patients in the sovateltide group (76.1%) had an improvement of mRS of ≥2 points from the baseline compared to the control group (52.8%) at 90 days of treatment. The scores on the mRS at 90 days show an ordinal shift that highly favors sovateltide therapy (56% in control vs. 81% in sovateltide shift in mRS range 0-2). The median score (IQR) of the NIHSS scale at 90 days was 3 (0 to 6) in the control and 1 (0 to 3) in the sovateltide group (p=0.003). An improvement of NIHSS of ≥6 points vs. baseline at 90 days of treatment was significant (p=0.033) in sovateltide (80.6%) compared to the control (64.2%) group. The median score (IQR) of the BI scale at 90 days was 85 (60 to 100) in control and 95 (80 to 100) in the sovateltide group (p=0.01). The number needed to treat (NNT) patients (having mRS of 2 or less at 90 days) with sovateltide was 5 (95% CI 2.6 to 12.2). All-cause mortality was 2.56% in the control and 5.00% in the sovateltide group. Conclusions: Sovateltide demonstrated a statistically significant and clinically meaningful improvement in neurological outcomes in patients with ACIS compared to the standard of care. In addition, sovateltide produced a remarkable ordinal shift in the mRS across the range at day 90 post-randomization.

Role of endothelin in the pathophysiology of migraine: A new view on an old player

There is cumulating evidence that endothelin-1 (ET-1) may play a role in migraine, however controversial findings still impede a conclusion to be drawn. Herein we tested the hypothesis that endothelin ETB receptors are major contributors to migraine-like responses. ET-1, IRL-1620 (selective ETB receptor agonist) or CGRP were injected into the trigeminal ganglion (TG) of female Wistar rats, and the development of periorbital mechanical allodynia was assessed hourly with von Frey hairs. Twenty-four hours later, rats were exposed to an aversive light for 1 h, after which the reactivation of periorbital mechanical allodynia (indicating photic sensitivity) was assessed up to 4 h. Moreover, the effect of systemic Bosentan (ETA/ETB receptors antagonist) or the selective antagonists of ETA (BQ-123) and ETB (BQ-788) receptors injected into the TG were evaluated against CGRP-induced responses. ET-1 and IRL-1620 injection into the TG induced periorbital mechanical allodynia and photic sensitivity. Bosentan attenuated periorbital mechanical allodynia but failed to affect photic sensitivity induced by CGRP. Selective blockade of ETB receptors in the TG fully prevented the development of periorbital mechanical allodynia and photic sensitivity induced by CGRP, but ETA receptor blockade caused only a slight reduction of periorbital mechanical allodynia without affecting photic sensitivity. ETB receptor-operated mechanisms in the TG may contribute to migraine-like responses in female rats.

Indoxyl sulfate enhances endothelin-1-induced contraction via impairment of NO/cGMP signaling in rat aorta.

The microbiome-derived tryptophan metabolite, indoxyl sulfate, is considered a harmful vascular toxin. Here, we examined the effects of indoxyl sulfate on endothelin-1 (ET-1)-induced contraction in rat thoracic aortas. Indoxyl sulfate (10-3 M, 60 min) increased ET-1-induced contraction but did not affect isotonic high-K+-induced contraction. The ET-1-induced contraction was enhanced by endothelial denudation in both control and indoxyl sulfate-treated groups. BQ123 (10-6 M), an ETA receptor antagonist, reduced the ET-1-induced contraction in both control and indoxyl sulfate groups. BQ788 (10-6 M), an ETB receptor antagonist, increased the contraction in the control group but had no effect on the indoxyl sulfate group. Conversely, indoxyl sulfate inhibited relaxation induced by IRL1620, an ETB receptor agonist. L-NNA, an NO synthase (NOS) inhibitor, increased the ET-1-induced contractions in both the control and indoxyl sulfate groups, whereas L-NPA (10-6 M), a specific neuronal NOS inhibitor, did not affect the ET-1-induced contraction in both groups. However, ODQ, an inhibitor of soluble guanylyl cyclase, increased the ET-1-induced contraction in both groups. Organic anion transporter (OAT) inhibitor probenecid (10-3 M) and antioxidant N-acetyl-L-cysteine (NAC; 5 × 10-3 M) inhibited the effects of indoxyl sulfate. A cell-permeant superoxide scavenger reduced the ET-1-induced contraction in the indoxyl sulfate group. The aortic activity of SOD was reduced by indoxyl sulfate. The present study revealed that indoxyl sulfate augments ET-1-induced contraction in rat aortae. This enhancement may be due to the impairment of NO/cGMP signaling and may be attributed to impairment of the antioxidant systems via cellular uptake through OATs.

