Angiotensin II-upregulated MAP kinase phosphatase-3 modulates FOXO1 and p21 in adrenocortical H295R cells.

MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes.

Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

17ß-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 ß, that catalyses cGMP formation. α1ß1 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 ß1 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 ß1 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 ß1 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 ß1 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.

Cadmium induced-oxidative stress in pituitary gland is reversed by removing the contamination source.

Cadmium (Cd(2+)) is one of the most important environmental contaminants and acts as an endocrine disruptor. Previously, we have demonstrated that the simultaneous administration of Cd(2+) and melatonin (Mel) in drinking water impaired metal-induced oxidative stress in rat anterior pituitary gland. The aim of this study was to investigate if a treatment started after the toxic manifestations of Cd( 2+) became evident could reverse the effects of the metal. Animals exposed to Cd(2+) (5 parts per million [ppm], 30 days) were treated with Mel or without the metal during the next 1 or 2 months. Cd(2+) exposure increased the expression of heme oxygenase-1 (HO-1), a biomarker of oxidative stress, and an a posteriori Mel treatment reversed oxidative stress induced by Cd(2+). This effect was also observed 1 month after metal removal. The Cd(2+)-induced increase in metallothionein-1 (MT-1) and nitric oxide synthase 1 (NOS1) expression were also reversed by metal removal. In addition, serum prolactin and luteinizing hormone levels affected by Cd( 2+) exposure were normalized. Considering that the manifestations of Cd(2+) intoxication become evident only after a certain period of metal accumulation, these results show that metal removal is enough to reverse Cd(2+) effects in anterior pituitary gland and bring to light the relevance of moving away the individual from the contamination source.

Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands.

17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.

Chromium VI administration induces oxidative stress in hypothalamus and anterior pituitary gland from male rats.

Hexavalent chromium (Cr VI)-containing compounds are known carcinogens which are present in industrial settings and in the environment. The major route of chromium exposure for the general population is oral intake. Previously we have observed that Cr VI affects anterior pituitary secretion and causes oxidative stress in vitro. The aim of the present work was to investigate if in vivo Cr VI treatment (100 ppm of Cr VI in drinking water for up 30 days) causes oxidative stress in hypothalamus and anterior pituitary gland from male rats. This treatment produced a 4-fold increase of chromium content in hypothalamus and 10-fold increase in anterior pituitary gland. Lipid peroxidation showed a significant increase in hypothalamus and anterior pituitary. Cr VI augmented superoxide dismutase activity in anterior pituitary gland and glutathione reductase activity in hypothalamus, but glutathione peroxidase and catalase activities remained unchanged in both tissues. Heme oxygenase-1 mRNA expression significantly rose in both tissues. Metallothionein 1 mRNA content increased in anterior pituitary and metallothionein 3 mRNA increased in hypothalamus. These results show, for the first time, that oral chronic administration of Cr VI produces oxidative stress on the hypothalamus and anterior pituitary gland which may affect normal endocrine function.

Altered synaptic synchrony in motor nerve terminals lacking P/Q-calcium channels.

The variance in synaptic delays among endplate potentials events (referred here as jitter) was measured to study the contribution of voltage dependent calcium channels to transmission synchronicity in neuromuscular synapses from wild type and alpha-1A knockout mice (i.e., lacking P/Q type calcium channels). Knockout synapses presented higher jitter values than wild type ones under a wide range of extracellular calcium concentration ([Ca2+]o) values. Accordingly, wild type synapses showed less synchronic neurotransmitter release when P/Q type calcium channels were partially blocked as well as under lower [Ca2+]o. In the knockout synapses, N-type calcium channels mediated neurotransmitter release in a more temporally precise way than the R-type ones. Our results suggest that the type of calcium channels mediating transmitter release influenced the degree of synaptic synchrony. Thus, these results provide insight on the mechanisms underlying several pathologies associated with P/Q type calcium channels.