Mechanisms involved in the antinociception induced by systemic administration of guanosine in mice.

BACKGROUND AND PURPOSE: It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines on pain transmission. The aim of this study was to investigate the effects of intraperitoneal (i.p.) and oral (p.o.) administration of guanosine on mice pain models. Additionally, investigation into the mechanisms of action of guanosine, its potential toxicity and cerebrospinal fluid (CSF) purine levels were also assessed. EXPERIMENTAL APPROACH: Mice received an i.p. or p.o. administration of vehicle (0.1 mM NaOH) or guanosine (up to 240 mg x kg(-1)) and were evaluated in several pain models. KEY RESULTS: Guanosine produced dose-dependent antinociceptive effects in the hot-plate, glutamate, capsaicin, formalin and acetic acid models, but it was ineffective in the tail-flick test. Additionally, guanosine produced a significant inhibition of biting behaviour induced by i.t. injection of glutamate, AMPA, kainate and trans-ACPD, but not against NMDA, substance P or capsaicin. The antinociceptive effects of guanosine were prevented by selective and non-selective adenosine receptor antagonists. Systemic administration of guanosine (120 mg x kg(-1)) induced an approximately sevenfold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by intraplantar capsaicin. CONCLUSIONS AND IMPLICATIONS: This study provides new evidence on the mechanism of action of the antinociceptive effects after systemic administration of guanosine. These effects seem to be related to the modulation of adenosine A(1) and A(2A) receptors and non-NMDA glutamate receptors.

Importance of schedule of administration in the therapeutic efficacy of guanosine: early intervention after injury enhances glutamate uptake in model of hypoxia-ischemia.

Perinatal cerebral hypoxia-ischemia (HI) is an important cause of mortality and neurological disabilities such as cerebral palsy, epilepsy, and mental retardation. The potential for neuroprotection in HI can be achieved mainly during the recovery period. In previous work, we demonstrated that guanosine (Guo) prevented the decrease of glutamate uptake by hippocampal slices of neonatal rats exposed to a hypoxic-ischemic (HI) insult in vivo when administrated before and after insult. In the present study, we compared the effect of Guo administration only after HI using various protocols. When compared with the control, a decrease of [(3)H] glutamate uptake was avoided only when three doses of Guo were administered immediately, 24 h and 48 h after insult, or at 3 h, 24 h, and 48 h after injury or at 6 h, 24 h, and 48 h after HI. These findings indicate that early Guo administration (until 6 h) after HI, in three doses may enhance glutamate uptake into brain slices after hypoxia/ischemia, probably resulting in decreased excitotoxicity.

Methylmercury increases S100B content in rat cerebrospinal fluid.

S100B, a calcium binding protein physiologically produced and released by astrocytes, has been used as a peripheral marker of brain damage. Here, we investigated the effects of subcutaneous injections of methylmercury chloride (MeHg-5mg/kg), an environmental neurotoxicant, on S100B protein content in cerebrospinal fluid (CSF) of adult rats. In addition, the performance of animals in an open field (number of squares crossing and rearings) was also analyzed in order to obtain a possible link between alteration in S100B protein content in CSF and parameters related to neurological injury. MeHg treatment increased serum mercury and S100B protein levels in the CSF. A decrease in the numbers of crossings and rearings was observed in MeHg-treated animals when compared to control group, which suggests a possible neurological injury. The present data show, for the first time, increased S100B levels in CSF after exposure to a neurotoxic metal. Authors discuss the possibility of astrocytic involvement in MeHg-induced neurotoxicity.

Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype.

Hepatic stellate cells are intralobular connective tissue cells expressing the myofibroblast or the lipocyte phenotypes. They participate in homeostasis of the liver extracellular matrix, repair, regeneration, and fibrosis under the former phenotype, and control the retinol metabolism, storage, and release under the latter one. They are heterogeneous in terms of their tissue distribution, function, and expression of cytoskeletal proteins. We have studied the expressions of intermediate filaments in the cloned GRX cell line representative of murine hepatic stellate cells, by immunolabeling, reverse transcription polymerase chain reaction (RT-PCR), immunoprecipitation and Western blots. GRX cells expressed vimentin, desmin, glial fibrillary acidic protein (GFAP), and smooth muscle alpha actin (SM-alphaA). Vimentin, desmin, and SMN-alphaA were expressed in all cultures. GFAP showed a heterogeneous intensity of expression and did not form a filamentous cytoskeletal network, showing a distinct punctuate cytoplasmic distribution. When activated by inflammatory mediators, GRX cells increased expression of desmin and GFAP. Retinol-mediated induction of the lipocyte phenotype elicited a strong decrease of intermediate filament protein expression and the collapse of the filamentous structure of the cytoskeleton. Quiescent hepatic stellate precursors can respond to physiologic or pathologic stimuli, expressing activated myofibroblast or lipocyte phenotypes with distinct patterns of cytoskeleton structure, metabolic function, and interaction with the tissue environment.

Methylmalonic and propionic acids increase the in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of young rats through NMDA glutamate receptors.

