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

OBJECTIVES: 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. METHODS: 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. KEY FINDINGS: 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. CONCLUSIONS: 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.

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.

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation.

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

Natriuretic response to renal medullary endothelin B receptor activation is impaired in Dahl-salt sensitive rats on a high-fat diet.

Renal medullary endothelin B receptors (ET(B)) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension have impaired renal medullary ET(B) function. We found that 4-week high-caloric diet elevated systolic BP in Dahl salt-sensitive (Dahl S) rats (126+/-2 vs. 143+/-3 mm Hg, p<0.05). We hypothesized that renal medullary ET(B) function is dysfunctional in DS rats fed a high-caloric diet. We compared the diuretic and natriuretic response to intramedullary infusion of ET(B) agonist sarafotoxin 6c (S6c) in DS rats fed either a normal or high-caloric diet for 4 weeks. Urine was collected during intramedullary infusion of saline for baseline collection followed by intramedullary infusion of either saline or S6c. We first examined the ET(B) function in DS rats fed a normal diet. S6c increased urine flow (2.7+/-0.3 microl/min during baseline vs. 5.1+/-0.6 microl/min after S6c; p<0.05; n=5) and sodium excretion (0.28+/-0.05 vs. 0.81+/-0.17 micromol/min; p<0.05), suggesting that DS rats have renal medullary ET(B) function. However, DS rats fed a high-caloric diet displayed a significant increase in urine flow (2.7+/-0.4 vs. 4.2+/-0.4 microl/min, baseline vs. S6c infusion, respectively; p<0.05, n=6), but no significant change in sodium excretion in response to S6c (0.32+/-0.06 vs. 0.45+/-0.10 micromol/min). These data demonstrate that renal medullary ET(B) function is impaired in DS rats fed a high-caloric diet, which may be contributed to the elevation of blood pressure during high-caloric feeding in this model.

Reduction of Uterine Perfusion Pressure Induced Redistribution of Endothelin Receptor Type-B Between the Intima and Media Contributes to the Pathogenesis of Pregnancy-Induced Hypertension.

BACKGROUND/AIMS: Studies have shown that a change in endothelin receptor expression in the artery is related to pregnancy-induced hypertension (PIH). However, the mechanism underlying this change remains unclear. METHODS: To test whether the distribution of endothelin receptor type-A (ETAR) and type-B (ETBR) plays an important role in PIH, a reduction of uterine perfusion pressure (RUPP) rat model was used to mimic some of the features of PIH; the resulting variable endothelin receptor expression was investigated in the media and intima of the aorta. Single vascular smooth muscle cells (VSMCs) were isolated from RUPP and normal pregnant (NP) rats to study the effect of ETAR and ETBR in smooth muscle cells. RESULTS: Compared with NP rats, RUPP rats had a significant redistribution of ETBR expression in the intima and media, while there was no significant difference in ETAR expression between the two groups. ETBR upregulation in VSMCs enhanced cellular contraction and contributed to PIH. The TNF-α plasma levels in RUPP rats were two-fold higher than those of NP rats, which upregulated the expression of ETBR in VSMCS through the NF-κB pathways in RUPP rats. CONCLUSION: Redistribution of ETBR between the media and intima played an important role in the pathogenesis of PIH.

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.

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.

Stimulation of endothelin B receptors by IRL-1620 decreases the progression of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment that ultimately leads to death. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies in our lab have shown that stimulation of ETB receptors provide significant neuroprotection following Aß1-40 administration. It is possible that IRL-1620 may be neuroprotective due to angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling following Aß1-40 administration has not been established. The purpose of this study was to determine the effect of stimulation of ETB receptors by IRL-1620 on vascular and neuronal growth factors after Aß1-40 administration. Rats were treated with Aß1-40 (day 1, 7 and 14) in the lateral cerebral ventricles using stereotaxically implanted cannula and received three intravenous injections of IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) at 2-h interval on day 8; experiments were performed on day 15. Rats were sacrificed for estimation of brain ETB receptors, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression using immunofluorescence and Western blot. In the Morris swim task, amyloid-ß (Aß)-treated rats showed a significant (p<0.0001) impairment in spatial memory. Rats treated with IRL-1620 significantly (p<0.001) reduced the cognitive impairment induced by Aß. BQ788 treatment completely blocked IRL-1620-induced improvement in cognitive impairment. IRL-1620 treatment enhanced the number of blood vessels labeled with VEGF compared to vehicle treatment. Additionally, cells showed increased (p<0.001) positive staining for NGF in IRL-1620-treated animals. ETB, VEGF and NGF protein expression significantly (p<0.001) increased in the brain of IRL-1620-treated rats as compared to vehicle. Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study demonstrate that IRL-1620 improves both acquisition (learning) and retention (memory) on the water maze task and enhances angiogenic and neurogenic remodeling. These findings indicate that the ETB receptor may be a novel therapeutic target for AD and other neurovascular degenerative disorders.

Selective Endothelin-B Receptor Stimulation Increases Vascular Endothelial Growth Factor in the Rat Brain during Postnatal Development.

Endothelin, vascular endothelial growth factor and nerve growth factor play important roles in development of the central nervous system. ET(B) receptors have been shown to promote neurovascular remodeling in the adult ischemic brain through an increase in VEGF and NGF. It is possible that ET(B) receptors may be involved in postnatal development of the brain through VEGF and NGF. In the present study, the brains of male rat pups on postnatal days 1, 7, 14 and 28 were analyzed for expression of ET(B) receptors, VEGF and NGF. In order to determine the effect of ET(B) receptor stimulation, a separate group of pups were administered saline or ET(B) receptor agonist, IRL-1620, on day 21, and their brains were analyzed on day 28. The intensity of ET(B) receptor and VEGF staining in the vasculature as well as the number of blood vessels of normal pups increased with age and was significantly higher on postnatal day 14 compared to day 1 and day 7. In contrast, both ET(B) and NGF staining intensity in the cortex and subventricular zones decreased (P<0.01) at postnatal day 14 compared to earlier time points. Stimulation of ET(B) receptors resulted in a significant increase in VEGF and ET(B) intensity both in the vasculature and the brain (P<0.05), however, IRL-1620 did not produce any change in NGF expression. Results indicate that ET(B) receptors appear to play a role in the development of the CNS and selective stimulation of ET(B) receptors enhances VEGF but not NGF in the postnatal rat brain.

Endothelin@25 - new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12.

Since the discovery of endothelin (ET)-1 in 1988, the main components of the signalling pathway have become established, comprising three structurally similar endogenous 21-amino acid peptides, ET-1, ET-2 and ET-3, that activate two GPCRs, ETA and ETB . Our aim in this review is to highlight the recent progress in ET research. The ET-like domain peptide, corresponding to prepro-ET-193-166 , has been proposed to be co-synthesized and released with ET-1, to modulate the actions of the peptide. ET-1 remains the most potent vasoconstrictor in the human cardiovascular system with a particularly long-lasting action. To date, the major therapeutic strategy to block the unwanted actions of ET in disease, principally in pulmonary arterial hypertension, has been to use antagonists that are selective for the ETA receptor (ambrisentan) or that block both receptor subtypes (bosentan). Macitentan represents the next generation of antagonists, being more potent than bosentan, with longer receptor occupancy and it is converted to an active metabolite; properties contributing to greater pharmacodynamic and pharmacokinetic efficacy. A second strategy is now being more widely tested in clinical trials and uses combined inhibitors of ET-converting enzyme and neutral endopeptidase such as SLV306 (daglutril). A third strategy based on activating the ETB receptor, has led to the renaissance of the modified peptide agonist IRL1620 as a clinical candidate in delivering anti-tumour drugs and as a pharmacological tool to investigate experimental pathophysiological conditions. Finally, we discuss biased signalling, epigenetic regulation and targeting with monoclonal antibodies as prospective new areas for ET research.

Endothelin B receptor agonist, IRL-1620, enhances angiogenesis and neurogenesis following cerebral ischemia in rats.

Endothelin B receptor agonist, IRL-1620, has been shown in previous studies, conducted in our lab, to provide significant neuroprotection at both 24h and 1 week following permanent cerebral ischemia. It is possible that IRL-1620 may be neuroprotective due to angiogenesis and neurogenesis. However, the effect of IRL-1620 on neurovascular remodeling following cerebral ischemia has not been established. The present study was conducted to determine the effect of IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)] on astrocytes, neurons, and vascular endothelial cells after induction of cerebral ischemia. Male Sprague-Dawley rats undergoing permanent middle cerebral artery occlusion (MCAO) received three intravenous injections of either vehicle or IRL-1620 at 2, 4, and 6h post occlusion. At 24h post occlusion, IRL-1620 treatment preserved neuronal numbers in the cortex, striatum and subventricular zone (SVZ) of the ischemic rat brain, while simultaneously enhancing the number of blood vessels labeled with vascular endothelial growth factor (VEGF) compared to vehicle treatment. By 1 week following MCAO, VEGF-positive vessels/30 µm brain slice in the IRL-1620 group numbered 11.33±2.13 versus 4.19±0.79 in the vehicle group (P<0.01). Additionally, animals receiving IRL-1620 displayed increased number of proliferating cells (P<0.0001) and cells positively staining for nerve growth factor (NGF; P<0.0001) in the infarcted brain. VEGF and NGF protein expression significantly increased at 1 week post MCAO in the infarcted hemisphere of IRL-1620 treated rats as compared to sham (P<0.01). Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study indicate that IRL-1620, administered on the day of infarct, is neuroprotective and enhances angiogenic and neurogenic remodeling following cerebral ischemia.

Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1.

BACKGROUND: Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes. METHODS: Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA. RESULTS: Treatment of cultured astrocytes with ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala(1,3,11,15)-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine. CONCLUSION: These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains.

Endothelin B receptor agonist, IRL-1620, provides long-term neuroprotection in cerebral ischemia in rats.

We have earlier shown that stimulation of endothelin B receptors by IRL-1620 provides significant neuroprotection at 24h following cerebral ischemia. However, the effect of IRL-1620 is not known in the subacute phase of cerebral ischemia, where development of cerebral edema further contributes towards brain damage. This study was designed to determine the effect of IRL-1620 on neurological functions, infarct volume, oxidative stress, and endothelin receptors following permanent middle cerebral artery occlusion for 7 days. Rats received three intravenous injections of either vehicle or IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)] at 2, 4, and 6h post occlusion. Treatment with IRL-1620 reduced infarct volume (54.06 ± 14.12 mm(3) vs. 177.06 ± 13.21 mm(3)), prevented cerebral edema and significantly improved all neurological and motor function parameters when compared to the vehicle-treated group. Vehicle-treated middle cerebral artery occluded rats demonstrated high levels of malondialdehyde and low levels of reduced glutathione and superoxide dismutase; these effects were reversed in IRL-1620 treated rats. No change in expression of endothelin A receptor was observed 7 days after induction of cerebral ischemia in vehicle or IRL-1620 treated rats. Rats receiving IRL-1620 demonstrated an upregulation of endothelin B receptor only in the infarcted hemisphere 7 days following occlusion. All effects of IRL-1620 were blocked by endothelin B receptor antagonist, BQ788. Results of the present study demonstrate that IRL-1620, administered on day 1, provides significant neuroprotection till 7 days after the induction of cerebral ischemia in rats. Selective endothelin B receptor activation may prove to be a novel therapeutic target in the treatment of cerebral ischemia.

G-protein-coupled receptor kinase interactor-1 (GIT1) is a new endothelial nitric-oxide synthase (eNOS) interactor with functional effects on vascular homeostasis.

Endothelial cell nitric-oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in the vasculature, undergoes extensive post-translational modifications that modulate its activity. Here we have identified a novel eNOS interactor, G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), which plays an unexpected role in GPCR stimulated NO signaling. GIT1 interacted with eNOS in the endothelial cell cytoplasm, and this robust association was associated with stimulatory eNOS phosphorylation (Ser(1177)), enzyme activation, and NO synthesis. GIT1 knockdown had the opposite effect. Additionally, GIT1 expression was reduced in sinusoidal endothelial cells after liver injury, consistent with previously described endothelial dysfunction in this disease. Re-expression of GIT1 after liver injury rescued the endothelial phenotype. These data emphasize the role of GPCR signaling partners in eNOS function and have fundamental implications for vascular disorders involving dysregulated eNOS.

IRL-1620, an endothelin-B receptor agonist, enhanced radiation induced reduction in tumor volume in Dalton's Lymphoma Ascites tumor model.

ETB receptor agonist, IRL-1620 (or SPI-1620) presently in US Phase 1 clinical trial, has been demonstrated to selectively and transiently increase tumor blood flow. The present study was conducted to determine the effect of IRL-1620 on radiation therapy in tumor bearing mice inoculated with Dalton's Lymphoma Ascites cells. Tumors were allowed to grow for 30 days to a size of 1.10-1.29 cm3 before starting the treatment. The animals with or without IRL-1620 treatment were exposed to radiation (4 Gy/dose) on every alternate day for a total of 5 doses. Tumor volume was determined twice every week till the end of study. Radiation alone did not affect the tumor volume; however, animals treated with IRL-1620 followed by radiation produced a significant (64%) reduction in tumor volume. Survival of mice improved from 0/10 at 56 days after tumor inoculation in vehicle plus radiation group to 6/10 at 70 days in IRL-1620 (9 nmol/kg) plus radiation group. It is concluded that IRL-1620 improves the efficacy of radiation treatment in tumor bearing mice. (These findings have been earlier presented as an abstract ).

Endothelin B receptor agonist, IRL-1620, reduces neurological damage following permanent middle cerebral artery occlusion in rats.

Endothelin and its receptors have long been considered therapeutic targets in the treatment of ischemic stroke. Recent studies indicate that ET(B) receptors may provide both vasodilatation and neuroprotection. The purpose of this study was to determine the effect of selectively activating the ET(B) receptors following permanent middle cerebral artery occlusion in rats. IRL-1620 [Suc-[Glu9,Ala11,15]-Endothelin-1(8-12)], a highly selective ET(B) agonist, was used alone and in conjunction with BQ788, an ET(B) antagonist, to determine the role of ET(B) receptors in cerebral ischemia. Rats were assessed for neurological deficit and motor function, and their brains were evaluated to determine infarct area, oxidative stress parameters, and ET receptor protein levels. Animals treated with IRL-1620 showed significant improvement in all neurological and motor function tests when compared with both vehicle-treated and BQ788-treated middle cerebral artery occluded groups. In addition, there was a significant decrease in infarct volume 24h after occlusion in animals treated with IRL-1620 (24.47±4.37mm(3)) versus the vehicle-treated group (153.23±32.18mm(3)). Blockade of ET(B) receptors by BQ788 followed by either vehicle or IRL-1620 treatment resulted in infarct volumes similar to those of rats treated with vehicle alone (163.51±25.41 and 139.21±15.20mm(3), respectively). Lipid peroxidation, as measured by malondialdehyde, increased and antioxidants (superoxide dismutase and reduced glutathione) decreased following infarct. Treatment with IRL-1620 reversed these effects, indicating that ET(B) receptor activation reduces oxidative stress injury following ischemic stroke. Animals pretreated with BQ788 showed similar oxidative stress damage as those in the vehicle-treated group. No significant difference was observed in ET(B) receptor levels in any of the groups. The present study demonstrates that ET(B) receptor activation may be a novel neuroprotective therapy in the treatment of focal ischemic stroke.

I.c.v administration of an endothelin ET(B) receptor agonist stimulates vascular endothelial growth factor-A production and activates vascular endothelial growth factor receptors in rat brain.

Vascular endothelial growth factors (VEGFs), a family of angiogenic factors, are upregulated by nerve injuries. To clarify the extracellular signals involved in VEGF production in the brain, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. I.c.v. administration of 500 pmol/d Ala(1,3,11,15)-ET-1, an ET(B) receptor agonist, increased the level of VEGF-A mRNA in the rat cerebrum, whereas those of VEGF-B, placental growth factor (PLGF), angiopoietin (ANG)-1, and ANG-2 mRNAs were not largely affected by Ala(1,3,11,15)-ET. The ET-induced increases in cerebrum VEGF-A mRNA were reduced by coadministration of 1 nmol/d BQ788, an ET(B) antagonist. Ala(1,3,11,15)-ET-1 also stimulated the production of VEGF-A proteins in the cerebrum. Immunohistochemical observations in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats showed that glial fibrillary acidic protein (GFAP)-positive astrocytes had VEGF-A immunoreactivity. Neurons, microglia, and brain capillary endothelial cells in the Ala(1,3,11,15)-ET-1-infused rats did not show VEGF-A reactivity. The i.c.v. administration of Ala(1,3,11,15)-ET-1 stimulated tyrosine phosphorylations of VEGF-R1 and R2 receptors in the rat cerebrum, whereas expression levels of total VEGF-R1 and R2 proteins were not largely changed. Immunoreactivity of tyrosine-phosphorylated VEGF-R1 was selectively shown in GFAP-positive astrocytes in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats. Tyrosine-phosphorylated VEGF-R2 proteins were present in astrocytes and brain capillary endothelial cells. These findings indicate that activation of brain ET(B) receptors increases production of VEGF-A and stimulates VEGF receptor signaling in the brain.

Human choroidal melanocyte signal transduction responses to various pharmacological agents: focus on endothelin receptors.

PURPOSE: The receptor-coupled signal transduction systems present in isolated human choroidal melanocytes (HCOMs) were investigated. METHODS: [(3)H]-inositol phosphates ([(3)H]-IPs) generated in the cells were measured by ion-exchange chromatography. cAMP generated in the cells was quantified using an enzyme immunoassay. RESULTS: Initially, HCOM cells were challenged with a relatively high concentration (e.g., 1 µM-1 mM) of a variety of pharmacological agents in order to determine which functional receptors were present in these cells. Full concentration-response pharmacological studies were subsequently conducted on endothelin receptors. While a number of prostaglandins (PGs) (e.g., PGD(2), PGE(2), PGF(2α), cloprostenol, latanoprost acid, U-46619), histamine, carbachol, bombesin, and arginine-vasopressin were essentially inactive at stimulating the phosphoinositide (PI) hydrolysis response, endothelin-1 (ET-1) potently and efficaciously generated [(3)H]-IPs. Concentration-response studies yielded the following potency (EC(50)) and efficacy (E(max) relative to ET-1) data: ET-1 EC(50) = 3.4 ± 1.4 nM, E(max) = 100%, n = 3; BQ-3020 (ET(B) receptor-selective agonist) EC(50) = 13 ± 4 nM, E(max) = 73 ± 2%, n = 3). The effects of ET-1 on [(3)H]-IPs production were blocked by the ET(B) receptor-selective antagonist, BQ-788 (IC(50) = 10 ± 5 nM, n = 3), while the ET(A) receptor-selective antagonist (BQ-610) was essentially inactive. In the adenylyl cyclase (AC) assay, while isoproterenol (10 µM), ET-1 (1 µM) and PGE(2) (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD(2), PGF(2α), PGI(2), latanoprost, latanoprost acid, U-46619 and BW245C [all at > 10 µM]) were inactive. CONCLUSIONS: It is concluded that HCOMs express functionally active ET(B) receptors that mediate the production of [(3)H]-IPs. Additionally, HCOMs generate cAMP in response to ET-1, PGE(2), and isoproterenol. These data may have relevance to the melanogenic activity of HCOM cells.

Endothelin type B receptor-induced sustained Ca2+ influx involves G(q/11)/phospholipase C-independent, p38 mitogen-activated protein kinase-dependent activation of Na+/H+ exchanger.

The mechanism for sustained Ca2+ influx activated by G protein-coupled receptors was examined. In Chinese hamster ovary cells expressing recombinant human endothelin type B receptor (ET(B)R) and endogenous P2Y receptor (P2Y-R), endothelin-1 elicited a sustained Ca2+ influx depending on G(q/11 )protein, phospholipase C (PLC), Na+/H+ exchanger (NHE), and p38 mitogen-activated protein kinase (p38MAPK), whereas P2Y-R-induced sustained Ca2+ influx was negligible. Functional studies showed that NHE activation by ET(B)R was mediated via p38MAPK but not G(q/11)/PLC, while that by P2Y-R involves only G(q/11)/PLC/p38MAPK. These results suggest that G(q/11)/PLC-independent NHE activation via p38MAPK plays an important role in ET(B)R- mediated sustained Ca2+ influx.

Peripheral endothelin B receptor agonist-induced antinociception involves endogenous opioids in mice.

Endothelin-1 (ET-1) produced by various cancers is known to be responsible for inducing pain. While ET-1 binding to ETAR on peripheral nerves clearly mediates nociception, effects from binding to ETBR are less clear. The present study assessed the effects of ETBR activation and the role of endogenous opioid analgesia in carcinoma pain using an orthotopic cancer pain mouse model. mRNA expression analysis showed that ET-1 was nearly doubled while ETBR was significantly down-regulated in a human oral SCC cell line compared to normal oral keratinocytes (NOK). Squamous cell carcinoma (SCC) cell culture treated with an ETBR agonist (10(-4)M, 10(-5)M, and 10(-6) M BQ-3020) significantly increased the production of beta-endorphin without any effects on leu-enkephalin or dynorphin. Cancer inoculated in the hind paw of athymic mice with SCC induced significant pain, as indicated by reduction of paw withdrawal thresholds in response to mechanical stimulation, compared to sham-injected and NOK-injected groups. Intratumor administration of 3mg/kg BQ-3020 attenuated cancer pain by approximately 50% up to 3h post-injection compared to PBS-vehicle and contralateral injection, while intratumor ETBR antagonist BQ-788 treatment (100 and 300microg/kg and 3mg/kg) had no effects. Local naloxone methiodide (500microg/kg) or selective mu-opioid receptor antagonist (CTOP, 500microg/kg) injection reversed ETBR agonist-induced antinociception in cancer animals. We propose that these results demonstrate that peripheral ETBR agonism attenuates carcinoma pain by modulating beta-endorphins released from the SCC to act on peripheral opioid receptors found in the cancer microenvironment.