María Cano Abad | Universidad Autónoma de Madrid (original) (raw)
Papers by María Cano Abad
Journal of Polymer Science Part B: Polymer Physics, 1996
The miscibility of polyetherimides (PEIs) with epoxy monomers based on diglycidylether of bisphen... more The miscibility of polyetherimides (PEIs) with epoxy monomers based on diglycidylether of bisphenol-A (DGEBA), and with reactive mixtures based on stoichiometric amounts of DGEBA and a n aromatic diamine (DA) {either 4,4'-diaminodiphenylsulfone (DDS) or 4,4'methylenebis[3-chloro 2,6-diethylaniline] (MCDEA)}; was experimentally studied. Cloudpoint curves (temperature vs. composition) are reported for PEI-DGEBA and PEI-DGEBA-DA initial mixtures. Cloud-point conversions are reported for the reactive mixtures, for various PEI amounts and polycondensation temperatures. A thermodynamic model based on the Flory-Huggins-Staverman approach, taking polydispersity of both components into account, was used to analyze the experimental information. A single relationship between the interaction parameter and temperature, X(T), could fit experimental results of mixtures of two commercial PEIs with DGEBA. The addition of DDS led to a decrease in miscibility whereas MCDEA improved the initial miscibility. In both cases, the interaction parameter decreased with conversion, meaning that PEI was more compatible with oligomeric species than with the mixture of starting monomers. The phase separation process in initially miscible rubberor thermoplastic-modified thermosetting polymers is the result of two factors: increase in the average molar size of the thermosetting oligomer (main driving force favoring demixing), and variation of the interaction parameter with conversion, which may act to increase or decrease the cloud-point conversion determined by the first factor.
American journal of physiology. Cell physiology, 1998
Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time... more Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from ECvalues, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Caconcentration ([Ca]), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake ofCa, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Caentry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca]. Veratridine induced oscillations of cytosolic Caconcentration ([Ca]) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal...
Advances in Pharmacology, 1997
Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... BM Olivera, G. Miljanich, J. Ramachandran and ME Adams, Calcium channel diversity and neurotransmitter release: The oM-conotoxins and ...
Cell Calcium, 2011
The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasma... more The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.
En la presente tesis nos planteamos esclarecer los mecanismos basicos de la muerte celular tras u... more En la presente tesis nos planteamos esclarecer los mecanismos basicos de la muerte celular tras un episodio isquemico cerebral y el posible rescate farmacologico. Es bien sabido que la sobrecarga de CA2+ produce muerte celular. En la isquimia, la falta de oxigeno y de glucosa, conduce a una despolarizacion incontrolada neuronal, liberandose a la hendidura sinaptica aminoacidos excitadores, tales como glutamato, que al interaccionar con sus receptores de membrana posinaptica cierran un circulo vicioso de hiperexcitabilidad neuronal donde el Ca2+ accede al interior celular a traves de los receptores de NMDA, canales de Ca2+ voltaje accede al interior celular a traves de los receptores de NMDA, canales de CA2+ voltaje dependiente (CCVD), de las bombas de Ca2+ trabajanod en sentido reverso y por el vaciamiento de los depositos intracelulares de Ca2+. Como modelo celular hemos empleado, la celula cromafin bovina. Esta expresa CCVD del tipo neuronal, L, N, P/Q, por lo que es un excelente ...
Molecular Pharmacology, 2003
We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence correspond... more We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 M; half-maximal current at 4 M) elicited inward currents at Ϫ60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at ϩ30 mV than at Ϫ60 mV. 2Ј,3Ј-O-(4-Benzoyl)-benzoyl-ATP and ␣-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 M, respectively). The currents were inhibited by suramin, pyr-idoxal-5-phosphate-6-azo-2Ј,4Ј-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 M, respectively; 2Ј,3Ј-O-(2Ј,4Ј,6Ј-trinitrophenol)-ATP (1 M) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X 5 receptor was permeable to calcium (P Ca /P Na ϭ 1.5) and N-methyl-D-glucamine (NMDG) (P NMDG /P Na ϭ 0.4); it was also permeable to chloride (P Cl /P Na ϭ 0.5) but not gluconate (P gluc /P Na ϭ 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X 7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X 5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP. A P2X 5 subunit cDNA was first isolated from libraries of rat sympathetic ganglia (Collo et al., 1996) and heart (Garcia-Guzman et al., 1996). The RNA is expressed predominantly in heart (Garcia-Guzman et al., 1996) but there is also expression in brain, spinal cord, and adrenal gland (Garcia-Guzman et al., 1996). Immunoreactivity for P2X 5 subunits has also been described in developing skeletal muscle of the rat (Meyer et al., 1999; Ryten et al., 2001); recently, the receptor has been implicated in the differentiation of satellite cells into mature multinucleated muscle fibers (Ryten et al., 2002). Mammalian skin (Groschel-Stewart et al., 1999) and endocrine (Glass and Burnstock, 2001) cells also show P2X 5 immunoreactivity.
Los digitalicos son incuestionablemente uno de los farmacos descubiertos mas valiosos de la Farma... more Los digitalicos son incuestionablemente uno de los farmacos descubiertos mas valiosos de la Farmacopea. La introduccion de la Digitalis fue el punto de referencia en la historia de la terapeutica de las enfermedades del corazon.
En la ultima edicion de premios Nobel en el area de Quimica han sido premiados los doctores Osamu... more En la ultima edicion de premios Nobel en el area de Quimica han sido premiados los doctores Osamu Shimomura, Martin Chalfie y Roger Y. Tsien (Figura 1). Tres cientificos dedicados al estudio de proteinas bioluminiscentes procedentes de una medusa llamada Aequorea victoria. El ingenio de estos cientificos ha revolucionado el modo de observar a la celula. La proteina fluorescente verde aislada por Shimomura, �Green Fluorescence Protein� (GFP) en la decada de los 60, llevo a Chalfie a la brillante idea de dirigirla geneticamente a las neuronas del gusano Caenorhabditis elegans. Tsien modifico el cromoforo de la GFP y creo una amplia gama de colores, permitiendo a todos los cientificos del mundo monitorizar eventos intracelulares, invisibles hasta el momento.
American journal of physiology. Cell physiology, 1998
Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time... more Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from ECvalues, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Caconcentration ([Ca]), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake ofCa, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Caentry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca]. Veratridine induced oscillations of cytosolic Caconcentration ([Ca]) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal...
Neuropharmacology, Jan 31, 2004
Galantamine is currently used to treat Alzheimer’s disease patients; it behaves as a mild blocker... more Galantamine is currently used to treat Alzheimer’s disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide β-amyloid1–40 and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against β-amyloid1–40 or thapsigargin-induced toxicity, was reversed by α-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of α7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer’s disease.
Advances in Pharmacology, 1997
Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... BM Olivera, G. Miljanich, J. Ramachandran and ME Adams, Calcium channel diversity and neurotransmitter release: The oM-conotoxins and ...
Journal of Biological Chemistry, 2004
We here describe a new molecularly engineered green fluorescent protein chimera that shows a high... more We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK a (pK a) around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca 2؉ ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca 2؉ accumulation by this organelle. Finally, we demonstrate that Ca 2؉ mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.
Cell Calcium, 2011
The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasma... more The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the ...
The Journal of pharmacology and experimental therapeutics, 2012
The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+)... more The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was...
Cell Calcium, 2008
Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked t... more Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.
The novel Ca 2+ channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca 2+ transi... more The novel Ca 2+ channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca 2+ transients ([Ca 2+ ] c) that regulate the production of amyloid beta (Ab). Its mutated channel P86L-CALHM1 has been associated to Alzheimer's disease (AD). Using cytosolic-and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca 2+ entering into Hela cells and the Ca 2+ released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca 2+ entry in the three cell types; however, the [Ca 2+ ] c and mitochondrial Ca 2+ transients [Ca 2+ ] m had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca 2+ released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca 2+ overload, suggesting how these cells may become more vulnerable to apoptotic stimuli.
…, Jan 1, 2004
Galantamine is currently used to treat Alzheimer&... more Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.
Journal of Polymer Science Part B: Polymer Physics, 1996
The miscibility of polyetherimides (PEIs) with epoxy monomers based on diglycidylether of bisphen... more The miscibility of polyetherimides (PEIs) with epoxy monomers based on diglycidylether of bisphenol-A (DGEBA), and with reactive mixtures based on stoichiometric amounts of DGEBA and a n aromatic diamine (DA) {either 4,4'-diaminodiphenylsulfone (DDS) or 4,4'methylenebis[3-chloro 2,6-diethylaniline] (MCDEA)}; was experimentally studied. Cloudpoint curves (temperature vs. composition) are reported for PEI-DGEBA and PEI-DGEBA-DA initial mixtures. Cloud-point conversions are reported for the reactive mixtures, for various PEI amounts and polycondensation temperatures. A thermodynamic model based on the Flory-Huggins-Staverman approach, taking polydispersity of both components into account, was used to analyze the experimental information. A single relationship between the interaction parameter and temperature, X(T), could fit experimental results of mixtures of two commercial PEIs with DGEBA. The addition of DDS led to a decrease in miscibility whereas MCDEA improved the initial miscibility. In both cases, the interaction parameter decreased with conversion, meaning that PEI was more compatible with oligomeric species than with the mixture of starting monomers. The phase separation process in initially miscible rubberor thermoplastic-modified thermosetting polymers is the result of two factors: increase in the average molar size of the thermosetting oligomer (main driving force favoring demixing), and variation of the interaction parameter with conversion, which may act to increase or decrease the cloud-point conversion determined by the first factor.
American journal of physiology. Cell physiology, 1998
Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time... more Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from ECvalues, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Caconcentration ([Ca]), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake ofCa, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Caentry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca]. Veratridine induced oscillations of cytosolic Caconcentration ([Ca]) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal...
Advances in Pharmacology, 1997
Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... BM Olivera, G. Miljanich, J. Ramachandran and ME Adams, Calcium channel diversity and neurotransmitter release: The oM-conotoxins and ...
Cell Calcium, 2011
The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasma... more The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.
En la presente tesis nos planteamos esclarecer los mecanismos basicos de la muerte celular tras u... more En la presente tesis nos planteamos esclarecer los mecanismos basicos de la muerte celular tras un episodio isquemico cerebral y el posible rescate farmacologico. Es bien sabido que la sobrecarga de CA2+ produce muerte celular. En la isquimia, la falta de oxigeno y de glucosa, conduce a una despolarizacion incontrolada neuronal, liberandose a la hendidura sinaptica aminoacidos excitadores, tales como glutamato, que al interaccionar con sus receptores de membrana posinaptica cierran un circulo vicioso de hiperexcitabilidad neuronal donde el Ca2+ accede al interior celular a traves de los receptores de NMDA, canales de Ca2+ voltaje accede al interior celular a traves de los receptores de NMDA, canales de CA2+ voltaje dependiente (CCVD), de las bombas de Ca2+ trabajanod en sentido reverso y por el vaciamiento de los depositos intracelulares de Ca2+. Como modelo celular hemos empleado, la celula cromafin bovina. Esta expresa CCVD del tipo neuronal, L, N, P/Q, por lo que es un excelente ...
Molecular Pharmacology, 2003
We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence correspond... more We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 M; half-maximal current at 4 M) elicited inward currents at Ϫ60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at ϩ30 mV than at Ϫ60 mV. 2Ј,3Ј-O-(4-Benzoyl)-benzoyl-ATP and ␣-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 M, respectively). The currents were inhibited by suramin, pyr-idoxal-5-phosphate-6-azo-2Ј,4Ј-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 M, respectively; 2Ј,3Ј-O-(2Ј,4Ј,6Ј-trinitrophenol)-ATP (1 M) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X 5 receptor was permeable to calcium (P Ca /P Na ϭ 1.5) and N-methyl-D-glucamine (NMDG) (P NMDG /P Na ϭ 0.4); it was also permeable to chloride (P Cl /P Na ϭ 0.5) but not gluconate (P gluc /P Na ϭ 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X 7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X 5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP. A P2X 5 subunit cDNA was first isolated from libraries of rat sympathetic ganglia (Collo et al., 1996) and heart (Garcia-Guzman et al., 1996). The RNA is expressed predominantly in heart (Garcia-Guzman et al., 1996) but there is also expression in brain, spinal cord, and adrenal gland (Garcia-Guzman et al., 1996). Immunoreactivity for P2X 5 subunits has also been described in developing skeletal muscle of the rat (Meyer et al., 1999; Ryten et al., 2001); recently, the receptor has been implicated in the differentiation of satellite cells into mature multinucleated muscle fibers (Ryten et al., 2002). Mammalian skin (Groschel-Stewart et al., 1999) and endocrine (Glass and Burnstock, 2001) cells also show P2X 5 immunoreactivity.
Los digitalicos son incuestionablemente uno de los farmacos descubiertos mas valiosos de la Farma... more Los digitalicos son incuestionablemente uno de los farmacos descubiertos mas valiosos de la Farmacopea. La introduccion de la Digitalis fue el punto de referencia en la historia de la terapeutica de las enfermedades del corazon.
En la ultima edicion de premios Nobel en el area de Quimica han sido premiados los doctores Osamu... more En la ultima edicion de premios Nobel en el area de Quimica han sido premiados los doctores Osamu Shimomura, Martin Chalfie y Roger Y. Tsien (Figura 1). Tres cientificos dedicados al estudio de proteinas bioluminiscentes procedentes de una medusa llamada Aequorea victoria. El ingenio de estos cientificos ha revolucionado el modo de observar a la celula. La proteina fluorescente verde aislada por Shimomura, �Green Fluorescence Protein� (GFP) en la decada de los 60, llevo a Chalfie a la brillante idea de dirigirla geneticamente a las neuronas del gusano Caenorhabditis elegans. Tsien modifico el cromoforo de la GFP y creo una amplia gama de colores, permitiendo a todos los cientificos del mundo monitorizar eventos intracelulares, invisibles hasta el momento.
American journal of physiology. Cell physiology, 1998
Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time... more Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from ECvalues, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Caconcentration ([Ca]), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake ofCa, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Caentry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca]. Veratridine induced oscillations of cytosolic Caconcentration ([Ca]) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal...
Neuropharmacology, Jan 31, 2004
Galantamine is currently used to treat Alzheimer’s disease patients; it behaves as a mild blocker... more Galantamine is currently used to treat Alzheimer’s disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide β-amyloid1–40 and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against β-amyloid1–40 or thapsigargin-induced toxicity, was reversed by α-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of α7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer’s disease.
Advances in Pharmacology, 1997
Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for ... more Note: This is a one-page preview only. Click here to download preview. ... Enable JavaScript for PDF Excerpt to view it inline. ... BM Olivera, G. Miljanich, J. Ramachandran and ME Adams, Calcium channel diversity and neurotransmitter release: The oM-conotoxins and ...
Journal of Biological Chemistry, 2004
We here describe a new molecularly engineered green fluorescent protein chimera that shows a high... more We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK a (pK a) around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca 2؉ ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca 2؉ accumulation by this organelle. Finally, we demonstrate that Ca 2؉ mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.
Cell Calcium, 2011
The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasma... more The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the ...
The Journal of pharmacology and experimental therapeutics, 2012
The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+)... more The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was...
Cell Calcium, 2008
Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked t... more Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.
The novel Ca 2+ channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca 2+ transi... more The novel Ca 2+ channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca 2+ transients ([Ca 2+ ] c) that regulate the production of amyloid beta (Ab). Its mutated channel P86L-CALHM1 has been associated to Alzheimer's disease (AD). Using cytosolic-and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca 2+ entering into Hela cells and the Ca 2+ released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca 2+ entry in the three cell types; however, the [Ca 2+ ] c and mitochondrial Ca 2+ transients [Ca 2+ ] m had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca 2+ released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca 2+ overload, suggesting how these cells may become more vulnerable to apoptotic stimuli.
…, Jan 1, 2004
Galantamine is currently used to treat Alzheimer&... more Galantamine is currently used to treat Alzheimer's disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimer's disease.