Effects of Thymoquinone on Urotensin-II and TGF-ß1 Levels in Model of Osteonecrosis in Rats.

Objectives: We aimed to investigate the therapeutic effects of thymoquinone (TMQ) treatment in osteonecrotic rats by evaluating protein levels, osteonecrosis (ON) levels, fatty acid degeneration, oxidative status, and plasma levels of Urotensin-II (U-II) and transforming growth factor-beta (TGF-ß1). Materials and Methods: 40 weight-matched adult male Wistar rats were grouped as control (n = 10), methylprednisolone acetate (MPA) (n = 10), thymoquinone (TMQ) (n = 10), and MPA + TMQ (n = 10). To induce ON, 15-week-old animals were subcutaneously injected with MPA at a dose of 15 mg/kg twice weekly for 2 weeks. TMQ was injected into 15-week-old rats via gastric gavage at a dose of 80 mg/kg per day for 4 weeks. The rats in the MPA + TMQ group were administered TMQ 2 weeks before the MPA injection. At the end of the treatments, cardiac blood samples and femur samples were collected for biochemical and histological evaluations. Results: In the control and TMQ groups, no ON pattern was observed. However, in tissues exposed to MPA, TMQ treatment resulted in significantly decreased ON levels compared to the MPA group. The number of cells that were positive for 8-OHdG and 4-HNE was significantly lower in the MPA + TMQ group than in the MPA group (p < 0.05). In terms of TGF-ß1 and U-II levels, we observed that both TGF-ß1 (367.40 ± 23.01 pg/mL vs. 248.9 ± 20.12 pg/mL) and U-II protein levels (259.5 ± 6.0 ng/mL vs. 168.20 ± 7.90 ng/mL) increased significantly in the MPA group compared to the control group (p < 0.001). Furthermore, TGF-ß1 (293.50 ± 14.18 pg/mL) and U-II (174.80 ± 4.2 ng/mL) protein levels were significantly decreased in the MPA + TMQ group compared to the MPA group (p < 0.05 and p < 0.01, respectively). There was a statistically positive correlation (p < 0.05) between the TGF-ß1 and U-II protein levels in all groups (p = 0.002, rcontrol = 0.890; p = 0.02, rTMQ = 0.861; p = 0.024, rMPA+TMQ = 0.868) except for the MPA group (p < 0.03, rMedrol = -0.870). Conclusions: As far as we know, this is the first study to demonstrate the curative functions of TMQ on ON by causing a correlated decrease in the expression of U-II and TGF-ß1 in the femoral heads of rats.

Urotensin II Receptor Modulation with 1,3,4-Benzotriazepin-2-one Tetrapeptide Mimics.

Urotensin II receptor (UT) modulators that differentiate the effects of the endogenous cyclic peptide ligands urotensin II (UII) and urotensin II-related peptide (URP) offer potential for dissecting their respective biological roles in disease etiology. Selective modulators of hUII and URP activities were obtained using 1,3,4-benzotriazepin-2-one mimics of a purported bioactive γ-turn conformation about the Bip-Lys-Tyr tripeptide sequence of urocontrin ([Bip4]URP). Considering an active ß-turn conformer about the shared Phe-Trp-Lys-Tyr sequence of UII and URP, 8-substituted 1,3,4-benzotriazepin-2-ones were designed to mimic the Phe-Bip-Lys-Tyr tetrapeptide sequence of urocontrin, synthesized, and examined for biological activity. Subtle 5- and 8-position modifications resulted in biased signaling and selective modulation of hUII- or URP-induced vasoconstriction. For example, p-hydroxyphenethyl analogs 17b-d were strong Gα13 and ßarr1 activators devoid of Gαq-mediated signaling. Tertiary amides 15d and 17d negatively modulated hUII-induced vasoconstriction without affecting URP-mediated responses. Benzotriazepinone carboxamides proved to be exceptional tools for elucidating the pharmacological complexity of UT.

Exploration of the urocontrin A scaffold yields new urotensinergic system allosteric modulator and competitive antagonists.

The urotensinergic system, involved in the development and/or progression of numerous pathological conditions, is composed of one G protein-coupled receptor (UT) and two endogenous ligands known as urotensin II (UII) and urotensin II-related peptide (URP). These two structurally related hormones, which exert common and divergent effects, are thought to play specific biological roles. In recent years, we have characterized an analog termed urocontrin A (UCA), i.e. [Pep4]URP, which is capable of discriminating the effects of UII from URP. Such an action could allow the delineation of the respective functions of these two endogenous ligands. In an effort to define the molecular determinants involved in this behavior and to improve the pharmacological profile of UCA, we introduced modifications from urantide, considered for some time as a lead compound for the development of UT antagonists, into UCA and assessed the binding, contractile activity and G protein signaling of these newly developed compounds. Our results show that UCA and its derivatives exert probe-dependent effects on UT antagonism, and we have further identified [Pen2, Pep4]URP as a Gq biased ligand with an insurmountable antagonism in our aortic ring contraction assay.

Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits.

Accumulated evidence shows that elevated urotensin II (UII) levels are associated with cardiovascular diseases. However, the role of UII in the initiation, progression, and regression of atherosclerosis remains to be verified. Different stages of atherosclerosis were induced in rabbits by a 0.3% high cholesterol diet (HCD) feeding, and either UII (5.4 µg/kg/h) or saline was chronically infused via osmotic mini-pumps. UII promoted atherosclerotic fatty streak formation in ovariectomized female rabbits (34% increase in gross lesion and 93% increase in microscopic lesion), and in male rabbits (39% increase in gross lesion). UII infusion significantly increased the plaque size of the carotid and subclavian arteries (69% increase over the control). In addition, UII infusion significantly enhanced the development of coronary lesions by increasing plaque size and lumen stenosis. Histopathological analysis revealed that aortic lesions in the UII group were characterized by increasing lesional macrophages, lipid deposition, and intra-plaque neovessel formation. UII infusion also significantly delayed the regression of atherosclerosis in rabbits by increasing the intra-plaque macrophage ratio. Furthermore, UII treatment led to a significant increase in NOX2 and HIF-1α/VEGF-A expression accompanied by increased reactive oxygen species levels in cultured macrophages. Tubule formation assays showed that UII exerted a pro-angiogenic effect in cultured endothelial cell lines and this effect was partly inhibited by urantide, a UII receptor antagonist. These findings suggest that UII can accelerate aortic and coronary plaque formation and enhance aortic plaque vulnerability, but delay the regression of atherosclerosis. The role of UII on angiogenesis in the lesion may be involved in complex plaque development.

2-{N-[(2,4,5-trichlorophenoxy) acetyl]-N-methylamino}-3-pyrrolidinepropanamide analogs as potential antagonists of Urotensin II receptor.

THE PURPOSE OF THE ARTICLE: To identify novel small molecule antagonists of Urotensin II receptor with acceptable pharmacological profile. MATERIALS AND METHODS: Structure-activity-relationship (SAR) studies on 2-{N-[(2,4,5-trichlorophenoxy) acetyl]-N-methylamino}-3-pyrrolidinepropanamide series were conducted and shortlisted compounds were synthesized and evaluated in in vitro cell-based assays. Human and mouse Urotensin II receptor overexpressing CHO cells were used for calcium release and radioligand binding assays. Initial molecules in this series had solubility and inter-species variability issue in the calcium release assay. We, therefore, conducted SAR to overcome these 2 issues and molecules with accepted in vitro profile were evaluated further in mouse pressor response model to generate the in vivo proof of concept for UII receptor antagonization. RESULTS AND CONCLUSIONS: We report herewith identification of 2-{N-[(2,4,5-trichlorophenoxy)acetyl]-N-methylamino}-3-pyrrolidinepropanamides series to obtain novel small molecule antagonists of Urotensin II receptor with acceptable pharmacological profile.

Dynamic changes of urotensin II and its receptor during ovarian development of olive flounder Paralichthys olivaceus.

Urotensin II (UII) is a kind of fish somatostatins cyclic peptide, which was originally extracted from the caudal neurosecretory system (CNSS). The system of UII and UII receptor (UIIR) has been reported to have multiple physiological regulatory functions, such as cardiovascular control, osmoregulation, and lipid metabolism. However, the effect of UII and UIIR on the ovarian development has not been covered. This study investigated the expression pattern of UII and UIIR in the ovarian follicles and explored their impact on ovarian development in olive flounder Paralichthys olivaceus. The results showed that the highest UII and UIIR mRNA levels were observed at stage II and stage III follicles during ovarian development, respectively. In situ hybridization revealed that a strong signal of UII was expressed in the oocyte nuclei of stage II follicles, however, UIIR was found in the follicle cells and oocyte cytoplasm of stage II and stage III follicles. Similarly, immunohistochemistry found positive signal of UII was detected in the oocyte nuclei of stage II follicles. The results from in vitro culture of olive flounder follicles suggested the expression of UII and UIIR mRNA levels significantly increased by 10 IU/ml human chorionic gonadotropin (hCG) for 9 h. Furthermore, the transcriptional expression of UII and UIIR was not statistically significantly changed by 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP). These results firstly suggested that UII and UII receptor may play vital roles in regulating ovarian growth in olive flounder.

Loss of urotensin II receptor diminishes hyperglycemia and kidney injury in streptozotocin-treated mice.

Beyond the CNS, urotensin II (UII) and its receptor (UT) are functionally expressed in peripheral tissues of the endocrine, cardiovascular, and renal systems. The expression levels of UII and UT in the kidney and circulating UII levels are increased in diabetes. UII also promotes mesangial proliferation and matrix accumulation in vitro. Here, we evaluate the effect of UT deletion on the development of hyperglycemia and diabetic kidney disease (DKD) in streptozotocin (STZ)-treated mice. Ten-week-old WT and UT knockout (KO) mice were injected with STZ for 5 days to induce diabetes. Blood glucose levels were measured weekly, and necropsy was performed 12 weeks after STZ injection. UT ablation slowed hyperglycemia and glucosuria in STZ-treated mice. UT KO also ameliorated STZ-induced increase in HbA1c, but not STZ-induced decrease in plasma insulin levels. However, STZ-induced increases in plasma glucagon concentration and immunohistochemical staining for glucagon in pancreatic islets were lessened in UT KO mice. UT ablation also protected against STZ-induced kidney derangements, including albuminuria, mesangial expansion, glomerular lesions, and glomerular endoplasmic reticulum stress. UT is expressed in a cultured pancreatic alpha cell line, and its activation by UII triggered membrane depolarization, T- and L-type voltage-gated Ca2+channel-dependent Ca2+influx, and glucagon secretion. These findings suggest that apart from direct action on the kidneys to cause injury, UT activation by UII may result in DKD by promoting hyperglycemia via induction of glucagon secretion by pancreatic alpha cells.

Urotensin II induces activation of NLRP3 and pyroptosis through calcineurin in cardiomyocytes.

Cell pyroptosis, a new type of programmed cell death, has been recently reported to play important roles in the development of cardiac remodeling. How cardiomyocyte pyroptosis is induced remains to be elucidated. Urotensin II (UII) has been known closely related to cardiac remodeling and the development of heart failure. Inhibition of UII receptors has been shown to be effective in the treatment of cardiac hypertrophy and remodeling. However, it is not clear whether UII might induce cardiomyocyte pyroptosis. We here examined the effect of UII treatment on pyroptosis in cultured cardiomyocytes. Treatment of cardiomyocyes of neonatal rats with UII (500 nmol/l) for 48 hours induced a significant pyroptosis as evidenced by not only increased cell death but also upregulated expression levels of NLR family pyrin domain containing 3 (NLRP3), caspase-1, IL-1ß, IL-18 and gasdermin D (GMDSD)-N which are important markers for the identification of cell pyroptosis. All these pyroptosis responses induced by UII were abrogated by an inhibitor of NLRP3. Moreover, the antagonist of UII receptor, Urantide abolished UII- induced cardiomyocyte pyroptosis. Additionally, inhibition of calcineurin by cyclosporin A rather than that of CaMKII by KN93 suppressed the UII-upregulated expression levels of those pyroptosis markers. We therefore demonstrate that UII might induce cardiomyocyte pyroptosis through calcineurin.

Urantide prevents CCl4­induced acute liver injury in rats by regulating the MAPK signalling pathway.

A number of drugs and other triggers can cause acute liver injury (ALI) in clinical practice. Therefore, identifying a safe drug for the prevention of liver injury is important. The aim of the present study was to investigate the potential preventive effect and regulatory mechanism of urantide on carbon tetrachloride (CCl4)­induced ALI by investigating the expression of components of the MAPK signalling pathway and the urotensin II (UII)/urotensin receptor (UT) system. Liver oedema and severe fatty degeneration of the cytoplasm were observed in ALI model rats, and the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were found to be significantly increased. Compared with those in the ALI model group, ALT and AST levels and the liver index did not significantly increase in each group given the preventive administration of urantide, and the liver tissue morphology was correspondingly protected. Moreover, the gene and protein expression levels of UII, G protein­coupled receptor (GPR14) and the oxidative stress­sensitive cytokines, α­smooth muscle actin and osteopontin were decreased, indicating that the protein translation process was effectively maintained. However, the expression levels of MAPK signalling pathway­related proteins and genes were decreased. It was found that urantide could effectively block the MAPK signalling pathway by antagonizing the UII/UT system, thus protecting the livers of ALI model rats. Therefore, it was suggested that ALI may be associated with the MAPK signalling pathway, and effective inhibition of the MAPK signalling pathway may be critical in protecting the liver.

Urantide alleviates the symptoms of atherosclerotic rats in vivo and in vitro models through the JAK2/STAT3 signaling pathway.

Atherosclerosis is the leading cause of human death, and its occurrence and development are related to the urotensin II (UII) and UII receptor (UT) system and the biological function of vascular smooth muscle cells (VSMCs). During atherosclerosis, impaired biological function VSMCs may promote atherosclerotic plaque formation. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway is an important mediator of signal transduction; however, the role of this signaling pathway in atherosclerosis and VSMCs remains unknown. This study aimed to investigate the effects of urantide on the JAK2/STAT3 signaling pathway in atherosclerosis. We examined the effect of urantide on the UII/UT system and the JAK2/STAT3 signaling pathway in a high fat diet induced atherosclerosis rat model and studied the effect and mechanism of urantide on the phenotypic transformation of VSMCs. We found that the UII/UT system and JAK2/STAT3 signaling pathway were highly activated in the thoracic aorta in atherosclerotic rats and in ox-LDL- and UII-induced VSMCs. After urantide treatment, the pathological changes in atherosclerotic rats were effectively improved, and the activities of the UII/UT system and JAK2/STAT3 signaling pathway were inhibited. Moreover, urantide effectively inhibited proliferation and migration and reversed the phenotypic transformation of VSMCs. These results demonstrated that urantide may control the JAK2/STAT3 signaling pathway by antagonizing the UII/UT system, thereby maintaining the biological function of VSMCs and potentially preventing and curing atherosclerosis.

Urantide decreases hepatic steatosis in rats with experimental atherosclerosis via the MAPK/Erk/JNK pathway.

Hepatic steatosis, an indicator of atherosclerosis (AS), is always accompanied by inflammatory responses and disturbances in lipid metabolism. The present study investigated the protective effect of urantide, a urotensin II (UII) receptor antagonist, on the liver of rats with AS with hepatic steatosis by regulating the MAPK pathway. AS was induced in rats via an intraperitoneal injection of vitamin D3 and the administration of a high­fat diet. Urantide treatment was then administered to the rats. Pathology, liver index, lipid levels and liver function were measured to determine liver injury. The expression levels of UII and G protein­coupled receptor 14 (GPR14) were determined using immunohistochemistry, reverse transcription­quantitative PCR and western blotting. The expression levels of MAPK­related proteins in hepatocytes from each group were quantified using western blotting and immunofluorescence staining. Rats with AS had typical pathological changes associated with AS and hepatic steatosis, which were significantly improved by urantide treatment. Blood lipid levels, body weight, liver index and liver function were recovered in rats with AS after urantide treatment. Urantide downregulated the expression levels of UII and GPR14 in the livers of rats with AS; concurrently, the phosphorylation of Erk1/2 and JNK was significantly decreased. Moreover, no significant changes were observed in the phosphorylation of p38 MAPK in AS rat livers. In conclusion, urantide inhibits the activation of Erk1/2 and JNK by blocking the binding of UII and GPR14, thereby alleviating hepatic steatosis in rats with AS, ultimately restoring lipid metabolism in the liver and alleviating AS lesions.

Urantide attenuates myocardial damage in atherosclerotic rats by regulating the MAPK signalling pathway.

OBJECTIVE: To explore the effect of urantide on atherosclerotic myocardial injury by antagonizing the urotensin II/urotensin II receptor (UII/UT) system and regulating the mitogen-activated protein kinase (MAPK) signalling pathway. METHODS: Atherosclerosis (AS) was established in rats by administering a high-fat diet and an intraperitoneal injection of vitamin D3. The effect of treatment with urantide (30 µg/kg), a UII receptor antagonist, for 3, 7, or 14 days on AS-induced myocardial damage was evaluated. RESULTS: The heart of rats with AS exhibited pathological changes suggestive of myocardial injury, and the serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased. Additionally, significant increases in the levels of UII, its receptor (G protein-coupled receptor 14, GPR14), p-P38, p-extracellular signal-regulated kinase (ERK) and p-c-Jun N-terminal kinase (JNK) were observed in the heart. Urantide improved pathological changes in the heart of rats with AS and reduced the serum CK and LDH levels. Additionally, the UII antagonist decreased the increased levels of UII, GPR14, p-P38, p-ERK and p-JNK in the heart. CONCLUSIONS: Urantide alleviates atherosclerotic myocardial injury by inhibiting the UII-GPR14 interaction and regulating the MAPK signalling pathway. We hypothesized that myocardial injury may be associated with the regulation of the MAPK signalling pathway.

Development of Novel 111-In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor Overexpressed in Solid Tumours.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.

The peptide compound urantide regulates collagen metabolism in atherosclerotic rat hearts and inhibits the JAK2/STAT3 pathway.

The aim of the present study was to investigate the effect of urantide on collagen metabolism in the hearts of rats with atherosclerosis (AS) by evaluating the expression of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway constituents. Urantide was delivered to rats with AS via tail vein injection for 3, 7 and 14 days. Serological indicators were identified by an automated biochemical analyzer. Histomorphological changes in the cardiac tissue of rats were observed by pathological staining techniques. The expression of genes and proteins was assessed using reverse transcription­quantitative PCR and western blot analysis, respectively. Localization of proteins was detected by immunofluorescence. Overexpression of urotensin II (UII) and its receptor, G protein­coupled receptor 14 (GPR14), was observed in the hearts of rats with AS and the expression of both proteins significantly declined after urantide administration. Triglyceride, total cholesterol, low­density lipoprotein, high­density lipoprotein and calcium levels were improved in rats with AS following treatment with urantide. Notably, urantide was able to antagonize the UII/GPR14 system. Urantide treatment resulted in markedly decreased expression levels of matrix metalloproteinase 2 (MMP­2), collagen type I/III, and genes and proteins in the JAK2/STAT3 pathway. By contrast, TIMP metallopeptidase inhibitor 2 (TIMP­2) levels were increased. In addition, the MMP­2/TIMP­2 protein ratio was significantly decreased in rats treated with urantide compared with AS rats with no urantide treatment. Constituents of the JAK2/STAT3 pathway and collagen type I/III were found to be localized in the diseased tissue and blood vessels of the hearts of rats with AS. In conclusion, urantide was able to effectively block the UII/GPR14 system by regulating the JAK2/STAT3 pathway and collagen metabolism. Inhibition of the UII/GPR14 system may prevent and potentially treat atherosclerotic myocardial fibrosis. Based on the current results, it was hypothesized that collagen metabolism may be associated with the JAK2/STAT3 pathway.

Microinjection of urotensin II into the pedunculopontine tegmentum leads to an increase in the consumption of sweet tastants.

The pedunculopontine tegmentum (PPTg) plays a role in processing multiple sensory inputs and innervates brain regions associated with reward-related behaviors. The urotensin II receptor, activated by the urotensin II peptide (UII), is selectively expressed by the cholinergic neurons of the PPTg. Although the exact function of cholinergic neurons of the PPTg is unknown, they are thought to contribute to the perception of reward magnitude or salience detection. We hypothesized that the activation of PPTg cholinergic neurons would alter sensory processing across multiple modalities (ex. taste and hearing). Here we had three aims: first, determine if cholinergic activation is involved in consumption behavior of palatable solutions (sucrose). Second, if so, distinguish the impact of the caloric value by using saccharin, a zero calorie sweetener. Lastly, we tested the UII-mediated effects on perception of acoustic stimuli by measuring acoustic startle reflex (ASR). Male Sprague-Dawley rats were bilaterally cannulated into the PPTg, then placed under food restriction lasting the entire consumption experiment (water ad lib.). Treatment consisted of a microinjection of either 1 µL of aCSF or 1 µL of 10 µM UII into the PPTg, and the rats were immediately given access to either sucrose or saccharin. For the remaining five days, rats were allowed one hour access per day to the same sweet solution without any further treatments. During the saccharin experiment rats were tested in a contact lickometer which recorded each individual lick to give insight into the microstructure of the consumption behavior. ASR testing consisted of a baseline (no treatment), treatment day, and two additional days (no treatment). Immediately following the microinjection of UII, consumption of both saccharin and sucrose increased compared to controls. This significant increase persisted for days after the single administration of UII, but there was no generalized arousal or increase in water consumption between testing sessions. The effects on ASR were not significant. Activating cholinergic PPTg neurons may lead to a miscalculation of the salience of external stimuli, implicating the importance of cholinergic input in modulating a variety of behaviors. The long-lasting effects seen after UII treatment support further research into the role of sensory processing on reward related-behaviors at the level of the PPTg cholinergic neurons.

Functional effects of urotensin-II on intracellular pH regulators in human radial artery smooth muscle cells.

The regulation of intracellular pH (pHi) plays a vital role in various cellular functions. We previously demonstrated that three different acid extruders, the Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC) and H+-linked monocarboxylate transporter (MCT), functioned together in cultured human radial artery smooth muscle cells (HRASMCs). However, the functions of acid-loading transporters in HRASMCs remain poorly understood. Urotensin II (U-II), one of the most potent vasoconstrictors, is highly expressed in many cardiovascular diseases. The aim of this present study was to determine the concentration effect of U-II (3 pM∼100 nM) on the functional activity of pHi regulators in HRASMCs. Cultured HRASMCs were derived from segments of human radial arteries obtained from patients undergoing bypass grafting. Changes in pHi recovery due to intracellular acidification and alkalization induced by NH4Cl prepulse and Na-acetate prepulse, respectively, were detected by microspectrofluorimetry with the pH-sensitive fluorescent dye BCECF. Our present study showed that (a) U-II increased the activity of NHE in a concentration-dependent manner but did not change that of NBC or MCT or resting pHi, (b) the Cl--OH- exchanger (CHE) facilitated base extrusion, and (c) U-II induced a concentration-dependent increase in the activity of CHE. In conclusion, for the first time, our results highlight a concentration-dependent increase in the activity of NHE and CHE, but not NBC and MCT, induced by U-II in HRASMCs.

Urotensin II and urantide exert opposite effects on the cellular components of atherosclerotic plaque in hypercholesterolemic rabbits.

Increasing levels of plasma urotensin II (UII) are positively associated with atherosclerosis. In this study we investigated the role of macrophage-secreted UII in atherosclerosis progression, and evaluated the therapeutic value of urantide, a potent competitive UII receptor antagonist, in atherosclerosis treatment. Macrophage-specific human UII-transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet for 16 weeks to induce atherosclerosis. Immunohistochemical staining of the cellular components (macrophages and smooth muscle cells) of aortic atherosclerotic lesions revealed a significant increase (52%) in the macrophage-positive area in only male transgenic rabbits compared with that in the nontransgenic littermates. However, both male and female transgenic rabbits showed a significant decrease (45% in males and 31% in females) in the smooth muscle cell-positive area compared with that of their control littermates. The effects of macrophage-secreted UII on the plaque cellular components were independent of plasma lipid level. Meanwhile the wild-type rabbits were continuously subcutaneously infused with urantide (5.4 µg· kg-1· h-1) using osmotic mini-pumps. Infusion of urantide exerted effects opposite to those caused by UII, as it significantly decreased the macrophage-positive area in male wild-type rabbits compared with that of control rabbits. In cultured human umbilical vein endothelial cells, treatment with UII dose-dependently increased the expression of the adhesion molecules VCAM-1 and ICAM-1, and this effect was partially reversed by urantide. The current study provides direct evidence that macrophage-secreted UII plays a key role in atherogenesis. Targeting UII with urantide may promote plaque stability by decreasing macrophage-derived foam cell formation, which is an indicator of unstable plaque.

Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure.

BACKGROUND/AIMS: Skeletal muscle atrophy is one of the main manifestations of protein energy wasting. We hypothesized that urotensin II (UII) can lead to skeletal muscle atrophy through upregulating autophagy and affecting Irisin precursor fibronectin type III domain containing 5 (FNDC5) expressions. METHODS: Three animal models (the sham operation, wild-type C57BL/6 mice with 5/6 nephrectomy, UII receptor (UT) gene knockout (UTKO) mice with 5/6 nephrectomy) were designed. Skeletal muscle weight, cross-sectional area (CSA) along with UII, FNDC5, LC3, and p62 expression were investigated. C2C12 cells were differentiated for up to 4 days into myotubes. These cells were then exposed to different UII concentrations (10-5 to 10-7 M) for 6-12 h and analyzed for the expressions of autophagic markers. These cells were also exposed to the same predetermined UII concentrations for 48-72 h and analyzed for the FNDC5 expression. Myotube diameter was measured. RESULTS: Upregulation of UII expression in skeletal muscle tissue was accompanied by reduced muscle weight and skeletal muscle CSA in the 2 posterior limbs, upregulated autophagy markers expression, and downregulated FNDC5 expression in 5/6 nephrectomy mice. The decrease of skeletal muscle weight, skeletal muscle CSA, downregulation of FNDC5 expression, and the upregulation of autophagy markers were inhibited in UTKO with 5/6 nephrectomy mice. Our in vitrostudy showed that UII could directly decrease myotube diameter, induce autophagy markers upregulation, and inhibit expression of FNDC5. When UII receptor gene was interfered by UT-specific siRNA, UII induced autophagy markers upregulation and FNDC5 downregulation were inhibited. CONCLUSION: We are the first to verify UII induces mice skeletal muscle atrophy associated with enhanced skeletal muscle autophagy and inhibited FNDC5 expression in chronic renal failure.

A Novel and Highly Potent Urotensin II Receptor Antagonist Inhibits Urotensin II-Induced Pressure Response in Mice.

This study was designed to characterize the pharmacological profile of DS37001789, which is a structurally novel piperazine derivative that acts as urotensin II (U-II) receptor antagonist. DS37001789 inhibited [I]-U-II binding to human GPR14, U-II receptor, with an IC50 value 0.9 nM. Its potency was superior to that of ACT-058362, a nonpeptide U-II receptor antagonist whose IC50 was 120 nM. Human U-II-induced vascular contraction was blocked by DS37001789. The dose-response curve of DS37001789 in rats and monkeys did not show species differences, and it shifted to the right without any effects on the maximum vascular response. Moreover, orally administered DS37001789 dose-dependently prevented human U-II-induced blood pressure elevation in mice, and this effect was significant at dose and higher dose (30 and 100 mg/kg), and its potency was superior to that of ACT-058362 (100 mg/kg). These results suggest that DS37001789 is a highly potent U-II receptor antagonist both in vitro and in vivo, with no marked species difference. DS37001789 would be a useful tool to clarify the physiological roles of U-II/GPR14 system. In addition, it can serve as a novel therapeutic agent for diseases in which the U-II/GPR14 system is upregulated, such as hypertension, heart failure, renal dysfunction, and diabetes.

Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides.

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.