Relaxin has beneficial effects on liver lipidome and metabolic enzymes.

Relaxin is an insulin-like hormone with pleiotropic protective effects in several organs, including the liver. We aimed to characterize its role in the control of hepatic metabolism in healthy rats. Sprague-Dawley rats were treated with human recombinant relaxin-2 for 2 weeks. The hepatic metabolic profile was analyzed using UHPLC-MS platforms. Hepatic gene expression of key enzymes of desaturation (Fads1/Fads2) of n-6 and n-3 polyunsaturated fatty acids (PUFAs), of phosphatidylethanolamine (PE) N-methyltransferase (Pemt), of fatty acid translocase Cd36, and of glucose-6-phosphate isomerase (Gpi) were quantified by Real Time-PCR. Activation of 5'AMP-activated protein kinase (AMPK) was analyzed by Western Blot. Relaxin-2 significantly modified the hepatic levels of 19 glycerophospholipids, 2 saturated (SFA) and 1 monounsaturated (MUFA) fatty acids (FA), 3 diglycerides, 1 sphingomyelin, 2 aminoacids, 5 nucleosides, 2 nucleotides, 1 carboxylic acid, 1 redox electron carrier, and 1 vitamin. The most noteworthy changes corresponded to the substantially decreased lysoglycerophospholipids, and to the clearly increased FA (16:1n-7/16:0) and MUFA + PUFA/SFA ratios, suggesting enhanced desaturase activity. Hepatic gene expression of Fads1, Fads2, and Pemt, which mediates lipid balance and liver health, was increased by relaxin-2, while mRNA levels of the main regulator of hepatic FA uptake Cd36, and of the essential glycolysis enzyme Gpi, were decreased. Relaxin-2 augmented the hepatic activation of the hepatoprotector and master regulator of energy homeostasis AMPK. Relaxin-2 treatment also rised FADS1, FADS2, and PEMT gene expression in cultured Hep G2 cells. Our results bring to light the hepatic metabolic features stimulated by relaxin, a promising hepatoprotective molecule.

Serelaxin (recombinant human relaxin-2) treatment affects the endogenous synthesis of long chain poly-unsaturated fatty acids and induces substantial alterations of lipidome and metabolome profiles in rat cardiac tissue.

BACKGROUND AND PURPOSE: Recombinant human relaxin-2, serelaxin, is being proved as a novel drug with therapeutic efficacy in some cardiovascular diseases, especially heart failure, a disease whose physiopathology and course are firmly correlated with important alterations in cardiac metabolism. The aim of our present work was to investigate changes in the cardiac metabolome following relaxin-2 treatment. EXPERIMENTAL APPROACH: Sprague-Dawley rats were treated with human recombinant relaxin-2 using osmotic minipumps at a dose of 0.4 mg/kg/day for 2 weeks. Body composition was measured with a nuclear magnetic resonance imaging system seven days after surgery and on the final day of the experiment. The last two days of treatment, respiratory quotient, locomotor activity and energy expenditure were measured with a calorimetric system. The plasma levels of relaxin-2, total cholesterol, high- and low- density lipoproteins (HDL, LDL), triglycerides and the hepatic enzymes glutamic-pyruvic transaminase (GTP) and gamma-glutamyltransferase (GGT) levels were analyzed. The metabolic profiling of both atria from relaxin-2-treated and control rats was carried out using two separate ultra-high performance liquid chromatography (UHPLC)-Time of Flight-MS based platforms analyzing methanol and chloroform/methanol extracts combined with a UHPLC-single quadrupole-MS based platform used to analyze aminoacids and with a methanol/water extract platform that covered polar metabolites. Identified ion features in the methanol extract platform included fatty acids, acyl carnitines, bile acids, monoacylglycerophospholipids, monoetherglycerophospholipids, free sphingoid bases, and oxidized fatty acids. The chloroform / methanol extract platform provided coverage over glycerolipids, cholesterol esters, sphingolipids, diacylglycerophospholipids, and acyl-ether-glycerophospholipids. Gene expression levels of the adipokines adiponectin, leptin and nesfatin-1 in visceral adipose tissue and cardiac gene expression levels of key enzymes of desaturation and elongation of n-6 and n-3 PUFAs were assessed by Real Time-PCR. KEY RESULTS: Twenty-eight metabolites out of three hundred sixty-two were significantly altered by human relaxin-2. These included fifteen glycerophospholipids: three phosphatidylethanolamines (PE) and twelve phosphatidylcholines (PC); eight sphingolipids: three ceramides (Cer) and five sphingomyelins (SM); and also five aminoacids and one carboxylic acid. Interestingly, the majority of changes correspond to lipid classes, twelve of them polyunsaturated diacylglycerophosphatidylcholines with long acyl chains, containing mainly docosahexaenoic acid (22:6) and arachidonic acid (20:4). Atrial levels of Elovl5 (Elongation of very long chain fatty acids protein 5), Fads1 (Δ5-fatty acid desaturase) and Fads2 (Δ6-fatty acid desaturase), key enzymes of elongation and desaturation of n-6 and n-3 PUFAs like arachidonic acid and DHA, respectively, were significantly increased by relaxin-2 treatment. Atrial tissues from rats treated with relaxin-2 showed a significant increase in the mRNA levels of Srebf1, a transcription factor that activates the gene expression of Elovl5, Fads1 and Fads2. The treatment with relaxin-2 significantly decreased the visceral fat mRNA expression levels of adiponectin, leptin and nesfatin-1, adipokines known to exert an important influence on the regulation of cardiovascular function. CONCLUSION AND IMPLICATIONS: Serelaxin (human recombinant relaxin-2) treatment induces significant changes in cardiac major components of the membrane lipid bilayer such as glycerophospholipids and sphingolipids, known to have structural roles but also very relevant regulatory effects in cardiac function. Serelaxin induced also modifications in several aminoacids of high influence in cardiac energy metabolism regulation. Our results highlight the need to further understand the role of relaxin-2 in the regulation of cardiac energy metabolism, in the context of the therapeutic strategies for the treatment of cardiometabolic pathologies as heart failure.

