Yasushi Masuda

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.

Solubilization of receptor for pituitary adenylate cyclase activating polypeptide from bovine brain

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP) was solubilized from bovine brain membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Saturable binding experiments using [125I]PACAP27 revealed that the solubilized extract contained a single class of binding sites with a Kd of 200 pM and a Bmax of 0.9 pmol/mg protein. The Kd did not change significantly after the solubilization. Competitive binding experiments confirmed that the solubilized receptor retained a specificity for PACAP. Thus, the solubilization of the receptor in an active form was successfully achieved by the present procedure.

A Short-Length Peptide YY Analogue with Anorectic Effect in Mice

Peripheral administration of PYY3–36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25–36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3–36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 μg/kg) in mice, suggesting the biologic potential of short-length PYY3–36 analogues with a potent anorectic effect.

Antiobesity Effect of a Short-Length Peptide YY Analogue after Continuous Administration in Mice

Gastrointestinal peptides such as peptide YY (PYY) can regulate appetite, which is relevant to the study of obesity. The intraperitoneal bolus administration of PYY3-36 and a 12-amino acid PYY analogue, benzoyl-[Cha27,28,36,Aib31]PYY25-36 (1), showed similar anorectic activity by activating the Y2 receptor (Y2R). However, food intake inhibition and body weight loss were not observed upon continuous subcutaneous administration of 1 with osmotic pumps in diet-induced obese (DIO) mice. N-Terminal elongation of 1, together with amino acid substitution at position 24, led to a hydrophilic 14-amino acid peptide, Ac-[d-Hyp24,Cha27,28,36,Aib31]PYY23-36 (18), that showed higher affinity and more potent agonist activity for Y2R and a robust anorectic activity with potency similar to that of PYY3-36. In addition, the continuous subcutaneous administration of 18 at 0.3 mg/(kg·day) induced significant body weight loss in DIO mice. These results suggest that a short-length PYY analogue can be a lead compound for antiobesity therapy in a sustained-release formulation.

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.