Biomedical subjects

M Pullen

Publications and source records attributed to M Pullen.

At least 37 records · Page 2Linked to original sources

PubMed1995

Identification of ETB receptor subtypes using linear and truncated analogs of ET.

Using canine spleen and lung membranes as model systems, we have shown the presence of subtypes of ETB receptors. This classification was done based on the binding profiles of various analogs of ET-1 that have been identified as ETB-selective. Saturation binding experiments performed with [125I] ET-3 and [125I] IRL-1260 (ETB-selective ligands) indicated that [125I] IRL-1620 labeled 80-90% of [125I] ET-3 binding sites in canine lung, whereas in canine spleen, the binding of [125I] IRL-1620 was 10-20% of [125I] ET-3 binding. In addition, competition binding experiments using ETB-selective agonists [Ala1,3,11,15]-ET-1 (also known as 4-Ala ET-1), N-acetyl-[Ala11,15] ET-1 (6-21) also known as BQ3020 and Suc-[Glu9, Ala11,15] ET-1 (8-21) also known as IRL-1620 indicated that all three ligands displaced [125I] ET-3 from canine lung membranes with approximately 500-1000 fold greater affinity than canine spleen membranes. On the other hand, ET-1, ET-3, S6a, S6b, and S6d displayed very similar IC50 values in both preparations, except S6c which was approximately 20 fold less potent in canine spleen compared to lung. These data indicate that ETB receptors present in canine spleen are different from those present in lung and that ETB-selective linear as well as truncated analogs of ET-1 are good tools to identify these subtypes of ETB receptors.

Animals↗

PubMed1995

[125I]-BQ3020, a potent ETB-selective agonist, displays species differences in its binding characteristics.

Binding of [125I]-BQ3020 to membranes prepared from rat cerebellum, dog lung and CHO cells stably transfected with the human endothelin ETB receptor clone was specific, saturable and of high affinity. The apparent dissociation constants (KdS) were 63, 53 and 112 pM for rat cerebellum, dog lung and human ETB, respectively. The maximum bindings (Bmax) were 3.8, 0.430 and 20 pmol/mg protein for rat cerebellum, dog lung and human clone, respectively. Competition binding experiments using [125I]-BQ3020 and unlabelled ET-1, ET-3, S6c and BQ123 indicated that ET-1, ET-3 and S6c competed for [125I]-BQ3020 binding with similar high affinity (IC50 approximately 0.3 nM), whereas BQ123 was ineffective in competing for [125I]-BQ3020 binding. Time course experiments suggested that the binding of [125I]-BQ3020 to all three preparations was rapid, and reached steady-state by approximately 40 min at 30 degrees C. Addition of excess unlabelled ET-1 resulted in partial dissociation (25-40%) from human ETB and dog lung receptors, whereas from rat cerebellum it was essentially irreversible. These data suggest that [125I]-BQ3020 is a potent and selective ETB agonist and its reversible binding property is species-dependent.

Animals↗

PubMed1995

Thrombin-mediated down-regulation of endothelin receptors in mesangial cells coincides with the down-regulation of neutral endopeptidase activity.

Thrombin-mediated down-regulation of endothelin (ET) receptors was studied in rat glomerular mesangial cells. Overnight incubation of mesangial cells with thrombin (10 nM) resulted in a significant decrease (67%) in the number of ET receptors, with no change in affinity. Northern analysis of the mRNA from these cells showed a corresponding decrease in the ETA receptor message. Such a decrease in ET receptors could result from an increase in ET levels caused by an increase in synthesis and/or a decrease in degradation. It has been previously reported that thrombin stimulates ET production in endothelial and mesangial cells. Because ET is known to be degraded by neutral endopeptidase (NEP), which is present at high levels in the kidney, the potential effects of thrombin on NEP activity were evaluated. There was a decrease of NEP activity in mesangial cells at 16 and 24 hr after treatment with 10 nM thrombin. This effect was specific for thrombin, because NEP activity was not altered after treatment with thrombin in the presence of hirudin, an inhibitor of thrombin activity. The thrombin-mediated decrease in NEP activity correlated with a decrease in NEP protein and mRNA levels, as determined by Western and Northern analyses, respectively. To determine whether the thrombin-mediated decrease in ET receptors had a functional corollary, ET-1-stimulated intracellular calcium mobilization was measured. Overnight incubation with 10 nM thrombin resulted in a significant inhibition of ET-stimulated intracellular calcium mobilization. This effect was specific for ET, because thrombin pretreatment did not affect vasopressin-stimulated intracellular calcium mobilization in mesangial cells. These results indicate that the thrombin-mediated down-regulation of ET receptors is due, in part, to a thrombin-stimulated increase in ET resulting from the down-regulation of NEP and the reported increase in ET synthesis. In addition, pretreatment of mesangial cells with ET-1 caused a significant decrease (85%) in ET receptor number and ET-1-mediated intracellular calcium release (84%), without affecting vasopressin- or thrombin-mediated responses.

Amino Acid Sequence↗

PubMed1995

Nonpeptide endothelin receptor antagonists: IV. Identification of receptors in rabbit colonic mucosa and smooth muscle and correlation with physiological effects.

Endothelin (ET) has been previously demonstrated to be a potent intestinal secretory stimuli and to elicit intestinal smooth muscle contractions. In rat distal colon, the ETA receptor antagonist cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp] abolishes the changes in ion transport (as measured by short-circuit current) elicited by ET-1. In this study, effects of ET-1, sarafotoxin 6c and "big" ET in the absence and presence of the endothelin receptor antagonists cyclo [D-Asp-L-Pro-D-Val-L-Leu-D-Trp] and (+/-)-3-[2-(carboxymethoxy)-4-methoxyphenyl]-1-[3,4- (methylenedioxy)phenyl]-5-(prop-1-yloxy)indan-2-carboxylic acid on ion transport in rabbit distal colon were assessed through measurement of short-circuit current changes in segments of muscle-stripped mucosa in Ussing chambers. In addition, changes in smooth muscle contraction in response to acetylcholine, ET-1, ET-3 and sarafotoxin 6c in the absence and presence of (+/-)-3-[2-(carboxymethoxy)-4-methoxyphenyl]-1-[3,4- (methylenedioxy)phenyl]-5-(prop-1-yloxy)indan-2-carboxylic acid and of cyclo [D-Asp-L-Pro-D-Val-L-Leu-D-Trp] were measured in mucosa-free preparations oriented along their longitudinal axes. Receptor binding studies with mucosal or smooth muscle homogenates were conducted with [125I]ET-1, with [125I]ET-3 and with [125I]Tyr13Suc-[Glu9,Ala11,15]-endothelin-1(8-21 ) to identify the subtypes of ET receptors present in these preparations. From studies with rabbit colonic mucosa, both binding studies and measurement of short-circuit current confirm that the ETB receptor subtype is the predominant subtype and is responsible for the changes observed in ion transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Identification and function of putative ETB receptor subtypes in the dog kidney.

Binding studies performed in dog kidney, lung, and spleen using [125I]endothelin (ET) 3 and a series of ETB-selective ligands indicated the presence of two subtypes of ETB receptors, an IRL-1620-sensitive (putative ETB1) and an IRL-1620-insensitive (putative ETB2) receptor. The IRL-1620-sensitive (but not IRL-1620-insensitive) ETB receptors displayed similar pharmacology to the cloned human ETB receptor and a high affinity for the ETB receptor antagonist RES-701. ETB1 receptors predominated in the dog kidney and lung and ETB2 receptors predominated in the dog spleen. Stimulation of ETB receptors in dogs in vivo inhibited sodium reabsorption, as ET-1 infusion in the presence of the ETA antagonist BQ123, but not the mixed ETA/ETB receptor antagonist, (+/-)SB 209670, resulted in increased sodium excretion. Furthermore, infusion of the ETB-selective agonist Sarafotoxin S6c (S6c) resulted in an increase in sodium excretion, a response that was attenuated by infusion of RES-701. These data indicate that the putative ETB1 receptor may mediate ET-induced inhibition of tubule sodium reabsorption in the dog. In addition, we observed no putative ETB2 receptor-mediated renal vasoconstriction, consistent with the apparent low abundance of this subtype in the dog kidney.

Animals↗

PubMed1994

Cloning and characterization of a novel endothelin receptor from Xenopus heart.

