Biomedical subjects

José M Medina

Publications and source records attributed to José M Medina.

At least 19 recordsLinked to original sources

PubMed2006

Glucose metabolism and proliferation in glia: role of astrocytic gap junctions.

Astrocytes play a well-established role in brain metabolism, being a key element in the capture of energetic compounds from the circulation and in their delivery to active neurons. Their metabolic status is affected in many pathological situations, such as gliomas, which are the most common brain tumors. This proliferative dysfunction is associated with changes in gap junctional communication, a property strongly developed in normal astrocytes studied both in vitro and in vivo. Here, we summarize and discuss the findings that have lead to the identification of a link between gap junctions, glucose uptake, and proliferation. Indeed, the inhibition of gap junctional communication is associated with an increase in glucose uptake due to a rapid change in the localization of both GLUT-1 and type I hexokinase. This effect persists due to the up-regulation of GLUT-1 and type I hexokinase and to the induction of GLUT-3 and type II hexokinase. In addition, cyclins D1 and D3 have been found to act as sensors of the inhibition of gap junctions and have been proposed to play the role of mediators in the mitogenic effect observed. Conversely, in C6 glioma cells, characterized by a low level of intercellular communication, an increase in gap junctional communication reduces glucose uptake by releasing type I and type II hexokinases from the mitochondria and decreases the exacerbated rate of proliferation due to the up-regulation of the Cdk inhibitors p21 and p27. Identification of the molecular actors involved in these pathways should allow the determination of potential therapeutic targets that could lead to the testing of alternative strategies to prevent, or at least slow down, the proliferation of glioma cells.

Animals↗

PubMed2006

Tolbutamide reduces glioma cell proliferation by increasing connexin43, which promotes the up-regulation of p21 and p27 and subsequent changes in retinoblastoma phosphorylation.

Our previous work has shown that tolbutamide increases gap junctional permeability in poorly coupled C6 glioma cells and that this effect is similar and additive to that found with dbcAMP, a well-known activator of gap junctional communication. Furthermore, the increase in gap junctional communication promoted by tolbutamide or dbcAMP is concurrent with the inhibition of proliferation of C6 glioma cells. In the present work, we show that tolbutamide and dbcAMP increase the synthesis of the tumor suppressor protein Cx43 and that they decrease the level of Ki-67, a protein expressed when cells are proliferating. These effects were accompanied by a reduction in the phosphorylation of pRb, mainly on Ser-795, a residue critical for the control of cell proliferation. The decrease in the phosphorylation of pRb is not likely to be mediated by a reduction in the levels of D-type cyclins, since instead of decreasing the expression of cyclins, D1 and D3 increased slightly after treatment with tolbutamide or dbcAMP. However, the Cdk inhibitors p21 and p27 were up-regulated after treatment with tolbutamide and dbcAMP, suggesting that they would be involved in the decrease in pRb phosphorylation. When Cx43 was silenced by siRNA, neither tolbutamide nor dbcAMP were able to up-regulate p21 and consequently to reduce glioma cell proliferation, as judged by Ki-67 expression. In conclusion, tolbutamide and dbcAMP inhibit C6-glioma cell proliferation by increasing Cx43, which correlates with a reduction in pRb phosphorylation due to the up-regulation of the Cdk inhibitors p21 and p27.

Animals↗

PubMed2006

Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes.

We showed previously that the inhibition of gap junctional communication in astrocytes increased bromodeoxyuridine (BrdU) incorporation and promoted changes in the metabolic phenotype destined to fulfil the requirements of cell proliferation. In the present study we investigated the changes in the cell cycle of astrocytes promoted by the inhibition of intercellular communication through gap junctions. Thus, the presence of endothelin-1 and carbenoxolone, two gap junction uncouplers, promoted an increase in the percentage of astrocytes found in the S, G2 and M phases of the cell cycle, with a concomitant decrease in G0 and G1 phases. In addition, the levels of Ki-67, a protein present during all active phases of the cell cycle but absent from resting cells, increased after the inhibition of gap junctional communication. These effects were not observed when the inhibition of gap junctions was prevented with tolbutamide, indicating that the inhibition of gap junctional communication promotes the entry of astrocytes into the cell cycle. The passage of the cells from a quiescent state to the cell cycle is ultimately regulated by the degree of retinoblastoma phosphorylation. Inhibition of gap junctions increased the phosphorylation of retinoblastoma at Ser 780 but not at Ser 795 or Ser 807/811. In addition, the levels of cyclins D1 and D3 increased, whereas those of p21 and p27 were not significantly modified. Because D-type cyclins are key regulators of retinoblastoma protein phosphorylation, it is suggested that the phosphorylation of retinoblastoma protein at Ser 780, observed under our experimental conditions, is a consequence of the increase in the levels of cyclins D1 and D3. Our work provides evidence for the involvement of cyclins D1 and D3 as sensors of the inhibition of gap junctional communication in astrocytes.

Animals↗

PubMed2006

Binocular interactions in random chromatic changes at isoluminance.

To examine the type of chromatic interactions at isoluminance in the phenomenon of binocular vision, I have determined simple visual reaction times (VRT) under three observational conditions (monocular left, monocular right, and binocular) for different chromatic stimuli along random color axes at isoluminance (simultaneous L-, M-, and S-cone variations). Upper and lower boundaries of probability summation as well as the binocular capacity coefficient were estimated with observed distributions of reaction times. The results were not consistent with the notion of independent chromatic channels between eyes, suggesting the existence of excitatory and inhibitory binocular interactions at suprathreshold isoluminance conditions.

Color↗

PubMed2006

Postreceptoral chromatic-adaptation mechanisms in the red-green and blue-yellow systems using simple reaction times.

Simple visual-reaction times (VRT) were measured for a variety of stimuli selected along red-green (L-M axis) and blue-yellow [S-(L + M) axis] directions in the isoluminant plane under different adaptation stimuli. Data were plotted in terms of the RMS cone contrast in contrast-threshold units. For each opponent system, a modified Piéron function was fitted in each experimental configuration and on all adaptation stimuli. A single function did not account for all the data, confirming the existence of separate postreceptoral adaptation mechanisms in each opponent system under suprathreshold conditions. The analysis of the VRT-hazard functions suggested that both color-opponent mechanisms present a well-defined, transient-sustained structure at marked suprathreshold conditions. The influence of signal polarity and chromatic adaptation on each color axis proves the existence of asymmetries in the integrated hazard functions, suggesting separate detection mechanisms for each pole (red, green, blue, and yellow detectors).

Adaptation, Physiological↗

PubMed2005

The increase in gap junctional communication decreases the rate of glucose uptake in C6 glioma cells by releasing hexokinase from mitochondria.

We have previously shown that the enhancement of glucose uptake caused by the inhibition of gap junctional communication is a consequence of the increase in astrocyte proliferation. Since C6 glioma cells are highly proliferative and are poorly coupled through gap junctions, we used these cells to investigate the effect of increasing gap junctional communication on the rate of glucose uptake. Previous work by us had shown that tolbutamide increases gap junctional communication in C6 glioma cells, as does dbcAMP, a classical activator of gap junctional communication. In this work, our results show that both tolbutamide and dbcAMP reduce the rate of glucose uptake in C6 glioma cells and that their effects are additive. The main glucose transporters expressed in C6 glioma cells are GLUT-1 and GLUT-3. Neither the expression nor the cellular localization of either GLUT-1 or GLUT-3 were modified by increasing gap junctional communication. The estimation of glucose uptake with 2-deoxyglucose includes not only glucose transport but also glucose phosphorylation, which in C6 glioma cells is mainly catalyzed by type I and type II hexokinase. Our results reveal that the increase in gap junctional communication caused by tolbutamide and dbcAMP is associated with a decrease in the activity of hexokinase. In agreement with this, tolbutamide and dbcAMP caused a rapid change in the localization of both type I and type II hexokinase, which were detached from the mitochondria to the cytosol.

Animals↗

PubMed2004

Antioxidant defence of the neonatal rat brain against acute hyperammonemia.

Oxidative stress associated with the presence of elevated concentrations of ammonia in the brain has been proposed as one possible mechanism involved in ammonia toxicity. In a previous study [Brain Res.973 (2003) 31], we reported that neonatal rats are more resistant to acute ammonia toxicity than adult rats. In the present work, we studied the antioxidant status of the brain in hyperammonemic neonatal rats. Increased activities of the antioxidant enzymes and enhanced glutathione content were found in the brains of the hyperammonemic neonatal rats as compared to the controls. In addition, no changes in brain reactive oxygen species (ROS) levels and lipid peroxidation due to hyperammonemia were found. Therefore, acute ammonia intoxication does not induce oxidative stress in neonatal rats, a fact that may explain the resistance against hyperammonemia shown by neonatal rats.

Ammonia↗

PubMed2004

The neurotrophic effect of oleic acid includes dendritic differentiation and the expression of the neuronal basic helix-loop-helix transcription factor NeuroD2.

We have shown recently that the presence of albumin in astrocytes triggers the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons. Thus, oleic acid promotes axonal growth together with the expression of the axonal growth-associated protein, GAP-43. Here we attempted to elucidate whether the neurotrophic effect of oleic acid includes dendritic differentiation. Our results indicate that oleic acid induces the expression of microtubule associated protein-2 (MAP-2), a marker of dendritic differentiation. In addition, the presence of oleic acid promotes the translocation of MAP-2 from the soma to the dendrites. The time course of MAP-2 expression during brain development coincides with that of stearoyl-CoA desaturase, the limiting enzyme of oleic acid synthesis, indicating that both phenomena coincide during development. The effect of oleic acid on MAP-2 expression is most probably independent of autocrine factors synthesized by neurons because this effect was also observed at low cellular densities. As oleic acid is an activator of protein kinase C, the possible participation of this transduction pathway was studied. Our results indicate that added oleic acid or oleic acid endogenously synthesized by astrocytes exerts its neurotrophic effect through a protein kinase C-dependent mechanism as the effect was inhibited by sphingosine or two myristoylated peptide inhibitors of protein kinase C. The transduction pathway by which oleic acid induces the expression of genes responsible for neuronal differentiation appears to be mediated by the transcription factor NeuroD2, a regulator of terminal neuronal differentiation.

Animals↗

PubMed2004

Endothelin-1 stimulates the translocation and upregulation of both glucose transporter and hexokinase in astrocytes: relationship with gap junctional communication.

We have previously shown that endothelin-1 increases glucose uptake in astrocytes. In the present work we investigate the mechanism through which endothelin-1 (ET-1) increases glucose uptake. Our results show that ET-1 activates a short-term and a long-term mechanism. Thus, ET-1 induced a rapid change in the localization of both GLUT-1 and type I hexokinase. These changes are probably aimed at rapidly increasing the entry and phosphorylation of glucose. In addition, ET-1 upregulated GLUT-1 and type I hexokinase and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and type II hexokinase. These changes provide astrocytes with the machinery required to sustain a high rate of glucose uptake for a longer period of time. Our previous work had suggested that the effect of ET-1 on glucose uptake was associated with the inhibition of gap junctions. In this work, we compare the effect of ET-1 with that of carbenoxolone, a classical inhibitor of gap junction communication. Carbenoxolone increased glucose uptake to the same extent as ET-1 following the same mechanisms. Thus, carbenoxolone induced a rapid change in the localization of both GLUT-1 and type I hexokinase, upregulated GLUT-1 and type I hexokinase and induced the expression of GLUT-3 and type II hexokinase. When the inhibition of gap junction was prevented by tolbutamide, neither ET-1 nor carbenoxolone were able to increase the levels of GLUT-1, GLUT-3, type I hexokinase or type II hexokinase, indicating that these events are closely related to gap junctions.

Animals↗

PubMed2003

Oleic acid induces GAP-43 expression through a protein kinase C-mediated mechanism that is independent of NGF but synergistic with NT-3 and NT-4/5.

We have recently shown that the presence of albumin in astrocytes triggers the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons. Thus, oleic acid promotes axonal growth, neuronal clustering, and the expression of the axonal growth-associated protein, GAP-43. In this work we show that oleic acid upregulates GAP-43 expression by a protein kinase C (PKC)-dependent mechanism. Since GAP-43 expression has been shown to be upregulated by several neurotrophins, we investigated the relationship between the effect of oleic acid and that of NGF, neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) on GAP-43 expression. Our results indicate that NGF is not involved in the neurotrophic effect of oleic acid because the addition of NGF did not modify the effect of oleic acid on GAP-43 expression. Neither NT-3 nor NT-4/5 alone modified GAP-43 expression. However, NT-3 and NT-4/5 acted synergistically with oleic acid to increase GAP-43 expression. The lack of effect of NGF as compared to other neurotrophins is not unexpected since we have not found TrkA expression under our experimental conditions. The effect of oleic acid on GAP-43 expression must be independent of autocrine factors synthesized by neurons because this effect was also observed at low cellular densities. In conclusion, our results indicate that oleic acid behaves as a neurotrophic factor, inducing GAP-43 expression through a PKC-mediated mechanism that is not mediated by other neurotrophic factors but that is strongly synergized by NT-3 and NT-4/5.

Animals↗

PubMed2003

Role of oleic acid as a neurotrophic factor is supported in vivo by the expression of GAP-43 subsequent to the activation of SREBP-1 and the up-regulation of stearoyl-CoA desaturase during postnatal development of the brain.

We have recently reported that albumin, a serum protein present in the developing brain, stimulates the synthesis of oleic acid by cultured astrocytes by inducing stearoyl-CoA 9-desaturase, the rate-limiting enzyme in oleic acid synthesis, through activation of the sterol regulatory element-binding protein-1. In this work, we offer evidence supporting the in vivo occurrence of this process during the postnatal development of the rat brain. Our results show that albumin reaches maximal brain level by day 1 after birth, coinciding with activation of the sterol response element binding protein-1, which is responsible for the transcription of the enzymes required for oleic acid synthesis. In addition, the developmental profile of stearoyl-CoA 9-desaturase-1 mRNA expression follows that of sterol regulatory element-binding protein-1 activation, indicating that these phenomena are tightly linked. In a previous work, we showed that oleic acid induces neuronal differentiation, as indicated by the expression of growth associated protein-43. Here, we report that the expression of growth associated protein-43 mRNA peaks at about day 7 after birth, following the maximal expression of stearoyl-CoA 9-desaturase-1 mRNA that occurs between days 3 and 5 postnatally. In conclusion, our results support the hypothesis that the synthesis of oleic acid is linked to neuronal differentiation during rat brain development.

Age Factors↗

PubMed2003

Tolerance of neonatal rat brain to acute hyperammonemia.

The aim of the present work was to study the effects of hyperammonemia on brain energy metabolism in neonatal rats. Rats were rendered hyperammonemic by ammonium acetate administration. This decreased brain ATP concentrations but enhanced brain ammonia and lactate levels in both adult and neonatal rats. In adult rats, the decrease in brain ATP concentrations was accompanied by a plunge in the respiratory control rate (RCR) of brain mitochondria. However, the ammonia-induced effect on RCR was not observed in neonatal rats, suggesting that the fall in ATP levels observed in neonatal rats would not be due to an impairment of mitochondrial respiratory efficiency. However, in neonatal rats the increase in blood and brain ammonia concentrations did not change brain glutamate concentrations but decreased glutamine contents. These results may be of relevance for the understanding of the resistance of neonatal rats observed in this work to acute ammonia toxicity

Acute Disease↗

PubMed2003

Effect of endothelin-1 on astrocytic protein content.

The astrocytic endothelin (ET) receptors, ET(A) and ET(B), modulate calcium signaling and the astrocytic gap junctional network. The nonselective ET receptor ligand ET-1 inhibits gap junction permeability, an effect that can be blocked by tolbutamide. This mechanism may play a role in pathophysiological conditions such as ischemic stroke, characterized by elevated tissue ET-1 levels and hypertrophic-appearing reactive astrocytes. Therefore, the effect of ET-1 on cellular protein content was investigated in confluent once-passaged rat astrocyte cultures under serum-free conditions, by the Lowry method. Gap junction permeability was determined by the dye transfer technique. ET-1 prevented the decrease in astrocytic protein content observed in controls. The effect of ET-1 on cellular protein content was most pronounced in cultures seeded at high density, but it was attenuated in ET(B)-deficient (sl/sl) astrocytes. This effect could be blocked by the nonselective ET antagonist LU 302872 (10 micro M), as well as by the protein synthesis inhibitor cycloheximide (10 micro M). This increase in astrocytic protein content was inhibited by the ATP-sensitive K(+) channel blocker tolbutamide, which also antagonized the ET-1-induced reduction of gap junction permeability and reversed the morphological changes observed in astrocytes upon ET-1 treatment. Cytosine arabinoside (10 micro M), a DNA synthesis blocker, inhibited the ET-1-induced BrdU uptake without affecting the ET-1-induced increase in astrocytic protein content. To conclude, ET-1 induces an increase in astrocytic protein content as well as changes in astrocyte morphology in vitro. This hypertrophic response involves uncoupling of the astrocytic gap junctional network and is not dependent on DNA synthesis.

Animals↗

PubMed2002

Flow cytometry of isolated mitochondria during development and under some pathological conditions.

Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed.

Aging↗

PubMed2002

Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture.

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.

Adenosine Triphosphate↗

PubMed2002

Albumin promotes neuronal survival by increasing the synthesis and release of glutamate.

It is well known that the presence of albumin within the brain and the CSF is developmentally regulated. However, the physiological relevance of this phenomenon is not well established. We have previously shown that albumin specifically increases the flux of glucose and lactate through the pyruvate dehydrogenase reaction in astrocytes. Here we show that, in neurones, albumin also increases the oxidation of glucose and lactate through the pyruvate dehydrogenase-catalysed reaction, the final purpose of this being the synthesis of glutamate. Thus, in neurones, the presence of albumin strongly increased the synthesis and release of glutamate to the extracellular medium. Our results also suggest that glutamate release caused by albumin is designed to promote neuronal survival. Thus, under culture conditions in which neurones die by apoptosis, the presence of albumin promoted neuronal survival and maintained the differentiation programme of these cells, as judged by the expression of the axonal protein, GAP-43. The effect of albumin on neuronal survival was counteracted by the presence of DNQX, an antagonist of non-NMDA-glutamate receptors, suggesting that the glutamate synthesized and released due to the presence of albumin is responsible for neuronal survival. In addition, the effect of albumin seemed to depend on the activity of the NGF receptor, TrkA, suggesting that the glutamate synthesized and released due to the presence of albumin promotes neuronal survival through the activity of TrkA.

Animals↗

PubMed2002

Oxidative stress in preterm rat brain is due to mitochondrial dysfunction.

Prematurity-mediated cerebral damage has been associated with oxidative stress. The aim of the present work was to study the possible role played by free oxygen radicals generated by mitochondrial respiratory function in cerebral injury in preterm neonates. Our results show that whereas total glutathione concentrations are similar in term and preterm neonates, the GSH/GSSG ratio decreases sharply in preterm neonates immediately after birth. This effect is not due to a lack of enzymes involved in GSH regeneration, such as glutathione reductase and glucose-6-phosphate dehydrogenase, but to a significant increase in free-radical generation in preterm rat brain as shown by the increase in lipoperoxidation. Because the mitochondrion is the main source of free radicals in the cell, mitochondrial respiratory function was studied in the brain of preterm neonates. Our results show that prematurity prevented the postnatal increases in complex II-III activity and ATP concentrations that occur in term neonates at 5 min after delivery. All these effects were counteracted by the oxygen supply, suggesting that the inhibition of mitochondrial function is caused by restricted oxygen availability. Consequently, cerebral damage associated with prematurity may be mediated by mitochondrial free-radical generation as a consequence of hypoxia undergone by preterm neonates at birth.

Animals↗

PubMed2001

Transcytosis of albumin in astrocytes activates the sterol regulatory element-binding protein-1, which promotes the synthesis of the neurotrophic factor oleic acid.

We have recently reported that albumin, a serum protein present in the developing brain, stimulates the synthesis of oleic acid by astrocytes, which promotes neuronal differentiation. In this work, we gain insight into the mechanism by which albumin induces the synthesis of this neurotrophic factor. Our results show that astrocytes internalize albumin in vesicle-like structures by receptor-mediated endocytosis. Albumin uptake was followed by transcytosis, including passage through the endoplasmic reticulum, which was required to induce the synthesis of oleic acid. Oleic acid synthesis is feedback-regulated by the sterol regulatory element-binding protein-1, which induces the transcription of stearoyl-CoA 9-desaturase, the key rate-limiting enzyme for oleic acid synthesis. In our research, the presence of albumin activated the sterol regulatory element-binding protein-1 and increased stearoyl-CoA 9-desaturase mRNA. Moreover, when the activity of sterol regulatory element-binding protein-1 was inhibited by overexpression of a truncated form of this protein, albumin did not affect stearoyl-CoA 9-desaturase mRNA, indicating that the effect of albumin is mediated by this transcription factor. The effect of albumin was abolished when traffic to the endoplasmic reticulum was prevented or when albumin was accompanied with oleic acid. In conclusion, our results suggest that the transcytosis of albumin includes passage through the endoplasmic reticulum, where oleic acid is sequestrated, initiating the signal cascade leading to an increase in its own synthesis.

Animals↗