In this study we investigated the effects of methylmalonic acid (MMA) and propionic acid (PA) on the phosphorylation of cytoskeletal proteins of cerebral cortex of rats. Slices of tissue were incubated with 32P-orthophosphate in the presence or absence of glutamate, MMA, PA and ionotropic or metabotropic glutamate receptor agonists. The cytoskeletal fraction was isolated and the radioactivity incorporated into the cytoskeletal proteins was measured. Results demonstrated that the acids, glutamate and NMDA increased the phosphorylation of the proteins studied. However, this effect was not observed for non-NMDA ionotropic agonists or metabotropic agonists. Experiments using glutamate receptor antagonists confirmed that MMA and PA at the same concentrations as found in tissues from propionic or methylmalonic acidemic children increase the phosphorylation of cytoskeletal proteins, possibly via NMDA glutamate receptors. Therefore, it is feasible that these findings may be related to the neurological dysfunction characteristic of these disorders.

Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27).

The effect of external Ca2+ ([Ca2+]e) on the incorporation of [32P] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total 32P-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total 32P-incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total 32P-incorporation. In zero [Ca2+]e the non-specific channel blocker Co2+ (1 mM) decreased total 32P-incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181-186] in which it was shown that in immature hippocampal slices zero [Ca2+]e compared with 1 mM [Ca2+]e increased 32P-incorporation into GFAP without changing total incorporation. The difference between the results for total 32P-incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+]e in the medium since in slices concentrations of [Ca2+]e higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 microM) and zero [Ca2+]e stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+]e the effect on the more acidic isoform was proportionally greater.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity.

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca(2+)-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca(2+)-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation state of GFAP and a consequent increase in the number of mitotic astrocytes.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+ -dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of thereceptor by glutamate inhibits Ca2+ entry into the astrocytes andconsequently down-regulates a Ca2+-dependent dephosphorylationcascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation state of GFAP and a consequent increase in the number of mitotic astrocytes.

Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus.

We studied the effect of external Ca2+ on the incorporation of [32P]phosphate into the astrocytic marker protein, glial fibrillary acidic protein (GFAP), in hippocampal slices from rats in the postnatal age range 12-16 days to +60 days (P12-P16 to +P60). At age P12-P16 the presence of Ca2+ in the incubation medium inhibited the incorporation of 32P into GFAP; this inhibition declined to near zero by P21 and subsequently 32P-incorporation became progressively more dependent on Ca2+ until by P60 no GFAP phosphorylation was observed in the absence of Ca2+. With tissue from immature rats inhibition of 32P-incorporation into GFAP started at a medium concentration of 7.5 microM Ca2+, reached 50% at 100 microM and then remained constant up to 1 mM; with adults maximal phosphorylation required 1 mM Ca2+ in the medium. The inorganic Ca(2+)-channel blockers, Co2+ and Ni2+, and a high concentration of the L-type blocker, nifedipine, reversed the effects of external Ca2+ on GFAP phosphorylation. The results suggest a late developmental change in the compartmental disposition of Ca2+ in astrocytes.

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.

Glutamate stimulates the phosphorylation of glial fibrillary acidic protein in slices of immature rat hippocampus via a metabotropic receptor.

Phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats (10-16 days postnatal) was strongly stimulated by glutamate in the presence of Ca2+. This effect apparently occurred via a metabotropic receptor since the specific agonist of metabotropic glutamate receptors, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), stimulated GFAP phosphorylation by 173% whilst the mixed agonists, ibotenate and quisqualate, stimulated to a lesser extent. Ionotropic agonists were mainly ineffective. The action of 1S,3R-ACPD was blocked by L(+)-2-amino-3-phosphonopropionic acid (L-AP3) a specific antagonist of the metabotropic glutamate receptor coupled to the hydrolysis of phosphoinositides and was reduced by 70% by preincubation of the slices with pertussis toxin. In contrast to these results with immature animals glutamate had little or no effect on the phosphorylation of GFAP in hippocampal slices from adult rats.

Effect of morphine, ACTH, epinephrine, Met-, Leu- and des-Tyr-Met-enkephalin on beta-endorphin-like immunoreactivity of rat brain.

Morphine (1,0 mg/kg), ACTH1-24 (10.0 micrograms/kg), epinephrine (12.0 micrograms/kg), Met-enkephalin (2.0 and 5.0 micrograms/kg), Leu-enkephalin (2.0 micrograms/kg) and des-Tyr-Met-enkephalin (2.0 micrograms/kg) all produced marked reductions of beta-endorphin-like immunoreactivity in the rat diencephalon. At a dose of 0.4 mg/kg, naloxone had no effect of its own and was unable to reverse the depleting effect of the other substances. The depletion of beta-endorphin-like immunoreactivity caused by the various treatments is attributable to release and subsequent degradation of beta-endorphin and/or of its precursors. The various behavioral effects of morphine, ACTH, epinephrine and the enkephalins may be explained by the release of endogenous beta-endorphin.