RESUMO
The cellular sources of nitric oxide in the hypothalamus are thought to be 'NOergic' neurones. Using free radical electrochemical sensors we investigated nitric oxide and superoxide radical release in primary hypothalamic cell cultures. We present evidence that under interleukin-1beta (IL-1beta) stimulation hypothalamic astrocytes rather than neurones release nitric oxide. Under L-arginine deprivation and IL-1beta stimulation a concentration-dependent release of superoxide was also observed, which was inhibited in the presence of nitric oxide synthase inhibitor nitro-L-argininemethyl-ester. These findings support the hypothesis that the balance between nitric oxide and superoxide may be of vital importance in hypothalamic pathophysiology.
Assuntos
Astrócitos/fisiologia , Hipotálamo/metabolismo , Interleucina-1/fisiologia , Neurônios/fisiologia , Óxido Nítrico Sintase/metabolismo , Animais , Astrócitos/enzimologia , Morte Celular/fisiologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Hipotálamo/citologia , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Consumo de Oxigênio/fisiologia , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismoRESUMO
Oxygen-derived free radicals play an important role in the physiology and pathophysiology of brain cell function. Because of their labile nature, however, it has been difficult to investigate their actions directly. This problem has been addressed, in primary rat brain cell cultures, in this study by utilization of two novel electrochemical sensors. It has been demonstrated that extracellular superoxide originates from the astrocytic subpopulation in a calcium/calmodulin dependent manner and responds to constitutive nitric oxide synthase inhibition. The results indicate a novel function for the astrocytic constitutive nitric oxide synthase in regulating extracellular superoxide release and, therefore, controlling neuronal nitric oxide availability.
Assuntos
Astrócitos/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Espaço Extracelular/metabolismo , Imuno-Histoquímica , Microglia/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I , RatosRESUMO
The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials.