Argirein alleviates stress-induced and diabetic hypogonadism in rats via normalizing testis endothelin receptor A and connexin 43.

AIM: Argirein (rhein-arginine) is a derivative of rhein isolated from Chinese rhubarb (Rheum Officinale Baill.) that exhibits antioxidant and anti-inflammatory activities. In the present study we investigated the effects of argirein on stress-induced (hypergonadotrophic) and diabetic (hypogonadotrophic) hypogonadism in male rats. METHODS: Stress-induced and diabetic hypogonadism was induced in male rats via injection of isoproterenol (ISO) or streptozotocin (STZ). ISO-injected rats were treated with argirein (30 mg·kg(-1)·d(-1), po) or testosterone replacement (0.5 mg·kg(-1)·d(-1), sc) for 5 days, and STZ-injected rats were treated with argirein (40-120 mg·kg(-1)·d(-1), po) or aminoguanidine (100 mg·kg(-1)·d(-1), po) for 4 weeks. After the rats were euthanized, blood samples and testes were collected. Serum hormone levels were measured, and the expression of endothelin receptor A (ETA), connexin 43 (Cx43) and other proteins in testes was detected. For in vitro experiments, testis homogenate was prepared from normal male rats, and incubated with ISO (1 µmol/L) or high glucose (27 mmol/L). RESULTS: ISO injection induced hyper-gonadotrophic hypogonadism characterized by low testosterone and high FSH and LH levels in the serum, whereas STZ injection induced hypogonadotrophic hypogonadism as evidenced by low testosterone and low FSH and LH levels in the serum. In the testes of ISO- and STZ-injected rats, the expression of ETA, MMP-9, NADPH oxidase and pPKCε was significantly increased, and the expression of Cx43 was decreased. Administration of argirein attenuated both the abnormal serum hormone levels and the testis changes in ISO- and STZ-injected rats, and aminoguanidine produced similar actions in STZ-injected rats; testosterone replacement reversed the abnormal serum hormone levels, but did not affect the testis changes in ISO-injected rats. Argirein (0.3-3 µmol/L) exerted similar effects in testis homogenate incubated with ISO or high glucose in vitro. CONCLUSION: Two types of hypogonadism of male rats exhibit increased expression of ETA and depressed expression of Cx43 in testes, despite different patterns of serum FSH and LH. Argirein alleviates the two types of male hypogonadism via normalizing ETA and Cx43 in testes.

Immunohistochemical assessment of endothelin-1 axis in psoriasis, basal cell carcinoma and squamous cell carcinoma.

AIM: Endothelin-1 is an autocrine growth factor for keratinocytes, an effect controlled by its A and B receptors, with no previous comparison of endothelin axis expression in inflammatory and neoplastic skin diseases showing keratinocyte proliferation. The aim of the study was to investigate endothelin-1 axis expression in skin lesions of psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). METHODS: This study included 40 subjects (8 patients with SCC, 12 patients with BCC, 10 patients with psoriasis, and 10 healthy controls). Biopsies from lesional skin of patients and normal skin of controls were examined immunohistochemically for endothelin-1 and its receptors A and B frequency and grade of expression. RESULTS: Endothelin-1 and receptor A were detected in all patients with SCC and psoriasis, with a higher frequency and grade of expression than controls and BCC. The frequency of receptor B expression was significantly lower while higher staining grade was found in BCC (8.3%) rather than other studied groups. CONCLUSION: A comparable higher frequency and grade of expression of endothelin-1 and its receptor A are documented in psoriasis and SCC than in BCC and controls denoting their involvement in keratinocyte proliferation in both diseases. Receptor A is the predominately expressed receptor in psoriasis and SCC.

Immunoreactive endothelin-1 and endothelin a receptor in basilar artery perivascular nerves of young and adult capybaras.

The purpose of this qualitative morphological study was the immunocytochemical and ultrastructural comparison of perivascular nerves of the basilar artery (BA) of young (6-month-old) and adult (12-month-old) capybaras - adult capybaras showed regression of the internal carotid artery (ICA). The study focused on immunolabeling for the vasoactive peptide endothelin-1 (ET-1) and endothelin A receptor (ETA) as well as for the synapse marker synaptophysin (SYP). In the BA of young capybaras, immunoreactivity for ET-1, ETA receptor and SYP was detected in perivascular nerve varicosities and/or intervaricosities. Immunoreactivity for ET-1 and ETA receptor was also displayed by some Schwann cells, which accompanied perivascular nerves. In addition to the presence of the above-described perivascular nerve characteristics, the BA of adult animals also revealed structurally altered perivascular nerves, where axon profiles were irregular in shape with dense axoplasm, while the cytoplasm of Schwann cells was vacuolated and contained myelin-like figures. These structurally altered perivascular nerves displayed immunoreactivity for ET-1, ETA receptor and SYP. These results show that the ET-1 system is present in some of the BA perivascular nerves and it is likely that this system is affected during animal maturation when ICA regression takes place. The role of ET-1 in cerebrovascular nerves is still unclear but its involvement in neural (sensory) control of cerebral blood flow and nerve function is possible.

Expression patterns of endothelin-1 and its receptors in colorectal cancer.

BACKGROUND AND OBJECTIVES: Endothelin-1 (ET-1), a potent vasoconstricting peptide, plays an important role in carcinogenesis. Previous in vitro studies have shown that colorectal cancer cells produce ET-1. METHODS: ET-1 and its receptors ET-A (ET(A) R) and ET-B (ET(B) R) were analyzed in colorectal cancer cell lines and tumors by Western blot and immunohistochemistry. Also, ET-1 levels were measured by ELISA in blood samples collected before and after tumor resection. RESULTS: ET-1 was immunohistochemically expressed by tumor cells at a variable level in 39 cases tested. The adjacent normal mucosa was negative for ET-1 expression. Strong ET(A) R expression observed in the deeper infiltrating areas at the periphery of neoplastic tissue correlated significantly with tumor stage. ET(B) R levels were very low or undetectable. Western blot analysis in paired (normal, tumor) fresh-frozen samples of colorectal cancers and in four colon carcinoma cell lines confirmed these findings. In addition, lower levels of ET-1 in the peripheral circulation after the tumor resection were found by ELISA as compared to those observed before surgery. CONCLUSIONS: ET-1 and ET(A) R, but not ET(B) R, are expressed at a higher level in primary and cultured colon carcinoma cells as compared to normal colon mucosa cells. Further functional studies are needed to explore the role of ET-1/ET(A) R axis in colon carcinogenesis.

Activation of endothelin-1 receptor signaling pathways is associated with neointima formation, neoangiogenesis and irreversible pulmonary artery hypertension in patients with congenital heart disease.

BACKGROUND: It is unclear why some patients, who undergo complete repair or palliative surgery for congenital heart disease (CHD), still develop irreversible pulmonary artery hypertension (PAH). There is no consensus to preoperationally assess the reversible and irreversible pulmonary vasculopathy seen in PAH. METHODS AND RESULTS: The peri-operative pulmonary hemodynamic data of 16 CHD patients (reversible PAH, n = 6; irreversible PAH, n = 10) were analyzed. The lung biopsies were also performed during surgery for defining histopathological characteristics as well as immunohistochemical expression of endothelin-1 (ET-1), endothelin-1 receptors (ETR), and its downstream signaling markers in the small pulmonary arteries and arterioles. Neointimal formation and neoangiogenesis was characterized by increased intimal layer immunoreactivity for α-SMA, Factor VIII, CD34, and VEGF. Neointimal formation was found in 90% of patients and neoangiogenesis was found in 80% of patients with irreversible PAH. Neither was present in the reversible PAH group and the control group. Expression of ET-1 and ETR in the neointimal layer of the pulmonary arterioles was upregulated in irreversible PAH, and immunoreactivity of phospho-Akt, phospho-ERK1/2, and phospho-mTOR was also increased in irreversible PAH. CONCLUSIONS: Increased expression of ET-1, ETR, and activation of signaling pathways were observed in the pulmonary arteries and arterioles of irreversible PAH patients associated with CHD. Activation of these pathways might in turn lead to neointimal formation and neoangiogenesis and thus might contribute to irreversible pulmonary vascular abnormalities.

