Relaxin causes changes of the liver. In vivo studies in rats.

During pregnancy, the liver undergoes metabolic adjustments directed to fulfil the needs of the mother and the growing fetus. This study was designed to verify whether relaxin, a hormone related to pregnancy, may induce histochemical and ultrastructural modifications of hepatocytes which can be related to metabolic changes. Estrogen-primed female rats were treated with relaxin (10 microg in repository vehicle) for 18 h. Additional male rats were treated with relaxin (10 microg/day in PBS) for 4 days. Appropriate vehicle-treated rats were used as controls. After fasting, the rats were killed and liver fragments were processed for light and electron microscopy and for computer-assisted morphometry of PAS-positive glycogen deposits and acid phosphatase-reactive organelles. In both sexes, the relaxin-treated rats underwent a significant decrease in the amount of glycogen in the hepatocytes as compared with the controls. These changes were accompanied by an increase in smooth endoplasmic reticulum, endocytosis vesicles and lysosomes. These findings show that relaxin promotes glycogen depletion and induces morphological changes of hepatocytes which are consistent with functional activation. It is suggested that relaxin might play an important role in hepatic metabolic adjustments occurring during pregnancy.

Relaxin up-regulates the nitric oxide biosynthetic pathway in the mouse uterus: involvement in the inhibition of myometrial contractility.

The uterus is a site of nitric oxide (NO) production and expresses NO synthases (NOS), which are up-regulated during pregnancy. NO induces uterine quiescence, which is deemed necessary for the maintenance of pregnancy. Relaxin is known to promote uterine quiescence. Relaxin has also been shown to stimulate NO production in several targets. In this study we investigated the effects of relaxin on the NO biosynthetic pathway of the mouse uterus. Estrogenized mice were treated with relaxin (2 microg) for 18 h, and the uterine horns were used for determination of immunoreactive endothelial-type NOS and inducible NOS. Moreover, uterine strips from estrogenized mice were placed in an organ bath, and the effect of relaxin on K+-induced contracture was evaluated in the presence or absence of the NOS inhibitor nitro-L-arginine. Relaxin increases the expression of endothelial-type NOS in surface epithelium, glands, endometrial stromal cells, and myometrium, leaving inducible NOS expression unaffected. Moreover, relaxin inhibits myometrial contractility, and this effect is blunted by nitro-L-arginine, thus indicating that the L-arginine-NO pathway is involved in the relaxant action of relaxin on the myometrium. Because relaxin is elevated during pregnancy, it is suggested that relaxin has a physiological role in the up-regulation of uterine NO biosynthesis during pregnancy.

Relaxin favors the development of activated human T cells into Th1-like effectors.

Differentiation of naive CD4+ helper T (Th) cells into Th1 or Th2 effectors, as characterized by their opposite pattern of cytokine production, can be influenced by several factors, including hormones. In this study, we demonstrate that porcine relaxin, at concentrations ranging from 10(-10) to 10(-6) M, favors the in vitro development of human antigen-specific T cells into Th1-like effectors and enhances both IFN-gamma mRNA expression and IFN-gamma production by established human T cell clones. The promoting effect of relaxin on the development of IFN-gamma-producing cells was not due to a relaxin-induced release of IL-12 and/or IFN-alpha by antigen-presenting cells. These results suggest that relaxin may contribute to the regulation of the immune homeostasis during pregnancy and may also play some role in counteracting Th2-dominated disorders.

Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart.

Myocardial injury caused by ischemia and reperfusion comes from multiple pathogenic events, including endothelial damage, neutrophil extravasation into tissue, platelet and mast cell activation, and peroxidation of cell membrane lipids, which are followed by myocardial cell alterations resulting eventually in cell necrosis. The current study was designed to test the possible cardioprotective effect of the hormone relaxin, which has been found to cause coronary vessel dilation and to inhibit platelet and mast cell activation. Ischemia (for 30 minutes) was induced in rat hearts in vivo by ligature of the left anterior descending coronary artery; reperfusion (for 60 minutes or less if the rats died before this predetermined time) was induced by removal of the ligature. Relaxin (100 ng) was given intravenously 30 minutes before ischemia. The results obtained showed that relaxin strongly reduces 1) the extension of the myocardial areas affected by ischemia-reperfusion-induced damage, 2) ventricular arrhythmias, 3) mortality, 4) myocardial neutrophil number, 5) myeloperoxidase activity, a marker of neutrophil accumulation, 6) production of malonyldialdehyde, an end product of lipid peroxidation, 7) mast cell granule release, 8) calcium overload, and 9) morphological signs of myocardial cell injury. This study shows that relaxin can be regarded as an agent with a marked cardioprotective action against ischemia-reperfusion-induced myocardial injury.

