Chronic Endothelin Research Articles

Prevention of the progression of renal injury in diabetic rodent models with preexisting renal disease with chronic endothelin A receptor blockade.

The endothelin (ET) system has emerged as a therapeutic target for the treatment of diabetic nephropathy (DN). The present study examined whether chronic endothelin A (ETA) receptor blockade with atrasentan prevents the progression of renal injury in two models of DN with preexisting renal disease that exhibit an increased renal ET-1 system compared with nondiabetic rats: streptozotocin-treated Dahl salt-sensitive (STZ-SS) and type 2 diabetic nephropathy (T2DN) rats. Nine week-old SS rats were treated with (STZ; 50 mg/kg ip) to induce diabetes. After 3 wk of diabetes, proteinuria increased to 353 ± 34 mg/day. The rats were then separated into two groups: 1) vehicle and 2) atrasentan (5 mg·kg-1·day-1) via drinking water. After 6 wk of treatment with atrasentan, mean arterial pressure (MAP) and proteinuria decreased by 12 and 40%, respectively, in STZ-SS rats. The degree of glomerulosclerosis and renal fibrosis was significantly reduced in the kidneys of atrasentan-treated STZ-SS rats compared with vehicle STZ-SS rats. Interestingly, treatment with atrasentan did not affect GFR but significantly increased renal blood flow by 33% and prevented the elevations in filtration fraction and renal vascular resistance by 23 and 20%, respectively, in STZ-SS rats. In contrast to the STZ-SS study, atrasentan had no effect on MAP or proteinuria in T2DN rats. However, treatment with atrasentan significantly decreased glomerular injury and renal fibrosis and prevented the decline in renal function in T2DN rats. These data indicate that chronic ETA blockade produces advantageous changes in renal hemodynamics that slow the progression of renal disease and also reduces renal histopathology in the absence of reducing arterial pressure and proteinuria.

Contrasting differences of chronic endothelin A receptor blockade during the progression of renal injury in type‐1 and type‐2 diabetic nephropathy (689.3)

Contrasting differences of chronic endothelin A receptor (ETA) blockade during the progression of renal injury in type‐1 and type‐2 diabetic nephropathy Denisha Spires1, Tiffani White2, Lateia Taylor2 and Jan M. Williams2. 1Tougaloo College, Tougaloo, MS, 2Department of Pharmacology, University of Mississippi Medical Center, Jackson, MS 39216 The endothelin (ET) system has been shown to play an important role in the development and progression of diabetic nephropathy (DN) via the endothelin A (ETA) receptor. Preliminary studies from our laboratory indicate that the induction of diabetes (STZ, 50 mg/kg, i.p.) in Dahl salt‐sensitive (STZ‐SS) rats promotes the development DN that was associated with an increase in ET‐1 excretion. Therefore, the present study examined whether chronic ETA blockade with ABT‐627 prevents the progression of renal injury in STZ‐SS rats with pre‐existing renal disease. After 6 weeks of ABT‐627 (5mg/kg/day) treatment, proteinuria was markedly decreased in ABT‐627 treated STZ‐SS rats versus vehicle treated rats without any changes in arterial pressure (310±32 vs. 517±68 mg/day, respectively). The degree of glomerulosclerosis and renal interstitial fibrosis was significantly reduced in the kidneys of ABT‐627 treated STZ‐SS rats compared to vehicle STZ‐SS rats. We next determined whether treatment with ABT‐627 would be beneficial in a type‐2 diabetic model (T2DN rat) with pre‐existing renal injury. In contrast to the STZ‐SS study, ABT‐627 had no effect on the progression of renal injury in T2DN rats. In conclusion, these data indicate that treatment with an ETA receptor blocker prevents the progression of renal injury in type‐1 diabetes but not in type‐2 diabetes.Grant Funding Source: Supported by P20GM104357

Chronic endothelin A receptor blockade attenuates contribution of sympathetic nervous system to salt hypertension development in adult but not in young Dahl rats

