Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes.

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.

Relaxin alters the plasma osmolality-arginine vasopressin relationship in the rat.

During pregnancy, in women and the rat, there is a resetting of the plasma osmolality-arginine vasopressin relationship (P(osmol)/PAVP) such that a decrease in P(osmol) is maintained without suppression of PAVP. This occurs at a time when relaxin is detectable in plasma. The hypothesis tested here was that relaxin could alter the P(osmol)/PAVP in the non-pregnant rat. One group of ovariectomized rats (n = 15) was treated for 7 days with intravenous synthetic human relaxin (10 micrograms/h) in 10 microliters 0.9% (w/v) NaCl. Controls were two groups of rats either with no treatment (n = 15) or treated with vehicle alone (n = 15). One-third of each group received hypertonic saline (0.4 mol NaCl/l, 2 ml/100 g body weight i.p.) on day 7, and one-third were deprived of water for the final 24 h. All rats were killed by decapitation and blood was collected rapidly (< 40 s) for hormone and osmolality assays. The P(osmol) in all relaxin-treated rats was significantly (P < 0.001) lower than that in both control groups, but the PAVP was unchanged. The log PAVP/P(osmol) regression line was significantly shifted in elevation (P < 0.001) but not in slope. Thus treatment of ovariectomized rats with relaxin caused changes in fluid balance which mimic those occurring in normal pregnancy.

A bioassay for inhibin using pituitary cell cultures.

A bioassay for inhibin based on the suppression of gonadotrophin releasing hormone (Gn-RH)-stimulated secretion of FSH by primary monolayer cultures of rat anterior pituitary cells is described. The cultures were exposed to standard or test materials for 3 days. The levels of FSH in the media were measured by radioimmunoassay after exposure for 6 h to a maximally stimulating concentration of Gn-RH (10 nmol/l). The standard was prepared from ovine testicular lymph. Several preparations of proteins from gonadal tissues or secretions suppressed the levels of FSH in parallel with the standard. The levels of LH were also reduced but higher doses of active material were required. Non-specificity from cell damage and inactivation of Gn-RH have been excluded. The secretion of gonadotrophins by the pituitary cells was also inhibited by androgens, but not in parallel with the standard and secretion of LH was affected more than that of FSH. Control lymph protein preparations from castrated sheep had no detectable activity. The assay was sensitive and had adequate precision and practicability. It has proved useful for monitoring preliminary steps in the purification of inhibin.

Inhibin from cultures of rat seminiferous tubules.

Medium from cultures of mature rat seminiferous tubules contained a substance which suppressed, in a dose-related manner, the luteinizing hormone releasing hormone (LH-RH)-stimulated secretion of FSH by cultured rat pituitary cells. The secretion of LH was suppressed to a lesser extent and the basal secretion of both LH and FSH was inconsistently affected. Gel filtration on Sephadex G-100 did not fractionate the activity. The active material did not inhibit the secretion of TSH or destroy LH-RH and the activity was not due to testosterone or oestradiol in the medium. Control media from liver cultures were inactive. It is concluded that inhibin is present in media from cultures of rat seminiferous tubules.

Relaxin in human pregnancy serum measured with an homologous radioimmunoassay.

This study reports serum levels of relaxin in normal and special-interest pregnancies using an homologous radioimmunoassay for human relaxin. The mean levels in uncomplicated antenatal patients were lower than those reported in studies using heterologous assays, but the trend in serum levels was similar. Serum levels peaked at ten weeks' gestation and decreased progressively to term. Relaxin was detectable in all pregnant subjects assessed at the time of the first missed menses. The mean relaxin levels in patients having in vitro fertilization and embryo transfer who subsequently delivered a single infant were significantly higher than those in normal antenatal patients at an equivalent gestational age. Patients with twin pregnancies after in vitro fertilization and embryo transfer generally had higher levels than patients with single pregnancies. Some pregnant patients who aborted after in vitro fertilization and embryo transfer had declining levels of relaxin before 40 days postlaparoscopy.

Relaxin levels in antenatal patients following in vitro fertilization.

The aim of this study was to investigate the relationship between serum relaxin and pregnancy outcome in a group of patients pregnant after in vitro fertilization (IVF). Patients who delivered a single live infant at term after IVF had mean serum levels of relaxin more than double the mean levels in normal antenatal patients. The high relaxin levels were compatible with delivery at term. However, because of the proposed role of relaxin in the process of cervical ripening, the high serum levels may help explain the high rate of preterm labor observed among IVF patients.

Characterization of a mutant type-1 angiotensin II receptor.

We have used PCR to amplify the rat AT1 receptor from liver mRNA. The receptor DNA was subcloned into pcDNAI and a number of individual clones were transiently expressed in COS-7 cells. These individual receptors were characterized by binding of [125I] Sar1, Ile8-angiotensin II. It was found that one of the AT1 receptor clones did not bind the radiolabeled angiotensin II ligand. The mutant clone was analyzed by nucleotide sequencing. This analysis revealed that the clone has a single amino acid substitution in the third transmembrane domain of the receptor; a leucine at position 112 was changed to a proline. At this stage it is not known whether the mutant receptor is the result of a PCR artifact or an allelic difference.

Cardiac effects of relaxin in rats.

Relaxin is usually considered to be a hormone of pregnancy, but porcine relaxin has been shown to increase heart rate in rats. We investigated the cardiac effects of synthetic human gene-2 relaxin (hRlx-2) in vitro in isolated rat atria. Synthetic hRlx-2 produced concentration-dependent positive chronotropic effects in spontaneously beating right atria (EC50 [concentration required to produce 50% of the maximal response] = 0.09 [SE 0.03] nmol/l) and concentration-dependent positive inotropic effects in electrically driven left atria (EC50 = 0.31 [0.02] nmol/l). The potency of hRlx-2 is greater than that of endothelin, angiotensin II, and (-)-isoprenaline in isolated rat atria. Relaxin has powerful chronotropic and inotropic effects on the heart that are probably mediated through a direct action on relaxin receptors.

Gonadotrophin releasing hormone causes a biphasic secretion of luteinizing hormone and follicle stimulating hormone by cultures of rat anterior pituitary cells.

Primary monolayer cultures of adult male rat anterior pituitary cells secreted both LH and FSH in a biphasic manner when incubated with gonadotrophin releasing hormone (GnRH). The peaks of secretion of LH and FSH were coincident: the first occurred between 15 and 30 min and the second between 1 and 3 h after the addition of GnRH. Approximately 20% of the total amount of gonadotrophins secreted in the 6 h treatment with GnRH was contained in the first peak. Inhibitors of the secretion of gonadotrophins affected LH and FSH secretion differently. Inhibin suppressed the secretion of FSH to a greater extent than that of LH, whereas testosterone and cycloheximide had a greater effect on LH. Neither phase of secretion of LH or FSH was reduced preferentially by inhibin or testosterone but the greater effect of cycloheximide was on the second phase of secretion.

Variability in the apparent molecular weight of inhibin.

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in 1 M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in 0.1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.

Antenatal serum levels of relaxin in patients having preterm labour.

This study is the first report of antenatal levels of relaxin measured by homologous radioimmunoassay in peripheral serum from patients who subsequently had a preterm delivery. Serial blood samples were collected antenatally from a group of subjects known to be at increased risk of preterm labour because of a past history of shortened pregnancy. Serum relaxin was measured using an homologous radioimmunoassay based on a synthetic bioactive analogue of the native hormone. In women whose pregnancies ended preterm most measurements were within the range of values previously established in normal antenatal patients although some measurements early in pregnancy were above the normal range. These findings suggest that low serum levels of relaxin are not causatively related to the onset of labour before term.

Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin.

A radioimmunoassay for relaxin was developed in which a synthetic analogue of human relaxin was used as standard, tracer, and immunogen. Relaxin could not be measured in sera from men or non-pregnant women, but was measurable in pregnant women from the tenth week of gestation until term. Concentrations ranged from 0.19-1.18 ng/ml, with highest levels measured in the first trimester.

Relaxin in sera during the luteal phase of in-vitro fertilization cycles.

To identify the time when relaxin can first be detected in peripheral sera after in-vitro fertilization (IVF) and embryo transfer, blood samples were collected from 20 women up to 14 days after oocyte retrieval. Sixteen women did not become pregnant and in eight of them relaxin (but not beta-human chorionic gonadotrophin, beta-hCG) was measurable for the first time at days 6 to 12. Concentrations of other hormones measured were also different in these eight women compared with the remaining eight non-pregnant women; their serum concentrations of 17 alpha-OH progesterone, progesterone and oestradiol were higher but concentrations of luteinizing hormone and follicle-stimulating hormone were lower. Three women became pregnant; relaxin and beta-hCG were first detected on the same day (10 to 12). The remaining woman had increased beta-hCG levels but did not develop a clinical pregnancy. Measurement of serum relaxin during IVF cycles may allow assessment of corpora luteal function before its identification by levels of steroid hormones.

Relaxin in paired samples of serum and milk from women after term and preterm delivery.

In a study to determine if relaxin could be measured in milk and if so to correlate concentrations in milk and serum, paired samples of milk and serum were collected from 12 women 3 days after term delivery (term group), from 16 women 3 days after preterm delivery (preterm group), and from some of these patients 6 weeks after delivery (eight term and six preterm). Relaxin was measured by specific human relaxin radioimmunoassay. Relaxin from milk and sera behaved similarly in the relaxin radioimmunoassay and reverse-phase high-performance liquid chromatography. Concentrations of relaxin in sera and milk collected 3 days after delivery did not differ significantly within the term or preterm groups. Neither were there differences in relaxin levels in sera and milk between the term and preterm groups. At 6 weeks postpartum, relaxin was not measured in any sera but was measured in milk from six of eight patients in the term group and five of six patients in the preterm group. Relaxin concentrations in milk were higher in the preterm group. The presence of relaxin in milk at 6 weeks postpartum suggests a nonluteal site of synthesis.