MEK1/2 inhibitor U0126 but not endothelin receptor antagonist clazosentan reduces upregulation of cerebrovascular contractile receptors and delayed cerebral ischemia, and improves outcome after subarachnoid hemorrhage in rats.

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia.

BACKGROUND AND PURPOSE: Increased expression of endothelin receptor type B (ETBR), a vasoactive receptor, has recently been implied in the reduced cerebral blood flow and exacerbated neuronal damage after ischemia-reperfusion (I/R). The study explores the regulatory mechanisms of ETBR to identify drug targets to restore normal cerebral artery contractile function as part of successful neuroprotective therapy. METHODS: We have employed in vitro methods on human and rat cerebral arteries to study the regulatory mechanisms and the efficacy of target selective inhibitor, Mithramycin A (MitA), to block the ETBR mediated contractile properties. Later, middle cerebral artery occluded (MCAO) rats were used to substantiate the observations. Quantative PCR, immunohistochemistry, western blot and wire myograph methods were employed to study the expression and contractile properties of cerebral arteries. RESULTS: Increased expression of specificity protein (Sp1) was observed in human and rat cerebral arteries after organ culture, strongly correlating with the ETBR upregulation. Similar observations were made in MCAO rats. Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels. It also significantly reduced the ETBR mediated cerebrovascular contractility. Detailed analysis indicated that ERK1/2 mediated phosphorylation of Sp1 might be essential for ETBR transcription. CONCLUSION: Transcription factor Sp1 regulates the ETBR mediated vasoconstriction in focal cerebral ischemia via MEK-ERK signaling, which is also conserved in humans. The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling.

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.

The ocular endothelin system: a novel target for the treatment of endotoxin-induced uveitis with bosentan.

PURPOSE: We compared the anti-inflammatory effects of bosentan and dexamethasone in endotoxin-induced uveitis (EIU). METHODS: Endotoxin-induced uveitis was induced by subcutaneous injection of lipopolysaccharide (LPS, 200 µg) in Wistar rats. Rats were divided randomly into 10 groups (n = 6). Bosentan at doses of 50 and 100 mg/kg were administered orally 1 hour before and 12 hours after LPS injection, and dexamethasone was administered by intraperitoneally 30 minutes before and 30 minutes after LPS injection at a dose of 1 mg/kg. Data were collected at two time points for each control and treatment; animals were killed at either 3 or 24 hours after LPS injection. Histopathologic evaluation and aqueous humour measurements of TNF-α level were performed, and endothelin-1 (ET-1), inducible nitric oxide synthase (iNOS), and endothelin receptor A and B (EDNRA and B) expression were analyzed. RESULTS: The group treated with 100 mg/kg bosentan at 24 hours displayed significantly milder uveitis and fewer inflammatory cells compared to LPS-injected animals, and there were similar findings in the dexamethasone-treated group at 24 hours. The TNF-α levels in the dexamethasone treatment group were lower than those in the LPS-induced uveitis control group (P < 0.05); however, there was no difference between the dexamethasone and bosentan treatment groups at 3 and 24 hours after LPS administration. Bosentan treatment at doses of 50 and 100 mg/kg significantly decreased iNOS expression compared to LPS-injected animals (P < 0.001). The ET-1 expression was suppressed significantly by bosentan and dexamethasone at 3 and 24 hours after LPS administration (P < 0.001). The EDNRA expression in the bosentan treatment groups was statistically significantly lower than that in the LPS-induced uveitis control group at 3 and 24 hours after LPS administration (P < 0.05). CONCLUSIONS: Bosentan reduces intraocular inflammation and has similar effects as dexamethasone in a rat model of EIU.

EDNRB and DCC salivary rinse hypermethylation has a similar performance as expert clinical examination in discrimination of oral cancer/dysplasia versus benign lesions.

