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 effects 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 effects 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 effect 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 effect 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 affect 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 pEC50 values for human gene 2 and porcine relaxins were lower than those in the atrial assay, but rat relaxin had similar pEC50 values in both atrial and uterine assays. Rat relaxin was significantly 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 differ from that in the uterus.
Relaxin is a hormone structurally related to insulin and insulin-like growth factor, which exerts its regulatory effect on the musculoskeletal and other systems through binding to its receptor in various tissues, mediated by different signaling pathways. Relaxin alters the properties of cartilage and tendon by activating collagenase. This hormone is also involved in bone remodeling and healing of injured ligaments and skeletal muscle. In this review, we have summarized the literature on the effect of relaxin in musculoskeletal system to provide a broad perspective for future studies in this field.
The metabolism of varying quantities of pregnenolone has been studied in nuclei-free homogenates from Macaca fascicularis testes by using capillary gas chromatography, after derivatization of metabolites as O-methyl oximes/trimethylsilyl ethers. Evidence was obtained indicating that both pathways for testosterone biosynthesis were operating. 5-Androstene-3 beta, 17 beta-diol was formed in especially high quantities. Two 16-androstenes, namely 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol, were also quantitatively important as metabolites. Co-incubation of stored homogenates with relaxin resulted in 80-100% reduction of the formation of all metabolites quantified except for 5 alpha-androst-16-en-3-one, which was stimulated. Freezing the homogenates at -10 degrees C for 3 weeks resulted in marked 4- to 6-fold reduction in the yields of testosterone and of the 5-ene and 4-ene metabolites from pregnenolone.
The effects of exogenously introduced oestradiol-17 beta (E) and relaxin (RLX) on cervical extensibility and collagen organisation were tested in rats ovariectomised in late pregnancy. When the cervices were stretched in vitro by 1 mm increments, it was found that those from rats given E alone generated significantly higher tensions than those from control rats, while cervices from rats given both E and RLX had tensions similar to controls. Examination of cervical sections under the light microscope and ultra-thin sections under the electron microscope showed that the collagen fibres in the cervices from E-treated rats were highly organised, whereas those from animals given E+RLX and control animals were disorganised and dispersed. It was concluded that E decreased cervical extensibility, while RLX counteracted the effect of E to maintain a soft and easily extensible cervix.
The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. The binding kinetics of [33P]-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+/-1 degrees C) and dissociated slowly. The association and dissociation rate constants were 4.31+/-0.34x10(8) M(-1) x min(-1) and 1.55+/-0.38x10(-3) min(-1) respectively. Thus, the kinetic dissociation constant was 3.46+/-0.59 pM. 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 affinities (pK(D)) of [33P]-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 non-reproductive tissues. In male rats, treatment with 17beta-oestradiol (20 microg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. 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 affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35+/-0.09 in atria, 8.22+/-0.07 in cerebral cortex and 8.48+/-0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17beta-oestradiol treatment produced complex effects 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 effect on the atria and cerebral cortex of the female rats.
The changes in knee laxity and relaxin receptor expression at different phases of rodent estrous cycle are not known. Here, changes in the parameter were investigated in rats at different phases of the estrous cycle. Estrous cycle phases of intact female rats were determined by cytological examination of the vaginal smear. Following phase identification, blood was collected for serum hormone analyses. Knee passive range of motion (ROM) was determined by using a digital miniature goniometer. The animals were then sacrificed and patellar tendon, collateral ligaments and hamstring muscles were harvested for relaxin/insulin-like family peptide receptor 1 and 2 (RXFP1/RXFP2) analyses. Knee passive ROM was the highest at proestrus followed by diestrus and the lowest at estrus. Estrogen level was the highest at proestrus while progesterone and relaxin levels were the highest at diestrus. A strong correlation was observed between relaxin and progesterone levels. At proestrus, expression of RXFP1 and RXFP2 proteins and mRNAs were the highest at proestrus followed by diestrus and estrus. The finding shows that higher level of progesterone and relaxin in diestrus might be responsible for higher laxity of knee joint in rats.
Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity.