Nitric oxide levels in cochlear fluids of guinea pig

PURPOSE: To describe a technique to obtain guinea pigs cochlear fluids and measure nitric oxide (NO) concentration. METHODS: Six guinea pigs were used and sacrificed. The cochlear fluids collected for measurement of NO, performed by chemiluminescence (NOA 280). RESULTS: Through the chemiluminescence was possible to analyze the concentration of NO in cochlear fluids obtained. Average levels of nitric oxide from guinea pigs was 12.55 µM. CONCLUSION: It is possible to obtain nitric oxide cochlear fluids, with this technique and nitric oxide concentration measure by chemiluminescence, a quantitative and more precise method

Cyclosporine A and NAC on the inducible nitric oxide synthase expression and nitric oxide synthesis in rat renal artery cultured cells

Background. the immunosuppressor cyclosporine A (CsA) presents the nephrotoxicity as its major side effect that is mostly attributed to a renal vasoconstriction. This may be due to an excessive generation of vasoconstrictors like reactive oxygen species (ROS), or due to a reduction of vasodilators such as the nitric oxide, which in turn, can be caused by increased amounts of ROS. We evaluated the effect of CsA and the antioxidant N-acetylcysteine (NAC) on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide synthesis, in rat renal artery vascular smooth muscle cells (rVSMCs) primary culture.Methods. in cells treated during 72 hours with CsA (10 mu g/mL), its vehicle (control) (10 mu L/mL), Escherichia coli lipopolysaccharide (LPS) (100 mu g/mL), CsA + LPS, NAC (6.13 mmol/L), or CsA + NAC, we determined the nitric oxide synthesis ( Griess and chemiluminescence methods), iNOS expression [reverse transcription-polymerase chain reaction (RTPCR)] and cell viability (acridine orange method).Results. in rVSMCs, LPS increased nitric oxide and iNOS expression; CsA decreased basal and LPS-induced nitric oxide and iNOS expression; NAC increased nitric oxide and blunted the nitric oxide reduction caused by CsA, with no effect on iNOS. CsA reduced cell viability.Conclusion. in this study, CsA reduced nitric oxide synthesis in rVSMCs, both through iNOS down-regulation and reduction of cell viability, which could be responsible for the vasoconstrictive effect of the CsA. in the effect of CsA on nitric oxide, probably a role is also played by free radical production, as this effect was blunted by NAC.UNIFESP, EPM, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Emergency Div, São Paulo, BrazilUNIFESP, EPM, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Emergency Div, São Paulo, BrazilWeb of Scienc

Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O2•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O2•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma