Endothelin Subtype A Research Articles

Effects of a new magnesium-rich artificial cerebrospinal fluid on contractile 5-hydroxytryptamine and endothelin receptors in rat cerebral arteries

ABSTRACTObjectives Cerebral vasospasm after subarachnoid haemorrhage (SAH) is associated with cerebrovascular contractile receptor upregulation resulted from haemolysis in the subarachnoid space. This study developed a new magnesium-rich artificial cerebrospinal fluid (MACSF) formula and investigated its effects on receptor-mediated contraction in rat basilar arteries.Methods Clear and haemorrhagic cerebrospinal fluid (CSF) were collected from patients with hydrocephalus or SAH. MACSF was freshly prepared using clinical intravenous injections. Rat basilar arteries were segmented and incubated with clear CSF, haemorrhagic CSF or MACSF. The contractile responses were studied by myograph. The messenger ribonucleic acid (mRNA) and protein expression of 5-hydroxytryptamine 1B (5-HT1B), endothelin subtype B (ETB) and endothelin subtype A (ETA) receptors were evaluated by real-time polymerase chain reaction (PCR) and Western blot analyses.Results Haemorrhagic CSF exposure shifted the contractile curves induced by 5-hydroxytryptamine (5-HT), sarafotoxins 6c (S6c) and endothelin-1 (ET-1) leftward with increased maximal contraction values. Furthermore, mRNA and protein expression were markedly elevated for 5-HT1B, ETB and ETA receptors on arteries exposed to haemorrhagic CSF. However, the contractile responses to 5-HT, S6c or ET-1 and expression of 5-HT1B, ETB and ETA receptors in rat cerebral arteries exposed to MACSF remained unaffected compared to those exposed to clear CSF. Besides, unlike normal saline which can inactive in-vitro vessels, MACSF can maintain their physiological activity.Conclusion Haemorrhagic CSF induces upregulation of 5-HT1B, ETB and ETA receptors in rat cerebral arteries. However, MACSF can maintain in-vitro rat basilar arteries in good physiological activity and normal expression of contractile 5-HT and ET receptors.

Airborne fine particulate matter alters the expression of endothelin receptors in rat coronary arteries

Exposure to airborne fine particulate matter (PM2.5) is associated with cardiovascular diseases. However, a comprehensive understanding of the underlying mechanisms by which PM2.5 induces or aggravates these diseases is still insufficiently clear. The present study investigated whether PM2.5 alters the expression of the endothelin subtype B (ETB) and endothelin subtype A (ETA) receptors in the coronary artery and examined the underlying mechanisms. Rat coronary artery segments were cultured with PM2.5 in the presence or absence of MEK/ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was measured by myography. ETB and ETA receptor expression was evaluated using RT-PCR, western blot and immunohistochemistry. Compared with fresh arteries, the cultured coronary arteries showed a significantly enhanced contraction mediated by the ETB receptor and an unaltered contraction mediated by the ETA receptor. Culture with PM2.5 significantly enhanced the contraction and the mRNA and protein expression levels of the ETB and ETA receptors in the coronary arteries, suggesting that PM2.5 induces an upregulation of ETA and ETB receptors. In addition, the PM2.5-induced increases in ETB- and ETA-mediated vasoconstriction and receptor expressions could be notably decreased by MEK1/2 inhibitor, U0126 and Raf inhibitor, SB386023, suggesting that the upregulation of ETB and ETA receptors is related with MEK/ERK1/2 pathway. In conclusion, PM2.5 induces the ETB and ETA receptor upregulation in rat coronary arteries, and the MEK/ERK1/2 pathway may be involved in this process.

Synthesis and In Vivo Evaluation of Novel PET Radioligands for Imaging the Endothelin-A Receptor

The endothelin subtype-A (ETA) receptor is a member of a family of G-protein-coupled receptors that plays a central role in vasoconstriction, cell proliferation, and hormone production. The aim of this study was to synthesize and evaluate in vivo (11)C- and (18)F-labeled analogs of the potent and selective ETA antagonist N-[[2'-[[(4,5-dimethyl-3-isoxazolyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]-2-yl]methyl]-N,3,3-trimethylbutanamide (BMS-207940). Protected precursors and authentic nonradioactive standards were synthesized by reductive amination and subsequent alkylation of protected aldehyde 1. Desmethyl precursor 2 was reacted with (11)C-methyl iodide followed by deprotection and high-performance liquid chromatography purification to produce (11)C-(N-[[2'-[[(4,5-dimethyl-3-isoxazolyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]-2-yl]methyl]-N,1-methylindolecarboxamide) ((11)C-BMS-5p) 3. Nitro precursor 4 was reacted with (18)F-fluoride and purified by high-performance liquid chromatography to produce (18)F-(N-[[2'-[[(4,5-dimethyl-3-isoxazolyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]-2-yl]methyl]-N,4-fluorobenzamide) ((18)F-FBzBMS) 5. Biodistribution was determined by injecting male CD-1 mice via the tail vein with either (11)C-BMS-5p 3 or (18)F-FBzBMS 5. Specific binding was determined by pretreatment with 1 mg of BMS-207940 per kilogram. PET scanning of a baboon using either (11)C-BMS-5p 3 or (18)F-FBzBMS 5 was performed at baseline and 40 min after intravenous administration of 1 mg of BMS-207940 per kilogram. (11)C-BMS-5p 3 was synthesized with 1.5% radiochemical yield in 36 min, with an average specific activity of 1,051 GBq/micromol (28,400 mCi/micromol; n=5) at the end of synthesis. (18)F-FBzBMS 5 was synthesized with 0.54% radiochemical yield in 130 min, with an average specific activity of 12.9 GBq/micromol (349 mCi/micromol, n=7) at the end of synthesis. In mice, the highest uptake of both radioligands was in the liver, lungs, and heart. Radioactivity in the liver washed out over time, and uptake in the lungs and heart remained relatively stable. Mice pretreated with 1 mg of BMS-207940 per kilogram showed greater than 64% specific binding in the lungs and kidneys at 60 min. Specific binding in the heart was determined to be 63% for (11)C-BMS-5p 3 and 81% for (18)F-FBzBMS 5. PET studies in a baboon showed high uptake of both radioligands in the myocardium. Between 35 and 85 min, the heart-to-blood ratio was 4.7 to 1. Pretreatment with a 1 mg/kg dose of BMS-207940 showed 85% specific binding in the myocardium at 85 min after injection. Both (11)C-BMS-5p 3 and (18)F-FBzBMS 5 bind selectively to the ETA receptor in vivo. Further development of these radioligands for imaging the ETA receptor in humans is warranted.

Role of Podocytes for Reversal of Glomerulosclerosis and Proteinuria in the Aging Kidney After Endothelin Inhibition

The cause of focal-segmental glomerulosclerosis as a consequence of physiological aging, which is believed to be inexorable, is unknown. This study investigated whether inhibition of endothelin-1, a growth-promoting peptide contributing to renal injury in hypertension and diabetes, affects established glomerulosclerosis and proteinuria in the aged kidney. We also determined the role of endothelin receptors for podocyte injury in vivo and in vitro. Aged Wistar rats, a model of spontaneous age-dependent glomerulosclerosis, were treated with the orally active endothelin subtype A (ET(A)) receptor antagonist darusentan, and evaluation of renal histology, renal function studies, and expression analyses were performed. In vitro experiments using puromycin aminonucleoside to induce podocyte injury investigated the role of ET(A) receptor signaling for apoptosis, cytoskeletal injury, and DNA synthesis. In aged Wistar rats, established glomerulosclerosis and proteinuria were reduced by >50% after 4 weeks of darusentan treatment, whereas blood pressure, glomerular filtration rate, or tubulo-interstitial renal injury remained unaffected. Improvement of structural injury in glomeruli and podocytes was accompanied by a reduction of the expression of matrix metalloproteinase-9 and p21Cip1/WAF1. In vitro experiments blocking ET(A) receptors using specific antagonists or RNA interference prevented apoptosis and structural damage to podocytes induced by puromycin aminonucleoside. In conclusion, these results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. The results further suggest that age-dependent glomerulosclerosis is not merely a "degenerative" but a reversible process locally confined to the glomerulus involving recovery of podocytes from previous injury.

Cardiac endothelin system impairs left ventricular function in renin-dependent hypertension via decreased sarcoplasmic reticulum Ca(2+) uptake.

We evaluated the role of the cardiac endothelin (ET) system in compensated hypertensive left ventricular (LV) hypertrophy (LVH) and after the transition toward LV dysfunction. Hypertensive transgenic rats overexpressing the Ren2 gene (Ren2 rats) were investigated between the ages of 10 and 30 weeks (Ren2-10 and Ren2-30 groups, respectively) and compared with age-matched normotensive Sprague-Dawley (SD) rats (SD-10 and SD-30 groups, respectively). Systolic blood pressure and LV weight were elevated in both Ren2 groups compared with their age-matched SD control groups (P:<0.0001). In Ren2-30 rats, LV end-diastolic pressure increased and -dP/dt(max) decreased compared with the values in SD-30 and Ren2-10 rats (P:<0.05). This was paralleled by an activation of LV mRNA expression of preproET-1 and ET-converting enzyme-1 and ET subtype A (ETA) receptor binding in Ren2-30 compared with Ren2-10 rats (P:<0.001). Cardiac fibrosis was increased and sarcoplasmic reticulum (SR) Ca(2+) reuptake was reduced in Ren2-30 compared with SD-30 and Ren2-10 rats (P:<0.05). Treatment of Ren2 rats with the selective ETA receptor antagonist Lu135252 between 10 and 30 weeks of age did not lower systolic blood pressure, heart weight, or cardiac fibrosis but completely prevented the deterioration of LV end-diastolic pressure and abolished alterations in -dP/dt(max) and SR Ca(2+) reuptake compared with no treatment in Ren2-30 and SD-30 rats (P:<0.05). Activation of the cardiac ET system accounts at least in part for the LV dysfunction that gradually develops in LVH. The protective effect of ETA antagonism can be attributed to the improvement of diastolic LV function that is due to normalization of impaired SR Ca(2+) uptake.

Stimulation of the renin-angiotensin system by endothelin subtype A receptor blockade in conscious dogs.

Previous studies in dogs have shown additive or even synergistic effects of combined angiotensin-converting enzyme inhibition and either nonselective endothelin subtype A/B (ETA/B) or selective endothelin subtype A (ETA) receptor blockade on renal vascular resistance and mean arterial blood pressure. A possible mechanism underlying this interaction may be a stimulation of the renin-angiotensin system during endothelin (ET) receptor blockade. We therefore investigated whether plasma renin activity and renin release are regulated by the ETA receptor. Experiments were made in conscious, chronically instrumented dogs receiving either saline or the selective ETA receptor antagonist LU 135252 (10 mg/kg IV). Eighty to 100 minutes after the administration of LU 135252 (n=5), heart rate (99+/-7 versus 81+/-6 bpm; P<0.05) and renal blood flow (327+/-40 versus 278+/-36 mL/min; P<0.05) were increased significantly, whereas mean arterial blood pressure tended to be lower (93+/-5 versus 105+/-7 mm Hg). These changes were associated with a 2-fold increase in plasma renin activity (0.74+/-0.12 versus 0.37+/-0.10 ng angiotensin I per milliliter per hour; P<0.05). Measurements of renin release at various renal perfusion pressures (n=5) with the use of a vascular occluder implanted around the left renal artery revealed that ETA receptor blockade did not alter renin release at resting renal perfusion pressure (78+/-25 versus 71+/-39 U/min) but strongly enhanced the sensitivity of pressure-dependent renin release <80 mm Hg approximately 2.2-fold. In conclusion, selective ETA receptor blockade is associated with a stimulation of the circulating renin-angiotensin system, which results from both a sensitization of pressure-dependent renin release and a larger proportion of blood pressure values falling into the low pressure range, where renin release is stimulated. These find-ings strengthen the view that ET and the renin-angiotensin system closely interact to regulate vascular resistance and provide a physiological basis for synergistic hypotensive effects of a combined blockade of both pressor systems.

Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats.

In isolated cardiac myocytes, the direct effects of angiotensin II on cellular growth and gene expression were shown to be mediated by endothelin via the endothelin subtype A (ETA) receptor. To determine whether this pathway is also involved in the cardiovascular adaptations to a chronic activation of the renin-angiotensin system in vivo, the effects of a selective ETA receptor antagonist (LU 127043) were investigated in adult rats with renal artery stenosis. Four groups of rats (n=107) were studied over a period of 10 days after surgery: (1) sham-operated animals with saline administration, (2) rats subjected to left renal artery clipping with saline administration, (3) sham-operated rats with LU 127043 administration, and (4) rats subjected to left renal artery clipping with LU 127043 administration. LU 127043 (50 mg/kg) or saline was given by gavage twice daily starting 1 day before the operation. In clipped rats with saline administration, plasma renin activity, the ratio of left ventricular weight to body weight, and mRNAs for beta-myosin heavy chain and atrial natriuretic peptide were significantly elevated as early as 2 days after surgery. Blood pressure started to rise on the third postoperative day and attained a steady state hypertensive level by day 6. Blockade of ETA receptors had no effects on plasma renin activity or the time course of hypertension in clipped animals but completely prevented left ventricular hypertrophy and the re-expression of the beta-myosin heavy chain and atrial natriuretic peptide genes on day 2. While the expressions of the beta-myosin heavy chain and atrial natriuretic peptide genes were not different from saline-treated, clipped animals after day 4, the development of left ventricular hypertrophy remained markedly blunted (-50%) during ETA receptor blockade until day 10. These results show that a continuous blockade of ETA receptors significantly attenuates the development of left ventricular hypertrophy and, more transiently, fetal gene expression in the early phase of renovascular hypertension. Since neither blood pressure nor the increase in plasma renin activity was significantly altered by ETA receptor blockade, the inhibitory influences of the ETA receptor antagonist on left ventricular hypertrophy and gene expression were mediated most likely through a direct blockade of myocardial ETA receptors.

Neurohormonal Activation and Progression of Heart Failure

The model for heart failure has changed radically over the past 20 years. No longer a simple hemodynamic paradigm of pump dysfunction, heart failure is now characterized as a complex clinical syndrome with release of many neurohormones and cytokines, which are believed to be most responsible for progression of disease. This change in our understanding of the pathophysiology of heart failure has important therapeutic implications. Drugs designed to influence the myocardial contractile state have been found over the past few decades to have either a neutral or an adverse effect on long-term survival, whereas agents designed to block the renin-angiotensin-aldosterone and other neurohormonal systems have proved to be remarkably effective treatment. Recently, drugs designed to block excessive sympathetic nervous system activity have been demonstrated in well-controlled studies to be safe and effective forms of therapy for heart failure. Carvedilol, a nonselective β-adrenergic blocker with α1-blocking and antioxidant properties, is associated with prevention of progression of heart failure as manifested by improvement in left ventricular (LV) function, reduction in heart size, and improved survival in patients with New York Heart Association functional Class II and III symptoms. This improvement is observed equally in patients with ischemic and non-ischemic heart failure. It is tempting to speculate that β-adrenergic blockers prevent the progression of heart failure by reducing LV mass and LV chamber size. In essence, carvedilol, and perhaps other β-adrenergic blockers, appear to abrogate relentless LV remodeling which is typically associated with progression of heart failure. The combination of angiotensin-converting enzyme inhibitors andβ-adrenergic blockers may be particularly effective in this regard, although more data on β-adrenergic blockers in patients with advanced heart failure are needed. Data from experimental heart failure animal models also suggest that endothelin(ET) subtype A (ETA) receptor blockers have the potential to lessen the pace of progressive LV remodeling. As our understanding of the neuroendocrine response to diminished cardiac performance improves, novel and even more imaginative neurohormonal and cytokine antagonists are likely to emerge as important new treatments for both hypertension and heart failure.

Chronic effects of FR139317 and enalapril on renal failure rats with moderate exercise.

We assessed the renal effects of moderate treadmill exercise in the spontaneously hypertensive rats (SHR) remnant kidney model of chronic renal failure (CRF). The effects of chronic administration of a specific endothelin (ET) subtype A (ETA) receptor antagonist, FR139317 (32 mg/kg/day i.p.) and an angiotensin-converting enzyme inhibitor, enalapril (2 mg/kg/day i.p.), in combination with moderate exercise were also investigated. Eight-week-old SHR were subjected to 5/6 nephrectomy. One week after surgery the rats were divided into five groups: (a) no treadmill running; (b) moderate treadmill running, 20 m/min for 60 min (Ex) per day; (c) Ex plus FR139317; (d) Ex plus enalapril; and (e) m-Ex plus enalapril in combination with FR139317, for 4 weeks. In SHR-CRF, Ex significantly attenuated the increase in urinary protein excretion. Enalapril significantly attenuated the increase in systolic blood pressure and urinary protein excretion. FR139317 at this dose did not show any antihypertensive or renal protective effect in this model. These results suggest that moderate exercise may protect renal function in SHR CRF. They also suggest that FR139317 may not have an additional antihypertensive and renal protective effect in this exercise model.

Structure and function of small arteries in salt-induced hypertension: effects of chronic endothelin-subtype-A-receptor blockade.

The involvement of endothelin in salt-induced hypertension is unclear. In the Dahl rat model, we studied the effects of a selective endothelin-subtype A (ET[A]) receptor antagonist, LU135252, on blood pressure, vascular structure, and function. Dahl salt-sensitive and salt-resistant rats were treated for 8 weeks with 4% NaCl alone or in combination with LU135252 taken orally (60 mg/kg per day). The geometry and reactivity of basilar and mesenteric arteries were studied in vitro under perfused and pressurized conditions using a video dimension analyzer. Chronic salt administration increased systolic blood pressure by 37 +/- 3 mm Hg and media-lumen ratio of the basilar and mesenteric arteries in salt-sensitive rats (P<.05). These structural changes were caused by eutrophic remodeling in basilar and hypertrophic remodeling in mesenteric arteries. Endothelium-dependent relaxations to acetylcholine and contractions to endothelin-1 were impaired in mesenteric arteries of salt-sensitive rats on a high NaCl diet. LU135252 prevented part of the increase in systolic blood pressure and structural and functional alterations but increased plasma endothelin 1 levels (P<.05 versus salt-treated, saltsensitive rats). LU135252 had no effect on these parameters in salt-resistant rats. These findings suggest that the long-term pressor effect of salt administration is mediated in part by the action of endogenous endothelin acting via ET(A) receptors. Thus, chronic ET(A) receptor blockade may be useful therapeutically to lower arterial pressure and prevent endothelial dysfunction and hypertrophic remodeling of resistance arteries in salt-sensitive forms of hypertension.

Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth.

Mesangial cell growth and accumulation of extracellular matrix proteins constitute key features of progressive glomerular injury. Endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictor agents, evoke a number of similar responses in mesangial cells. In rat mesangial cells, we compared ET-1 and Ang II effects on matrix protein production and cell proliferation as well as the potential interaction between the two hormones. When cells in 0.5% fetal calf serum were incubated for 24 hours with various concentrations of ET-1 or Ang II, both peptides stimulated, in a dose-dependent manner, fibronectin and type IV collagen mRNA expression, fibronectin synthesis, and mitogenesis. Incubation with specific receptor antagonists of both hormones demonstrated that endothelin subtype A (ETA) and angiotensin type 1 (AT1) receptors were involved. Preincubation of cells with two different protein kinase C inhibitors or with a neutralizing anti-transforming growth factor-beta antibody, but not an unrelated IgG, diminished the peptide-induced fibronectin synthesis. A dual interrelation seems to exist between ET-1 and Ang II. Thus, the AT1 receptor antagonist losartan and the angiotensin-converting enzyme inhibitors quinaprilat and captopril diminished the ET-1-mediated effects, whereas, the ETA receptor antagonist BQ-123 diminished the Ang II-induced fibronectin synthesis and mesangial cell proliferation. Our results suggest that ET-1 and Ang II stimulate matrix protein synthesis and mesangial cell mitogenesis through ETA and AT1 receptors, respectively, by complicated mechanisms, implicating protein kinase C activation, synthesis of transforming growth factor-beta, and release of one peptide by the other. These data could be important for a better understanding of the participation of vasoactive substances in the pathogenesis of glomerulosclerosis.

A specific endothelin subtype A receptor antagonist protects against injury in renal disease progression

We have recently reported that renal preproendothelin-1 gene is up-regulated in rats with renal mass reduction (RMR) and that time-dependent increase in urinary excretion of the corresponding peptide correlates with renal disease progression. Here we evaluated whether a specific endothelin subtype A (ETA) receptor antagonist, FR139317, reduced signs of disease activity in this model. Two groups of rats were given FR139317 or its vehicle (saline) from day 7 to day 60 after the surgical procedure. Sham-operated animals were the control group. Blood pressure, urinary protein excretion and serum creatinine were evaluated at days 0, 7 (before FR139317 or saline administration), 30, 45 and 60. At sacrifice, histological evaluation of renal tissue was performed. The results showed that ETA receptor blocker reduced the abnormal permeability to proteins, limited glomerular injury and prevented renal function deterioration thus confirming the working hypothesis. These findings suggest that this class of compounds may eventually prove useful in the treatment of human progressive nephropathies.