Relaxin counteracts myocardial damage induced by ischemia-reperfusion in isolated guinea pig hearts: evidence for an involvement of nitric oxide.

Relaxin was previously shown to cause coronary vasodilation and to inhibit mast cell activation through a stimulation of endogenous nitric oxide production. This suggests that relaxin may have beneficial effects on ischemia-reperfusion-induced myocardial injury, which is triggered by endothelial damage and impaired nitric oxide generation. In this study, we tested the effect of relaxin on isolated and perfused guinea pig hearts subjected to ischemia and reperfusion. Ischemia was induced by ligature of the left anterior descending coronary artery; removal of the ligature induced reperfusion. Relaxin, at the concentration of 30 ng/ml of perfusion fluid, causes: a significant increase in coronary flow and in nitric oxide generation; a significant decrease in malonyldialdehyde production and in calcium overload, both markers of myocardial injury; an inhibition of mast cell granule exocytosis and histamine release, which are known to contribute to myocardial damage; a reduction of ultrastructural abnormalities of myocardial cells; an improvement of heart contractility. The beneficial effects of relaxin were blunted by the NO synthase inhibitor L-NMMA. The current study provides first experimental evidence that relaxin has a powerful protective effect on the heart undergoing ischemia and reperfusion acting through a nitric oxide-driven mechanism.

Relaxin counteracts asthma-like reaction induced by inhaled antigen in sensitized guinea pigs.

In previous studies, the peptide hormone relaxin (RLX) was found to inhibit mast cell secretion and platelet activation. It has been established that the release of mediators from these cells plays a central pathogenic role in allergic asthma. This prompted us to ascertain whether RLX may counteract the respiratory and histopathological abnormalities of the asthma-like reaction to inhaled antigen in sensitized guinea pigs. Guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received RLX (30 microg/kg BW, twice daily for 4 days) before antigen challenge. Other animals received inactivated RLX in place of authentic RLX. Respiratory abnormalities, such as cough and dyspnea, were analyzed as were light and electron microscopic features of lung specimens. RLX was shown to reduce the severity of respiratory abnormalities, as well as histological alterations, mast cell degranulation, and leukocyte infiltration in sensitized guinea pigs exposed to ovalbumin aerosol. RLX was also found to promote dilation of alveolar blood capillaries and to reduce the thickness of the air-blood barrier. This study provides evidence for an antiasthmatic property of RLX and raises the possibility of new therapeutic strategies for allergic asthma in humans.

Relaxin activates the L-arginine-nitric oxide pathway in human breast cancer cells.

Recently, we demonstrated that relaxin (RLX), a peptide hormone of ovarian origin, inhibits growth and promotes differentiation of MCF-7 breast adenocarcinoma cells. We also showed that RLX stimulates the production of nitric oxide (NO) in several cell types. NO has been reported to have antitumor activity by inhibiting proliferation, promoting differentiation, and reducing the metastatic spread of some tumor cell types. In this study, we aimed at evaluating whether RLX influences the L-arginine-NO pathway in MCF-7 cells. The cells were grown in the absence or presence of RLX at different concentrations, and cell proliferation, constitutive and inducible NO synthase activities, nitrite production, and intracellular levels of cyclic GMP were investigated. The results obtained indicate that RLX increases inducible NO synthase activity and potentiates NO production. This was accompanied by an elevation of intracellular cyclic GMP, which is known to mediate the cell response to NO. The RLX-induced activation of the L-arginine-NO pathway in the MCF-7 cells was inversely related to the rate of cell proliferation. These results suggest that the cytostatic effect of RLX on MCF-7 breast cancer cells may rely on its ability to stimulate endogenous production of NO.

Relaxin depresses platelet aggregation: in vitro studies on isolated human and rabbit platelets.

BACKGROUND: Relaxin, a peptide hormone of ovarian origin, has been shown to cause a striking dilatory action on microvessels in different organs. In our recent studies, relaxin has been shown to stimulate the production of nitric oxide, a powerful vasodilatory agent, in several targets. Nitric oxide also inhibits platelet aggregation. This prompted us to search for a role of relaxin in platelet function. EXPERIMENTAL DESIGN: The effect of relaxin on platelet aggregation was studied in isolated human and rabbit platelets. The samples were incubated with relaxin at different concentrations and then stimulated with collagen or thrombin. Aggregation and intracellular levels of cGMP and Ca2+ were determined. In some experiments, inhibitors or potentiators of nitric oxide activity were also used to clarify whether the mechanism of action of relaxin involves the L-arginine-nitric-oxide pathway. Electron microscopy of platelets treated and not treated with relaxin was also carried out. RESULTS: Preincubation of the platelets with relaxin before stimulation with proaggregants resulted in a significant, concentration-dependent inhibition of platelet aggregation, accompanied by an elevation of intraplatelet cGMP and a decrease in the rise of cytosolic Ca2+ levels. The effect of relaxin appeared to be mediated through nitric oxide. Ultrastructurally, relaxin was shown to hinder the conformational changes and granule exocytosis usually occurring in platelets during aggregation. CONCLUSIONS: This newly recognized antiaggregatory property of relaxin, together with the vasodilatory and hypotensive activities of the peptide demonstrated in previous studies, allows for this hormone to be regarded as a protective agent against thrombotic and hypertensive disorders of pregnancy and cardiovascular diseases.

Helicobacter pylori potentiates histamine release from rat serosal mast cells induced by bile acids.

In the present study we have experimentally addressed the effects of Helicobacter pylori on the bile acid capability of histamine release. Bile acids alone were confirmed to be able to induce in vitro histamine release from rat serosal and mucosal mast cells. On the contrary, no significant histamine release was obtained when incubating any Helicobacter pylori preparations alone with mast cells. However, histamine release induced by bile acids was significantly enhanced, without any significant increase in lactate dehydrogenase activity, when whole washed or formalin-killed bacterial cells or crude cell walls were incubated with mast cells in the presence of cholic (0.3 mM), deoxycholic (0.3 mM), or lithocholic (0.3 mM) acids, chenodeoxycholylglycine (0.3 mM), and deoxycholyltaurine (3 mM). The electron microscopic features of mast cells incubated with Helicobacter pylori were consistent with an exocytotic secretion. The release of histamine induced by 0.3 mM deoxycholic acid in the presence of Helicobacter pylori was inhibited by the preincubation of the cells with dimaprit (an H2 agonist) and potentiated by the H2 antagonist, ranitidine. The current results suggest a link between human Helicobacter pylori infection and histamine release and a possible involvement of gastric mucosal mast cells in the pathogenesis of Helicobacter pylori-associated gastritis.

Platelet aggregation and histamine release by immunological stimuli.

Platelet aggregation and histamine release were evaluated in normal and IgE pretreated human platelets exposed in vitro to IgE, anti-IgE and thrombin. The response of platelets from atopic donors directly stimulated with anti-IgE was also evaluated. Histamine release was measured by fluorimetric analysis of histamine content in platelets and in supernatants. The morphology of platelets exposed to immunological and non-immunological stimuli was recorded using an electron microscope. A detectable amount of histamine was measured in quiescent platelets. Their exposure to varying concentrations of thrombin produces a progressive aggregation which runs parallel to histamine release. The effects were significantly enhanced in platelets pretreated with IgE. Incubation of normal platelets with increasing concentrations of IgE myeloma protein, or with anti-human IgE antibody was ineffective on both aggregation and histamine release. However, incubation of platelets passively sensitized with IgE-myeloma protein with different concentrations of anti-human IgE antibody produces a concentration-dependent increase both in aggregation and histamine release. The same effects were obtained using platelets from atopic donors directly stimulated with anti-IgE. The electron microscopic pattern of platelet aggregation induced by thrombin was indistinguishable from that evoked by anti-IgE in IgE pretreated platelets. Loratadine, a non-sedative H1-receptor blocker, significantly abated platelet aggregation and histamine release induced by anti-IgE in IgE pretreated platelets.