A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation.

In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5. Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10-100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose-response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.

Novel drug development opportunity for relaxin in acute myocardial infarction: evidences from a swine model.

The hormone relaxin has been shown to cause coronary vasodilation and to prevent ischemia/reperfusion-induced cardiac injury in rodents. This study provides evidence that relaxin, used as an adjunctive drug to coronary reperfusion, reduces the functional, biochemical, and histopathological signs of myocardial injury in an in vivo swine model of heart ischemia/reperfusion, currently used to test cardiotropic drugs for myocardial infarction. Human recombinant relaxin, given at reperfusion at doses of 1.25, 2.5, and 5 microg/kg b.wt. after a 30-min ischemia, caused a dose-related reduction of key markers of myocardial damage (serum myoglobin, CK-MB, troponin T) and cardiomyocyte apoptosis (caspase 3, TUNEL assay), as well as of cardiomyocyte contractile dysfunction (myofibril hypercontraction). Compared with the controls, relaxin also increased the uptake of the viability tracer 201Thallium and improved ventricular performance (cardiac index). Relaxin likely acts by reducing oxygen free radical-induced myocardial injury (malondialdehyde, tissue calcium overload) and inflammatory leukocyte recruitment (myeloperoxidase). The present findings show that human relaxin, given as a drug to counteract reperfusion-induced cardiac injury, affords a clear-cut protection to the heart of swine with induced myocardial infarction. The findings also provide background to future clinical trials with relaxin as adjunctive therapy to catheter-based coronary angioplasty in patients with acute myocardial infarction.

Basic progress and future therapeutic perspectives of relaxin in ischemic heart disease.

Relaxin has been validated as a cardiotropic hormone, being produced by the heart and acting on specific heart receptors. Evidence is accumulating that it could hamper the pathophysiologic mechanisms of ischemic heart disease. Time is ripe to study relaxin as a cardiotropic drug, as recombinant human relaxin (hrRLX) is now available and previous clinical trials have shown a virtual lack of toxicity and adverse side effects, even at high doses. Our recent observations suggest that relaxin, besides being a preventative agent, may also be effective in the treatment of acute myocardial infarction and may be an adjuvant for precursor cell grafting to repair postinfarct myocardium. In a swine model of myocardial infarction currently used to test cardiotropic drugs due to its similarities with human ischemic heart disease, hrRLX, given at reperfusion upon 30 min of ischemia, markedly reduced serum and tissue markers of myocardial injury, cardiomyocyte apoptosis and leukocyte recruitment, resulting in overall improvement in cardiac performance compared with the controls. In in vitro mixed cultures of mouse skeletal myoblasts and adult rat cardiomyocytes, relaxin increased gap junction formation and potentiated gap junction-mediated intercellular exchanges and signaling between the coupled cells. In view of the therapeutic use of myoblast grafting for cardiac repair, relaxin could hence favor the electromechanical coupling of grafted myoblasts with the resident cardiomyocytes and facilitate their transdifferentiation towards a cardiac phenotype. Relaxin, therefore, shows promising therapeutic potential in cardiology and cardiac surgery.

Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells.

Relaxin (RLX) is a reproductive hormone with vasodilatatory properties on several organs, including the heart. RLX-induced vasodilatation appears to depend on the stimulation of endogenous NO production. Here, we investigate whether RLX acts on rat coronary endothelial (RCE) cells in vitro by inducing changes of NO generation and, if so, to clarify the possible mechanism of action. RCE cells were treated for 24 h with vehicle (controls) or RLX, alone or in association with inhibitors of NO synthesis or dexamethasone, which inhibits transcription of NO synthase gene. In some experiments, inactivated RLX was given in the place of authentic RLX. Expression of NO synthase isozymes II and III was analyzed by immunocytochemistry, Western blot, and RT-PCR. NO production was evaluated by the Griess reaction for nitrite and the NO-sensitive fluorophore DAF-2/DA. Agonist-induced changes of intracellular Ca2+ transient were studied with the Ca2+-sensitive fluorophore Fura 2-AM. RLX was found to up regulate NOS II mRNA and protein and to stimulate intrinsic NO generation, likely through the activation of a dexamethasone-sensitive transcription factor, and to decrease agonist-induced intracellular Ca2+ transient. Conversely, RLX had negligible effects on NOS III expression. By these biological effects, RLX may afford significant protection against cardiovascular disease.

Human recombinant relaxin reduces heart injury and improves ventricular performance in a swine model of acute myocardial infarction.

This study shows that relaxin can be effective in the treatment of acute myocardial infarction. In a swine model of heart ischemia-reperfusion currently used to test cardiotropic drugs because of its similarities with human myocardial infarction, human recombinant relaxin (2.5 and 5 microg/kg body weight), given at reperfusion after a 30-min ischemia, markedly reduced the main serum markers of myocardial damage (myoglobin, CK-MB, and troponin T) and the metabolic and histopathologic parameters of myocardial inflammation and cardiomyocyte injury, resulting in overall improvement of ventricular performance (increased cardiac index) compared to the controls. These results provide a background for future clinical trials with human relaxin as adjunctive therapy to catheter-based coronary angioplasty in patients with acute myocardial infarction.

Inhibitory effects of relaxin on human basophils activated by stimulation of the Fc epsilon receptor. The role of nitric oxide.

This study investigates whether relaxin (RLX), a hormone previously shown to inhibit mast cell function and to stimulate endogenous nitric oxide (NO) biosynthesis, counteracts the activation of isolated human basophils stimulated with anti-IgE or phorbol ester, and, if so, whether NO is involved. RLX reduced dose-dependently the expression of the activation marker CD63, the release of histamine and the rise of intracellular Ca2+ levels which triggers granule release by stimulated basophils. RLX also blunts the ultrastructural signs of anaphylactic granule release. The effects of RLX appear to depend upon activation of Ca2+/calmodulin-dependent NO synthase and endogenous NO production. They were reproduced by the NO donor sodium nitroprusside (SNP) and were reverted by the NO synthase inhibitor N(omega)-monomethyl-L-arginine, or by the NO scavenger oxyhemoglobin, or by blocking the NO physiological target guanylate cyclase with ODQ. In conclusion, RLX appears to play a role in down-regulating basophil function upon immunologic and nonimmunologic activation.

Efficacy of relaxin on functional recovery of post stroke patients.

BACKGROUND: Relaxin is a peptide hormone that exerts specific effects on cardiovascular system and human brain, leading to the hypothesis that this hormone may play a protective role against CVD and integration and modulation of behavioral activation. We aimed to demonstrate the efficacy of Relaxin on functional recovery of post-stroke patients. METHODS: Patients admitted within a Rehabilitation Unit suffering from stroke have been evaluated. Patients have been randomized to RLX (40 mcg/d) plus rehabilitation vs a control group that underwent only rehabilitation. A preliminary analysis of 36 patients at 20 and 40 days was made using the mRS for global function, the Functional Independent Measure (FIM) for daily activity and Trail Making Test (TMT) for cognitive function. RESULTS: Eighteen patients (age 72 (64-79), M 56%) randomized to RLX plus rehabilitation were compared to 18 patients (age 68 (64-78), M 50%) that underwent only rehabilitation. There was no difference between the two groups in terms of risk factors, stroke syndromes and etiology. At admission the two groups showed the same characteristics in terms of functional aspects (mRS, FIM; p ns) and cognitive function (TMT; p ns). After 20 days (T1) the treatment group (RLX+rehabilitation) showed no differences between the two groups (FIM 78 vs 69; p ns), while after 40 days (T2) patients treated with RLX+R showed an excellent recovery (FIM 96 vs 75; p0.001). In terms of cognitive function patients RLX+R revealed a better performance at T1 (TMT 3.5 vs 2; p 0.002) and still better at T2 (TMT 4 vs 2; p 0.001). These results have been confirmed in terms of global function both at T1 (mRS 2.5 vs 3; p0.001) and T2 (mRS 2 vs 3; p < 0.001). CONCLUSION: Relaxin showed in this analysis a positive effects on stroke patient's recovery, thus offering the broad therapeutic potential role of RLX as new drug in post-stroke patients.

Efficacy and safety of oral porcine relaxin (pRLX) in adjunct to physical exercise in the treatment of peripheral arterial disease (PAD).

INTRODUCTION: PAD medical therapy has a number of limitations. RLX showed promises in experimental model mainly through NO release. Our study is the first to evaluate the efficacy and safety of RLX in PAD. MATERIALS-METHODS: Eligible PAD La fontaine IIa-IIb patients were randomized in 2 groups. Group A was treated with physical therapy plus oral pRLX, 20 ug b.i.d for 12 weeks, group B received physical therapy alone. Pain Free Walking Distance (PFWD) and Maximum Walking Distance (MWD) at 3 and 12 wks and at follow up 3 months after treatment interruption were performed. RESULTS: The percentage increases of PFWD in group B were 23 +/- 9, 65 +/- 17, and 35 +/- 4 respectively at 3 and at 12 weeks, and 3 months after termination. In Group A showed significantly higher percentage increases: 74 +/- 16 p < 0.01, 168 +/- 28 p < 0.001, and 122 +/- 15 p < 0.001 at the corresponding time points. The percentage increases of MWD in the B group were 29 +/- 7, 55 +/- 10 and 54 +/- 8 at the above time points, while in the A group were 55 +/- 10 p < 0.001, and 99 +/- 12 p < 0.001. The RLX patients referred a better physical and mental status. No adverse events during or after the treatment were recorded. COMMENT: RLX resulted very effective in PAD. Our results may suggest that the observed functional benefits should come not only from hemodynamic improvement but also from positive vascular remodeling.

Relaxin as a cardiovascular drug: a promise kept.

Relaxin (RLX), formerly known for its effects on reproduction and pregnancy, has been later shown to be a pleiotropic hormone, capable of also targeting numerous non-reproductive organs of the cardiovascular, nervous, respiratory, tegumental, excretory and digestive systems. Most of these effects have been studied in animal models, but there is compelling evidence that RLX also acts in humans. In more recent years, human luteal-type (H2) RLX synthesised by recombinant DNA technology has been investigated in clinical trials, mostly oriented to assess its therapeutic potential in cardiovascular disease. This indication was based on the accumulating pre-clinical evidence that RLX possesses prominent biological effects on systemic and coronary blood vessels, cardiomyocyte growth and differentiation, and cardiac/vascular connective tissue remodelling. This mini-review was intended as an update of our previous article that appeared in this journal in 2009, as the last 2 years have been characterised by fundamental achievements on the clinical profile of RLX. Eventually, after many years of inconclusive studies, RLX appears to be about to reach a recognised dignity as a cardiovascular drug.

