Cerebellar Cytosol Research Articles

2-Halopropionic Acid-induced Cerebellar Granule Cell Necrosis in the Rat: In Vivo and In Vitro Studies

Daily oral administration of 2.3 mmol/kg l-2-chloropropionic acid ( l-2-CPA), dl-2-bromopropionic acid (2-BPA) or dl-2-iodopropionic acid (2-IPA) but not dl-2-fluoropropionic acid (2-FPA) produced cerebellar granule cell necrosis in the rat. Twenty four hours after three doses of l-2-CPA or two doses of 2-BPA, animals showed clinical signs of motor incoordination and reduced hindlimb function which was associated with marked cerebellar oedema and cerebellar granule cell necrosis. Biochemical analyses showed a marked increase in cerebellar water and Na + content, and a reduction in cerebellar glutamate and aspartate. 2-IPA at this dose was toxic, the animals not surviving a second dose, histopathology showed hepatic and renal necrosis with mild cerebellar granule cell necrosis. 2-FPA was not neurotoxic after four daily doses. A marked decrease in hepatic and cerebellar non-protein sulphydryl (NP-SH) content was observed 4 h after a single dose of 2.3 mmol/kg l-2-CPA, 2-BPA and 2-IPA but not 2-FPA. Daily doses of 2-BPA for 3 days produced a sustained 50% depletion in cerebellar NP-SH. In vitro, l-2-CPA, 2-BPA and 2-IPA produced glutathione (GSH) depletion in the presence of rat liver cytosol, while 2-FPA did not. Depletion of GSH in the presence of cerebellar cytosol was only observed with 2-IPA. Studies using primary cultures of rat cerebellar granule cells, showed that all analogues produced a concentration dependent loss of cell viability. Mean EC 50 values for 2-FPA, l-2-CPA, 2-BPA and 2-IPA toxicity were 1.7, >10, 0.5 and 0.3 mM, respectively, for 24 h continuous exposure. MK-801 and Vitamin E afforded protection against l-2-CPA-induced cytotoxicity but not against the other analogues. In summary, in addition to l-2-CPA, both 2-BPA and 2-IPA produce cerebellar granule cell necrosis in the rat. Depletion of GSH in the cerebellum may be contributory factor in the cascade of events leading to neurotoxicity.

Effects of kyotorphin ( l-tyrosyl- l-arginine) on [ 3H]N G-nitro- l-arginine binding to neuronal nitric oxide synthase in rat brain

l-Tyrosyl- l-arginine (kyotorphin) is known as an endogenous analgesic neuropeptide. We examined whether kyotorphin and other arginine-containing neuropeptides were endogenous substrates for neuronal nitric oxide synthase (NOS) in the rat brain. Cytosol fractions of the rat cerebellum contained higher concentrations of neuronal NOS (nNOS) than endothelial NOS. In rat cerebellar cytosol, the binding activity of [ 3H]N G-nitro- l-arginine (NNA) was inhibited equally by l-arginine ( l-Arg), kyotorphin, and l-leucyl- l-Arg (a kyotorphin receptor antagonist). Binding activities were inhibited to lesser degrees by fibronectin active fragments, bradykinin, and dynorphin A, but were not inhibited by l-tyrosyl- d-Arg or substance P. Interestingly, the inhibition of [ 3H]NNA binding by kyotorphin was attenuated by inhibitors of kyotorphin-hydrolyzing peptidases (KTPases) such as bestatin and arphamenine B. These results suggest that kyotorphin is degraded to l-Arg by KTPases, which in turn may act as substrate for nNOS.

Changes in some pro-and antioxidants in rat cerebellum after chronic alcohol intake

Some pro- and antioxidants were measured in the cerebellum from ethanol-fed rats using ethanol administration in drinking water as a model of moderate alcohol intoxication. After 4 weeks of ethanol intake, a 30% increase in the nonheme iron content in the cerebellum occurred in ethanol-fed rats as compared to control animals. The low-molecular-weight chelated iron (LMWC-Fe) content as well as the percentage of total nonheme iron represented by LMWC-Fe were increased in the cerebellar cytosol after chronic ethanol administration. Cerebellar copper and selenium concentrations were lower and zinc concentration higher in ethanolfed rats than in controls. Ethanol consumption decreased the cerebellar vitamin E level. Glutathione Stransferase [EC 2. 5. 1. 18]activity was higher, whereas glutathione peroxidase [glutathione: H 2O 2 oxidoreductase, EC 1. 11. 1. 9]activity was not altered by ethanol treatment. No significant changes in cerebellar lipid peroxidation, carbonyl protein content, or glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming) EC 6. 3. 1. 2]activity were observed. These results suggest that adaptative increases in some elements of the antioxidant defense may counteract the increase in LMWC-Fe, a pro-oxidant factor, and prevent the occurrence of overt cellular lipid and protein damage. However, after 8 weeks of ethanol intake, the activity of glutamine synthetase, an enzyme specially sensitive to inactivation by oxygen radicals, was decreased, suggesting that this prevention was not totally achieved.

