Heterozygous knock-Out of ET(B) receptors induces BQ-123-sensitive hypertension in the mouse

Homozygous knock-out of ET(A) or ET(B) receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ET(A) or ET(B) receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ET(B) (+/-) knock-out mice showed a significantly higher mean arterial blood pressure than the ET(A) (+/-) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ET(B) agonist, IRL-1620, was significantly reduced in the ET(A) (+/-) knock-out mice. In ET(B) (+/-) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ET(A) and ET(B) receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ET(A) antagonist) or BQ-788 (ET(B) antagonist). Also, ET(A)-selective or mixed ET(A)/ET(B)- but not ET(B)-selective antagonists reversed the hypertensive state of the ET(B) (+/-) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ET(B) (+/-) but not ET(A) (+/-) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ET(B) receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1-dependent pressor effects in the mouse.     

The impact of endothelin on renal hemodynamics in conscious rats

Endothelin (ET), a peptide recently isolated from the supernatant of cultured endothelial cells, is the most potent vasoconstrictive and hypertensive agent known up till now. We have examined the effect of ET-1 intravenous injection on regional hemodynamics in conscious unrestrained rats. Normal rats are instrumented with an arterial catheter for measurement of mean arterial pressure (MAP) and with pulsed Doppler flow probes on renal and mesenteric arteries and the abdominal aorta for simultaneous recording of blood flow velocities (V). These parameters allow calculation of vascular resistance (R) (R = MAP/V). Thus, ET-1 induces an initial and sharp hypotension, concomitant with tachycardia and a marked vasoconstriction of renal and mesenteric arteries, but a vasodilatation of aorta. This response is followed by a dose-dependent and long-lasting increase of MAP and of renal, mesenteric and aortic vascular resistances accompanied by a decrease of heart rate. The greatest impact of ET-1 constrictive effects is seen on the renal vascular bed whereas the abdominal aorta appears to be far less sensitive. In fact, the dose of 2 nmol/kg of ET-1 induces a dramatic and long-lasting fall of renal blood flow (-86%) resulting from an important vasoconstriction (+1818%). Finally, an elevation of proteinuria is revealed in ET-1 (2 nmol/kg) treated rats, but not in those treated with the same dose of Angiotensin II. This proteinuria is characterized by the appearance of proteins with a molecular weight from 20,000 to 140,000 and sometimes 280,000, and an increase of excreted albumin, seeming to reflect an alteration of glomerular permeability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism in experimental hepatopulmonary syndrome

BACKGROUND/AIMS: In experimental hepatopulmonary syndrome (HPS), hepatic endothelin-1 (ET-1) release during common bile duct ligation (CBDL) and ET-1 infusion in pre-hepatic portal hypertension after portal vein ligation (PVL) initiate vasodilatation through an endothelin B receptor mediated increase in pulmonary endothelial nitric oxide synthase (eNOS). We evaluated if pulmonary ET receptor expression changes in experimental cirrhosis and portal hypertension and confers susceptibility to HPS.METHODS: In normal, PVL and CBDL animals, lung ET receptor expression and localization were assessed and ET receptor levels and functional analysis of ET-1 effects on eNOS levels were evaluated in intralobar pulmonary artery (PA) and aortic (AO) segments. Normal rats underwent evaluation for HPS after ET-1 infusion.RESULTS: There was a selective increase in ET(B) receptor expression in the pulmonary vasculature from PVL and CBDL animals. ET-1 stimulated NO production and an ET(B) receptor mediated increase in eNOS levels in PA segments from PVL and CBDL animals, but not normal animals. ET-1 did not alter lung eNOS levels or cause HPS in normal rats.CONCLUSIONS: ET(B) receptor expression and ET-1 mediated eNOS and NO production are enhanced in the lung vasculature in cirrhotic and portal hypertensive animals and correlate with in vivo susceptibility to ET-1 mediated HPS.     

Aortic smooth muscle cell phenotypic modulation and fibrillar collagen deposition in angiotensin II-dependent hypertension

BACKGROUND: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). METHODS: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ET(A)/ET(B) antagonist bosentan (n=6) or the ET(A)-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ET(A) and ET(B) receptor density were also assessed with quantitative autoradiography. RESULTS: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ET(A) receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ET(A) receptors. CONCLUSIONS: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels.     

Blood pressure-independent effect of angiotensin AT1 receptor blockade on renal endothelin-1 production in hypertensive uremic rats

OBJECTIVE: We recently reported that treatment of uremic rats with reduced renal mass with the angiotensin II (Ang II) subtype 1 receptor (AT1) antagonist losartan reduces endothelin-1 (ET-1) levels in blood vessels and in glomeruli. Although this suggests an important role for Ang II in the modulation of ET-1 production, the concomitant decrease in blood pressure may also be involved. The present study was designed to investigate whether the modulation of ET-1 production in uremic rats is related to tissue-specific effects of AT1 receptor blockade or to the antihypertensive effect of losartan. DESIGN: One week after renal mass reduction, uremic rats were treated with the conventional triple therapy (TRx) [reserpine (5 mg/l), hydralazine (80 mg/l) and hydrochlorothiazide (25 mg/l)] or losartan (20 mg/kg per day) for 6 weeks. Immunoreactive-ET-1 (ir-ET-1) levels in plasma and urine, as well as in vascular and renal tissues were measured by a specific radioimmunoassay after sample extraction and purification. RESULTS: Before treatment, systolic blood pressure was significantly higher in uremic animals compared to sham-operated controls (165+/-4 versus 123+/-2 mmHg, respectively; P < 0.01). Treatment with the TRx or with losartan normalized systolic blood pressure in uremic rats, whereas it was further increased in untreated uremic animals. At week 6, serum creatinine, proteinuria and urinary ET-1 and transforming growth factor-beta1 (TGF-beta1) excretion, as well as vascular and glomerular ET-1 content were increased in uremic rats compared to the controls (P < 0.01). Treatment of uremic rats with the TRx or with losartan reduced ET-1 content in the thoracic aorta and the mesenteric arterial bed (P < 0.01). However, losartan, but not the TRx, significantly attenuated the rise of serum creatinine, proteinuria and urinary ET-1 and TGF-beta1 excretion, as well as ET-1 content in glomeruli of uremic rats. Compared with the controls, renal preproET-1 mRNA expression was also significantly higher in uremic rats. Treatment of uremic rats with losartan prevented renal preproET-1 mRNA overexpression, indicating that changes in glomerular ET-1 content and urinary ET-1 excretion were related to modulation of renal ET-1 production. CONCLUSIONS: These findings indicate that the effect of losartan on ET-1 production in peripheral blood vessels may be mediated, in part, by the reduction of blood pressure. In contrast, the reduction of renal ET-1 production is mediated by tissue-specific effects of AT1 receptor blockade, and may contribute to the renal protective effects of losartan.     

Chronic nitric oxide inhibition aggravates hypertension in erythropoietin-treated renal failure rats

Alterations in nitric oxide (NO) and endothelin-1 (ET-1) production have recently been reported in erythropoietin (r-HuEPO)-induced hypertension in renal failure rats. The present study was designed to evaluate the effect of NO synthase inhibition with the L-arginine analog NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP) and ET-1 production in control and in uremic rats treated or not treated with r-HuEPO. Renal failure was induced by a two-stage 5/6 nephrectomy. Control and uremic rats were studied separately and subdivided into four groups: vehicle, r-HuEPO, L-NAME + vehicle and L-NAME + r-HuEPO. L-NAME (100 mg/kg/day), r-HuEPO (100 U/kg, subcutaneously, three times per week), the vehicle or both were administered during 4 weeks in control rats and during 2 weeks in uremic rats. Systolic BP was recorded before and after the onset of treatment at weeks 2 and 4 in control rats and at weeks 1 and 2 in uremic rats. Hematocrit, serum creatinine, plasma, blood vessel (thoracic aorta and mesenteric artery bed) and renal cortex immunoreactive (ir) ET-1 concentrations were measured at the end of the protocol. L-NAME enhanced BP in control and uremic rats and the increase was significantly higher in uremic rats under r-HuEPO therapy (222 +/- 7 mmHg vs 198 +/- 6 mmHg, p<0.05). L-NAME induced an increase in thoracic aorta ir-ET-1 concentrations in control and uremic rats. In contrast, ir-ET-1 concentrations were unchanged in the mesenteric arterial bed and the renal cortex of control and uremic animals. R-HuEPO increased thoracic aorta ir-ET-1 contents in L-NAME treated control and uremic rats. These results underline the important role of NO release in opposing the action of vasopressors on blood vessel tone which appears more important in uremic rats treated with r-HuEPO. L-NAME treatment increased large vessel, but not small resistance artery ir-ET-1 concentrations, suggesting differential regulation of ET-1 production in different vascular beds under chronic NO synthase inhibition.     

Importance of local production of endothelin-1 and of the ET(B)Receptor in the regulation of pulmonary vascular tone

Interaction between locally released endothelin-1 (ET-1) and the endothelial ET(B)receptor could modulate pulmonary vascular tone. We evaluated pulmonary ET-1 clearance and ET-1-ET(B)receptor interaction in the modulation of pulmonary vascular tone. Controls and rats with Monocrotaline (MCT)-induced pulmonary hypertension (PH) were studied. Lungs were isolated and perfused under constant pressure. The effect of the selective ET(B)antagonist BQ-788 (10(-12)-10(-8)mole) on perfusion flow rate and(125)I-ET-1 extraction was determined. Baseline(125)I-ET-1 extraction was reduced from 62+/-5% in controls to 49+/-10% in PH (P=0.012). BQ-788 inhibited extraction with a higher half-inhibitory dose in the MCT group (-Log ID(50)= 8.9+/-0.4 vs. 9.5+/-0.1, P=0.03). BQ-788 induced a mild reduction in perfusion flow rate of 0.7+/-0.3 ml/min in controls. In the MCT group, this occurred at a lower dose and was more pronounced with a maximal reduction of 3.3+/-0.7 ml/min (P<0.01 vs. control). ET-1 was undetectable in the effluent at baseline but was present in similar concentrations in both groups after ET(B)blockade. Addition of 2 pg/ml ET-1 to lung perfusate did not modify pulmonary ET-1 clearance or the effect of BQ788 on perfusion flow rate in control lungs. In normal rat lungs, the ET(B)receptor plays a minor regulatory role on vascular tone. In MCT hypertension however, despite a reduction in ET(B)mediated extraction, luminal production of ET-1 attenuates the increase in pulmonary vascular tone.     

Antihypertensive and renal protective effects of renin-angiotensin system blockade in uremic rats treated with erythropoietin

BACKGROUND: Correcting anemia with recombinant human erythropoietin (rhEPO) in chronic renal failure has been associated with an increased blood pressure (BP), which may accelerate the decline in renal function. This has been attributed, in part, to the activation of the renin-angiotensin system. The present study was designed to investigate the protective effect of the angiotensin II-receptor blocker losartan compared with the angiotensin-converting enzyme inhibitor captopril and conventional triple therapy (TRx) in uremic rats receiving rhEPO therapy. METHODS: Renal failure was induced by renal mass ablation followed by a 3-week stabilization period. Uremic rats were then divided into five groups with similar systolic BP: vehicle; rhEPO (100 U/kg, subcutaneously, three times per week); rhEPO + losartan (20 mg/kg/d); rhEPO + captopril (20 mg/kg/d); and rhEPO + TRx (reserpine 5 mg/L, hydralazine 80 mg/L, hydrochlorothiazide 20 mg/L). Systolic BP as well as blood and renal parameters were assessed before and after a 3-week treatment period. Renal histology was evaluated at the end of the study. RESULTS: The uremic rats developed hypertension, anemia, proteinuria, and increased urinary endothelin-1 (ET-1) excretion. The rhEPO corrected the anemia but aggravated the hypertension (P < .01), glomerular sclerosis, tubular atrophy, and interstitial fibrosis. Treatment with losartan, captopril, and the TRx prevented the rhEPO-induced increased in systolic BP. The TRx was less effective in preventing histologic injuries despite similar systolic BP reduction. CONCLUSIONS: Blockade of the renin-angiotensin system is highly effective in preventing both hypertension and renal histologic damage in rhEPO-treated uremic rats and this benefit seems to extend beyond the antihypertensive effect.     

Evidence against the hypothesis that endothelial endothelin B receptor expression is regulated by relaxin and pregnancy

The endothelial endothelin B (ET(B)) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET(B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 mug/h or vehicle for 5 d or 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET(B) expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET(B) protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET(B) expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET(B) expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET(B) receptor protein has little or no role in relaxin stimulation of the endothelial ET(B)/nitric oxide vasodilatory pathway.     

CHRONIC NITRIC OXIDE INHIBITION AGGRAVATES HYPERTENSION IN ERYTHROPOIETIN-TREATED RENAL FAILURE RATS

Alterations in nitric oxide (NO) and endothelin-1 (ET-1) production have recently been reported in erythropoietin (r-HuEPO)-induced hypertension in renal failure rats. The present study was designed to evaluate the effect of NO synthase inhibition with the L-arginine analog N^G-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP) and ET-1 production in control and in uremic rats treated or not treated with r-HuEPO. Renal failure was induced by a two-stage 5/6 nephrectomy. Control and uremic rats were studied separately and subdivided into four groups: vehicle, r-HuEPO, L-NAME + vehicle and L-NAME + r-HuEPO. L-NAME (100 mg/kg/day), r-HuEPO (100 U/kg, subcutaneously, three times per week), the vehicle or both were administered during 4 weeks in control rats and during 2 weeks in uremic rats. Systolic BP was recorded before and after the onset of treatment at weeks 2 and 4 in control rats and at weeks 1 and 2 in uremic rats. Hematocrit, serum creatinine, plasma, blood vessel (thoracic aorta and mesenteric artery bed) and renal cortex immunoreactive (ir) ET-1 concentrations were measured at the end of the protocol. L-NAME enhanced BP in control and uremic rats and the increase was significantly higher in uremic rats under r-HuEPO therapy (222 ± 7 mmHg vs 198 ± 6 mmHg, p<0.05). L-NAME induced an increase in thoracic aorta ir-ET-1 concentrations in control and uremic rats. In contrast, ir-ET-1 concentrations were unchanged in the mesenteric arterial bed and the renal cortex of control and uremic animals. R-HuEPO increased thoracic aorta ir-ET-1 contents in L-NAME treated control and uremic rats. These results underline the important role of NO release in opposing the action of vasopressors on blood vessel tone which appears more important in uremic rats treated with r-HuEPO. L-NAME treatment increased large vessel, but not small resistance artery ir-ET-1 concentrations, suggesting differential regulation of ET-1 production in different vascular beds under chronic NO synthase inhibition.     

Adrenergic and endothelin B receptor-dependent hypertension in dopamine receptor type-2 knockout mice

Polymorphism of the dopamine receptor type-2 (D(2)) gene is associated with essential hypertension. To assess whether D(2) receptors participate in regulation of blood pressure (BP), we studied mice in which the D(2) receptor was disrupted. In anesthetized mice, systolic and diastolic BPs (in millimeters of mercury) were higher in D(2) homozygous and heterozygous mutant mice than in D(2)+/+ littermates. BP after alpha-adrenergic blockade decreased to a greater extent in D(2)-/- mice than in D(2)+/+ mice. Epinephrine excretion was greater in D(2)-/- mice than in D(2)+/+ mice, and acute adrenalectomy decreased BP to a similar level in D(2)-/- and D(2)+/+ mice. An endothelin B (ET[B]) receptor blocker for both ET(B1) and ET(B2) receptors decreased, whereas a selective ET(B1) blocker increased, BP in D(2)-/- mice but not D(2)+/+ mice. ET(B) receptor expression was greater in D(2)-/- mice than in D(2)+/+ mice. In contrast, blockade of ET(A) and V(1) vasopressin receptors had no effect on BP in either D(2)-/- or D(2)+/+ mice. The hypotensive effect of an AT(1) antagonist was also similar in D(2)-/- and D(2)+/+ mice. Basal Na(+),K(+)-ATPase activities in renal cortex and medulla were higher in D(2)+/+ mice than in D(2)-/- mice. Urine flow and sodium excretion were higher in D(2)-/- mice than in D(2)+/+ mice before and after acute saline loading. Thus, complete loss of the D(2) receptor results in hypertension that is not due to impairment of sodium excretion. Instead, enhanced vascular reactivity in the D(2) mutant mice may be caused by increased sympathetic and ET(B) receptor activities.     

Endothelins, their receptors, and retinal vascular dysfunction in galactose-fed rats

PURPOSE: Endothelins are potent vasoactive factors that have been implicated in the pathogenesis of several vascular disorders. This study was conducted to determine the role that endothelins play in the development of retinal microangiopathy under hyperhexosemic conditions induced by galactose feeding. Methods: Retinal blood flow was determined using Doppler sonography in galactose fed rats with or without an endothelin receptor antagonist (Bosentan) treatment and were compared to control rats after 1 and 6 months of follow-up. Levels of endothelin-1, endothelin-3, (ET-1, ET3) and receptors endothelin A, endothelin B, (ET(A), ET(B)) mRNA expression were determined by semiquantitative RT-PCR. Immunohistochemical distribution of ET-1 and ET-3, ligand binding, and autoradiography to determine ET receptor distribution were carried out. RESULTS: Retinal vasoconstriction measured by an increase in resistivity index (RI) was present in 1 month galactose feeding compared to controls, which was prevented by Bosentan treatment. After 6 months of follow up all animal groups exhibited higher RI compared to their 1 month counterpart, although they were not different from each other. Compared to the controls, after 1 month levels of mRNA for ET-1, ET-3, and ET(A) were increased in galactose-fed rats, whereas ET(B) mRNA production remained similar to controls. After 6 months, all four genes exhibited increased levels compared to the controls, and no effect of Bosentan treatment on gene expression was evident. Increased immunoreactivity of ET-1 and ET-3 was determined, as well as increased ET receptor concentration was further present in the retina of galactose-fed animals. CONCLUSION: The data suggests that endothelin production is increased under hyperhexosemic conditions and that the endothelins play an important role in regulating the hemodynamics of retinal blood flow.     

Endothelin-1 induces vasoconstriction on portal-systemic collaterals of portal hypertensive rats

Portal hypertension is associated with increased hepatic and collateral resistance to an increased portal blood flow. Endothelin-1 (ET-1) can induce intrahepatic vasoconstriction and consequently increase portal pressure. It is unknown if ET-1 also modulates portal pressure by a direct vasoconstrictive effect on collaterals. This study investigated the collateral vascular responses to ET-1, the receptors in mediation, and the regulation of ET-1 action by nitric oxide and prostaglandin. The portal-systemic collaterals of partially portal vein-ligated rats were tested by in situ perfusion. The concentration-response curves of collaterals to graded concentrations of ET-1 (10(-10)-10(-7) mol/L) with or without BQ-123 (ET(A) receptor antagonist, 2 x 10(-6) mol/L), BQ-788 (ET(B) receptor antagonist, 10(-7) mol/L) or both were recorded. In addition, the collateral responses to ET-1 with preincubation of n(omega)-nitro-L-arginine (NNA; 100 mol/L), indomethacin (INDO; 10 mol/L), or in combination were performed. ET-1 increased the perfusion pressure of collaterals and its effect was significantly suppressed by BQ-123 alone and BQ-123 plus BQ-788, but not BQ-788 alone (P <.05). Incubation with NNA, INDO, or both significantly enhanced the response of collaterals to ET-1 (P < .05). These results show that ET-1 produces a direct vasoconstrictive effect on the collateral vessels of portal hypertensive rats. This effect is mediated by ET(A,) but not ET(B), receptors. Both nitric oxide and prostaglandin modulate the collateral vascular response to ET-1 and may therefore participate in the development and maintenance of portal hypertension.     

Increased O2*- production and upregulation of ETB receptors by sympathetic neurons in DOCA-salt hypertensive rats

Superoxide anion (O2*-) production is elevated in the vasculature of hypertensive animals but it is not known if O2*- production is also elevated in the sympathetic nervous system. We measured O2*- levels in prevertebral sympathetic ganglia of deoxycorticosterone acetate (DOCA)-salt hypertensive rats using the dihydroethidine (DHE) fluorescence method. O2*- was elevated in ganglia from DOCA-salt rats compared with normotensive sham rats. Treatment of ganglia with endothelin (ET)-1 (3x10(-8) mol/L) resulted in a 200% increase in fluorescence intensity in neurons, which was attenuated by the ET(B) receptor antagonist BQ788 (10(-7) mol/L). ET-1 also increased the O2*- induced fluorescence in dissociated sympathetic neurons and PC-12 cells via activation of ET(B) receptors, but not ET(A) receptors. To evaluate whether elevated ET-1 levels in the ganglia might contribute to the elevated O2*- found in ganglia we measured the amount of ET-1 using an ELISA assay. ET-1 levels in sham rat celiac ganglia were 695.6+/-40.9 picogram per gram; they were not different than ET-1 levels in ganglia from DOCA-salt rats. We then compared ET(B) receptor levels in ganglia from sham and DOCA-salt animals. ET(B) receptor mRNA levels were 32% higher and ET(B) receptor protein levels were 20% higher in celiac ganglia from DOCA-salt rats than from sham rats separately. In conclusion, O2*- is elevated in prevertebral sympathetic ganglia in DOCA-salt hypertension, and ET-1 is a potent stimulus for the elevation of O2*- levels in sympathetic ganglia, an effect that may be mediated by the upregulation of ET(B) receptors.     

Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes

Endothelin (ET)-1 and suppressor of cytokine signaling (SOCS)-3 were respectively found to regulate energy metabolism and hormone signaling in fat cells. Although ET-1 can also regulate the expression of SOCS-3-stimulating hormones, it is still unknown whether ET-1 regulates SOCS-3 gene expression. This study investigated the pathways involved in ET-1's modulation of SOCS-3 gene expression in 3T3-L1 adipocytes. ET-1 upregulated SOCS-3 mRNA and protein expression in dose- and time-dependent manners. The concentration of ET-1 that increased SOCS-3 mRNA levels by 250-400% was ～100nM with 2-4h of treatment. Treatment with actinomycin D prevented ET-1-stimulated SOCS-3 mRNA expression, suggesting that the effect of ET-1 requires new mRNA synthesis. Pretreatment with the ET type A receptor (ET(A)R) antagonist, BQ-610, but not the ET type B receptor (ET(B)R) antagonist, BQ-788, prevented the stimulatory effect of ET-1 on SOCS-3 gene expression. The specific inhibitors of either MEK1 (U-0126 and PD-98059), JAK (AG-490), JNK (SP-600125), or PI3K (LY-294002 and wortmannin) reduced ET-1-increased levels of SOCS-3 mRNA and respectively inhibited ET-1-stimulated activities of MEK1, JAK, JNK, and PI3K. These results imply that the ET(A)R, ERK, JAK, JNK, and PI3K are functionally necessary for ET-1's stimulation of SOCS-3 gene expression. Moreover, ET-1 was observed to upregulate expressions of SOCS-1, -2, -3, -4, -5, and -6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein-1 mRNAs. This suggests that ET-1 selectively affects particular types of SOCS family members. Changes in SOCS gene expressions induced by ET-1 may help explain the mechanism by which ET-1 modulates hormone signaling of adipocytes.     

Neutralization of transforming growth factor-beta attenuates hypertension and prevents renal injury in uremic rats

OBJECTIVE: We investigate the role of transforming growth factor-beta (TGF-beta) in hypertension and renal failure progression in uremic rats, and whether it modulates the endothelin (ET) system. DESIGN: Following renal mass reduction, uremic rats (Nx) received the pan-specific TGF-beta neutralizing antibody 1D11 (0.5 mg/kg, three times/week), the isotype control antibody 13C4 or the AT1 antagonist losartan (10 mg/kg per day) for 6 weeks. RESULTS: Before treatment, the blood pressure was higher in Nx rats and increased further over time in Nx+13C4 rats. At the end of the study, Nx+13C4 rats exhibited increased serum creatinine, proteinuria and renal expression and excretion of TGF-beta1 and ET-1. ET-1 concentrations were greater in vascular and renal tissues, whereas the ETB receptor expression was reduced. Renal injuries were comprised of blood vessel hypertrophy, glomerular sclerosis, tubular atrophy and interstitial fibrosis, which was associated with increased alpha-smooth muscle actin expression. Treatment of uremic rats with the 1D11 antibody attenuated the increase in blood pressure and the decline in renal function. Losartan normalized the blood pressure and significantly attenuated the increase in serum creatinine and proteinuria. However, both treatments prevented renal TGF-beta1 and ET-1 overexpression, and prevented all renal histological injuries. The 1D11 antibody only improved ETB receptor expression. CONCLUSIONS: Neutralization of TGF-beta attenuates hypertension and renal failure progression in uremic animals, in part, by preventing renal injury processes. These effects may be related to the modulation of the ET system, preventing renal ET-1 overproduction and the reduction of ETB receptor expression. Our data also suggest that TGF-beta1 is involved, at least in part, in the pathological effects related to angiotensin II in chronic renal failure.     

Administration of exogenous endothelin-1 following vascular balloon injury: early and late effects on intimal hyperplasia.

Administration of exogenous endothelin-1 (ET-1) has been shown to stimulate neointimal hyperplasia following arterial balloon angioplasty (BA). However, the specific effects of ET-1 on the cellular and extracellular matrix response of the vessel wall after balloon injury and the persistence of these ET-1 effects have not been studied. The objectives of this study were to determine the acute (1 week) and long term (10 weeks) effects of administering exogenous ET-1 after arterial BA on neointimal hyperplasia, collagen synthesis and content, cellular proliferation, and ET(A) and ET(B) receptor expression. Thirty-one rabbits were randomized to receive subcutaneous ET-1 (500 pmol/kg/day for 1 week) or placebo time-release pellets and sacrificed at either 1 or 10 weeks after BA. At 1 week, there was a significant two-fold increase in intimal cross-sectional area (CSA) in ET-1 treated animals compared with placebo. ET-1 treated animals showed significant increases in collagen synthesis (ten-fold) and collagen content (three-fold) compared to placebo treated animals. ET-1 treated animals also had a significant increase (two-fold) in proliferation rates. In addition, ET(A) and ET(B) receptor expression were significantly upregulated in ET-1 treated animals. By 10 weeks these stimulatory effects on intimal CSA and collagen content were no longer evident with a 'catch up' phenomenon observed in the placebo treated animals. Similarly, ET(A) and ET(B) mRNA levels had declined significantly in both groups. Therefore, exogenous ET-1 acutely stimulates extracellular and cellular processes including increased expression of ET(A) and ET(B) receptors contributing to intimal hyperplasia. However, these effects are transient and not maintained long term after withdrawal of exogenous ET-1 stimulation.