Nora Riveros

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro

Kainate (0.62-5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl-DL-aspartate (50-1000 microM), L-glutamate (0.62-5 mM), DL-homocysteate (0.62-2.5 mM), and ibotenate (6-170 microM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino acids N-acetyl-L-aspartate, and alpha-methyl-DL-aspartate (both 1.25-5 mM), did not increase such influx. We suggest that enhanced calcium uptake may represent the basis for the neurotoxic effects of these compounds.

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

Dihydropyridine Receptors as Voltage Sensors for a Depolarization-evoked, IP3R-mediated, Slow Calcium Signal in Skeletal Muscle Cells

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations

The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

Differential gene expression in skeletal muscle cells after membrane depolarization

Skeletal muscle is a highly plastic tissue with a remarkable capacity to adapt itself to challenges imposed by contractile activity. Adaptive response, that include hypertrophy and activation of oxidative mechanisms have been associated with transient changes in transcriptional activity of specific genes. To define the set of genes regulated by a depolarizing stimulus, we used 22 K mouse oligonucleotide microarrays. Total RNA from C2C12 myotubes was obtained at 2, 4, 18, and 24 h after high K+ stimulation. cDNA from control and depolarized samples was labeled with cyanine 3 or 5 dyes prior to microarray hybridization. Loess normalization followed by statistical analysis resulted in 423 differentially expressed genes using an unadjusted P‐value ≤ 0.01 as cut off. Depolarization affects transcriptional activity of a limited number of genes, mainly associated with metabolism, cell communication and response to stress. A number of genes related to Ca2+ signaling pathways are induced at 4 h, reinforcing the potential role of Ca2+ in early steps of signal transduction that leads to gene expression. Significant changes in the expression of molecules involved in muscle cell structure were observed; K+‐depolarization increased Tnni1 and Acta1 mRNA levels in both differentiated C2C12 and rat skeletal muscle cells in primary culture. Of these two, depolarization induced slow Ca2+ transients appear to have a role only in the regulation of Tnni1 transcriptional activity. We suggest that depolarization induced expression of a small set of genes may underlie Ca2+ dependent plasticity of skeletal muscle cells. J. Cell. Physiol. 210: 819–830, 2007.

CFTR mutations in Chilean cystic fibrosis patients

An analysis of five of the most common cystic fibrosis (CF) mutations worldwide (ΔF-508, R-553X, G-551D, N-1303K and G-542X) was performed in 36 Chilean patients. Polymerase chain reaction (PCR) amplification of the DNA followed by allele specific restriction enzyme analysis was used for detection. The overall frequencies of the mutations in the chromosomes analyzed were 29.2% for ΔF-508 and 4.2% for R-553X (n=72). The G-542X, G-551D and N-1303 K mutations were absent in the Chilean sample. Our data suggest however that ΔF-508 is not the most common CF mutation in Chilean patients. ΔF-508 and R-553X account for only 33.4% of the alleles; 66.6% of them do not respond to the probes used and still remain uncharacterized.

Membrane depolarization induces calcium-dependent upregulation of Hsp70 and Hmox-1 in skeletal muscle cells

Heat shock proteins (HSPs) are a conserved family of cytoprotective polypeptides, synthesized by cells in response to stress. Hsp70 and heme oxygenase 1 (Hmox-1) are induced by a variety of cellular stressors in skeletal muscle, playing a role in long-term adaptations and muscle fibers regeneration. Though HSPs expression after exercise has been intensely investigated, the molecular mechanisms concerning Hsp70 and Hmox-1 induction are poorly understood. The aim of this work was to investigate the involvement of calcium in Hsp70 and Hmox-1 expression upon depolarization of skeletal muscle cells. We observed that depolarization of myotubes increased both mRNA levels and protein expression for Hsp70 and Hmox-1. Stimulation in the presence of intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70-induced expression. It is known that inositol-1,4,5-trisphophate (IP(3))-mediated slow Ca(2+) transients, evoked by membrane depolarization, are involved in the regulation of gene expression. Here we demonstrated that inhibition of IP(3)-dependent calcium signals decreased both Hsp70 mRNA induction and Hsp70 and Hmox-1 protein expression. Inhibitors of calcium-dependent protein kinase C also abolished Hsp70 mRNA induction. Our results provide evidence that membrane depolarization increases Hsp70 and Hmox-1 expression in cultured skeletal muscle cells, which the effect is critically dependent on Ca(2+) released from IP(3)-sensitive intracellular stores and that it involves PKC as an upstream effector in Hsp70 mRNA-induced expression.

Abnormal distribution of inositol 1,4,5-trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy-derived cells

Inositol 1,4,5‐trisphosphate (IP3) receptors (IP3Rs) drive calcium signals involved in skeletal muscle excitation‐transcription coupling and plasticity; IP3R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP3R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP3Rs (IP3R1) and type 2 IP3Rs (IP3R2) show a higher expression in type II fibers, whereas type 3 IP3Rs (IP3R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP3R2 label, whereas 24 ± 7% of type II fibers have lost the IP3R1 label. RCDMD cells show 5‐fold overexpression of IP3R2 and down‐regulation of IP3R3 compared with RCMH cells. A tetanic stimulus induces IP3‐dependent slow Ca2+ transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation‐driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP3‐dependent Ca2+ release may play a significant role in DMD pathophysiology.—Cárdenas, C., Juretić, N., Bevilacqua, J. A., García, I. E., Figueroa, R., Hartley, R., Taratuto, A. L., Gejman, R., Riveros, N., Molgó, J., Jaimovich, E. Abnormal distribution of inositol 1,4,5‐trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy‐derived cells. FASEB J. 24, 3210–3221 (2010). www.fasebj.org

Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1β in dystrophic skeletal muscle cell lines.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by reduced or no expression of dystrophin, a cytoskeletal protein that provides structural integrity to muscle fibres. A promising pharmacological treatment for DMD aims to increase the level of a structural dystrophin homolog called utrophin. Neuregulin-1 (NRG-1), a growth factor that potentiates myogenesis, induces utrophin expression in skeletal muscle cells. Microarray analysis of total gene expression allowed us to determine that neuregulin-1β (NRG-1β) is one of 150 differentially expressed genes in electrically stimulated (400 pulses, 1 ms, 45 Hz) dystrophic human skeletal muscle cells (RCDMD). We investigated the effect of depolarization, and the involvement of intracellular Ca2+ and PKC isoforms on NRG-1β expression in dystrophic myotubes. Electrical stimulation of RCDMD increased NRG-1β mRNA and protein levels, and mRNA enhancement was abolished by actinomycin D. NRG-1β transcription was inhibited by BAPTA-AM, an intracellular Ca2+ chelator, and by inhibitors of IP3-dependent slow Ca2+ transients, like 2-APB, Ly 294002 and Xestospongin B. Ryanodine, a fast Ca2+ signal inhibitor, had no effect on electrical stimulation-induced expression. BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca2+-dependent PKC isoforms) abolished NRG-1β mRNA induction. Our results suggest that depolarization induced slow Ca2+ signals stimulate NRG-1β transcription in RCDMD cells, and that Ca2+-dependent PKC isoforms are involved in this process. Based on utrophin´s ability to partially compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up-regulation could be important for new therapeutic strategies design.

Neuropathological changes in the rat brain cortex in vitro: effects of kainic acid and of ion substitutions

The ionic mechanisms that may contribute to the neurotoxicity of kainic acid, were studied in a system of rat thin neocortical slices superfused in vitro. Slices superfused for 3 h under control conditions showed an essentially normal aspect when studied by light microscopy. Presence of 30 microM kainate in the superfusion fluid induced neuronal swelling, nuclear condensation and signs of necrosis in some cells, while other neurons, especially in deeper layers, appeared dark and condensed, with microvacuolation. The neuropil presented numerous profiles of swollen dendrites. When the slices were superfused with chloride-free medium, a large number of pyknotic neurons was seen. This was further enhanced by 30 microM kainate, which produced no swelling in this medium. These effects of Cl-free medium were almost entirely prevented in Cl-free medium without calcium and with 0.1 mM of EGTA. Sodium-free medium induced a marked neuronal swelling that was not much changed by kainate. When calcium in an otherwise normal superfusion fluid was reduced to 0.1 mM, a large number of pyknotic neurons, some with incrustations, were seen. Kainate (30 microM) in this low calcium medium led to a very large swelling and destruction of neurons, and to a spongy neuropil. These effects of kainate were greatly intensified in calcium-free-EGTA (0.1 mM) medium. Ca-free-EGTA medium by itself induced considerable neuronal and neuropil swelling. It is concluded that kainate induces neuronal swelling by a sodium- and chloride-dependent mechanism, and the enhancement of swelling in low calcium is due to an increased sodium uptake.(ABSTRACT TRUNCATED AT 250 WORDS)