Bioassay of Relaxin Using a Reference Standard: A Simple and Reliable Method Utilizing Direct Measurement of Interpubic Ligament Formation in Mice

Steinetz, Bernard G.; Beach, V. L.; Kroc, Robert L.; Stasilli, Neil R.; Nussbaum, R. E.; P, Nemith; Dun, R. E.

doi:10.1210/endo-67-1-102

Cited by 128 publications

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“…Synthetic human relaxin H2 was supplied by E. Rinderknecht (Genentech). It was active in the mouse pubic symphysis assay (16) and a cAMP bioassay (14,17). The concentration of relaxin was determined by amino acid analysis.…”

Section: Methodsmentioning

confidence: 99%

Relaxin binding in the rat heart atrium.

Osheroff¹,

Cronin²,

Lofgren³

1992

Proc. Natl. Acad. Sci. U.S.A.

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Relaxin is a member of the insulin family of polypeptides that is best known as a reproductive hormone. In an effort to elucidate the mechanism of action of relaxin we previously localized the specific binding sites of a 32P-labeled relaxin in the rat uterus and brain. These studies suggested that, in addition to its classical role in pregnancy, relaxin might have other physiological functions. In the present paper we describe the specific and high-afminity binding of relaxin to the cardiac atrium of both male and female rats. The relaxin binding could not be displaced by peptides belonging to the same family [insulin, insulin-like growth factor I (IGF-I)] or by peptides that were identified in the atrium or were known to have cardiovascular functions (atrial natriuretic peptide, angiotensin II). The dissociation constant for relaxin in the atrium was estimated to be 1.4 nM, which was similar to that found in the uterus (1.3 nM) and the brain (1.4 nM). In view of the close association of relaxin with reproduction, an experiment was also performed to compare the relaxin binding in the uterus and heart after gonadectomy and sex steroid treatment. It was found that the relaxin binding in the rat uterus was diminished by 53% overall following ovariectomy but was restored to 90% of normal levels when treated with estrogen (but not with testosterone). In contrast, the relaxin binding in the rat heart was not affected by castration or sex steroid treatment. We conclude that specific and high-affinity relaxin receptors exist in the atrium of both the male and female rat heart and that these are regulated differently than the relaxin receptors in the uterus.Relaxin is structurally related to insulin and insulin-like growth factors and is best known as a pregnancy-associated hormone (1, 2). In addition to its classical actions on cervical ripening, uterine contraction, and pubic symphysis elongation (1, 2), relaxin has been suggested to participate in the regulation of blood pressure (3-8) and neuropeptide release, notably oxytocin and vasopressin (9-13). However, the results from these studies were conflicting, and the site of action of relaxin was undetermined. Biochemical support for these hypotheses was provided when it was shown that, in addition to the uterus and cervix (14), relaxin bound with specificity and high affinity (Kd 1 nM) to several sites in the male and female rat brain (15). Some of these sites, in particular two of the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminalis) and the magnocellular hypothalamic nuclei (paraventricular and supraoptic nucleus) are known to be involved in the control of blood pressure, fluid balance, and the release of neuropeptides. To further investigate the mechanism of action of relaxin, we continued the search for relaxin target tissues and were surprised to observe the specific and high-affinity binding of relaxin to the atrium of the rat heart. We also show that the atrial binding is regulated differently than the uterus. ...

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Section: Methodsmentioning

confidence: 99%

Relaxin binding in the rat heart atrium.

Osheroff¹,

Cronin²,

Lofgren³

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…Relaxin was purified from freshly frozen pregnant sow ovaries using the procedures described by Sherwood & O'Byrne (1974). This material corresponded to the peptide designated as CM-a' by Sherwood & O'Byrne (1974) and it had a potency of 2-87 NIH-R-P1 (442 GPU/mg) when measured in the mouse interpubic ligament bioassay (Steinetz et al, 1960). An antiserum to CM-a' was raised in one rabbit using the immunization method described by Vaitukatis, Robbins, Neischlag & Ross (1971).…”

Section: Gel Filtration Offollicularfluidmentioning

confidence: 99%

Identification of relaxins in porcine follicular fluid and in the ovary of the immature sow

Matsumoto¹,

Chamley²

1980

Reproduction

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“…Mouse interpubic ligament assays were carried out as described by Steinetz et al [24], using ovariectomized virgin female mice. Mice were primed with 5 pg estrogen cypionate in 100 p1 sesame oil and five days later were injected, subcutaneously, with rat relaxin or rat relaxin analogs in 100 pl 1 % benzopurpurin 4 8 .…”

Section: Methodsmentioning

confidence: 99%

The Chemical Synthesis of Rat Relaxin and the Unexpectedly High Potency of the Synthetic Hormone in the Mouse

Büllesbach

Schwabe

1996

European Journal of Biochemistry

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Rat relaxin, as isolated from ovaries, has been described in the literature as a low potency hormone in the mouse symphysis pubis assay. Searching for an explanation, a helix-breaking glycine residue in the B chain seemed to be the most auspicious perturbation. Rat relaxin was chemically synthesized and analyzed by reverse-phase high performance liquid chromatography, amino acid composition, mass spectrometry and circular dichroic spectroscopy. Analogs of rat relaxin were synthesized either with aspartic acid in place of the helix-breaking glycine residue in the receptor-binding region of the B chain or with Asp-Leu-Val instead of Gly-Tyr-Val at positions B14-B16. In receptor-binding assays [B14D, B15L, B16VIrelaxin was a better ligand than rat relaxin, whereas the [B14D]relaxin was less potent. In the mouse symphysis pubis assay, both analogs were less potent than unmodified rat relaxin, but the [B14D, B15L, B16VIrelaxin was better than [B14D]relaxin. In contrast to previous reports on native rat relaxin, the chemically synthesized rat relaxin proved to be as active as human and porcine relaxin with respect to the standard mouse assay system. Glycine, which is considered to be a perturbator in an a helix, is not only tolerated in the B14 position but is required for full biological potency.Keywords: relaxin ; insulin; structurelfunction relationship; peptide synthesis.Relaxin, a hormone of parturition, has an insulin-like structure regarding molecular mass, chain arrangement [I], disulfide distribution [2, 31, and tertiary structure [4]. In contrast to insulins, relaxins from various species vary significantly in their primary structures [5]. Human relaxin I1 [6], for instance, and pig relaxin [7, 81 vary by 57% whereas mouse relaxin [9] and rat relaxin [ 101 vary by 23 % from each other. For comparison, differences between porcine and human insulins are 2% and rat insulins I and I1 are identical to mouse insulins I and I1 [ll]. In spite of the high sequence variations, all relaxins trigger the same biological effects in at least one of the small laboratory animals, i.e. rats, mice or guinea pigs [5, 121. Human relaxin I1 and porcine relaxin are equipotent in standard mouse symphysis pubis assays [13, 141 and compete for the same receptor on crude membrane preparations of mouse brain and mouse uteri [15, 161. Isolated ovarian rat relaxin displayed relatively low potency [ 17, 181 as compared to porcine relaxin. The presence of glycine in the a-helix loop that lies between the two binding-site arginine residues in the major helix of the B chain provided a plausible explanation for this observation. In this paper, we report on the chemical synthesis of rat relaxin and two derivatives that particularly address the problem of B-chain integrity. MATERIALS AND METHODSAmino acid derivatives used for peptide synthesis were purchased from either Bachem Bioscience or Bachem CaliCorrespondence to C. Schwabe, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charle...

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Bioassay of Relaxin Using a Reference Standard: A Simple and Reliable Method Utilizing Direct Measurement of Interpubic Ligament Formation in Mice

Cited by 128 publications

References 0 publications

Relaxin binding in the rat heart atrium.

Relaxin binding in the rat heart atrium.

Identification of relaxins in porcine follicular fluid and in the ovary of the immature sow

The Chemical Synthesis of Rat Relaxin and the Unexpectedly High Potency of the Synthetic Hormone in the Mouse

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