Endothelin (ET) receptors display subtype heterogeneity and so far three subtypes of ET receptors, namely ETA, ETB, and ETC, have been identified, cloned, sequenced, and characterized. Based on the binding profile of ET and related peptides, a novel ET receptor (ETAX) was identified in the follicular membranes of Xenopus laevis oocytes (Kumar, C. S., Nuthulaganti, P., Pullen, M., and Nambi, P. (1993). Mol. Pharmacol. 44, 153-157). Here we report the cloning and characterization of this ETAX subtype from X. laevis heart. A cDNA was isolated that encodes a protein of 415 amino acids that shares 74, 60, and 51% identities with human ETA, human ETB, and Xenopus ETC receptors, respectively. Competition binding studies of the cloned receptor expressed in COS cells using ET-related peptides suggested that this receptor is pharmacologically identical to that expressed in Xenopus oocyte follicular, heart, and lung membranes. Phosphoinositide turnover and oocyte electrophysiological studies indicated that the cloned receptor is functionally coupled to a second messenger system.

Amino Acid Sequence↗

PubMed1994

Identification of a novel endothelin receptor in Xenopus laevis liver.

Membranes prepared from Xenopus liver displayed high density of high affinity endothelin (ET) binding sites. These sites have the same affinity for [125I] ET-1 and [125I] ET-3. Scatchard analysis of the specific binding from saturation binding experiments revealed an apparent dissociation constant (Kd) of 93.1 and 70.9 pM and maximum binding (Bmax) of 602 and 651 fmol/mg protein for [125I] ET-1 and [125I] ET-3, respectively. Competition binding experiments using [125I] ET-1 and unlabelled ET-1, ET-3, S6c, and BQ123 indicated that ET-1 and ET-3 were the most potent in displacing [125I] ET-1 binding from these membranes (IC50 1 and 0.3 nM, respectively), whereas S6c BQ123, selective for ETB and ETA receptors, respectively, did not have any inhibitory effect up to 1 microM. These data clearly indicate that the ET receptors present in Xenopus liver membranes belong to a new subtype of ET receptor. Because it resembled mammalian ETB receptors in its affinities for ET-1 and ET-3, we propose that this receptor be called the ETBX receptor.

Animals↗

PubMed1994

Distribution and functional role of renal ET receptor subtypes in normotensive and hypertensive rats.

Experiments were designed to compare the distribution and physiological roles of endothelin (ET) receptor subtypes, ETA and ETB, in the kidneys of normotensive Sprague-Dawley (SD) and spontaneously hypertensive (SH) rats. Using [125I] ET-1 and subtype-selective ligands sarafotoxin 6c (S6c, ETB-selective agonist) and BQ123 (ETA-selective antagonist), the distribution of ETA and ETB receptors in SD rat kidney cortex, outer medulla and papilla was calculated to be 50:50, 30:70 and 10:90, respectively. The ET receptor subtypes in outer medulla and papilla of age-matched SH rats were similar to those of SD. However, in the cortex of SH rats, the ratio of ETA to ETB was 25:75 compared to 50:50 in SD rats. In addition, the affinity of the ET receptors was also higher in SH rats (117 pM vs. 235 pM). In the conscious SD rats, bolus i.v. injections of ET-1 and S6c elicited similar dose-dependent decrease in renal blood flow (RBF), which were unaffected by the infusion of the selective ETA receptor antagonist, BQ123. The SH rats were more sensitive to the renal vasoconstrictor effect of S6c and ET-1. Also, the dose-response curve to S6c was shifted to the left when compared to ET-1; however, BQ123 infusion abolished this difference. In renal clearance studies, BQ123 infusions decreased RBF and glomerular filtration rate (GFR) only in SH rats, and the fractional excretion of sodium only in SD rats. The combined data indicate that the distribution and functional roles of ETA and ETB receptor subtypes are altered in the kidneys of SH rats.

Animals↗

PubMed1994

Endotoxin-mediated changes in plasma endothelin concentrations, renal endothelin receptor and renal function.

The purpose of these studies was to examine the changes in renal endothelin (ET) receptor, renal function and plasma ET (ET-1) concentration in male Sprague-Dawley rats injected with nonlethal doses of Escherichia coli endotoxin (LPS). Prior to the injection of LPS, kidney ET receptor density was 59 +/- 5 fmol/mg protein (n = 20). At 24 h after the injection of 1 or 3 mg/kg LPS, [125I]ET-1 binding to kidney membranes was increased by 70% in both LPS groups (p < 0.001). Scatchard analysis of the saturation binding experiments confirmed that the increase in [125I]ET-1 binding was due to an increase in receptor density with no change in affinity (202 pmol/l at baseline and 168 pmol/l and 246 pmol/l at 24 h after the injection of 1 and 3 mg/kg LPS, respectively). At 7 days after the injection of LPS, kidney ET-1 receptor density was still increased by 30 +/- 5% and 58 +/- 16%, respectively (p < 0.05, compared to the baseline value). Baseline values for Na+ and K+ excretion were approximately 115 muEq/h and 214 +/- mu/Eq/h respectively, and were decreased with LPS. Maximal decreases in Na+ and K+ excretion occurred at 48 h (-85%) and 30 h (-82%), respectively, following the injection of 3 mg/kg LPS and returned to baseline levels in 7 days. Following the injection of 3 mg/kg LPS, plasma immunoreactive ET-1, as measured by radioimmunoassay, increased in a time-dependent manner: the maximal increase of 60% occurred within 1 h after the injection of LPS (p < 0.05), and thereafter returned to baseline levels. Kidney tissue levels of ET-1 increased from baseline values of 2.6 fmol/mg protein to a peak of 4.6 fmol/mg protein 1 h after the injection of LPS. Tissue ET-1 levels were still significantly elevated at 6 h but not 24 h after LPS injection. These studies suggest that ET-1, either by increases in plasma concentration and/or altered receptor density, may be involved in the LPS-induced impairment of renal function.

Animals↗

PubMed1994

Characterization of a functional angiotensin II receptor in Xenopus laevis heart.

High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1994

Nonpeptide endothelin receptor antagonists. I. Effects on binding and signal transduction on human endothelinA and endothelinB receptors.

The effect of a series of endothelin (ET) receptor antagonists, including the novel nonpeptide receptor antagonist, SB 209670, on [125I]ET-1 binding to human ET receptors (ETA and ETB) cloned and stably expressed in Chinese hamster ovary cells was studied. SB 209670 was found to compete for [125I]ET-1 binding with apparent Ki values of 0.43 +/- 0.09 and 14.7 +/- 3.0 nM for ETA and ETB receptors, respectively. This inhibition was competitive because addition of SB 209670 in saturation binding experiments resulted in decreased affinity, with no change in maximum binding. In addition, SB 209670 inhibited ET-1-mediated accumulation of inositol phosphates and intracellular calcium release in a concentration-dependent manner. The racemic mixtures, (+/-)-SB 209670 and (-)-SB 209670, were approximately 1.5 and at least 200-fold less potent than SB 209670. The binding affinity of (+/-)-SB 209670 therefore resides in (+)-antipode. The peptide antagonists, BQ123 (ETA-selective) and RES 701 (ETB-selective), were 40- and 6-fold less potent than SB 209670 in inhibition of [125I] ET-1 binding to ETA and ETB receptors, respectively. The nonselective peptide antagonist, PD 142893, was 75- and 10-fold less potent than SB 209670, whereas the nonpeptide antagonist, bosentan, was approximately 80- and approximately 30-fold less potent than SB 209670 in inhibition of [125I]ET-1 binding to ETA and ETB receptors, respectively. Thus, the present studies indicate clearly that SB 209670 is the most potent nonpeptide ET receptor antagonist yet described.

Animals↗

PubMed1994

Characterization of canine renal endothelin receptor subtypes and their function.

Binding and renal functional studies were conducted to characterize endothelin (ET) receptors in the dog kidney. Binding studies that were performed in renal cortical membranes by using [125I]-ET-1 and [125I]-ET-3 and the ETA- and ETB-selective ligands, BQ123 (cyclo [D-Trp-D-Asp-L-Pro-D-Val-L-Leu]) and sarafotoxin 6c (S6c), respectively, revealed that the ratios of ETA to ETB receptors in cortical, medullary and papillary membranes were 22:78, 39:61 and 50:50, respectively. In vivo studies in the anesthetized dog demonstrated that an intrarenal artery infusion of ET-1 (0.3-10 ng kg-1 min-1) resulted in a dose-dependent decrease in renal blood flow (RBF) and glomerular filtration rate (GFR). At a dose of 10 ng kg-1 min-1 of ET-1, RBF and GFR decreased by 82 +/- 6% and 89 +/- 6%, respectively. An infusion of BQ123 (10 micrograms kg-1 min-1) into the renal artery resulted in a significant inhibition of the ET-1-induced renal vasoconstriction. At identical doses as ET-1, S6c had little effect on either RBF (-3 +/- 6%) or GFR (-6 +/- 16%). ET-1 decreased urine flow and had little effect on fractional sodium excretion, whereas S6c increased both urine flow and fractional sodium excretion. These data indicate that ET-1-induced renal vasoconstriction in the dog is mediated by ETA receptors; however, ETB receptor stimulation may inhibit sodium reabsorption.

Amino Acid Sequence↗

PubMed1994

Species differences in the binding characteristics of [125I]IRL-1620, a potent agonist specific for endothelin-B receptors.

[125I]Tyr13-Suc-[Glu9,Ala11,15]-Endothelin-1 (8-21) ([125I]IRL-1620) displayed specific, saturable and high affinity binding to membranes prepared from rat and dog cerebellum, rat and human lung and cloned human endothelin-B (ETB) receptors. The apparent dissociation constant (Kd) for rat cerebellum, dog cerebellum, rat lung, human lung and human ETB clone were 69.1, 34.8, 128, 71.9, and 115 pM, respectively. The maximum binding (Bmax) to these membranes were 3.2, 0.71, 1.67, 1.85 and 62.3 pmol/mg of protein for rat cerebellum, dog cerebellum, rat lung, human lung and human ETB clone, respectively. Unlabeled endothelin-1 (ET-1), ET-3 and sarafotoxin 6c (S6c) displaced [125I]IRL-1620 binding to these membranes with similar affinity (IC50 = 0.1-2.5 nM), whereas cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu), a selective antagonist for ETA receptors, had no effect on [125I]IRL-1620 binding up to 1 microM. Time course experiments of association and dissociation indicated that [125]IRL-1620 binding to dog and human tissues and human ETB clone was rapid and reversible, whereas in rat tissues, the binding was rapid but irreversible, suggesting that this might be due to species difference. [125I]ET-1 binding was irreversible in all the tissues tested. No binding of [125I]IRL-1620 was detectable in rat vascular smooth muscle cells or cloned human ETA receptors. These data indicate that [125I]IRL-1620 is highly selective for ETB receptors and the reversible binding characteristics of this ligand appears to be species-dependent.

Amino Acid Sequence↗

PubMed1993

Transient forebrain ischemia alters acutely endothelin receptor density and immunoreactivity in gerbil brain.

Pharmacologic studies suggest that endothelin (ET) plays an important role in the pathophysiology of hemorrhagic and ischemic stroke. In the gerbil, transient forebrain ischemia (10 min) resulted in profound motor deficits and a 15% reduction in ET receptor density in the hippocampus at 60 min post-reperfusion. A significant 2-fold increase in forebrain immunoreactive ET accompanied the maximum post-ischemic decrease in ET receptor density. These results suggest that the synthesis and availability of ET are increased acutely in the forebrain following transient cerebral ischemia.

Analysis of Variance↗

PubMed1993

Sex difference in the antidiuretic activity of vasopressin in the rat.

A possible gender difference in the antidiuretic activity of vasopressin was studied in male and female Sprague-Dawley rats. Infusion of vasopressin (3-100 pg.kg-1.min) into conscious, chronically instrumented water-loaded rats resulted in a dose-dependent antidiuresis in both male and female rats. Male rats, however, were more than three times more sensitive to vasopressin than female rats. Thus the effective doses of vasopressin (pg.kg-1.min-1) to decrease urine flow to 30 microliters.min-1.100 g-1 (18 +/- 5 in males; 58 +/- 12 in females), to increase urine osmolality to 600 mosmol/kgH2O (35 +/- 5 in males; 119 +/- 15 in females), and to decrease free water clearance to 30 microliters.min-1.100 g-1 (8 +/- 3 in males; 28 +/- 7 in females) were significantly (P < 0.05) lower in males. Furthermore, in vitro studies in papillary collecting duct cells demonstrated a significantly higher density of vasopressin V2 receptors and a greater ability of vasopressin to stimulate adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in males than in females. Vasopressin V2-receptor density (maximum binding) was 359 +/- 47 and 238 +/- 22 fmol/mg in male and female rats, respectively (P < 0.05). There was no difference in apparent dissociation constants (Kd). Vasopressin resulted in a dose-dependent increase in cAMP accumulation in papillary collecting duct cells, and at the highest concentration of vasopressin used (10(-8) M) cAMP increased from 44 +/- 10 to 182 +/- 51 fmol/micrograms protein in males and from 30 +/- 4 to 91 +/- 18 fmol/micrograms protein in females (P < 0.05). (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Endothelin inhibits vasopressin action in rat inner medullary collecting duct via the ETB receptor.

We characterized the endothelin (ET) receptor subtype responsible for the inhibition of vasopressin (AVP)-induced increases in osmotic water permeability (Pf) and cAMP accumulation in rat inner medullary collecting ducts (IMCD). ET-1 (10 nM) produced a rapid and transient decrease in AVP-stimulated Pf from 1241 +/- 112 to 224 +/- 38 microns/sec. At the same concentration (10 nM), the selective ETB receptor agonist sarafotoxin 6c (S6c) produced the same degree of inhibition with a time course identical to that of ET-1. Exposure of IMCDs to the ETA-selective antagonist BQ123 (100 nM) had no effect on ET-1-induced inhibition of AVP-dependent Pf. In suspensions of IMCD cells, ET-1, ET-3 or S6c produced concentration-dependent inhibition of AVP-stimulated cAMP accumulation to the same extent and with similar potencies (IC50 = 10-30 nM). BQ123 (1 nM to 10 microM) had no effect on ET-1-induced inhibition of AVP-stimulated cAMP formation. Saturation binding experiments with radiolabeled ET-1 and the selective ETB agonist IRL1620 and competition binding studies with selective ETA and ETB receptor ligands demonstrated that > or = 80% of the ET-1 binding sites in IMCD membranes were of the ETB subtype. Therefore, results from functional, biochemical and binding studies suggest that the ETB receptor is the ET receptor subtype that inhibits AVP action in the rat IMCD.

Animals↗

PubMed1993

Novel endothelin receptors in the follicular membranes of Xenopus laevis oocytes mediate calcium responses by signal transduction through gap junctions.

Follicular oocytes from Xenopus laevis display saturable and high affinity endothelin (ET)-1 binding sites. Competition binding experiments using unlabeled ET-1, ET-3, and sarafotoxin 6c indicated that these ET receptors belong to the ETA receptor subtype. However, the ETA receptor-selective antagonist BQ123 [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)] failed to inhibit 125I-ET-1 binding to these receptors, suggesting that these receptors belong to a novel subtype of ETA receptors (ETAX) distinct from the human ETA receptors. These endogenous receptors are present in the follicular layer, because defolliculated oocytes did not show any 125I-ET-1 binding. Addition of ET-1 to follicular oocytes led to Ca2+ mobilization, which was reversibly blocked by treatments that uncouple gap junctions, suggesting that these ETAX receptors mediate their response by transferring signals through gap junctions. On the other hand, the expressed human ETA receptor-mediated Ca2+ mobilization was not blocked by inhibitors of gap junctions. In agreement with the binding data, the endogenous ETAX receptor-mediated response was not inhibited by BQ123 even at 100 nM, whereas the expressed human ETA receptor-mediated response was inhibited by 50% at concentrations as low as 10 nM. This further confirms that the amphibian ETAX receptors are different from mammalian ETA receptors. Finally, ET-1 enhanced the rate of progesterone-induced maturation of follicular oocytes, implying the involvement of these endogenous ETAX receptors in an in vivo maturation process.

Amino Acid Sequence↗

PubMed1993

Rat kidney endothelin receptors in ischemia-induced acute renal failure.

The current study was designed to assess the possible changes in kidney endothelin (ET) receptors in a model of ischemia-induced acute renal failure. In unilaterally nephrectomized (right kidney) Sprague-Dawley rats, the left renal artery was occluded for 30 min under pentobarbital anesthesia (40 mg/kg i.p.). Body temperature was maintained at 37 degrees C. Sham-operated rats were used as control. Plasma creatinine levels were measured and ET receptors were quantitated by binding of [125I]ET-1 to membranes prepared from the renal cortex at 0, 2, 5 and 24 hr after reperfusion. There was a time-dependent increase in plasma creatinine levels as well as in [125I]ET-1 binding to cortical membranes at 2, 5 and 24 hr postreperfusion. Saturation binding experiments using [125I]ET-1 and [125I]ET-3 indicated that this increase in binding was due to an increase in affinity without significant change in maximum binding. The affinities were 219, 134, 100, 69 and 80 pM for [125I]ET-1 and 320, 273, 130, 168 and 80 pM for [125I]ET-3 and, for sham, 0, 2, 5 and 24 hr postreperfusion, respectively. These data support the hypothesis of ET involvement in the pathophysiology of acute renal failure in rats.

Acute Kidney Injury↗