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Biomedical subjects

V Ceña

Publications and source records attributed to V Ceña.

At least 19 recordsLinked to original sources
PubMed2005

Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions.

Minocycline, a semisynthetic derivative of tetracycline, displays beneficial activity in neuroprotective in models including, Parkinson disease, spinal cord injury, amyotrophic lateral sclerosis, Huntington disease and stroke. The mechanisms by which minocycline inhibits apoptosis remain poorly understood. In the present report we have investigated the effects of minocycline on mitochondria, due to their crucial role in apoptotic pathways. In mitochondria isolated suspensions, minocycline failed to block superoxide-induced swelling but was effective in blocking mitochondrial swelling induced by calcium. This latter effect might be mediated through dissipation of mitochondrial transmembrane potential and blockade of mitochondrial calcium uptake. Consistently, minocycline fails to protect SH-SY5Y cell cultures against reactive oxygen species-mediated cell death, including malonate and 6-hydroxydopamine treatments, but it is effective against staurosporine-induced cytotoxicity. The effects of this antibiotic on mitochondrial respiratory chain complex were also analyzed. Minocycline did not modify complex IV activity, and only at the higher concentration tested (100 microM) inhibited complex II/III activity. Other members of the minocycline antibiotic family like tetracycline failed to induce these mitochondrial effects.

Animals↗
PubMed2004

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture.

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the alpha1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.

Adrenal Medulla↗
PubMed2003

Mitochondrial control of neuron death and its role in neurodegenerative disorders.

Genetic or functional mitochondrial alterations can result in the initiation of cell death programs that are believed to contribute to cell death in diabetes, ageing and neurodegenerative disorders. Mitochondria are being considered the main link between cellular stress signals activated during acute and chronic nerve cell injury, and the execution of nerve cell death. This second function of mitochondria is regulated by several families of proteins that can trigger an increase in permeability of the outer and/or inner mitochondrial membrane. One example of this is the formation of the mitochondrial permeability transition pore (MPTP). This process can trigger the release of cell death-inducing factors from mitochondria, as well as a dissipation of the mitochondrial transmembrane potential, depletion of ATP, and increased free radical formation. Among the factors released from mitochondria are cytochrome c, the apoptosis inductor factor (AIF), and caspases. We review the role of the MPTP in diverse physiological and pathological processes, including neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS). The design of drugs that could interfere with the functions of the MPTP could allow novel therapeutic approaches for the treatment of acute and chronic nerve cell injury.

Cell Death↗
PubMed2003

Role and regulation of p53 in depolarization-induced neuronal death.

The tumor suppressor gene p53 is a potent transcriptional regulator for genes involved in many cellular activities including cell cycle arrest and apoptosis. In this study, we examined the role of p53 in neuronal death induced by the sodium channel modulator veratridine. We also analyzed the involvement of Ca2+, mitochondria and reactive oxygen species in p53 activation. Exposure of hippocampal neurons to veratridine (0.3-100 microM) resulted in a dose-dependent neuronal death, measured 24 h after treatment. p53-Like immunoreactivity, undetectable in neurons under control conditions, was observed in about 25% of neurons, 7 h after veratridine exposure. Treatments that modified the alkaloid-induced Ca2+ influx including tetrodotoxin or Ca2+ removal, prevented either veratridine-induced cell death or p53 immunoreactivity. Mitochondria were involved in veratridine-induced cell death, as the alkaloid collapsed inner transmembrane mitochondrial potential in a Ca2+ influx dependent manner. Treatments of neuronal cultures with the permeability transitory pore blockers cyclosporin A and bongkrekic acid prevented veratridine-induced p53 immunoreactivity and neuronal death, placing mitochondria upstream of veratridine-induced p53 immunoreactivity. Reactive oxygen species also participated in veratridine-induced neurotoxicity and p53 activation. Antisense knockdown of p53 resulted in a significant increase in neuronal survival after veratridine treatment. This protective effect was maintained on N-methyl-D-aspartate or ischemia-induced death but not on staurosporine cytotoxicity. These results together suggest that p53-expression is involved in veratridine-induced neuronal death and that p53 might be a link between toxic stimuli of different types and neuronal death.

Animals↗
PubMed2001

Naloxone inhibits nicotine-induced receptor current and catecholamine secretion in bovine chromaffin cells.

Nicotine-induced catecholamine (CA) secretion and inward ionic currents were inhibited by the opioid antagonist naloxone in cultured bovine chromaffin cells. Naloxone inhibited nicotine-induced CA secretion, as detected by an on-line real-time electrochemical technique, in a dose-dependent manner (IC(50)=29 microM). In voltage-clamped chromaffin cells, nicotine (10 microM) evoked an average peak inward current of -146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 microM). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 microM). This provides strong evidence that naloxone modulation of nicotine-induced CA secretion does not involve opioid receptors but results from the direct interaction with the nicotinic receptor itself, which might also be the case for other related opioid compounds.

Animals↗
PubMed2001

Glutamate N-methyl-D-aspartate receptor blockade prevents induction of GAP-43 after focal ischemia in rats.

Growth associated protein-43 (GAP-43) gene induction may be involved in reactive events that follow cerebral ischemic damage. Antagonists of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors are thought to ameliorate functional outcome after ischemic injury. To assess whether glutamate NMDA receptor blockade could alter GAP-43 postischemic induction we performed immunocytochemistry in rat brains that had been subjected to middle cerebral artery occlusion. Cortical cells did not constitutively express GAP-43, yet focal ischemia induced its expression, with an intense signal generated in cells over the lesioned area at 6 h, increasing at 24 h postischemia. This signal was effectively decreased by pretreatment with the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (0.1 mg/kg s.c.), but not by the glutamate release blocker riluzole (8 mg/kg i.v.), suggesting that overactivation of NMDA receptor during ischemia is linked to GAP-43 expression.

Animals↗
PubMed2001

An activity-dependent switch from facilitation to inhibition in the control of excitotoxicity by group I metabotropic glutamate receptors.

Activation of group I metabotropic glutamate receptors (mGlu1 or -5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a "neuroprotective mode". The characteristics of the activity-dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1-45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype-selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity-dependent switch in regulating excitotoxic neuronal death and, therefore, the recent "history" of these receptors is critical for the response to agonists or antagonists.

Animals↗
PubMed2000

Veratridine induces apoptotic death in bovine chromaffin cells through superoxide production.

The molecular mechanisms involved in veratridine-induced chromaffin cell death have been explored. We have found that exposure to veratridine (30 microM, 1 h) produces a delayed cellular death that reaches 55% of the cells 24 h after veratridine exposure. This death has the features of apoptosis as DNA fragmentation can be observed. Calcium ions play an important role in veratridine-induced chromaffin cell death because the cell permeant Ca(2+) chelator BAPTA-AM and extracellular Ca(2+) removal completely prevented veratridine-induced toxicity. Following veratridine treatment, there is a decrease in mitochondrial function and an increase in superoxide anion production. Veratridine-induced increase in superoxide production was blocked by tetrodotoxin (TTX; 10 microM), extracellular Ca(2+) removal and the mitochondrial permeability transition pore blocker cyclosporine A (10 microM). Veratridine-induced death was prevented by different antioxidant treatments including catalase (100 IU ml(-1)), N-acetyl cysteine (100 microM), allopurinol (100 microM) or vitamin E (50 microM). Veratridine-induced DNA fragmentation was prevented by TTX (10 microM). Veratridine produced a time-dependent increase in caspase activity that was prevented by Ca(2+) removal and TTX (10 microM). In addition, calpain and caspases inhibitors partially prevented veratridine-induced death. These results indicate that chromaffin cells share with neurons the molecular machinery involved in apoptotic death and might be considered a good model to study neuronal death during neurodegeneration.

Animals↗
PubMed2000

Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells.

A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Apoptosis↗
PubMed1999

Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection.

The role of group-I metabotropic glutamate receptors (mGlu1 and 5) in neurodegeneration is still controversial. While antagonists of these receptors are consistently neuroprotective, agonists have been found to either amplify or attenuate excitotoxic neuronal death. At least three variables affect responses to agonists: (i) the presence of the NR2C subunit in the NMDA receptor complex; (ii) the existence of an activity-dependent functional switch of group-I mGlu receptors, similar to that described for the regulation of glutamate release; and (iii) the presence of astrocytes expressing mGlu5 receptors. Thus, a number of factors, including the heteromeric composition of NMDA receptors, the exposure time to drugs or to ambient glutamate, and the function of astrocytes clearing extracellular glutamate and producing neurotoxic or neuroprotective factors, must be taken into account when examining the role of group-I mGlu receptors in neurodegeneration/neuroprotection.

Animals↗
PubMed1997

Tricyclic antidepressants block cholinergic nicotinic receptors and ATP secretion in bovine chromaffin cells.

Nicotine-induced ATP secretion from chromaffin cells was blocked by imipramine and desipramine. This blocking action took place on both, fast and slow, components of ATP secretion. Exposure of chromaffin cells to nicotine (10 microM) for 4 s induced an inward current of about -155 pA. Imipramine and desipramine blocked, in a concentration-dependent manner, both peak inward current and total charge influx in response to nicotine. In addition, imipramine and desipramine partially (40%) blocked depolarization-induced ATP secretion and Ca2+ currents evoked by high K+. This suggests that tricyclic antidepressants block nicotine-induced ATP secretion by acting on two targets: one is the nicotinic receptor itself and the second one are voltage-dependent Ca2+ channels.

Adenosine Triphosphate↗
PubMed1996

Extracellular calcium has distinct effects on fast and slow components of the depolarization-induced secretory response from chromaffin cells.

An increase in extracellular Ca2+ concentration from 0.25 to 10 mM enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K+ concentration (75 mM). The increment in extracellular Ca2+ concentration also increased the observed peak inward Ca2+ current in response to long (10-s) depolarizing pulses from a holding potential of -55 mV to +5 mV, from about -26 to -400 pA. However, the total amount of Ca2+ influx into the cell only increased when the extracellular Ca2+ concentration was raised from 0.25 to 1 mM and then remained constant up to 10 mM extracellular Ca2+. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 mM extracellular Ca2+, of approximately 4 and 41 s, being the fast component of secretion produced by the exocytosis of approximately 220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca2+ concentration.

Adenosine Triphosphate↗
PubMed1995

omega-Conotoxin GVIA blocks nicotine-induced catecholamine secretion by blocking the nicotinic receptor-activated inward currents in bovine chromaffin cells.

We have studied the contribution of N-type voltage-dependent Ca2+ channels to both norepinephrine and epinephrine secretion from bovine chromaffin cells induced by high K+ or nicotine using omega-conotoxin GVIA, a selective blocker of N-type voltage-dependent Ca2+ channels. We found that high K+ (75 mM) induced catecholamine secretion was not affected by exposure of bovine chromaffin cells to omega-conotoxin GVIA (1 microM). However, nicotine-induced both norepinephrine and epinephrine secretion were similarly blocked (about 25%) by omega-conotoxin GVIA (1 microM). This effect could be explained by a potent (about 80%) and reversible blockade of the inward current induced by nicotine receptor activation in bovine chromaffin cells. The results indicate that besides the blockade of N-type voltage-dependent channels, omega-conotoxin GVIA is a potent and reversible blocker of the nicotinic receptor-induced currents in chromaffin cells.

Acetylcholine↗
PubMed1995

omega-Agatoxin IVA blocks nicotinic receptor channels in bovine chromaffin cells.

