Biomedical subjects

Michel P Rathbone

Publications and source records attributed to Michel P Rathbone.

12 recordsLinked to original sources

PubMed2007

Guanosine inhibits CD40 receptor expression and function induced by cytokines and beta amyloid in mouse microglia cells.

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-gamma (10 ng/ml) plus TNF-alpha (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta(1-42); 500 nM). Culture pretreatment with guanosine (10-300 microM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-gamma/TNF-alpha challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-gamma-induced phosphorylation on Ser(727) and translocation of STAT-1alpha to the nucleus as well as TNF-alpha-/Abeta-induced IkappaBalpha and NF-kappaB p65/RelA subunit phosphorylation, thus inhibiting NF-kappaB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-gamma/TNF-alpha-induced CD40 expression and function as well as on STAT-1alpha or NF-kappaB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed2005

Nucleoside transporter expression and function in cultured mouse astrocytes.

Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755-760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1-4 degrees C to study diffusional uptake and 1-60-min uptakes at 37 degrees C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na(+)-independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na(+)-dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine.

Adenosine↗

PubMed2004

Acceleration of blood-brain barrier formation after transplantation of enteric glia into spinal cords of rats.

Enteric glia share morphological, biochemical, and functional properties with astrocytes. Thus, like astrocytes, transplantation of enteric glia into the central nervous system (CNS) might facilitate the development of the characteristics of the blood brain barrier (BBB) in endothelial cells. This study explored this possibility by examining barrier formation after implantation into the spinal cord of rats. Phaseolus vulgaris leucoagglutin (PHAL)-treated enteric glia suspensions were injected into the spinal cord at the T11-T12 level of adult Wistar female rats. Control animals were injected with either 3T3 fibroblast, glioma C6 cells, or culture medium. Evan's blue, a dye excluded by the BBB, was injected intravenously from 1 week to 2 months after implantation. Leakage of dye was determined macroscopically and the ultrastructure of the capillaries was examined. During the first week leakage of dye correlated ultrastructurally with predominantly non-overlapping endothelial cell junctions, even with clefts between adjacent cells. Tight junctions were fully formed by 2 months and no dye leaked. Electron microscopic analysis showed that enteric glia had end-feet in close contact with endothelial cells. In contrast, the injection sites in all control animals leaked dye until 2 months, and most of the tight junctions that did form were incomplete. Furthermore, most 3T3 or C6 control cells had died at 2 months and those that survived, unlike enteric glia, had no anatomical relationship to blood vessels. These data demonstrate that implantation of enteric glia accelerates the formation of the characteristics of the BBB in spinal cord capillaries.

Animals↗

PubMed2004

Guanosine protects SH-SY5Y cells against beta-amyloid-induced apoptosis.

Apoptosis is implicated in the pathophysiology of Alzheimer's disease. Extracellular guanosine inhibits staurosporine-induced apoptosis in astrocytes. We examined whether guanosine protects SH-SY5Y human neuroblastoma cells against beta-amyloid(betaA)-induced apoptosis. Addition of betaA (fragment 25-35, 5 microM for 24 h) to SH-SY5Y cells increased the number of apoptotic cells, as evaluated by oligonucleosome ELISA. Guanosine pre-treatment decreased betaA-induced apoptosis (maximal effect after 24 h, 300 microM, p<0.05). The anti-apoptotic effect of guanosine was reduced by LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) (p<0.05). Guanosine increased phosphorylation of Akt/PKB, and this was abolished by inhibiting PI3K or MEK, (p<0.001, 5 min). Thus, the protective effect of guanosine against betaA-induced apoptosis of SH-SY5Y cells is mediated via activation of the PI3K/Akt/PKB and MAPK pathways.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2004

The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes.

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.

Animals↗

PubMed2003

Guanosine promotes myelination and functional recovery in chronic spinal injury.

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Administration of guanosine (8 mg/kg/day, i.p.) for 7 consecutive days, starting 5 weeks after moderate SCI in rats, improved locomotor function and spinal cord remyelination. Myelinogenesis was associated with an increase in the number of mature oligodendrocytes detected in guanosine-treated spinal cord sections in comparison with controls. These data indicate that guanosine-induced remyelination resulted, at least in part, from activation of endogenous oligodendrocyte lineage cells. These findings may have significant implications for chronic demyelinating diseases.

Animals↗

PubMed2003

Enteric glia promote functional recovery of CTM reflex after dorsal root transection.

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

3T3 Cells↗

PubMed2003

Enteric glia promote regeneration of transected dorsal root axons into spinal cord of adult rats.

After spinal cord injury axonal regeneration is poor, but may be enhanced by the implantation of olfactory ensheathing glia (OEG). Enteric glia (EG) share many properties of OEG. Transected dorsal root axons normally do not regenerate through the central nervous system myelin into the spinal cord. We tested whether EG, like OEG, could promote regeneration in this paradigm. Three weeks after EG implantation, numerous regenerating dorsal root axons reentered the spinal cord. Ingrowth of dorsal root axons was observed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal gray commissure. No axonal ingrowth was observed in control animals, indicating that transplanted EG enabled regeneration of the injured dorsal root axons into the adult spinal cord. Thus, EG implantation may be beneficial in promoting axonal growth after central nervous system injury.

3T3 Cells↗

PubMed2002

Glial cells express multiple ATP binding cassette proteins which are involved in ATP release.

Rat brain astrocyte and microglia cultures express different members of ATP-binding-cassette (ABC) proteins. RT-PCR analysis showed that astrocytes are equipped with P-glycoprotein (mdr1a, mdr1b), multidrug resistance-associated-protein (mrp1, mrp4, mrp5) and cystic fibrosis transmembrane conductance regulator (CFTR). No transcripts for mrp5 and CFTR were detected in microglia. The ABC protein functional activities are shown by the following results: (i) cyclosporin A (50 microM), verapamil (50 microM), probenecid (1 mM) or sulfinpyrazone (2 mM) enhanced [3H]vincristine accumulation; (ii) cyclosporin A or verapamil but not probenecid or sulfinpyrazone enhanced [3H]digoxin accumulation; (iii) glibenclamide (100 microM) inhibited 36Cl efflux from astrocytes. ATP release from glial cells was inhibited by the pretreatment with ABC protein inhibitors indicating that ABC proteins are involved in nucleotide efflux from glial cells which represent the main source of cerebral extracellular purines.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2002

Mechanisms of apoptosis induced by purine nucleosides in astrocytes.

Astrocytes release adenine-based and guanine-based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration-dependent manner up to 100 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2-3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine-based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 microM) or propentophylline (100 microM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S-adenosyl-L-homocysteine (SAH) and greatly reduced the ratio of S-adenosyl-L-methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.

Adenine↗

PubMed2002

Specific [(3)H]-guanosine binding sites in rat brain membranes.

1. Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine-based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) of 0.57 +/- 0.03 pmol mg(-1) protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [(3)H]-guanosine was: guanosine=6-thio-guanosine > inosine > 6-thio-guanine > guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. 5. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

Animals↗

PubMed2002

A method for purifying enteric glia from rat myenteric plexus.

The enteric nervous system is a large and complex division of the peripheral nervous system. The glia associated with it share some characteristics with the olfactory-ensheathing glia, astrocytes and Schwann cells. To facilitate studies of rat enteric glia, we have developed a method for preparing them in large quantities with a high degree of homogeneity. The enteric glia were isolated from the small intestine of Wistar rats by enzymatic digestion with dispase. The cell isolate was added to a mitotically arrested layer of 3T3 cells. Subsequent separation of the enteric glia from the 3T3 cells was done enzymatically, with unavoidable loss of many enteric glia and potential contamination of enteric glia cultures with the 3T3 cells. Therefore, 3T3 cells were cultured in Nunc 0.2-microm tissue culture inserts that could be readily removed from the wells when no longer needed. There was no loss of the enteric glia. The cultures consisted entirely of GFAP-labeled cells, presumptive enteric glia. This method permits the culturing of large numbers of highly purified enteric glia without the use of expensive growth factors and complement-mediated cytolysis.

3T3 Cells↗