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.

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.

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In this study we report on the establishment and characterization of two novel lymphoma cell lines (CRO‐AP/3 and CRO‐AP/5) which carry infection by human herpesvirus type‐8 (HHV‐8) and have derived from AIDS‐related primary effusion lymphoma (PEL). These two cell lines are representative of different virologic subtypes of PEL, i.e. HHV‐8+/EBV− PEL in the case of CRO‐AP/3 and HHV‐8+/EBV+ PEL in the case of CRO‐AP/5. Consistent with the diagnosis of PEL, both CRO‐AP/3 and CRO‐AP/5 expressed indeterminate (i.e. non‐B, non‐T) phenotypes although immunogenotypic studies documented their B‐cell origin. Both cell lines are devoid of genetic lesions of c‐MYC, BCL‐2 and p53 as well as gross rearrangements of BCL‐6. Detailed histogenetic characterization of these novel PEL cell lines suggests that PEL may derive from a post‐germinal centre B cell which has undergone pre‐terminal differentiation. The CRO‐AP/3 and CRO‐AP/5 cell lines may provide a valuable model for clarifying the pathogenesis of PEL. In particular, these cell lines may help understand the relative contribution of HHV‐8 and EBV to PEL growth and development and may facilitate the identification of recurrent cytogenetic abnormalities highlighting putative novel cancer related loci relevant to PEL.

Melatonin is endowed with a growth inhibitory effect in MCF-7 breast cancer cells whose mechanism has been related to an antiestrogenic activity exerted by inhibition of binding of the estradiol-estrogen receptor complex to its DNA responsive element. Looking for downstream gene determinants of this effect, we performed a transcriptome profiling by high-density microarrays of estrogen-treated MCF-7 cells exposed or not to melatonin. We found that cyclin D1 was one of the main downregulated genes by melatonin. Validation experiments clearly confirm that in MCF-7 cells the estrogen-induced growth inhibitory activity of melatonin is consistently associated with inhibition of estrogen-elicited cyclin D1 induction. This effect is almost purely transcriptional. Reporter gene assays indicate that the same portion of the cyclin D1 promoter which confers estrogen sensitivity, encompassing a potential cAMP responsive element binding site, is repressed by melatonin. Transcriptional downregulation of cyclin D1 is the key molecular event for melatonin's antiproliferative activity, as this activity can be completely and selectively rescued by transient cyclin D1 overexpression. Finally, we provide indirect evidence that the effect of melatonin on the cyclin D1 promoter is mediated by the c-jun and ATF-2 proteins, known to bind the minimal estrogen-sensitive cyclin D1 promoter element. These findings establish for the first time a molecular link between melatonin and its effects on the cell cycle, providing at the same time a rationale for its use in adjuvant chemotherapy.

The hormone relaxin (RLX), which can be detected in human venous cord blood, has been shown to be a potent vasodilator, acting through increased expression of inducible nitric oxide synthase (NOS II) and nitric oxide (NO) generation. This study aims at clarifying whether RLX, at concentrations of 100 and 1000 ng/ml for 6 or 12 h of exposure, can influence the expression of NOS isoforms in human umbilical vein endothelial cells (HUVEC) cultured in vitro. NOS mRNA expression was studied by quantitative real-time RT-PCR, NOS protein expression and activity was studied by Western blot and nitrite assay, and immunoreactive NOS localization was performed by confocal microscopy. Untreated HUVEC expressed all the NOS isoforms, especially the constitutive, endothelial-type NOS III and, to a lesser extent, NOS II and NOS I. RLX-treated cells showed an increased expression of NOS II, attaining a maximum with 1000 ng/ml RLX, which gave rise to increased NO generation, as shown by nitrite assay. This effect of RLX appears to be mediated by activation of NOS II transcription factor NF-kappaB, since it was abolished by the NF-kappaB inhibitors curcumin-95 and dexamethasone. These findings suggest that RLX in the umbilical vein might contribute to the NO-dependent regulation of vascular tone.

1 Our previously published data indicate that an endogenously produced 5-lipoxygenase metabolite can strongly contract isolated endothelium-preserved rat aortic strips when cyclo-oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl-containing leukotrienes (Cys Lts) are involved in this endothelium-dependent contraction. 2 The isometric contraction of endothelium-preserved rat aortic strips was recorded in preparations preincubated with 5 mM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK-571, selective type 1 Cys Lts receptor (Cys Lt 1 ) antagonists and the Cys Lt 1 /Cys Lt 2 (type 2 Cys Lts receptor) antagonist BAYu9773 dose-dependently prevented ACh-induced contraction, their IC 50 s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC 50 1.5 mM).

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.