Relaxin: A pleiotropic hormone

Gen. Pharmac. Vol. 28, No. 1, pp. 13-22, 1997 Copyright @ 1997 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/97 $17.00 + .00 PII S0306-3623(96)00171-1 All rights reserved ELSEVIER

REVIEW

Relaxin: A Pleiotropic Hormone Daniele Bani DIPARTIMENTO DI ANATOMIA UMANA E ISTOLOGIA, SEZlONE DI ISTOLOGIA,V. LEG. P1ERACC1NI6, 1-50139 FIaENZE,ITALY

[TEL: (39"55) 414710; FAX (39"55) 4221948; E'MAIL: [email protected]] ABSTRACT. 1. Relaxin is a peptide hormone of about 6000 Da belonging to the insulin family. Like insulin, relaxin is composed by two disulfide-linked chains, termed the A and B chains, the B chain bearing the receptor interaction site. 2. Relaxin is produced primarily by the corpus luteum, in both pregnant and nonpregnant females. It attains the highest plasma levels during pregnancy. In this condition, relaxin is also produced by the decidua and placenta. In males, relaxin is synthesized in the prostate and released in the seminal fluid. An additional source of relaxin has recently been identified in the heart atria. 3. Relaxin has a broad range of biologic activities, some of which have been known for a long time. These latter ones include: (a) the induction of collagen remodeling and consequent softening of the tissues of the birth canal in view of delivery; (b) the inhibition of uterine contractile activity; (c) the stimulation of growth and differentiation of the mammary gland. 4. In more recent years, novel sites of relaxin action have been recognized. In particular, it has been shown that relaxin: (a) regulates growth and differentiation of breast cancer cells in culture; (b) promotes dilation of blood vessels in several organs and tissues, including the uterus, the mammary gland, the lung and the heart; (c} has a chronotropic action on the heart; (d) inhibits the release of histamine by mast cells, thus being able to counteract experimental allergic asthma; (d) depresses aggregation of platelets and their release by megakaryocytes; (e) influences the secretion of hormones by the pituitary gland; and (f) contributes to the regulation of fluid balance. 5. Concerning the mechanisms of action of relaxin, stimulation of nitric oxide generation, with consequent rise in intraceUular cyclic GMP levels, and stimulation of cyclic AMP production have been demonstrated to occur in the target cells and organs. 6. It may be expected that the next decade will provide answers about the utility of relaxin, in terms of insight into the actual physiologic functions of relaxin in the animal kingdom and especially in man, in view of possible therapeutic use of relaxin or relaxin-derived drugs in human disease, especially considering that human recombinant relaxin is now available for clinical experimentation. Copyright © 1997 Elsevier Science Inc. CEN PHARMAC28;1:13--22, 1997. KEY WORDS. Relaxin, nitric oxide, breast cancer, vasodilation, mast cells RELAXIN'S " C U R R I C U L U M

VITAE'"

Relaxin was first investigated in 1926, when Frederick Hisaw first reported on the relaxation of the interpubic ligament of female guinea pigs following injections of serum from pregnant guinea pigs or rabbits (Hisaw, 1926). Four years later, Hisaw and his group (Fevold et al., 1930) obtained a crude aqueous extract from sow corpora lutea with chemical characteristics of a peptide and which retained the property to elongate the interpubic ligament. This substance was identified as a new hormone and was given the name "Relaxin." Relaxin research spread slowly in the next 15-20 years, hindered by technical limitations in peptide isolation and lack of reliable bioassays. In 1945, and even more in early 1950s, there was a surge in relaxin research, when pioneering discovoies concerning the biologic effects of this hormone on the female reproductive apparatus were made. In fact, relaxin was found to promote the growth of the mammary gland (Hamolsky and Sparrow, 1945), to inhibit spontaneous contractile activity of the myotnetrium (Krantz et al., 1950), and to soften the cervix uteri (Graham and Dracy, 1953). Although Received 13 February 1996; accepted 11 March 1996.

in the above studies impure porcine relaxin was employed, these findings have been repeatedly confirmed in the following years, when purified relaxin preparations became available. Only 10 years later was a simple and specific quantitative bioassay for relaxin set up by Steinetz et al. (1960), using the measurement of pubic symphisis elongation in mice. Relaxin entered its later life from 1970s onward, along with the development of reliable methods for its isolation and purification (Sherwood and OByrne, 1974; Sherwood, 1979). The availability of purified relaxin in relatively large amounts allowed the identification of the amino-acid sequence of the peptide (Schwabe et al., 1976; James et al., 1977; Schwabe et al., 1977), and hence the determination of its structure and biochemical properties, as well as the setup of sensitive radioimmunoassays (Bryant, 1972; Sherwood et al., 1975; Sherwood and Crnekovic, 1979), thus overcoming the difficulties of measuring relaxin in blood, where its levels are too low for detection by the bioassay, and allowing identification of relaxin and determination of its plasma levels in a variety of species, including man (Schwabe et al., 1978). On these grounds, relaxin research gained great momentum in the following years. There have been many significant advances over this period, which have been exten-