PURPOSE: Promoter hypermethylation has been recently proposed as a means for head and neck squamous cell carcinoma (HNSCC) detection in salivary rinses. In a prospective study of a high-risk population, we showed that endothelin receptor type B (EDNRB) promoter methylation in salivary rinses is a useful biomarker for oral cancer and premalignancy. EXPERIMENTAL DESIGN: Using that cohort, we evaluated EDNRB methylation status and 8 additional genes. Clinical risk assessment by expert clinicians was conducted and compared with biomarker performance in the prediction of premalignant and malignant disease. Methylation status of 9 genes was analyzed in salivary rinses of 191 patients by quantitative methylation-specific PCR. RESULTS: HOXA9, EDNRB, and deleted in colorectal cancer (DCC) methylation were associated (P = 0.012; P < 0.0001; P = 0.0005) with premalignant or malignant disease. On multivariable modeling, histological diagnosis was only independently associated with EDNRB (P = 0.0003) or DCC (P = 0.004) methylation. A subset of patients received clinical risk classification (CRC) by expert clinicians based on lesion examination. CRC, DCC, and EDNRB were associated with diagnosis of dysplasia/cancer on univariate (P = 0.008; P = 0.026; P = 0.046) and multivariate analysis (P = 0.012; P = 0.037; P = 0.047). CRC identified dysplasia/cancer with 56% of sensitivity and 66% of specificity with a similar area under curve [AUC; 0.61, 95% confidence interval (CI) = 0.60-0.81] when compared to EDNRB and DCC combined AUC (0.60, 95% CI = 0.51-0.69), sensitivity of 46% and specificity of 72%. A combination of EDNRB, DCC, and CRC was optimal AUC (0.67, 95% CI = 0.58-0.76). CONCLUSIONS: EDNRB and/or DCC methylation in salivary rinses compares well to examination by an expert clinician in CRC of oral lesions. These salivary biomarkers may be particularly useful in oral premalignancy and malignancy screening in clinical care settings in which expert clinicians are not available.

Effects of cyclic intermittent hypoxia on ET-1 responsiveness and endothelial dysfunction of pulmonary arteries in rats.

Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular disorders and in some cases is complication of pulmonary hypertension. We simulated OSA by exposing rats to cyclic intermittent hypoxia (CIH) to investigate its effect on pulmonary vascular endothelial dysfunction. Sprague-Dawley Rats were exposed to CIH (FiO2 9% for 1 min, repeated every 2 min for 8 h/day, 7 days/wk for 3 wk), and the pulmonary arteries of normoxia and CIH treated rats were analyzed for expression of endothelin-1 (ET-1) and ET receptors by histological, immunohistochemical, RT-PCR and Western Blot analyses, as well as for contractility in response to ET-1. In the pulmonary arteries, ET-1 expression was increased, and ET-1 more potently elicited constriction of the pulmonary artery in CIH rats than in normoxic rats. Exposure to CIH induced marked endothelial cell damage associated with a functional decrease of endothelium-dependent vasodilatation in the pulmonary artery. Compared with normoxic rats, ETA receptor expression was increased in smooth muscle cells of the CIH rats, while the expression of ETB receptors was decreased in endothelial cells. These results demonstrated endothelium-dependent vasodilation was impaired and the vasoconstrictor responsiveness increased by CIH. The increased responsiveness to ET-1 induced by intermittent hypoxia in pulmonary arteries of rats was due to increased expression of ETA receptors predominantly, meanwhile, decreased expression of ETB receptors in the endothelium may also participate in it.

Tamoxifen modulates cell migration and expression of angiogenesis-related genes in human endometrial endothelial cells.