Endothelin-1 decreases the expression of Ephrin-A and B subtypes in cultured rat astrocytes through ETB receptors

Ephrin family proteins are cell surface molecules that regulate several cellular functions through cell-cell interactions. During nervous tissue repair after injury, the expression of ephrin subtypes in astrocytes is altered, affecting the axonal elongation and migration of neuronal precursors. However, the mechanism regulating the expression of ephrin subtypes in astrocytes has not been investigated. Herein, we studied the effects of endothelin-1 (ET-1) on the expression of ephrin subtypes in cultured rat astrocytes. Our results showed that ET-1 (100 nM) treatment for 1−24 h reduced the expression of ephrin-A2, -A4, -B2, and -B3 mRNA and protein in astrocytes, whereas the expression of ephrin-A1, -A3, -A5, and -B1 mRNA were not affected. Sarafotoxin S6c, a selective ETB receptor agonist, decreased the expression of ephrin-A2, -A4, -B2, and -B3 in cultured astrocytes. The decrease in ephrin-A2, -A4, -B2, and -B3 expression by ET-1 treatment was reduced in the presence of BQ788, an ETB receptor antagonist, while FR139317, an ETA receptor antagonist, had no effects. These results suggest that ET-1 is a signaling molecule that downregulates ephrin-A2, -A4, -B2, and -B3 expression in astrocytes.

Central endothelin ETB receptor activation reduces blood pressure and catecholaminergic activity in the olfactory bulb of deoxycorticosterone acetate-salt hypertensive rats

Endothelins regulate catecholaminergic activity in the olfactory bulb (OB) in normotensive and hypertensive animals. Administration of an endothelin ETA receptor antagonist decreases blood pressure in deoxycorticosterone acetate-salt (DOCA-salt) rats along with a reduction in tyrosine hydroxylase (TH) activity and expression. In the present work, we sought to establish the role of brain endothelin ETB receptor on blood pressure regulation and its relationship with the catecholaminergic system within the OB of DOCA-Salt rats. Sprague-Dawley male rats were divided into control and DOCA-Salt groups. Blood pressure, heart rate and TH activity as well as neuronal nitric oxide synthase (nNOS) expression were assessed following IRL-1620 (selective endothelin ETB receptor agonist) applied to be brain. IRL-1620 significantly reduced systolic, diastolic, and mean arterial pressure in DOCA-Salt hypertensive rats. It also decreased TH activity, TH total and phosphorylated forms expression as well as its mRNA in the OB of hypertensive animals. The expression of phospho-Ser1417-nNOS, which reflects nNOS activation, was significantly decreased in the of OB of DOCA-salt rats, but it was enhanced by IRL-1620. These findings suggest that DOCA-Salt hypertension depends on endogenous central endothelin ETA receptor activity, rather than on ETB, and that low endothelin ETB stimulation is essential for blood pressure elevation in this animal model. The effect of endothelin ETA receptor antagonism may also result from endothelin ETB receptor overstimulation. The present study shows that endothelin receptors are involved in the regulation of TH in the OB and that such changes are likely implicated in the hemodynamic control and sympathetic outflow.

Anti-apoptotic activity of ETB receptor agonist, IRL-1620, protects neural cells in rats with cerebral ischemia

