A study of cyclic AMP-dependent protein phosphorylation in Schistocerca gregaria CNS: a comparison to that in mammalian CNS

1. The effect of cyclic AMP (10 microM) on the incorporation of 32P into protein was studied in cell-free preparations of Schistocerca gregaria cerebral ganglia. 2. Cyclic AMP-dependent phosphorylation of total protein was maximal after 60 sec, had a pH optimum of 7 to 8, was not affected by temperature (22-37 degrees C) and had a Km of 77 microM ATP. 3. Cyclic AMP increased the phosphorylation of total and specific protein in soluble fractions greater than synaptosomal greater than microsomal greater than crude membrane fractions. 4. In a direct comparison of locust brain to rat cerebral cortex, cyclic AMP stimulated the increased phosphorylation of only three protein bands, whereas in identical fractions of locust brain the phosphorylation of at least 12 protein bands was observed.     

Distribution of the thy-1 antigen in cellular and subcellular fractions of adult mouse brain.

By using a cytotoxicity inhibition assay employing AKR anti-C3H thymocyte antiserum, we have determined the degree of expression of the thy-1 antigen in fractions of adult mouse brain. As expressed as cytotoxicity inhibitory capacity per mg protein with C3H whole brain arbitrarily assigned a value of 1.0, the following values were found: C3H cerebral cortex, 5.8; C3H cerebral cortex synaptosomes, 2.5: C3H whole brain myelin, 0.65; C3H cerebral cortex neurons, 0.16; and C3H cerebral cortex mitochondria 0.10. Neither C1300 neuroblastoma cells nor any AKR neural fraction had detectable levels of thy-1. The findings indicate that the thy-1 antigen is found mainly in mouse cerebral cortex and in synaptosomal fractions, whereas myelin fractions contain lower but perhaps significant amounts of thy-1. Cerebral cortex neurons, isolated by a method requiring a 90-min mild trypsinization at 37 degrees C, did not display significant amounts of the thy-1 antigen. These results lend themselves to further study in the area of differentiation and development of central nervous system components and in the area of central nervous system immunopathology.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanvalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.This work was supported by, the Polish Academy of Sciences within the project 10.4.     

Distribution of the Glucose Transporter in the Mammalian Brain

We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.     

Regional and Subcellular Distribution of Thy-1 in Human Brain Assayed by a Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay, specific for the monomeric form of human Thy-1, was developed and used for quantitation of the Thy-1 antigen in human brain tissue. Determination of Thy-1 in homogenates of 12 anatomically defined brain regions showed that Thy-1 is present throughout the human brain. However, significant variation was found in the expression of the glycoprotein in different regions. Thy-1 appears to be generally enriched within gray matter: caudate nucleus, cerebral cortex, and putamen were found to contain the highest Thy-1 concentration (approximately 2.5 micrograms Thy-1/mg protein). Interestingly, the cerebellar cortex contained only 25% of the Thy-1 concentration of cerebral gray matter. Cerebral subcortical white matter contained half the amount of Thy-1 compared to cerebral cortex. Determination of Thy-1 in subcellular fractions prepared from human brain biopsy tissue indicated that the highest relative concentration of Thy-1 is associated with synaptosomal membranes and myelin/axonal membrane fractions.     

Influence of exogenous gangliosides (GM1, GD1a, GMix) on a Ca-activated Mg-dependent ATPase in cellular and subcellular brain fractions of the djungarian dwarf hamster (Phodopus sungorus)

The influence of 150 nM exogenously-added gangliosides (G_M1, G_D1a, G_Mix) on a Ca²⁺-activated, Mg²⁺-dependent ATPase was investigated in cellular and subcellular fractions (P1-fraction, synaptosomal fraction, synaptic membranes) from whole brain, cortex, cerebellum and brain stem of the djungarian dwarf hamster (Phodopus sungorus). Gangliosides are effective at this concentration in stimulating the enzyme activity in all fractions from whole brain. Inhomogenous results (stimulation, inhibition and no effects), however, were obtained in the different individual brain regions.     

Late signals are required for the stimulation of DNA synthesis in rat mammary fibroblasts by growth factors

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the lag period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1–2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9–18 h by EGF may be different to those produced by bFGF.     

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.     

EFFECTS OF ENVIRONMENTAL COMPLEXITY ON AMINO ACID INCORPORATION INTO RAT CORTEX AND HIPPOCAMPUS IN VIVO

Abstract— Amino acid incorporation in vivo was investigated in the cortex and hippocampus of rats raised in enriched and deprived environments for various periods of time following weaning. At early times after weaning (7 days), the incorporation of l -[³H]leucine into all sub-cellular fractions of both cortex and hippocampus was higher in enriched than in deprived rats. At 16 days, incorporation into synaptosomal sub-cellular fractions was higher in enriched than in deprived hippocampus, and lower in enriched than in deprived cortex; incorporation into perikaryal fractions of both brain regions was the same in the two groups of animals. Incorporation into subcortical nuclear protein fractions was higher in enriched rats at this time. At 35 days, the only difference between enriched and deprived rats was a lower incorporation into cortical synaptosomal sub-fractions in the former. Experiments involving double labelling and electrophoresis indicate that there is no stimulation or inhibition of the synthesis of any particular protein in hippocampal nuclear and synaptosomal sub-fractions of enriched rats. Synaptosomal proteins of cortex have a greater half-life in enriched than in deprived rats; proteins of perikaryal fractions of cortex, and of all fractions of hippocampus, are turning over at the same rate in enriched and deprived animals.     

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post-natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period. Qualitatively these maturational changes were not affected by neonatal thyroidectomy. Quantitative changes, which applied equally to the cerebral cortex and brain as a whole, were observed. At the age of 35 days, the weights of the cerebral hemispheres and cerebellum were reduced by thyroidectomy by 20 per cent; the overall DNA content per organ did not change, but the amounts of protein and RNA relative to DNA decreased significantly. It is therefore inferred that thyroid deficiency affects the size of the cells in brain and cerebellum rather than their total number. Conversely, the cell population of the liver was only a quarter of that in the control. There was a small but significant decrease in the hepatic protein and RNA content in the hypothyroid animal. The activities of the following enzymes which served as markers for subcellular fractions in homogenates of cerebral cortex were determined: lactate dehydrogenase for the supernatant, glutamate dehydrogenase for the mitochondrial and glutamate decarboxylase for the synaptosomal fractions. When the activities were expressed on a fresh weight basis a significant decrease by comparison with the control values was observed only in the case of glutamate decarboxylase (—15 per cent at the age of 17–32 days); when the activities were based on DNA content all values were reduced, probably as a result of the general decrease in cell size. Pyrimidine metabolism of brain and liver, studied after the administration of [6-¹⁴C]-orotic acid, was not affected in either tissue by neonatal thyroidectomy. A small but significant reduction in the incorporation of labelled pyrimidine nucleotides in liver RNA was observed, but no significant decrease in the incorporation in cerebral RNA was found in the hypothyroid rats.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

Equilibration of Halothane with Brain Tissue In Vitro: Comparison to Brain Concentrations During Anesthesia

A method was devised for reproducing anesthetic concentrations of halothane in slice and membrane preparations of rat brain in vitro. Rats were anesthetized with varying concentrations of halothane, responsiveness was tested, and brain halothane content was determined by heptane extraction and gas chromatography. The inspired concentration of halothane at which half of all animals were unresponsive was 1.05%. At 1.25% halothane, all animals were unresponsive and brain halothane was determined to be 41 +/- 1.3 nmol/mg lipid. No significant differences in halothane concentration between whole brain and a variety of brain regions were detected. To obtain similar concentrations in vitro, membranes or slices of cerebral cortex were incubated in Krebs-Ringer bicarbonate buffer (KRB) that had been preequilibrated with anesthetic. Halothane equilibrated rapidly with the buffer and the tissues. The partition coefficient between gas and KRB was found to be 0.78, and between brain slices and KRB approximately 12. Slightly lower gas concentrations were necessary in vitro than in vivo to obtain the same tissue levels of anesthetic. Using this method, it was shown that there was no effect of anesthetic concentrations of halothane on the uptake of [3H]norepinephrine or [3H]choline into slices of rat cerebral cortex.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Calcium-dependent phosphorylation of specific synaptosomal fraction proteins: Possible role of phosphoproteins in mediating neurotransmitter release

Calcium ions caused a marked increase in the level of endogenous phosphorylation of specific proteins from synaptosomal fractions prepared from rat cerebral cortex. The levels of phosphorylation of these specific proteins were dependent upon the presence of calcium and regulated by small changes in the concenrration of calcium ions. The effect of calcium was independent of ATP concentration over a wide range of concentrations. The results are compatible with the hypothesis that some of the effects of calcium on synaptic transmission might be mediated by the effect of calcium on the phosphorylation of specific synaptosomal proteins.     

COMPARISON OF THE BINDING’ OF β-ALANINE AND y-AMINOBUTYRIC ACID IN SYNAPTOSOMAL-MITOCHONDRIAL FRACTIONS OF RAT BRAIN1

Abstract— Na⁺-dependent ‘binding’ of β-alanine and GABA was examined with synaptosomal-mitochondrial fractions of rat brain incubated for 10 min at 0°C. GABA was bound to a much greater extent than β-alanine to particles of cerebral cortex, whole cerebellum and brain stem. For cerebral cortex, the binding capacity (B_max) for GABA was about 18 limes greater than that for β-alanine. and the affinity of the particles for GABA was about 2′ times greater than for β-alanine. The order of potency of GABA binding to brain regions was cerebral cortex > cerebellum > brain stem, whereas that for β-alanine was the reverse. If the binding of β-alanine is taken to indicate the glial component of the Na⁺-dependent binding process for GABA, then most of the GABA was bound to neuronal elements under the conditions employed.     

Development of a synaptosomal model to determine drug-induced in vivo changes in GABA-levels of nerve endings in 11 brain regions of the rat

An experimental procedure was developed which allowed the simultaneous measurement of GABA in synaptosomes from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Post-mortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. Experiments with addition of aminooxyacetic acid (AOOA, 1 mM) to the homogenizing medium indicated that GABA was not being degraded during synaptosome preparation. The presence of exogenous GABA (1 mM) did not alter the GABA levels in the organelles, indicating that no significant redistribution of GABA occurred during subcellular fractionation. On the basis of these findings, it was suggested that synaptosomal fractions could be used as a model to monitor indirectly the drug-induced changes in GABA levels of nerve endings in discrete brain areas of the intact animal. In vivo experiments with AOAA (30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosmal GABA levels in different brain regions. Although AOAA was more potent than VPA in increasing GABA in whole tissue of most brain regions, significant increases of synaptosomal GABA levels after AOAA were only determined in olfactory bulbs and frontal cerebral cortex. In contrast, VPA induced significant synaptosomal GABA increases in olfactory bulbs, hypothalamus, superior and inferior colliculus, substantia nigra, and cerebellum. The data indicate that the synaptosomal model can provide useful information on the in vivo effects of drugs on GABA levels in nerve terminals and their ability to exert this effect in specific brain areas.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes

Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+-dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87-kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87-kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, gamma-aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+-dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus-secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.