The selective estrogen receptor modulator tamoxifen is used for the prevention and treatment of breast cancer. The adverse effects of tamoxifen include vaginal endometrial bleeding, endometrial hyperplasia, and cancer, conditions associated with angiogenesis. The aim of this study was to examine the effects of tamoxifen on cell migration and angiogenesis-related gene expression in human endometrial endothelial cells (HEECs). The regulatory effects of tamoxifen on endometrial stromal cells and HEECs were also examined. HEECs and stromal cells were isolated and grown in monocultures or co-cultures, and incubated with 0.1 to 100 µmol/L tamoxifen for 48 hours. Quantitative PCR demonstrated that tamoxifen decreased the mRNA expression of vascular endothelial growth factor-A (VEGF-A) and increased the mRNA expression of VEGF receptor-1 and placental growth factor (PLGF) in HEECs. Tamoxifen's effects on VEGF-A were inhibited when HEECs were co-cultured with stromal cells. In addition, tamoxifen reduced VEGF-induced HEEC migration. The tamoxifen-metabolizing enzymes CYP1A1 and CYP1B1 were detected by immunohistochemistry in and around endometrial blood vessels and by quantitative PCR in HEECs. Our data suggest that tamoxifen changes the regulation of angiogenesis in the endometrium, likely by reducing angiogenic activity. The results also indicate that endometrial stromal cells regulate some of tamoxifen's effects in HEECs, and the presence of tamoxifen-metabolizing enzymes suggests tamoxifen bioactivation in the endometrial vasculature in vivo. These findings may help to elucidate the mechanism of the bleeding disturbances associated with tamoxifen treatment.

Endothelial endothelin B receptor-mediated prevention of cerebrovascular remodeling is attenuated in diabetes because of up-regulation of smooth muscle endothelin receptors.

Structure and function of the cerebrovasculature is critical for ischemic stroke outcome. We showed that diabetes causes cerebrovascular remodeling by activation of the endothelin A (ET(A)) receptors. The goal of this study was to test the hypotheses that vasculoprotective endothelial ET(B) receptors are decreased and pharmacological inhibition of the ET(B) receptor augments vascular remodeling of middle cerebral arteries (MCAs) in type 2 diabetes. MCA structure, matrix metalloprotease (MMP) activity, and matrix proteins as well as ET(A) and ET(B) receptor profiles were assessed in control Wistar and diabetic Goto-Kakizaki rats treated with vehicle, the ET(B) receptor antagonist (2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-[(2,6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxyphenyl)pyrrolidine-3-carboxylic acid (A192621) (30 mg/kg/day), or the dual ET receptor antagonist bosentan (100 mg/kg/day) for 4 weeks. Diabetes increased vascular smooth muscle (VSM) ET(A) and ET(B) receptors; the increase was prevented by chronic bosentan treatment. MCA wall thickness was increased in diabetes, and this was associated with increased MMP-2 activity and collagen deposition but reduced MMP-13 activity. Because of up-regulation of VSM ET receptors in diabetes, selective ET(B) receptor antagonism with A192621 blunts this response, and combined ET(A) and ET(B) receptor blockade with bosentan completely prevents this response. On the other hand, A192621 treatment augmented remodeling in control animals, indicating a physiological protective role for this receptor subtype. Attenuation of changes in ET receptor profile with bosentan treatment suggests that ET-1 has a positive feedback on the expression of its receptors in the cerebrovasculature. These results emphasize that ET receptor antagonism may yield different results in healthy and diseased states.

Aging-related kidney damage is associated with a decrease in klotho expression and an increase in superoxide production.

The purpose of this study was to determine changes in klotho, endothelin (ET) receptors, and superoxide production in kidneys of aged rats and whether these changes are exacerbated in aged rats with cognitive impairment. Twenty aged rats (male, 27 months) were divided into an Old Impaired group (n=9) and an Old Intact group (n=11) according to a cognitive function test. A group of 12-month-old rats (n=10) was used as a Young Intact group. Serum creatinine was increased significantly in the Old Impaired group, suggesting impaired renal function. Aged rats showed glomerulosclerosis and tubulointerstitialfibrosis. These pathological changes were markedly aggravated in the old cognitively impaired than in the old cognitively intact animals. Notably, aged rats demonstrated a significant decrease in klotho protein expression in renal cortex and medulla. Protein expression of IL-6, Nox2, ETa receptors and superoxide production were increased whereas mitochondrial SOD (MnSOD) and ETb receptors expression were decreased in kidneys of the aged rats. Interestingly, these changes were more pronounced in the old impaired than in the old intact rats. In conclusion, the aging-related kidney damage was exacerbated in aged rats with cognitive impairment. Klotho, ETB, and MnSOD were downregulated but ETa, IL-6, Nox2, and superoxide production were upregulated in the aging-related kidney damage. These changes were more pronounced in rats with cognitive impairment.

