Biomedical subjects

R Rodnight

Publications and source records attributed to R Rodnight.

At least 19 recordsLinked to original sources

PubMed2004

Role of intracellular calcium stores on the effect of metabotropic glutamate receptors on phosphorylation of glial fibrillary acidic protein in hippocampal slices from immature rats.

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.

Aging↗

PubMed2003

Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures.

Organotypic hippocampal cultures have been recently used to study in vitro ischaemic neuronal death. Sub-lethal periods of ischaemia in vivo confer resistance to lethal insults and many studies have demonstrated the involvement of heat shock proteins in this phenomenon. We used organotypic hippocampal cultures to investigate the involvement of heat shock protein (HSP) 27 in preconditioning to oxygen and glucose deprivation. Neuronal damage was assessed using propidium iodide fluorescence; HSP27 phosphorylation and immunocontent were obtained using (32)Pi labelling followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. We observed that immunocontent of HSP27 was increased after lethal or sub-lethal treatment, indicating it is a response to metabolic stress. Treatments with 5 or 10 min of oxygen and glucose deprivation (OGD) or 1- microM N-methyl-D-aspartate (NMDA) induced tolerance to 40 min of OGD associated with an increase in HSP27 immunocontent and phosphorylation. These data suggest that, in vitro, phosphorylated HSP27 might be involved in preconditioning, probably acting as a modulator of actin filaments or by the blockage of neurodegenerative processes.

Animals↗

PubMed2003

The influence of the extracellular matrix on the morphology and intracellular pH of cultured astrocytes exposed to media lacking bicarbonate.

In previous work we showed that the polygonal shape of hippocampal astrocytes cultured on poly-L-lysine changes to a stellate morphology with loss of actinomyosin stress fibers on exchanging the culture medium for saline buffered with HEPES [Brain Res 946 (2002)12]. By contrast, in bicarbonate-buffered saline containing Ca(2+) astrocytes remained polygonal and continued to express stress fibers. Evidence suggests that stellation induced by saline buffered with HEPES is related to intracellular acidification due to the absence of bicarbonate. Here we studied the influence of the matrix used in preparing astrocyte cultures. Stellation in HEPES-saline occurred on a matrix of fibronectin, but not on matrices of collagen I or IV provided Ca(2+) was present. Laminin partially prevented stellation in HEPES-saline. Further, the intracellular acidification induced by HEPES-saline observed in astrocytes cultured on polylysine was abolished in cells cultured on collagens and was attentuated on a matrix of laminin. Two observations suggested the involvement of integrins and focal adhesions. (1) Treatment of cultures on collagens with a blocking antibody to the beta1 integrin subunit abolished protection against HEPES-induced stellation. (2) Compared with polylysine, astrocytes cultured on collagens expressed increased contents of phosphotyrosine proteins, focal adhesion proteins vinculin and paxillin, the beta1 integrin subunit and increased numbers of focal adhesions labelled with anti-vinculin. The observation that astrocytes cultured on collagen I or IV, in contrast to polylysine, express stress fibers and a constant intracellular pH in the absence of buffering by bicarbonate may be related to the fact that in the intact brain astrocytic processes (or end-feet) encounter and bind to collagen IV and laminin in the basement membrane of the endothelial cells which surround the cerebral capillaries. It is also possible that astrocytes retain this capacity from early development when fibrous matrix proteins are present.

Actins↗

PubMed2001

An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation.

Brain ischemia results in cellular degeneration and loss of function. Here we investigated the neuroprotective effect of lithium in an in vitro model of ischemia. Organotypic hippocampal slice cultures were exposed to oxygen and glucose deprivation. Cellular death was quantified by measuring uptake of propidium iodide (PI). Lithium chloride (0.2-1.2 mM) was added to the medium before, during and after lesion induction. A decrease in incorporation of PI was observed, indicating a neuroprotective effect in all doses tested. We also studied the effect of lithium on the phosphorylation of HSP27, a heat shock protein involved in cellular protection in its dephosphorylated state. In the lesioned hippocampus, 0.4 mM lithium chloride decreased the proportion of phosphorylated HSP27 to total HSP27. These results suggest that lithium may be useful in the treatment of brain ischemia.

Animals↗

PubMed2001

Effects of global cerebral ischemia and preconditioning on heat shock protein 27 immunocontent and phosphorylation in rat hippocampus.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.

Animals↗

PubMed2001

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures.

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:

Adenosine Triphosphate↗

PubMed2000

Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology.

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process-bearing phenotype of astrocytes.

Animals↗

PubMed2000

Extracellular S100B protein modulates ERK in astrocyte cultures.

S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.

Animals↗

PubMed2000

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions.

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca(2+) increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.

Adenosine Triphosphate↗

PubMed2000

Identification of brain ecto-apyrase as a phosphoprotein.

Ecto-apyrase is a transmembrane glycoprotein that hydrolyzes extracellular nucleoside tri- or diphosphates. Apyrase activity is affected by several physiological and pathological conditions indicating the existence of regulatory mechanisms. Considering that apyrase presents consensus phosphorylation sites, we studied the phosphorylation of this enzyme. We found an overlay of the immunoblotting and phosphorylated bands in three different preparations from rat brain: (a) hippocampal slices, (b) synaptic plasma membrane fragments and (c) cultured astrocytes. In addition, two-dimensional electrophoresis separations with human astrocytoma cells were done to identify unequivocally the coincidence between the immunodetected and phosphorylated protein. These observations indicate that apyrase can be detected as a phosphoprotein, with obvious implications in the regulation of this enzyme.

Animals↗

PubMed2000

P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes.

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.

Adenosine Diphosphate↗

PubMed2000

Time-dependent enhancement of inhibitory avoidance retention and MAPK activation by post-training infusion of nerve growth factor into CA1 region of hippocampus of adult rats.

Several studies have demonstrated that chronic intracerebroventricular nerve growth factor (NGF) infusion has a beneficial effect on cognitive performance of lesioned as well as old and developing animals. Here we investigate: (i) the effect of post-training infusion of NGF into the CA1 region of hippocampus on inhibitory avoidance (IA) retention in rats; (ii) the extension of the effect, in time and space, of NGF infusion into CA1 on the activity of mitogen-activated protein kinase (MAPK, syn: ERK1/2, p42/p44 MAPK). NGF was bilaterally injected into the CA1 regions of the dorsal hippocampus (0.05, 0.5 or 5.0 ng diluted in 0.5 microL of saline per side ) at 0, 120 or 360 min after IA training in rats. Retention testing was carried out 24 h after training. The injection of 5.0 and 0.5, but not 0.05, ng per side of NGF at 0 and 120 min after IA training enhanced IA retention. The highest dose used was ineffective when injected 360 min after training. The infusion of 0. 5 microL of NGF (5.0 ng) induced a significant enhancement of MAPK activity in hippocampal microslices; this enhancement was restricted to a volume with 0.8 mm radius at 30 min after injection. The MAPK activation was still seen 180 min after NGF infusion, although this value showed only a tendency. In conclusion, localized infusion of NGF into the CA1 region enhanced MAPK activity, restricted in time and space, and enhanced IA retention in a time- and dose-dependent manner.

Age Factors↗

PubMed1999

The mGluR stimulating GFAP phosphorylation in immature hippocampal slices has some properties of a group II receptor.

In a previous study we showed that phosphorylation of the astrocytic marker glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats (P12-P16) is regulated by a metabotropic glutamate receptor (mGluR). The subtypes of these receptors are divided into three groups and exhibit two distinct transduction signals: activation of phospholipase C and liberation of internal calcium (group I) or modulation of cAMP synthesis (groups II and III). Here we investigated the subtype of mGluR involved. Phosphorylation was strongly stimulated by the selective group II agonists DCG IV, L-CCG-I and 1S,3S-ACPD, whereas the group I agonist 3,5-DHPG and the group III agonist L-AP4 had no effect. These results show that the receptor regulating GFAP phosphorylation in the immature hippocampus has some of the properties of a group II mGluR.

Animals↗

PubMed1999

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.

Age Factors↗

PubMed1999

Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus.

Transient global cerebral ischemia induced in rats by four-vessel occlusion for 20 min produced an increase in the immunocontent of glial fibrillary acidic protein and a protein phosphorylation response that was different in the CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (one, four, seven, 14 and 30 days) and observed that the immunocontent and in vitro rate of phosphorylation of glial fibrillary acidic protein in the CA1 region was significantly increased at all intervals after the ischemic insult, indicating that the astrocytic response was maintained for at least 30 days. After reperfusion for 14 days a significant increase in the ratio "in vitro phosphorylation rate/immunocontent" in the CA1 region was observed when compared to control values, to other intervals and to the dentate gyrus, suggesting a hyperphosphorylation of this intermediate filament protein at this interval. In the dentate gyrus, an area less vulnerable to the insult, labelling and immunocontent of glial fibrillary acidic protein were equally increased from four days of reperfusion and the increase remained significant until 30 days, confirming that neuronal death is not the only determining factor for gliosis to occur. In control sham-operated animals, neither the CA1 region nor the dentate gyrus showed significant increases in labelling or immunocontent. Changes in the phosphorylation of glial fibrillary acidic protein may be essential for the plastic response of astrocytes to neuronal damage, as neurons and astrocytes can act as functional units involved in homeostasis, plasticity and neurotransmission.

Animals↗

PubMed1998

Lithium treatment causes gliosis and modifies the morphology of hippocampal astrocytes in rats.

The biological basis of the clinical efficacy of lithium in the treatment of mental illness has been extensively studied in neurones, but little is known about the effects of the drug on glia. Recently we showed that treatment of rats with clinically relevant doses of lithium chloride results in a 35% increase in the immunocontent of the astrocyte marker GFAP in the hippocampus. Here we studied the cytology of this phenomenon. Rats were treated for 4 weeks with a lithium diet which resulted in serum Li+ concentrations of 0.6-1.2 mmol/l. GFAP immunocytochemistry of the hippocampus revealed a mild gliosis in the CA1 area and the dentate gyrus which was associated with a change in the orientation of astrocytic processes. In control animals astrocyte processes were mainly orientated perpendicular to the stratum pyramidale, whereas in treated animals the cells were predominantly stellar in appearance.

Animals↗

PubMed1998

Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus.

The effect of glutamate and lack of external Ca2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12-16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517-523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181-186), glutamate strongly stimulated GFAP phosphorylation in media with Ca2+ and in media lacking Ca2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices.

Animals↗

PubMed1998

The S100B protein inhibits phosphorylation of GFAP and vimentin in a cytoskeletal fraction from immature rat hippocampus.

The S100B protein belongs to a family of small Ca2+-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S 100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 microM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca2+ alone, Ca2+ plus calmodulin or Ca2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca2+/calmodulin-dependent and a Ca2+/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.

Animals↗