Effects of portacaval anastomosis on pancreatic islets of the rat.

The pancreatic islets of rats with surgically constructed end-to-side portacaval anastomosis were studied immunocytochemically, morphometrically, and ultrastructurally, and the plasma levels of glucose, insulin, and glucagon determined. The results of the current study show that, four weeks after surgery, hypoglycemia, normal insulinemia, and hyperglucagonemia occur and that these hematologic changes are associated with immunocytochemical and ultrastructural signs of impairment of the secretory activity of islet B cells and normal secretion pattern of the remaining islet cell types. The causes and the meaning of the hematologic and islet cell changes are discussed, and the hypothesis has been drawn that they are primarily related to the functional deterioration of the liver, which follows the diversion of the portal blood in the systemic circulation.

Relaxin influences growth, differentiation and cell-cell adhesion of human breast-cancer cells in culture.

The effects of different concentrations of relaxin (RLX) on growth and differentiation of a human breast-cancer cell line (MCF-7) have been studied after various times of exposure. The cells were cultured for 4 and 7 days in the absence (control) and the presence of highly purified porcine RLX at concentrations of 10(-9) M and 10(-6) M. (3H)-Thymidine uptake assay was used to evaluate cell proliferation. Electron microscopy and immunocytochemistry for the cell-cell adhesion molecule E-cadherin were carried out to evaluate cell differentiation. Analysis of DNA changes associated with apoptosis was performed to clarify whether RLX induces active cell death in the MCF-7 cells. The findings obtained show that RLX, when applied at micromolar concentrations, or even at nanomolar concentrations for long exposure times, suppresses proliferation, stimulates differentiation, and enhances expression of the surface molecule E-cadherin. Growth inhibition is not accompanied by apoptosis. The results of this study show that RLX can be recognized as a novel agent active in influencing growth and differentiation of MCF-7 breast-cancer cells. When applied at appropriate concentrations and exposure times, the peptide has a growth-inhibitory action, thus reversing the growth-stimulatory effect exerted at low concentrations for short exposure times, and promotes differentiation and cell-cell adhesion. These last-mentioned properties might result in a decrease in invasiveness of breast adenocarcinoma cells.

Relaxin influences the growth of MCF-7 breast cancer cells. Mitogenic and antimitogenic action depends on peptide concentration.

BACKGROUND: The effects of relaxin (RLX) on the human breast adenocarcinoma cell line MCF-7 have been evaluated. METHODS: The cells were maintained in culture with Dulbecco's modified Eagle's medium with 1% and 10% fetal calf serum. Highly purified porcine RLX was added at concentrations ranging between 10(-11) and 10(-6) M, and, after 96-hour incubation in the presence of the peptide, cell proliferation, intracellular cyclic adenosine monophosphate (cAMP) content, percent cycling cells, and structural and ultrastructural pattern were studied. RESULTS: The results indicate that RLX is a direct modulator of MCF-7 cell proliferation, stimulating growth at low concentrations and inhibiting growth at high concentrations. Determinations of percent cycling cells and intracellular cAMP accumulation agree with the results of the growth studies. Addition of different concentrations of 8-Br-cAMP to the culture medium results in a dose-related stimulation of MCF-7 cell proliferation. Morphologic examination shows that, in the current experiments, RLX does not induce any clear-cut signs of differentiation of MCF-7 cells in terms of activation of secretion or intracellular lipid deposition. CONCLUSION: These findings indicate that RLX should be regarded as a novel agent involved in the control of growth of human breast cancer cells.

Morphological changes in the human endocrine pancreas induced by chronic excess of endogenous glucagon.

