1 The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [ 33 P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. 2 The binding kinetics of [ 33 P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23+18C) and dissociated slowly. The association and dissociation rate constants were 4.31+0.34610 8 M 71 min 71 and 1.55+0.38610 73 min 71 respectively. Thus, the kinetic dissociation constant was 3.46+0.59 pM. 3 Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding a nities (pK D ) of [ 33 P]-human gene 2 relaxin (B33) were 8.92+0.09 in atrial myocardium and 8.79+0.04 in cerebral cortex of male rats, and 8.79+0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in nonreproductive tissues. 4 In male rats, treatment with 17b-oestradiol (20 mg in 0.1 ml sesame oil s.c., 18 ± 24 h) signi®cantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no signi®cant e ect in atria and cerebral cortex, but it signi®cantly increased the density of relaxin receptors in uterine myometrium. 5 Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90+0.02), and cerebral cortex (8.90+0.03) and uterine myometrium (8.89+0.03) with a nities not signi®cantly di erent from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with a nities (8.35+0.09 in atria, 8.22+0.07 in cerebral cortex and 8.48+0.06 in uterine myometrium) signi®cantly less than human gene 2 (B33) and porcine relaxins. 6 Quantitative receptor autoradiography is the method of choice for measurement of a nities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17b-Oestradiol treatment produced complex e ects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no e ect on the atria and cerebral cortex of the female rats.

1 The receptors for relaxin in the rat atria and uterus were investigated and compared by use of a series of synthetic and native relaxin analogues. The assays used were the positive chronotropic and inotropic e ects in rat spontaneously beating, isolated right atrium and electrically driven left atrium and the relaxation of K + precontracted uterine smooth muscle. 2 Relaxin analogues with an intact A-and B-chain were active in producing powerful chronotropic and inotropic e ects in the rat isolated atria at nanomolar concentrations. Single-chain analogues and structural homologues of relaxin such as human insulin and sheep insulin-like growth factor I had no agonist action and did not antagonize the e ect of the B29 form of human gene 2 relaxin. 3 Shortening the B-chain carboxyl terminal of human gene 1 (B2 ± 29) relaxin to B2 ± 26 reduced the activity of the peptide and removal of another 2 amino acid residues (B2 ± 24) abolished the activity. This suggests that the B-chain length may be important for determination of the activity of relaxin. More detailed studies are needed to determine the e ect of progressive amino acid removal on the structure and the bioactivity of relaxin. 4 Porcine prorelaxin was as active as porcine relaxin on a molar basis, suggesting that the presence of the intact C-peptide did not a ect the binding of the prorelaxin to the receptor to produce functional responses. 5 Relaxin caused relaxation of uterine longitudinal and circular smooth muscle precontracted with 40 mM K + . The pEC 50 values for human gene 2 and porcine relaxins were lower than those in the atrial assay, but rat relaxin had similar pEC 50 values in both atrial and uterine assays. Rat relaxin was signi®cantly less potent than either human gene 2 or porcine relaxin in the atrial assay, but in the uterine assay they were equipotent. The results suggest that the relaxin receptor or the signalling pathway in rat atria may di er from that in the uterus.

No abstract

The primary structure of ovine Leydig cell insulin-like peptide (Ley I-L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I-L suggested that the peptide might possess a relaxin-like function. Ovine Ley I-L was assembled by Fmoc-solid-phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain-combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin-like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I-L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I-L and relaxin, we prepared a synthetic analogue of ovine Ley I-L containing a single replacement of B-chain residue 12, His, with Arg. This was found to possess significant relaxin-like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I-L is similar to that of relaxin and highlighting the key requirement for the five-residue sequence, Arg-X-X-X-Arg, to be present in position B12-16 for characteristic relaxin activity.