Browsing by Author "Gutierrez-Merino, Carlos"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- Actin cytoskeleton disruption is an early event upon exposure of cerebellar granule neurons to SIN-1-induced oxidative stressPublication . Tiago, Teresa; Silva, D.; Samhan-Arias, A. K.; Aureliano, Manuel; Gutierrez-Merino, CarlosIn this work we have studied the alterations of the actin cytoskeleton in cultured cerebellar granule neurons during exposure to the peroxynitritereleasing agent SIN-1 for less than 2 hours. Actin polymerization state was assessed by fluorescence microscopy ratio images using double labelling for actin filaments (phallacidin) and monomers (DNase-I). In addition, agonists and antagonists of L-type Ca2+ channels and NMDA receptors were used in order to find out whether these compounds were able to attenuate or potentiate the effects of oxidative stress on the perturbation of the actin cytoskeleton. The results reveal that a flux of peroxynitrite as low as 0.5 ;M/min during 1h is sufficient to promote alterations of actin dynamics leading to partial actin cytoskeleton disruption and suggest that this is an early event linked to cytosolic calcium concentration changes.
- Gold compounds inhibit the Ca2+-ATPase activity of brain PMCA and human neuroblastoma SH-SY5Y cells and decrease cell viabilityPublication . Berrocal, Maria; Cordoba-Granados, Juan J.; Carabineiro, Sónia A. C.; Gutierrez-Merino, Carlos; Aureliano, Manuel; Mata, Ana M.Plasma membrane calcium ATPases (PMCA) are key proteins in the maintenance of calcium (Ca2+) homeostasis. Dysregulation of PMCA function is associated with several human pathologies, including neurodegenerative diseases, and, therefore, these proteins are potential drug targets to counteract those diseases. Gold compounds, namely of Au(I), are well-known for their therapeutic use in rheumatoid arthritis and other diseases for centuries. Herein, we report the ability of dichloro(2-pyridinecarboxylate)gold(III) (1), chlorotrimethylphosphinegold(I) (2), 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidenegold(I) chloride (3), and chlorotriphenylphosphinegold(I) (4) compounds to interfere with the Ca2+-ATPase activity of pig brain purified PMCA and with membranes from SH-SY5Y neuroblastoma cell cultures. The Au(III) compound (1) inhibits PMCA activity with the IC50 value of 4.9 µM, while Au(I) compounds (2, 3, and 4) inhibit the protein activity with IC50 values of 2.8, 21, and 0.9 µM, respectively. Regarding the native substrate MgATP, gold compounds 1 and 4 showed a non-competitive type of inhibition, whereas compounds 2 and 3 showed a mixed type of inhibition. All gold complexes showed cytotoxic effects on human neuroblastoma SH-SY5Y cells, although compounds 1 and 3 were more cytotoxic than compounds 2 and 4. In summary, this work shows that both Au (I and III) compounds are high-affinity inhibitors of the Ca2+-ATPase activity in purified PMCA fractions and in membranes from SH-SY5Y human neuroblastoma cells. Additionally, they exert strong cytotoxic effects.
- Inhibition of SERCA and PMCA Ca2+-ATPase activities by polyoxotungstatesPublication . Aureliano, Manuel; Fraqueza, Gil; Berrocal, Maria; Cordoba-Granados, Juan J.; Gumerova, Nadiia I.; Rompel, Annette; Gutierrez-Merino, Carlos; Mata, Ana M.Plasma membrane calcium ATPases (PMCA) and sarco(endo) reticulum calcium ATPases (SERCA) are key proteins in the maintenance of calcium homeostasis. Herein, we compare for the first time the inhibition of SERCA and PMCA calcium pumps by several polyoxotungstates (POTs), namely by Wells-Dawson phospho-tungstate anions [P2W18O62]6-(intact, {P2W18}), [P2W17O61]10-(monolacunary, {P2W17}), [P2W15O56]12-(trilacunary, {P2W15}), [H2P2W12O48]12-(hexalacunary, {P2W12}), [H3P2W15V3O62]6- (trivanadium-substituted, {P2W15V3}) and by Preyssler-type anion [NaP5W30O110]14-({P5W30}). The speciation in the solu-tions of tested POTs was investigated by 31P and 51V NMR spectroscopy. The tested POTs inhibited SERCA Ca2+- ATPase activity, whereby the Preyssler POT showed the strongest effect, with an IC50 value of 0.37 mu M. For {P2W17} and {P2W15V3} higher IC50 values were determined: 0.72 and 0.95 mu M, respectively. The studied POTs showed to be more potent inhibitors of PMCA Ca2+-ATPase activity, with lower IC50 values for {P2W17}, {P5W30} and {P2W15V3}.
- Unveiling the agonistic properties of Preyssler-type Polyoxotungstates on purinergic P2 receptorsPublication . Poejo, Joana; Gumerova, Nadiia I.; Rompel, Annette; Mata, Ana M.; Aureliano, Manuel; Gutierrez-Merino, CarlosThe Preyssler-type polyoxotungstate ({P5W30}) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P5W30} on the cytosolic Ca2+ concentration. Changes in cytosolic Ca2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca2+ indicator Fluo3. 31P-Nuclear magnetic resonance measurements of {P5W30} indicate its stability in the medium used for cytosolic Ca2+ measurements for over 30 min. The findings reveal that addition of {P5W30} to the extracellular medium induces a sustained increase of the cytosolic Ca2+ concentration within minutes. This Ca2+ increase is triggered by extracellular Ca2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 μM {P5W30}. In addition, after the {P5W30}-induced cytosolic Ca2+ increase, the transient Ca2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 μM {P5W30}. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P5W30} in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P5W30} could serve as a novel agonist of purinergic P2 receptors.