14

D. Bani

sively reviewed by several researchers in the relaxin field (BryantGreenwood, 1982; Kemp and Niall, 1984; Schwabe and Btillesbach, 1988; 1990; Bani et al., 1991; Bryant-Greenwood, 1991; Weiss, 1991; Bryant-Greenwood and Schwabe, 1994; Sherwood, 1994; Bigazzi et al., 1995a). Significant achievements include: the isolation and characterization of relaxins from many animal species; the recognition of corpus luteum, decidua and placenta as the primary sources of relaxin; the elucidation of the role of relaxin in the physiology of female reproductive organs in pregnancy and out of pregnancy, including the mammary gland; the identification, sequencing and cloning of relaxin genes in different animal species, including man; the production of human relaxin by both chemical synthesis and recombinant technology; and the identification and characterization of relaxin binding sites in the target organs. Corroborated by these achievements, relaxin has become a respectable hormone of the female reproductive system, with an outstanding role in the maintenance of pregnancy, in parturition and in mammary gland development. These issues have been treated in detail in a recent overview by Sherwood (1994). In the last few years, evidence is accumulating that relaxin is more than just a hormone of reproduction, being able to act on cells, tissues and organs other than those of the female reproductive system. Indeed, relaxin is now emerging as a pleiotrophic hormone with multiple, often interlocked actions on several systems, such as the cardiovascular, the respiratory and the hemostatic systems. Relaxin appears to be involved in the regulation of functional adjustments of these systems which take place during pregnancy and, possibly, in the physiologic control of their function in nonpregnant females and even in males. In the final part of this review, I will concentrate on the more recent achievements in this field.

vary from species to species (reviewed in Sherwood, 1994). Recently, an additional source of relaxin has been identified in atrial cardiocytes (Taylor and Clark, 1994). In humans, the bulk of circulating relaxin is produced by the corpus luteum, although minor amounts of the hormone are also produced by the decidua and placenta (Bigazzi et al., 1980; Sakbun et al., 1990; Johnston et al., 1991). The maximum concentrations can be measured in the first trimester of normal pregnancy (900 pg/ml at week 10 of gestation), after which there is a decline until term (Bell et al., 1987). In nonpregnant women, relaxin is produced mainly by the corpus luteum, which is primarily responsible for the small but consistent rise in plasma relaxin (30-150 pg/ml) observed 9-10 days after the LH surge (Stewart et al., 1990). Besides the corpus luteum, the endometrial glands, the thecal cells of ovarian follicles and the mammary gland parenchymal cells have been identified as minor sources of relaxin (see Sherwood, 1994). Relaxin is also produced in the male. In men, the primary source of relaxin is the prostate (Ivell et al., 1989; Sokol et al., 1989; Hansell et al., 1991), from which the hormone is secreted mainly, and perhaps solely, in the seminal plasma (Winslow et al., 1992). In fact, no evidence has been provided that relaxin is also released in the blood. In humans, two molecular forms of relaxin with high sequence homologies, designated H1 and H2 relaxins, and encoded by two distinct genes, have been identified (Hudson et al., 1983, 1984). Of these, H2 relaxin is the main form and corresponds to circulating relaxin produced by the ovary. H1 relaxin seems to be only produced by decidua and trophoblast, together with H2 relaxin, and might have a paracrine function at the placental site (Bryant-Greenwood, 1991; Hansell et al., 1991).

STRUCTURE, BIOSYNTHESIS, AND TISSUE SOURCES OF RELAXIN

BIOLOGICAL EFFECTS OF RELAXIN E f f e c t of r e l a x i n o n connective tissue components

Along with identification of the primary structure of relaxins from different animal species, it appeared that relaxin should be viewed as a member of a larger family of related molecules, which also include insulin and insulinlike growth factors (Blundell and Humbel, 1980). Relaxin, like insulin, has a molecular weight of about 6000 Da and consists of two chains, termed the A and B chains. Both chains are approximately the same size, and are covalently linked by two interchain disulfide bonds with an intradisulfide link in the A chain (Schwabe et al., 1978). When comparing relaxins from different species, their sequences differ largely by about 30-60%. Nevertheless, in all species, location of disulfide bonds and/or cystine pairings are found in similar positions, thus suggesting that all relaxins have similar tertiary structure (Bryant-Greenwood and Schwabe, 1994; Schwabe and Biillesbach, 1994). The receptor interaction site is located in the B chain and involves two active arginine residues. On the other hand, the a chain seems to be necessary to determine the specific binding of relaxin to its own receptors and not to receptors for the other hormones of the same family (Schwabe and Biillesbach, 1994). Relaxin, like insulin, is synthesized in a precursor form, termed preprorelaxin, which is processed to the mature hormone by sequential proteolytic digestion of a signal peptide and of a connecting peptide between the two chains (Kemp and Niall, 1984). Studies in several mammalian species showed that relaxin is produced in highest levels by female reproductive organs during pregnancy. The primary sources are the corpus luteum, decidua and placenta, which account for the production of most if not all relaxin circulating in the peripheral blood. The individual contribution of these organs to the whole amount of relaxin released in blood may

Traditionally, the first biologic activity attributed to relaxin is the lengthening of the interpubic ligament and softening of the tissues of the birth canal (cervix and vagina), which has a net effect of facilitating the passage of the fetus at birth (reviewed by Sherwood, 1994). This effect is synergistic with that of ovarian steroids and is likely mediated through the induction by relaxin of collagen remodeling. However, the exact mechanism of action on collagen metabolism is still unclear, due to controversial reports. In fact, similar in vitro studies on human fibroblasts showed that relaxin promotes collagen secretion on one hand (Wiqvist et al., 1984), whereas it causes a decrease in collagen production and an increase in collagenase synthesis and collagen breakdown on the other hand (Unemori and Amento, 1990). Possibly, these conflicting results depend on different responsiveness to relaxin of fibroblasts coming from different anatomic sites. Anyhow, these findings raise the possibility that relaxin acts through an interplay between de novo synthesis and demolition of collagen. Relaxin may have an outstanding role at delivery by inducing the metabolic changes in the connective tissue of cervix and interpubic ligament needed for collagen remodeling. Indeed, in some species, such as pigs and rats, there is a prepartum relaxin surge, which is held responsible for successful dilation of the birth canal (Sherwood, 1994). Because there is no prelabor systemic relaxin surge in women, it has been suggested that local relaxin produced by the decidua and placenta and/or increased sensitivity of target cells to relaxin may be involved in the induction of the above changes (Bryant-Greenwood and Schwabe, 1994). The ability of relaxin to induce collagen remodeling may have a physiologic relevance not only in pregnancy but also in nonpreg-

Relaxin: A Pleiotropic Hormone

15

nant cycles. In fiact, relaxin produced by the theca interna of the ovarian follicle is regarded as one of the hormones regulating the local production of the collagenolytic enzymes which weaken the follicle wall before its rupture at ovulation (Bryant-Greenwood, 1982). The collagenolytic property of relaxin has also raised the interest of clinicians toward this hormone as a therapeutic tool for induction of cervical ripening (MacLennan et al., 1986a), both for treatment of scleroderma (Casten and Boucek, 1958), and for facilitating skin distension upon implantation of skin expanders (Kibblewhite et al., 1991 ). In turn, this has prompted pharmaceutical companies toward the production of human H2 relaxin for clinical use, by either recombinant technology or chemical synthesis. Besides collagen, relaxin can also influence molecules of the extracellular matrix of connective tissues. It has been reported that relaxin, in combination with estrogen, increases the content of glycosaminoglycans in rat uterus and cervix (Vasilenko and Mead, 1987). More recently, researchers from our group provided in vivo evidence for a prominent role of relaxin in promoting the synthesis of laminin by endometrial stromal cells of mice given relaxin systemically in combination with ovarian steroids (Bani e4 al., 1995a). Laminin is known to be expressed by mouse endometrial stromal cells after they have become decidual cells of pregnancy (Wever et al., 1986), and is thought to play a key role in allowing trophoblast adhesion to decidual components and, hence, blastocyst implantation (Glasser, 1990; Michie and Head, 1994). This suggests that relaxin may be regarded as a physiologic agent favoring embryo implantation into the uterus. If the same also holds true for humans, the possibility is raised for the clinical use of relaxin in the preparation of women undergoing assisted in vivo and in vitro fertilization. E f f e c t s of r e l a x i n o n u t e r i n e m o t i l i t y

The inhibition of myometrial contractility is a classic effect of relaxin. Relaxin has been repeatedly found to reduce the frequency and/or amplitude of spontaneous or electrically driven uterine contractions in several animal species, especially guinea pigs, rats and pigs (reviewed in Sherwood, 1994). The mechanism of action of relaxin on myometrial smooth muscle cells is not completely understood at present. It has been shown that relaxin increases intracellular cAMP in a dose-dependent fashion (Sanborn et al., 1980; Hsu et al., 1985), and it seems feasible that this eventually results in a decrease in myosin light-chain kinase phosphorylation (Sanborn, 1986) and hence relaxation of the cell contractile apparatus. A further mechanism seems to involve the reduction in the rise in cytosolic Ca 2+ levels, required for the formation of the Ca2+-calmodulin complex--needed for myosin light-chain kinase activation (Anwer et al., 1989). Surprisingly, both porcine and human H1 and H2 relaxins have slight or no inhibitory effect on the contraction of human myometrium in vitro (MacLennan et al., 1986b; MacLennan and Grant, 1991; Petersen etal., 1991; Bryant-Greenwood and Schwabe, 1994). These findings account for the failure of relaxin when used as a drug for prevention of threatened premature labor or for inhibition of labor at term (Kelly and Posse, 1956; Decker et al., 1958), and provide support for the view that relaxin's putative quiescent effect on the myometrium is of little physiologic or clinical relevance in the human. E f f e c t s of r e l a x i n o n t h e m a m m a r y

gland

The discovery that relaxin, in synergism with ovarian steroids, has a growth-promoting effect on the mammary gland was made by Hamolsky and Sparrow (1945). Nevertheless, interest in the mammary gland as a target for relaxin was sporadic until the early 1980s, when

conclusive experiments aimed at evaluating the mammotrophic properties of purified relaxin preparations were carried out by several research groups, including ours (reviewed in Bani et al., 1991; Bigazzi et al., 1995a). In particular, our studies in ovariectomized mice given purified porcine relaxin provided light and electron-microscopic evidence that the hormone promotes growth and differentiation of the parenchymal components of the mammary gland (e.g., epithelial and myoepithelial cells), which result in extensive branching and elongation of the duct system and in the appearance of a true lobule-alveolar organization of the gland (Bani and Bigazzi, 1984; Bani et al., 1985, 1986). This effect could only be obtained upon estrogen priming, which is needed to induce relaxin receptors in the mammary tissues. Relaxin was also shown to have a positive effect on the stromal components of the mammary gland, promoting growth and differentiation of fibroblasts and adypocytes, loosening of the collagen framework and imbibition of the amorphous ground substance (Bani and Bigazzi, 1984; Bianchi et al., 1986; Bani Sacchi et al., 1987). Moreover, relaxin is also required to allow a normal development of the nipple (Kuenzi and Sherwood, 1992; Kuenzi et al., 1995). Despite its mammotrophic action, relaxin has never been found to be lactogenic, with prolactin being needed to induce milk secretion by the mammary gland (Smith, 1954). The need for endogenous relaxin to induce normal mammary development has been expertly clarified by researchers of Dr. Sherwood's team, who caused disruption of the entire mammary apparatus by administration of neutralizing antirelaxin antibodies throughout the second half of pregnancy in rats (Hwang et al., 1991; Kuenzi and Sherwood, 1992). The mammotrophic property of relaxin has also been confirmed by studying its effects on the pigeon crop sac, which is functionally analogous to the mammary gland in its responsiveness to mammotrophic hormones and had been previously used for prolactin bioassay (Nicoll, 1967). As it does on the mammary gland, relaxin markedly stimulates growth and differentiation of crop sac epithelial cells (Bigazzi et al., 1988; Bani et al., 1990). It is conceivable that relaxin has a physiologic role in mammary gland development in the human, even if no definitive evidence has been provided. Interestingly, immunoreactive relaxin has been found in normal and neoplastic human mammary tissues, suggesting the possibility of a paracrine control of mammary growth by relaxin (Mazoujian and Bryant-Greenwood, 1990). Indeed, it seems feasible that relaxin is involved in the regulation of growth of benign and malignant mammary gland neoplasms. High relaxin concentrations have been found in the cyst fluid of patients with mammary dysplasia (Nardi et al., 1983) and in the plasma of a woman with a giant fibroadenoma of the breast (M. Bigazzi, unpublished observations). With the aim to provide further insight into the putative influence of relaxin on the growth of mammary tumors, we focused our interest on the effects of this hormone on human breast adenocarcinoma cells of the MCF-7 line. In a series of experiments, we could demonstrate that relaxin is a modulator of MCF-7 breast cancer cell growth. When added to the culture medium of the MCF-7 cells for short exposure times (4 days) relaxin had a biphasic effect, stimulating cell proliferation at low, nanomolar concentrations and inhibiting it at high, micromolar concentrations (Bigazzi et al., 1992). Over longer time periods (7 days), relaxin had a marked growthinhibitory effect on MCF-7 cells at any concentration assayed, and concurrently promoted cell differentiation and expression of E-cadherin, a cell--cell adhesion molecule whose expression on cancer cells is inversely related to their malignancy and spreading ability (Bani Sacchi eta/., 1994). The positive effect of relaxin on MCF-7 cell differentiation was even enhanced when these cells were cocultured together with myoepithelial cells (Bani et al., 1994). Interestingly, myoepithelial cells are known to envelope the mammary ducts from

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F I G U R E 1. Effect of relaxin on MCF-7 breast adenocarcinoma cells after a 96-h incubation. Along with the increase in inducible nitric oxide synthase activity (black circles), cell proliferation (white circles) increases and then decreases under the basal value. Significance of differences: *p
F I G U R E 2. Effect of relaxin on the coronary flow in isolated and perfused guinea pig hearts. Perfusion with relaxin (5 x 10 9 M) causes a striking increase in the coronary flow, an effect even more potent than that of acetylcholine (10 -s M), or of sodium nitroprusside (SNP; 10 -7 M). ( I ) RLX; (O) A C H ; (A) SNP. Significance of differences: *pO.O1.

which mammary carcinoma arises and their presence around neoplastic buds in fairly large numbers has been associated with a more favorable prognosis of the disease (Ahmed, 1974). Concerning the mechanism of action of relaxin on MCF-7 cells, it is possible that a cAMP-dependent mechanism mediates the mitogenic effect of the peptide found at nanomolar concentrations for short exposure times (Bigazzi et al., 1992). O n the other hand, the growth-inhibitory and differentiation-promoting actions of relaxin appear to be mediated through the stimulation of nitric oxide synthases, especially the inducible isoform (Fig. 1), which leads to elevated, long-lasting generation of endogenous nitric oxide. Of note, nitric oxide has been shown to have a powerful cytostatic action on tumor cells by inhibiting DNA synthesis and causing oxidative injury, as well as to promote differentiation and to hinder the metastatic potential of cancer cells (see Bani et al., 1995b). In the MCF-7 cells, the relaxininduced elevation of nitric oxide is paralleled by a rise of intracellular cGMP (Bani et al., 1995b), which is the mediator of the response of the cells to nitric oxide (lgnarro, 1991). Taken together, the above findings open the possibility that relaxin, at appropriate concentrations and times of exposure, may also reveal a beneficial effect against mammary cancer in vivo. If this is the case, relaxin could be included among the drugs for hormonal therapy of breast carcinoma.

mesocaecum (Bigazzi et al., 1986). The vasodilatory action of relaxin agrees with other reports that this hormone decreases blood pressure and blunts the response of mesenteric vessels to vasoconstrictors in spontaneously hypertensive rats (St. Louis and Massicotte, 1985; Massicotte et al., 1989), and also fits well with pioneer clinical studies of Casten et al. (1958, 1960), who found dramatic, albeit transient, amelioration of peripheral vascular disease and Raynaud's syndrome following intramuscular injection of impure porcine relaxin. Recently, we gained a deeper knowledge of the vasodilatory effect of relaxin and its mechanisms of action. In a first series of experiments, we tested the ability of relaxin to influence the vasculature of the heart by assaying the effects of the hormone on the coronary flow of guinea pig and rat hearts isolated and perfused in a Langendorff apparatus. Relaxin caused a prompt, concentration-dependent increase in the coronary flow, an effect even more potent than that of other well-known vasodilatory agents such as acetylcholine or sodium nitroprusside (Fig. 2). The relaxin-induced increase in the coronary flow was paralleled by the increase in the endogenous production of nitric oxide, a powerful vasodilatory agent (Ignarro et al., 1987; Pahner et al., 1987), thus indicating that relaxin acts on the coronary vessels through a nitric oxide-driven mechanism (Bani Sacchi et al., 1995). From these achievements, further experiments have been carried out to ascertain which are the target cells for relaxin at the vascular level. Recently, we could demonstrate that bovine vascular smooth muscle cells in vitro respond to relaxin by increasing the production of nitric oxide and the intracellular levels of cGMP (Fig. 3), which is the mediator of the cell response to nitric oxide (Ignarro, 1991). This is accompanied by a concurrent attenuation of the rise of cytosolic free Ca 2+ induced by vasoconstrictors, such as thrombin or cal-

Effects of relaxin

on the cardiovascular system

Substantial evidence has been provided that relaxin causes blood vessel dilation in several organs and tissues, such as rat and mouse uterine endometrium and myometrium (Vasilenko et al., 1986; Bani et al., 1995a), mouse mammary gland (Bani and Bigazzi, 1984; Bani et al., 1988), pigeon crop sac mucosa (Bigazzi et al., 1988) and rat

Relaxin: A Pleiotropic Hormone

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cium ionophore A23187, and by morphologic changes in cell shape and in cytoskeletal contractile microfilaments consistent with cell relaxation (manuscript in preparation). Our findings on bovine vascular smooth muscle cells are in close keeping with those of Anwer et al. (1989) on rat uterine smooth muscle cells, in which relaxin was found to cause a reduction of the rise in cytosolic Ca > levels that is required for cell contraction. It is worth noting that, besides smooth muscle cells from blood vessels and myometrium, relaxin has also been shown to inhibit spontaneous contraction of the smooth muscle wall of the ileum (Del-Angel-Meza et al., 1991). Taken together, the above findings suggest that relaxin acts as a relaxing factor on smooth muscle cells from different organs, including the vascular wall, the myometrium, and the intestinal wall. A possible common mechanism of action can also be hypothesized, which involves primarily a stimulation of the endogenous production of nitric oxide and, consequently, an increase of intracellular cGMP, a depression of the rise of cytosolic Ca 2+ in response to constrictor stimuli and, finally, an inactivation of the cell contractile machinery leading to cell relaxation. In the search for target cells for the vasodilatory action of relaxin, we also focused our attention on mast cells, which are known to be involved in the regulation of vascular tone and microvascular permeability through the release of vasoactive mediators, such as histamine. Interestingly, our previous morphol0gic studies on the mammary gland revealed that the relaxin-induced vasodilation was not accompanied by a concurrent release of histamine-storing granules by mast cells, thus suggesting that the action of relaxin is independent of the release of vasoactive mediators by mast cells (Bani et al., 1988). This prompted us to assay the effect of relaxin on mast cells, using rat and guinea pig serosal mast cells in vitro and in vivo. Our results show that relaxin causes a dose-dependent inhibition of granule exocytosis and histamine release by mast cells challenged with either immunologic or nonimmunologic stimuli. O n the other hand, relaxin powerfully stimulates endogenous production of nitric oxide by these cells and attenuates the rise of cytosolic Ca > concentration induced by secretagogues (Masini et al., 1994). The re-

laxin-induced activation of the nitric oxide pathway in mast cells provides a valid explanation for its inhibitory action on histamine release, with endogenous nitric oxide being an autocrine inhibitor of mast cell function (Salvemini et al., 1991). It is likely that increased nitric oxide generation by perivascular mast cells may also contribute to the vasodilatory action of relaxin. At variance with other vasodilatory agents, such as histamine, which also promote angiogenesis (see Norrby and Wooley, 1993), relaxin seems not to be angiogenic. In fact, using the quantitative mesenteric-window assay in rats (Norrby, 1992), no angiogenic effect could be found upon systemic administration of relaxin (manuscript in preparation). This is not surprising if it is taken into account that relaxin stimulates nitric oxide production by ceils of the vascular wall, and that nitric oxide has been shown to inhibit proliferation of vascular smooth muscle cells (Garg and Hassid, 1989). In recent years, evidence is accumulating that the heart is a target for relaxin. In fact, high affinity relaxin receptors have been identified in the heart atria of rats (Osheroff et al., 1992; Osheroff and Ho, 1993), and several reports agree that relaxin exerts positive chronotropic effects on rat and guinea pig hearts (Parry et al., 1990; Kakouris et al., 1992; Ward et al., 1992, Bani Sacchi et al., 1995), whereas a positive inotropic effect is still matter of debate. Based on these findings, Kakouris et al. (1992) suggested that relaxin may be responsible for the elevation of cardiac output observed during human pregnancy, when plasma relaxin levels also become elevated (Bell et al., 1987). Recently, we found that relaxin, administered systemically to rats, causes a depletion of immunoreactive atrial natriuretic peptide (ANP) by atrial cardiocytes (manuscript in preparation). Therefore, a further physiologic action of relaxin on the heart might be the regulation of ANP release and, hence, of body fluid balance. In a recent report, Taylor and Clark (1994) provided evidence for the local production and release of relaxin by rat atrial cardiocytes, and suggested that cardiac relaxin may act as an autocrine or paracrine physiologic regulator of cardiac function and/or growth and development. Additional putative functions of cardiac relaxin will be discussed in a later section. Effects of relaxin on hemostasis

Relaxin has been shown to exert its vasodilatory action through the stimulation of nitric oxide production in its target cells. Of note, nitric oxide is a potent vasodilator as well as a potent inhibitor of platelet adhesion and aggregation (Radomski et al., 1987). O n these grounds, we were prompted to investigate the possibility of a role for relaxin in platelet function. The results obtained show that preincubation of isolated human and rabbit platelets with relaxin before stimulation with proaggregants causes a concentration-dependent inhibition of platelet aggregation (Fig. 4). Once again, this effect of relaxin on platelets appears to be mediated through an elevation of the endogenous production of nitric oxide, with concurrent increase in intracellular cGMP and attenuation of the rise of cytosolic Ca 2+ evoked by proaggregants (Bani et al., 1995c). In addition, further experiments have provided evidence that relaxin interferes with the release of platelets by megakaryocytes. In fact, systemic administration of relaxin to rats caused a reduction of circulating platelets and induced morphologic changes of spleen megakaryocytes consistent with a depression of thrombocytopoiesis (Bani et al., 1995d). These findings speak in favor of an antithrombotic activity of relaxin. This may have a physiologic function in counteracting the hypercoagulable state that occurs during normal pregnancy (Bonnar, 1981). Going a step further, it is possible that deficient produc-

18

D. Bani R e l a x i n as a growth factor

tion and/or receptor sensitivity to relaxin may contribute to the onset of disorders of pregnancy such as pre-eclampsia and intravascular disseminated coagulation.

As a member of the insulin family, relaxin shares trophic and growth-promoting properties with other cognate molecules, such as insulin and insulinlike growth factors. Several lines of evidence indicate that relaxin stimulates the growth of target organs and tissues in various animal species, alone or in combination with other growth effectors, such as ovarian steroids. Relaxin has been shown to cause the growth of uterine cervical epithelimn (Datta et al., 1968), to increase weight, protein and DNA content in the uterus (Vasilenko et al., 1980; Bylander et al., 1987; Hall et al., 1990, 1992); to promote hyperplasia of endometrial stromal cells (Bani et al., 1995a); to stimulate the growth of epithelial, myoepithelial, stromal and adipose cells of the mammary gland (see Bani et al., 1991; Bigazzi et al., 1995a); to cause hyperplasia of pigeon crop sac epithelium (Bigazzi et al., 1988); and to stimulate proliferation of follicular granulosa cells in vitro (Zhang and Bagnell, 1993). Indeed, relaxin is a peculiar growth factor, since it can exhibit a biphasic action, depending on both peptide concentrations and times of exposure. As shown by us on MCF-7 breast cancer cells (Bigazzi et al., 1992) and by other investigators on mammary gland fibroblasts (Sheffield and Anderson, 1984), incubation of cells with low relaxin concentrations increased, whereas high relaxin concentrations suppressed 3H-thymidine incorporation into DNA. Moreover, in MCF-7 cells, we found that relaxin at low concentration is mitogenic in short exposure times, and antimitogenic in longer exposure times (Bani Sacchi et al., 1994). This behavior of relaxin is likely related to the actual state of differentiation of target cells.

Effects of relaxin on the respiratory system

E f f e c t s of r e l a x i n o n t h e m a l e reproductive system

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TR 40 mU/ml RLX 30 ng/ml

RLX 10 ng/ml Control FIGURE 4. Effect of relaxin on human washed platelets shown by aggregometric recording. A marked, concentration-dependent depression of platelet aggregation evoked by thrombin (TR) can be observed upon 10-min preincubation with relaxin.

The reader will recall that relaxin has been demonstrated to inhibit granule exocytosis and histamine release by mast cells challenged with immunologic stimuli (Masini et al., 1994) and to inhibit platelet activation (Bani et al., 1995c) through a nitric oxide-mediated mechanism. Because IgE-dependent release of inflammatory mediators by mast cells and platelets is known to play a major role in the pathogenesis of allergic asthma, the possibility that relaxin may counteract antigen-induced asthma intrigued us. To verify this hypothesis, guinea pigs were sensitized with ovalbmnin and challenged with the aerosolized antigen in a device which allowed evaluation of the respiratory activity. Some animals received relaxin systemically for 4 days before antigen challenge. In these animals, relaxin was found to reduce markedly cough severity and bronchospasm and to prevent dyspnea as compared with their control counterparts not given relaxin. Histologic examination of the lungs showed that relaxin reduced bronchoconstriction, mast cell degranulation and accumulation of inflammatory leukocytes, especially eosinophils (Bani et al., in press). The antiasthmatic effect of relaxin may explain previous clinical reports of an improvement of asthma during pregnancy (White et al., 1989), when the circulating levels of relaxin are elevated (Bell et al., 1987). In our study, relaxin was also found to promote dilation of alveolar blood capillaries and attenuation of the air-blood barrier, thus suggesting that this hormone increases lung perfusion and gas exchanges. Besides contributing to the amelioration of asthmatic symptoms, this property of relaxin may provide a possible interpretation for a physiologic role of cardiac relaxin produced and secreted by atrial cardiocytes (Taylor and Clark, 1994). It is possible that, once secreted by right atrial cardiocytes, relaxin may reach the pulmonary circulation and act as a regulatory agent of lung perfusion.

The physiologic role of relaxin in seminal plasma is not clearly defined. Addition of neutralizing antirelaxin antibodies to seminal plasma decreases the motility of human sperm (Sarosi et al., 1983; Sokol et al., 1988). Moreover, there is limited evidence that relaxin improves motility of spermatozoa, but this improvement only occurs on hypomotile spermatozoa and within a limited range of dosage of the hormone (see Weiss, 1989, 1995). Claims that relaxin may contribute to the normal penetration of cervical mucus by sperm are still controversial. Recently, we found that local injection of pure porcine relaxin in the cavernous body of human volunteers can increase blood flow in the deep penile artery, as evaluated by an echo Doppler apparatus (Bigazzi et al., 1995b). Judged on the data available at present, relaxin's usefulness as a therapeutic agent in either assisted reproduction or male infertility is a tempting perspective, but remains an open question that requires additional study. E f f e c t s of r e l a x i n on the brain and the pituitary gland

Relaxin binding sites have been identified in several regions of the brain, especially those known to be involved in the control of systemic blood pressure, fluid balance and secretion of hypothalamic hormones (Osheroff et al., 1990; Osheroff and Phillips, 1991; Osheroff and Ho, 1993). The actual physiologic actions of endogenous relaxin on the central nervous system remain to be established. However, investigations into the effects of exogenous relaxin on posterior pituitary secretion indicate that this hormone stimulates vasopressin secretion (Parry et al., 1990; Parry and Summerlee, 1991) and triggers thirst, thus increasing water intake (Robertson and Summerlee, 1991 ). These findings led to the suggestion that relaxin may have a role in controlling fluid volume during pregnancy

Relaxin: A Pleiotropic Hormone (Summerlee et al., 1995). On the other hand, a role of relaxin in influencing the release of oxytocin is controversial, both inhibition and potentiation of oxytocin secretion having been observed after relaxin administration (see Sherwood, 1994). Relaxin can also affect the release of hormones from the anterior pituitary. Several reports agree that the hormone promotes prolactin secretion, both in vitro and in vivo (Bethea et al., 1989; Sortino et al. , 1989), and there is limited evidence that relaxin stimulates growth hormone secretion (Bethea e t a / . , 1989) and inhibits luteinizing hormone secretion (Summerlee et al., 1991). Effects of relaxin on fluid balance

It is known that pregnancy is accompanied by an expansion of maternal extracellular fluid volume accomplished by sodium and water retention. In humans, as in other species, water retention predominates, so that the total body osmolality falls (Lindheimer et al., 1989). It is likely that relaxin plays a role in the regulation of the adjustments of fluid balance that occur during pregnancy. Indeed, relaxin receptors have been identified in areas of the brain known to be involved in the control of body fluid homeostasis (Osheroff and Phillips, 1991). Moreover, chronic infusion of relaxin has been shown to cause water retention and to decrease plasma osmolality in rats (Weisinger et al., 1993). Finally, our own findings on rat hearts show that relaxin promotes the release of ANP, which has important actions on body fluid homeostasis (Brenner et al., 1990). Of note, ANP attains high levels in plasma during pregnancy (Hirai et al., 1988), coincidentally with the elevation in circulating relaxin (Bell et al., 1987). CONCLUSIONS AND PERSPECTIVES Seventy years have elapsed since the discovery of relaxin. For many years since the first report by Hisaw (1926), relaxin has been regarded mainly as a "nice-to-have" hormone for which a real need could not be demonstrated. From the late 1970s onwards, along with a resurgence of the interest in relaxin research, it has become clear that this hormone plays a major role in female reproductive physiology. In more recent years, experimental evidence has been accumulating that relaxin is a pleiotropic hormone, with a broad range of biologic activities on various organs and apparatuses in pregnant and nonpregnant females and in males, in physiologic and pathologic conditions. These achievements offer tempting perspectives for future research on relaxin, likely directed toward verifying the actual importance of relaxin in human physiology, as well as the possible therapeutic applications for this hormone. Judging from the findings of our more recent studies on the effects of relaxin on coronary vessels, MCF-7 breast cancer ceils, mast cells, platelets and vascular smooth muscle cells (Masini et al., 1994; Bani Sacchi et al., 1995; Bani et al., 1995b, 1995c), the hypothesis can be put forward that relaxin can evoke the response of its different targets through a common mechanism of action; that is, the stimulation of endogenous production of nitric oxide and the consequent elevation of intracellular cGMP, which is the mediator of the biologic action of nitric oxide (Ignarro, 1991). According to this view, relaxin can be regarded as a physiologic stimulant of nitric oxide production, putatively capable of influencing many biologic processes involving nitric oxide. In this context, it is of particular interest that relaxin has been shown to have potent vasodilatory, antihypertensive and antithrombotic properties (St. Louis and Massicotte, 1985; Massicotte et al., 1989; Bani Sacchi et al., 1995; Bani et al., 1995b), which can be recognized as classicul properties of nitric ox-

