Endothelin Receptor Density Research Articles

Darusentan is a potent inhibitor of endothelin signaling and function in both large and small arteriesThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Endothelin is a potent vasoconstrictor often up-regulated in hypertension. Endothelin vasoconstriction is mediated via the G-protein coupled endothelin A (ETA) receptor present on vascular smooth muscle. Endothelin receptor antagonists (ERAs) have been shown to antagonize ET-induced vasoconstriction. We describe the primary pharmacology of darusentan, a propanoic acid based ERA currently in phase 3 clinical trials for resistant hypertension. Darusentan was tested in membrane-, cell-, and tissue-based assays to determine its biochemical and functional potency. Rat aortic vascular smooth muscle cells (RAVSMs) were characterized using flow cytometry. RAVSM membrane fractions tested in saturation experiments exhibited moderate endothelin receptor density. Receptor counting revealed that >95% of the endothelin receptors in these fractions were the ETA subtype. (S)-Darusentan competed for radiolabeled endothelin binding in RAVSM membranes with single-site kinetics, exhibiting a Ki = 13 nmol/L. (R)-Darusentan exhibited no binding activity. In cultured RAVSMs, endothelin induced increases in inositol phosphate and Ca2+ signaling, both of which were attenuated by (S)-darusentan in a concentration-dependent manner. In isolated endothelium-denuded rat aortic rings, (S)-darusentan inhibited endothelin-induced vascular contractility with a pA2 = 8.1 +/- 0.14 (n = 4 animals; mean +/- SD). (R)-Darusentan had no effect. The vasorelaxant potency of (S)-darusentan did not change when determined in isolated denuded rat mesenteric arterioles, suggesting a similar mode of action in both conductance and resistance arteries. In vascular smooth muscle, (S)-darusentan is an ERA with high affinity for the ET receptor, which in this preparation is predominantly ETA receptors. (S)-Darusentan inhibits endothelin-induced signaling related to pro-contractile activity and is a potent inhibitor of vasoconstriction in large and small arteries.

Vagally mediated cholestatic and choleretic effects of centrally applied Endothelin-1 through ET A receptors

The role of Endothelin-1 (ET-1) in the central nervous system is not fully understood yet although several studies strongly support its neuromodulatory role. A high density of endothelin receptors is present in the dorsal vagal complex that is the major site for the regulation of the digestive function. Therefore in the present study we sought to establish the role of ET-1 in the central regulation of bile secretion in the rat. Intracerebroventricular ET-1 injection exhibited opposite behaviors on spontaneous bile secretion according to the dose administered. Lower doses of ET-1 (1 fM) increased bile flow and bicarbonate excretion whereas higher doses (1 nM) decreased bile flow and bile acid output. Both the choleretic and the cholestatic effects of ET-1 were abolished in animals pretreated with icv BQ-610 (selective ET A antagonist) but not with BQ-788 (selective ET B antagonist). In addition, truncal vagotomy but not adrenergic blockade abolished ET-1 effects on bile secretion. Brain nitric oxide was not involved in ET-1 response since l-NAME pretreatment failed to affect ET-1 actions on the liver. Portal venous pressure was increased by centrally administered ET-1 being the magnitude of the increase similar with low and high doses of the peptide. These results show that centrally applied ET-1 modified different bile flow fractions independent of hemodynamic changes. Lower doses of ET-1 increased bile acid independent flow whereas higher doses decreased bile acid dependent flow. Vagal pathways through the activation of apparently distinct ET A receptors mediated the cholestatic as well as the choleretic effects induced by ET-1. Present findings show that ET-1 participates in the central regulation of bile secretion in the rat and give further insights into the complexity of brain–liver interaction.

Participation of endogenous endothelin and ETA receptor in premicturition contractions in rats with bladder outlet obstruction

A relationship between endogenous endothelins and bladder overactivity has recently been suggested, but the related endothelin receptor subtype has not been identified. Here, to evaluate the involvement of endothelin-1 and its receptors in bladder overactivity, we investigated endothelin-1 levels and the expression of its receptors in the bladder of rats with bladder outlet obstruction (BOO), a model for bladder overactivity. We also investigated the effects of a selective endothelin ET(A) receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on bladder functions in conscious BOO rats. Partial obstruction of the urethra led to a progressive increase in bladder weight from weeks 1 to 6. Binding assays performed using plasma membranes prepared from these bladders to estimate endothelin receptor density from the maximum [(125)I]endothelin-1 binding showed increased endothelin receptor density (about double) at 1, 2, and 6 weeks after the operation in the BOO bladder. The densities of endothelin ET(A) receptors in the bladder of sham-operated and BOO rats at 2 weeks after operation were about 3.5 and 5 times those of endothelin ET(B) receptors respectively. Furthermore, the endothelin-1 level was also increased in the BOO bladder. Two weeks after operation, BOO rats showed an increase in maximum bladder capacity and micturition volume and the generation of premicturition contractions. The frequency of premicturition contractions was dose-dependently reduced by YM598 (0.1-3 mg/kg, i.v.) without any effect on other voiding parameters in BOO rats. These data suggest that endothelin-1 and endothelin ET(A) receptors might be involved in the generation of premicturition contractions in BOO rats, and that endothelin ET(A) receptor antagonists such as YM598 may have ameliorating effects in patients with bladder overactivity associated with BOO.

Interaction of Endothelin with Renal Nerves Modulates Kidney Function in Spontaneously Hypertensive Rats

Background and Methods: We investigated kidney function, renal endothelin-1 concentration, prepro-endothelin-1 mRNA as well as endothelin receptor A and B mRNA expression and receptor properties in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) with intact renal nerves and 7 days after renal denervation. In addition, responses of renal function to the non-selective ET_A/ET_B receptor blocker bosentan (10 mg/kg i.v. bolus injection) were studied. Results: In SHR, renal papillary prepro-endothelin-1 mRNA expression, endothelin-1 tissue concentrations and endothelin receptor density were significantly lower than in normotensive rats. Renal denervation was associated with a decrease in papillary tissue prepro-endothelin-1 mRNA and in WKY rats also with a significant reduction in papillary endothelin-1 content without affecting ET receptor density. Bosentan did not alter renal blood flow or glomerular filtration rate but decreased urine flow rate in both intact normotensive and hypertensive rats, whereas it decreased urine sodium and potassium excretion only in intact WKY. Bosentan had no effects on renal function in renal denervated rats. Conclusion: Since renal papillary endothelin-1 appears to counteract the fluid and sodium retaining effects of renal nerve activity, an impaired renal endothelin-1 synthesis in SHR may contribute to excessive sodium retention and thus to the pathogenesis of hypertension in SHR.

Synthesis, in vitro pharmacology and biodistribution studies of new PD 156707-derived ET A receptor radioligands

It is assumed that the regulation of cardiac endothelin (ET) receptor density is abnormal in heart diseases. From that perspective, an ET receptor radioligand is needed to assess ET receptor density in vivo. The nonpeptidyl ET A receptor antagonist PD 169390 was labelled with radioiodine to give a putative radioligand for SPECT. Labelling with [ 125I]iodide and [ 123I]iodide was accomplished with good to excellent radiochemical yields. The affinities of the nonradioactive reference and those of selected precursor compounds for ET A receptors were determined, using [ 125I]iodine labelled endothelin-1 with mouse ventricular membranes. All employed substances exhibited potent in vitro pharmacological characteristics with K i values comparable to that of the lead compound PD 156707. Biodistribution studies and scintigraphic imaging experiments in mice, however, showed no significant uptake of the [ 123I] derivative in the heart.

Role of endothelin axis in progression to aggressive phenotype of prostate adenocarcinoma

Mitogenic and anti-apoptotic actions of endothelin-1 (ET-1) are mediated through endothelin A (ET(A)) receptors. We investigated endothelin receptor expression in increasingly aggressive phenotype and in vivo effects of combination therapy using ET(A) antagonist with paclitaxel. Dunning prostate cancer cells ranged in aggressiveness from non-tumorigenic G, to tumorigenic, non-metastatic AT-1, and to tumorigenic and metastatic MLL. Binding assays were performed alongside Q-PCR to assess receptor density. MLL xenografts were treated with vehicle, atrasentan, paclitaxel, and paclitaxel+atrasentan. Saturation binding assays demonstrated endothelin receptor density of MLL and AT-1 cells seven- and threefold higher than G cells, respectively. Q-PCR showed 9- and 4.5-fold greater ET(A) mRNA expression in MLL and AT-1 than G cells, respectively and no endothelin receptor B (ET(B)) expression. Combination therapy had significant effect on reduction of tumor volume than paclitaxel or atrasentan alone. ET(A) expression increases in aggressive prostate carcinoma. ET(A) blockade combined with paclitaxel may reduce tumor growth in advanced prostate carcinoma.

Use of a mixed endothelin receptor antagonist in portopulmonary hypertension: A safe and effective therapy?

Portopulmonary hypertension (PPHTN), a severe complication of portal hypertension is observed in 3%-6% of patients evaluated for liver transplantation. Endothelin-1, a potent vasoconstrictor, is likely to play a role in the pathogenesis of primary pulmonary hypertension, and, in 2 recent trials, the dual endothelin receptor antagonist bosentan has shown beneficial effects in this disease. A role for endothelins in the development of both pulmonary hypertension and cirrhosis has been suggested. We therefore hypothesized that endothelin receptor blockade may be beneficial in the treatment of PPHTN. We report a case of a 42-year-old patient with PPHTN and alcoholic cirrhosis treated with the mixed endothelin receptor antagonist bosentan. The patient rapidly improved from NYHA IV to stage II, experienced a remarkable improvement of 6-minute walking distance from 0 to 590 m within 6 months, and resumed working full-time as a locksmith after 7 months of treatment. Improvement of cardiovascular parameters included a reduction of pulmonary vascular resistance by 60%, a decrease of mean pulmonary artery pressure (mPAP) from 55 to 44 mm Hg at 9 months, and a decline of plasma B-type natriuretic peptide (BNP) from 339 pg/mL to 19 pg/mL after 1 year. There were no adverse events except for a transient decrease in systemic blood pressure. To our knowledge, this is the first report of a patient with PPHTN treated with an endothelin receptor antagonist. The marked and sustained improvement supports the undertaking of controlled studies of the safety and efficacy of bosentan in PPHTN.

Evidence that Morphine Tolerance May Be Regulated by Endothelin in the Neonatal Rat

Opioids are widely used in the neonatal intensive care units for analgesia and sedation. Management of tolerance and withdrawal symptoms in neonates remains a major challenge. The present study investigates the involvement of a central endothelin (ET) mechanism in the development of tolerance to morphine in neonatal rats. Pregnant female rats were rendered tolerant to morphine and rat pups were delivered at term by cesarean section. The affinity (Kd) and density (Bmax) of ET receptors was determined by [125I]ET-1 binding in the brains of neonatal rats. Changes in G-protein stimulation were determined in placebo and morphine-tolerant neonatal rats by [35S]-guanosine-5'-o-(3-thio)triphosphate ([35S]GTPgammaS)-binding assay. Morphine tolerance did not affect the characteristics (affinity and density) of the ET receptors in the neonatal rat brains. Morphine as well as ET-1 produced significantly lower (p < 0.05) maximal stimulation of [35S]GTPgammaS binding in morphine-tolerant neonatal rats compared to the placebo group. The ETA receptor antagonist, BMS182874, produced significantly higher stimulation of G proteins in the morphine-tolerant compared to the placebo group. The ETB receptor agonist, IRL1620, produced a similar effect in both placebo and morphine-tolerant rats. This is the first report indicating the involvement of the G-protein-coupled ETA receptor in neonatal morphine tolerance.

Age-Related Changes in the Properties of the Endothelin Receptor System at Protein and mRNA Levels in the Rat Vas Deferens

As age-related changes occur in the properties of the endothelin (ET) receptor system in several mammalian tissues, and as there are significant amounts of functional ET receptors in the vas deferens, we investigated the age-related changes in the ET receptor system at the protein and mRNA levels in the rat vas deferens. The ET system was investigated in the vasa deferentia of 3 weeks, 3 months and 22 months old rats. ET receptors were characterized and quantified at the protein level by radioligand receptor binding, and gene transcript levels of ET-1, ET-3, ET converting enzyme-1 (ECE-1), and ETA and ETB receptor subtypes were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). The results of radioligand receptor binding assays demonstrate that there is a higher density of total ET receptors in the vas deferens of 3 weeks old rats than in 3 months and 22 months old rats, and that the predominant ET receptor is of the ETA subtype in all three ages. Real-time RT-PCR data show that the predominant mRNA expression of ETs and their receptors in all age groups studied are ET-1 and the ETA receptor subtype, respectively. Furthermore, ET-1, ET-3, ECE-1, and ETA and ETB receptor subtype mRNAs are expressed at higher levels in the 3 weeks old rats as compared with the other two age groups. These results demonstrate the presence of age-related changes in the properties of the ET receptor system at both protein and mRNA levels in the rat vas deferens.

Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin A receptor activation.

Degradation of the extracellular matrix proteins by matrix metalloproteinases (MMP) is an important regulatory step in the vascular remodeling process. Recent studies demonstrated that ETA receptors regulate cardiac MMP activity and fibrosis in DOCA-salt hypertension. However, little is known about endothelin (ET)-1 regulation of vascular MMP activity in hypertension. Thus early changes in ET-1-mediated regulation of MMP activity were measured in borderline hypertensive rats that develop impaired vasorelaxation and hypertension with chronic exposure to stress. Experiments were performed after 10 days of exposure to the behavioral stressor, air-jet stress, but before the onset of stress-induced hypertension. Study groups were 1) control (n = 8); 2) air-jet stress for 10 days (n = 8); 3) control plus ETA antagonist ABT-627 (n = 4), and 4) air-jet stress plus ETA antagonist (n = 4). MMP activity in the thoracic aorta was assessed by gelatin zymography. MMP protein and tissue ET-1 levels were evaluated by immunohistochemistry, and ET receptor density was determined by immunoblotting. Exposure to stress caused a twofold increase in plasma ET-1 levels (P < 0.05), and there was increased ET-1 staining at the tissue level. Total MMP activity and expression of MMP-2 and MMP-9 were increased in the stress group. ETA receptor antagonism prevented the increase in MMP expression and activation in the stress group. These results provide evidence that the MMP system is activated before the development of hypertension and ET-1 mediates these early events in vascular remodeling.

Developmental changes in the functional, biochemical and molecular properties of rat bladder endothelin receptors.

The effect of aging on functional, biochemical, anatomical and molecular properties of endothelin (ET) receptors in bladder smooth muscle of the 3-week-, 3-month- and 22-month-old rats was examined using isolated muscle bath techniques, radioligand binding on membrane particulates and slide mounted tissue sections, and real-time reverse transcription polymerase chain reaction (RT-PCR). ET-1 induced significantly larger contractile responses in bladder dome muscle strips from 3-week- than from 3-month- and 22-month-old rats. The expression level of total ET receptors, determined by saturation binding experiments with [(125)I]ET-1, was higher in detrusor from 3-week- than 22-month-old rats. Inhibition studies with BQ123, a selective ET(A) receptor antagonist, indicated the predominance of the ET(A) receptor subtype and a similar proportion of ET(A) to ET(B) receptor subtypes in the rat detrusor at all ages studied. Autoradiographic data support the age-dependent decrease in the density of ET receptors and also indicate that the ET(A) receptor subtype is primarily located in the smooth muscle layer, whereas the ET(B) receptor subtype is located in both the urothelial and smooth muscle layers. Determined by real-time RT-PCR, ET 1, ET-3, ECE-1 and ET receptor subtype (ET(A) and ET(B)) mRNAs were shown to be higher in bladders of 3-week- compared to 3-month- or 22-month-old rats. This study indicates age-dependent alterations in the ET receptor system at both gene transcript and protein levels in the Fischer rat detrusor.

Altered regulation of endothelin A receptor subtype in the cerebral arterioles in response to a Japanese-style diet, in stroke-prone hypertensive rats.

To investigate the expression of endothelin (ET)-1 and its receptors in the cerebral arterioles of stroke-prone (spSHR) and control spontaneously hypertensive rats (SHRs) and the changes in endothelin receptor subtypes A and B density elicited by a stroke-permissive diet, before the development of stroke. Six-week-old SHRs (n=11) and spSHRs (n=11) were assigned to either a regular or a "Japanese"-style diet, in addition to 1% NaCl in the drinking water, for 4 weeks. Cryosections (10 microm thick) of rat brain were assessed for endothelin receptor distribution and density by autoradiography with [125I]ET-1 (10(-10) mol/l) in the presence of cold ET-1 (10(-6) mol/l) or the peptide antagonists BQ-123 (10(-6) mol/l) or BQ-788 (10(-6) mol/l). Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect specific mRNAs and localize immunoreactive ET-1 and ET(A) and ET(B). In both strains, immunoreactive ET-1 was detected in the endothelium of cerebral arterioles, and RT-PCR and autoradiography demonstrated the coexistence of both receptor subtypes in brain homogenates and the cerebral arteriole walls, respectively. With the regular diet, the ET(A) receptor density was lower in SHRs than in spSHRs (P = 0.007), whereas the ET(B) receptor density was similar (P = NS). The Japanese-style diet increased the density of ET(A) receptors (P = 0.006) in SHRs, but decreased it (P = 0.019) in spSHRs. No effect was seen on ET(B) receptor density. ET(A) and ET(B) receptor subtypes are expressed in the wall of cerebral arterioles of SHRs and spSHRs. The latter strain showed a marked increase in ET(A) receptor density under a regular diet, in addition to an altered regulation in response to a stroke-permissive diet.

Impact of parainfluenza-3 virus infection on endothelin receptor density and function in guinea pig airways.

We examined the impact of parainfluenza-3 (P-3) respiratory tract viral infection on the density and function of endothelin (ET) receptor subtypes (ET(A) and ET(B)) in guinea pig tracheal smooth muscle. Total specific binding of [(125)I]ET-1 and the relative proportions of ET(A) and ET(B) binding sites for this ligand were assessed at day 0 (control) and at 2, 4, 8 and 16 days post-inoculation. At day 0, the proportions of ET(A) and ET(B) binding sites were 30% and 70% respectively. Total specific binding was significantly reduced at day 4 post-inoculation (32% reduction, n=8-12, P<0.05) and was largely due to a corresponding fall in ET(B) receptor density at this time point (38% reduction, n=8-12, P<0.05). The density of ET(A) receptors also fell significantly at day 8 post-inoculation (33% reduction, n=6-12, P<0.05). By day 16 post-inoculation, the densities of ET(A) and ET(B) receptors had recovered to control values. The ratio of ET(A):ET(B) receptor subtypes did not alter with P-3 infection. While P-3 infection reduced the density of tracheal smooth muscle ET(A) and ET(B) receptors, the contractile sensitivity and maximum response to carbachol and ET-1 was not altered in tissue from day 4 post-inoculation compared with the control. There seems to be a significant functional reserve for both receptor subtypes in this species that buffers the impact of P-3 infection on airway smooth muscle responsiveness to ET-1.

Effect of castration on endothelin receptors.

Endothelin (ET) plays a pivotal role in the pathogenesis of cell growth disorders such as cancer. Atrasentan (ABT-627), a selective antagonist for the ET receptor A (ET(A)), has shown benefit in controlling disease progression in men with hormone refractory prostate cancer who have undergone aggressive hormone ablation therapy. It is not known how hormone ablation affects ET-binding sites, although ET-1 and ET(A) expression are found to be elevated in prostate cancer patients. In this study, we examined the effect of castration on ET receptor binding in male beagle dogs. Three dogs were surgically castrated and another three sham-operated. The animals were sacrificed 1 week after operation and membranes were prepared from the prostate, heart, brain, kidney, liver and lung for ET-1, ET-3 and angiotensin II (A-II, as a control) binding studies. No significant difference in A-II binding was observed between castrated and sham-operated animals. However, ET-1 and ET-3 binding to prostate and brain membranes were altered significantly. From saturation binding studies using ET-1 in the prostate, the K(d) and B(max) values increased from 0.043 nM and 0.094 pmol/mg respectively in sham-operated dogs to 0.104 nM and 0.311 pmol/mg respectively in castrated animals. These results indicate that surgical castration in dogs produces a change in the ET receptor density in the prostate and brain, and may have implications for the effect of hormone ablation therapy on ET receptor expression in prostate cancer patients.

The effect of castration on endothelins, their receptors and endothelin converting enzyme in rat prostate.

We previously have shown that experimental diabetes in rats causes prostatic involution, reduces serum testosterone levels, and causes an upregulation in prostatic endothelin (ET) receptors. Furthermore, insulin treatment normalizes these changes (Saito et al., Mol Cell Biochem 210:1-12, 2000). Since experimental diabetes-induced reduction in serum testosterone may be a factor in the alteration of the ET receptors and of prostatic growth, we investigated the effect of castration, another means of involuting the prostate and decreasing serum testosterone levels, on the expression of ET receptors in ventral and dorsolateral regions of the rat prostate.Three-month-old Sprague-Dawley rats were surgically castrated or sham operated, and then killed on the 7th post-operative day. Biochemical and pharmacological properties, and localization of ET receptors in the rat prostate, were determined by performing a series of binding experiments with [(125)I]ET-1 and by light microscopy autoradiography, respectively. The expression levels of ET-1, ET-3, ET receptor subtypes and endothelin converting enzyme-1 (ECE-1) mRNAs were assessed by relative multiplex reverse transcription polymerase chain reaction (RT-PCR). The total density of ET receptors increases 3.7-fold in the ventral and 2.1-fold in the dorsolateral regions of the castrated rat prostate compared to sham operated animals. Castration causes a 2.4-fold increase in the density of alpha(1)-adrenoceptors (alpha(1)-ARs) in the ventral region of the prostate, but no change in the density of alpha(1)-ARs in the dorsolateral region of the rat prostate. The predominant ET receptor subtype in the rat prostate is the ETA subtype, which is mainly located in the prostatic stroma. In addition, RT-PCR data show an upregulation in the expression of ETB receptor subtype, ET-1 and ECE-1 mRNA in both regions, and a downregulation in the expression of ETA receptor subtype mRNA in the dorsolateral region of the castrated rat prostate. There is no change in the expression of ET-3 mRNA in either region. Castration does not cause significant changes in the pharmacological properties of prostatic ET receptors, i.e., the predominance of ETA receptors in either region of the prostate, or the expression of ETA receptor subtype mRNA in the ventral region of the castrated rat prostate. These results suggest the existence of a region/lobe-specific regulatory role for testosterone in the expression of the ET receptor system in the rat prostate.

Endothelin Receptors in the Aetiology and Pathophysiology of Varicose Veins

Introduction: varicose veins are tortuous and poorly contractile. Their aetiology remains unclear. Neovascularisation has been suggested as a possible explanation. Endothelins are mitogenic, promoting proliferation and migration of endothelial cells via endothelin-B receptors. We hypothesise that endothelial cells and endothelin receptor density and distribution may play a role in the development of varicosis. Methods: saphenous vein segments from nine patients with varicose veins were compared to six controls. Slide-mounted sections were incubated in radioactive labelled endothelin-1 and receptor subtype-selective ligands and binding sites assessed using autoradiography. Endothelin-1 and endothelial cells were identified by immunohistochemistry and CD31-positive staining cells counted. Results: radioactive labelled endothelin-1 and endothelin-B receptor binding was reduced in varicose compared to control veins (p=0.04). Endothelin-A receptor binding was diffuse, with no difference in density in both groups (p=0.58). Endothelin-B receptor binding was diffuse with superimposed clusters. Although the density of medial endothelin-B receptor binding was reduced in the varicose group, more clusters were identified in this group compared to controls (p=0.005). CD-31 staining identified these clusters as endothelial cells. Conclusion: the reduced endothelin-1 binding and endothelin-B receptor density may be partially responsible for the reduced vasocontractility in varicose veins. We speculate that the increase in endothelin-B receptor binding CD31-positive endothelial cells in varicose veins may potentially stimulate mitogenesis and migration, leading to new vessel formation.

Time-dependent up-regulation of endothelin-A receptors and down-regulation of endothelin-B receptors and nitric oxide synthase binding sites in the renal medulla of a rabbit model of partial bladder outlet obstruction: potential clinical relevance.

To assess the density of endothelin (ET) receptors (ET-1 is a potent vasoconstrictor peptide acting on two known receptors, ETA and ETB ) and nitric oxide synthase (NOS) binding sites in the kidney of a rabbit model of bladder outlet obstruction (BOO). Partial BOO was created in adult New Zealand White rabbits; after 1, 3, 4 and 6 weeks of BOO, kidney sections were incubated with radioligands for ET-1, ETA, ETB receptors and with [3H]-NOARG (a ligand for NOS). Autoradiographs were generated and analysed densitometrically. Sections were also assessed by NADPH histochemistry. Plasma creatinine, urea and electrolyte levels were regularly monitored. The control and 6-week BOO kidneys were also evaluated ultrastructurally by electron microscopy. There was no significant change in plasma creatinine, urea and electrolyte levels. ETA and ETB receptor density was significantly greater in the medulla than in the cortex (P<0.001) in all animals. There was an up-regulation of ETA receptors (P=0.03) and down-regulation of ETB receptors (P=0.03) and NOS binding sites (P<0.001), as well as decreased NADPH staining in the medulla of 6-week partial BOO kidneys. Electron microscopy detected glomerular disruption of the obstructed kidneys. The time-dependent changes in ETA and ETB receptors, NOS binding sites and NADPH staining in the renal medulla, as well as ultrastructural changes, occur despite normal renal function. These changes appear to be an early event and may play a role in the development of renal failure. Hence, the use of ETA receptor antagonists at this early stage may prevent the development of renal failure/impairment in BOO.

Developmental changes in the biochemical and functional properties of endothelin receptors in rabbit renal pelvis.

To examine the effect of age on the biochemical and functional properties, and regional distribution of endothelin (ET) receptors in the rabbit renal pelvis. The properties of ET receptors in 6-week-old and 6-month-old male rabbit renal pelves were examined using isolated muscle-bath and radioligand receptor-binding techniques. ET-1 caused a significant increase in the contractile force in muscle strips from all regions of the renal pelvis from both age groups, with the following rank order: upper=middle>lower. The magnitude of the ET-1-induced contractile responses were similar in the lower pelvic regions in both ages, but the responses in the upper and middle regions were significantly greater in younger rabbits. ET-1 increased the frequency of spontaneous activity in a concentration-dependent manner in the upper and middle pelvic regions in both age groups, with significantly smaller ED50 values in the younger than in the older rabbits. In both age groups the lower pelvic region lacked spontaneous activity. The density of total ET receptors was higher in the upper and middle regions of the renal pelvis than in the lower renal pelvis of both ages, with the density in the upper and middle regions being greater in older than in younger rabbits. In all regions, ET subtype selective compounds inhibited [125I]ET-1, binding consistent with the predominance of the ETA receptor subtype, except in the lower region of the older rabbits, in which the densities of ETA and ETB subtypes were similar. In all regions, the younger renal pelvis contained a higher proportion of ETA receptors than in older tissues. Light microscopic autoradiographic data indicated the presence of ETA and ETB receptors in smooth muscle and epithelial cells, respectively. These data indicate the presence of regional differences in the density of ET receptors and in the contractile responses to ET-1 in rabbit renal pelvis, and that although older rabbit renal pelvis contains more total ET receptors than younger renal pelvis, the latter had a higher portion of the ETA receptor subtype and the younger tissues were more responsive to ET-1.

Endothelin-1, endothelin-1 receptors and cardiac natriuretic peptides in failing human heart

Endothelin (ET)-1 is a potent vasoconstrictor peptide produced in the myocardium that can exert important effects on cardiac myocyte growth and phenotype; cardiac natriuretic peptides (ANP and BNP) are known to act as physiological antagonists of ET-1. In this study a comparative determination of ET-1 receptors and of the local productions of ET-1 and of ANP and BNP was made in different sites of failing and nonfailing hearts. Tissue from right and left atrium, right and left ventricle and interventricular septum from seven adult heart transplant recipients with end-stage idiopathic dilated cardiomyopathy (functional class III and IV, with ejection fraction < 35%) and from four post-mortem subjects without cardiac complications was analyzed. In failing hearts we observed a tendency to increase of density of binding sites, most evident in left ventricle (62.6 ± 22.6 fmol/mg protein vs. 29.0 ± 3.3, mean ± SEM, p = ns). A prevalence of ET-A subclass, observed in all samples, resulted more pronounced in failing hearts where this increase, found in all the cardiac regions, was more evident in left ventricle (p = 0.0007 vs nonfailing hearts). The local concentrations of ET-1, ANP and BNP resulted significantly increased in failing hearts with respect to controls in all sides of the heart. In failing hearts we have observed a tendency to increase in endothelin receptor density mainly due to a significant upregulation of ET-A subtype and a parallel increase of the tissue levels of ANP, BNP and ET-1 indicating an activation of these systems in heart failure.

Endothelin synthesis and receptors in porcine kidney.

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.