Martínez Laorden, Elena

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Martínez Laorden, Elena

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Farmacología

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Expression of heat shock protein 27 and troponin T andtroponin I after naloxone-precipitated morphine withdrawal
(0005-11-15) Pilar Almela , María-Victoria Milanés , María-Luisa Laorden; Martínez Laorden, Elena; Farmacología
Open Access
Conditioned aversive memory associated with morphine withdrawal increases brain derived neutrophic factor in dentate gyrus and basolateral amygdala
(2019-07) Navarro-Zaragoza, Javier; Milanés, María-Victoria; Laorden, María-Luisa; Almela, Pilar; Martínez Laorden, Elena; Farmacología
Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of conditioned naloxone-precipitated morphine withdrawal on BDNF and its precursor protein, proBDNF. We used the conditioned-place aversion (CPA) paradigm to evaluate the role of corticotropin-releasing factor (CRF)/CRF1 receptor signalling on the BDNF expression and corticosterone plasma levels after CPA expression and extinction. Male mice were rendered dependent on morphine and injected acutely with naloxone before paired to confinement in a naloxone-associated compartment. The expression of BDNF and proBDNF in the dentate gyrus (DG) and basolateral amygdala (BLA) was measured in parallel with the corticosterone plasma levels with and without CRF1 receptor blockade. Mice subjected to conditioned naloxone-induced morphine-withdrawal showed an increased expression of BDNF (in DG and BLA) in parallel with an enhancement of corticosterone plasma levels. These results demonstrated that BDNF expression together with the increased activity of hypothalamic-pituitary-adrenocortical (HPA) axis are critical to the acquisition of aversive memory. However, we have observed a decrease in corticosterone plasma levels and BDNF expression after CPA extinction reaffirming the importance of BDNF in the maintenance of aversive memory. In addition, the pre- treatment with the CRF1 receptor antagonist CP-154,526 before naloxone conditioning session impaired morphine withdrawal-induced aversive memory acquisition, the increased corticosterone plasma levels and the expression of BDNF observed after CPA expression in the DG and BLA. Altogether present results suggesting a clear connexion between HPA axis and BDNF in the formation and extinction of aversive memory.
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Corticotropin-releasing factor (CRF) receptor-1 is involved in cardiac noradrenergic activity observed during naloxone-precipitated morphine withdrawal
(0010-02-14) Martínez Laorden, Elena; García Carmona, Juan Antonio; Baroja Mazo, Alberto; Romecín, Paola; Atucha, Noemi M; Milanés, María Victoria; Laorden, María Luisa; Farmacología
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Morphine withdrawal activates hypothalamic-pituitary-adrenal axis and heat shock protein 27 in the left ventricle: the role of extracellular signal-regulated kinase
(American Society for Pharmacology and Experimental Therapeutics (ASPET), 2012-05-29) Hurle, M. A.; Milanés, M. V.; Almela Rojo, Pilar; Martínez Laorden, Elena; Farmacología; Facultades de la UMU::Facultad de Medicina
The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [e.g., noradrenergic activity, induction of the hypothalamo-pituitary-adrenocortical (HPA) axis, and the expression and activation of heat shock proteins (Hsps)]. The present study investigated the role of extracellular signal-regulated protein kinase (ERK) and β-adrenoceptor on the response of stress systems to morphine withdrawal by the administration of [amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327), a selective inhibitor of ERK activation, or propranolol (a β-adrenoceptor antagonist). Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by the injection of naloxone (2 mg/kg s.c.). Plasma concentrations of adrenocorticotropin and corticosterone were determined by radioimmunoassay; noradrenaline (NA) turnover in left ventricle was determined by high-performance liquid chromatography; and catechol-O-methyl transferase (COMT) and Hsp27 expression and phosphorylation at Ser82 were determined by quantitative blot immunolabeling. Morphine-withdrawn rats showed an increase of NA turnover and COMT expression in parallel with an enhancement of adrenocorticotropin and plasma corticosterone concentrations. In addition, we observed an enhancement of Hsp27 expression and phosphorylation. Pretreatment with SL327 or propranolol significantly reduced morphine withdrawal-induced increases of plasma adrenocorticotropin and Hsp27 phosphorylation at Ser82 without any changes in plasma corticosterone levels. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of HPA axis in parallel with an enhancement of Hsp27 expression and Hsp27 phosphorylation at Ser82 and suggest a role for β-adrenoceptors and ERK pathways in mediating morphine-withdrawal activation of the HPA axis and cellular stress response.