Clinical profile of relaxin, a possible new drug for human use.

Relaxin (RLX) belongs to the relaxin hormone super-family, which in humans includes 3 RLX molecules and 4 insulin-like peptides. Of these, luteal RLX is the main circulating form in animals and man. RLX, formerly known for its effects on reproduction and pregnancy, has been demonstrated to act on numerous other targets, including the cardiovascular, central and peripheral nervous, respiratory, tegument, excretory and digestive systems. Most of these effects have been studied in animal models, but there is compelling evidence that RLX also acts in the human. Over time, RLX, as a crude or purified extract from corpora lutea or as a pure molecule produced by chemical synthesis or recombinant DNA technology, has been the object of clinical studies aimed at identifying its possible therapeutic fields of application. The availability of human recombinant RLX and the most recent achievements on its biological effects, especially on connective tissue remodelling and cardiovascular physiopathology, have sparkled a novel peak of interest of clinicians to the therapeutic potential of RLX. This review of the existing clinical studies with RLX is focused at its pharmacological and toxicological features, with the aim to support interest of clinicians and pharmaceutical companies to continue the pathway which may eventually succeed in translating RLX from the laboratory bench to the bedside.

Relaxin as a cardiovascular hormone: physiology, pathophysiology and therapeutic promises.

Relaxin is a hormone belonging to the so-called relaxin superfamily, which also includes insulin-like peptides. Relaxin is best known for its effects on the female reproductive system, which primarily include lengthening of the pubic symphysis and softening of the tissues of the birth canal, stimulating mammary and endometrial development, and maintenance of myometrial quiescence. In recent years, evidence has been accumulating that relaxin can have multiple and diverse effects on both reproductive and nonreproductive organs, tissues and cells, thus acting as a sort of 'manager' hormone to optimize the many physiological changes taking place during pregnancy. Among the specific relaxin targets, there are the blood vessels and the heart. In fact, relaxin is a potent vasodilatator of the systemic and coronary circulation, by a mechanism of action involving nitric oxide, and can influence cardiac beating rate. Identification of the numerous possible roles of relaxin in the pathophysiology of cardiovascular diseases, not to mention the possible therapeutical applications of relaxin, remains a difficult task. Based on the known biological effects of relaxin, it is becoming increasingly evident that the potential fields of clinical investigation of human relaxin as a cardiovascular drug could be in ischemic heart disease (acute and chronic myocardial infarction), in cardiac fibrosis, in cell transplantation for cardiac repair, and in obliterative peripheral arterial disease. Availability of the homologous human peptide, knowledge of its pharmacological, pharmacokinetic and pharmacodynamic profiles, and increasing interest of clinicians and pharmaceutical companies should synergistically act to transfer relaxin from the laboratory bench to the bedside.

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus.

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.

Depression by relaxin of neurally induced contractile responses in the mouse gastric fundus.

The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.

Relaxin depresses small bowel motility through a nitric oxide-mediated mechanism. Studies in mice.

Gastrointestinal motility is reduced and the incidence of functional gastrointestinal disorders is increased in pregnancy, possibly due to hormonal influences. This study aims to clarify whether the hormone relaxin, which attains high circulating levels during pregnancy and has a nitric oxide-mediated relaxant action on vascular and uterine smooth muscle, also reduces bowel motility and, if it does, whether nitric oxide is involved. Female mice in proestrous or estrous were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Isolated ileal preparations from both groups were used to record contractile activity, either basal or after acute administration of relaxin (5 x 10(-8) M). Drugs inhibiting nitric oxide biosynthesis or neurotransmission were used in combination with relaxin. Expression of nitric oxide synthase isoforms by the ileum was assessed by immunocytochemistry and Western blot analysis. Relaxin caused a clear-cut decay of muscle tension and a reduction in amplitude of spontaneous contractions upon either chronic administration to mice or acute addition to isolated ileal preparations. These effects were significantly blunted by N(G)-nitro-L-arginine, but not by the neural blockers we used. Moreover, relaxin increased the expression of nitric oxide synthases II and III, but not synthase I. Relaxin markedly inhibits ileal motility in mice by exerting a direct action on smooth muscle through the activation of intrinsic nitric oxide biosynthesis.