Porcine and human relaxin bioactivity: bioactivities of porcine relaxin and human relaxin do not differ in mice and rats.

This study compares the bioactivity of porcine relaxin-1 to that of recombinant human relaxin-2 in mice and rats. The effects of the two hormone preparations on elongation of the mouse interpubic ligament and both the wet weight and the extensibility of the rat cervix were compared. No difference in bioactivity was detected between porcine relaxin-1 and recombinant human relaxin-2 in either rodent. Therefore, decisions concerning which of the two available forms of relaxin to employ for in vivo experimentation in mice and rats can be made without concerns about relative bioactivity.

Clinical use of relaxin to facilitate birth: reasons for investigating the premise.

In the United States, both medical and nonmedical factors have driven the cesarean section rate to over 26% of all deliveries. In addition to questions of increased cost associated with operative delivery, some have questioned the ethics of performing cesarean section for nonmedical reasons. Reduction of both the duration and the pain associated with vaginal delivery would likely bring about a decline in the rate of both medical and nonmedical cesarean sections. This chapter summarizes recent findings that support the premise that through its growth-promoting and softening effects on the cervix, short-term subcutaneous administration of pharmacologic amounts of relaxin to women at term holds promise as a means of reducing the duration and discomfort associated with delivery. Two recent studies conducted in pregnant rats demonstrated that the cervix is highly responsive to relaxin during the antepartum period and that short-term subcutaneous administration of the hormone to relaxin-deficient animals not only promotes growth and softening of the cervix, but also reduces the duration of labor and delivery. Moreover, recent human clinical trials examining the influence of 24 weeks of continuous subcutaneous administration of recombinant human relaxin for the treatment of scleroderma provided evidence not only that the human reproductive tract is responsive to relaxin, but also that the administration of the hormone does not cause serious adverse side effects. It is concluded that recent findings provide an impetus for an investigation into relaxin's potential for cervical remodeling and facilitating birth in women.

The extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy in rats.

Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5'-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.

Relaxin is essential for renal vasodilation during pregnancy in conscious rats.

Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.

Evidence that relaxin inhibits apoptosis in the cervix and the vagina during the second half of pregnancy in the rat.

The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19-21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.

Inhibition of nitric oxide synthase activity diminishes the acute effects of relaxin on growth, but not softening, of the cervix in the rat.

Relaxin promotes growth and softening of the cervix during pregnancy in the rat. This study examined the hypothesis that nitric oxide (NO) mediates the effects of relaxin on the rat cervix. To test that hypothesis, N omega-nitro-L-arginine methyl ester (L-NAME) was used to inhibit NO synthase, the enzyme that converts arginine to NO and L-citrulline. Nonpregnant rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy each animal was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provide blood levels similar to those during late pregnancy. Rats were assigned to three treatment groups. The control group OPE (n = 6 rats) received 0.5 ml L-NAME vehicle (PBS) sc at 6-h intervals from 0600 h on day 7 through 1200 h on day 8 and 0.5 ml relaxin vehicle (PBS) sc at 0600 and 1200 h on day 8. Group OPER (n = 6 rats) was treated in the same way as group OPE, except that 20 microg porcine relaxin were administered. Group OPERI (n = 7 rats) was treated in the same way as group OPER, except that L-NAME was administered at a dose of 100 mg/kg x 6 h. Between 1400-1500 h on day 8, the cervices were removed and weighed. Cervical wet weight and extensibility were markedly greater (P < 0.01) in relaxin-treated group OPER rats than in group OPE controls. Treatment with L-NAME diminished relaxin's effects on cervical wet weight, but not cervical extensibility. In conclusion, this study provides evidence that NO contributes to the acute effects of relaxin on the growth, but not the softening, of the rat cervix.

Relaxin stimulates uterine edema via activation of estrogen receptors: blockade of its effects using ICI 182,780, a specific estrogen receptor antagonist.

Relaxin's ability to stimulate uterine growth is well established. The mechanisms by which relaxin exerts this effect, however, remain unclear. In light of previous work demonstrating peptide growth factor activation of estrogen receptors (ERs), the present study was conducted to determine if relaxin similarly stimulates ERs. Twenty-five day-old female Sprague-Dawley rats were bilaterally ovariectomized and treated with estradiol or porcine relaxin alone or in combination with the ER antagonist ICI 182,780. Following treatment with 17beta-estradiol or relaxin alone, the uterine weight/body weight ratio (UtW/BW) increased significantly over control values (+98% and +77% respectively, p<0.0003). Pre-treatment of animals with ICI 182,780 (3 microg/g BW) prior to either estradiol or relaxin treatment completely inhibited the hormone-induced increases in uterine weight (p<0.0005). ICI 182,780 alone had no significant effect. Histological analysis of uterine cross-sections revealed that the edema present in the endometrium of animals treated with estradiol or relaxin alone was completely absent in the uteri of animals pre-treated with ICI 182,780. These data indicate that relaxin-induced uterine edema and growth is mediated by ERs.

Relaxin is a potent renal vasodilator in conscious rats.

The kidneys and other nonreproductive organs vasodilate during early gestation; however, the "pregnancy hormones" responsible for the profound vasodilation of the renal circulation during pregnancy are unknown. We hypothesized that the ovarian hormone relaxin (RLX) contributes. Therefore, we tested whether the administration of RLX elicits renal vasodilation and hyperfiltration in conscious adult, intact female rats. After several days of treatment with either purified porcine RLX or recombinant human RLX 2 (rhRLX), effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) increased by 20%-40%. Comparable renal vasodilation and hyperfiltration was also observed in ovariectomized rats, suggesting that estrogen and progesterone are unnecessary for the renal response to rhRLX. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester completely abrogated the increase in ERPF and GFR elicited by chronic administration of purified porcine RLX. In contrast, the renal vasoconstrictory response to angiotensin II was attenuated by the RLX treatment. Short-term infusion of purified porcine RLX to conscious rats over several hours failed to increase ERPF and GFR. Plasma osmolality was consistently reduced by the chronic administration of both RLX preparations. In conclusion, the renal and osmoregulatory effects of chronic RLX administration to conscious rats resemble the physiological changes of pregnancy in several respects: (a) marked increases in ERPF and GFR with a mediatory role for nitric oxide; (b) attenuation of the renal circulatory response to angiotensin II; and (c) reduction in plasma osmolality.

Monoclonal antibodies specific for rat relaxin. X. Endogenous relaxin induces changes in the histological characteristics of the rat vagina during the second half of pregnancy.

This study employed morphometric analysis to evaluate changes in the histological characteristics that accompany relaxin-induced growth and softening of the vagina during the second half of rat pregnancy. There were three treatment groups (N = 4/group). Five milligrams of a monoclonal antibody for rat relaxin, designated MCA1, were injected i.v. daily on days 12-21 of gestation to treatment group MCA1. Control groups received either 5 mg of monoclonal antibody for fluorescein (MCAF; monoclonal antibody control) or 0.5 ml PBS (vehicle control). Vaginas were removed on day 22 of pregnancy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissue sections (5 microm) were stained with Gomori's trichrome to visualize collagen, or orcein to visualize elastin. Measurements were performed with a light microscope equipped with a video camera connected to a computer. Within the vaginal stroma, the density of collagen fiber bundles was lower, the length of elastin fibers was shorter, and the cross-sectional area and wall thickness of arteries were greater in relaxin-replete control rats than in relaxin-deficient MCA1-treated rats. These relaxin-induced changes in the stroma appear to account, at least in part, for the hormone's softening effect on the vagina. Within the epithelium, there were approximately 2-fold more basal and mucus-secreting cells in relaxin-replete control rats than in MCA1-treated rats. The relaxin-induced accumulation of epithelial cells appears to contribute to vaginal growth. We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery in pregnant rats.

Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissue at term.

Ripening and dilation of the rat cervix at term involves a widespread reduction in the density and organization of collagen fibers following a polymorphonuclear eosinophilic invasion. These are hormonally regulated events; however, the correlation between hormonal milieu and these morphological changes is not well understood. To investigate the role of preparturient relaxin and estradiol-17ss (E2) in cervical collagen remodeling and eosinophilic infiltration, pregnant rats were either sham-ovariectomized (group C) or ovariectomized in the morning of Day 22. Ovariectomized rats were treated with E2 (group OE), relaxin (group OR), E2 plus relaxin (group OER), or hormone vehicles (group O). There were 4 or 5 animals per group. Cervices were taken from animals killed approximately 1 h before expected parturition. Five-micrometer serial sections of paraffin-embedded cervices were stained with either Sirius Red in alkaline solution to measure eosinophil infiltration or in Picrosirius to measure collagen birefringence. Ovariectomized rats treated with E2 (group OE or OER) showed high levels of eosinophilic infiltration that did not differ from those in group C intact controls. However, the values of eosinophilic infiltration in ovariectomized rats treated with relaxin or hormone vehicles (groups OR and O) were low and far below (p < 0.01) those of groups OE and C. In ovariectomized rats treated with E2 alone (group OE), the widespread reduction in collagen organization that occurred in group C controls was not observed. It was only in ovariectomized rats treated with relaxin or E2 + relaxin (groups OR and OER) that the values of birefringence were low, and they were as low as in control group C. In conclusion, this study indicates that eosinophilic infiltration and collagen remodeling in the rat cervix at term are under the control of different hormones: E2 stimulates eosinophilic invasion, and relaxin promotes a widespread reorganization of collagen fibers.

Identification of specific relaxin-binding cells in the human female.

Relaxin is secreted during pregnancy, but it has no verified effects in humans. The objective of the present study was to identify the cells containing specific relaxin-binding sites in the uterine cervix, vagina, uterus, mammary glands, mammary nipples, and term placenta in the human. The uterine cervix, vagina, and uterus were obtained from hysterectomy specimens. Mammary glands and nipples were obtained after modified radical mastectomy. Placenta was obtained after normal delivery. Tissue samples were cut into slices (0.5-3 cm3), frozen in liquid nitrogen, and cryosectioned (8 microm). Cells that bind relaxin were identified by sequential application of biotinylated porcine relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver enhancement for signal amplification. Relaxin bound with specificity to epithelial cells, smooth muscle cells, and blood vessels in the cervix, vagina, uterus, and mammary nipples; to epithelial cells and blood vessels in the mammary glands; and to skin of the mammary nipples. In addition, relaxin bound to individual cell types within the term placenta (amnion epithelium, syncytiotrophoblasts, blood vessels), and to sebaceous glands within the nipples. We conclude that the specific relaxin-binding cells probably contain relaxin receptors. Identification of putative relaxin receptors may provide insight into physiological and/or therapeutic roles of relaxin in the human.

Relaxin increases the accumulation of new epithelial and stromal cells in the rat cervix during the second half of pregnancy.

Both cervical and vaginal growth are relaxin dependent during rat pregnancy. We recently reported a relaxin-dependent 1.5-fold increase in cervical and vaginal DNA content from midpregnancy until term. This finding indicated that relaxin probably promotes cervical and vaginal growth at least in part by promoting cellular proliferation. The objective of this study was to identify and quantify cells in the cervix and vagina that proliferate during the second half of rat pregnancy in response to relaxin. Primiparous pregnant rats were ovariectomized or sham ovariectomized (group C; n = 8) on day 9 of pregnancy (D9). Ovariectomized rats were then treated with physiological doses of progesterone plus estrogen (n = 7) or progesterone, estrogen, and porcine relaxin (n = 7). Cellular proliferation was determined by continuously administering a low dose of 5-bromo-2'-deoxyuridine (BrdU) via miniature osmotic pump from D9-D22. On D22, cervices and vaginas were collected, fixed in formalin, paraffin embedded, and serially sectioned (4 microm). Adjacent serial sections were either immunostained for BrdU to assess cell proliferation or stained with hematoxylin to determine total cell number. Cell proliferation was evaluated by counting BrdU-positive nuclei and total nuclei in the same area on adjacent sections. Cell counts were determined using computerized digital morphometric analysis at x575. In control rats, nearly 75% of the epithelial cells and 55% of the stromal cells within the cervix at term had proliferated during the second half of pregnancy. The accumulation of approximately half of the new cells was relaxin dependent. Within the cervical stroma, relaxin increased the accumulation of cells associated with blood vessels and also the number of isolated cells (probably fibroblasts). Relaxin did not appear to affect smooth muscle cell proliferation in the cervix. In contrast to the cervix, a minority of vaginal epithelial cells (45%) and stromal cells (20%) proliferated during the second half of pregnancy. Although relaxin appeared to have a tendency to increase the accumulation of new vaginal epithelial and stromal cells, morphometric analysis did not provide support for such an effect. In conclusion, this study demonstrates that relaxin promotes a marked increase in the accumulation of new epithelial cells and stromal cells within the cervix. The relaxin-induced increase in new epithelial and stromal cells probably contributes to relaxin's effects on growth and remodeling of the cervix that are required for rapid and safe delivery.

Localization of specific relaxin-binding cells in the ovary and testis of pigs.

It is not known whether relaxin has physiological roles in the gonads in mammalian species. Limited evidence indicates that relaxin may act locally to regulate ovarian function in pigs. The possibility of a role for relaxin in testicular function in pigs has not been investigated. A major initial step toward the establishment of direct effects of relaxin on the ovary and/or testis is to demonstrate that relaxin binds with specificity to the gonads. Accordingly, the first objective of this study was to employ an immunohistochemical localization technique to determine whether relaxin-binding cells are present in the ovaries and/or testis of pigs. Once they were found to be present, the second objective was to determine whether relaxin-binding sites noticeably change either within the ovary at different stages of the estrous cycle and pregnancy, or within the testis at sexual maturity. Ovaries were collected from four stages of the estrous cycle (midfollicular, late follicular, early luteal, and midluteal) and three stages of the pregnancy (Day 40, Day 80, and Day 110). Two gilts were used for each of the stages of the estrous cycle and pregnancy. Testes were collected from a 5-mo-old immature boar and a 36-mo-old mature boar. Tissues were cut into cubes (3-4 cm3), frozen in liquid nitrogen, and cryosectioned (8 microm). Specific cell types that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. In the ovary, specific relaxin-binding sites were localized in both the theca and granulosa cells of developing follicles, luteal cells, and blood vessels. In the testis, specific relaxin-binding sites were localized in the Leydig cells. There were no apparent differences in relaxin-binding distribution within the ovary at different stages of the estrous cycle and pregnancy in gilts, or within the testis at sexual maturity in boars. We conclude that the specific relaxin-binding cells within the ovary and testis of the pig may contain relaxin receptors. Therefore, relaxin may have effects in the ovary and testis of pigs.

Evidence that relaxin's effects on growth and softening of the cervix are not mediated through prostaglandins in the rat.

Relaxin plays a major role in promoting the growth and softening of the cervix that occurs during the second half of pregnancy in the rat. There is limited evidence that prostaglandins play a role in cervical softening in mammalian species. Accordingly, this study was conducted to determine if prostaglandins mediate relaxin's effects on the rat cervix. To attain that objective, indomethacin was used to inhibit cyclooxygenase, the key enzyme in the conversion of arachidonic acid to prostaglandins. Twenty-six nonpregnant female rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy (O), each rat was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provided blood levels similar to those during late pregnancy in rats. Rats were randomly assigned to three treatment groups. Group OPE controls (n = 8 rats) received 2 ml indomethacin vehicle (0.5% methyl cellulose, 0.025 Tween 80 in water) via gavage at 0900 h on days 8 and 9 and 0.5 ml relaxin vehicle (0.9% NaCl) s.c. at 6-h intervals from 1200 h on day 8 through 0600 h on day 10. Group OPER (n = 9 rats) was treated as group OPE except that 20 microg highly purified porcine relaxin was administered. Group OPERI (n = 9 rats) was treated as group OPER except that indomethacin was administered at a dose (20 mg/kg BW) that reduced cervical PGE2 levels by more than 90%. Between 0800 h and 1000 h on day 10, the cervices were removed, trimmed of fat, weighed, and placed in ice-cold Krebs-Ringer bicarbonate buffer, pH 7.5. Cervical extensibility (degree of softening) was determined within 4 h of tissue collection. Both the mean cervical wet weight and the mean cervical extensibility in the relaxin-treated group OPER rats were markedly greater (P < 0.01) than in the group OPE controls. Treatment with indomethacin did not diminish relaxin's effects on either cervical wet weight or cervical extensibility. In conclusion, this study provides evidence that relaxin's effects on cervical growth and softening in the rat are not mediated through prostaglandins.

Evidence that systemic relaxin promotes moderate water consumption during late pregnancy in rats.

The protein hormone relaxin is secreted by the ovaries throughtout the second half of the 23 day pregnancy in the rat. We recently reported that neutralization of endogenous relaxin with monoclonal antibodies for rat relaxin decreases water consumption during the daily light period during the second half of pregnancy in rats. The apparent effects of relaxin on water consumption, however, were extremely modest. One explanation for the failure to observe a greater relaxin-dependent effect on water consumption is failure of the monoclonal antibody for rat relaxin to neutralize all circulating relaxin. A second explanation is that circulating relaxin has only slight effects on water consumption. This investigation was conducted with an experimental model in which circulating relaxin was removed in order to re-examine the effects of relaxin on water consumption during the daily light period in late pregnancy in rats. On day 9 (D9) of pregnancy, before the presence of relaxin (R) in the circulation, primiparious pregnant rats were ovariectomized (O) or sham ovariectomized (C). Throughout the remainder of pregnancy, rats were treated with combinations of either progesterone (P) and estrogen (E, group OPE) or progesterone, estrogen and porcine relaxin (group OPER) in doses that restore physiological parameters to values similar to those that occur during the second half of pregnancy in intact rats. Progesterone and estrogen were administered by Silastic tubing implants and porcine relaxin was administered via miniature osmotic pump. Sham-ovariectomized animals received either the hormone vehicles (group SC) or no implants (group IC). Water consumption was measured daily from D4 to D20 at both 0700 and 2100 h which was when the lights went on and off respectively. Water consumption increased as pregnancy continued from D10 to D20 during the daily 10 h dark periods (P < 0.01), but not during the 14 h light periods for all four groups. Daily water consumed by rats in group OPE was significantly lower (P < 0.05) than that consumed by shamovariectomized rats from D17 to D20 and lower than that consumed by rats in group OPER on D20. During the dark period there was no difference in water consumption among groups. During the light period, however, group OPE consumed significantly less water (P < 0.05) than group C from D18 to D22. Moreover, there was a consistent tendency (P < 0.13) for the water consumption to be greater in rats in group OPER than in those in the relaxin-deficient group OPE during the daily light period from D11 to D20 of pregnancy. We conclude that the increase in water consumption that occurs during the daily dark periods during the second half of pregnancy is not attributable to circulating relaxin. Circulating relaxin promotes only modest increases in water consumption during the daily light periods during late pregnancy in the rat.