Open Access
Cambios adaptativos cardiacos durante la dependencia de morfina. Implicación e la proteína de choque térmico Hsp27
(2014-05-19) Martínez Laorden, Elena; Laorden Carrasco, Mª Luisa; Almela Rojo, Pilar; Facultad de Medicina
Actualmente se sabe que hay una estrecha relación entre adicción y desórdenes cardiacos. Sin embargo, se conoce muy poco acerca de los mediadores y mecanismos implicados. Nuestros objetivos han sido: 1. Valorar la actividad simpática cardiaca, determinando los niveles de NA y normetanefrina (NMN). Como posibles mecanismos implicados evaluamos: -la expresión de las dos isoformas de la enzima COMT y - la expresión y fosforilación en serina 40 y 31 de la proteína TH. 2. Evaluar el grado de estrés que se produce durante la adicción a morfina valorando la respuesta del eje HHA y la puesta en marcha de mecanismos cardioprotectores. Se cuantificaron los niveles plasmáticos de ACTH y corticosterona y se valoró la expresión de Hsp27 y la fosforilación de la misma. 3. Investigar un posible nexo de unión entre Hsp27 y troponinas para ello valoramos la expresión de Hsp27, TnT y TnI, así como su co-localización. 4. Valorar el papel de los receptores de CRF1 en la actividad del sistema noradrenérgico cardiaco y del eje HHA. También se valoró la implicación de estos receptores en la expresión y fosforilación de Hsp27. Para ello se utilizó el antagonista selectivo de receptores de CRF1 (CP-154,526) y ratones genéticamente deficientes del receptor de CRF1 (CRF-/-, knockout, KO). Metodología Se han utilizado ratas (Sprague-Dawley) y ratones (B6,129 CRHtklee) macho (WT, CRF1R+/+) y homocigotos recesivos (KO, CRF1R-/-). La dependencia de morfina se indujo mediante la implantación subcutánea de pellets de morfina base (75mg) o mediante inyección i.p. de dosis crecientes de morfina (10-60 mg/kg). Para la precipitación del síndrome de abstinencia se utilizó naloxona (1 o 2 mg/kg, s.c.). El análisis de ACTH y corticosterona se realizó mediante un kit de radioinmunoanálisis (RIA). Se utilizó cromatografía liquida de alta resolución (HPLC) con detector electroquímico para la determinación de NA y NMN. Las proteínas Hsp27 total y fosforilada, las troponinas y la mu-calpaina se determinaron mediante western-blot. Se realizaron experimentos con inmunofluorescencia para determinar Hsp total y las troponinas TnT y TnI; así como para la co-localización de Hsp27 con TnT y TnI. Resultados y conclusiones -Durante el síndrome de abstinencia a morfina se produce un aumento de la expresión de las dos isoformas de COMT, que serían las responsables del aumento de NMN y, por tanto, del turnover de NA. Asimismo, observamos un aumento de la expresión y de la fosforilación de TH . Estos últimos resultados indicarían la puesta en marcha de mecanismos post-transcripcionales. -El síndrome de abstinencia a morfina cursa con una hiperactivación del eje HHA, como indica los elevados niveles plasmáticos de ACTH y corticosterona. Propranolol y SL-327 fueron capaces de antagonizar el incremento de ACTH y no de corticosterona, lo que sugiere que mecanismos independientes de la ACTH hipofisaria mediarían la respuesta de las suprarrenales. -Durante la dependencia de morfina se produce un aumento en la expresión de Hsp27, lo que corrobora que la adicción a opioides provoca un importante estrés celular. Además, nuestro estudio demuestra que los receptores β-adrenérgicos y la vía de señalización ERK estaría implicada en la fosforilación de Hsp27 y, por tanto, en los efectos cardioprotectores que induce esta proteína. Así mismo se evidencia una co-localización entre Hsp27 y cTnT, lo que sugiere la existencia de algún tipo de interacción entre ambas proteínas. -Estos resultados demuestran que el bloqueo selectivo de los receptores de CRF1 y la deleción genética de los receptores de CRF1 antagonizan los cambios adaptativos cardiacos, lo que sugiere que los fármacos antagonistas que tengan como diana el receptor de CRF1 podrían mejorar el estrés y los efectos nocivos cardiacos inducidos durante el síndrome de abstinencia a morfina. ABSTRACT There is a close relationship between addiction and cardiac disorders. However, very little is known about the mediators and mechanisms implicated. Our aims were: 1) To evaluate the cardiac sympathetic activity by determining the levels of NA and normetanephrine (NMN). Asses possible mechanisms involved:-expression of the two isoforms of COMT and - the expression and phosphorylation of TH at serine 40 and 31. 2) To assess the degree of stress that occurs during addiction to morphine assessing the response of the HPA axis and cardioprotective mechanisms. Plasma ACTH and corticosterone levels were quantified and Hsp27 expression and phosphorylation of TH was evaluated. 3) To investigate a possible link between Hsp27 and troponin. For it expression of Hsp27, TnT and TnI, and their co-localization was determined. 4) To evaluate the role of CRF1 receptors in the activity of cardiac noradrenergic system and the HPA axis. The involvement of these receptors in the expression and phosphorylation of Hsp27 was also assessed. Selective CRF1 receptor antagonist (CP-154, 526) and mice genetically deficient CRF1 receptor (CRF-/- knockout, KO) was used. Methods We used rats (Sprague-Dawley) and mice (B6, 129 CRHtklee) male (WT, CRF1R + / +) and homozygous recessive (KO, CRF1R-/ -). Morphine dependence was induced by subcutaneous implantation of pellets of morphine base (75mg) or by ip injection increasing doses of morphine (10-60 mg / kg). For precipitation of withdrawal naloxone (1 or 2 mg / kg, sc) was administered. The ACTH and corticosterone analysis was done using a radioimmunoassay kit (RIA). We used liquid chromatography (HPLC) with electrochemical detector for NA and NMN determination. Total and phosphorylated Hsp27 proteins, troponins and mu-calpain were determined by western-blot. Immunofluorescence experiments were performed to determine the total Hsp TnT and TnI and troponin, as well as for the co-localization of Hsp27 with TnT and TnI. Results and conclusions - During morphine withdrawal we observed an increased expression of the two isoforms of COMT, which are responsible of increased NMN and therefore the NA turnover. We also observed increased expression and phosphorylation of TH. These latter results indicate the involvement of post-transcriptional mechanisms. - Morphine withdrawal induced an activation of the HPA axis, as indicated by elevated plasma levels of ACTH and corticosterone. Propranolol and SL-327 were able to antagonize the increase in ACTH but not in corticosterone, suggesting that separate mechanisms mediate pituitary ACTH adrenal response. - Morphine dependence produced an increased Hsp27 expression, corroborating that opioid addiction causes major cellular stress. Furthermore, our study demonstrates that the β-adrenergic receptors and ERK signaling would be involved in the phosphorylation of HSP27 and therefore in the cardioprotective effects induced by this protein. Also colocalization between Hsp27 and cTnT is evidence suggesting the existence of some interaction between the two proteins. - These results demonstrate that selective blockade of CRF1 receptors and genetic deletion of CRF1 receptors antagonized the cardiac adaptive changes observed during morphine withdrawal, suggesting that CRF1 receptor antagonists may improve stress and harm cardiac induced during morphine withdrawal.
Open Access
Cardiac Protective Role of Heat Shock Protein 27 in the Stress Induced by Drugs of Abuse
(0021-05-20) Javier Navarro-Zaragoza * , María Victoria Milanés, María Luisa Laorden and Pilar Almela; Martínez Laorden, Elena; Farmacología
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Conditioned aversive memory associated with morphinewithdrawal increases brain‐derived neurotrophic factor indentate gyrus and basolateral amygdala
(Wiley, 2019-07-08) Martínez Laorden, Elena; Navarro Zaragoza, Javier; Milanés, María-Victoria; Laorden, María-Luisa; Farmacología
Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of conditioned naloxone-precipitated morphine withdrawal on BDNF and its precursor protein, proBDNF. We used the conditioned place aversion (CPA) paradigm to evaluate the role of corticotropin-releasing factor (CRF)/CRF1 receptor signaling on the BDNF expression and corticosterone plasma levels after CPA expression and extinction. Male mice were rendered dependent on morphine and injected acutely with naloxone before paired to confinement in a naloxone-associated compartment. The expression of BDNF and proBDNF in the dentate gyrus (DG) and basolateral amygdala (BLA) was measured in parallel with the corticosterone plasma levels with and without CRF1 receptor blockade. Mice subjected to conditioned naloxone-induced morphine withdrawal showed an increased expression of BDNF (in DG and BLA) in parallel with an enhancement of corticosterone plasma levels. These results demonstrated that BDNF expression together with the increased activity of hypothalamic-pituitary-adrenocortical (HPA) axis are critical to the acquisition of aversive memory. However, we have observed a decrease in corticosterone plasma levels and BDNF expression after CPA extinction reaffirming the importance of BDNF in the maintenance of aversive memory. In addition, the pre-treatment with the CRF1 receptor antagonist CP-154 526 before naloxone conditioning session impaired morphine withdrawal-induced aversive memory acquisition, the increased corticosterone plasma levels, and the expression of BDNF observed after CPA expression in the DG and BLA. Altogether, present results are suggesting a clear connection between HPA axis and BDNF in the formation and extinction of aversive memory.
Open Access
Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal
(2013-12-27) Almela Rojo, Pilar; García-Carmona, Juan-Antonio; Martínez Laorden, Elena; Milanés, María-Victoria; Laorden, María Luisa; Farmacología
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Naloxone-precipitated morphine withdrawal evokes phosphorylation of heat shock protein 27 in rat heart through extracellular signal-regulated kinase
(Elsevier, 2011-04-17) Almela, P.; Martínez Laorden, Elena; Atucha, N.M.; Milanés, María Victoria; Laorden, María Luisa; Farmacología; Facultades de la UMU
Heat shock protein 27 (Hsp27) is a well-known stress response protein that becomes phosphorylated through extracellular signal-regulated kinase (ERK). Different drugs of abuse, such as morphine and/or its withdrawal, induce severe stress situations. In this study, we investigated Hsp27 and phospho-Hsp27 expression during morphine dependence and withdrawal and evaluated the involvement of ERK in the phosphorylation of Hsp27 in the rat right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg/kg, s.c.). ERK1/2, Hsp27 and phospho-Hsp27 at Ser15 were determined by quantitative blot immunolabeling using specific antibodies. Hsp27 expression was increased 30, 60, 90 and 120 min (144.5 ± 14.2%, P < 0.0001; 128.9 ± 4.6%, P = 0.04; 177.4 ± 12.7, P < 0.0001; and 136.2 ± 11.0%, P = 0.042, respectively) after saline injection to rats dependent on morphine. Naloxone-precipitated morphine withdrawal also increased the phosphorylation of Hsp27 at Ser15 at those time points (146.8 ± 19.8%, P = 0.034; 143.9 ± 17.9%, P = 0.032; 161.2 ± 33.3%, P = 0.029; and 152.2 ± 25.5%, P = 0.008, respectively). However, there were no changes in Hsp27 phosphorylation in the morphine dependent group injected with saline. In addition, there was an increase in the phosphorylation of ERK 60 min after naloxone injection in morphine dependent rats (pERK1: 116.3 ± 4.2%, P = 0.015 and pERK2: 117.2 ± 1.5%, P = 0.05). Pretreatment with SL327, an inhibitor of ERK phosphorylation, decreased activation (phosphorylation) of both ERK and Hsp27 (pERK1: 4.5 ± 3.6%, P < 0.0001; pERK2: 42.3 ± 3.3%, P < 0.0001; and pHsp27: 97.6 ± 1.5%, P = 0.008), suggesting that ERK activation triggers Hsp27 phosphorylation. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of Hsp27 in the heart and suggest that phosphorylation of Hsp27 is closely linked to and also dependent on the ERK pathway.
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Cardiac adverse effects of naloxone-precipitated morphine withdrawal on right ventricle: Role of corticotropin-releasing factor (CRF) 1 receptor
(Elsevier, 2014-01-05) Navarro Zaragoza, Javier; Martínez Laorden, Elena; Mora, L.; Hidalgo, J.; Milanés, María Victoria; Laorden, María luisa; Farmacología
Opioid addiction is associated with cardiovascular disease. However, mechanisms linking opioid addiction and cardiovascular disease remain unclear. This study investigated the role of corticotropin-releasing factor (CRF) 1 receptor in mediating somatic signs and the behavioural states produced during withdrawal from morphine dependence. Furthermore, it studied the efficacy of CRF1 receptor antagonist, CP-154,526 to prevent the cardiac sympathetic activity induced by morphine withdrawal. In addition, tyrosine hydroxylase (TH) phosphorylation pathways were evaluated. Like stress, morphine withdrawal induced an increase in the hypothalamic–pituitary–adrenal (HPA) axis activity and an enhancement of noradrenaline (NA) turnover. Pre-treatment with CRF1 receptor antagonist significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropic hormone (ACTH) levels, NA turnover and TH phosphorylation at Ser31 in the right ventricle. In addition, CP-154,526 reduced the phosphorylation of extracellular signal-regulated kinase (ERK) after naloxone-precipitated morphine withdrawal. In addition, CP-154,526 attenuated the increases in body weight loss during morphine treatment and suppressed some of morphine withdrawal signs. Altogether, these results support the idea that cardiac sympathetic pathways are activated in response to naloxone-precipitated morphine withdrawal suggesting that treatment with a CRF1 receptor antagonist before morphine withdrawal would prevent the development of stress-induced behavioural and autonomic dysfunction in opioid addicts.