Endothelin-B receptor agonist, IRL-1620, provides significant neuroprotection following cerebral ischemia in rats. Whether this neuroprotection is due to inhibition of apoptosis is unknown. IRL-1620-treated rats following permanent middle cerebral artery occlusion (MCAO) showed significant improvement in neurological and motor functions along with a decrease in infarct volume at 24 h (−81.3%) and day 7 (−73.0%) compared to vehicle group. Cerebral blood flow (CBF) significantly improved in IRL-1620-treated animals compared to vehicle by day 7 post MCAO. IRL-1620-treated rats showed an increase in phospho-Akt and decrease in Bad level 7 h post-occlusion compared to vehicle, while Akt and Bad expression was similar in cerebral hemispheres at 24 h post-MCAO. The phospho-Bad level was lower in vehicle- but not in IRL-1620-treated rats at 24 h. Anti-apoptotic Bcl-2 expression decreased, while pro-apoptotic Bax expression increased in vehicle-treated MCAO rats, these changes were attenuated (P < 0.01) by IRL-1620. Mitochondrial membrane-bound Bax intensity significantly decreased in IRL-1620 compared to vehicle-treated MCAO rats. IRL-1620 treatment reduced (P < 0.001) the number of TUNEL-positive cells compared to vehicle at 24 h and day 7 post MCAO. The results demonstrate that IRL-1620 is neuroprotective and attenuates neural damage following cerebral ischemia in rats by increasing CBF and reducing apoptosis.

Minimally modified low-density lipoprotein upregulates the ETB and α1 receptors in mouse mesenteric arteries in vivo by activating the PI3K/Akt pathway

The current study aimed to explore whether minimally modified low-density lipoprotein (mmLDL) via tail vein injection upregulates the ETB and α1 receptors in mouse mesenteric arteries by activating the PI3K/Akt pathway. The contraction curves of the mesenteric arteries caused by sarafotoxin 6c (S6c, ETB receptor agonist) and phenylephrine (PE, α1 receptor agonist) were measured by a myograph system. Serum oxLDL was detected using enzyme-linked immunosorbent assays. The levels of the ETB receptor, the α1 receptor, PI3K, p-PI3K and p-Akt were detected using real-time polymerase chain reaction and Western blot analyses. Minimally modified low-density lipoprotein noticeably enhanced the contraction effect curves of S6c and PE, with significantly increased Emax values (P<0.01), compared to those of the control group. This treatment significantly increased the mRNA expression and protein levels of the ETB and α1 receptors and the protein levels of p-PI3K and p-Akt in the vessel wall (P<0.01). LY294002 inhibited the effect of mmLDL. An increase in mmLDL activated the PI3K/Akt pathway, which upregulated the expression of the ETB and α1 receptors and enhanced the ETB and α1- receptor-mediated contractile function.

Constriction of Retinal Venules to Endothelin-1: Obligatory Roles of ETA Receptors, Extracellular Calcium Entry, and Rho Kinase.

PurposeEndothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca2+), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules.MethodsPorcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools.ResultsRetinal venules developed basal tone and constricted concentration-dependently to ET-1. The ETA receptor (ETAR) antagonist BQ123 abolished venular constriction to ET-1, but ETB receptor (ETBR) antagonist BQ788 had no effect on vasoconstriction. The ETBR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca2+, venules lost basal tone and ET-1–induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ETARs and ETBRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules.ConclusionsExtracellular Ca2+ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ETARs and extracellular Ca2+ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.

Up-regulation of contractile endothelin receptors by airborne fine particulate matter in rat mesenteric arteries via activation of MAPK pathway.

Fine particle matters (PM2.5) is a well-known risk factor for cardiovascular diseases. However, the underlying molecular mechanisms are largely unknown. Vascular hyper-reactivity plays an important roles in the pathogenesis of cardiovascular diseases. The present study was designed to investigate a hypothesis that PM2.5 up-regulated endothelin receptors in mesenteric artery and the potential underlying mechanisms. Rat mesenteric arteries were cultured with PM2.5. The artery contractile responses were recorded by a sensitive myograph. ETB and ETA receptor expressions of mRNA and protein were assessed by quantitative real-time PCR, Western blotting, and immunohistochemistry, respectively. Results showed that ETB receptor agonist, sarafotoxin 6c induced a negligible contraction in fresh artery segments, while ETA receptor agonist, ET-1 induced an obvious contraction. After organ culture, the contraction curve mediated by ETB and ETA receptors were shifted toward the left. PM2.5 1.0μg/ml cultured for 16h further enhanced ETB and ETA receptor-mediated contractile responses with a markedly increased maximal contraction. The organ culture enhanced ETB and ETA receptor mRNA and protein levels from fresh arteries, which were further increased by PM2.5. The U0126 (MEK/ERK1/2 inhibitor) and SB203580 (p38 inhibitor) significantly attenuated both organ cultured-induced and PM2.5-induced up-regulation of ETB receptor. U0126 also suppressed organ culture-increased and PM2.5-increased expressions of ETA receptor. SB203580 only suppressed PM2.5-induced enhanced expressions of ETA receptor In conclusion, airborne PM2.5 up-regulates ETB and ETA receptors of mesenteric artery via p38 MAPK and MEK/ERK1/2 MAPK pathways.

Attenuation of opioid tolerance by ETB receptor agonist, IRL-1620, is independent of an accompanied decrease in nerve growth factor in mice.

AimETA receptor antagonists reverse opioid tolerance but the involvement of ETB receptors is unknown. In morphine or oxycodone tolerant mice we investigated (1) the effect of ETB receptor agonist, IRL-1620, on analgesic tolerance; (2) changes in expression of the brain ETA and ETB receptors; and (3) alterations in the brain VEGF, NGF, PI3K and notch-1 expression.Main methodsBody weight, body temperature, and tail-flick latency were assessed before and after a challenge dose of morphine or oxycodone in vehicle or IRL-1620 treated mice. Expression studies were carried out using Western blots.Key findingsTail flick latency to a challenge dose of opioid was significantly increased by IRL-1620 from 39% to 100% in morphine tolerant and from 8% to 83% in oxycodone tolerant mice. Morphine or oxycodone did not alter ETA or ETB receptor expression. IRL-1620 had no effect on ETA however it increased (61%) expression of ETB receptors. IRL-1620-induced increase in ETB receptor expression was attenuated by morphine (39.8%) and oxycodone (51.8%). VEGF expression was not affected by morphine or oxycodone and was unaltered by IRL-1620. However, NGF and PI3K expression was decreased (P < 0.001) by morphine and oxycodone and was unaffected by IRL-1620. Notch-1 expression was not altered by morphine, oxycodone or IRL-1620.SignificanceETB receptor agonist, IRL-1620, restored analgesic tolerance to morphine and oxycodone, but it did not affect morphine and oxycodone induced decrease in NGF/PI3K expression. It is concluded that IRL-1620 attenuates opioid tolerance without the involvement of NGF/PI3K pathway.

High salt intake increases endothelin B receptor function in the renal medulla of rats

AimsEndothelin (ET)-1 promotes natriuresis via the endothelin B receptor (ETB) within the renal medulla. In male rats, direct interstitial infusion of ET-1 into the renal medulla has no effect on renal sodium and water excretion but is associated with endothelin A receptor (ETA)-dependent reductions in medullary blood flow. Loss of ETB function leads to salt-sensitive hypertension. We hypothesized that HS intake would increase the natriuretic and diuretic response to renal medullary infusion of ET peptides. Main methodsMale Sprague–Dawley (SD) rats were fed a normal (NS) or high (HS) salt diet for 7days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. Medullary infusion of a low dose of ETB receptor agonist, sarafotoxin 6c (S6c; 0.15μg/kg/h), or ET-1 (0.45μg/kg/h) was used to determine changes in sodium excretion (UNaV). Key findingsIn HS fed rats, intramedullary infusion of a low dose of S6c induced a significant increase in UNaV, roughly 2-fold over baseline, compared to no response to this low dose in NS fed rats. In HS fed rats, intramedullary infusion of ET-1 induced a significantly greater increase in UNaV compared to NS fed rats, although this increase was not different from the HS time control studies. SignificanceWe conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor activation in vivo consistent with a role for the ETB receptor in maintaining fluid-electrolyte homeostasis.

Airborne fine particulate matter induces an upregulation of endothelin receptors on rat bronchi

Airborne fine particulate matter (PM2.5) is a risk factor for respiratory diseases. However, little is known about the effects of PM2.5 on bronchi. The present study investigated the effect of airborne PM2.5 on rat bronchi and the underlying mechanisms. Isolated rat bronchial segments were cultured for 24 h. Endothelin (ET) receptor-mediated contractile responses were recorded using a wire myograph. The mRNA and protein expression levels of ET receptors were studied using quantitative real-time PCR, Western blotting, and immunohistochemistry. The results demonstrated that ETA and ETB receptor agonists induced remarkable contractile responses on fresh and cultured bronchial segments. PM2.5 (1.0 or 3.0 μg/ml) significantly enhanced ETA and ETB receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction compared to the DMSO or fresh groups. PM2.5 increased the mRNA and protein expression levels of ETA and ETB receptors. U0126 (a MEK1/2 inhibitor) and SB203580 (a p38 inhibitor) significantly suppressed PM2.5-induced increases in ETB receptor-mediated contractile responses, mRNA and protein levels. SP600125 (a JNK inhibitor) and SB203580 significantly abrogated the PM2.5-induced enhancement of ETA receptor-mediated contraction and receptor expression. In conclusion, PM2.5 upregulates ET receptors in bronchi. ETB receptor upregulation is associated with MEK1/2 and p38 pathways, and the upregulation of ETA receptor is involved in JNK and p38 pathways.

Evaluation of liposomal nanocarriers loaded with ETB receptor agonist, IRL-1620, using cell-based assays

One common feature of most neurodegenerative diseases, including Alzheimer’s disease (AD) and stroke, is the death of neuronal cells. Neuronal cell death is associated with apoptosis, generation of reactive oxygen species and oxidative stress. Neuronal cell death pathways can be reversed by endothelin B receptor agonist, IRL-1620, which was found to enhance neuroprotection by promoting vascular and neuronal growth in a rodent stroke model. Previous studies conducted at our institution indicated that the treatment with IRL-1620 significantly improved neurological and motor function while reducing oxidative stress and overall infarct area. IRL-1620 is a hydrophilic, 15 amino acid peptide and has a molecular weight of 1820Da. In this study, we have encapsulated IRL-1620 in PEGylated liposomes in order to enhance its efficacy. Each batch of liposomes encapsulating IRL-1620 was evaluated for particle size, polydispersity index, and charge (zeta potential) over a period of time to determine their stability. A dose–response bar graph was plotted based on the effect of neuroprotection by free IRL-1620 on differentiated neuronal PC-12 cells. The 1nM concentration was found to have the highest cell viability. The liposomes loaded with IRL-1620 were tested on differentiated neuronal PC-12 cells for their neuroprotective ability against apoptosis caused by removal of nerve growth factor (NGF) against free (non-encapsulated) IRL-1620. The liposomal IRL-1620 was found to proliferate the growth of serum-deprived differentiated PC-12 cells significantly (p<0.0001). In the western blot analysis, the expression of the anti-apoptotic marker, BCL-2 was found to be increased, and that of pro-apoptotic marker, BAX was found to be decreased with liposomal IRL-1620. The effects were found to be independent of the NGF levels. Finally the free IRL-1620 was found to cause neuronal outgrowth equivalent to the 75ng/ml NGF treatment.

Increased cerebrovascular sensitivity to endothelin-1 in a rat model of obstructive sleep apnea: a role for endothelin receptor B.

Obstructive sleep apnea (OSA) is associated with cerebrovascular diseases. However, little is known regarding the effects of OSA on the cerebrovascular wall. We tested the hypothesis that OSA augments endothelin-1 (ET-1) constrictions of cerebral arteries. Repeated apneas (30 or 60 per hour) were produced in rats during the sleep cycle (8 hours) by remotely inflating a balloon implanted in the trachea. Four weeks of apneas produced a 23-fold increase in ET-1 sensitivity in isolated and pressurized posterior cerebral arteries (PCAs) compared with PCAs from sham-operated rats (EC50=10(-9.2) mol/L versus 10(-10.6) mol/L; P<0.001). This increased sensitivity was abolished by the ET-B receptor antagonist, BQ-788. Constrictions to the ET-B receptor agonist, IRL-1620, were greater in PCAs from rats after 2 or 4 weeks of apneas compared with that from sham-operated rats (P=0.013). Increased IRL-1620 constrictions in PCAs from OSA rats were normalized with the transient receptor potential channel (TRPC) blocker, SKF96365, or the Rho kinase (ROCK) inhibitor, Y27632. These data show that OSA increases the sensitivity of PCAs to ET-1 through enhanced ET-B activity, and enhanced activity of TRPCs and ROCK. We conclude that enhanced ET-1 signaling is part of a pathologic mechanism associated with adverse cerebrovascular outcomes of OSA.