We have studied the contribution of P-type voltage-dependent Ca2+ channels to both catacholamine (CA) and ATP secretion from bovine chromaffin cells induced by high K+ or nicotine using omega-agatoxin IVA, a selective blocker of P-type voltage-dependent Ca2+ channels. We found that high K+ (75 mM) induced the release of about 13% of norepinephrine, 5% epinephrine and 11% ATP, and that omega-agatoxin (100 nM) did not affect this secretion. However, both nicotine-induced CA and ATP secretion were significantly blocked (about 50%) by omega-agatoxin IVA (100 nM). In addition, this toxin also reversibly blocked (about 70%) the inward current induced by nicotine in bovine chromaffin cells. The results suggest that, besides its known action of blocking P-type voltage-dependent channels, omega-agatoxin is a potent and reversible blocker of the nicotinic receptor channel in chromaffin cells, and that this action would explain the blockade of nicotine-induced secretion.

Adenosine Triphosphate↗
PubMed1995

Catecholamine secretion, calcium levels and calcium influx in response to membrane depolarization in bovine chromaffin cells.

In this paper, we show that exposure of chromaffin cells to high K+ (75 mM) for 5 min releases about 15% of total norepinephrine and 8% of total epinephrine contained in chromaffin cells. The measured resting membrane potential of these cells was -55 mV. Long (10 s) depolarizing electrical pulses applied from a holding potential of -55 mV to 5 mV, that would produce a depolarization similar to exposure to high K+ (75 mM), induced an inward Ca2+ current that inactivated with a time constant of about 0.8 s and promoted the influx of about 1 fmol of Ca2+ into the cell. Both high K+ and electrically-induced depolarization increased intracellular Ca2+ levels to a similar value (about 350 nM). Extrapolation would indicate that total Ca2+ influx in high K+ (75 mM)-stimulated 10(6) chromaffin cells would amount to 1 nmol which would promote the secretion of about 4.9 nmol of norepinephrine and 3.5 nmol of epinephrine from 10(6) chromaffin cells. The results indicate that Ca2+ influx in response to depolarization is short-lived, likely due to Ca(2+)-dependent inactivation of voltage-dependent Ca2+ channels. However, intracellular Ca2+ levels remain high as long as depolarization is present and long after Ca2+ influx has ceased. This would suggest that some processes related to either Ca2+ buffering or extrusion from the cell may be voltage dependent.

Animals↗
PubMed1993

Catecholamine secretion induced by tetraethylammonium from cultured bovine adrenal chromaffin cells.

The resting potential in adrenal medullary chromaffin cells is maintained by the activity of different K(+)-channels. Blockade of K(+)-channels should, at least in principle, lead to membrane depolarization, and the ensuing activation of voltage-gated Ca(2+)-channels should promote Ca2+ entry and catecholamine (CA) secretion. In support of this mechanism we found and report here that the K(+)-channel blocker tetraethylammonium (TEA) depolarized the chromaffin cell membrane, induced a substantial elevation in cytosolic [Ca2+], and a dose-dependent CA secretion reaching a maximum at 50 mM of approx. 10% of the total CA in the cells. In addition, TEA-induced CA secretion was found to be absolutely dependent on [Ca2+]o. In the presence of [Ca2+]o, TEA-stimulated CA release was blocked completely by elevated [MgCl2]o (12 mM), and inhibited in part by the Ca(2+)-channel antagonist nifedipine. The Ca(2+)-channel agonist Bay K-8644 markedly enhanced TEA-evoked CA release suggesting the involvement of L-type Ca(2+)-channels. Since, external application of TEA (30-50 mM) markedly blocked outward K+ currents but not inward currents carried by Na+ and Ca2+, we concluded that TEA stimulates CA secretion by blocking those K(+)-channels involved in the maintenance of the resting membrane potential.

Adrenal Medulla↗
PubMed1992

Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla.

Using a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. These cDNAs encode a 660-amino acid protein that has a molecular weight of 73,288 kDa and no amino-terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat-shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non-transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms-pulses, can be described as a Ik(A) potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell.

Adrenal Medulla↗