RESUMEN

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell viability, modifications of NO, intracellular Ca(2+) concentration [Ca(2+)](i) and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca(2+)](i) immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O(2) hypoxia induces variations of NO and [Ca(2+)](i) with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.

Asunto(s)

Hipoxia de la Célula/fisiología , Neuroblastoma/metabolismo , Oxígeno/farmacología , Adenosina Trifosfato/metabolismo , Aldehídos/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , F2-Isoprostanos/metabolismo , Humanos , Hierro/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Carbonilación Proteica

RESUMEN

Nerve cells are very susceptible to hypoxia responsive for mitochondrial dysfunctions involved in the subsequent oxidative stress, apoptosis and necrosis. In this paper, we examined the effect of 12 h incubation of U-373 MG astrocytes in hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell proliferation, modifications of NO and ATP production, intracellular Ca(2+) concentration [Ca(2+)](i), membrane potential, desferoxamine-chelatable free iron, esterified F2-isoprostanes levels and the production of phosphorylated ERK. The same parameters were evaluated also after a following re-oxygenation period of 24 h. Immediately after hypoxia the NO concentration increased significantly and returned to values similar to those of controls after the re-oxygenation period. At the same time, ATP levels remained similar to controls and the cell proliferation significantly decreased. This involved a significant increase of [Ca(2+)](i) immediately after hypoxia and the value remained significantly elevated after the following re-oxygenation period. Moreover, after hypoxia, astrocytes were slightly although not significantly depolarized. Indeed iron and F2-isoprostanes levels increased significantly after hypoxia. Finally ERK proteins increased slowly and not significantly after hypoxia and the same trend was observed after the re-oxygenation period. On the whole, our results indicate that 2% O(2) hypoxia induces a moderate oxidative stress, well tolerated by U-373 MG cells, remaining the ATP production, mitochondrial membrane potential and activated ERK proteins, similar to the values of controls.

Asunto(s)

Astrocitos/citología , Hipoxia/patología , Oxígeno/administración & dosificación , Adenosina Trifosfato/metabolismo , Astrocitos/enzimología , Astrocitos/metabolismo , Western Blotting , Calcio/metabolismo , Línea Celular , Proliferación Celular , Deferoxamina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quelantes del Hierro/metabolismo , Isoprostanos/metabolismo , Potenciales de la Membrana , Fosforilación

RESUMEN

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.

Asunto(s)

Calcio/fisiología , Calmodulina/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Mitosis , Neuroglía/citología , Neuronas/citología , Óxido Nítrico/fisiología , Animales , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , GMP Cíclico/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , ADN/biosíntesis , Humanos , Espacio Intracelular/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Proteína Quinasa 3 Activada por Mitógenos/biosíntesis , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Ratas , Transducción de Señal

RESUMEN

The translationally controlled tumor protein (TPT1, also known as TCTP) is a highly conserved, abundantly expressed protein found in mammals as well as in a wide range of other organisms of both the animal and plant kingdom. Initially considered as a growth-related protein, later studies showed TPT1 is endowed with multiple biological activities, including calcium binding. The present study aimed to evaluate the expression of TPT1 in the human placenta and to examine the functional role of the protein in the calcium binding and homeostasis of trophoblast cells. Samples were analyzed by Western blot, reverse transcription-polymerase chain reaction and immunohistochemistry. The effect of TPT1 knockdown by small interfering RNA (siRNA) on calcium uptake and buffering was assessed in the HTR-8/SVneo cell line. TPT1 protein and mRNA were detected in first-trimester and term placenta. In the tissue, TPT1 was localized in the villous trophoblast. TPT1 expression significantly increased during gestation, with the higher protein and mRNA levels reached at term. Recombinant placental TPT1 bound calcium in vitro, while downregulation of the protein levels by siRNA in HTR-8/SVneo cells was associated with a reduced cellular calcium-uptake activity and buffering capacity. These data demonstrate, for the first time, the expression of TPT1 in the human placenta and support a direct role of the protein in placental calcium transport.

Asunto(s)

Calcio/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Biomarcadores de Tumor , Western Blotting , Células Cultivadas , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Embarazo , Trimestres del Embarazo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína Tumoral Controlada Traslacionalmente 1

RESUMEN

We investigated whether a combination of static electromagnetic field (EMF) at a flux density of 4.75 T together with pulsed EMF at a flux density of 0.7 mT generated by an NMR apparatus (NMRF), could promote movements of Ca(2+), cell proliferation, and the eventual production of proinflammatory cytokines in human lymphocytes as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 micro g/ml phytohaemagglutinin (PHA) immediately before the exposure period. Our results clearly demonstrate that NMRF exposure increases the [Ca(2+)](i), without any proliferative, or activating, or proinflammatory effect on both normal and PHA stimulated lymphocytes. Accordingly, the levels of interferon gamma, tumor necrosis factor alpha, interleukin-1beta, interleukin-2, and interleukin-6 remained unvaried after exposure. Exposure of Jurkat cells statistically decreased the [Ca(2+)](i) and the proliferation. This is consistent with the low levels of IL-2 measured in supernatants of these cells after exposure. On the whole our data suggest that static and pulsed NMRF exposure contribute synergistically in the increase of the [Ca(2+)](i) without any activating or proinflammatory effect either in normal or in PHA challenged lymphocytes. In Jurkat cells, by changing the properties of cell membranes, NMRF exposure can influence Ca(2+) transport processes and hence Ca(2+) homeostasis, causing a marked decrease of proliferation.

Asunto(s)

Calcio/metabolismo , Citocinas/metabolismo , Campos Electromagnéticos , Linfocitos/fisiología , Linfocitos/efectos de la radiación , Espectroscopía de Resonancia Magnética , División Celular/efectos de los fármacos , División Celular/fisiología , División Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Células Jurkat , Linfocitos/citología , Linfocitos/efectos de los fármacos , Fitohemaglutininas/farmacología , Dosis de Radiación

RESUMEN

(1) Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (TAG), 2-aminoethylarsonic (AEA), 2-hydroxyethanesulfonic (ISE) and (+/-)cis-2-aminocyclohexane sulfonic acids (CAHS) have been shown to displace [(3)H]taurine binding from rabbit brain synaptic membrane preparations, without interacting either with GABA-ergic systems, nor with taurine uptake mechanism, thus behaving like direct taurinergic agents. (2) To answer the question whether the role of taurine as an endogenous cryogen depends on the activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in combination and their effects on rectal (RT)- and ear-skin temperature and gross motor behavior (GMB) were monitored. (3) Taurine (1.2 x 10(-6)-4.8 x 10(-5) mol) induced a dose-related hypothermia, vasodilation at ear vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 x 10(-5) mol) induced a taurine-like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine, induced a dose-related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused a dose-related hyperthermia, but at doses higher than 1.2 x 10(-7) mol caused death within 24 h after treatment. (4) CAHS (4.8 x 10(-5) mol) antagonized the hyperthermic effect induced by TAG (1.2 x 10(-6) mol), AEA (1.2 x 10(-8) mol) or ISE (4.8 x 10(-5) mol). (5) In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation.

Asunto(s)

Regulación de la Temperatura Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Taurina/metabolismo , Taurina/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Regulación de la Temperatura Corporal/fisiología , Encéfalo/metabolismo , Inyecciones Intraventriculares , Masculino , Conejos

RESUMEN

1. The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. 2. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [(3)H]muscimol. Saturation experiments of the binding of [(3)H]GABA to GABA(B) receptors showed that SM possess two binding components; twice Triton X-100-treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [(3)H]taurine in a saturable manner (K(d)=249.0+/-6.3 nM and B(max)=3.4+/-1.0 pmol mg(-1) prot). 3. Among the 19 structural analogues of taurine, 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (TAG), 2-aminoethylarsonic (AEA), 2-hydroxyethanesulfonic (ISE) and (+/-)cis-2-aminocyclohexane sulfonic acids (CAHS) displaced [(3)H]taurine binding (K(i)=0.13, 0.13, 13.5 and 4.0 micro M, respectively). These analogues did not interact with GABA(A) and GABA(B) receptors and did not affect taurine- and GABA-uptake systems and GABA-transaminase activity. 4. 3-Aminopropanesulfonic acid (OMO), beta-alanine, pyridine-3-sulfonic acid, N,N,N-trimethyltaurine (TMT), 2-(guanidino)ethanesulfonic acid (GES), ethanolamine-O-sulphate, N,N-dimethyltaurine (DMT), taurine and (+/-)piperidine-3-sulfonic acid (PSA) inhibited [(3)H]muscimol binding to GABA(A) receptors with different affinities (K(i)=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 micro M, respectively). Taurine, 2-aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [(3)H]GABA to GABA(B) receptors with K(i)'s in the micro M range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC(50)=3.72 micro M) and PSA GABA transaminase activity (IC(50)=103.0 micro M). 5. In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Asunto(s)

Encéfalo/metabolismo , Taurina/análogos & derivados , Taurina/farmacocinética , Ácido gamma-Aminobutírico/farmacocinética , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , 4-Aminobutirato Transaminasa/metabolismo , Animales , Sitios de Unión/fisiología , Interacciones Farmacológicas , Isomerismo , Estructura Molecular , Muscimol/farmacocinética , Piperidinas/metabolismo , Piperidinas/farmacología , Conejos , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Relación Estructura-Actividad , Membranas Sinápticas/química , Sinaptosomas/metabolismo , Taurina/síntesis química , Tritio

RESUMEN

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.

Asunto(s)

Calcio/metabolismo , Cuerpo Estriado/metabolismo , Fura-2/análogos & derivados , Hipoxia/metabolismo , Óxido Nítrico/metabolismo , Animales , Anticoagulantes/farmacología , Astrocitoma , Línea Celular Tumoral , Cuerpo Estriado/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Fura-2/metabolismo , Heparina/farmacología , Humanos , Hidroliasas/metabolismo , Hipoxia/fisiopatología , Imidazoles/farmacología , Técnicas In Vitro , Espacio Intracelular/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Perfusión/métodos , Ratas , Ratas Sprague-Dawley , Rutenio/farmacología , Rianodina/farmacología

RESUMEN

We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation.

Asunto(s)

Campos Electromagnéticos , Células Jurkat/efectos de la radiación , Linfocitos/efectos de la radiación , Cafeína/farmacología , Calcio/metabolismo , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Células Cultivadas , Citocinas/metabolismo , Humanos , Células Jurkat/citología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/efectos de la radiación , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Espectroscopía de Resonancia Magnética/efectos adversos , Exposición Profesional , Fitohemaglutininas/farmacología , Traumatismos por Radiación/etiología , Valores de Referencia

RESUMEN

Interleukin-1beta (IL-1beta) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca2+ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1beta and other pyrogens. Data from different labs indicate that Ca2+ and Na+ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca2+ and PGE2 concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1beta-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204-212) peptide counteract IL-1beta-induced fever and abolish changes in Ca2+ and PGE2 concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca2+ mobilization in response to IL-1beta and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP3)-sensitive pools are the main source of the mobilized Ca2+. It is concluded that the NO/cGMP/Ca2+ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1beta.

Asunto(s)

Señalización del Calcio/fisiología , GMP Cíclico/fisiología , Interleucina-1/fisiología , Óxido Nítrico/fisiología , Pirógenos/fisiología , Animales , Humanos