Endothelin-1, but not angiotensin II, induces afferent arteriolar myosin diphosphorylation as a potential contributor to prolonged vasoconstriction

Bolus administration of endothelin-1 elicits long-lasting renal afferent arteriolar vasoconstriction, in contrast to transient constriction induced by angiotensin II. Vasoconstriction is generally evoked by myosin regulatory light chain (LC20) phosphorylation at Ser19 by myosin light chain kinase (MLCK), which is enhanced by Rho-associated kinase (ROCK)-mediated inhibition of myosin light chain phosphatase (MLCP). LC20 can be diphosphorylated at Ser19 and Thr18, resulting in reduced rates of dephosphorylation and relaxation. Here we tested whether LC20 diphosphorylation contributes to sustained endothelin-1 but not transient angiotensin II-induced vasoconstriction. Endothelin-1 treatment of isolated arterioles elicited a concentration- and time-dependent increase in LC20 diphosphorylation at Thr18 and Ser19. Inhibition of MLCK or ROCK reduced endothelin-1-evoked LC20 mono- and diphosphorylation. Pretreatment with an ETB but not an ETA receptor antagonist abolished LC20 diphosphorylation, and an ETB receptor agonist induced LC20 diphosphorylation. In contrast, angiotensin II caused phosphorylation exclusively at Ser19. Thus, endothelin-1 and angiotensin II induce afferent arteriolar constriction via LC20 phosphorylation at Ser19 due to calcium activation of MLCK and ROCK-mediated inhibition of MLCP. Endothelin-1, but not angiotensin II, induces phosphorylation of LC20 at Thr18. This could contribute to the prolonged vasoconstrictor response to endothelin-1.

ASSA14-03-11 Role of GRK4γ variant 142V in the regulation of renal ETB receptor in hypertension

Background The endothelin receptor B (ETBR) regulates blood pressure and water and electrolyte balance by engendenring natriuresis. In hypertensive states, the ETBR mediated diureis and natriuresis is impaired. However, the underlying mechanisms are not clear. G protein-coupled receptor kinase 4 (GRK4), whose gene locus 4p16.3 is linked to essential hypertension, cause sodium retention and increase blood pressure via impairment of renal dopamine receptor and enhancement of renin-angiotensin system functions. Due to the higher activity of GRK4 in kidney from spontaneously hypertensive rats (SHRs) and hypertensive patients, we hypothesise that GRK4 might be the cause of ETBR impairment in hypertension. Method Experiments were carried out in male anaesthetic spontaneously hypertensive rats (SHR)and in normotensive Wistar-Kyoto (WKY) rats. The ETB receptor agonist, BQ-3020 (0.1, 0.5, 1.0 ug/kg/min) were infused via supra-renal artery at a rate of 0.04 ml/min for 40 min. The same experiments were conducted in GRK4 A142V and GRK4 Wild Type transgenic mice. The ETBR function were also checked in the wild-type and A142V transfected renal proximal tubule (RPT) cells from mice. Results We found that diuresis and natriuresis of ETBR agonist, BQ3020, in Wistar-Kyoto (WKY) rats, which was impaired in SHRs. The GRK4 expression was higher in renal cortex from SHRs as compared with WKY rats. In GRK4 A142V transgenic mice, it resulted that ETBR-mediated diuresis and natriuresis was impaired compared with Wild type. In wild-type transfected cells, activation of ETBR inhibited Na + -K + -ATPase activity; while in A142V transfected cells, the inhibitory effect was lost. There are co-localization and co-immunoprecipitation between ETBR and GRK4 in RPT cells. The linkage of ETBR/GRK4 was higher in wilde-type cells than in A142V cells. Similar phenomenon was found in the kidney from WKY and SHRs, SHRs had higher ETBR/GRK4 linkage, accompanied with higher ETBR phosphorylation, which might account for the impaired ETBR function in hypertension. Conclusion This study provides a mechanism by which GRK4, via regulation of renal ETBR function, participates in the pathogenesis of hypertension.

Endothelin-1 increases the expression of VEGF-R1/Flt-1 receptors in rat cultured astrocytes through ETB receptors.

