Relaxin stimulates bronchial epithelial cell PKA activation,migration, and ciliary beating

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.     

Serum immunoreactive relaxin in women during a 24-h period

Serum relaxin concentrations were measured every 30 min during a 24-h period in nonpregnant and pregnant women. Relaxin was undetectable in all serum samples obtained from 3 nonpregnant women. Relaxin was detectable in all serum samples obtained from 2 pregnant women. However, neither episodic secretion of relaxin nor a 24-h rhythm in relaxin secretion was discernible in these women.     

Relaxin activity in the pregnant cat

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.     

Relaxin

1. Relaxin is a hormone of reproduction that appears to affect parturition, uterine accommodation, and sperm motility to varying degrees in many species. 2. All relaxins have the same two chain, disulfide-linked insulin-like structure and two arginine residues in the midregion of the B chain. 3. The active relaxin molecule is produced by excision of a connecting peptide from the prohormone. 4. The biosynthetic pathways of insulin and relaxin are alike, but the relaxin prohormone is about twice as large as the corresponding proinsulin. 5. The primary structures of relaxins from apparently closely related species differ significantly in their amino acid compositions and do not fit into the traditional scheme of molecular evolution.     

Developmental expression and serotonergic regulation of relaxin 3/INSL7 in the nucleus incertus of rat brain

Relaxin 3 or insulin like peptide 7 has been identified as a new member of the insulin/relaxin superfamily. We recently reported that relaxin 3 was dominantly expressed in the brain, particularly in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons and that it might act as a neurotransmitter. In the present study we investigated the developmental expression and serotonergic regulation of relaxin 3 gene in the rat brain. Relaxin 3 mRNA appeared at embryonic day 18 in the near region of the fourth ventricle, and was shown to have increased its density and the number of expressing neurons by in situ hybridization and RT-PCR examination. Relaxin 3 peptide was detected after birth by immunocytochemistry. Since the NI is located just caudal to the dorsal raphe nucleus where abundant serotonin (5-HT) neurons are present, we examined if 5-HT effects on the expression of relaxin 3. Relaxin 3 gene expression in the NI significantly increased after 5-HT depletion by p-chlorophenylalanine (PCPA) administration. We also observed the 5-HT1A receptor localization in relaxin 3 positive neurons of the NI. This result suggests that 5-HT negatively regulates the expression of relaxin 3 gene in the NI. The function of relaxin 3 neurons in the brain is influenced by the serotonergic activity.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Relaxin Treatment in an Ang-II-Based Transgenic Preeclamptic-Rat Model

Relaxin is a peptide related to pregnancy that induces nitric oxide-related and gelatinase-related effects, allowing vasodilation and pregnancy-related adjustments permitting parturition to occur. Relaxin controls the hemodynamic and renovascular adaptive changes that occur during pregnancy. Interest has evolved regarding relaxin and a therapeutic principle in preeclampsia and heart failure. Preeclampsia is a pregnancy disorder, featuring hypertension, proteinuria and placental anomalies. We investigated relaxin in an established transgenic rat model of preeclampsia, where the phenotype is induced by angiotensin (Ang)-II production in mid pregnancy. We gave recombinant relaxin to preeclamtic rats at day 9 of gestation. Hypertension and proteinuria was not ameliorated after relaxin administration. Intrauterine growth retardation of the fetus was unaltered by relaxin. Heart-rate responses and relaxin levels documented drug effects. In this Ang-II-based model of preeclampsia, we could not show a salubrious effect on preeclampsia.     

Pregnant mouse corpora lutea: immunocytochemical localization of relaxin and ultrastructure

Relaxin was localized in corpora lutea of pregnant mouse ovaries by using the unlabeled antibody peroxidase-antiperoxidase technique and a highly specific rabbit antirat relaxin serum. Relaxin immunostaining was first observed in luteal cells located at the periphery of corpora lutea on Day 10 of gestation. The number of relaxin immunostained cells and the intensity of the stain gradually increased to reach a maximum between Days 16 and 18 of gestation. While a few luteal cells were specifically stained for relaxin on Day 1 postpartum, no luteal cells were stained on Day 2 postpartum. Ultrastructural studies of luteal cells from pregnant mouse ovaries revealed the presence of a distinct electron-dense, membrane-bound granule population, which was first observed on Day 12 of gestation. The granules increased in number to reach a maximum between Days 16 and 18 of gestation, and were absent by Day 2 postpartum. The appearance and disappearance of this granule population closely paralleled the relaxin immunostaining in the luteal cells. We suggest that the granules may be the subcellular sites of relaxin storage in the pregnant mouse ovary.     

Physiologic role of relaxin on mammary gland growth in rats

The role of relaxin in stimulating growth of the mammary gland was assessed in ovariectomized and intact male rats for a period of 20 days. In addition to relaxin alone, the ovarian mammogenic hormones estradiol and progesterone were used in combination with relaxin and with each other to evaluate responses of mammae. Indices for mammary growth included wet weight, dry fat-free tissue, DNA, RNA, total protein, and collagen. Quantitative estimates of DNA and collagen represented the best indicators of parenchymal and stromal growth, respectively. Because changes in body weights were significantly different among hormonally administered groups, these were included as well. In Ovariectomized young rats, relaxin alone and in combination with estradiol and progesterone increased all indices significantly (P less than 0.01). The collagenous portion of total protein was high for the group receiving relaxin alone (62%) compared with the control group (46%). Relaxin administered along with estradiol and progesterone increased collagen accumulation to 73%, compared with 54% in the estradiol + progesterone group. Relaxin did not significantly increase growth indices when administered to male rats at 10 and 20 micrograms/day, while 30 micrograms stimulated a significant increase in total protein (P less than 0.05), suggesting that 30 micrograms of relaxin/day may be considered the basal concentration needed to induce a physiologic response in males. Relaxin induced a growth effect on mammae by synergizing with progesterone and estradiol in order to stimulate parenchymal proliferation, as noted by a DNA increase, and to increase stromal distensibility of the mammary pad by invoking accumulation of collagen and total protein in substituting for mammary adipose tissue.     

Rabbit endometrial relaxin: immunohistochemical localization during preimplantation, pregnancy, and lactation

Relaxin was localized in rabbit endometrium (but not ovary) on Days 4-30 of pregnancy and Days 2-5 of lactation. The hormone was not observed on Days 2 and 3 post coitus. Relaxin was found in endometrial glands throughout the length of the uterus on Days 4-9 post coitus. Later, on Days 11-23, relaxin was localized in both uterine endometrial gland cells and luminal epithelial cells. At this time, staining was observed only in the endometrium directly associated with implantation sites. Areas between implantation sites were devoid of staining. On Days 25-30 of pregnancy, relaxin was found mainly in uterine luminal epithelial cells. Few glands were observed with relaxin. During the first week of lactation, the staining profile was the same as that observed on Days 25-30. Relaxin was not found in the endometrium of pseudopregnant rabbits (Days 1, 4, 8, 12, and 16). The early appearance of uterine relaxin at the time the blastocyst migrates into the uterine cavity coupled with the hormone's later confinement to implantation sites suggests that the blastocyst initiates and the conceptus maintains uterine relaxin.     

Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.     

Restricted,but abundant,expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5' RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.     

