Biomedical subjects

P Brachet

Publications and source records attributed to P Brachet.

At least 37 records · Page 2Linked to original sources

PubMed1997

Noradrenaline inhibits the programmed cell death induced by 1,25-dihydroxyvitamin D3 in glioma.

The rat glioma cell line C6.9 has been recently reported to respond to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by the induction of a programmed cell death. Since, in vivo, glial cells are thought to be exposed to several neurotransmitters, we investigated the possibility of a neurotransmitter-mediated inhibition of this active cell death process. Noradrenaline and the beta-adrenoceptor agonist isoproterenol showed significant inhibition of the 1,25(OH)2D3-induced programmed cell death. The beta-adrenoceptor antagonist propanolol reversed this inhibition, while the alpha-adrenoceptor antagonist yohimbin was devoid of any effect. This suggests that the efficiency of antiproliferative vitamin D-related therapies could be influenced by endogenous levels of noradrenaline.

Adrenergic beta-Agonists↗

PubMed1997

1,25-Dihydroxyvitamin D3 inhibits the expression of inducible nitric oxide synthase in rat central nervous system during experimental allergic encephalomyelitis.

The inducible form of nitric oxide synthase (iNOS) generates nitric oxide of which the excessive production is associated with central nervous system (CNS) inflammatory diseases. The investigation of iNOS expression during experimental allergic encephalomyelitis (EAE) of the Lewis rat demonstrated iNOS immunoreactivity and mRNA both during inflammatory bursts (days 12 and 23 post-immunization) and during the remission phase (day 18). iNOS expression was region-specific and expanded with time along a caudo-rostral axis, thus, correlating with the development of inflammatory infiltrates. Whereas cells of the monocyte/macrophage lineage continuously contributed to iNOS expression, astrocytes only expressed iNOS immunoreactivity or mRNA during the relapse (day 23). In order to investigate possible regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-D3) on iNOS expression, rats were treated with the hormone after the beginning of clinical signs (days 11, 13, 19, 21 and 23 post-immunization), and areas of the CNS were examined at day 23. 1,25-D3 exerted a drastic inhibitory effect on iNOS expression, both at the protein and the mRNA levels. However, this effect was region-specific, and was most pronounced in the cerebellum and brainstem, but non-existent in cerebral cortex. iNOS down-regulation occurred in macrophages, activated microglia and astrocytes. The inhibition of iNOS expression in some CNS structures could account for the improvement of clinical signs observed in EAE-rats treated with 1,25-D3. Since 1,25-D3 can be synthesized by activated macrophages or microglia, our results support the hypothesis that this hormone might be implicated in the control of the CNS-specific immune responses. 1,25-D3 or its analogues could, thus, be of therapeutic value in the management of iNOS-associated diseases of the CNS.

Aging↗

PubMed1996

1,25-Dihydroxyvitamin D3 induces programmed cell death in a rat glioma cell line.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a seco-steroid hormone with potential antitumoral activities, has been recently reported to exert cytotoxic effects on C6 glioma cells. However, the molecular mechanisms which trigger this cell death remain unknown. We show here that this 1,25(OH)2D3-induced cell death is dependent upon protein synthesis and is accompanied by the expression of c-myc, p53, and gadd45 genes. Two other genes, coding for interleukin-6 and vaso-endothelial growth factor, are also upregulated after addition of 1,25(OH)2D3. This programmed cell death can be suppressed when cells are treated with forskolin, a drug which increases intracellular cAMP concentration, or with genistein, an inhibitor of tyrosine protein kinases. However, in spite of the demonstration of fragmented DNA in 1,25(OH)2D3-treated cells, the C6.9 cells used in this study do not show the classical morphological features of apoptosis. These results provide the first evidence for the existence of a programmed cell death triggered by 1,25(OH)2D3 in glioma cells and may provide a basis for the development of new therapeutic strategies. In addition, these data also suggest that the treatment of C6.9 cells with 1,25(OH)2D3 may be a useful model to study the molecular mechanisms involved in the programmed cell death of a cell of glial origin.

Animals↗

PubMed1996

Differential expression of M-CSF, LIF, and TNF-alpha genes in normal and malignant rat glial cells: regulation by lipopolysaccharide and vitamin D.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the expression of macrophage colony-stimulating factor (M-CSF), leukemia inhibitory factor (LIF), and tumor necrosis factor-alpha (TNF-alpha) genes in primary rat astrocytes and C6 glioma cells was examined. The results show that the hormone differentially regulates the cytokine mRNA in the two cell types. 1,25-(OH)2D3 augments M-CSF and LIF mRNA in C6 glioma cells, while lipopolysaccharide (LPS) has minimal effects. When LPS and 1,25-(OH)2D3 are used in combination, a strong synergistic effect upon the induction of M-CSF and LIF genes is observed. No TNF-alpha transcript has been detected in C6 glioma cells under any stimulus conditions used. In contrast, 1,25-(OH)2D3 has no pronounced effect on M-CSF, LIF, and TNF-alpha transcripts in primary astrocytes when used as a sole stimulus, while treatment with LPS strongly enhances the levels of the three cytokines. However, when 1,25-(OH)2D3 is used in combination with LPS, a partial reduction in LPS-induced levels of M-CSF and TNF-alpha mRNA is observed. The overall results indicate that genes coding for some inflammatory cytokines obey distinct regulatory mechanisms in C6 cells and in primary astrocytes. They also suggest that 1,25-(OH)2D3, by altering the response of astrocytes to an inflammatory stimulus, could participate in the regulation of the CNS immune response.

Animals↗

PubMed1996

1,25-Dihydroxyvitamin D3 regulates gamma 1 transpeptidase activity in rat brain.

gamma-Glutamyl transpeptidase (gamma-GT), primarily described as a kidney enzyme, is also expressed in several cell types of the central nervous system (CNS). It is involved in the glutathione cycle and in cysteine transport. Here we report that the specific activity of this enzyme is transiently increased in the rat brain, following a treatment with 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. In vitro experiments showed that this positive regulatory effect does not affect endothelial cells of the brain microvessels, but does affect pericytes and parenchymal astrocytes. Changes in the specific activity of gamma-GT were not correlated with any important modification of brain amino acid concentrations. Since gamma-GT is though to participate in the scavenging of reactive oxygen species, these data suggest that 1,25-D3 could be an effector controlling detoxification processes in the brain.

Amino Acids↗

PubMed1996

1,25-Dihydroxyvitamin D3 regulates the expression of the low-affinity neurotrophin receptor.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is known to regulate the expression of neurotrophins [45,46]. Here, we report that 1,25-(OH)2D3 does not influence the expression of truncated or full-length forms of trkB and trkC receptors mRNAs in primary cultures of astrocytes and in C6 glioma cells. In contrast, low concentrations of 1,25-(OH)2D3 increased low-affinity neurotrophin receptor (P75NTR) mRNA and protein levels in C6 glioma cells. Putative vitamin D responsive elements (VDRE) in the P75NTR promoter have been investigated by transfecting plasmids containing sequences from P75NTR promoter fused to a cat reporter gene. A region between -610 and -860 bp upstream from the translation start codon was found to respond to 1,25-(OH)2D3. Interestingly, 1,25-(OH)2D3 does not regulate P75NTR in primary cultures of astrocytes even at concentration as high as 10(-7) M. Since long-term treatment of 1,25-(OH)2D3 induces cell death in C6 glioma cells but not in primary astrocytes [41], the possible involvement of P75NTR in 1,25-(OH)2D3-induced cell death is discussed. Finally, in-vivo studies show that treatment of 15-day-old and adult rats with 1,25-(OH)2D3 leads to a decrease in the level of P75NTR mRNA in the spinal cord but does not influence its expression in dorsal root ganglion or sciatic nerve. These results suggest that 1,25-(OH)2D3 may have a role in the specific regulation of P75NTR in vivo.

Animals↗

PubMed1996

1,25-Dihydroxyvitamin D3, an inducer of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has significant therapeutic potentials, in particular for neurodegenerative disorders. To determine factors that would enhance GDNF expression, we analysed the effect of 1,25-(OH)2 D3 in C6 glioma cells. Treatment of C6 cells with 10(-7) M, 1,25-(OH)2 D3 for 48 h elicited an 18.5-fold increase in the level of GDNF mRNA. In addition, our results indicate that 1,25-(OH)2 D3 is effective at concentrations as low as 10(-10) M and that retinoic acid has additive effects. These data indicate that 1,25-(OH)2 D3 is a potent inducer of GDNF expression and suggest that 1,25-(OH)2 D3 may contribute to the regulation of GDNF in vivo.

Animals↗

PubMed1996

Cytotoxic effects of 1 alpha,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line.

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3) has recently been reported to exert a toxic effect on both rat and human glioma cell lines. However the potential clinical use of 1 alpha,25(OH)2D3 in the treatment of glioma is impaired by its potent hypercalcemic effects. We have therefore investigated the effects on glioma cell growth of several vitamin D3 analogues which have previously been shown to be less calcemic in vivo than 1 alpha,25(OH)2D3. The present study shows that several analogues are able to induce, in vitro, the death of rat glioma cells (C6.9). The compound KH 1060 appears to be the most effective in the induction of cell death, while MC 1288 and CB 1093 are as potent as 1 alpha,25(OH)2D3. EB 1089 was somewhat less effective than 1 alpha,25(OH)2D3 and MC 903, which is currently used in the treatment of psoriasis, has only a weak activity on C6.9 cells. The effective doses used are around 10(-9) M for 1 alpha,25(OH)2D3 and 10(-10) M for KH 1060. Interestingly, the toxic effect exerted by 1 alpha,25(OH)2D3 and its analogues is accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene. These findings, together with the fact that the therapies currently available for glioma are only palliative, suggest that 1 alpha,25(OH)2D3 analogues such as KH 1060, EB 1089 or CB 1093, alone or in combination with other therapeutic approaches, could be of potential interest in the treatment of brain glial tumors.

Animals↗

PubMed1996

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Animals↗

PubMed1996

Hepatic and renal D-amino acid oxidase activities in the growing rat after ten days of protein undernutrition and refeeding.

The activity levels of hepatic and renal D-amino acid oxidase (EC 1.4.3.3), a key enzyme for D-amino acid utilization in mammals, were determined in growing rats after a 10 day period of protein undernutrition and subsequent refeeding. Reducing the protein intake for 10 days (3% casein diet) resulted in an 8% loss of the animals' mean body weight and a 50% decrease in the mean size of the liver and kidney as compared to the control animals fed on a 22% casein diet during the same period. When the undernourished rats were refed with the normal protein diet, their weight increased about four fold as compared with that of the control animals, and after a ten day period of refeeding, the lost body and tissue weights were completely recovered. As far as the specific activity of D-amino acid oxidase was concerned, a 44% reduction took place in the liver of rats subjected to protein undernutrition for 10 days. During the period of refeeding, however, the enzyme activity level increased slowly in comparison with the overall hepatic protein level, since its specific activity on day 10 was still 28% below that of the control rats. In sharp contrast, no significant change in the kidney enzyme level was observed throughout these nutritional manipulations. This study strongly suggests that the synthesis and/or catabolism of D-amino acid oxidase may depend on the dietary protein content of the liver but not on that of the kidney. This suggests that D-amino acid oxidase may possibly play distinct physiological roles in these two body organs.

Animals↗

PubMed1996

1,25 Dihydroxyvitamin D3 exerts regional effects in the central nervous system during experimental allergic encephalomyelitis.

1,25-dihydroxyvitamin D3 (1,25-D3) is already known to prevent clinical signs of experimental allergic encephalomyelitis when animals are treated during the immunization phase. In the present work we have evaluated the ability of 1,25-D3 to inhibit chronic relapsing experimental allergic encephalomylitis (EAE) of the Lewis rat, when administered after the beginning of clinical signs. We observed a significant clinical improvement in 1,25-D3-treated rats. This effect was accompanied by a profound inhibition of CD4 antigen expression by central nervous system (CNS) infiltrating monocytes/macrophages and parenchymal microglia. In addition, immunohistochemical analysis performed at the time of the second attack evidenced a region-specific distribution of inflammatory cells. In the same way, some aspects of the effects exerted by 1,25-D3 appeared to vary depending on the region considered, namely spinal cord, brainstem, cerebellum, midbrain or anterior brain. Thus, in 1,25-D3-treated rats, we observed an almost complete inhibition of CD4 antigen expression in the granule cell layer and the adjacent white matter of the cerebellum as well as a marked decrease in the number of OX42-positive cells (macrophages and activated microglia) in anterior brain sections. We conclude that 1,25-D3 can exert immunomodulatory effects inside the CNS during an ongoing immune process and may thus represent a promising therapy for multiple sclerosis.

Animals↗

PubMed1996

Modulation of putrescine transport in rat intestinal brush-border membrane vesicles by fasting and refeeding.

Fasting and refeeding dramatically alter small intestinal mucosal growth which is greatly dependent on polyamine biosynthesis and transport. The aim of this study was therefore to examine the uptake of the diamine putrescine by brush-border membrane vesicles from the small intestine of rats fasted for 3 days or refed a standard diet after a PERIOD OF FASTING. WHILE THE MICHAELIS-MENTEN CONSTANT KM WAS essentially unaltered, the maximum velocity (Vmax) for putrescine uptake was 1.85-fold higher in fasted animals than in ad libitum-fed controls. Refeeding fasted rats for 24 h caused a 31% decrease in the Vmax value that, however, remained 1.27-fold higher than in control RATS, WHILE THE KM VALUE WAS STILL UNCHANGED. FASTING RATS OR refeeding rats after a period of fasting caused only a 13 or 17% increase, respectively, in the value of the constant for the nonsaturable component (P) of putrescine transport relative to the corresponding control condition. Our study also confirms that both the mucosal polyamine biosynthesis and intestinal content are altered by fasting. We suggest that an increased uptake activity may have a conservative role by preventing a substantial loss of tissue polyamines during fasting.

Animals↗

PubMed1995

Expression of the nerve growth factor gene is controlled by the microtubule network.

Colchicine, nocodazol, and vinblastine, three microtubule-disrupting drugs, were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell-secreted NGF protein in L929 cells, with levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubule-stabilizing agent Taxotere, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down-regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene.

Amyloid beta-Protein Precursor↗

PubMed1995

Phosphatidylcholine-phospholipase C mediates the induction of nerve growth factor in cultured glial cells.

Addition of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) to cultured glial cells increased the levels of nerve growth factor (NGF) mRNA and the amount of cell-secreted NGF. The effect of PC-PLC was 2.5 times higher than that elicited by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. In cells in which protein kinase C (PKC) was fully inhibited or downregulated, the effect of PC-PLC was reduced-though still evident-and similar to that exerted by sphingosine. Results thus indicate that PC-PLC induces the synthesis of NGF by glial cells by a PKC-dependent and PKC-independent mechanisms.

Animals↗

PubMed1995

Interactions between second messenger pathways influence NGF synthesis in mouse primary astrocytes.

Primary mouse brain astrocytes were stimulated with phorbol 12-myristate 13-acetate (PMA), serum, forskolin and ionophore A23187, in order to investigate the effect of distinct signalling pathways on the expression of the nerve growth factor (NGF) gene and of proto-oncogenes encoding transcription factors of the Fos and Jun families. PMA, and to a lesser extent serum, induced a marked accumulation of NGF transcripts, in agreement with published observations [Brain Res., 570 (1992) 316-322]. The effect of A23187 was less pronounced and that of forskolin barely detectable. No relationship was observed between the expression of NGF gene and that of c-fos, fos-B, fra-1, jun-B proto-oncogenes. In contrast, changes in the levels of NGF transcripts were associated with corresponding modifications of the levels of c-jun transcripts, a fact which suggests that the c-Jun protein exerts a regulatory role on the expression of the NGF gene. In these cells, however, the regulation of NGF synthesis appears complex, since a pretreatment with forskolin or ionophore A23187 interfered with the promoting effect elicited by PMA or serum in inducing an early decline of the levels of NGF transcripts. This phenomenon was accompanied by a corresponding decrease in the amounts of cell-secreted NGF in cells treated with forskolin and PMA. A23187 had a much more striking effect on the production of mature NGF since this compound maintained the level of cell-secreted NGF to basal values, irrespective of the presence of PMA. A similar inhibitory effect was observed with thapsigargin, another compound able to increase the cytosolic concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Transport and steady-state accumulation of putrescine in brush-border membrane vesicles of rabbit small intestine.

Absorption of polyamines from the lumen is essential for cell proliferation in small intestine but also in other rapidly growing body tissues and tumors. Intestinal uptake of polyamines is thought to involve one or more transport systems, but the characteristics of these systems have not yet been clearly elucidated. Because high levels of putrescine have been identified in intestinal lumen, we explored kinetic, physiochemical, and structural features of uptake of this diamine across rabbit intestinal brush-border membrane vesicles (IBBMV) prepared by CaCl2 or MgCl2 precipitation procedure. Initial rates of putrescine influx were measured during 5-min incubations at 25 or 37 degrees C (optimal temperature) for concentrations of 0.45-145 microM. At both temperatures, kinetics of putrescine transport fitted a model with a single Michaelis-Menten uptake component plus a nonsaturable uptake component. At 37 degrees C, the kinetic parameters for the saturable component of putrescine uptake, Km,app and Vmax,app, were 16.8 +/- 4.7 microM and 19.9 +/- 2.8 pmol.mg protein-1.min-1, respectively. The value of the constant for the nonsaturable component of putrescine uptake (P = 0.45 +/- 0.06 x 10(-8) l.mg protein-1.s-1) suggested this component represented essentially nonspecific binding of putrescine to IBBMV. Cadaverine, spermidine, and spermine were competitive inhibitors of putrescine transport, with inhibition constants equal to 47, 117, and 219 microM, respectively. When effects of a variety of alkyldiamines and structural analogues of polyamines (1 mM) on influx of 5.6 microM putrescine were compared, cadaverine, methylglyoxal bis(guanylhydrazone) (MGBG), and cyclic derivatives of MGBG were found to exhibit the highest inhibitory potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

1,25-dihydroxyvitamin D3 regulates NT-3, NT-4 but not BDNF mRNA in astrocytes.

The effect of 1,25-dihydroxyvitamin D3 on neurotrophin mRNA expression was studied in primary cultures of astrocytes. In addition to its known effects on NGF expression, 1,25-dihydroxyvitamin D3 was shown to upregulate NT-3 mRNA levels, while NT-4 expression was slightly but significantly downregulated. No effect was observed on BDNF mRNA expression. These data clearly show a differential regulation of the four neurotrophins by 1,25-dihydroxyvitamin D3 in primary cultures of astrocytes and suggest that 1,25-dihydroxyvitamin D3 may participate in the expression of NGF, NT-3 and NT-4 in the central nervous system.

Animals↗

PubMed1994

Alterations in intestinal uptake of putrescine and tissue polyamine concentrations in tumor-bearing rats.

Intestinal absorption of putrescine and tissue metabolism of polyamines were investigated in rats grafted with the rapidly growing Mat-Lylu prostatic tumor. These animals exhibited a dramatic 21% decrease in weight and protein, but not DNA, content of their intestinal mucosa, relative to healthy rats reared under similarly controlled nutritional conditions. No significant variation in the specific activities of intestinal brush-border membrane enzymes was observed, however, suggesting a comparable differentiation state of intestinal cells exists in both groups. Putrescine uptake by brush-border membrane vesicles prepared from cancerous or healthy rat intestine was a time dependent process at 25 degrees C. Equilibrium uptake was much greater than could be explained by equilibration of the vesicle space with putrescine, indicating that the diamine was bound to membrane sites. Kinetics of putrescine uptake at 2 min revealed that the process involves two components, a saturable Michaelis-Menten carrier and passive diffusion. With respect to the kinetic parameters of putrescine transport, no significant changes were observed between the tumor-bearing and the control rats. After correction for nonspecific binding to the membranes, putrescine accumulation at equilibrium (75 min) was concentration-dependent and fit a single-site saturable model. Maximum accumulation of the diamine at equilibrium (Bmax) was increased by more than 46% in the cancerous rats relative to the controls, but the dissociation constant (Kd) was unchanged. Efflux of putrescine from the vesicles was slightly slower in the tumor-bearing group, but the differences were generally not significant. No change was observed with respect to the specific activity of ornithine decarboxylase and the concentration of polyamines in the intestinal mucosa. In Mat-Lylu grafted rats fed a standard diet supplemented with [14C]putrescine, about 19% of body radioactivity was recovered in the tumor within 24 h. This was concomitant with a decrease in the percentage of radioactivity retained in the intestinal, renal and hepatic tissues, relative to that retained in the same tissues of healthy rats. Our findings indicate that the presence of the tumor evolves an adaptive response in the small intestine of the rat, involving an increased capacity of the brush-border membrane to accumulate putrescine.

Adenocarcinoma↗