Kinetic analyses demonstrate that the equilibrium assumption does not apply to [125I]endothelin-1 binding data

doi:10.1016/0024-3205(92)90038-Q

Life Sciences

Volume 51, Issue 24, 1992, Pages 1869-1876

https://doi.org/10.1016/0024-3205(92)90038-Q Get rights and content

Abstract

The kinetics of [¹²⁵I]Endothelin-1 ([¹²⁵I]ET-1) binding were studied using membranes from rat heart, rat lung, rat brain, and porcine vascular smooth muscle at 37°C in 0.05M Tris-HCl buffer (pH=7.4). The dissociation half-life ( $t 12, diss.$ ) for bound [¹²⁵I]ET-1 was in excess of 30 hours for each tissue studied. Equilibrium-time requirements for proper Scatchard analysis of [¹²⁵I]ET-1 were also far in excess of 30 hours for each tissue. These data suggest that determination of dissociation constants, K_d, and receptor concentrations, B_max, by conventional Scatchard analysis is not feasible with [¹²⁵I]ET-1. Kinetic analyses may provide a more accurate means for determining [¹²⁵I-ET-1] binding characteristics including K_d and B_max.

References (17)

W. Fischli et al.
Life Sci.
(1989)
A. Hemsén et al.
Eur. J. Pharmacol.
(1990)
E.R. Martin et al.
J. Biol. Chem.
(1990)
M.M. Bradford
Anal. Biochem.
(1976)
G.A. McPherson
J. Pharmacol. Methods
(1985)
P.J. Munson et al.
Anal. Biochem.
(1980)
G.A. Weiland et al.
Life Sci.
(1981)
M. Yanagisawa et al.
Nature
(1988)

There are more references available in the full text version of this article.

Cited by (83)

Endothelin-1 Treatment Induces an Experimental Cerebral Malaria–Like Syndrome in C57BL/6 Mice Infected with Plasmodium berghei NK65
2016, American Journal of Pathology
Citation Excerpt :
Although the plasma half-life of ET-1 is 1 to 7 minutes,70 the effect on blood pressure after a low-dose bolus either by injection or continuous infusion (1 nmol/kg in rats, 4 pmol/kg per minute in humans) lasted for >1 hour after exogenous administration.71,72 This is likely because of the fact that uptake of the peptide and into various tissues and its eventual dissociation from its cognate binding sites in those tissue is extremely slow (ie, >30 hours).73,74 Herein, we demonstrated that mice infected with PbN (non-ECM model) developed vascular alterations with corresponding neurological impairments, resulting in an ECM-like syndrome, when administered exogenous ET-1.
Plasmodium berghei ANKA infection of C57BL/6 mice is a widely used model of experimental cerebral malaria (ECM). By contrast, the nonneurotropic P. berghei NK65 (PbN) causes severe malarial disease in C57BL/6 mice but does not cause ECM. Previous studies suggest that endothelin-1 (ET-1) contributes to the pathogenesis of ECM. In this study, we characterize the role of ET-1 on ECM vascular dysfunction. Mice infected with 10⁶ PbN-parasitized red blood cells were treated with either ET-1 or saline from 2 to 8 days postinfection (dpi). Plasmodium berghei ANKA–infected mice served as the positive control. ET-1–treated PbN-infected mice exhibited neurological signs, hypothermia, and behavioral alterations characteristic of ECM, dying 4 to 8 dpi. Parasitemia was not affected by ET-1 treatment. Saline-treated PbN-infected mice did not display ECM, surviving until 12 dpi. ET-1–treated PbN-infected mice displayed leukocyte adhesion to the vascular endothelia and petechial hemorrhages throughout the brain at 6 dpi. Intravital microscopic images demonstrated significant brain arteriolar vessel constriction, decreased functional capillary density, and increased blood-brain barrier permeability. These alterations were not present in either ET-1–treated uninfected or saline-treated PbN-infected mice. In summary, ET-1 treatment of PbN-infected mice induced an ECM-like syndrome, causing brain vasoconstriction, adherence of activated leukocytes in the cerebral microvasculature, and blood-brain barrier leakage, indicating that ET-1 is involved in the genesis of brain microvascular alterations that are the hallmark of ECM.
Thermostabilization of the Human Endothelin Type B Receptor
2016, Journal of Molecular Biology
Citation Excerpt :
In the thermostabilization of A2AR, NTSR1, and CRF1R, receptor mutations that allowed for efficient retention of the radioactive ligands after heat treatment in an agonist- or antagonist-bound form were selected to bias the conformation toward either state in addition to the thermostabilization. The ETBR binds agonist ET-1 in a virtually irreversible manner [17], but our crystallization trials were unsuccessful. We therefore surveyed thermostable alanine mutants after heat treatment in a ligand-free form by ET-1 binding, which revealed several potential candidates.
The peptide hormone endothelin, produced by the vascular endothelium, is involved in several physiological functions, including maintenance of vascular tone and humoral homeostasis. Endothelin transmits signals through the endothelin receptor, a G-protein-coupled receptor. Structural studies of the endothelin type B receptor (ET_BR) have been unsuccessful due to its structural flexibility and instability in detergent-solubilized solution. To overcome these problems, we explored thermostabilization of human ET_BR by establishing an ET_BR expression system in Escherichia coli, followed by systematic alanine scanning mutagenesis. Among 297 point mutations, 11 thermostabilizing residues were selected and further mutated to other amino acids. The thermostability indices of these residues, represented by the ratios of endothelin-1 (ET-1) binding activities with or without heat treatment at 27 °C for 30 min in a ligand-free form, were compared. The ligand affinity and apparent melting temperature (T_m) of the five most thermostable mutants, R124Y, D154A, K270A, S342A, and I381A, were then examined. The apparent T_m of three single mutants, R124Y, D154A, and K270A, was approximately 7 °C higher than that of the wild type. The apparent T_m value of a combination of the five residues, named the Y5 ET_BR mutant, was 17 °C higher than that of the wild type. The Y5 ET_BR mutant exhibited an affinity for ET-1 and activated G_q similar to the wild type. Further investigation of the pharmacological properties affected by combinatorial mutations of ET-1, ET-3, TxET-1, and K8794 suggested that Y5 ET_BR is highly suitable for representing a ligand-free form of ET_BR and is potentially applicable for studying an ET-1-bound form.
Introduction: Basic Biology of the Renal Endothelin System
2015, Seminars in Nephrology
Desensitization and internalization of endothelin receptor a; Impact of g protein-coupled receptor kinase 2 (GRK2)-mediated phosphorylation
2013, Journal of Biological Chemistry
Citation Excerpt :
Furthermore, the binding characteristics of the maximally mutated variant ETA-14PD did not differ significantly from those of ETA-WT (Table 1). Because of the nearly irreversible binding of ET-1 to ETA (20, 21), pre-stimulation with ET-1 to induce receptor desensitization was not possible. Ca2+ flux experiments revealed desensitization of ETA-mediated Ca2+ release (data not shown), but they are not suited for monitoring specific ETA desensitization because the inositol trisphosphate receptor itself is rapidly desensitized (22).
Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.
Background: The involvement of phosphorylation of endothelin receptor A (ET_A) in its desensitization and internalization is unclear.
Results: Phosphorylation-deficient ET_A mutants display defective regulation, whereas receptor internalization is not affected.
Conclusion: GRK2-mediated phosphorylation contributes to ET_A desensitization, but not to ET_A internalization.
Significance: This work contributes to the understanding of the complex GRK2-mediated ET_A regulation involving multiple mechanisms.
Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca<sup>2+</sup> in adult cardiac myocytes
2013, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
To test this hypothesis, we analyzed the internalization and trafficking of endothelin receptors in ventricular myocytes. Since the half-life of the ET:ETR complex is greater than 20 h [38,41], we followed the internalization of ET:ETR complexes using fluorescent endothelin analogs in conjunction with confocal fluorescence microscopy. Fig. 2 shows FAM-ET-1 fluorescence (left, green), LysoTracker (middle, red), or an overlay of both signals (right).
Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca^2 + signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB²¹]ET-1) evoked an increase in nucleoplasmic Ca^2 + ([Ca^2 +]_n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca^2 +]_n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca^2 + signaling.
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Kinetic analyses demonstrate that the equilibrium assumption does not apply to [125I]endothelin-1 binding data

Abstract

Life Sci.

Eur. J. Pharmacol.

J. Biol. Chem.

Anal. Biochem.

J. Pharmacol. Methods

Anal. Biochem.

Life Sci.

Nature

Kinetic analyses demonstrate that the equilibrium assumption does not apply to [¹²⁵I]endothelin-1 binding data