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Expressions of VEGF-R1 and -R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF-R3 and Tie-2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF-R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF-E, a selective VEGF-R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF-R1 and -R2. Treatment with ET-1 increased VEGF-R1 mRNA and protein levels. The effects of ET-1 on VEGF-R1 mRNA were mimicked by Ala(1,3,11,15) -ET-1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET-1 did not affect the mRNA levels of VEGF-R2, -R3, and Tie-2. Pre-treatment with ET-1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues. To clarify the regulatory mechanisms of vascular endothelial growth factor (VEGF) receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Effects of selective VEGF-R1 and R2 agonist showed that these receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment with ET-1 increased expression of VEGF-R1, which was mediated by ETB receptors. Pre-treatment with ET-1 potentiated the VEGF-R1-mediated activations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes.

Heart ischaemia–reperfusion induces local up‐regulation of vasoconstrictor endothelin ETB receptors in rat coronary arteries downstream of occlusion

Background and PurposeEndothelins act via two receptor subtypes, ETA and ETB. Under physiological conditions in coronary arteries, ETA receptors expressed in smooth muscle cells mediate vasoconstriction whereas ETB receptors mainly found in endothelial cells mediate vasorelaxation. However, under pathophysiological conditions, ETB receptors may also be expressed in vascular smooth muscle cells mediating vasoconstriction. Here, we have investigated whether vasoconstrictor ETB receptors are up‐regulated in coronary arteries after experimental myocardial ischaemia in rats.Experimental ApproachMale Sprague‐Dawley rats were subjected to either heart ischaemia–reperfusion (15 min ischaemia and 22 h reperfusion), permanent ischaemia (22 h) by ligation of the left anterior descending coronary artery, or sham operation. Using wire myography, the endothelin receptor subtypes mediating vasoconstriction were examined in isolated segments of the left anterior descending and the non‐ligated septal coronary arteries. Endothelin receptor‐mediated vasoconstriction was examined with cumulative administration of sarafotoxin 6c (ETB receptor agonist) and endothelin‐1 (with or without ETA or ETB receptor blockade). The distribution of ETB receptors was localized with immunohistochemistry and quantified by Western blot.Key ResultsEndothelin ETB receptor‐mediated vasoconstriction and receptor protein levels were significantly augmented in coronary arteries situated downstream of the occlusion after ischaemia–reperfusion compared with non‐ischaemic arteries. In contrast, the ETA receptor‐mediated vasoconstriction was unaltered in all groups.Conclusions and ImplicationsIschaemia–reperfusion induced local up‐regulation of ETB receptors in the smooth muscle cells of coronary arteries in the post‐ischaemic area. In contrast, in non‐ischaemic areas, ETB receptor function was unaltered.

Nuclear Factor‐kappaB‐Mediated Endothelin Receptor Up‐Regulation Increases Renal Artery Contractility in Rats

Increased renal artery contractility leads to renal vasospasm and ischaemia as well as kidney damage. This study was designed to examine the hypothesis that organ culture of renal arteries induces transcriptional up-regulation of endothelin type A (ETA ) and type B2 (ETB2 ) receptors in the smooth muscle cells via activation of nuclear factor-kappaB (NF-κB) and subsequently increases renal artery contractility. Rat renal artery segments were organ-cultured for 6 or 24hr to increase endothelin receptor-mediated contraction. To dissect molecular mechanisms involved in this process, inhibitors for NF-κB signalling pathway (MG-132 and BMS345541), transcription (actinomycin D) and translation (cycloheximide) were used during organ culture. Endothelin receptors were studied using a sensitive myograph (functional contractility), real-time PCR (mRNA analysis) and immunohistochemistry (protein localization). Compared with fresh segments, contractile responses to endothelin-1 (non-selective endothelin receptor agonist) and sarafotoxin 6c (selective ETB receptor agonist) were significantly increased in the segments after 24hr of organ culture; ETB2 receptor-mediated maximal contraction increased from 2.7±0.5 to 135.3±5.1 (p<0.001), and potency (pEC50 ) of ETA receptor agonist increased from 8.20±0.04 to 8.72±0.07 (p<0.001). This was in parallel with increased corresponding mRNA and protein expression for ETA and ETB2 receptors. BMS345541, MG-132, actinomycin D or cyclohexamide, respectively, suppressed the up-regulation of ETA and ETB2 receptors. Immunostaining performed with specific antibody showed that IκB was phosphorylated during organ culture. In conclusion, activation of NF-κB mediates up-regulation of ETA and ETB2 receptors and subsequently increases renal artery contractility, which may contribute to renal vasospasm and ischaemia as well as kidney damage.

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.