Motohiro Kondoh

Two distinct types of endothelin receptors are present on chick cardiac membranes

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.

Two different forms of endothelin receptors in rat lung

We provided evidence for the presence of two distinct types of rat endothelin (ET) receptors having different molecular masses by affinity labelling of rat lung membranes with 125I‐ET‐1, ET‐2 and ET‐3 and SDS‐PAGE followed by autoradiography. One type, which is 44 kDa has a higher affinity for ET‐1 and ‐2 than ET‐3, whereas the other type, which is 32 kDa, preferably interacts with ET‐3 rather than ET‐1 and ‐2.

Identification of the endothelin-1 receptor in the chick heart

Summary This study suggests that binding sites for endothelin-1 (ET-1) are distinct from those for dihydropyridine (DHP)-sensitive, voltage-dependent Ca2+ channels and that ET-1 has its own specific receptors in chick cardiac membranes.

Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.

Characterization of endothelin receptor subtypes.

Scatchard-plot analysis of [125I]ET-1 and [125I]ET-3 binding to rat lung membranes exhibited almost the same Kd values whereas the concentration of the binding sites of ET-1 is approximately four times higher than that of ET-3. This result suggests the presence of at least two distinct subtypes of ET receptors: an ET-1-specific type and an ET-3-specific or ET-1 and ET-3 nonselective type. On the other hand, in rat brain membranes, a curvilinear Scatchard plot was obtained for [125I]ET-3 in contrast to a linear plot for [125I]ET-1. This finding also demonstrates the existence of the two different receptor subtypes having the same affinity for ET-1, but one of which has a high affinity and the other has a low affinity for ET-3. Moreover, these data indicate that an ET receptor subtype exists in rat brain different from the subtype in rat lung. To obtain the bases for a further detailed characterization of the receptor, we have purified the rat lung ET receptor to homogeneity by ET-1-affinity chromatography. The purified receptor exhibits a molecular mass of 45 kDa, in good agreement with that estimated from the affinity labeling.

Endothelin Receptors and Receptor Subtypes

Endothelin, originally isolated from culture media of porcine aortic endothelial cells, is one of the most potent vasoactive peptide known to date and consists of 21 amino acids containing two intracellular disulfide bonds.21 In addition to vasoconstrictor activity, it has been found that endothelin has a variety of biological actions: it increases blood pressure;21,22 stimulates release of atrial natriuretic peptide,4 prostacyclin, thromboxane A2, and endothelial cell—derived relaxing factor3; and inhibits renin release.12 Moreover, three isopeptides of endothelin have been identified based on the analysis of a human genomic library and named endothelin-1, endothelin-2, and endothelin-3.5 These findings raise the question of how the endothelin isopeptides and their various biological actions are related. It is also of great interest to know whether the three endothelin isopeptides have their own specific receptors, respectively, or bind in common to a homologous population of endothelin receptors. The elucidation of these problems is essential for understanding the physiological roles of endothelin.