Upregulation Of ET-1 Research Articles

Tissue factor–thrombin signaling enhances the fibrotic activity of myofibroblasts in systemic sclerosis through up‐regulation of endothelin receptor A

The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)-thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ET(A) ) were investigated. Collagen production and migratory activity of HCMFs were further assessed. HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ET(A) expression. ET(A) overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. Tissue factor-thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET(A) expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.

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.

NFATc3 contributes to intermittent hypoxia-induced arterial remodeling in mice

Sleep apnea (SA) is defined as intermittent respiratory arrest during sleep and affects up to 20% of the adult population. SA is also associated with an increased incidence of hypertension and peripheral vascular disease. Exposing rodents to intermittent hypoxia during sleep mimics the cyclical hypoxia/normoxia of SA. We have previously shown that in mice and rats intermittent hypoxia induces ET-1 upregulation and systemic hypertension. Furthermore, intermittent hypoxia (IH) in mice increases nuclear factor of activated T cells isoform 3 (NFATc3) transcriptional activity in aorta and mesenteric arteries, whereas the calcineurin/NFAT inhibitor cyclosporin A prevents IH-induced hypertension. More importantly, NFATc3 knockout (KO) mice do not develop IH-induced hypertension. The goals of this study were to determine the role of NFATc3 in IH-induced arterial remodeling and whether IH-induced NFATc3 activation is mediated by ET-1. Oral administration of both a dual (bosentan) and a selective endothelin receptor type A antagonist (PD155080) during 2 days of IH exposure attenuated NFAT activation in aorta and mesenteric arteries. Rho kinase inhibition with fasudil also prevented IH-induced NFAT activation. Mesenteric artery cross-sectional wall thickness was increased by IH in wild-type (WT) and vehicle-treated mice but not in bosentan-treated and NFATc3 KO mice. The arterial remodeling in mesenteric arteries after IH was characterized by increased expression of the hypertrophic NFATc3 target smooth muscle-alpha-actin in WT but not in KO mice. These results indicate that ET-1 is an upstream activator of NFATc3 during intermittent hypoxia, contributing to the resultant hypertension and increased wall thickness.

Influence of Endothelin 1 Receptor Inhibition on Functional, Structural and Molecular Changes in the Rat Heart after Irradiation

Radiation-induced heart disease is a severe side effect of thoracic radiotherapy. Studies suggest that mast cells play a protective role in radiation-induced heart disease and that the endothelin (ET) system mediates protective effects of mast cells in other disorders. This study examined whether mast cells modulate the cardiac ET system and examined the effects of ET receptor inhibition in a rat model of radiation-induced heart disease. Mast cell-deficient (Ws/Ws), mast cell-competent (+/+) and Sprague-Dawley rats received 18 Gy irradiation to the heart. Left ventricular mRNA of ET1 and its receptors (ETA and ETB) was measured in Ws/Ws and +/+ rats at 1 week and 3 months. Sprague-Dawley rats were treated with the ETA/ETB antagonist bosentan, and at 6 months cardiac changes were assessed using the Langendorff perfused rat heart preparation, immunohistochemistry and real-time PCR. Ws/Ws and +/+ rat hearts did not differ in baseline mRNA. In contrast, +/+ rats hearts exhibited up-regulation of ET1 after irradiation, whereas Ws/Ws rats hearts did not, suggesting the possibility of interactions between mast cells and the cardiac ET system. Bosentan induced reductions in left ventricular systolic pressure, developed pressure and +dP/dtmax but did not affect fibrosis. Because of the known opposing effects of ETA and ETB, studies with selective antagonists may clarify the role of each receptor.

Lipid-Soluble Smoke Particles Upregulate Vascular Smooth Muscle ETB Receptors via Activation of Mitogen-Activating Protein Kinases and NF-kappaB Pathways

Cigarette smoke is a strong risk factor for cardiovascular disease. However, the underlying molecular mechanisms that lead to cigarette smoke-associated cardiovascular disease remain elusive. With functional and molecular methods, we demonstrate for the first time that lipid-soluble cigarette smoke particles (dimethylsulfoxide-soluble cigarette smoke particles; DSP) increased the expression of endothelin type B (ET(B)) receptors in arterial smooth muscle cells. The increased ET(B) receptors in arterial smooth muscle cells was documented as enhanced contractility (sensitive myograph technique), elevated levels of ET(B) receptor mRNA (quantitative real-time PCR), and protein expressions (immunohistochemistry and Western blotting). Intracellular signaling was studied with Western blotting and phosphoELISA; this revealed that DSP induced extracellular-regulated protein kinases 1 and 2 (ERK1/2), p38, and nuclear factor-kappaB (NF-kappaB) phosphorylation within 3 h. Blocking ERK1/2, p38, or NF-kappaB activation by their specific inhibitors significantly attenuated the DSP-induced upregulation of ET(B) receptor-mediated contraction and both ET(B) receptor mRNA and protein expression. In addition, dexamethasone abolished the DSP-induced upregulation of ET(B) receptor-mediated contraction. In conclusion, upregulation of ET(B) receptors by DSP in arterial smooth muscle cells involves activation of mitogen-activating protein kinases (ERK1/2 and p38) and the downstream transcriptional factor NF-kappaB pathways.

Endothelin‐1 (ET‐1) and ET‐A receptors mediate chronic hypoxia‐(CH)‐induced inflammation in rat carotid body

CH in rat carotid body induces an inflammatory response which is characterized by immune cell invasion and the expression of pro‐inflammatory cytokines. In the present study, we have investigated the role of ET‐1 and ET‐A receptors in the development of CH‐induced inflammation. Following 7 days of CH (380 Torr), immunocytochemical (ICC) studies demonstrated elevated levels of ET‐1 and ET‐A receptors in O2‐sensitive type I cells. In addition, double‐label ICC showed that following CH, ET‐A receptors were also expressed on invasive CD45+ immune cells distributed in tissue surrounding chemosensory cell lobules. Concurrent treatment with the ET‐A/B receptor antagonist, bosentan (200mg/kg/day), blocked ED‐1+ immune cell invasion and the up‐regulation of cytokines, including interleukin‐1β (IL‐1β), IL‐6 and tumor necrosis factorα (TNFα). Moreover, bosentan treatment blocked CH‐induced increased expression of acid sensitive ion channels (ASICs) in chemoafferent neurons in the petrosal ganglion (PG), and drug treatment significantly reduced chemoreceptor hypersensitivity. Our findings are consistent with the hypothesis that CH‐induced inflammation involves the up‐regulation and release of ET‐1 from type I cells; ET‐1 acts in an autocrine/paracrine mechanism via ET‐A receptors on chemosensory type I cells, and immune cells to promote an inflammatory response. USPHS Grants NS 12636 and NS 07938.

Therapeutic Effects of Endothelin-A Receptor Antagonist on Bladder Overactivity in Rats with Chronic Spinal Cord Injury

We investigated the effects of suppression of endothelin-A (ET(A)) receptors on bladder function and ET-1 levels in the bladder in rats with chronic spinal cord injury (SCI). We transected the spinal cord of female Sprague-Dawley rats at the level of Th 8-9. Awake cystometrograms were performed 4 weeks after spinal cord transection. We evaluated cystometric parameters such as mean amplitudes of nonvoiding contractions (NVCs), the number of NVCs, voided volume, voiding efficiency, and micturition pressure before and after intravenous (i.v.) injection of ABT-627, an ET(A) antagonist, or A-19261, an ET(B) antagonist, in SCI animals. Four weeks after spinalization, we also measured the protein and mRNA levels of ET-1 in the bladder using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). ABT-627 (1 mg/kg, i.v.) but not A-192621 (10 mg/kg, i.v.) significantly decreased the amplitude of NVCs and the number of NVCs in SCI rats. There were no significant changes in pressure threshold, maximum voiding pressure, voided volume, or voiding efficiency. ELISA analysis for ET-1 showed significantly elevated protein concentrations in SCI rats compared with spinal cord intact rats. Significant upregulation of the ET-1 mRNA was also noted in SCI bladders. These results suggest that upregulation of ET-1 is involved in the mechanism inducing bladder overactivity in chronic SCI rats, and that an ET(A) receptor antagonist can suppress SCI-induced bladder overactivity as indicated by a reduction in NVCs. Thus, ET(A) receptor inhibition could be an effective treatment for neurogenic bladder overactivity in pathological conditions such as SCI.

Examination of mRNA expression in rat hearts and lungs for analysis of effects of exposure to concentrated ambient particles on cardiovascular function

Epidemiological studies have suggested that fine particulate matter (f-PM) is associated with adverse effects on cardiovascular health. However, these effects on the cardiovascular system have not yet been fully elucidated. Using mRNA expression and correlation analyses, we designed the present study to elucidate (1) translocation of chemicals in inhaled f-PM to the heart, (2) induction of oxidative stress, one of the causes of cardiovascular diseases (CVDs), (3) mRNA expression related to CVDs, and (4) correlations among mRNA expression of various molecules and cardiovascular function. Wistar Kyoto male rats were exposed to concentrated ambient particles (CAPs, 0.6–1.5 mg/m 3) in Yokohama for 4 days (4.5 h/day) or to filtered air for 3 days and CAPs for 1 day or to filtered air for 4 days. Messenger RNA expression and cardiovascular function were measured after the 4-day exposure. In samples of heart tissue, the mRNAs of cytochrome P450 (CYP) 1B1, a biomarker of exposure to chemicals; heme oxygenase-1 (HO-1), a marker of oxidative stress; and endothelin A (ET A) receptor, a receptor of vasoconstrictors, were up-regulated by CAPs; their levels were significantly correlated with the cumulative weight of CAPs in the exposure chamber. The up-regulation of ET A receptor mRNA was significantly correlated with the increase in HO-1 mRNA and weakly with the increase in mean blood pressure (ΔMBP). These results suggest the possibility that chemicals in CAPs might be translocated to the heart, where they induce oxidative stress and activate endothelin signaling, resulting in an increase in the blood pressure. The exposure to f-PM might thus affect cardiovascular function through activation of endothelin signaling.

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor κB and Ets

Conclusion: Hypertension accelerates progression of experimental abdominal aortic aneurysms (AAAs) by upregulation of Ets and nuclear factor κB (NF κB). Summary: The pathogenesis of AAA is complex but appears to involve upregulation of certain matrix metalloproteinases (MMPs). Experimental AAAs are characterized by upregulation of transcription factors, NF κB, which regulate transcription, and Ets, which regulates expression of MMPs. The role of hypertension in pathogenesis of AAA is controversial, with some studies suggesting it contributes to aneurysm formation and other studies suggesting it is not an independent risk factor for aneurysm formation. In this study, the authors used an experimental model of AAA to investigate the effects of hypertension on transcription factors NF κB and Ets. They also investigated the effects of hypertension on progression of AAA. Experimental AAAs were produced by elastase perfusion in both normotensive and hypertensive rats. Compared with normotensive rats, the size of the AAAs increased more rapidly in the hypertensive rats. Western blot analysis indicated the expression of MMP-2, MMP-3, MMP-9, and MMP-12, as well as that of intercellular adhesion molecule, was increased in hypertensive rats. This was accompanied by upregulation of NFκB and Ets. There was also increased activity of MMPs in the aortas of hypertensive rats vs those of normotensive rats. Chimeric decoy oligodeoxynucleotide (ODN) inhibits expression of NFκB and Ets. Transfection of the ODN resulted in significant inhibition of aortic dilatation in both hypertensive and normotensive rats. Transfection with chimeric decoy ODN resulted in significant inhibition of degeneration of elastic fibers in aortas of both normotensive and hypertensive rats. Expression of MMP-2, MMP-3, MMP-9, and MMP-12 and intercellular adhesion molecule was decreased by chimeric decoy ODN. This was accompanied by inhibition of macrophage migration. Comment: The study indicates hypertension accelerates progression of AAA in an experimental rat model. Perhaps inhibition of NF κB and Ets may someday be used as a method of decreasing aneurysm expansion in both normotensive and hypertensive patients.

Endothelin and endothelial dysfunction.

Nitric oxide (NO) and endothelin (ET) produced in endothelial cells are leading molecules which regulate vascular function. Failure of the physiological balance between these two molecules is usually referred to as endothelial dysfunction. ET was initially identified as a potent vasoconstrictive peptide. Three ET isoforms and two ET receptors have been identified. One of the isoforms, ET-1, plays a significant role in many cardiovascular diseases. On the other hand, oxidized low-density lipoprotein (oxLDL) is known to induce endothelial dysfunction. The endothelial receptor for oxLDL was cloned, and named lectin-like oxidized receptor-1 (LOX-1). Activation of LOX-1 generates reactive oxygen species (ROS), and acivates a transcriptional factor, nuclear factor κB (NFκB), resulting in down-regulation of NO and up-regulation of ET-1. LOX-1 might be a key molecule in the generation of endothelial dysfunction. In endothelial dysfunction, ET-1 is an aggravating factor of cardiovascular diseases.

Gender hormones and the progression of experimental polycystic kidney disease

Male gender is a risk factor for progression of autosomal-dominant polycystic kidney disease (ADPKD), clinically and in the Han:SPRD rat model. Orchiectomy limits progression, but mechanisms of the detrimental effect of androgen, and/or beneficial effects of estrogen, are not known. This protocol tested the hypothesis that male gender (intact androgen status) promotes progression, while female gender (intact estrogen status) is protective; and that these disease-modifying effects are due to changes in expression of known fibrotic mediators. Studies were performed in male and female noncystic control (+/+) and cystic (+/-) rats subjected to orchiectomy, ovariectomy, or sham operation. At 12 weeks of age, renal function was measured. Blood and kidneys were taken for measurement of plasma and renal renin, endothelin (ET-1), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF), using biochemical, protein expression, and immunohistochemical methods. Cystic male rats exhibited significantly reduced glomerular filtration (GFR) and effective renal plasma flow (ERPF) rates, with suppression of plasma and renal renin, up-regulation of renal ET-1 and eNOS, and down-regulation of renal VEGF expression. Orchiectomy attenuated the fall in GFR and ERPF, while numerically limiting changes in eNOS and VEGF. Female rats exhibited less cystic growth, with normal renin status, lesser elevation of renal ET-1, and proportionately lesser changes in VEGF and eNOS. Ovariectomy led to higher blood pressure and reduced GFR and ERPF, with a trend toward upregulation of ET-1, and significant down-regulation of VEGF and eNOS. Female gender is protective, but ovariectomy attenuates the protective effect of female gender, in association with changes in renal expression of ET-1, VEGF, and eNOS. The accelerated disease in male rats can be attenuated by orchiectomy and consequent changes in expression of disease mediators.

Endothelin‐mediated remodeling in aortas of diabetic rats

Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.

Mechanisms underlying enhanced contractile response to endothelin-1 in diabetic rat basilar artery

We investigated the influence of streptozotocin-induced diabetes on the responsiveness of the rat basilar artery to endothelin-1 (ET-1) and nitric oxide (NO), which is known to counteract ET-1. In basilar arteries isolated from diabetic rats: (a) the ET-1-induced contraction was enhanced, (b) the contraction induced by N G-nitro- l-arginine [a nitric oxide synthase (NOS) inhibitor] was weaker, and (c) the levels of the mRNAs for ET A/ET B receptors and prepro-ET-1, but not for NOS, were significantly elevated (all versus age-matched controls). These data indicate that ET-1-induced vasoconstriction may be increased in the diabetic rat basilar artery, and that this hyper-reactivity to ET-1 may be due to an overproduction of ET-1, an up-regulation of ET A/ET B receptors, and a defect in the bioavailability of NO.

Attenuation of iNOS mRNA exacerbates hypoperfusion and upregulates endothelin-1 expression in hippocampus and cortex after brain trauma

Nitric oxide (NO, a vasodilator) and endothelin-1 (ET-1, a powerful vasoconstrictor) participate in the regulation of brain’s microcirculation influencing each other’s expression and synthesis. Following injury to the brain, NO is derived largely from the inducible form of nitric oxide synthase (iNOS). We used Marmarou’s model of traumatic brain injury (TBI) to study the cerebral blood flow and expression (mRNA) of ET-1 in rats that were pretreated with antisense iNOS oligodeoxynucleotides (ODNs). Intracerebroventricular application of iNOS ODNs resulted in reduced synthesis of iNOS as detected by Western blot analysis. The cerebral blood flow (measured by laser Doppler flowmetry), generally decreased after TBI, was further markedly reduced in the treated animals and remained at low levels up to 48 h post-TBI. The expression of ET-1 (detected by in situ hybridization in cortex and hippocampus) was increased 2–3-fold following TBI alone and this increase reached 5–6-fold in animals pretreated with antisense iNOS ODNs. The results indicate that most likely, NO, generated primarily by iNOS, suppresses ET-1 production and that a decrease of NO results in upregulation of ET-1 via transcriptional and translational mechanisms. Increased availability of ET-1 at the vascular bed and the neuropil may contribute to the altered microvascular reactivity and reduced perfusion of the brain following TBI.

The role of the sodium hydrogen exchanger-1 in mediating diabetes-induced changes in the retina.

The sodium hydrogen exchanger (NHE) is a transmembrane protein responsible for alkalinization and control of intracellular acidosis by the removal of hydrogen and the subsequent influx of sodium. Our investigation attempts to determine the role of NHE-1 in the pathogenesis of early retinal microangiopathy due to diabetes. Diabetes was induced in male Sprague-Dawley rats with a single intravenous streptozotocin injection (65 mg/kg). To examine the duration-dependent changes in NHE-1 expression, retinas from 1-, 6- and 12-week diabetic animals were analyzed. To examine the functional consequences of NHE-1 inhibition in comparison with good blood glucose control, diabetic rats were randomly assigned to poorly controlled diabetic, well-controlled diabetic, poorly controlled diabetic with cariporide groups and were compared with nondiabetic controls after six weeks. Cariporide is an orally active inhibitor of NHE-1 (6000 ppm in rat chow). At the end of the treatment period, color Doppler ultrasound was used to determine the resistivity index (RI) of the central retinal artery. The mRNA expression of endothelin (ET) isoforms 1 and 3, inducible and endothelial nitric oxide synthase (iNOS and eNOS respectively) and NHE-1 were examined. NHE-1 distribution was localized with immunohistochemistry. All diabetic animals showed hyperglycemia, increased glycated hemoglobin and lower body weight gain compared to nondiabetic controls. Diabetes caused an increased RI, indicative of retinal vasoconstriction, which was corrected by both cariporide treatment and good glucose control. NHE-1 was localized in the endothelium of the retinal microvasculature and the neuronal and glial components. NHE-1 mRNA expression was unchanged after 1 week and increased after 6 and 12 weeks of diabetes. Furthermore, a diabetes-induced upregulation of ET-1 and ET-3 mRNA expression after six weeks was corrected with cariporide treatment. NHE-1 inhibition of diabetic animals upregulated iNOS mRNA levels, although expression of eNOS and iNOS mRNA were not altered in poorly controlled diabetes. Improved blood glucose control with higher doses of insulin also corrected diabetes-induced increased RI by upregulating eNOS and iNOS mRNA expression. The results of the study suggest that NHE-1 may be involved in the regulation of several vasoactive modulators that contribute to functional alterations in diabetic retinal microangiopathy.

Activation of the right ventricular endothelin (ET) system in the monocrotaline model of pulmonary hypertension: response to chronic ETA receptor blockade.

Although activation of the endothelin (ET) system contributes to pulmonary hypertension, modifications of the cardiopulmonary ET system and its responses to chronic ET receptor blockade are not well known. To investigate this, rats were injected with monocrotaline (60 mg/kg intraperitoneal) or saline, followed with treatment with the selective ETA receptor antagonist LU135252 (LU; 50 mg.kg(-1).day(-1)) or with saline. After 3 weeks, haemodynamics, cardiac hypertrophy, ET-1 levels and cardiopulmonary ET-receptor-binding profile were evaluated. Monocrotaline (n =7) elicited marked pulmonary hypertension and right ventricular hypertrophy compared with controls (n =8). Both variables were substantially attenuated by LU therapy (n =8; P <0.05 for both). After monocrotaline, right ventricular ET-1 levels were more significantly increased than in the left ventricle (+198% compared with +127%; P <0.05). ETB receptor density was augmented (3-fold) in the right ventricle, whereas that of ETA receptors was not affected. LU treatment also significantly attenuated these alterations (P <0.05). In the lungs, ET-1 levels were not increased after monocrotaline, whereas the balance of ETB to ETA receptors was altered, with a trend toward a lower percentage of ETB than in the control rats. LU treatment did not affect these variables in the lungs. Therefore monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy are associated with the up-regulation of ET-1 and ETB receptors in the right ventricle. These alterations are attenuated with the reduction of pulmonary hypertension and right ventricular hypertrophy after chronic blockade of the ETA receptors, supporting the role of the ET system in right ventricular hypertrophy.

Role of mitogen-activated protein kinases in endothelin ET B receptor up-regulation after organ culture of rat mesenteric artery

Organ culture of isolated arteries results in increased levels of endothelin ET B (ET B) receptor mRNA and in enhanced ET B receptor mediated contraction. The present study was designed to pinpoint the mitogen-activated protein kinase (MAPK) subtype involved in up-regulation of ET B receptors after organ culture of rat mesenteric arteries. Western blot and selective antibodies towards constitutional and phosphorylated MAPKs revealed the appearance of phosphorylated MAPK of the extracellular signal-regulated kinases (ERK) 1/2 type at 3 h of organ culture. The functional ET B receptor and its mRNA expression were up-regulated after 24 h of organ culture. Following incubation with the MEK 1/2 specific inhibitor SB408039 or the raf inhibitor SB386023b the up-regulation was attenuated both for ET B receptor responses and in ET B receptor mRNA expression in the vessel segments. Neither Western blot nor myograph or mRNA analysis showed involvement of the other MAPKs studied. Our results suggest that the ERK1/2 MAPKs are involved in the endothelin ET B receptor up-regulation following organ culture.

Differential expression of endothelin receptors in regenerating spinal motor neurons in mice

On day 4 after sciatic nerve crush injury, expression and localization of endothelin receptors ET A and ET B in the lumbar spinal cord were examined. Immunohistochemical staining with antibodies to ET A and ET B receptors showed cytoplasmic distribution of ET A receptors in motor neurons, whereas ET B receptors were localized in the perinuclear region. On the injured side of the lumbar spinal cord, when compared to contralateral, results demonstrated an up-regulation of ET B and a down-regulation of ET A receptors expression at the level of both mRNA and protein. These results suggest that ET B receptors may play a role in the regeneration of axotomized motor neurons.

Distinct intragraft response pattern in relation to graft size in liver transplantation.

The molecular mechanism of small-for-size graft injury remains unclear. The aim of this study is to investigate the gene expression pattern of acute phase response in relation to graft size in a rat-liver transplantation model. A rat orthotopic liver transplantation model using 30%, 50%, and whole grafts was used. The graft survival rates and liver morphology were compared among the three groups. Two transcription factors, nuclear factor (NF)-kappaB (p65) and early growth response (Egr-1), and their downstream genes were compared. According to the graft size, the rats were grouped as follows: group 1 (n=20), 32% (24-47%); group 2 (n=10), 56% (50-65%); and group 3 (n=10), 104% (89-120%). The 7-day survival rates were 20% (P=0.039 vs. group 2, P=0.000 vs. group 3), 60%, and 100% in groups 1, 2, and 3, respectively. Dilation of hepatic sinusoids and vacuolization of hepatocytes were observed in group 1. Up-regulation of Egr-1 and endothelin (ET)-1 and over-expression of nitric oxide synthase (iNOS) was found in group 1, but heme oxygenase (HO)-1 and A20 were down-regulated. At 24 hours after reperfusion, the intragraft protein level of heat-shock protein (Hsp)-70 was significantly lower in group 1 than that in group 3 (12.4 vs. 17.0 ng/mL, P=0.04). More apoptotic nuclei were found in group 1. Small-for-size graft injury was related to early over-expression of Egr-1 associated with up-regulation of ET-1 and deterioration of intracellular homeostasis reflected by down-regulation of Hsps and A20.

The Role of Endothelin in Hormone-Refractory Prostate Cancer

Aggressive chemotherapy has made only a limited contribution to improvements in patient prognosis and well-being in hormone-refractory prostate cancer (HRPC). Such poor progress results from the biological basis of the disease, localisation of the tumour and the relatively high age of affected men, and leaves patients with a dismal prognosis. Given the palliative role of current treatments, attention has focused on the development of therapies targeted at non-androgenic mediators of prostate growth. Endothelin-1 (ET-1), a 21-amino-acid peptide produced by endothelial cells and prevalent in seminal fluid, has been identified as one such mediator. In addition to its potent mitogenic and vasoconstrictive properties, ET-1 has been shown to suppress apoptosis and induce angiogenesis. In HRPC cells, increased levels of ET-1 have been observed. ET-1 mediates its effects through two receptors, of which the endothelin-A (ET A) receptor is most important in prostate cancer. An up-regulation of ET A receptor levels and decreased expression of endothelin-B (ET B) receptors is observed in HRPC cells. Taken together, these factors are thought to play a significant role in the progression of the disease. Research has, therefore, focused on development of ET-1 antagonists to disrupt the mitogenic and angiogenic effects of ET-1 and slow disease progression. As ET-1 is also an important factor in the development of new bone, ET-1 antagonists may potentially inhibit the development of skeletal metastases and associated pain, which characterise this disease. Atrasentan, a highly specific ET A receptor antagonist, is currently in clinical development. Data are awaited from clinical trials to confirm the role of this agent in the treatment of HRPC.