Effects of relaxin on rat atrial myocytes. I. Inhibition of I(to) via PKA-dependent phosphorylation.

The peptide hormone relaxin has direct, positive inotropic and chronotropic effects on rat hearts in vivo and in vitro. Relaxin's effects on the electrophysiological properties of single quiescent atrial cells from normal rats were investigated with a whole cell patch clamp. Relaxin had a significant inhibitory effect on outward potassium currents. The outward potassium current consisted of a transient component (I(to)) and a sustained component (I(S)). The addition of 100 ng/ml of relaxin inhibited the peak I(to) in a voltage-dependent manner (74% inhibition at a membrane potential of -10 mV to 30% inhibition at +70 mV). The time to reach peak I(to) and the apparent time constant of inactivation of I(to) were increased by relaxin. Dialysis with the protein kinase A inhibitor 5-24 amide (2 microM) prevented relaxin's effects, suggesting an obligatory role for this kinase in the relaxin-dependent regulation of the potassium current.

Effects of relaxin on rat atrial myocytes. II. Increased calcium influx derived from action potential prolongation.

Relaxin produces positive inotropic and chronotropic effects in rat hearts. The effect of relaxin on the action potential duration (APD) of single quiescent rat atrial cells was investigated with a whole cell patch clamp. Relaxin induced a significant, dose-dependent prolongation of the APD. This effect was maximal at 200 ng/ml (nominal concentration of 33.6 nM), which caused, on average, a 57% increase in the time taken to reach 90% repolarization. The effect of relaxin was blocked by the protein kinase A inhibitor 5-24 amide, indicating that its effect is mediated by an adenosine 3',5'-cyclic monophosphate-dependent mechanism. The increased APD induced by relaxin caused an enhanced entrance of calcium, with the charge carried through voltage-activated calcium channels increased by approximately 25%. This increase was not due to a direct modulation of calcium currents (20); rather, it was a consequence of the longer period of cellular depolarization. Our findings that relaxin increased the APD and therefore increased the calcium influx in atrial myocytes could explain the positive inotropic effects induced by relaxin in atrial preparations.

Evidence that endogenous relaxin promotes growth of the vagina and uterus during pregnancy in gilts.

Recently, it was demonstrated that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats and that administration of porcine relaxin promotes growth of the uterus in nonpregnant or early pregnant gilts. This study examined the effects of circulating relaxin on growth of both the vagina and uterus during the last two thirds of the 114-day gestation period in gilts. Furthermore, this study employed an in vitro immunohistochemical localization technique to determine whether the vagina and uterus in pigs have specific relaxin-binding sites. Three groups of pregnant gilts were used: sham-ovariectomized controls (group C; n = 8), ovariectomized progesterone-treated (group OP; n = 6), and ovariectomized progesterone- plus relaxin-treated (group OPR; n = 7). Gilts were either sham ovariectomized or ovariectomized on day 40 of gestation. Hormone replacement therapy with progesterone (group OP), progesterone plus relaxin (group OPR), or hormone vehicles (group C) began on day 38 (progesterone) or day 40 (relaxin) and continued until day 110. On day 110, the vagina and uterus were collected, and wet weight, dry weight, and percent hydration were determined. Small pieces (2-3 cm3) of the vagina and uterus from groups C and OP were frozen and cryosectioned for the immunohistochemical localization of relaxin-binding sites. Relaxin promoted growth of both the vagina and uterus. The wet weights of both the vagina and uterus in relaxin-deficient gilts (group OP) were lower (P < 0.05) than those in controls (group C), and relaxin replacement therapy (group OPR) restored the wet weights of both tissues to values that did not differ from those in controls. The mean dry weights and percent hydrations in the vagina and uterus did not differ among treatments. Immunohistochemical localization studies in the vagina and uterus demonstrated that specific and saturable binding of relaxin was localized in the same cell types of both tissues, namely epithelial cells (luminal in vagina, and both luminal and glandular in uterus), smooth muscle cells (both circular and longitudinal in vagina, and myometrial in uterus), and cells associated with blood vessels. In conclusion, this study provides evidence that circulating relaxin promotes growth of both the vagina and uterus during pregnancy in the pig. Furthermore, this study provides evidence that both the vagina and uterus contain specific and saturable relaxin-binding sites in epithelial cells, smooth muscle cells, and cells associated with blood vessels. We conclude that these cells probably initiate relaxin's effects on the vagina and uterus of the pregnant pig.

