Biomedical subjects

M Pullen

Publications and source records attributed to M Pullen.

At least 19 recordsLinked to original sources

PubMed2000

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.

Animals↗

PubMed1999

Mechanism of adrenomedullin-stimulated hyaluronic acid release in rat mesangial cells.

Adrenomedullin is a potent vasodilatory peptide that increases cAMP in a number of different systems including rat mesangial cells. Since mesangial cells play a significant role in glomerular matrix production, we evaluated the effects and molecular mechanisms of adrenomedullin action on hyaluronic acid release, an important extracellular matrix component. Adrenomedullin increased hyaluronic acid release in mesangial cells in a concentration-dependent manner. Forskolin, an adenylate cyclase activator, and dibutyryl-cAMP, a cell permeable cAMP analog, also increased hyaluronic acid release significantly. Adrenomedullin-stimulated hyaluronic acid release was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Inhibition of protein kinase A with H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride)]], a potent protein kinase A inhibitor did not affect adrenomedullin-stimulated hyaluronic acid release; however, H89 [[N-[2-(( p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]] inhibited forskolin- and dibutyryl-cAMP-induced hyaluronic acid production. In addition, SB203580 [[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-im idazole), a P38 mitogen-activated protein kinase (P38 MAPK) inhibitor attenuated adrenomedullin-, forskolin-, and dibutyryl-cAMP-stimulated hyaluronic acid release. Hyaluronic acid release induced by adrenomedullin, forskolin and dbcAMP was also inhibited by wortmannin [[1S-(1alpha, 6balpha, 9abeta, 11alpha, 11bbeta)]-11-(Acetyloxy)-1, 6b, 7, 8, 9a, 10, 11, 11b-octahydro-1-(methoxymethyl)-9a, 11b-dimethyl-3H-furo[4, 3, 2-de]indeno[4, 5-h]-2-benzopyran-3, 6, 9-trione]. We conclude that adrenomedullin, forskolin and dbcAMP cause an increase in hyaluronic acid release in rat mesangial cells through a pathway that involves activation of wortmannin-sensitive kinase and P38 MAPK. Although cAMP stimulation and protein kinase A activation can induce hyaluronic acid release. adrenomedullin-stimulated hyaluronic acid release appears to be independent of protein kinase A activation. These data provide the first demonstration of the involvement of P38 MAPK- and wortmannin-sensitive kinase pathways in the stimulation of hyaluronic acid production by rat mesangial cells.

Adrenomedullin↗

PubMed1999

Down regulation of kidney neutral endopeptidase mRNA, protein and activity during acute renal failure: possible mechanism for ischemia-induced acute renal failure in rats?

Neutral endopeptidase (NEP, 24.11) is an ectoenzyme involved in the degradation of peptide hormones such as endothelin (ET), atrial natriuretic factor and enkephalins. The current study was designed to assess the involvement of NEP in ischemia-induced acute renal failure (ARF). In unilaterally nephrectomized Sprague-Dawley rats, the left renal artery was occluded for 30 min under pentobarbital anesthesia (40 mg/kg, i.p.) at 37 degree C. In addition to plasma creatinine levels, NEP activity was determined in renal cortical membranes at 0, 2, 5, and 24 h following reperfusion. Plasma creatinine levels significantly increased at 2, 5 and 24 h. There was a significant decrease in NEP activity as early as 2 h following reperfusion that was maintained up to 24 h (57.9 +/- 4%) with a concomitant loss of enzyme protein shown by Western analysis. Northern analysis of kidney cortical RNA, probed with an NEP cDNA, showed a 45% decrease in NEP mRNA level by the end of the ischemic period and decreased further during reperfusion. Thus, decrease in NEP mRNA levels preceded the changes in protein level, enzyme activity and plasma creatinine levels. These data, along with the reported increase in the tissue level of ET in kidney cortex, and the beneficial effect of ET antibody as well as ET receptor antagonist in ARF, suggest that down regulation of NEP, one of the mechanisms leading to increased tissue level of ET, may be a contributing factor to ARF.

Acute Kidney Injury↗

PubMed1999

Distribution of neutral endopeptidase activity along the rat and rabbit nephron.

Neutral endopeptidase (NEP) activity was measured in various nephron segments dissected from rat and rabbit kidney. In the rat, only the proximal straight tubule and glomerulus had measurable NEP activity of 86 +/- 11.3 pmol/min/mm tubule length and 5.8 +/- 1.5 pmol/min/glomerulus, respectively. In the rabbit, significant activity was observed in both the proximal convoluted tubule (70.8 +/- 7.2 pmol/min/mm) and proximal straight tubule (29.6 +/- 2.3 pmol/min/mm) as well as in the glomerulus (12.8 +/- 2.2 pmol/min/glomerulus). In the rat proximal tubule, phosphoramidon and thiorphan inhibited NEP activity, with IC50 values of 26.6 +/- 6.0 and 6.9 +/- 1.6 nmol/l, respectively. Incubation of rat proximal tubules with phorbol 12-myristate 13-acetate resulted in a 50% reduction in membrane-associated NEP activity. The results demonstrate that in both the rat and rabbit NEP is restricted to the glomerulus and proximal tubule. This localized distribution of NEP and its potential regulation by the protein kinase C pathway may play a key role in determining local concentrations of important regulatory peptides in the kidney.

Animals↗

PubMed1999

Endothelin B receptor modulates inflammatory pain and cutaneous inflammation.

The role of endothelin B (ET(B)) receptors in inflammation and nociception was examined using ET(B) receptor knockout mice. Genotyping studies were used with tissues from ET(B)((+/+)), ET(B)((+/-)), and ET(B)((-/-)) mice to confirm the loss of ET(B) receptors. Algesia induced by phenylbenzoquinone was evident in the (+/+) mice, reduced by approximately 80% in the (+/-) mice, and absent in the (-/-) mice. Phenylbenzoquinone-induced algesia in (+/+) mice was inhibited 74% by the ET(B) receptor-selective antagonist A192621 (25 mg/kg p.o.), but unaffected by the ET(A) receptor-selective antagonist SB 234551 (25 mg/kg p.o.). Noninflammatory pain, induced by hotplate, was equivalent between (+/+) and (-/-) mice. The cutaneous inflammatory response to topical arachidonic acid (AA) also was evaluated. Whereas (+/+) mice had a marked inflammatory response to AA, the (+/-), and (-/-) mice had significantly reduced fluid phase responses (37 and 65% inhibition, respectively). Neutrophil infiltration also was reduced in the (+/-) and (-/-) mice (51 and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) in (+/+) mice inhibited AA-induced swelling (39%), whereas SB 234551 (500 microg/ear) was without effect. Collectively, these results implicate the ET(B) receptor in mediation of inflammatory pain and cutaneous inflammatory responses in mice.

Animals↗

PubMed1998

Effects of endothelin receptor antagonism and angiotensin-converting enzyme inhibition on cardiac and renal remodeling in the rat.

The effects of a nonpeptide, orally active mixed endothelin (ET) ETA/ETB receptor antagonist, SB 217242, and an angiotensin-converting enzyme (ACE) inhibitor, ramipril, were evaluated after inter-renal aortic banding in the rat. Separate sham, vehicle, and treatment groups were compared in each study. In vehicle-treated animals in the ramipril group, aortic banding for 4 weeks produced significant cardiac hypertrophy (247 +/- 5 mg/100 g bw vs. 305 +/- 11 mg/100 g bw; p < 0.001), right (upstream) renal hypertrophy (380 +/- 6 mg/100 g bw vs. 559 +/- 28 mg/100 g bw; p < 0.001), and significant left (downstream) renal atrophy (405 +/- 4 mg/100 g bw vs. 192 +/- 25 mg/100 g bw; p < 0.001). Continuous ramipril treatment (1 mg/kg p.o. once daily), begun 3 days before aortic banding, inhibited cardiac hypertrophy (305 +/- 11 mg/100 g bw vs. 266 +/- 7 mg/100 g bw; p < 0.05) but did not alter renal hypertrophy or atrophy. In a similarly designed study, SB 217242 (30 mg/kg p.o. b.i.d.) had no effect on the development of cardiac hypertrophy (298 +/- 7 mg/100 g bw vs. 310 +/- 12 mg/100 g bw) or renal hypertrophy (561 +/- 15 mg/100 g bw vs. 575 +/- 19 mg/100 g bw), but abolished the development of renal atrophy (158 +/- 16 mg/100 g bw vs. 395 +/- 19 mg/100 g bw; p < 0.001). [125I]ET-1 radioligand binding experiments indicated that the density of both ETA and ETB receptors was increased dramatically (three- to fourfold) in the atrophic kidney cortex compared to sham or hypertrophic kidneys. In situ hybridization studies indicate an upregulation of ETB receptor mRNA in the glomeruli of atrophic kidneys within 5 days of aortic banding. In conclusion, an angiotensin-dependent mechanism may mediate cardiac hypertrophy associated with aortic banding, whereas ET-dependent mechanisms may mediate an atrophic response in the hypoperfused kidney, perhaps through an interaction with upregulated ETA and/or ETB receptors.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

SB 234551, a novel endothelin--A receptor antagonist, unmasks endothelin-induced renal vasodilatation in the dog.

Infusion of endothelin-1 (ET-1) into conscious, chronically instrumented dogs (10 ng/kg.min i.v.) resulted in a significant increase in mean arterial pressure and significant reductions in renal plasma flow, glomerular filtration rate, and sodium excretion. Intravenous infusion of SB 209670 (30 micrograms/kg.min, i.v.) abolished these responses, whereas infusion of SB 234551 (30 micrograms/kg.min, i.v.) resulted in significant increases in renal plasma flow and urinary sodium excretion. These data indicate that SB 234551 can unmask ETB receptor-induced renal vasodilatation and inhibition of sodium reabsorption.

Animals↗

PubMed1998

Coronary arterial lesions in dogs treated with an endothelin receptor antagonist.

Structurally and pharmacologically diverse vasodilators are known to lower blood pressure, increase heart rate, and produce acute injury to right coronary arteries in the dog. Administration of low concentrations of endothelin-1 (ET-1) to anesthetized dogs causes coronary vasoconstriction and reductions in coronary blood flow. Therefore, pharmacologic blockade of endothelin receptors (ETA and ETB) with the mixed ET receptor antagonist SB 209670 could lead to coronary vasodilatation. In toxicology studies, continuous administration of SB 209670 to dogs for 5 days at 50 micrograms/kg/min was associated with minor but sustained increases in heart rate (10-30 beats/min), slight decreases in mean arterial pressure (10-15 mm Hg), and medial hemorrhage and necrosis of extramural coronary arteries in the right atria. Doses of 10 micrograms/kg/min had no effect. The lesions in the right atrium were associated with the highest density of ET receptors, approximately 470 fmol/mg compared to 170-200 fmol/mg in the ventricles and septum. Because changes in systemic cardiovascular parameters are minimal, the coronary arterial lesion is most likely due to a local vasodilatory effect in the coronary bed.

Animals↗

PubMed1997

Binding characteristics of [125I]TTA 386, ETA-selective antagonist.

The data presented in this manuscript describe the binding characteristics of the ETA-selective antagonist, [125I]TTA 386 (hexamethylenelmino carbonyl-Leu-Tri-Ala-beta-Ala-Tyr-Phe). This radioligand bound with high affinity and specificity to cloned human ETA receptors and rat mesenteric artery ETA receptors. The apparent dissociation constants (KdS) and maximum binding capacities were 1.0 nM and 8.5 pmol/mg for cloned human ETA receptors and 0.8 nM and 170 fmol/mg for rat mesenteric artery membranes respectively. Binding of [125I]TTA 386 was fast reaching equilibrium by 45 min and 15 min for human ETA and rat mesenteric artery membrane, respectively. Addition of excess unlabeled ligand resulted in the dissociation of bound radioligand from both preparations. Competition of [125I]TTA 386 binding by unlabeled ET-1, ET-3, TTA 386 and BQ123 revealed appropriate ETA pharmacology. This radioligand did not display any binding to cloned human ETB receptors.

Animals↗

PubMed1997

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene.

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed W138 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and W138 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed W138 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylated RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.

3T3 Cells↗

PubMed1997

Identification and characterization of a novel endothelin receptor that binds both ETA- and ETB-selective ligands.

This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ETA- and ETB-selective ligands. This subtype has been identified in canine spleen membranes using ETB-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ETA-selective antagonists BQ123 and related cyclic pentapeptides. Binding of 125I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (Bmax) of 240.0 fmol/mg protein. Although the apparent affinities obtained with 125I-ET-1 and 125I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with 125I-ET-3 was approximately 35% of that obtained with 125I-ET-1, which indicates that canine spleen possesses both ETA and ETB receptors in the ratio 65:35. Competition binding experiments using 125I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for 125I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ETA-selective antagonist, displaced 125I-ET-3 binding from canine spleen with an IC50 value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)+ RNA indicated that the ETB receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ETB receptor present in this tissue is similar to that of the cloned human ETB receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ETB receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ETB receptors displayed different molecular weight bands and different pharmacology. In addition, the ETB receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ETB receptor in different tissues as well as different species including human.

Animals↗

PubMed1996

Molecular characterization of a novel human endothelin receptor splice variant.

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

Alternative Splicing↗

PubMed1996

Correlation between guanine nucleotide effect and reversible binding property of endothelin analogs.

[125I]-IRL-1620 and [125I]-ET-1 (readily reversible and essentially irreversible endothelin (ET) receptor agonists, respectively) were used to demonstrate the relationship between the reversible binding nature of ET receptor agonists and guanine nucleotide effect using ETB receptors as the model system. Addition of increasing concentrations of GTP gamma s to membranes prepared from Chinese hamster ovary (CHO) cells stably transfected with human ETB receptors, dog lung and pig lung decreased [125I]-IRL-1620 binding to these membranes between 50% and 60%, whereas [125I]-ET-1 binding to these receptors was unaffected by GTP gamma s. Saturation binding experiments in the absence and presence of 100 microM GTP gamma s indicated that the apparent dissociation constant [Kd(apparent)] for [125I]-IRL-1620 was increased 2 to 2.4-fold in all 3 membrane preparations in the presence of GTP gamma s compared to its absence. There was no difference in the apparent dissociation constants of [125I]-ET-1 in the presence and absence of GTP gamma s in these membrane preparations. This inhibitory effect was specific for guanosine triphosphate since adenine nucleotides failed to decrease the affinity of [125I]-IRL-1620 for the receptors. The correlation between guanine nucleotide effect and reversible binding property of the agonist was further strengthened by the observation that in rat cerebellum and rat renal papilla, where [125I]-IRL-1620 binding was irreversible, guanine nucleotides had no effect on the binding of this ligand. These data clearly indicate that there is a good correlation between the reversible binding property of the ET receptor agonist and the guanine nucleotide effect on the binding of the agonist.

Animals↗

PubMed1996

Evidence for the existence of endothelin-B receptor subtypes and their physiological roles in the rat.

The physiological roles of endothelin-B (ETB) receptor subtypes in systemic and renal hemodynamics were assessed in conscious Sprague-Dawley rats. Mean arterial pressure, hindlimb flow, and renal blood flow were measured via an implanted catheter and pulsed Doppler flow probes. Bolus intravenous injections of sarafotoxin 6c (S6c), a selective ETB agonist, elicited transient dose-dependent vasodilation, followed by sustained vasoconstriction in the systemic bed, but only vasoconstriction in the renal bed. RES-701-1, a selective ETB antagonist, blocked the dilator and potentiated the constrictor effect; SB-209670, a mixed ET receptor antagonist, attenuated both responses to S6c. In follow-up studies, the role of endogenous ET was assessed by administration of the antagonists alone: RES-701-1, SB-209670, and the ETA-selective antagonist BQ-123. RES-701-1 unmasked a significant systemic and renal vasoconstriction, which was attenuated by SB-209670 but not by BQ-123. SB-209670 and BQ-123 had no effect on basal hemodynamic parameters. Data from radioligand binding experiments showed that RES-701-1 binds with high affinity to the cloned human ETB receptor but poorly to the ETB receptor predominant in the rat kidney. Collectively, the results indicate that 1) the vascular effects of ET in the rat are mediated by two ETB receptor subtypes: an RES-701-1-sensitive subtype, mediating vasodilation, and an RES-701-1-insensitive subtype, mediating vasoconstriction; 2) the predominant role of endogenous ET is vasodilation; and 3) the ETA receptor plays a negligible role in the control of vascular tone in the rat.

Animals↗

PubMed1996

Nonpeptide endothelin receptor antagonists. VII: Binding characteristics of [3H]SB 209670, a novel nonpeptide antagonist of endothelin receptors.

The data presented in this manuscript describes the binding characteristics of [3H]SB 209670, a potent nonpeptide tritium-labeled endothelin (ET) receptor antagonist. The binding of this antagonist to cloned human ETA and ETB receptors was specific, saturable and of high affinity. The apparent dissociation constants were 0.20 and 1.0 nM for ETA and ETB receptors, respectively. The maximum binding was 4.7 and 22.5 pmol/mg protein for ETA and ETB receptors, respectively. Unlike [125]ET-1, the binding of [3H]SB 209670 was reversible. The half-times (T1/2) for dissociation of this ligand from ETA and ETB receptors were approximately 60 and 10 min, respectively. Competition binding studies using [3H]SB 209670 and unlabeled agonists ET-1, ET-3 and S6c indicated that these agonists displayed similar affinities for human ETB receptors, whereas with ETA receptors, ET-1 was approximately 50-fold and 1500-fold more potent than ET-3 and S6c, respectively. Of the peptide antagonists tested, BQ123 (ETA-selective peptide antagonist), displayed Ki values of 40 and > 2300 nM for ETA and ETB, whereas RES701 (ETB-selective antagonist) displayed Ki values of > 1600 and 81 nM for ETA and ETB receptors, respectively. The nonselective peptide antagonist, PD 142893, was approximately 2-fold more potent for ETA compared with ETB receptors. Similar observations were made with nonselective nonpeptide antagonists, Bosentan, (+/-) SB 209670, SB 209670, and (-) SB 209670. All these compounds were 2 to 10 times more potent for ETA than ETB receptors.

Animals↗

PubMed1995

Developmental expression of endothelin receptors in cerebellar neurons differentiating in culture.

We describe the identification and expression of endothelin (ET) receptor subtypes in differentiating cultured cerebellar neurons. Using [125I]ET-1 and the subtype-selective ligands BQ-123 and sarafotoxin 6c as selective ligands for the ETA and ETB receptors, respectively, we found that cerebellum from 8-day-old rats displayed only the ETB receptor subtype. We next cultivated cerebellar granule cell neurons to study ET receptor differentiation between 2 and 22 days in vitro. Using the above reagents, we found that while unlabeled ET-1 displayed monophasic competition curves, BQ-123 and sarafotoxin 6c displayed partial displacement curves, indicating the presence of both ETA and ETB receptors on these neurons. The proportion of ETB receptors gradually decreased from day 2 onwards the proportion of ETA receptors gradually increased. On days 2, 3, 4, and 5 of culture, the ETB:ETA receptor ratios were 90:10, 70:30, 60:40, and 40:60, respectively. There was no further change in receptor subtype ratio beyond day 5 and up to day 22. Northern blot analysis showed that ETB receptor message expression was 6.9-fold higher than that of ETA receptor expression on day 2, but steadily decreased with time, whereas ETA receptor message expression was minimal on day 2 and maximal by day 3 and 4. By day 7, receptor message was of equal abundance, which was in good agreement with the binding studies. This novel, developmentally regulated process predicts the existence of endogenous mediators of neuronal ET receptor expression.

Animals↗

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↗