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D F Condorelli

Publications and source records attributed to D F Condorelli.

At least 73 records · Page 4Linked to original sources
PubMed1993

Growth conditions differentially regulate the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured neurons.

We have studied the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K+ (10, 25, or 40 mM) with or without 100 microM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K+ (K10) and increased dramatically in cultures grown in 25 mM K+ (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 + NMDA). In cultures grown in 40 mM K+ (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated 45Ca2+ influx and 4 beta-[3H]phorbol 12,13-dibutyrate binding in intact cells. Both functional parameters increased along with the K+ concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Mechanisms underlying developmental changes in the expression of metabotropic glutamate receptors in cultured cerebellar granule cells: homologous desensitization and interactive effects involving N-methyl-D-aspartate receptors.

Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.

Aging↗
PubMed1992

Development profile of metabotropic glutamate receptor mRNA in rat brain.

We have studied the expression of metabotropic glutamate receptor (mGluR) mRNA by Northern blot analysis with a specific cDNA probe (the pmGR1 probe). In 1-day-old rats, the steady state levels of mRNA were higher in the hypothalamus and olfactory bulb, with intermediate levels in the cerebellum and low levels in the hippocampus and cerebral cortex. In the olfactory bulb, hypothalamus, and cerebral cortex, the expression of mGluR mRNA remained constant at 8 and 30 days of postnatal life. In contrast, in the cerebellum and hippocampus, mRNA levels increased progressively with age. There was no correlation between levels of mGluR mRNA and stimulation of polyphosphoinositide hydrolysis by 1-aminocyclopentane-1S,3R-dicarboxylic acid (trans-ACPD), which was much greater in brain slices from 8-day-old rats and was nearly absent in the adult cerebellum and olfactory bulb, where we have found the highest levels of mRNA. In addition, mGluR mRNA was detectable in cultured cerebellar granule cells but not in cultured neurons from cerebral hemispheres or in cultured astrocytes, which responded to trans-ACPD with an increased formation of [3H]inositol monophosphate. The discrepancies between levels of mGluR mRNA detected with the pmGR1 probe and trans-ACPD-stimulated polyphosphoinositide hydrolysis suggest either that different subtypes of mGluRs exist or that mRNA levels are not critical for the dynamic changes in the activity of mGluRs during development.

Aging↗
PubMed1990

Glial fibrillary acidic protein messenger RNA and glutamine synthetase activity after nervous system injury.

The level of the mRNA for glial fibrillary acidic protein (GFAP), the major protein of the intermediate filaments of astroglial cells, and the activity of glutamine synthetase (GS), an enzyme selectively localized in astrocytes, were measured at different times after a unilateral mechanical lesion in the rat cerebral cortex. A rapid and early increase (6 hours post-lesion) in GFAP mRNA was observed; GFAP mRNA level reached a peak at 1-3 days and then decreased. Moreover, an astrocytic activation in cortical zones far from the injury site and in the contralateral hemisphere was detected. No change of GS activity was observed in the same model of brain injury, showing that this astroglial marker is not modified during the reactive gliosis obtained with this experimental model. GFAP mRNA has also been detected in the rat sciatic nerve; however, its level was not modified after nerve transection, suggesting a different regulation of GFAP expression in the peripheral nervous system.

Animals↗
PubMed1990

ADP-ribosylation of proteins in brain regions of rats during postnatal development.

Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP-ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression. In the present study the process of ADP-ribosylation of chromosomal proteins (histone and non-histone proteins) in some rat brain regions during postnatal development was investigated; also the effect of epidermal growth factor (EGF) on this process in fetal brain slices was studied. It has been found that the process of ADP-ribosylation of total histones extracted from rat cerebral cortex and cerebellum at 1, 10 and 30 days of age, increases from 1 to 10 days of age (i.e. the period of maximal cell proliferation) and decreases thereafter, while the process of ADP-ribosylation of non-histone proteins (NHPs) sharply decreases during the same developmental period. The addition of EGF to fetal brain slices causes a significant increase of ADP-ribosylation of total histones (particularly of the histone H1 fraction) and also of NHPs and microsomal proteins. This result is in agreement with the effect of EGF as a mitogen factor, previously shown in astroglial cell cultures.

Adenosine Diphosphate Ribose↗
PubMed1990

Excitatory amino acids stimulate inositol phospholipid hydrolysis and reduce proliferation in cultured astrocytes.

Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate [( 3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12-h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl-3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12-tetradecanoate 13-acetate. Glutamate and ibotenate reduced [methyl-3H]thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2-amino-4-phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Activation of excitatory amino acid receptors reduces thymidine incorporation and cell proliferation rate in primary cultures of astrocytes.

Addition of quisqualate (a heterocyclic analogue of glutamate) reduced [methyl-3H]thymidine incorporation and cell proliferation in primary cultures of rat cortical astrocytes. The inhibitory action of quisqualate was mimicked by glutamate and ibotenate, whereas kainate, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were inactive. These results suggest that activation of a specific class of excitatory amino acid receptors contributes to the regulation of growth and proliferation of glial cells in primary culture.

Animals↗
PubMed1989

Induction of protooncogene fos by extracellular signals in primary glial cell cultures.

In the present study various extracellular factors, acting through different second messenger systems, were examined for their capacity to increase the level of c-fos mRNA in primary glial cell cultures. In particular EGF, 12-O-tetradecanoylphorbol 13-acetate, the beta-adrenergic agonist isoproterenol, and the glutamate agonists, ibotenic and quisqualic acid, were studied. All the extracellular stimuli tested induced a rapid and transient increase in c-fos mRNA level in glial cell cultures regardless of the signal transduction pathway and the final effect on cell proliferation.

Animals↗
PubMed1989

Age-dependent changes of nucleic acid labeling in different rat brain regions.

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-14C]thymidine incorporation into DNA of cerebral cortex and cerebellum during aging was observed. The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging. In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A) RNA and poly(A) RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.

Aging↗
PubMed1989

Effect of trophic factors, released after hippocampal injury, on astroglial cell proliferation.

An increase in astrocyte mitogenic factors and in some specific astroglial enzymatic activities after neuronal injury has been observed. Our study is concerned with the effect of the intracerebral administration of ibotenic acid (IBO) into the rat hippocampus. IBO injection causes a selective degeneration of neurons while sparing afferent fibers. We observed a transient increase in glutamine synthetase activity, a well-known astroglial marker, reaching a peak at 9-15 days after injury in lesioned hippocampus. We investigated the presence of astrocyte mitogenic factors at various times after toxin injection. Crude extracts, prepared from lesioned hippocampi 4, 9, and 14 days after IBO injection, were tested for the ability to stimulate [methyl-3H]thymidine incorporation into rat astroglial cell cultures. Crude extracts prepared 9 and 14 days after IBO injection showed a higher mitogenic activity compared to extracts prepared 4 days after lesion. Mitogenic activity of injured brain extracts was suppressed by heat inactivation (100 degrees C for 10 min).

Animals↗
PubMed1989

Antioxidant enzymatic activities and resistance to oxidative stress in primary and subcultured rat astroglial cells.

The survival of neural tissues depends in part on the balance between the formation of free radicals due to oxidative metabolism and the transformation of the free radicals to non-toxic compounds. Serial subculture of rat glial cells as described here resulted in a decrease of the specific activities of several antioxidant enzymes and a glial specific marker for astrocytes. Thus, there was an increased susceptibility to oxidative stress in cultures by the third passage. These subcultured glial cell cultures may represent a useful model for the study of free radical induced neural damage that may be relevant to CNS trauma and aging.

Animals↗
PubMed1988

Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures.

The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15-30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5,6-3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37 degrees C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr. The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4-10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied. Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures.

Animals↗
PubMed1988

Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures.

This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

Amino Acids↗
PubMed1988

Protein synthesis rates in rat brain regions and subcellular fractions during aging.

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

Aging↗
PubMed1986

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures.

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.

Animals↗
PubMed1986

Acetylation and phosphorylation of histones and nonhistone chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of developing rat brain.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.

Acetylation↗