19 ide (Ignarro eta/., 1987; Palmer eta/., 1987; Radomski et al., 1987). On these grounds, it is conceivable that relaxin can play an important role in protection against ischemic cardiac diseases in women, given that the incidence of coronary heart disease is very low among women during fertile life and increases markedly after menopause (Kannel and Abbott, 1987; Manson et al., 1992), along with the suppression of ovarian activity and, hence, of relaxin secretion. Going a step further, the possibility arises for the future use of relaxin or relaxin-derived drugs for the prevention and treatment of ischemic heart disease, hypertension and thromboembolic disorders. One of the major problems that relaxin investigators have dealt with is the setting up of easy, reliable and sensitive methods for relaxin bioassay. In fact, the commonly used guinea pig and mouse interpubic ligament bioassays, and mouse and rat uterine contractility bioassays, which have given indispensable data concerning the biologic activity of relaxin for more than 30 years, are neither precise nor sensitive, and they are expensive. A step forward has recently been made with the description of in vitro relaxin bioassays that are based on the stimulation of cAMP in established primary cultures of either human endometrial cells (Fei et al., 1990) or monkey uterine cells (Kramer et al., 1990), and that are sufficiently precise, specific, and at least 100-fold more sensitive than the in vivo bioassays, being able to detect relaxin concentrations of about 1 ng/ml. Nevertheless, even these in vitro relaxin bioassays have important limitations. The responsiveness to relaxin varied among different cell clones, as well as among different culture passages of the same clone. A transformed cell line that is responsive to relaxin, stable and widely available indefinitelywould offer substantial advantages over these cell culture bioassays. In our opinion, the human breast adenocarcinoma cell line MCF-7 can fulfill all the above qualifications. In fact, the MCF-7 cells have been shown to respond to relaxin, at concentrations ranging from 10-9 M (6 ng/ml) to 10-6 M (6 Izg/ml), by increasing intracellular cGMP in a dose-dependent fashion (Bani et al., 1995b). Therefore, the measurement of intracellular cGMP in MCF-7 cells, by either radioimmunoassay or ELISA, can be proposed as a valid in vitro bioassay for relaxin. It would have the same characteristics of previous in vitro assays in terms of precision, sensitivity and specificity, with the additional advantage of a better standardization of results, because it is based on a stable, widely distributed and available tumor cell line. Another fundamental question to be answered concerns the chemistry and physiology of relaxin receptors, in order to provide a better understanding of the mechanisms of action of relaxin through structure-function analysis of receptor action and signal transduction. Since the development of labeled relaxin preparations (Btillesbach and Schwabe, 1990; Osheroff et al., 1990), high-affinity binding sites or true specific relaxin receptors have been identified in the main target organs, and even in organs and tissues not formerly considered targets for relaxin. Efforts to isolate and characterize relaxin receptors are in their early stages (Biillesbach et al., 1995). However, substantial evidence has been provided that relaxin acts on target cells through at least two distinct pathways: the stimulation of cAMP production (Braddon, 1978; Sanborn et al., 1980; Hsu et al., 1985; Cronin et al., 1987; Chen et al., 1988; Fei et al., 1990; Kramer et al., 1990; Bigazzi et al., 1992), and the stimulation of nitric oxide production and consequent increase in cGMP (Masini et al., 1994; Bani Sacchi et al., 1995; Bani et al., 1995b, 1995c). Likely, the forthcoming research may help provide a deeper understanding of the properties of the relaxin receptor---or receptors--thus discovering the missing link; that is, the mechanisms that transduce the signal operated by relaxin on its targets and which regulate AMP cy-

20 clase and nitric oxide synthase activities and, possibly, additional intracellular metabolic pathways. In conclusion, it is this reviewer's feeling that relaxin research is about to gain great momentum, based on the many tempting perspectives which have arisen from the most recent achievements in this field. In particular, it may be expected that the next decade will provide answers about the utility of relaxin in terms of insight into the understanding of its actual physiologic functions in the animal kingdom and especially in man, in view of possible therapeutic use of relaxin or relaxin-derived drugs in human disease.

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