Identification of specific relaxin-binding cells in the cervix, mammary glands, nipples, small intestine, and skin of pregnant pigs.

We previously demonstrated that relaxin promotes growth and softening of the cervix and development of the mammary glands in the pregnant pig. An important aspect of understanding relaxin's mechanism of action in these tissues is to identify the specific cell type(s) that contains relaxin receptors, that is, to identify those cells that initiate relaxin's effects. The objective of the present study was to identify relaxin-binding cells in tissues known to respond to relaxin (cervix and mammary gland) as well as in tissues suspected of being responsive to relaxin (nipple, small intestine, and skin) in the pregnant pig. To accomplish that objective we developed an in vitro modification of an immunohistochemical technique recently developed for identification of relaxin-binding cells. Two groups of pregnant gilts were used: intact control (group C) and ovariectomized progesterone-treated (group OP). Group OP was ovariectomized on Day 40 of gestation (Day 40) and treated with progesterone (50 mg/2 ml corn oil i.m., twice daily) until Day 110 to maintain pregnancy. On Day 110, tissues from both groups were removed, cut into cubes (2-3 cm3), frozen in liquid nitrogen, and cryosectioned (8 microns). Specific cell types that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. The study demonstrates for the first time that relaxin binds with specificity to 1) blood vessels (cervix, mammary glands, nipples, small intestine); 2) smooth muscles in small intestine (circular, longitudinal, muscularis mucosa); and 3) skin from sites other than the mammary nipples (back, ear, thigh, leg). In addition, consistent with previous findings in the rat, prominent labeling was observed in epithelial cells in the cervix, mammary glands, and nipples; in smooth muscle cells in the cervix and mammary nipples; and in the skin of the nipples. There were no apparent differences in relaxin binding between group C and group OP. We conclude that the specific relaxin-binding cells in the cervix, mammary glands, nipples, small intestine, and skin of the pregnant pig probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues.

Effects of relaxin on lactational performance in ovariectomized gilts.

In gilts, mammary lobulo-alveolar growth begins on about Day 80 of gestation and continues progressively until term. Relaxin in concert with estrogen plays a major role in promoting this mammary gland growth. The present study was conducted to determine the importance for lactational performance of prepartum relaxin-dependent growth of the mammary glands in ovariectomized gilts given progesterone to maintain pregnancy. Twenty-four gilts were either sham ovariectomized or bilaterally ovariectomized and assigned to four treatment groups: sham-ovariectomized control, ovariectomized progesterone-treated, ovariectomized progesterone- and (starting at Day 80) relaxin-treated, and ovariectomized progesterone- and (starting at Day 100) relaxin-treated. Piglets were delivered by cesarian section, and gilts were given uniform colostrum-replete foster litters (born of untreated mothers) to nurse from Day 1 to Day 28 of lactation. Prepartum mammary development appeared by visual examination to be greatly reduced in relaxin-deficient gilts. Stimulus of the mammary nipples by the nursing piglets, however, appeared to overcome relaxin-dependent differences in mammary development among treatments. There was no effect of treatment on the time piglets spent at the udder, piglet mortality, piglet weight at Day 21 of lactation, milk composition, mammary cross-sectional area, or sow weight change during lactation. We conclude that gilts devoid of circulating luteal relaxin can display normal lactational performance when given colostrum-replete foster litters.