Endothelin-1 stimulates human adipocyte lipolysis through the ET A receptor.

OBJECTIVE: Levels of the vascular peptide endothelin-1 (ET-1) are significantly elevated in obesity. Adipose tissue-derived ET-1 attenuates insulin-mediated antilipolysis in human visceral adipocytes through the activation of the ET receptor B (ET(B)R), thereby linking ET-1 to insulin resistance. Whether ET-1 has direct effects on lipolysis in human adipocytes is not known. RESEARCH DESIGN AND SUBJECTS: Endothelin-1 receptor (ETR) mRNA expression was determined by quantitative PCR in 130 non-obese and obese subjects. ET-1 mRNA in different adipose tissue regions was also assessed. ETR protein expression was analyzed by western blotting in 37 subjects. The effect of ET-1 on lipolysis was assessed in freshly isolated adipocytes and in vitro differentiated adipocytes from human donors. RESULTS: Freshly isolated human adipocytes incubated with different concentrations of ET-1 showed no acute effect on lipolysis. In contrast, a 24 h incubation in primary cultures of human adipocytes resulted in a significant 50% increase in lipolysis. This effect was concentration dependent and could be mimicked by an agonist of the ET(A) receptor but not with a selective ET(B)R agonist. Adipocyte differentiation was not affected by any of the agonists. In subcutaneous (s.c.) adipose tissue from 19 non-obese and 18 obese subjects, the protein expression of ET(A)R was significantly higher in obese subjects whereas there was no difference in ET(B)R expression. Interestingly, the differences in protein expression were not observed at the mRNA level as ET(A)R expression was similar between lean and obese subjects. CONCLUSION: Long-term but not acute incubation of human adipocytes with ET-1 results in a significant increase in lipolysis. This appears to be mediated through the activation of ET(A)R, demonstrating a yet another receptor-specific effect of ET-1. In addition, the protein expression of ET(A)R is increased in s.c. adipose tissue in obesity, possibly through post-transcriptional mechanisms. An increased effect of ET-1 could be a mechanism that contributes to increased basal lipolysis in human obesity.

Expression and bioactivities of endothelin-1 in gingiva during cyclosporine A treatment.

BACKGROUND AND OBJECTIVE: This study aimed to evaluate the expression and bioactivities of endothelin-1 (ET-1) in gingiva during cyclosporine A (CsA) treatment. MATERIAL AND METHODS: After establishing edentulous ridges, experimental rats were fed 30 mg/kg/day CsA while control animals received mineral oil for 4 weeks, after which a reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemistry was used to examine the expression of ET-1, its receptors, proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) in gingivae. The roles of the endothelin receptors A and B (ET(A) and ET(B)) in CsA-enhanced expression of PCNA and iNOS were examined in cultured human gingival fibroblasts pretreated with receptor antagonists, by immunocytochemistry and RT-PCR, respectively. RESULTS: The mRNA expression of ET-1, ET(A) and ET(B), as well as of PCNA and iNOS, was significantly greater in edentulous gingiva that received CsA compared with control gingiva. Immunohistochemistry revealed more cells positively stained for ET-1 and its receptors in the tissues of CsA-treated rats than in those of control rats. In fibroblast cultures, enhanced mRNA expression of ET-1, ET(A) and ET(B) was observed after CsA treatment at the concentrations of 10 and 100 ng/mL. Cyclosporine A-enhanced PCNA expression was somewhat reduced by blockade of ET(A), but not ET(B), whereas iNOS expression was somewhat reduced by blockade of ET(B). CONCLUSION: Based on the present findings, we suggest that: (1) CsA upregulates the gingival expression of ET-1 and its receptors; and (2) ET(A) and ET(B) have different bioactivities, ET(A) being involved in cell proliferation and ET(B) being associated with iNOS expression.

Role of endothelin receptor type A on catecholamine regulation in the olfactory bulb of DOCA-salt hypertensive rats: Hemodynamic implications.

Increasing evidence shows that the olfactory bulb is involved in blood pressure regulation in health and disease. Enhanced noradrenergic transmission in the olfactory bulb was reported in hypertension. Given that endothelins modulate catecholamines and are involved in the pathogenesis of hypertension, in the present study we sought to establish the role of the endothelin receptor type A on tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in the olfactory bulb of DOCA-salt hypertensive rats. Sprague-Dawley male rats, randomly divided into Control and DOCA-Salt hypertensive groups, were used to assess endothelin receptors by Western blot and confocal microscopy, and their co-localization with tyrosine hydroxylase in the olfactory bulb. Blood pressure and heart rate as well as tyrosine hydroxylase expression and activity were assessed following BQ610 (ETA antagonist) applied to the brain. DOCA-Salt hypertensive rats showed enhanced ETA and decreased ETB expression. ETA co-localized with tyrosine hydroxylase positive neurons. Acute ETA blockade reduced blood pressure and heart rate and decreased the expression of total tyrosine hydroxylase and its phosphorylated forms. Furthermore, it also diminished mRNA tyrosine hydroxylase expression and accelerated the enzyme degradation through the proteasome pathway as shown by pretreatment with MG132, (20s proteasome inhibitor) intracerebroventricularly applied. Present findings support that the brain endothelinergic system plays a major role through ETA activation in the increase of catecholaminergic activity in the olfactory bulb of DOCA-Salt hypertensive rats. They provide rationale evidence that this telencephalic structure contributes in a direct or indirect way to the hemodynamic regulation in salt dependent hypertension.

Expression and function of ETA and ETB receptors in SSc.

Endothelin (ET) receptors are widely expressed within the human body with one of their major functions being regulation of the vascular tone. Pulmonary arterial hypertension and other complications associated with SSc are related to the function and or dysregulation of these receptors. As ET receptors also play a crucial role in SSc, this review will discuss the expression and physiological functions of ET receptors in the human organism, their signalling pathways, the complications of diseases they are associated with and their importance as a therapeutic target in SSc.

Effect of erythropoietin on blood pressure and on the vascular endothelial ET-1/ETB receptor system.

BACKGROUND: Recombinant human erythropoietin (rhEPO) increases blood pressure (BP) and the vascular production of endothelin-1 in renal failure rats. This study was designed to investigate the effect of rhEPO on BP and on the ET-1/ET(B)R system in rats with normal renal function. To further characterize the effect of rhEPO on the ET-1/ET(B)R system, we also studied heterozygous (+/-) ET(B)R knockout (KO) mice. METHODS: The animals received either the vehicle or rhEPO (100 U/kg subcutaneously three times per week). ET(B)R(+/-) mice were compared with ET(A)R(+/-) and wild-type (WT) mice. In rats, the ET(B)R mRNA expression was assessed in blood vessels as well as the vascular ET(B)R density using immunohistochemistry. In mice, ET-1 concentration was measured in the thoracic aorta. RESULTS: RhEPO administration increased hematocrit levels in all treated animals. This therapy had no effect on BP in normal rats, but it did increase vascular and renal cortex ET(B)R mRNA expression. Immunohistochemistry confirmed that the ET(B)R density was increased in blood vessel endothelium in these normal rats. In contrast, rhEPO increased BP in ET(B)R(+/-) mice and this pressor effect was associated with higher ET-1 concentrations in the thoracic aorta. CONCLUSIONS: RhEPO exerts a pleotropic effect on the endothelial ET-1/ET(B)R system. The increase in endothelial ET(B)R expression may contribute to maintaining normal BP during rhEPO administration in normal animals. Conversely, conditions with deficient ET(B)R expression, such as in ET(B)R(+/-) mice, may lead to hypertension while receiving the same therapy.

Age-related changes in endothelin-1 receptor subtypes in rat heart.

Density, affinity, and subtype distribution of endothelin-1 (ET-1) binding sites were determined in rat cardiac tissue as a function of age in order to evaluate the association of alterations in the endothelin receptor system and aging in the heart. A significant decrease in the receptor subtype ET-A, which represents 70% to 80% of the total receptor population in cardiac tissue of 3- and 12-month-old rats, was observed in 24-month-old rats with respect to the younger groups. These findings indicate an alteration in ET-1 cardiac receptors associated with aging, mainly due to a variation in the receptor subtype distribution.

Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus.

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression.

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Endothelin-2 induces oviductal contraction via endothelin receptor subtype A in rats.

Proper function of the oviduct is critical to reproductive success with regulated contraction and relaxation facilitating transportation of the germ cells to the site of fertilization. Endothelin-2 (EDN2) is a potent vasoconstrictor produced by granulosa cells of the preovulatory follicle at the time of ovulation; however, whether this gonadotropin surge-induced peptide played a role in facilitating germ cell transportation by inducing oviductal contraction was unknown. The objectives of these experiments were (1) to determine whether the endothelin receptor system was present in the oviduct, (2) to test the hypothesis that EDN2 induces oviductal contraction via a specific endothelin receptor subtype, (3) to determine, as a possible alternate source of the ligand, whether mRNA for EDN2 was expressed in cumulus-oocyte complexes (COCs) within the oviduct, and (4) to determine whether EDN2 could overcome prostaglandin E(2) (PGE(2))-induced oviductal relaxation. Microarray and real-time PCR analysis indicated that mRNA for both the endothelin receptor subtypes (ET(A) and ET(B)) was present in the oviduct, whereas immunohistochemical examination revealed that ET(A) protein was the dominant isoform, present in the luminal epithelial cells of the oviduct. Real-time PCR analysis demonstrated that mRNA for EDN2 was expressed in COCs after ovulation. Isometric tension analysis indicated that EDN2 was a potent oviductal constrictor and that the contractile effect of EDN2 was mediated by the ET(A) and not the ET(B) receptor subtype. The oviductal contraction induced by EDN2 also reversed oviductal relaxation induced by PGE(2). In summary, ET(A) receptor-specific EDN2-induced contraction as a facilitator of oviductal function suggests a novel pathway involved in germ cell transport and hence mammalian fertility.

Expression and prognostic relevance of endothelin-B receptor in vulvar cancer.

Overexpression of endothelin (ET)-1 and its receptors, ETAR and ETBR, commonly referred to as the 'ET-axis', has been demonstrated to play a role in cancer progression for various human tumours. Based on these results we propose a similar role of the expression of the ET-axis in vulvar cancer. Expression of the ET-axis was investigated immunohistochemically using tissue microarrays with tumour samples of 68 vulvar cancer patients. Samples were obtained from patients undergoing local excision or radical vulvectomy. ET-1 expression of tumour cells correlated highly significantly with early stages of vulvar cancer (p=0.004), whereas neither ETAR nor ETBR expression showed any association with TNM stages. High staining levels of ETBR in the tumour tissue were significantly related to tumour progression (p=0.01) and early metastases (p=0.09); low ETBR staining intensity correlated with longer relapse-free survival (p=0.019). In patients with ETBR overexpressing low-stage tumours (pT1-2) we observed a significantly reduced overall survival and disease-free survival (p=0.036 and 0.021, respectively). ETAR expression and ETBR expression were significantly correlative (p=0.018). Accordingly, co-expression of both receptors was related to tumour progression (p=0.022) and an increased risk for local recurrence (p=0.005). These results suggest that, in addition to established histological and clinical prognostic factors, analysis of ET-receptor and, in particular, of ETBR expression by means of simple immunohistochemical analysis might improve prediction of the prognosis for patients with vulvar squamous cell carcinoma.

Endothelin receptors and endothelin-1 in developing rat teeth.

INTRODUCTION: The endothelins are a family of small peptides with multiple roles in a variety of tissues. Signaling is mediated through two receptor subtypes, the endothelin A receptor (ET(A)) specific for Et-1 and the non-specific endothelin B receptor (ET(B)). OBJECTIVE: Our goal was to determine the location of immunoreactivity (IR) for ET(A) and ET(B) in developing and mature rat teeth as indicators of endothelin (Et) regulatory sites and to compare this to the Et-1 (ligand)-IR expression patterns. DESIGN: We used immunohistochemistry to study developing and mature rat molars and continuously developing incisors. RESULTS: We demonstrate ET(A), ET(B), and Et-1 expression patterns in teeth, for the first time. ET(A) was found in developing molar root pulp, pulpal vasculature, and preodontoblasts, and then persisted in odontoblasts or cellular cementocytes at the root apices of mature teeth. ET(B) was found at the molar (Hertwig's) root sheath during root formation and in molar ameloblasts, nerve fibers and odontoblasts of immature and mature teeth. In incisors, ET(B)-IR was associated with ameloblasts and the stem cell niche of the cervical loop while ET(A) was located in the substratum layer. Et-1 was found throughout the dental and periodontal tissues with higher concentrations associated with odontoblasts, nerves and incisor layers that expressed ET(B). CONCLUSION: The patterns of ET(A) and ET(B) in teeth differ from each other and from those of adjacent tissues suggesting multiple tooth-specific functions for endothelin during development and mature dental function.

Role of angiotensin II and endothelin-1 receptors in aging-related functional changes in rat cardiovascular system.

Angiotensin II (AII) and endothelin-1 (ET-1) are regarded as key players in the age-related changes in cardiovascular function. They are known to be involved in the pathogenesis of cardiac fibrosis and coronary vascular atherosclerosis. AII- and ET-induced vasoconstriction was augmented in coronary arteries of Langendorff-perfused heart from aged rats. In papillary muscles, ET-1-induced positive inotropic effect (PIE) was diminished by aging. On the other hand, both ET-1 and AII caused greater vasoconstriction in aged rat coronary arteries compared to those in the young rat. To further elucidate the mechanism of these age-dependent changes in cardiovascular effects of ET-1 and AII, we examined the expression of AII and ET-1 receptors in young (2-month-old) and aged (24-month-old) rats. Total RNA was isolated from left ventricles. For determination of the gene expression of AT(1) receptor and ET(A)/ET(B) receptor mRNA, competitive RT-PCR and Northern blot analysis were performed, respectively. [(125)I]ET-1 receptor assay was carried out in left ventricular membrane fraction. AT(1)-receptor, ET(A)-, and ET(B)-receptor mRNA were upregulated in the left ventricles of senescent rats compared with young ones. The affinity of ET-1-receptor was not changed, but receptor density was significantly increased in aged rats. Although the precise mechanism for the upregulation of AT(1) receptor and ET-1 receptor in the aged rat heart has not been clarified yet, these findings suggest that the activation of the renin-angiotensin system as well as ET receptor may be important for the physiological changes in aged hearts.

Effects of chronic cold exposure on the endothelin system.

Cold temperatures have adverse effects on the human cardiovascular system. Endothelin (ET)-1 is a potent vasoconstrictor. We hypothesized that cold exposure increases ET-1 production and upregulates ET type A (ETA) receptors. The aim of this study was to determine the effect of cold exposure on regulation of the ET system. Four groups of rats (6-7 rats/group) were used: three groups were exposed to moderate cold (6.7 +/- 2 degrees C) for 1, 3, and 5 wk, respectively, and the remaining group was maintained at room temperature (25 degrees C) and served as control. Cold exposure significantly increased ET-1 levels in the heart, mesenteric arteries, renal cortex, and renal medulla. Cold exposure increased ETA receptor protein expression in the heart and renal cortex. ET type B (ETB) receptor expression, however, was decreased significantly in the heart and renal medulla of cold-exposed rats. Cold exposure significantly increased the ratio of ETA to ETB receptors in the heart. An additional four groups of rats (3 rats/group) were used to localize changes in ETA and ETB receptors at 1, 3, and 5 wk of cold exposure. Immunohistochemical analysis showed an increase in ETA, but a decrease in ETB, receptor immunoreactivity in cardiomyocytes of cold-exposed rats. Increased ETA receptor immunoreactivity was also found in vascular smooth muscle cells of cold-exposed rats. Cold exposure increased ETA receptor immunoreactivity in tubule epithelial cells in the renal cortex but decreased ETB receptor immunoreactivity in tubule epithelial cells in the renal medulla. Therefore, cold exposure increased ET-1 production, upregulated ETA receptors, and downregulated ETB receptors.

The Role of MAPK Pathways in Airborne Fine Particulate Matter-Induced Upregulation of Endothelin Receptors in Rat Basilar Arteries.

Airborne fine particulate matter (PM(2.5)) increases the risk of cerebrovascular diseases. However, existing experimental data do not sufficiently explain how PM(2.5) affects cerebral vessels. This study sought to examine whether PM(2.5) alters endothelin (ET) receptor expression on rat cerebral arteries and the potential underlying mechanisms. Isolated rat basilar arteries were cultured with PM(2.5) aqueous suspension in the presence of mitogen-activated protein kinase (MAPK) pathway inhibitors. ET receptor-mediated vasomotor functions were recorded by a sensitive myograph. ET(A) and ET(B) receptor mRNA and protein expressions were assessed using quantitative real-time PCR, Western blotting, and immunohistochemistry, respectively. Compared with fresh and culture alone arteries, PM(2.5) significantly enhanced ET(A) and ET(B) receptor-mediated contractions and increased receptor mRNA and protein expressions in basilar arteries, indicating PM(2.5) upregulates ET(A) and ET(B) receptors. Culturing with SB386023 (MEK/ERK1/2 inhibitor), U0126 (ERK1/2 inhibitor), SP600125 [c-Jun N-terminal kinase (JNK) inhibitor], or SB203580 (p38 inhibitor) attenuated PM(2.5)-induced ETB receptor upregulation. PM(2.5)-induced enhancement of ET(A) receptor-mediated contraction and receptor expression was notably inhibited by SB386023 or U0126. However, neither SP600125 nor SB203580 had an effect on PM(2.5)-induced ET(A) receptor upregulation. In conclusion, PM(2.5) upregulates ET(A) and ET(B) receptors in rat basilar arteries. ET(B) receptor upregulation is involved in MEK/ERK1/2, JNK, and p38 MAPK pathways, and ET(A) receptors upregulation is associated with MEK/ERK1/2 pathway.

Endothelin-1 and endothelin receptors in the basilar artery of the capybara.

Little is known about cerebral vasculature of capybara, which seems may serve as a natural model of studying changes in cerebral circulation due to internal carotid artery atrophy at animal sexual maturation. This is the first study of the light- and electron-immunocytochemical localisation of endothelin-1 (ET-1) and ETA and ETB endothelin receptors in the basilar artery of capybaras (6 to 12-month-old females and males) using an ExtrAvidin detection method. All animals examined showed similar patterns of immunoreactivity. Immunoreactivity for ET-1 was detected in the endothelium and adventitial fibroblasts, whilst immunoreactivity for ETA and ETB receptors was present in the endothelium, vascular smooth muscle, perivascular nerves and fibroblasts. In endothelial cells immunoreactivity to ET-1 was pronounced in the cytoplasm or on the granular endoplasmic reticulum. Similar patterns of immunolabelling were observed for ETA and ETB receptors, though cytoplasmic location of clusters of immunoprecipitate seems dominant. These results suggest that the endothelin system is present throughout the wall of the basilar artery of capybara.