Relaxin: a product of the human corpus luteum of pregnancy.

Plasma samples from peripheral and ovarian veins were obtains from women at cesarean section. A peptide that immunologically cross-reacts with a specific antiserum to porcine relaxin is present in all samples. Its concentration is four times higher in the ovarian vein draining the ovary, which contains the corpus luteum of pregnancy, than in either the peripheral vein or the contralateral ovarian vein. Secretion of ovarian relaxin correlates with secretion of ovarian progesterone, thus providing another index of luteal function.

The source of relaxin in pregnant Syrian hamsters.

Serum immunoreactive relaxin (IR) was measured on days 8, 10, and 14 of gestation in intact and ovariectomized (day 8 of pregnancy) hamsters. In intact hamsters, IR increased from 3-4 ng/ml on day 8 to 20 ng/ml by day 14 of pregnancy. After ovariectomy on day 8, pregnancy failed, and IR decreased rapidly to 0.29 ng/ml on day 14. However, when pregnancy was maintained in ovariectomized hamsters by daily injections of 0.1 microgram 17 beta-estradiol and 4 mg progesterone, serum IR rose to levels similar to those in intact hamsters on days 10 and 14 of pregnancy (i.e. 15 and 20 ng/ml, respectively). Placentas were obtained from other groups of hamsters on days 11, 14, and 15 of pregnancy and homogenized for bioassay by the classical guinea pig pubic symphysis palpation bioassay. Homogenates of placentas obtained on days 14 and 15 contained, respectively, 4 and 10 micrograms eq porcine relaxin/serum relaxin/g fresh tissue. The placenta, rather than the ovary, appears to be the source of during pregnancy in the hamster.

Characterization of the circulating form of porcine relaxin: biological activity and terminal amino acids.

Relaxin is structurally related to insulin, and it induces pregnancy-related changes in the reproductive tract of several mammalian species. Relaxin isolated from the ovaries of pregnant sows has been used for primary structure determination, for much of the biological characterization of the hormone, and for the development of RIAs. Immunoreactive (IR) relaxin is found in peripheral blood during pregnancy in pigs and other species, but it has not been established that the substance identified by RIA is structurally or biologically equivalent to the native ovarian hormone. IR relaxin was, therefore, isolated from peripheral plasma of late pregnant gilts (days 112-114) for bioassay and determination of terminal amino acid residues. IR relaxin was monitored by a specific homologous RIA during fractionation of plasma by gel filtration, cation exchange, and hydrophobic binding to octadecysilica. IR relaxin circulates unbound and is equipotent with ovarian relaxin in the mouse pubic ligament bioassay. Amino acids released from IR relaxin by pyroglutamic aminopeptidase and carboxypeptidase-Y were converted to their 4-dimethylamino-azo-4'-sulfonyl derivatives for identification by HPLC. The B-chain of IR relaxin had an amino-terminal pyroglutamic acid. Amino acids sequentially released from the carboxy-terminal indicated a chain length of 28-30 amino acids, suggesting a heterogeneity reminiscent of that of ovarian relaxin isolated by other methods. Arginine was released from the free amino-terminal by dimethylaminozaobenzene-isothiocyanate degradation, indicating an intact A-chain of 22 amino acids. Blood immunoreactive relaxin in pigs is, therefore, a secreted biologically active form of relaxin with an amino acid composition similar to that of the form stored in the corpus luteum.

Serum immunoreactive relaxin and softening of the uterine cervix in pregnant hamsters.

Serum immunoreactive relaxin levels and ripening of the cervix were measured throughout pregnancy in hamsters. RIA relaxin rose from an undetectable level on day 7 to a maximum value of 29 ng/ml on day 15 of gestation and then fell prior to parturition. The cervix became progressively more dilatable from the 12th to the 16th day of pregnancy. It is suggested that the endogenous relaxin measured by RIA may induce the cervical softening. The absolute levels of immunoreactive relaxin appear to be 10 to 15-fold higher than those previously observed in rats, mice and guinea pigs.

Relaxin bioactivity and immunoactivity in human corpora lutea.

Relaxin-like activity in extracts of corpora lutea (CL) from pregnant and non-pregnant women was determined by radioimmunoassay and by guinea pig pubic symphysis palpation assay. The biologically determined activity paralled the immunoactivity of extracts of CL of pregnancy. The relaxin content of CL of non-pregnant women was too low for detection by the bioassay.

Isolation and characterization of relaxin from the sand tiger shark (Odontaspis taurus).

A peptide with relaxin activity in guinea pigs but not in mice has been extracted from the ovaries of pregnant sand tiger sharks (Odontaspis taurus). The structural similarity of this peptide to porcine relaxin includes molecular size approximately 6000 daltons), number of chains, and possibly, disulfide cross-links. The relaxin-type peptide isolated from shark ovaries contains the amino acid residues tyrosine, proline, and histidine, which are absent in the porcine hormone. The amino acid composition of shark relaxin, therefore, resembles that of porcine insulin to a greater extent than does the amino acid composition of porcine relaxin. This finding supports the idea that shark relaxin may be a primitive relaxin that has undergone fewer mutations than porcine relaxin since the putative duplication of the insulin gene. The data presented here suggest that the putative duplication of the insulin gene, which might have given rise to relaxin, has occurred much earlier than the separation of sharks from the general branch of animals that eventually gave rise to mammals.

Plasma immunoreactive relaxin levels in pregnant and nonpregnant women.

Immunoreactive relaxin was measured in plasma samples obtained from human volunteers utilizing the RIA procedure of Sherwood et al., as modified by O'Byrne and Steinetz for heterologous plasma samples. Immunoreactive hormone was not detected in samples obtained from men, and only rarely in plasma of nonpregnant women. Immunoreactive relaxin was present as early as the fourth week of pregnancy and was detectable throughout the course of gestation. Immunoreactive relaxin tended to be higher early in pregnancy, and there was no peak just before parturition as occurs in many other species. Our results are at variance with those of Bryant and coworkers, who reported high levels of immunoreactive relaxin in men and nonpregnant as well as pregnant women. The possible reasons for this discrepancy are presented.

Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits.

Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.

Distribution of relaxin in women during pregnancy.

The purpose of this study was to determine by RIA the concentrations of relaxin in various compartments and tissues in pregnant women. Ten pairs of maternal venous and cord blood were studied. The mean relaxin concentrations, in immunoreactive equivalents of porcine relaxin, were 0.683 ng/ml in maternal serum and 0.009 ng/ml in cord serum. Relaxin was undetectable in 8 of 9 samples of amniotic fluid. Mean concentrations of relaxin in pg immunoreactive equivalents of porcine hormone per mg protein, from maternal tissues at term pregnancy were as follows: fat 96 (N = 5), myometrium 47 (N = 4), skin 62 (N = 5), placenta 51 (N = 9), and corpus luteum 13,000 (N = 8). These data suggest that little relaxin crosses the placenta and little is produced in the fetus. Contrary to prior suggestions that relaxin may also be a placental product, relaxin appears to be solely produced in the corpus luteum, making it the only peripherally measured hormone that can be used as an index of luteal activity in pregnancy.

Relaxin in human decidua and term placenta.

Using both a homologous porcine RIA and 3 bioassays, we investigated the relaxin content of human decidua from first trimester and term gestations and of placentas at term. Aqueous extracts of these tissues yielded only between 211 and 1090 pg immunoreactive relaxin/g fresh tissue. Neither acetonehexane extraction, octadecylsilica cartridge elution nor molecular sieve chromatography increased these relaxin levels. Compared with term decidua, first trimester decidua contained significantly higher levels of immunoreactive relaxin. In vitro studies did not evidence significant relaxin production by either decidual or placental tissues, even in the presence of decidual prolactin production. These results are in direct contrast to earlier reports of high concentrations of relaxin in decidua and placenta.

Increased intra-articular substance P and prostaglandin E2 following injection of interleukin-1 in rabbits.

Inflammation was induced by intra-articular injection of 100 ng recombinant human interleukin-1 alpha (rhIL-1) into rabbit knees. Substance P (SP) and prostaglandin E2 (PGE2) were measured by radioimmunoassay (RIA) in the joint fluid at 4, 24 and 48 h after rhIL-1 injection. SP was increased by 4 h, further increased at 24 h and remained elevated at 48 h. PGE2 concentration was highest at 4 h and remained elevated at 48 h after rhIL-1 injection. Because of the proinflammatory activities of SP and PGE2, our studies suggest that the elevated SP and PGE2, in the joint may amplify or sustain an initial receptor-mediated inflammatory response to IL-1.

Radioimmunoassay (RIA) of relaxin in sera of various species using an antiserum to porcine relaxin.

We have investigated the application of a RIA for porcine relaxin to the assay of relaxinlike substances in the blood of various other mammalian species. The cross-reactivity between antiporcine relaxin antibody and the relaxinlike substances in the blood of other mammals during pregnancy was sufficiently high to permit the assay of 0.1-0.5 ml of serum or plasma samples. Nonspecific reactivity was controlled by adding similar volumes of serum or plasma obtained from ovariectomized female or intact male subjects to RIA tubes containing known porcine standards. RIA relaxin levels rose markedly during the last third of pregnancy in rats, mice, and guinea pigs. RIA relaxin was also found in late pregnancy in dog, rhesus and java monkeys, and human beings. The apparent blood levels of hormone found in each species will depend upon their degree of cross-reactivity with the antiporcine relaxin antibody as well as upon their actual concentration. Thus, absolute blood level values should not be taken literally. However, the fluctuations in RIA relaxin observed during the course of gestation in any given species would appear to reflect accurately the relative blood concentrations of the hormone.

Catabolin/interleukin-1 regulation of cartilage and chondrocyte metabolism.

Catabolin/interleukin-1 effects on metabolism were studied in bovine nasal cartilage organ culture and articular chondrocyte cell culture. Keratan sulfate (KS) and hyaluronic acid (HA) were determined by an ELISA; prostaglandin E2 by RIA, sulfated glycosaminoglycan using dimethylmethylene blue and proliferation by incorporation of tritiated thymidine. Gel filtration of untreated 4-day organ culture media indicated that large sulfated and KS-containing proteoglycans were released and eluted in the void volume. Catabolin/interleukin-1 increased release of sulfated glycosaminoglycans and these were of lower molecular weight with an altered distribution of KS. Catabolin/interleukin-1 treatment of chondrocytes caused a decrease in KS production and proliferation but an increase in HA and in prostaglanding E2 production. Alterations of the chondrocyte metabolism by catabolin/interleukin-1 causing proteoglycan matrix degradation and modulation of chondrocyte glycosaminoglycan biosynthesis and proliferation may play a role in cartilage erosion and failure to repair in arthritic diseases.

Effects of relaxin on early pregnancy in rats.

PIP: In several mammalian species, the polypeptide hormone, relaxin, prepares the reproductive tract for parturition. In rats, relaxin is secreted by the ovaries during the last week of a 22-day pregnancy. There is a failure of parturition in ovariectomized pregnant rats. In this study relaxin was injected into rats in the periimplantation period . The effects of relaxin on the response of the pregnant rat to a subabortifacient dose of prostaglandin F2-alpha (PGF2-alpha) was tested. Swine ovarian relaxin preparations were injected sc as a single dose on days 3,4, or 5 of pregnancy or as multiple doses on Days 3-5 or 4 and 5. Progesterone was injected sc on Days 3-6 or 2-9 of pregnancy. Dexamethasone was given orally on Days 4-6. Indomethacin was given by gavage on Days 3-6. PGF2-alpha was injected as a single dose on Day 5, alone or in combination with relaxin. Rats were killed on Day 10 and the uterine contents observed. Injections of .5 mg (but not of .1 mg) relaxin daily on Days 3-5 of pregnancy induced resorption of a large number of fetuses. Single doses of 1 mg relaxin given on Day 4 or 5, but not when given on Day 3, terminated pregnancy in rats. Progesterone, but not indomethacin, given on Days 3-5 prevented termination of pregnancy by relaxin given on Day 5. Resorption of litters induced by .5 mg doses of relaxin given on Days 4 or 5 prevented by daily injections of progesterone given from Days 2 to 9. Injections of 10 or 30 mcg of dexamethasone on Days 4, 5, and 6 partially or completely reversed the adverse effects of relaxin on litter survival. A single injection of 1 mg of PGF2-alpha on Day 5 failed to affect the pregnancy, but when given following a .1 mg dose of relaxin given on Days 3, 4, and 5 nearly complete resorption of litters resulted. These 2 compounds are therefore thought to act synergistically. Serum progesterone levels 24 hours after a single injection of 1 mg of purified relaxin were the same as controls. It is suggested that relaxin may antagonize the progesterone "block" to coordinated uterine contractions.^ieng

Elevated substance P and accelerated cartilage degradation in rabbit knees injected with interleukin-1 and tumor necrosis factor.

Cytokines, interleukin-1 (IL-1), tumor necrosis factor alpha, and the neurotransmitter, substance P, have been implicated in the pathogenesis of arthritis because they stimulate synovial cells to secrete prostaglandin E2 and collagenase in vitro. We investigated in vivo changes in intraarticular substance P and the degradation of cartilage proteoglycan in response to intraarticular cytokine injections in rabbits. Twenty-four hours after a single injection of 10 ng, 30 ng, or 100 ng of recombinant human IL-1 alpha (rHuIL-1 alpha) per joint, the mean +/- SEM levels of substance P detected in the cell-free joint lavage fluid were 250 +/- 67 fmoles, 480 +/- 60 fmoles, and 530 +/- 130 fmoles (n = 4-5), respectively. The level of substance P in the contralateral knees injected with diluent was 58 +/- 8 fmoles (n = 12). The level of substance P had increased by 2 hours after IL-1 injection and remained elevated in the joint 48 hours after injection. Cytokine-induced proteoglycan depletion was also time- and dose-dependent. Proteoglycan concentrations in articular cartilage dissected from the weight-bearing condyles were calculated as the ratio of sulfated glycosaminoglycan measured using 1,9-dimethylmethylene blue: hydroxyproline. After 48 hours, 10 ng, 30 ng, or 100 ng of rHuIL-1 alpha per joint decreased proteoglycan levels by 9 +/- 4%, 14 +/- 4%, and 21 +/- 3% (n = 8), respectively. Likewise, the injection of recombinant human tumor necrosis factor alpha induced depletion of intraarticular substance P and cartilage proteoglycan.