Endothelin-1 (ET-1) plays an important role in the pathogenesis of salt-dependent forms of hypertension in adult rats, but its participation in salt hypertension elicited in immature rats is still unknown. Therefore, we compared ET-1 role in the development or the maintenance of salt hypertension induced in young (4-week-old) or adult (12-week-old) Dahl rats. The contribution of pressor ET-1 effects to the maintenance of high blood pressure (BP) was studied using acute injection of ET(A) receptor antagonist ambrisentan (BSF 208075, 1 mg kg(-1) iv) to young or adult rats with established salt hypertension. Furthermore, using chronic ambrisentan treatment (30 mg kg(-1) day(-1) in the drinking fluid during 5 weeks of high salt intake), we investigated the age-dependent involvement of ET(A) receptors in salt hypertension development in these two age groups. Acute ET(A) receptor blockade lowered BP in both age groups of salt hypertensive Dahl rats more than in rats fed a low-salt diet (but without any age-dependent difference). Chronic ET(A) receptor blockade strongly attenuated the development of salt hypertension and cardiac hypertrophy in adult rats, but it had no significant effects on salt hypertension in young animals. Pronounced BP reduction induced in adult salt hypertensive rats by chronic ambrisentan treatment was attributed to attenuated sympathetic BP component, without changes in nitric oxide (NO)-dependent BP regulation. In contrast, chronic ambrisentan treatment of young animals did not modify sympathetic BP component but substantially attenuated NO-dependent vasodilatation. ET(A) receptor-mediated ET-1 effects play an important role in salt hypertension of adult but not young Dahl rats.

Chronic endothelin A receptor blockade attenuates contribution of sympathetic nervous system to salt hypertension development in adult but not in young Dahl rats

Chronic endothelin A receptor blockade attenuates contribution of sympathetic nervous system to salt hypertension development in adult but not in young Dahl rats

Effects of Chronic Endothelin ETA Receptor Blockade on Blood Pressure and Vascular Formation of Cyclic Guanosine-3’,5’-monophosphate in Spontaneously Hypertensive Rats

Endothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation via nitric oxide (NO) release and formation of cyclic guanosine-3',5'-monophosphate (cGMP). In spontaneously hypertensive rats (SHR) the cGMP-forming NO-receptor guanylyl cyclase (sGC) is downregulated. It is unclear whether ET contributes to the hypertensive phenotype of SHR, and whether this involves the disturbed cGMP signaling. The selective ETA receptor antagonist darusentan (CAS 171714-84-4), given orally via drinking water (10 mg kg(-1) d(-1)) for 12 weeks, significantly lowered systolic blood pressure of SHR as determined by radiotelemetry. Neither impaired endothelium-dependent relaxation to acetylcholine was restored nor sGC expression and activity affected when compared to control SHR. While these findings show a role for ETA receptors in blood pressure regulation in genetically elevated blood pressure, downregulation of sGC expression and cGMP-mediated vasorelaxant response in SHR were shown to be independent of ETA receptors. The findings suggest distinct mechanisms of gene expression affecting ET and cGMP mediated vasomotor functions.

Comparison of selective versus dual endothelin receptor antagonism on cerebrovascular dysfunction in diabetes

<title/>Objectives: Cerebrovascular tone plays a key role in controlling cerebral blood flow. Our studies have demonstrated that the endothelin system is upregulated in type 2 diabetes leading to increased sensitivity to endothelin-1 and decreased relaxation in basilar artery. While chronic endothelin A receptor blockade restored relaxation, selective endothelin B receptor blockade caused paradoxical constriction in diabetes. Whether this effect was due to activation of endothelin A receptors in the presence of endothelin B receptor blockade or due to the loss of vasculoprotective effects of endothelin B receptors remained unknown. The current study hypothesizes that due to the antagonism of the vasculoprotective endothelin receptor B, dual blockade will not be as effective as selective endothelin receptor A antagonism in improving cerebrovascular dysfunction in type 2 diabetes.Methods: These studies were done in non-obese, type 2 diabetic Goto-Kakizaki rats administered either vehicle, selective endothelin receptor A antagonist Atrasentan (5 mg/kg) or dual endothelin antagonist Bosentan (100 mg/kg) for 4 weeks. At termination, basilar arteries were collected and mounted on a wire myograph and cumulative dose-response curves to endothelin-1 (1-500 nM) and acetylcholine (1 nM-5 μm) were studied.Results: Basilar artery was highly sensitive to endothelin-1-mediated constriction in diabetic animals. While neither Atrasentan nor Bosentan affected endothelium-dependent vascular relaxation in control animals, both treatments improved the maximum dilatation in diabetes and Atrasentan also improved sensitivity to acetylcholine.Conclusion: In light of our previous data which showed that endothelin B receptors are vasculoprotective and blockade of this receptor worsens relaxation, current findings suggest that when blocked simultaneously with the endothelin receptor A, the endothelin receptor B antagonism is protective by reducing the hyperreactivity and improving cerebrovascular function in diabetes.

Preventive Effect of Chronic Endothelin Type A Receptor Antagonist on Coronary Microvascular Spasm Induced by Repeated Epicardial Coronary Artery Endothelial Denudation in Pigs

We investigated the role of endothelin-1 (ET-1) in coronary microvascular spasm in a porcine model. A flowmeter was implanted around the left anterior descending coronary artery (LAD), and epicardial coronary artery endothelial denudation (ED) was performed just distal to the flowmeter every 2 weeks (W) until 6 W in 10 pigs (ED group). Ten pigs were chronically treated with endothelin type A receptor (ET(A)) antagonist (TA-0201, 0.1 mg/kg/day, ED+ET(A) group), while neither ED nor administration of ET(A) antagonist was performed in 12 pigs (Control group). We assessed changes in LAD blood flow and the denuded site diameter induced by acetylcholine (ACh, 0.05 microg/kg i.c.). In the ED group, administration of ACh to LAD induced zero flow without LAD diameter reduction at 8 W. In the ED+ET(A) group, the decrease in LAD blood flow response to ACh was suppressed. Chronic administration of TA-0201 suppressed ROS generation in the myocardium. Decreases of eNOS and intimal thickening were smaller in the TA-0201 administration group than the non-TA-0201 administration group. Chronic administration of ET(A) receptor antagonist is effective to prevent coronary microvascular spasm.

Role of Endothelin in Mediating Soluble fms-Like Tyrosine Kinase 1–Induced Hypertension in Pregnant Rats

Although soluble fms-like tyrosine kinase 1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor, has been implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms whereby enhanced sFlt-1 production leads to hypertension remain unclear. Both sFlt-1 and endothelin 1 productions are elevated in women with preeclampsia and in placental ischemic animal models of preeclampsia; however, the importance of endothelin 1 and sFlt-1 interactions in the control of blood pressure during pregnancy is unknown. The purpose of this study was to determine the role of endothelin 1 in mediating sFlt-1-induced hypertension in pregnant rats. To achieve this goal, sFlt-1 (3.7 microg/kg per day for 6 days) was infused into normal pregnant rats and pregnant rats treated with a selective endothelin type A receptor antagonist, ABT 627 (5 mg/kg per day for 6 days). Plasma concentration of sFlt-1 increased from 735+/-34 pg/mL in normal pregnant rats to 2498+/-645 pg/mL (P<0.05) with infusion of sFlt-1. Arterial pressure increased from 100+/-1 mm Hg in normal pregnant rats to 122+/-3 mm Hg (P<0.05) in sFlt-1-infused rats. Chronic increases in plasma sFlt-1 in normal pregnant rats increased preproendothelin mRNA expression in the renal cortices by approximately 3-fold. In addition, chronic endothelin type A receptor blockade completely abolished the blood pressure response to sFlt-1 in pregnant rats (104+/-3 versus 100+/-1 mm Hg; P<0.05), whereas the endothelin A receptor antagonist had no effect on arterial pressure in NP rats (105+/-2 versus 100+/-1 mm Hg). In conclusion, this study demonstrates that endothelin 1, via endothelin type A receptor activation, plays an important role in mediating the hypertension in response to excess sFlt-1 during pregnancy.

Endothelin antagonism in portal hypertensive mice: implications for endothelin receptor-specific signaling in liver disease

Endothelin-1 (ET-1), a potent vasoactive peptide, plays an important role in the pathogenesis of liver disease and portal hypertension. Two major endothelin receptors (ET-A and ET-B) mediate biological effects, largely on the basis of their known downstream signaling pathways. We hypothesized that the different receptors are likely to mediate divergent effects in portal hypertensive mice. Liver fibrosis and cirrhosis and portal hypertension were induced in 8-wk-old male BALB/c mice by gavage with carbon tetrachloride (CCl4). Portal pressure was recorded acutely during intravenous infusion of endothelin receptor antagonists in normal or portal hypertensive mice. In vivo microscopy was used to monitor sinusoidal dynamics. Additionally, the effect of chronic exposure to endothelin antagonists was assessed in mice during induction of fibrosis and cirrhosis with CCl4 for 8 wk. Intravenous infusion of ET-A receptor antagonists into normal and cirrhotic mice reduced portal pressure whereas ET-B receptor antagonism increased portal pressure. A mixed endothelin receptor antagonist also significantly reduced portal pressure. Additionally, the ET-A receptor antagonist caused sinusoidal dilation, whereas the ET-B receptor antagonist caused sinusoidal constriction. Chronic administration of each the endothelin receptor antagonists during the induction of fibrosis and portal hypertension led to reduced fibrosis, a significant reduction in portal pressure, and altered sinusoidal dynamics relative to controls. Acute effects of endothelin receptor antagonists are likely directly on the hepatic and sinusoidal vasculature, whereas chronic endothelin receptor antagonism appears to be more complicated, likely affecting fibrogenesis and the hepatic microcirculation. The data imply a relationship between hepatic fibrogenesis or fibrosis and vasomotor responses.

Colfibrate attenuates blood pressure and sodium retention in DOCA-salt hypertension

Clofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR alpha) agonist, increases renal tubular cytochrome P450 4a (Cyp4a) expression thereby increasing 20-hydroxyeicosatetraenoic acid (20-HETE) production. To determine if clofibrate affects blood pressure regulation we studied mice with DOCA-salt induced hypertension in wild-type and PPAR alpha knockout mice. Wild-type mice treated with DOCA-salt had higher mean arterial pressures and higher cumulative sodium balance, but lower renal 20-HETE production than did vehicle-treated mice. Treating DOCA-salt mice with clofibrate attenuated the increase in mean arterial pressure and cumulative sodium balance while increasing 20-HETE production and renal Cyp4a expression. In contrast the PPAR alpha knockout mice treated with clofibrate and DOCA-salt showed no attenuation in the increase of blood pressure, cumulative sodium balance, renal 20-HETE production or Cyp4a protein expression. Expression of the PPAR alpha protein was greater in proximal tubules than in renal microvessels. Our results show that PPAR alpha pathway induces renal tubular 20-HETE production which affects sodium retention and blood pressure regulation in DOCA-salt-treated mice.

Pharmacology of Macitentan, an Orally Active Tissue-Targeting Dual Endothelin Receptor Antagonist

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A) receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

Atrasentan Treatment of Pulmonary Vascular Disease in Piglets With Increased Pulmonary Blood Flow

We studied the effect of chronic endothelin A receptor blockade by atrasentan on the pulmonary endothelin-1 system and vascular endothelial growth factor (VEGF) expression in piglets with high pulmonary blood flow. Twenty-five 4-week-old piglets with high pulmonary blood flow were randomized to three groups: sham operated (n = 8), placebo (water) (n = 7), or treatment with atrasentan (2 mg/kg per day) (n = 10). After 3 months, mean pulmonary arterial pressure (PAP) was higher in the placebo group than in the sham group [18 +/- 2 mm Hg versus 14 +/- 1 mm Hg; P < 0.05 (ANOVA)]. Atrasentan treatment was associated with lower cardiac output, PAP (14 +/- 1 mm Hg), and medial wall thickness of pulmonary arteries (diameter: 50-150 microM) compared with placebo [13.6 +/- 3.0% versus 18.1 +/- 4.2%; P < 0.05 (ANOVA)]. Quantitative real-time polymerase chain reaction for endothelin-1, endothelin B receptor, and endothelin-converting enzyme-1 mRNA in lung tissue did not differ. However, immunostaining as well as mRNA for VEGF were lower in atrasentan-treated animals (relative gene expression: atrasentan versus placebo: 0.8 +/- 0.3 versus 1.5 +/- 0.3; P = 0.009). Atrasentan treatment effectively reduces medial hypertrophy in piglets with chronic pulmonary hyperperfusion. Chronic endothelin A receptor blockade by atrasentan may interfere with the expression of VEGF.

Effects of endothelin ETAreceptor blocker LU 135252 on cardiac remodeling and survival in a hypertensive rat model of chronic heart failure

To investigate whether the endothelin ETA receptor blocker provides similar benefit on cardiac remodeling and survival in a hypertensive rat model of chronic heart failure (CHF). Male stroke-prone spontaneously hypertensive (SHR-SP) rats were subjected to permanent ligation of the left coronary artery and were treated for 6 weeks with the endothelin ETA receptor blocker LU 135252 (30 mg.kg(-1).d(-1)) starting 24 h after ligation or untreatment. Sham-operated rats served as normal controls. The mean arterial blood pressure (MAP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular contractility (LV dp/dt(max)), left ventricular inner diameter (LVD) and circumference (LVC), septal thickness, left ventricular interstitial collagen content (ICC) and heart weight (HW) were measured at the end of the treatment. Compared with the untreated group, LU 135252 tended to increase HW (1.43 +/-0.03 vs 1.38 +/-0.04 g; P> 0.05), increased LVD (7.65+/-0.24 mm vs 6.58+/-0.14 mm; P<0.05), markedly increased LVC (30.11+/-0.83 mm vs 24.82+/-0.85 mm; P< 0.01) and reduced left ventricular ICC (3.79%+/-0.09% vs 6.71%+/-0.11%; P< 0.01), slightly lowered MAP (132+/-6 mmHg vs 142+/-4 mmHg; P>0.05), reduced LVEDP (14 4 mmHg vs 27+/-4 mmHg; P<0.05) and improved LV dp/dtmax (4230+/-450 mmHg/s vs 1950+/-400 mmHg/s; P<0.05); survival was not prolonged significantly (13% vs 11%; P=NS). In this hypertensive rat model of CHF, chronic endothelin ETA receptor blockade with LU 135252 improves cardiac hemodynamics, however, it does not affect long-term survival and worsens cardiac remodeling. Thus, endothelin ETA receptor antagonists are unlikely to have an important role in the management of patients with CHF.

Does Malfunction of Arachidonic Acid Epoxygenase Explain Salt-Sensitive Hypertension?: Salt-Sensitive Hypertension Is Associated with Dysfunctional Cyp4a10 Gene and Kidney Epithelial Sodium Channel. J Clin Invest 116: 1696-1702, 2006.

Does Malfunction of Arachidonic Acid Epoxygenase Explain Salt-Sensitive Hypertension?: Salt-Sensitive Hypertension Is Associated with Dysfunctional Cyp4a10 Gene and Kidney Epithelial Sodium Channel. J Clin Invest 116: 1696-1702, 2006.

Effects of endothelin receptor blockade on hypervasoreactivity in streptozotocin-diabetic rats: vessel-specific involvement of thromboxane A2

Increased vasoconstrictor response to norepinephrine (NE) and endothelin (ET)-1 in arteries from diabetic animals is ameliorated by chronic endothelin receptor blockade with bosentan and was absent in endothelium-denuded arteries, suggesting the involvement of ET-1 and an endothelium-derived contracting factor such as thromboxane A2 (TxA2). To examine this possibility, we determined the effects of acute blockade of ET receptors or inhibition of TxA2 synthesis on the vascular function of superior mesenteric arteries (SMA) and renal arteries (RA) isolated from nondiabetic and 11-week streptozotocin (STZ) diabetic rats chronically treated with either bosentan or vehicle. Both in vitro incubation with bosentan and a selective ETA receptor blocker, BQ123, eradicated the increase in NE contractile responses in diabetic SMA. Additionally, in vitro incubation with the thromboxane synthase inhibitor, dazmegrel, abrogated the exaggerated NE and ET-1 contractile responses in diabetic SMA. Conversely, in RA, no significant acute effect of bosentan, BQ123, nor dazmegrel on vascular responses to NE was observed. Dazmegrel incubation attenuated the maximum contractile responses to ET-1 in diabetic RA; however, these responses in diabetic RA remained significantly greater than those of other groups. Diabetic RA but not SMA exhibited an enhanced contractile response to the TxA2 analogue U46619, which was corrected by chronic bosentan treatment. Immunohistochemical analyses in diabetic SMA revealed an increase in ETA receptor level that was normalized by chronic bosentan treatment. These data indicate that an interaction between ET-1 and TxA2 may be involved in mediating the exaggerated vasoconstrictor responses in diabetic arteries. Furthermore, the underlying mechanisms appear to be vessel specific.

Chronic endothelin receptor blockade reduces end-organ damage independently of blood pressure effects in salt-loaded heterozygous Ren-2 transgenic rats.

The present study was performed to evaluate the role of an interaction between the endothelin (ET) and the renin-angiotensin systems (RAS) in the development and maintenance of hypertension and in hypertension-associated end-organ damage in heterozygous male and female transgenic rats harboring the mouse Ren-2 renin gene (TGR). Twenty-eight days old heterozygous TGR and age-matched transgene-negative normotensive Hannover Sprague-Dawley rats (HanSD) were randomly assigned to groups with normal-salt (NS) or high-salt (HS) intake. Nonselective ET(A)/ET(B) receptor blockade was achieved with bosentan (100 mg.kg(-1).day(-1)). All male and female HanSD as well as heterozygous TGR on NS exhibited 100 % survival rate until 180 days of age (end of experiment). HS diet in heterozygous TGR induced a transition from benign to malignant phase hypertension. The survival rates in male and in female heterozygous TGR on the HS diet were 46 % and 80 %, respectively, and were significantly improved by administration of bosentan to 76 % and 97 %, respectively. Treatment with bosentan did not influence either the course of hypertension (measured by plethysmography in conscious animals) or the final levels of blood pressure (measured by a direct method in anesthetized rats) in any of the experimental groups of HanSD or TGR. Administration of bosentan in heterozygous TGR fed the HS diet markedly reduced proteinuria, glomerulosclerosis and attenuated the development of cardiac hypertrophy compared with untreated TGR. Our data show that the ET receptor blockade markedly improves the survival rate and ameliorates end-organ damage in heterozygous TGR exposed to HS diet. These findings indicate that the interaction between the RAS and ET systems plays an important role in the development of hypertension-associated end-organ damage in TGR exposed to salt-loading.

Effect of chronic endothelin blockade on PKC isoform distribution in mesenteric arteries from diabetic rats

Hemodynamic changes, including increased vasoconstriction and reduced blood flow have been detected in both human diabetic patients and in animal models of diabetes. We previously demonstrated that the endothelin (ET) system was upregulated and involved in mediating the exaggerated vasoconstrictor responses in superior mesenteric artery (SMA) from diabetic rats. Chronic treatment of diabetic rats with the dual endothelin receptor antagonist, bosentan abolished the enhanced contractile responses in diabetic SMA. The biological actions of ET-1 have been shown to be coupled to the hydrolysis of phosphotidylinositol 4,5-biphosphate and phosphotidylcholine and the subsequent production of diacylglycerol (DAG). DAG is an activator of the classical and novel isoforms of PKC. Increases in PKC activity, associated with translocation of specific PKC isoforms from the cytosol to the membrane, have been implicated in the vasoconstrictor effect of ET-1. The goal of the present study was to determine whether chronic treatment of diabetic rats with bosentan influences the activation of specific PKC isoforms in SMA from diabetic rats. Elevated levels of PKCbeta2 in both the cytosol and membrane fractions and PKCepsilon in the membrane fraction were detected in SMA from diabetic rats. However, neither the levels nor the distribution between the cytosol and membrane fractions of any of these PKC isoforms were affected by the treatment of the diabetic rats with bosentan. These observations indicate that bosentan improves vascular reactivity in STZ-diabetic rats by mechanisms other than correction of increased activities of PKC isoforms.

Metabolic responses with endothelin antagonism in a model of insulin resistance

Atrasentan, an endothelin antagonist, would have beneficial effects on metabolic responses in a model of insulin resistance. Zucker lean or fatty rats were maintained either on regular (lean and fatty control, n = 12) or atrasentan-treated water (5 mg/kg/d, fatty atrasentan, n = 13) for 6 weeks. There was no significant difference in water intake and body weight with the atrasentan-treated group compared with fatty controls. Although atrasentan had no effect on 3-hour fasting glucose levels, it reduced fasting insulin levels between weeks 2 and 4 of treatment by 53% (fatty control vs fatty atrasentan, P < .01). Atrasentan decreased the incremental area under the plasma glucose response curve ( ΔAUC) after a nutritionally complete meal tolerance test (MTT), by 28% in the atrasentan-treated group compared with fatty controls ( P < .05), and decreased the MTT-induced insulin ΔAUC by 63% in treated animals compared with the fatty control group ( P < .01). In addition, atrasentan significantly decreased the MTT-induced glucose-insulin index ΔAUC by 58% in treated rats compared with fatty controls ( P < .01). In summary, in the Zucker fatty rat, atrasentan significantly reduces (1) 3-hour fasting insulin levels at 4 weeks, (2) glucose and insulin MTT-induced ΔAUCs, and (3) the MTT-induced glucose-insulin index ΔAUC. These results demonstrate an improvement in hyperinsulinemia as well as in glucose tolerance and insulin sensitivity with chronic endothelin antagonism in a model of insulin resistance and suggest that chronic endothelin antagonism may have benefits in the treatment of insulin resistance and/or diabetes.

Chronic endothelin exposure inhibits connexin43 expression in cultured cortical astroglia

Severe brain lesions are accompanied by sustained increases in endothelin (ET) levels, which in turn profoundly affect brain microcirculation and neural cell function. A known response of astrocytes to acute increases in ET levels is the rapid and transient closure of gap junctions and the subsequent decrease of gap junction-mediated intercellular communication (GJIC). Because evidence exists that the loss of GJIC alters astrocytic gene expression, we analyzed the effects of chronic ET exposure on astrocytic gap junction coupling. We found that within 24 hr, cultured cortical astrocytes respond to low nanomolar concentrations (2-10 nM) of either ET-1 or ET-3 with a robust inhibition of connexin (Cx)43 expression, the major junctional protein in astrocytes, and a subsequent decline of GIJC. We further observed that in the continuous presence of ETs, Cx43 expression remained inhibited for at least 7 days. In addition, a similar decrease of Cx43 expression occurred in cultured spinal cord astrocytes maintained with ET-1 for 3 days. Applying ETs in combination with the highly selective ETA and ETB receptor antagonists, BQ123 and BQ788, respectively, revealed that the inhibitory influences on astrocytic Cx43 expression depend on activation of ETB receptors. We suggest that the observed ET-dependent inhibition of Cx43 expression and the resulting decline of GJIC might represent a major pathway by which ETs regulate astrocytic gene expression in the injured brain.

Chronic endothelin A receptor blockade in lambs with increased pulmonary blood flow and pressure.

Endothelin receptor blockade is an emerging therapy for pulmonary hypertension. However, hemodynamic and structural effects and potential changes in endogenous nitric oxide (NO)-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in pulmonary hypertension secondary to congenital heart disease are unknown. Therefore, the objectives of this study were to determine hemodynamic and structural effects and potential changes in endogenous NO-cGMP and endothelin-1 signaling of chronic endothelin A receptor blockade in a lamb model of increased pulmonary blood flow following in utero placement of an aortopulmonary shunt. Immediately after spontaneous birth, shunt lambs were treated lifelong with either an endothelin A receptor antagonist (PD-156707) or placebo. At 4 wk of age, PD-156707-treated shunt lambs (n = 6) had lower pulmonary vascular resistance and right atrial pressure than placebo-treated shunt lambs (n = 8, P < 0.05). Smooth muscle thickness or arterial number per unit area was not different between the two groups. However, the number of alveolar profiles per unit area was increased in the PD-156707-treated shunt lambs (190.7 +/- 5.6 vs. 132.9 +/- 10.0, P < 0.05). Plasma endothelin-1 and cGMP levels and lung NOS activity, cGMP, eNOS, preproendothelin-1, endothelin-converting enzyme-1, endothelin A, and endothelin B receptor protein levels were similar in both groups. We conclude that chronic endothelin A receptor blockade attenuates the progression of pulmonary hypertension and augments alveolar growth in lambs with increased pulmonary blood flow.