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

Abstract

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.

Protection from cigarette smoke‐induced vascular injury by recombinant human relaxin‐2 (serelaxin)

“…This property of RLX fits well with previous reports that this hormone can up‐regulate NOS expression in vascular endothelial cells 26, 27, 28, 29, 30. However, RLX may also promote arterial dilatation by a direct effect on vascular smooth muscle cells, as reported 29.…”

“…This working hypothesis is based on the following mainstays: ( i ) blood vessels are a physiological target of RLX and their cells express the specific RLX receptor RXFP1 25; ( ii ) RLX up‐regulates NOS expression and nitric oxide production in vascular endothelial cells 26, 27, 28, 29, 30; ( iii ) RLX improves inflammation‐induced endothelial dysfunction and NOS fall 31, 32; and ( iv ) RLX reduces oxidative stress and nitric oxide failure in different animal models of vascular injury 33, 34, 35, 36, 37.…”

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

“…1A); accordingly, 40 lM was selected. Importantly, this concentration is at least fourfold higher than the concentration required to measure levels of intracellular NO in mammalian cells which ranged from 0.1 to 10 lM (8,28,29,35). This may be attributed to inherently lower amounts of NO in the parasite or altered esterase activity or even variations in their membrane permeability.…”

“…Methods for detecting NO include trapping of NO with hemoglobin, chemiluminescence assays, electron paramagnetic resonance spectroscopy, or using electrochemical electrodes (7). Other indirect methods include estimation of nitric oxide synthase (NOS) activity, protein and mRNA expression of NOS (8) as also estimation of nitrate/nitrite levels. Unfortunately, these methods are limited by their inability to define the population of interest and analyze intracellular events at a single cell level.…”

Effect of N-acetyl-l-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

“…To test whether NAC can interfere with NO production, nitrite concentration was measured using a standard Griess micromethodology as described [32]. Guinea pig lung macrophages were used for this test as described [29].…”