Soluble guanylyl cyclase α1 subunit is a key mediator of proliferation, survival, and migration in ECC-1 and HeLa cell lines

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme constituted by two subunits, α1 and β1. Previously we have shown that 17β-estradiol (E2) exerts opposite effects on these subunits by increasing α1 and decreasing both β1 expression and enzymatic activity. To date, the physiological relevance of E2-induced sGC subunits’ imbalance has not been addressed. Also, increased levels strongly correlate with E2-induced proliferation in E2-dependent tissues. The aim of the present study was to investigate the role of sGCα1 in proliferation, survival, and migration in two E2-responsive and non-responsive tumour cell lines. Here we showed that E2 stimulated sGCα1 expression in ECC-1 endometrial cancer cells. sGCα1 knock-down significantly reduced E2-dependent cell proliferation. Moreover, sGCα1 silencing caused G1 arrest together with an increase in cell death and dramatically inhibited cell migration. Surprisingly, disruption of sGCα1 expression caused a similar effect even in absence of E2. Confirming this effect, sGCα1 knock-down also augmented cell death and decreased proliferation and migration in E2-unresponsive HeLa cervical cancer cells. Our results show that sGCα1 mediated cell proliferation, survival, and migration in ECC-1 and HeLa cells and suggest that sGCα1 can not only mediate E2-tumour promoting effects but can also be involved in hormone-independent tumour progression.Fil: Ronchetti, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; ArgentinaFil: Pino, María Teresa Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; ArgentinaFil: Cordeiro, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; ArgentinaFil: Bollani, Sabrina Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; ArgentinaFil: Ricci, Analía Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; ArgentinaFil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; Argentin

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

17β-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 α1- and decreasing β1-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 α-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC α-subunit are expressed. Here we show for the first time that sGCα2 and sGCα2 inhibitory (α2i) isoforms are expressed in this gland, but only sGCα2i 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. sGCα1, -β1, and -α2i 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. Copyright © 2009 the American Physiological Society.Fil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Ronchetti, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nudler, Silvana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Miler, Eliana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Quinteros, Alnilan Fernanda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

El cadmio como citotóxico y como metalohormona: efectos sobre el eje hipotálamo-hipofisario

En las últimas décadas, la contaminación ambiental ha aumentado considerablemente y junto con ella la preocupación concerniente a los efectos adversos de diferentes tóxicos ambientales. Entre ellos, el cadmio (Cd), un metal pesado ampliamente usado en la industria y presente en altas concentraciones en el humo del cigarrillo, ha sido objeto de numerosos estudios. Con una vida media muy larga dentro del organismo y una notoria capacidad de bioacumulación, el Cd es en sí una grave amenaza para la salud. Como muchos tóxicos, los efectos del Cd en el organismo dependen de la concentración del metal, del tiempo de exposición al mismo y de la susceptibilidad diferencial de los tejidos a su acción. Generalmente el Cd a concentraciones micromolares -tanto in vivo como in vitro- provoca en órganos endocrinos estrés oxidativo, muerte celular por apoptosis y desbalance hormonal. A concentraciones nanomolares, el Cd es capaz de mimetizar los efectos del estrógeno -hormona clave en la reproducción con acción en tejidos como útero y mama- con potenciales implicancias en la aparición y desarrollo de patologías neoplásicas hormona-dependientes. Este manuscrito se enfoca en los resultados de las investigaciones de nuestro laboratorio con respecto a los efectos tanto citotóxicos como proliferativos del Cd sobre el sistema hipotálamo-hipofisario y se discute sobre posibles tratamientos para revertir sus efectos deletéreos.In the last decades, environmental pollution has considerably increased and also the concern regarding the adverse effects of many environmental toxicants. Among them, cadmium (Cd), a heavy metal widely used in industry and present in high concentrations in cigarette smoke, has been the subject of numerous studies. Once in the organism it shields a very long halflife and a remarkable ability to bioaccumulate, which makes of Cd a serious threat to health. As many toxics, Cd effects in the body depend on the metal concentration, exposure time and differential susceptibility of tissues to its action. Generally, Cd at micromolar concentrations in endocrine organs causes oxidative stress, cell death by apoptosis and hormonal imbalance both, in vivo and in vitro. At nanomolar concentrations, Cd is able to mimic the effects of estrogen -reproductive hormone with key actions in tissues such as uterus and breast- with potential implications for the onset and progression of hormonedependent neoplastic diseases. This manuscript focuses on the results from our laboratory regarding to both, proliferative and cytotoxic effects of Cd on the hypothalamus-pituitary system and discusses possible treatments to reverse its deleterious effects.Fil: Duvilanski, Beatriz Haydee. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina; ArgentinaFil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

Effect of ionotropic and metabotropic glutamate agonists and D-aspartate on prolactin release from anterior pituitary cells

Although the presence of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors has been demonstrated in the anterior pituitary, recent reports on the direct effect of glutamate on prolactin (PRL) secretion by anterior pituitary cells have presented contradictory results. Hence, the aim of this study was to determine the effect of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor agonists on prolactin (PRL) release. In addition, since D-Aspartate (D-Asp) is found in the pituitary and is involved in neuroendocrine regulation, we also studied the direct action of D-Asp on PRL secretion. Finally, since the posterior pituitary participates in the regulation of PRL secretion, we examined the influence of the posterior pituitary on the effects of NMDA and D-Asp on PRL release. - Glutamate (1000 μM) increased PRL secretion from cultured anterior pituitary cells. Both NMDA (100 μM) and kainate (100 μM) increased PRL secretion and these effects were blocked by a specific NMDA receptor antagonist. AMPA did not modify PRL release in these cultures. The group I and II mGluR agonist, trans-ACPD (1000 μM), and a specific group II mGluR agonist, L-CCG-I (100-1000 μM), inhibited whereas specific group I and III mGluR agonists, 3-HPG and L-AP4 respectively, had no effect on PRL release. Finally, D-Asp (100-1000 μM) stimulated PRL secretion and this effect was reduced by a NMDA receptor antagonist. When anterior pituitary cells were cultured in the presence of posterior pituitary cells, NMDA did not modify PRL or GABA release, while D-Asp increased PRL secretion and decreased GABA release in these cocultures. - In conclusion, our results show that L-glutamate has a differential direct effect on PRL release: it exerts a stimulatory action via iGluRs and an inhibitory effect via mGluRs. D-Asp could directly stimulate PRL release through NMDA receptors. D-Asp may also stimulate PRL release by decreasing GABA release from the posterior pituitary.Fil: Pampillo, M.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Theas, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Seilicovich, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; ArgentinaFil: Lasaga, Mercedes Isabel. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Biología Celular e Histología. Centro de Investigación en Reproducción; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Adverse effects induced by chromium VI, cadmium and arsenic exposure on hypothalamus-pituitary physiology

Environmental contamination with some metalloids and heavy metals (M/HM) raises concern due to well known adverse effects on health. Among these pollutants, chromium VI (Cr VI), cadmium (Cd) and arsenic(As) are frequently present as a result of natural sources or due to industrial activities. They are able to easily enter the organism and negatively affect many organs and systems. In vivo (exposure to Cr VI, Cd or As through drinking water) and in vitro experiments (primary pituitary cell cultures) were performed in male Wistar rats to address their actions on hypothalamus-pituitary axis. All the M/HM accumulated in hypothalamus and pituitary gland and decreased pituitary cell viability and prolactin release mostly by generation of reactive oxygen species, since it was partially prevented by antioxidant treatment. In the pituitary, they increased lipid peroxydation and the expression of several oxidative stress markers. Cell death was mainly due to caspase-dependentapoptosis. Lactotrophs (prolactin-secreting cells) were the most affected pituitary population. Cd-driven cell death was also partially calcium- and calpain-dependent. Parallely, Cd stimulated the production of low levels of nitric oxide which exerted cytoprotective actions. These results showed that these M/HM display deleterious actions in hypothalamic-pituitary physiology by altering hormone release and promoting cell death.Fil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Ronchetti, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

Role of nitric oxide in the metabolism of arachidonic acid in the rat anterior pituitary gland

Nitric oxide (NO) affects cyclooxygenase (COX) and lipooxygenase (LOX) activities in several tissues. The aim of this study was to investigate the effect of NO on the AA metabolism in the anterior pituitary. LOX and COX products from anterior pituitaries of Wistar male rats were determined by [14C]-AA radioconversion method. Sodium nitroprusside (NP, 0.5 mM) and DETA NONOate (1 mM), NO donors, decreased 5-hydroxy-5,8,11,14-eicosatetraenoic acid (5-HETE) synthesis (P<0.05), effects that were reversed by hemoglobin. L-arginine also inhibited LOX activity. To the contrary, the inhibition of NO synthase by L-NAME (0.5 mM) or aminoguanidine (0.5 mM) increased 5-HETE production (P<0.05). COX activity was slightly stimulated by NP and L-arginine. However, DETA NONOate induced a stimulation of the synthesis of all prostanoids (P<0.05), this effect being reversed by hemoglobin. Neither NOS inhibitors nor hemoglobin modified basal prostanoids synthesis. These results indicate that NO inhibits LOX activity and stimulates COX activity in the anterior pituitary gland. The inhibition of LOX by NO may be another mechanism involved in the effects of NO on hormone release in the anterior pituitary.Fil: Velardez, Miguel Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Ogando, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

In vivo and in vitro arsenic exposition induces oxidative stress in anterior pituitary gland

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereasluteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 micromol/L iAssignificantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin releaseboth in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.Fil: Ronchetti, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Bianchi, Maria Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

Mechanisms of Chromium VI-induced apoptosis in anterior pituitary cells

Hexavalent chromium (Cr (VI)) is a highly toxic metal. Exposure to Cr (VI) compounds may affect reproductive functions. Due to the importance of anterior pituitary hormones on reproductive physiology we have studied the effects of Cr (VI) on anterior pituitary. We previously demonstrated that, after in vivo Cr (VI) administration, Cr accumulates in the pituitary gland and affects prolactin secretion. In vitro, Cr (VI) causes apoptosis in anterior pituitary cells due to oxidative stress generation. To better understand the mechanisms involved in Cr (VI)-induced apoptosis we studied: (a) whether Cr (VI) affects the intracellular antioxidant response and (b) which of the apoptotic factors participates in Cr (VI) effect. Our results show that Cr (VI) treatment induces a decrease in catalase and glutathione peroxidase (GPx) activity but does not modify glutathione reductase (GR) activity. Cr (VI) exposure causes an increase of GSH levels. p53 and Bax mRNA are also upregulated by the metal. Pifithrin alpha, a p53 transcriptional inhibitor, increases Cr (VI) cytotoxicity, suggesting a role of p53 as a survival molecule. The antioxidant N-acetyl-cysteine (NAC) could prevent Bax mRNA increase and caspase 3 activation, confirming that Cr (VI)-induced apoptosis involves oxidative stress generation.Fil: Quinteros, Alnilan Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Machiavelli, Leticia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Miler, Eliana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cabilla, Jimena Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Duvilanski, Beatriz Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin