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R Rodnight

Publications and source records attributed to R Rodnight.

At least 37 records · Page 2Linked to original sources
PubMed1997

Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping.

In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in hippocampal slices from adult rats is dependent on external Ca2+, whereas in slices from immature rats aged 12-16 days postnatal 32P incorporation into GFAP is inhibited by external Ca2+. The nature of this late developmental change in Ca2+ sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with [gamma-32P]ATP and from slices labelled with [32P]phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca2+/calmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca2+/calmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca2+/calmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca2+ and immature slices labelled in the absence of Ca2+ were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca2+/calmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca2+, kinase activity directly or indirectly dependent on Ca2+ was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca2+ sensitivity of GFAP phosphorylation during development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism(s) whereby Ca2+ controls the relative activity of these enzymes.

Animals↗
PubMed1997

Evidence for a role for calcium ions in the dephosphorylation of glial fibrillary acidic protein (GFAP) in immature hippocampal slices and in astrocyte cultures from the rat.

Evidence was sought for a role for Ca2+ in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in immature hippocampal slices. Although previous work showed that the main phosphatase dephosphorylating GFAP in this preparation is a Ca2+-independent type 1 enzyme, a role for Ca2+ was suggested by the observation that the incorporation of [32P]phosphate into GFAP in immature slices is inhibited by external Ca2+. This inhibition is strikingly different to the situation in mature slices where GFAP phosphorylation is completely dependent on Ca2+. Pure astrocyte cultures were probed by immunoblotting for the presence of the Ca2+-dependent phosphatase calcineurin. An enzyme content, amounting to about 2% of that found in fresh hippocampal tissue, was detected for both the catalytic (alpha) and regulatory (beta) subunits. The direct or indirect association of calcineurin with GFAP was suggested by observations showing that FK506, a specific inhibitor of calcineurin, increased the phosphorylation state of GFAP in immature slices and of GFAP and vimentin in astrocyte cultures.

Animals↗
PubMed1997

Temporal profiles of the in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein (GFAP) after kainic acid-induced lesions in area CA1 of the rat hippocampus: demonstration of a novel phosphoprotein associated with gliosis.

The in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein was studied in slices of area CA1 of the rat hippocampus after stereotaxic injection of 1 nmol of kainic acid. For controls the contralateral hippocampus was injected with saline. Hippocampal tissue was incubated with [32P]phosphate and analysed by two-dimensional electrophoresis for phosphorylation rate and by immunoblotting for immunocontent. Both these parameters decreased during the first 4 days after injection and then started to increase at 10 days and continued to increase until at least 84 days. Except for a small excess of phosphorylation rate at 28 days, the relationship between immunocontent and in vitro phosphorylation rate of glial fibrillary acidic protein remained constant, indicating that the reactive gliosis was not associated with hypo- or a major hyperphosphorylation of this protein. Histology showed a pronounced loss of CA1 pyramidal cells 1 day after injection. At 28 days after injection the pyramidal cells had disappeared and only a few abnormal neurones were present. In contrast, immunocytochemistry after 28 days showed a marked increase in astrocytes reacting positive to the antibody in the strata radiatum and lacunosum moleculare. Besides glial fibrillary acidic protein the expression of several other proteins was upregulated as a result of the injection of kainic acid. These included phosphovimentin and an unknown phosphoprotein designated pp25 which co-migrated on 2-D gels with a prominent phosphoprotein expressed in primary cultures of astrocytes. Pp25 was expressed in lesioned tissue more frequently than phosphovimentin and with a time course that started earlier. Of particular interest was the expression of pp25 in the contralateral saline-injected hippocampus 1 day after injection of kainic acid. It is possible that pp25 will prove to be a sensitive marker of gliosis.

Animals↗
PubMed1997

Wernicke-Korsakoff syndrome.

Alcohol abuse is one of the most serious problems in public health and the Wernicke-Korsakoff syndrome is one of the gravest consequences of alcoholism. The pathology is often undiagnosed in its less evident presentations, therefore an accurate diagnostic approach is a critical step in treatment planning. Treatment is based on restoration of thiamine, although this is insufficient to prevent the psychological decline of a great number of patients. The cognitive impact of the pathology is derived from the interaction of alcoholic neurotoxicity, thiamine deficiency and personal susceptibility. In this article, the literature concerning Wernicke-Korsakoff syndrome is reviewed.

Alcohol Amnestic Disorder↗
PubMed1997

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.

Animals↗
PubMed1996

The dephosphorylation of glial fibrillary acidic protein (GFAP) in the immature rat hippocampus is catalyzed mainly by a type 1 protein phosphatase.

We used protein phosphatase inhibitors to study the phosphatase activity involved in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in a cytoskeletal fraction and in slices prepared from hippocampi of immature rats. Cytoskeletal proteins were labelled with [gamma 32P]ATP in the presence of Ca2+ and calmodulin and then allowed to dephosphorylate through the activity of bound protein phosphatases. Dephosphorylation was inhibited by the protein phosphatase inhibitors okadaic acid and microcystin-LR, but not by EGTA. Maximal inhibition was given by 1 microM okadaic acid and 10 nM microcystin-LR. This difference of two orders of magnitude in the sensitivity of the dephosphorylation to the inhibitors indicates that the bound dephosphorylating activity in the cytoskeletal fraction was due to a type 1 protein phosphatase, rather than protein phosphatase 2A which is equally sensitive to okadaic acid and microcystin-LR. To investigate the dephosphorylation of GFAP in intact tissue we incubated slices with various concentrations of the cell-permeable inhibitor okadaic acid in the presence of [32P] phosphate. Net [32P]-incorporation into GFAP was increased by okadaic acid due to inhibition of dephosphorylation; the minimum effective concentration was 25 nM. Since the IC50 for inhibition of protein phosphatase 2A by okadaic acid is 0.1 nM, this result indicates that in intact tissue GFAP dephosphorylation is primarily due to a type 1 protein phosphatase.

Adenosine Triphosphate↗
PubMed1996

A developmental study of protein phosphorylating systems stimulated by phorbol dibutyrate in micro-slices of rat brain.

Age-dependent changes in the activity of protein phosphorylating systems stimulated by phorbol dibutyrate (M(r) range 40-80 kDa) were studied in micro-slices from rat brain labelled with [32P]orthophosphate. In adult animals phorbol stimulated the phosphorylation of the known substrates of protein kinase C (B-50/GAP-43 and MARCKS) and 3 unknown polypeptides: a basic molecule of 74 kDa (pp74B) and acidic molecules of 60 kDa (pp60A) and 42 kDa (pp42C). In neonatal animals the labelling of MARCKS was relatively very high as previously reported. Labelling of pp60A was present at birth, but increased during development; labelling of pp74B was only detected after days 8-10. The activity of the system labelling pp42C was very high during the first 2 weeks postnatal and then declined rapidly. The labelling of pp74B was considerably higher in slices from the cerebral cortex compared with the hippocampus; no regional differences in the labelling of pp60A were observed. Compared to B-50/GAP-43 and MARCKS, stimulation of phosphorylation of pp74B and pp60A required a higher concentration of phorbol. The substrates of all the phorbol-responsive systems, with the exception of pp74B, were soluble in 40% acetic acid. The possible identity of pp60A, pp74B and pp42C is discussed.

Aging↗
PubMed1995

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.

Age Factors↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Phosphorylation in vitro of glial fibrillary acidic protein is increased in rat hippocampus by administration of 2,5-hexanedione.

Rats were treated with 2,5-hexanedione (2,5-HD) daily for 30 days. Hippocampal microslices were then incubated with [32P]phosphate and the in vitro rate of phosphorylation and the immuno-content of glial acidic fibrillary protein (GFAP) were measured. Exposure to 2,5-HD decreased by 25% the immuno-content of GFAP and increased by 35% its rate of phosphorylation, resulting in an increase of 88% in the ratio phosphorylation rate/immuno-content for this protein.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Chronic administration of lithium chloride increases immunodetectable glial fibrillary acidic protein in the rat hippocampus.

We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li+ in serum reached 0.6-1.2 mM, a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [32P]phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more 32P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.

Animals↗
PubMed1994

Properties of phosphorylated glial fibrillary acidic protein in brain slices of guinea pig, mouse and rat.

1. Brain micro-slices from guinea pig, mouse and rat were incubated in Krebs-Ringer Na-HEPES buffered medium containing [32P]-phosphate and characterized by immunoblotting with polyclonal antibody to glial fibrillary acidic protein (GFAP). 2. GFAP presented small differences in two-dimensional electrophoretic mobility. 3. The phosphorylation of GFAP was dependent on Ca2+ in the incubation medium in adult animals. 4. Both the immunocontent and level of phosphorylation of GFAP were higher in hippocampus than in cerebral cortex of all three species.

Animals↗
PubMed1993

Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride.

A simple method for the transfer of 32P-labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS-buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.

Amino Acids↗