Up-regulation of G-protein-coupled receptors for endothelin and thromboxane by lipid-soluble smoke particles in renal artery of rat.

Up-regulation of G-protein-coupled receptors (GPCR) plays key roles in renal hypertension and cardiovascular disease pathogenesis. The present study was designed to examine if lipid-soluble cigarette smoking particles (DSP), nicotine and endotoxin (LPS), induce GPCR up-regulation for thromboxane A(2) (TP), endothelin type A (ET(A) ) and type B (ET(B) ) receptors in renal artery, and if intracellular signal mechanisms are involved. Renal artery segments of rats were exposed to DSP, nicotine or LPS, in organ culture for up to 24 hr. The GPCR-mediated contractions were recorded by using a myograph system. Expression of the GPCR was examined by real-time PCR and immunohistochemistry at mRNA and protein levels. Sarafatoxin 6c (S6c, selective ET(B) receptor agonist), endothelin-1 (ET-1, non-selective ET(A) and ET(B) receptor agonist) and 9,11-Dideoxy-9a,11a-methanoepoxy prostaglandin F(2a) (U46619, a TP receptor agonist) induced contractions were significantly increased after the arterial segments exposed to DSP in a concentration-dependent (0.1-0.4 µl/ml) manner, and S6c also induced a time-dependent contraction, compared to control (dimethyl sulfoxide). This was in parallel with enhanced mRNA expression for ET(B) receptor but not ET(A) and TP receptors, while increased protein expression for ET(A) , ET(B) and TP receptors was seen. The specific nuclear factor-kappa B (NF-κB) signal pathway inhibitor BMS345541 was applied to link DSP effects to the GPCR up-regulation. It totally abolished ET(B) receptor up-regulation, but not ET(A) and TP receptor up-regulations. Our results suggest that DSP transcriptionally up-regulated ET(B) receptor expression in rat renal artery via NF-κB signal pathways, whereas up-regulation of ET(A) and TP receptor-mediated contraction may involve post-transcriptional mechanisms.

Increased expression of the endothelin system in arterial lesions from patients with giant-cell arteritis: association between elevated plasma endothelin levels and the development of ischaemic events.

OBJECTIVE: Approximately 15-20% of patients with giant-cell arteritis (GCA) develop ischaemic complications often preceded by transient ischaemia. The expression of the endothelin (ET) system in GCA lesions was investigated to assess its relationship with the development of ischaemic complications. METHODS: Plasma ET-1 was quantified by immunoassay in 61 patients with biopsy-confirmed GCA and 16 healthy donors. ET-1, endothelin-converting enzyme (ECE-1) and endothelin receptor (ET(A)R and ET(B)R) messenger RNA were measured by real-time quantitative reverse transcriptase-PCR in temporal arteries from 35 of these patients and 19 control arteries. Proteins were measured by immunoassay and Western blot. RESULTS: ET-1 concentration was increased at the protein level in temporal artery samples from GCA patients compared with controls (0.98 (SEM 0.32) vs 0.28 (SEM 0.098) fmol/mg, p = 0.028). ECE-1, ET(A)R and ET(B)R/actin ratios (Western blot) were also significantly higher in GCA patients. Intriguingly, mRNA expression of ET-1, ECE-1 and both receptors was significantly reduced in GCA lesions compared with control arteries. When investigating mechanisms underlying these results, platelet-derived growth factor and IL-1beta, present in GCA lesions, were found to downregulate ET-1 mRNA in cultured human temporal artery-derived smooth muscle cells. Glucocorticoid treatment for 8 days did not result in significantly decreased endothelin tissue concentration (0.87 (SEM 0.2) vs 0.52 (SEM 0.08); p = 0.6). Plasma endothelin concentrations were higher in patients with ischaemic complications (1.049 (SEM 0.48) vs 1.205 (SEM 0.63) pg/ml, p = 0.032). CONCLUSIONS: The endothelin system is increased at the protein level in GCA lesions creating a microenvironment prone to the development of ischaemic complications. Recovery induced by glucocorticoids is delayed, indicating persistent exposure to endothelin during initial treatment.

Endothelin-1 enhances proliferation of lung cancer cells by increasing intracellular free Ca2+.

Endothelin-1 (ET-1), the most potent vasoconstrictor, has been shown to be mitogenic in many tumor cells as well as in vascular cells. It was previously reported that the mRNA of ET-1 and endothelin receptors (ETRs) are expressed in lung cancer cells. However, their biological role in lung cancer remains to be explored. The purpose of this study was to determine whether ET-1 stimulates proliferation of the human lung adenocarcinoma cell SPC-A1 and probe its cellular mechanism. Reverse-transcription polymerase chain reaction and Western blot analysis showed that both the mRNA and protein of ET-1, ET A R and ET B R are expressed in SPC-A1 cells. Application of ET-1 at 10(-15)-10(-8) M caused a dose-dependent cell proliferation and an increase in intracellular free Ca2+ concentration ([Ca2+]i). This ET-1-induced cell proliferation and [Ca2+]i increase were completely abolished by BQ123, a selective ET A R antagonist, but not by BQ788, a selective ET B R antagonist. Furthermore, it was significantly reduced by U73122, a specific inhibitor of phospholipase C (PLC), but not by U73433, the structural isomer of U73122. Chelating extracellular Ca2+ or blocking voltage dependent calcium channels by nifedipine also significantly reduced the mitogenic effect of ET-1 and [Ca2+]i increase in SPC-A1 cells. These results indicate that ET-1 acts as an autocrine growth factor and enhances proliferation of SPC-A1 cells via activation of ET A R. The phosphoinositol/Ca2+ pathway and Ca2+ influx through voltage dependent Ca2+ channels activated by ET A R contribute to this process.

Regulation and expression of endothelin-1 (ET-1) and ET-receptors in rat epithelial cells of renal and intestinal origin.

The hormone endothelin-1 (ET-1) is involved in many functions of the kidney and intestine. In addition to its vasoactive and proliferative effects, ET-1 is involved in the maintenance of water and salt balance, and in drug excretion by influencing the activity of different transporters in the epithelial cells of these two organs. To study ET-1 function and its role in pathophysiological processes in epithelial cells in vitro, we investigated ET-1 and ET-receptor expression and inducibility of ET-1 excretion by cytokines in three rat cell lines of intestinal (IEC-6) and renal (NRK-52E and GERP) origin. Immunocytochemistry showed that all three cell lines express ET-1 and the ET-A and ET-B receptor. ET-1 was expressed intracellularly, and also the ET-A receptor showed a punctate intracellular staining pattern. The ET-B receptor was localized in the membrane, which was confirmed by Western blot analysis. Real-time RT-PCR and ELISA showed that exposure of IEC-6 cells to the cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha), induced ET-1 mRNA expression and excretion, while IL-2 was ineffective. In NRK-52E cells, IL-1beta and TNFalpha induced ET-1 excretion as well. In GERP cells, adequate measurement of cytokine effects on ET-1 excretion was not possible, since ET-1 excretion under non-stimulated conditions was around the lowest level of detection. In conclusion, we showed ET-1 and ET-receptor expression, and inducibility of ET-1 by cytokines in IEC-6, NRK-52E, and GERP cells. These rat intestinal and renal cell lines appear to be suitable for further characterisation of ET-1 function and its role in pathophysiological processes in epithelial cells.

Expression of endothelins and their receptors in glioblastoma cell lines.

The endothelins (ETs) are a family of three peptides named ET-1, ET-2 and ET-3 that have been initially isolated as potent vasoactive peptides; ETs are synthesized as precursor proteins (preproETs) and are activated by proteolytic cleavage. ETs exert their biological effects through the activation of two receptors subtypes, ETA and ETB. Recent studies have shown that, besides its vascular effects, ET-1 appears to play a major role also in the growth and progression of various types of cancers and ETA or ETB are alternatively indicated as mediators of the ET-1 biological effects. In this study we have investigated the expression and the amounts of preproET-1, preproET-2, ETA and ETB receptors mRNA by classical RT-PCR and quantitative real-time PCR in one human low grade astrocytoma cell line and eight human glioblastoma cell lines. PCR products corresponding to ETB receptor and preproET-1 were detectable in all the cell lines whilst ETA receptor and preproET-2 were only detected in five cell lines. Quantitative real-time PCR experiments showed wide differences in the amounts of mRNAs among the cell lines examined. Range values were 0.23-4860.51 fg/mug total cDNA for preproET-1; 0.13-3330.18 fg/mug total cDNA for preproET-2; 0.63-286.12 fg/mug total cDNA for ETA and 14.40-6720.67 fg/mug total cDNA for ETB. We measured the ET-1 released in the extracellular medium by an ELISA assay and we found an excellent correlation (correlation coefficient r = 0.9526, P = 0.0003) between the amounts of preproET-1 mRNA and released ET-1 peptide. Finally, in the 1321N1 cell line ETB receptors are functionally coupled to intracellular signaling pathways because the stimulation of ETB receptors by ET-1 induces the phosphorylation of the extracellular regulated kinases (ERKs). Although the majority of glioblastoma cell lines in culture express ET isoforms and ET receptors, we conclude that ET-1 and the ETB receptors are likely to mediate the effects of the ET system in glioblastoma cell lines.

Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans.

OBJECTIVE: The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. METHODS: Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. RESULTS: At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. CONCLUSIONS: These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.

Renocortical mRNA expression of vasoactive factors during spironolactone protective effect in chronic cyclosporine nephrotoxicity.

We showed that spironolactone reduced structural damage and prevented renal dysfunction in chronic cyclosporine (CsA) nephrotoxicity. These findings evidenced an aldosterone renal vascular effect under this condition. To investigate aldosterone's role in modulating renal vascular tone, renocortical vasoactive pathways mRNA levels in chronic CsA nephrotoxicity as well as spironolactone's effect on renal function in acute CsA nephrotoxicity were evaluated. Two experimental sets were designed. For chronic nephrotoxicity, rats fed with low-sodium diet were divided into groups receiving vehicle, spironolactone (Sp), CsA, and CsA+Sp, for 21 days. Creatinine clearance, survival percentage, and renocortical mRNA levels of pro-renin, angiotensinogen (Ang), angiotensin receptors (AT(1A), AT(1B), and AT(2)), preproendothelin, endothelin receptors (ET(A), ET(B)), cyclooxygenase-2 (COX-2), and adenosine receptors (Ad(1), Ad(2A), Ad(2B), and Ad(3)) were analyzed. For acute nephrotoxicity, similar groups fed with a standard chow diet for 7 days were included. Serum potassium and sodium, glomerular filtration rate (GFR), and renal blood flow (RBF) were determined. In chronic model, CsA produced pro-renin and ET upregulation, altered adenosine receptors expression, and reduced Ang, AT(1A), AT(1B), ET(B), and COX-2 mRNA levels. Spironolactone protective effect in chronic nephrotoxicity was associated with prevention of pro-renin upregulation and increased AT(2), together with ET(B) reduction. In acute nephrotoxicity, spironolactone completely prevented GFR and RBF reduction induced by CsA. Our results suggest that aldosterone contributes to renal vasoconstriction observed in CsA nephrotoxicity and that renoprotection conferred by spironolactone was related to modification of renocortical vasoactive pathways expression, in which pro-renin normalization was the most evident change in chronic nephropathy. Finally, our data point to spironolactone as a potential treatment to reduce CsA nephrotoxicity in transplant patients.

[Therapeutic effects of the combination of traditional Chinese medicine and western medicine on patients with peptic ulcers].

OBJECTIVE: To explore the therapeutic effects and mechanisms of the combination of traditional Chinese medicine and western medicine on patients with peptic ulcers. METHODS: One hundred and twenty patients were randomly divided into 6 groups. Another 10 patients as the control group were confirmed with no peptic ulcers by endoscope, but had digestive tract symptoms. The clinical effects were compared among each group after the one month treatment. RESULTS: The clinical effects of the combination of Jianweiyuyang granules and ranitidine capsules were better than those of western medicine, with improvement in symptoms and syndrome (P < 0.01 to 0.05), but there was not significant difference with the rate of ulcer healing and the Hp clearance among the combination of Jianweiyuyang granules and ranitidine capsules, Jianweiyuyang granules, and ranitidine capsules (P > 0.05). The combination of Jianweiyuyang granules and ranitidine capsules could significantly upregulate the expression of MUCSAC mRNA (P < 0.01), while downregulate the expression of ETAR mRNA (P < 0.01). CONCLUSION: There is obvious advantage in treating peptic ulcers by the combination of Jianweiyuyang granules and ranitidine capsules, and its mechanisms may be to protect the gastric mucosal barrier by up-regulating the expression of MUCSAC mRNA and to improve the gastric mucosal blood flow by down-regulating the expression of ETAR mRNA.

Receptor changes in cerebral arteries after subarachnoid haemorrhage.

Subarachnoid haemorrhage (SAH), occurring with a delay of 4-10 days is linked to cerebral vasospasm (CVS), a pathological constriction of the cerebral arteries. Several agents have been suggested as being responsible - amongst these perhaps 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) are the most prominent, given their ability to elicit powerful constriction of arteries. Investigating both 5-HT and ET receptors we observed distinct changes in the receptor phenotype after experimental SAH - namely upregulation of the ETB and 5-HT1B receptors - linked to a higher sensitivity to the endogenous agonists. This multiple receptor upregulation may explain the failure in treating CVS using single receptor antagonists, and may also significantly change our understanding of the effector mechanism behind CVS. So far only the ET and 5-HT receptors have been studied in this regard, but other receptor systems may also undergo changes.

Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET.

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.

Alteration of endothelin system and calcium handling protein in left ventricles following drug treatment in dilated cardiomyopathy rats.

AIM: To investigate the changes of cardiac calcium handling proteins and endothelin system in dilated cardiomyopathy (DCM) rats and the effects of perindopril and bisoprolol on the remodeling ventricles. METHODS: DCM rats were employed using a 2-kidney, 1-clip hypertensive and diabetic model. Some of the DCM rats were treated with perindopril and bisoprolol for 3 months, respectively. The ratio of left ventricular weight to body weight (LVW/BW), mRNA expressions of calcium handling proteins and endothelin receptors were determined. The alterations of maximum binding capacity (Bmax) and equilibrium dissociation constant (KD) values of cardiac endothelin receptors (ETR) and its subtypes were detected. RESULTS: Compared with those of normal control, blood pressure, and LVW/BW in the DCM rats were elevated. Sarcoplasmic reticulum calcium pump (SERCA) mRNA expression and SERCA activity decreased in the left ventricle. The ETR Bmax decreased, especially the endothelin receptor A. Endothelin converting enzyme activity and expression were elevated, and mRNA expressions of beta1-adrenoreceptor and inositol-3-phosphate receptor in some hearts increased as well. The administration of perindopril and bisoprolol could reverse myocardial hypertrophy and restore the imbalance of calcium handling proteins and endothelin system. CONCLUSION: The disorder of calcium handling proteins and endothelin system existed in the hearts of DCM rats. Treatment of perindopril and bisoprolol could reverse myocardial hypertrophy and changes in DCM rats.