55 research outputs found
Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland
17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway
Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling
Reactive oxygen and nitrogen species change cellular responses through diverse mechanisms that are now being defined. At low levels, they are signalling molecules, and at high levels, they damage organelles, particularly the mitochondria. Oxidative damage and the associated mitochondrial dysfunction may result in energy depletion, accumulation of cytotoxic mediators and cell death. Understanding the interface between stress adaptation and cell death then is important for understanding redox biology and disease pathogenesis. Recent studies have found that one major sensor of redox signalling at this switch in cellular responses is autophagy. Autophagic activities are mediated by a complex molecular machinery including more than 30 Atg (AuTophaGy-related) proteins and 50 lysosomal hydrolases. Autophagosomes form membrane structures, sequester damaged, oxidized or dysfunctional intracellular components and organelles, and direct them to the lysosomes for degradation. This autophagic process is the sole known mechanism for mitochondrial turnover. It has been speculated that dysfunction of autophagy may result in abnormal mitochondrial function and oxidative or nitrative stress. Emerging investigations have provided new understanding of how autophagy of mitochondria (also known as mitophagy) is controlled, and the impact of autophagic dysfunction on cellular oxidative stress. The present review highlights recent studies on redox signalling in the regulation of autophagy, in the context of the basic mechanisms of mitophagy. Furthermore, we discuss the impact of autophagy on mitochondrial function and accumulation of reactive species. This is particularly relevant to degenerative diseases in which oxidative stress occurs over time, and dysfunction in both the mitochondrial and autophagic pathways play a role
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Quantification of mitochondrial DNA mutation load
Mitochondrial DNA (mtDNA) mutations are an important
cause of genetic disease and have been proposed to play
a role in the ageing process. Quantification of total
mtDNA mutation load in ageing tissues is difficult as
mutational events are rare in a background of wild-type
molecules, and detection of individual mutated molecules
is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to
document the incidence of mtDNA point mutations in
ageing include post-PCR cloning, single-molecule PCR and
the random mutation capture assay. The mtDNA mutation
load obtained by these different techniques varies by
orders of magnitude, but direct comparison of the three
techniques on the same ageing human tissue has not
been performed. We assess the procedures and practicalities
involved in each of these three assays and discuss the
results obtained by investigation of mutation loads in
colonic mucosal biopsies from ten human subjects
Complex I Is Rate-limiting for Oxygen Consumption in the Nerve Terminal*
Metabolic control analysis was used to determine the spread of control
exerted by the electron transport chain complexes over oxygen consumption
rates in the nerve terminal. Oxygen consumption rates and electron transport
chain complex activities were titrated with appropriate inhibitors to
determine the flux control coefficients and the inhibition thresholds in rat
brain synaptosomes. The flux control coefficients for complex I, complex
II/III, complex III, and complex IV were found to be 0.30 ± 0.07, 0.20
± 0.03, 0.20 ± 0.05, and 0.08 ± 0.05, respectively.
Inhibition thresholds for complex I, complex II/III, complex III, and complex
IV activities were determined to be ∼10, ∼30, ∼35, and
50–65%, respectively, before major changes in oxygen consumption rates
were observed. These results indicate that, of the electron transport chain
components, complex I exerts a high level of control over synaptosomal
bioenergetics, suggesting that complex I deficiencies that are present in
neurodegenerative disorders, such as Parkinson disease, are sufficient to
compromise oxygen consumption in the synaptosomal model of the nerve
terminal
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