吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Zn2+, mitochondria and neuronal injury

In addition to its familiar role as a component of metalloproteins, zinc is also sequestered in the presynaptic vesicles in 'zinc-containing' neurons. The best-established physiological role of synaptically released zinc is the tonic modulation of brain excitability through modulation of amino acid receptors; prominent pathological effects include acceleration of plaque deposition in Alzheimer's disease and exacerbation of excitotoxic neuron injury. Synaptically released zinc functions as a conventional synaptic neurotransmitter or neuromodulator being released into the cleft then recycled into the postsynaptic neurons during synaptic events, functioning analogously to calcium in this regard, as a transmembrane neural signal. To stimulate comparisons of zinc signals with calcium signals, we have compiled a list of the important parameters of calcium signals and zinc signals. More speculatively, we hypothesize that zinc signals may loosely mimic phosphate signals in the sense that signal zinc ions may commonly bind to proteins in a lasting manner, as a result changing their structure and function.     

REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES OF CEREBRAL CORTEX BY Ca2+ AND CYCLIC GMP

Cyclic nucleotide phosphodiesterase activity of porcine cerebral cortical extracts was measured with 0.1–100 μM-cyclic AMP and cyclic GMP and found to be dependent on both Ca²⁺ and added cyclic nucleotides. With decreasing substrate concentration activity with cyclic GMP became more dependent on Ca²⁺ whereas hydrolysis of cyclic AMP became less dependent. Cyclic GMP at 3 μM stimulated the hydrolysis of 0.1–10μM-cyclic AMP in the absence of Ca²⁺ (< 10^-10M) but inhibited activity with 200 μM-Ca²⁺ present. This differential, substrate- and Ca²⁺-dependent regulation was attributed to the presence of at least two types of phosphodiesterase distinguishable by DEAE-column chromatography. In the absence of Ca²⁺, activity with 1 μM-cyclic GMP eluted in one minor peak followed by two major peaks, D-I and D-II. Activity with 1 μM-cyclic AMP eluted almost entirely in D-II. Hydrolysis of cyclic AMP in D-II was activated by cyclic GMP. With added Ca²⁺ plus a Ca²⁺-dependent regulator (CDR), activity with 1 μM-cyclic GMP was markedly increased and eluted entirely at D-I. Total activity with 1 μM-cyclic AMP was only moderately increased and eluted as D-I with a shoulder at D-II. Elution profiles with 100 μM-substrate were relatively independent of substrate, with D-I predominant with Ca²⁺·CDR present and D-II predominant in its absence. Kinetic analysis of rechromatographed D-I showed a 20- to 40-fold activation by Ca²⁺·CDR that was largely due to an increase in V_max, with only 50% decreases in K_m Both substrates competitively inhibited hydrolysis of the other with K_i values equal to their respective K_m values (1.7 μM for cyclic GMP and 48 μM for cyclic AMP with Ca²⁺-CDR present). Studies with theophylline and trifluoperazine indicate differential, substrate-dependent inhibitions of both enzymes. These findings demonstrate that phosphodiesterase activity in neural tissue is subject to regulation by Ca²⁺, cyclic GMP, and inhibitors in a complex, substrate-specific and concentration-dependent manner.     

IN VITRO Ca2+ DEPENDENT PROTEOLYSIS IN BRAIN TISSUES: A POSSIBLE SOURCE OF ERROR IN TRANSMITTER METABOLISM STUDIES

—The effects of Ca²⁺ ions on the metabolism of [³H]serotonin and [³H]-labelled catecholamines have been examined in hippocampal slices or synaptosomes. The formation of [³H]-5 hydroxyindoles ([³H]serotonin + [³H]-5 hydroxyindoleacetic acid) from [³H]tryptophan and that of [³H]-labelled catecholamines from [³H]tyrosine were increased when Ca²⁺ was omitted from the incubating medium. However, the total synthesis of 5-HT from tryptophan and that of catecholamines from tyrosine did not seem to be significantly changed. Altered formation of tritiated amines were due to changes in the specific activities of respective precursor amino acids. This reflected altered sizes of the free amino acid pools caused by Ca²⁺-dependent in vitro proteolysis. This must be taken into consideration when studying in vitro Ca²⁺ dependency of neutrotransmitter metabolism.     

Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet ( Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding β-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.     

DEVELOPMENTAL CHANGES IN Na+, K+ AND ATP AND IN THE LEVELS AND TRANSPORT OF AMINO ACIDS IN INCUBATED SLICES OF RAT BRAIN

Abstract—

• 1 Upon incubation, slices of brain tissue took up fluid; the degree of swelling increased with increasing age. No sweiling occurred in slices from foetal brain. Since this swelling was associated with increases in the inulin space, the percentage of inulin space in slices at the end of incubation increased during brain development.
• 2 Most of the capacity for ion transport seemed to be absent from foetal brain. In vivo and in slices, Na⁺ was very high and K⁺ was very low in comparison to levels at other ages. There was a rapid change around birth, but no significant change at later ages. Upon incubation, Na⁺ levels increased in other slices, but not in slices of foetal brain.
• 3 Upon incubation of the slices, ATP levels were restored to levels close to those in the living brain; there were no significant alterations in available energy during development to explain changes in amino acid transport.
• 4 The composition of the free pool of cerebral amino acids in vivo changed with development, with some compounds (glutamic acid and related compounds) increasing, others (mostly‘essential’amino acids) decreasing, with age. These changes were not linear with time, and the level of a compound might exhibit several peaks during development.
• 5 The uptake (influx) of taurine, glutamate and glycine into brain slices increased rapidly during the foetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age and then declined to adult levels. The levels of steady-state uptake with glycine also exhibited a maximal peak at 2-3 weeks of postnatal age. Steady-state uptake of taurine and glutamate reached adult levels by about 3 weeks of age.
• 6 The pattern of inhibition of amino acid transport by two specific amino acid analogues changed during development for some amino acids (GABA, glycine and glutamate), indicating an alteration in substrate specificity.
• 7 The results demonstrate complex changes in cerebral amino acid transport during development, with several maxima or minima and with changes in specificity for at least some compounds.

     

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD-like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca²⁺ increases accompany all forms of SD, but persistently elevated Ca²⁺ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca²⁺ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca²⁺ influx via P/Q type channels is important for SD generated by localized application of high K⁺ solutions. In contrast, SD-like events recorded in in vitro ischemia models are not usually prevented by Ca²⁺ removal, but under some conditions, Zn²⁺ influx via L-type channels contributes to SD initiation. This review addresses different roles of Ca²⁺ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn²⁺ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.     

THE EFFECTS OF AGE AND ALCOHOL ON (Na++ K+)-ATPASE ACTIVITY OF WHOLE HOMOGENATE AND SYNAPTOSOMES PREPARED FROM MOUSE AND HUMAN BRAIN

Whole homogenates of mouse brain and nerve-ending fractions of mouse and human brain were obtained at various age levels representative of maturity and old age. The mice were 3, 8 and 26–29 months old and the humans ranged in age from 19 to 84 y. Measurements of (Na⁺+ K⁺)-ATPase in whole brain homogenate of mouse did not reveal any significant differences in relation to age. However, the ability of ethanol at various concentrations to inhibit membrane-bound synaptosomal (Na⁺+ K⁺)-ATPase was significantly greater in older mice and humans. The data are interpreted as indicating a change in the property of synaptic membranes as a consequence of advancing age.     

Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

THE DISTRIBUTION OF AMINO ACIDS, Na+ AND K+ FROM SURFACE TO CENTRE IN INCUBATED SLICES OF MOUSE BRAIN

—The effect of tissue damage on the uptake of amino acids by brain slices was investigated by measuring uptake in slices of different thickness and measuring the distribution of [¹⁴C]-labelled amino acid on the surface and in the centre of incubated slices. The uptake of glutamate, aspartate, and GABA was greater in 0.1 mm-thick slices than in 0.42 mm-thick slices in short and in long (up to 120 min) incubations; the uptake of other amino acids was equal or greater in the 0.42 mm-thick slices. The water content of incubated slices did not change greatly from surface to centre; inulin space was greater at the surface, and in slices from cortex, especially higher at the cut surface. Na⁺ and K⁺ concentrations were also higher at the surface. In the rest of the slice space, inulin, Na⁺ and K⁺ distribution was quite uniform. The distribution of ATP was inhomogeneous: in thinner slices the centre concentration was higher; in thicker slices the centre concentration was lower. Amino acid uptake initially (at 5 min) was higher at the surface, especially in the thicker slices; after longer time (30 min) incubation, the distribution of lysine and leucine was uniform, and glutamate uptake was greater at the surface. The inhomogeneity of distribution increased with increasing thickness of the slices. We concluded that the uptake of some amino acids (perhaps those for which, beside a low affinity transport, also a higher affinity transport system exists) is greater in thinner slices and greater on the surface of slices, and there is an initially inhomogeneous distribution during amino acid uptake. The uptake on the surface constitutes only a small portion of the total uptake, and tissue damage does not explain the greater uptake of amino acids by slices in comparison to the brain in vivo. This shows the higher transport capacity of cells in the brain and emphasizes the importance of mechanisms controlling the metabolite composition of the extracellular fluid in finally influencing the metabolite composition of the brain itself.     

溴氰菊酯对鸡脑突触膜蛋白磷酸化和Ca-ATP酶活力的影响

研究了神经毒性杀虫剂———溴氰菊酯对体内源性蛋白质磷酸化作用的影响。结果表明,浓度为１０－５ｍｏｌ／Ｌ溴氰菊酯明显抑制正常鸡和经三甲基苯基磷酸酯处理的鸡脑突触膜中５５ｋＤ和６０ｋＤ两种蛋白的磷酸化。而０２５ｍｍｏｌ／ＬＣａ２＋加０２５ｍｍｏｌ／Ｌ的钙调蛋白则明显地促进这两种蛋白质的磷酸化,但较低浓度（１０－６ｍｏｌ／Ｌ）时,溴氰菊酯明显抑制４８ｋＤ蛋白的磷酸化。而００３ｍｍｏｌ／ＬＣａ２＋加００３ｍｍｏｌ／Ｌ的钙调蛋白则明显地增强４８ｋＤ和４５ｋＤ两种蛋白的磷酸化。此外,还发现溴氰菊酯可抑制鸡脑突触膜中ＣａＡＴＰ酶活力。     

Ｃu^2＋、Zn^2＋诱导稀有Ｊu鲫应激蛋白质的研究

以稀有Ju鲫为材料,研究了应激蛋白质作为生物学指标的敏感性。结果表明,在无可观察效应浓度下,经５d亚慢性胁迫暴露,以Ｃu^2 为胁迫因子,稀有Ju鲫被诱导出约５４KDa的应激蛋白质;以Ｚn^2 为胁迫因子,稀有Ju鲫被诱导出约９４ＫＤa,67KDa和４０ＫＤa的应激蛋白质。应激蛋白质有可能成为一种生物学指标运用于生态风险性早期预警。     

Stage Specific Effects of Zn2+ on Sea Urchin Embryogenesis

The treatment of sea urchin embryos by Zn²⁺ followed by culture with Zn²⁺-specific chelators such as ethylenediamine-N, N'-diacetic acid and N-hydroxyethylethylenediamine-N, N', N'-triacetic acid, was performed at various developmental stages to find out specific stages for Zn²⁺ to induce abnormal differentiation. The treatment with 1 mM ZnSO₄ at 20°C during a period including two spans of development between 0 and 8 hr and between 14 and 16 hr post fertilization yielded permanent blastulae. Zn²⁺-treatment during the former span produced abnormal prisms and plutei with small archenteron. The treatment for a period including only the latter span failed to produce abnormal ones. Zn²⁺-treatment during a period including the gastrula stage also produced abnormal spherical embryos. Without the culture with these chelators, abnormal embryos were produced by Zn²⁺-treatment performed at any stages before gastrulation. A high zinc amount in the embryos just after the treatment became as low as in normal embryos soon after the culture with these chelators and was maintained during the culture without them. These results indicate that zinc retention occurs in the Zn²⁺-treated embryos and causes abnormal differentiation when the treated embryos develop in normal sea water through the Zn²⁺-specific periods of development.     

CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

PURIFICATION AND CHARACTERIZATION OF OX BRAIN NAD+ -DEPENDENT ISOCITRATE DEHYDROGENASE

Abstract— The NAD⁺ -dependent isocitrate dehydrogenase from ox brain has been purified about 130-fold by a method involving affinity chromatography on an NAD⁺ -derivative of agarose. The enzyme preparation is not homogeneous but it is free from contaminating enzyme activities that could interfere with kinetic studies. The kinetic properties of the enzyme did not appear to have been altered by the purification procedure involved. The initial velocity of the reaction showed a sigmoid dependence on the concentration of isocitrate, and ADP behaved as an allosteric activator. The kinetics with NAD⁺ as the substrate were hyperbolic. The molecular weight of the purified enzyme was found to be 285,000 ± 25,000.     

BRAIN ACETYLCHOLINE AND CHOLINESTERASE: EFFECT OF PHENOTHIAZINES AND PHYSOSTIGMINE INTERACTION IN RATS1

Abstract— The effect of phenothiazines either alone or in combination with physostigmine on whole brain acetylcholine concn and cholinesterase activity has been investigated in male rats. Phenothiazines (chlorpromazine, trifluperazine and thioridazine) when injected alone had no significant effect on brain acetylcholine concentration. Pretreatment with chlorpromazine and thioridazine significantly enhanced the physostigmine-induced increase in brain acetylcholine concn and inhibition of cholinesterase activity. However, trifluperazine had no significant effect on the physostigmine-induced increase in brain acetylcholine concentration and inhibition of cholinesterase activity. The potentiation of the physostigmine-induced increase in brain acetylcholine concn by phenothiazines may be due to (1) increased acetylcholine turnover secondary to the blockade of dopamine receptors by neuroleptic drugs and.