The non-tumoral endocrine pancreas from a patient with elevated plasma levels of glucagon due to a malignant glucagonoma was studied immunocytochemically, ultrastructurally and morphometrically. Compared with normal pancreatic islets from control subjects, those of the pancreas from the patient with a glucagonoma showed an almost complete disappearance of A cells, a decrease in immunoreactive insulin in B cells associated with cytological features indicating enhanced synthesis and secretion of this hormone, and an increase in immunoreactive somatostatin and pancreatic polypeptide (PP) accompanied by unusually high numbers of D and PP cells. In addition, numerous B cells were found outside the islets, either forming micro-islets or scattered in the exocrine tissue (nesidioblastosis). The possible mechanisms involved in determining the changes in the secretory activity of B cells and the alterations in the cell composition of the islets are discussed.

Response of the pigeon crop sac to mammotrophic hormones: comparison between relaxin and prolactin.

The effects of relaxin (RLX), a hormone that has previously been demonstrated to have mammotrophic properties, were studied in the pigeon crop sac, a well-known target organ for mammotrophic and lactogenic hormones, and compared with the effects produced by prolactin (PRL). The two hormones were injected directly over the crop at different doses and the response was evaluated after differing times of exposure. RLX causes a dose-related increase in wet and dry weights and [3H]thymidine and [3H]uridine uptake by the crop mucosa, as well as morphological changes indicating growth and differentiation of the epithelial cells similar to those occurring during physiological activation in incubation and hatching. At the doses assayed, the effects of RLX were nearly identical to those obtained following PRL in the short-term experiments, but differences in functional responses were found in the long-term experiment.

Histamine release from rat mast cells induced by metabolic activation of polyunsaturated fatty acids into free radicals.

Polyunsaturated fatty acids (PUFA: arachidonic and linoleic acid) release histamine from isolated purified rat serosal mast cells only in the presence of oxidizing systems such as phenobarbital-induced rat liver microsomes, prostaglandin-H-synthetase (PHS) or soybean lipoxygenase. The release of mast cell histamine by activated PUFA has a long time-course and the electron microscopical features are consistent with an exocytotic secretion in the case of arachidonic acid and cell lysis in the case of linoleic acid. The phenomenon is associated with a significant increase in malonyldialdehyde (MDA) and conjugated diene generation, suggesting a relationship between histamine release and membrane lipid peroxidation. The secretion of histamine was inhibited by anti-free radical interventions such as D-mannitol, reduced glutathione and alpha-tocopherol. Some cyclooxygenase and lipoxygenase inhibitors, cimetidine and carnitine derivatives, are differentially active in the inhibition of mast cell histamine release by activated arachidonic acid. These results suggest that free radical derivatives of PUFA, generated by metabolic activation, trigger mast cell histamine release.

The exocrine pancreas in patients with hyperinsulinemic hypoglycemia. A morphometrical and ultrastructural study.

The exocrine pancreas has been studied morphometrically and ultrastructurally in 12 patients with insulin-producing tumors in comparison with control patients free from gastrointestinal or pancreatic diseases and with normal glucose homeostasis. Light microscopical examination and morphometrical data revealed that in the insulinoma-bearing patients acinar cells undergo a marked and significant decrease in zymogen granule content, together with a slight reduction in the mean cell area. Such changes were accompanied by ultrastructural features indicating increased production and exocytosis of zymogen granules. The findings concerning centroacinar/ductular cells are consistent with an increase in their number and mean cell area, and an enhancement of their functional activity. In synthesis, both the enzyme- and bicarbonate-secreting cells appear to be greatly stimulated in conditions of chronic hyperinsulinemic hypoglycemia owing to the presence of insulinomas.

Relaxin: a mammotropic hormone promoting growth and differentiation of the pigeon crop sac mucosa.

The pigeon crop sac is responsive to local administration of mammotropic hormones independently of systemic hormonal influences. We showed previously in mice that relaxin promotes growth of the mammary gland only after pre-treatment with estrogen. In this study we investigated whether relaxin, locally administered, has a stimulatory effect without estrogen pre-treatment. The results of this study show that relaxin stimulates the crop sac mucosa causing changes in its gross appearance and histological modifications indicating enhanced growth and differentiation of the mucosal epithelium. The effects of relaxin are dose-related, as demonstrated by the increases of the wet and dry weights, the [3H]thymidine and [3H]uridine uptakes of the mucosa, and the histochemically detectable DNA content of the mucosal epithelium. These results strengthen the idea that relaxin should be included in the list of mammotropic hormones.