Relaxin Does Not Improve Angiotensin II-Induced Target-Organ Damage

Relaxin is a corpus-luteum produced protein hormone with vasodilatatory, anti-fibrotic, and angiogenic properties that are opposite to angiotensin (Ang) II. We investigated whether or not relaxin ameliorates Ang II-induced target-organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGR) that develop severe hypertension, target-organ damage, and die untreated within 7–8 weeks. Recombinant relaxin at a low (26 μg/kg/d) and a high dose (240 μg/kg/d) was given to 4 week-old dTGR and age-matched Sprague-Dawley rats (SD). Systolic blood pressure increased progressively in untreated dTGRs from 162±3 mmHg at week 5 to 225±5 mmHg at week 7. Relaxin had no effect on blood pressure whereas SD rats were normotensive (106±1 mmHg). Untreated and relaxin-treated dTGR had similarly severe cardiac hypertrophy indices. Relaxin did not ameliorate albuminuria and did not prevent matrix-protein deposition in the heart and kidney in dTGR. Finally, relaxin treatment did not reduce mortality. These data suggest that pharmacological doses of relaxin do not reverse severe effects of Ang II.     

Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Anti-relaxin and relaxin on prolactin and gonadotropin secretion in vitro from porcine pituitary cells

This study examines the possibility of a feedback interaction between gonadal relaxin and the pituitary by investigating the impact of exogenous relaxin and ablation of endogenous with relaxin anti-relaxin serum on pituitary hormone secretion in vitro. Three wells were assigned to treatments: 0, 100 and 1000 ng ml⁻¹ of relaxin, 1:100, 1:1000 and 1:10000 titer of anti-relaxin. Relaxin significantly enhanced prolactin (PRL) secretion (P < 0.05) in long-term culture but had no effect on luteinizing hormone and follicle stimulating hormone secretion. Relaxin anti-serum stimulated a dose dependent increase (P < 0.05) in gonadotropin secretion at 48, 72 and 96 h. Luteinizing hormone and follicle stimulating hormone increased two-fold in 48 h cultures in response to 1:100 anti-relaxin serum in comparison with untreated controls. Anti-relaxin serum at 1:100 completely suppressed PRL secretion after either 48, 72, and 96 h of culture. At 48 h all levels of anti-relaxin serum completely suppressed PRL secretion. These results indicate that endogenous relaxin may be involved at the adenohypophysial level in modulating gonadotropin and PRL release in the pig.     

Relaxin in the marmoset monkey: secretion pattern in the ovarian cycle and early pregnancy.

Relaxin is a peptide hormone with a broad range of biological activities, related not only to parturition and lactation but possibly also to decidualization, implantation, and early pregnancy. The present study was designed to investigate the secretion pattern of relaxin throughout the cycle and early pregnancy in the common marmoset monkey in relation to ovarian function and the systemic hormone milieu. First, a novel relaxin ELISA was developed and validated to confirm the pattern of relaxin secretion during pregnancy. Secondly, serum relaxin profiles were determined through nonconceptive and conceptive cycles and analyzed in relation to the concentration of other hormones and to the development of ovarian follicles and corpora lutea (CL). Blood samples were collected 2-3 times per week from the experimental animals and analyzed for relaxin, progesterone, and LH. The animals from the conceptive cycles were also ultrascanned at these time points to determine the ovarian status up to Day 25 of pregnancy. During early pregnancy, the relaxin levels in serum were approximately 1 ng/ml, increasing up to 15 ng/ml in the second trimester, at a time when progesterone levels had declined. In the third trimester, when progesterone levels were increasing again, the levels of relaxin decreased, returning to basal levels by term of pregnancy. In early pregnancy there was a parallel increase in both relaxin and LH/hCG, with the relaxin rise in the conceptive cycle appearing sooner than in the nonconceptive cycle, suggesting that, like chorionic gonadotropin (CG), relaxin may be a useful and early marker for pregnancy. Unlike the situation in the human, there was no correlation between the levels of either hormone and the number of CL detected, infants born, mother's age, or parity. Relaxin levels increased in early pregnancy before bioactive LH/CG, implying that relaxin is not directly regulated by this gonadotropin. Furthermore, hCG applied to nonconceptive females during the expected time of implantation caused an increase in progesterone but not in relaxin concentrations. In summary, the results obtained indicate that relaxin may be a reliable indicator of early pregnancy status in the common marmoset, but it is independent of direct CG influence.