N G-nitro-l-[ 3h]arginine binding properties of neuronal nitric oxide synthase in rat brain

N G-Nitro-L-arginine (L-NNA), a derivative of L-arginine (L-Arg), is known as a pseudo-substrate and inhibitor for nitric oxide synthase (NOS). To clarify the regulatory mechanism of substrate-binding domain in neuronal NOS (nNOS), we examined the characteristics of N G-nitro-L-[ 3H]Arg (L-[ 3H]NNA) binding using the cytosolic fraction and purified nNOS from the rat cerebellum, in comparison with L-[ 14C]citrulline formation from L-[ 14C]Arg. The L-[ 3H]NNA binding was inhibited by L-NNA > N G-methyl-L-Arg > diphenyleneiodonium > L-Arg, but was not inhibited by L-citrulline and D-Arg. Thus, L-NNA seems to bind the substrate-binding domain in the nNOS with high affinity rather than L-Arg. Even in the absence of NADPH, tetrahydrobiopterin (BH 4) and Ca 2+, the L-[ 3H]NNA binding activity was observed in the cerebellar cytosol, although L-[ 14C]citrulline could not be produced from L-[ 14C]Arg. L-[ 3H]NNA binding was increased by BH 4 alone and was markedly enhanced by NADPH plus BH 4 (NADPH/BH 4), but not by Ca 2+/CaM. In contrast, L-[ 14C]citrulline was formed only in the presence of NADPH/BH 4 and Ca 2+. Similar results were obtained in purified nNOS. These results suggest that L-[ 3H]NNA seems to bind the substrate-binding domain in the nNOS but the binding affinity of L-Arg was lower than the affinity of L-NNA. Although the substrate binding is necessary to BH 4 and NADPH, Ca 2+/CaM are further necessary for the formation of NO and L-citrulline.

Assay of neuronal nitric oxide synthase by HPLC determination of citrulline

Biological membranes from different tissue sources were incubated for nitric oxide synthase (NOS) activity under standard conditions at 20°C and compared with rat cerebellar cytosol. NOS activity was monitored as the formation of l-citrulline from l-arginine. Samples were purified on Amprep CBA cation-exchange minicolumns prior to derivatization with o-phtaldialdehyde (OPA) and HPLC analysis. The OPA derivatives of l-citrulline and l-arginine eluted well separated within 15 min during isocratic elution at room temperature. A linear relation between peak height and quantity of l-citrulline was seen down to the detection limit at 0.1 pmol l-citrulline. Formation of l-citrulline was measurable in rat cerebellar cytosol as well as in preparations not previously assayed for NOS activity, including rat colon, cat oesophagus and crayfish ( Pacifastacus leniusculus) brain. The method provides a sensitive and non-radioactive method for assaying NOS activity in small tissue samples and in tissues with low to moderate levels of NOS activity.

Calcium-induced degradation of the Inositol (1,4,5)-trisphosphate receptor/Ca 2+-channel

Ca 2+-induced degradation of the neuronal inositol (1,4,5)-trisphosphate receptor, a protein which regulates Ca 2+-release from intracellular stores, has been examined. The IP 3-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca 2+-activated neutral protease (calpain). Incubation of membranes or immunopurified IP 3-receptor with Ca 2+ and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca 2+-channel-containing part of the protein. These data suggest that activation of the IP 3-receptor, by causing increases in intracellular [Ca 2+], might result in degradation of the N-terminal, IP 3-binding part of the receptor.

(6 R)-5,6,7,8-Tetrahydro- l-biopterin modulates nitric oxide-associated soluble guanylate cyclase activity in the rat cerebellum

(6 R)-5,6,7,8-Tetrahydro- l-biopterin (R-THBP) is a cofactor not only for aromatic amino acid hydroxylases in mammalian tissues but also for nitric oxide synthase (NOS) induced by endotoxins or cytokines in some kinds of cells. Recently it has been reported that nitric oxide (NO) has biological activity in endothelium and in brain as well. NO activates soluble guanylate cyclase (sGC). Superoxide reacts with NO easily and shortens the halflife of NO actions. We found, in a study using rat cerebellar cytosol fraction, that R-THBP itself did not directly activate sGC, but activated sGC at concentrations ranging from 0.1 to 10 μ m only under NO generating conditions of activated NOS and in the presence of sodium nitroprusside. In addition, R-THBP (1 μ m) did not alter the NOS activity, which was determined by l-citrulline formation. These results suggest that R-THBP may regulate sGC activity associated with NO formation in the central nervous system.

The developmental changes of mRNA levels for a cerebellar protein (spot 35 protein) in rat brains

Our previous papers described a protein called spot 35 found in the cerebellar cytosol of adult rats by two-dimensional gel electrophoresis and localized in the Purkinje cells by immunohistochemical methods. Here we describe the biosynthesis of this spot 35 protein using a reticulocyte lysate cell-free system containing rat cerebellar mRNA. The developmental changes of mRNA-dependent protein biosynthesis were also examined. During postnatal 10–30 days, a rapid increase of mRNA levels for spot 35 protein was observed. The application of the new 45Ca-binding assay procedure revealed that this protein is a Ca-binding protein.

Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase

The regional and cellular distribution of G-substrate, a 23,000-dalton protein substrate specific for guanosine 3',5'-cyclic monophosphate-dependent protein kinase, has been examined in mammalian brain using immunoprecipitation, radioimmunoassay, and peptide-mapping techniques. In rabbit brain, G-substrate was found to be highly concentrated in the cerebellum. The concentration of G-substrate in cerebellar cytosol was 27.2 pmol/mg. The concentrations of G-substrate in cortex, hippocampus, and caudate were only 1 to 2% of that found in cerebellum. Studies of neurological mutant mice lacking either Purkinje cells (PCD and nervous) or granule cells (weaver) suggested that, within the cerebellum, G-substrate is localized almost exclusively in Purkinje cells. A phosphoprotein present in noncerebellar brain regions, which co-migrated with G-substrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown by peptide mapping to consist predominantly of phosphatase inhibitor-1. Phosphatase inhibitor-1, a potent inhibitor of protein phosphatase-1, is known to share several physicochemical properties with G-substrate. In contrast to the results obtained with G-substrate, the concentration of phosphatase inhibitor-1 was significantly lower in cerebellum than in other major brain regions. These and other data suggest that G-substrate may be a Purkinje cell-specific protein phosphatase inhibitor.

Isolation and immunohistochemical localization of a cerebellar protein

Our previous report described that a protein called spot 35 is found in the cerebellar cytosol of adult rats by two-dimensional gel electrophoresis. In this paper we isolated this protein from the soluble fraction of bovine and rat cerebella and then prepared an antiserum against the bovine protein. This protein shows p I around 5.3 and M r around 27 kdalton. Determination of the amino acid composition of this protein shows high glutamic acid, aspartic acid and leucine contents. Using the antiserum we examined the immunohistochemical localization of this protein by the peroxidase-antiperoxidase method. Purkinje cells, their dendrites and axons were immunohistochemically stained in the cerebella of adult rats, rabbits and humans. Other cells, such as granule cells and glial cells, and myelin did not react to the antiserum.

Ontogeny of microtubule-associated protein 2 in rat cerebellum: Differential expression of the doublet polypeptides

We have examined the ontogeny of the microtubule-associated protein, MAP2 in rat cerebellum using biochemical and immunocytochemical techniques. In adult animals MAP2 can be resolved as a doublet of polypeptides (referred to here as MAP2a and MAP2b). In young rats only the lower molecular weight MAP2b was present in cerebellar cytosol; MAP2a appeared by 20 days postnatally. The identification of the polypeptides as MAP2a and MAP2b in heat-stable fractions of cytosol was confirmed by peptide mapping which also demonstrated that MAP2a and MAP2b shared virtually identical peptide maps. Using a rabbit anti-MAP2 antibody that was characterised by immunoblotting of cerebellar homogenates, the localisation of MAP2 was examined in 10-day-old and adult cerebella. In 10-day-old animals, MAP2 immunoreactivity was detected in the external germinal layer, granular layer and most strikingly in Purkinje cell bodies and dendrites. Parallel fibre axons were unstained. In contrast both axons and dendrites were stained by an antibody against α-tubulin. In adult animals MAP2 immunoreactivity was largely confined to Purkinje cell dendrites. The absence of assembled microtubules from Purkinje cell dendrites of young animals despite the presence of MAP2 and tubulin immunoreactivity may be explained by the presence of only a single MAP2 polypeptide (MAP2b) whereas the presence of the MAP2 doublet polypeptides in the adult may facilitate polymerization. Since MAP2b does not co-assemble with microtubules efficiently in vitro it may represent a form of MAP2 unable to stimulate microtubule assembly. Therefore, the possibility exists that MAP2b may play an alternative role during dendritic growth.