Rat Brain Synaptic Plasma Membranes Research Articles

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca(2+) concentrations [Ca(2+) ](i) is critical for normal neuronal function, and alterations in Ca(2+) homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca(2+) ](i) is the plasma membrane Ca(2+) -ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca(2+) . We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto-5′-nucleotidase (CD73; EC 3.1.3.5, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K m and V max, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency ( V max/ K m). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.

Clustering of plasma membrane-bound cytochrome b 5 reductase within ‘lipid raft’ microdomains of the neuronal plasma membrane

Plasma membrane redox centres play a major role in neuronal defence against oxidative stress and survival. In cerebellar granule neurons in culture (CGN) a large pool of the flavoproteins are associated with the plasma membrane, and the intensity of CGN green/orange autofluorescence correlated with the levels of expression of cytochrome b 5 reductase. Regionalization of cytochrome b 5 reductase in the plasma membrane of CGN by fluorescence resonance energy transfer points out the close proximity between cytochrome b 5 reductase and the ‘lipid raft’ markers cholera toxin B and caveolin-2. This study unravels that membrane-bound cytochrome b 5 reductase is largely enriched at interneuronal contact sites in the neuronal soma and associated with ‘lipid rafts’ of the CGN plasma membrane. We also show that cytochrome b 5 reductase makes a large contribution to the NADH oxidase activity and to the red-shifted flavine fluorescence of purified rat brain synaptic plasma membranes. In conclusion, membrane-bound cytochrome b 5 reductase forms a large mesh of redox centres associated with the neuronal plasma membrane.

Time-dependent inhibition of Na+/K+-ATPase induced by single and simultaneous exposure to lead and cadmium

Time-dependent interactions of Na+/K+-ATPase, isolated from rat brain synaptic plasma membranes (SPMs), with Cd2+ and Pb2+ ions in a single exposure and in a mixture were investigated in vitro. The interference of the enzyme with these metal ions was studied as a function of different protein concentrations and exposure time. The aim of the work was to investigate the possibility of selective recognition of Cd2+ and Pb2+ ions in a mixture, on the basis of the different rates of their protein-ligand interactions. Decreasing protein concentration increased the sensitivity of Na+/K+-ATPase toward both metals. The selectivity in protein-ligand interactions was obtained by variation of preincubation time (incubation before starting the enzymatic reaction).

Effect of neonatal hypothyroidism on Ca 2+-ATPase activity in the rat brain

Effect of neonatal hypothyroidism on kinetic properties of Ca 2+-ATPase from rat brain synaptic plasma membranes and microsomes were examined. Neonatal hypothyroidism resulted in significant decrease in the enzyme activity in both the membrane systems. The synaptic membranes in control group displayed presence of one kinetic component whereas a low affinity component became evident in the hypothyroid group. In the microsomes, both control as well as hypothyroid groups showed presence of two kinetic components with the latter group showing two-fold increase in the K m. The Ca 2+ binding characteristics were generally unaltered in the enzyme from both the membrane systems. Our results suggest that impairment in the Ca 2+-ATPase activity together with altered kinetic properties could be one of the underlying biochemical mechanisms leading to CNS dysfunction as a consequence of thyroid hormone deprivation during critical stages of brain development.

Molecular interaction of NMDA receptor subunit NR3A with protein phosphatase 2A.

The catalytic subunit of the serine-threonine protein phosphatase 2A (PP2A) was previously found to bind to the carboxyl domain of NMDA receptor (NMDAR) subunit NR3A. We now report that NR3A constitutively associates with the PP2A holoenzyme, but not the core enzyme in rat brain synaptic plasma membranes. We also identified critical amino acids in NR3A required for binding to PP2A. We performed alanine-scanning mutagenesis in the PP2A-binding domain of the NR3A C-terminal (NR3Ac), then co-expressed the mutants together with the PP2A catalytic subunit in a yeast two-hybrid system and human embryonic kidney (HEK) 293 cells. We found that mutation of leucine 958, leucine 973 or histidine 974 or deletion of a spacer sequence of more than six amino acids between leucine 958 and histidine 974 disrupted the NR3A/PP2A interaction.

Adenosine A 1 receptor agonist treatment up-regulates rat brain metabotropic glutamate receptors

Chronic R- N 6-phenylisopropiladenosine (R-PIA) subcutaneous injection for 6 days significantly increased total glutamate receptor number (180% of control) in rat brain synaptic plasma membranes (SPM), without affecting receptor affinity. A higher increase in metabotropic glutamate (mGlu) receptor number (258% of control) was also detected, indicating that mGlu is the main type of glutamate receptor affected by this treatment. On the other hand, the observed increase in basal and calcium- and Gpp(NH)p-stimulated phospholipase C (PLC) activity after treatment was associated with a significant increase in PLC β 1 isoform, detected in SPM by immunoblotting assays. Moreover, an increase in PLC activity stimulation with trans-ACPD, in the absence and in the presence of Gpp(NH)p, was detected after R-PIA treatment. These results show that mGlu receptors and its effector system, PLC activity, are up-regulated by chronic exposure to an adenosine A 1 receptor agonist and suggest the existence of a cross-talk mechanism between both signal transduction pathways in rat brain.

Immobilization of Na, K-ATPase isolated from rat brain synaptic plasma membranes

Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM) was immobilized by adsorption on nitrocellulose (NC) polyvinylidene fluoride (PVDF) and glass fiber (GF) membranes. Partial SDS solubilization increased the enzyme activity by 40%. With regard to the preservation of the enzyme activity, nitrocellulose was shown to be the optimal support for the immobilization. The enzyme showed the highest percentage activity (14%) after 30 min of SPM adsorption, at 20?C under the vaccum, with 25 ?g of proteins per NC disc filter. In addition adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20?C. After adsorption, the sensitivity of the enzyme to HgCl2 and CdCl2 inhibition was higher. The results show that immobilized Na,K-ATPase SPMcan be used as a practical model for the detection of metal ions in different samples.

Effect of long-term aluminum feeding on lipid/phospholipid profiles of rat brain synaptic plasma membranes and microsomes.

Long-term exposure to AlCl_3 resulted in 57% phospholipid (TPL) content of synaptic plasma membranes from rat brain with significant decrease in the proportion and content of most of the phospholipid classes (32%- decreases was of lesser magnitude for phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The microsomal TPL and cholesterol (CHL) content decreased by 74% composition. However, the contents of all phospholipids decreased uniformly by about 70%. fluidized in Al fed rats. The results suggest that long-term exposure of rats to Al can alter the structure/function of synaptic plasma membranes and microsomes.

Chemical aspect of the influence of cobalt ions on atpase activity

The influence of Co 2+ ions on the activities of Na+/K+-ATPase and Mg2+ -ATPase, enzymes from rat brain synaptic plasma membrane, was studied. The aim of this study was to investigate the inhibition of both ATPases activities byexposure tocobalt ions as a function of experimentally added CoSO4. The "free" Co2+ concentrations in the reaction mixturewere also calculated and discussed. CoSO4 induced a dose-dependent inhibition of both enzymes. The IC50 values of Co 2+, as calculated from the experimental curves, were 168 mM for Na+/K+-ATPase and 262 mMfor Mg 2+-ATPase, and for the recalculated free Co 2+ concentration 75.4 mM for Na+/K+-ATPase and 136 mM for Mg 2+-ATPase. The obtained linear Dixon's plot for Na+/K+-ATPase implies equilibium binding of cobalt with inhibitory sites on the enzyme. The kinetic parameters for both enzymes in presence and absence of CoSO4 were calculated from the experimental data. The results of the kinetic analysis show that inhibition of Na+/K+-ATPase induced by CoSO4 is non-competitive, and for Mg 2+-ATPase that there are two sites of different sensitivities or two different enzymes.

Prevention and recovery of CuSO 4-induced inhibition of Na +/K +-ATPase and Mg 2+-ATPase in rat brain synaptosomes by EDTA

Enzymatic activities of Na +/K +-ATPase and Mg 2+-ATPase from rat brain synaptic plasma membrane were studied in the absence and presence of EDTA. The aim of the study was to examine the ability of this strong chelator to prevent and recover the CuSO 4-induced inhibition. The influence of experimentally added CuSO 4 and EDTA on MgATP 2− complex and ‘free’ Cu 2+ concentrations in the reaction mixture was calculated and discussed. CuSO 4 induced dose-dependent inhibition of both enzymes in the absence and presence of 1 mM EDTA. In the absence of EDTA, the IC 50 values of Cu 2+, as calculated from the experimental curves, were 5.9×10 −7 M for Na +/K +-ATPase and 3.6×10 −6 M for Mg 2+-ATPase. One millimolar EDTA prevented the enzyme inhibition induced by CuSO 4, but also reversed the inhibited activity, in a concentration-dependent manner, following exposure of the enzymes to the metal ion, by lowering ‘free’ Cu 2+ concentration. Kinetic analysis showed that CuSO 4 inhibits both the Na +/K +-ATPase and Mg 2+-ATPase, by reducing their maximum enzymatic velocities ( V max), rather than apparent affinity for substrate MgATP 2− ( K 0.5), implying the noncompetitive nature of enzyme inhibition induced by the metal. The kinetic analysis also confirmed two distinct Mg 2+-ATPase subtypes activated in the presence of low and high MgATP 2− concentrations. K 0.5 and V max were calculated using a computer-based program. The results of calculation showed that MgATP 2− concentration in the kinetic experiments exceeded three times the apparent K 0.5 value for the enzyme activation.

Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-atpase

Plasma membrane Ca 2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca 2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca 2+ transport 30 to 40 %. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca 2+-ATPase.

Membrane topology of the rat brain Na +–Ca 2+ exchanger

To provide experimental evidence for the topology of the Na +–Ca 2+ exchanger protein NCX1 in the membrane, indirect immunofluorescence studies using site specific anti-peptide antibodies and Flag-epitope insertion into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 and AbO-8 were raised against peptide segments present in a large hydrophilic loop of about 500 amino acids, which separates the hydrophobic amino terminal part of the protein from the hydrophobic carboxy terminal. AbO-10 was raised against the C-terminal tail of the protein. All three antibodies bound to the exchanger protein expressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those NCX1 isoforms that contained the epitope against which they were raised. Detection of the exchanger protein in transfected cells in situ required the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6, AbO-8 and AbO-10 bind. The Flag epitope was inserted into ten putative extramembraneous segments along the exchanger protein. For topology studies, only the Flag-mutants that retained Na +–Ca 2+ exchange activity in whole HeLa cells, were used. Immunofluorescence studies indicated, that the N-terminal of the protein is extracellular, the first hydrophilic loop separating transmembrane helices 1 and 2 as well as the C-terminal, are intracellular.

Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments

NMDA receptors are excitatory neurotransmitter receptors critical for synaptic plasticity and neuronal development in the mammalian brain. These receptors are found highly concentrated in the postsynaptic membrane of glutamatergic synapses. To investigate the molecular mechanisms underlying NMDA receptor localization, we used the yeast two-hybrid system to identify proteins expressed in the brain that interact with the NMDA receptor subunit NR1. Here we report that the 68 kDa neurofilament subunit NF-L directly interacts with the NR1 subunit. This interaction occurs between the cytoplasmic C-terminal domain of NR1 and the rod domain of NF-L. However, NR1 splice variants lacking the first C-terminal exon cassette (C1) failed to associate with NF-L. Immunogold electron microscopy revealed a preferential localization of NR1 at the ends of in vitro-assembled neurofilaments. Overexpression of C1 cassette-containing NR1 constructs in fibroblast cells disrupted the assembly of recombinant neurofilaments. In addition, NR1 and NF-L cofractionated in detergent-treated rat brain synaptic plasma membranes. Furthermore, NR1 and NF-L colocalize in the dendrites and growth cones of cultured hippocampal neurons. These results demonstrate the splice variant-specific association of NR1 with neurofilaments and suggest a possible mechanism for anchoring or localizing NMDA receptors in the neuronal plasma membrane.

Evidence That Synaptobrevin Is Involved in Fusion between Synaptic Vesicles and Synaptic Plasma Membrane Vesicles

We have developed a model system, consisting of rat brain synaptic vesicles and rat brain synaptic plasma membrane vesicles, to study the fusion process associated with the exocytotic release of neurotransmitters. Our results show a significant increase in the extent of fusion when the reaction takes place in cytosol compared to that obtained when fusion is carried out in buffer. This effect is mediated by cytosolic proteins, althoughN-ethylmaleimide-sensitive factor does not play a role in fusion. We also registered an almost complete inhibition of fusion when synaptic vesicles were pre-incubated with botulinum toxin B, indicating that synaptobrevin plays an important role in the coalescence of membrane lipids of the interacting membranes.

ATP-Sensitive Anion Channel from Rat Brain Synaptosomal Membranes Incorporated into Planar Lipid Bilayers

An anion channel was incorporated from rat brain synaptic plasma membrane fractions into planar lipid bilayers. The single-channel conductance was found to be 48.5 pS in choline-Cl solution (300 microM cis/100 microM trans). The anion selectivity of the channel was rather low (PCl/Pcholine = 1.7). The gating rate of the channel did not change with membrane potential over the range of -50 mV to 50 mV. Several drugs, which are known as inhibitors of anion channels, were found to be efficient inhibitors for the synaptosomal anion channel. 4-Acetoamino-4'-isothiocyanostilbene-2,2'-disulfonic acid, ethacrynic acid, indanyloxyacetic acid, and 5-nitro-2-(3-phenylpropylamino) benzoic acid inhibited the channel from the cis side of the membrane, corresponding to the cytoplasmic side of the plasma membrane. We found that the channel is regulated by intracellular ATP at millimolar concentrations. Other nucleotides, ADP and GTP, inhibited the channel as well. Glibenclamide, which is known as an inhibitor of an ATP-regulated potassium channel, inhibited the channel at micromolar concentrations from the trans side of the membrane. It is likely that the synaptosomal anion channel is a member of the ATP-binding cassette superfamily.

Nonclassical and endogenous cannabinoids: effects on the ordering of brain membranes.

The effects of several nonclassical cannabinoids and the endogenous cannabinoid ligand, anandamide on the lipid ordering of rat brain synaptic plasma membranes (SPM) were examined and compared to delta 9-tetrahydrocannabinol (delta 9-THC). SPM order was determined using fluorescence polarization. All compounds tested affected membrane ordering. delta 9-THC, CP-55,940, CP-55,244 and WIN-55212 decreased lipid ordering in SPM. Some stereospecificity was observed with delta 9-THC and WIN-55212, but not other compounds. Anandamide also decreased lipid order as did its putative precursor, arachidonic acid. In contrast to these compounds, levonantradol increased SPM lipid order. Although all pharmacologically active cannabinoids affect SPM lipid order, potency on this measure does not correlate well with their pharmacological potency. The results of this study suggest that membrane perturbation (either increases or decreases in lipid order) may be a necessary characteristic for cannabinoid pharmacological activity, but it is not a primary or sufficient determinate of action for this class of drugs.

Inhibition of ATPase activity in rat synaptic plasma membranes by simultaneous exposure to metals

Inhibition of Na +/K +-ATPase and Mg 2+-ATPase activities by in vitro exposure to Cd 2+, Pb 2+ and Mn 2+ was investigated in rat brain synaptic plasma membranes (SPMs). Cd 2+ and Pb 2+ produced a larger maximal inhibition of Na +/K +-ATPase than of Mg 2+-ATPase activity. Metal concentrations causing 50% inhibition of Na +/K +-ATPase activity (IC 50 values) were Cd 2+ (0.6 μM) < Pb 2+ (2.1 μM) < Mn 2+ (approximately 3 mM), and the former two metals were substantially more potent in inhibiting SPM versus synaptosomal Na +/K +-ATPase. Dixon plots of SPM data indicated that equilibrium binding of metals occurs at sites causing enzyme inhibition. In addition, IC 50 values for SPM K +-dependent p-nitrophenyl-phosphatase inhibition followed the same order and were Cd 2+ (0.4 μM) < Pb 2+ (1.2 μM) < Mn 2+ (300 μM). Simultaneous exposure to the combinations Cd 2+/Mn 2+ or Pb 2+/Mn 2+ inhibited SPM Na +/K +-ATPase activity synergistically (i.e., greater than the sum of the metal-induced inhibitions assayed separately), while Cd 2+/Pb 2+ caused additive inhibition. Simultaneous exposure to Cd 2+/Pb 2+ antagonistically inhibited Mg 2+-ATPase activity while Cd 2+/Mn 2+ or Pb 2+/Mn 2+ additively inhibited Mg 2+-ATPase activity at low Mn 2+ concentrations, but inhibited antagonistically at higher concentrations. The similar IC 50 values for Cd 2+ and Pb 2+ versus Mn 2+ inhibition of Na +/K +-ATPase and the pattern of inhibition/activation upon exposure to two metals simultaneously support similar modes of interaction of Cd 2+ and Pb 2+ with this enzyme, in agreement with their chemical reactivities.

On the concept of a bivalent pharmacophore for SKCa channel blockers: synthesis, pharmacological testing, and radioligand binding studies on mono-, bis-, and tris-quinolinium compounds.

The dissociation equilibrium constants (Kd values) of dequalinium (2) and the monoquinolinium compounds 1a and 1b have been determined from competition equilibrium radioligand binding with [125I]apamin on rat brain synaptic plasma membranes (SPMs). Dequalinium binds to the channel with 2 orders of magnitude higher affinity than 1a or 1b, suggesting that both quinolinium groups are needed for potent and selective SKCa channel blockade. The trisquinolinium compound 3 (1,1'-[5-[4-(4- aminoquinolinium-1-yl)but-1-yl]non-4-en-1,9-diyl]-bis-(4- aminoquinolinium)) has been synthesized and tested for inhibition of the afterhyperpolarization of rat sympathetic neurones and on the binding assay. Compound 3 shows approximately one order of magnitude higher potency than 2, being the most potent non-peptidic SKCa channel blocker reported so far (Kd approximately 30 nM). The higher affinity of 3 compared with 2 may be due to direct binding of the third quinolinium group to the channel or may arise from a reduction of the unfavorable entropy of binding via an increase of the "local concentration" of quinolinium groups.

Distribution of six transplasma membrane NADH-dehydrogenases in rat brain tissue

Transplasma membrane redox plays a significant role in cellular activation and growth. Six isoenzymes could be prepared from purified rat brain synaptic plasma membrane. Polyclonal antibodies have been prepared against six transplasma membrane oxydoreductases (PMO-I to PMO-VI) and the tissue distribution of the various iso-enzymes have been investigated in adult rat brains by means of immunohistochemistry. PMO-I is densely observed in layers I, IV and V of the parietal cortex, in CA1 of the hippocampus (except for the molecular layer), in the caudate putamen, in the dorsal, granular and ventral parts of the auditory nuclei, in some loci of the vestibular nuclei as well as in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-II is mainly located in the polymorphic layer of the dendate gyrus and in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-III is abundant in the piriform cortex, in the pyramidal layers of both CA1 and CA2, in the diagonal band of the basal ganglia, in the supraoptic nucleus and in various loci of the magnetocellular paraventricular nucleus of the hippothalamus as well as in the vestibular nuclei from the brain stem. In addition PMO-III is also densely present in motor nuclei (oculomotor, facial, hypoglossal and ambiguus nuclei), in the reticular formation and in the deep cerebellar nucleus as well as in the Purkinje layer of the cerebellar cortex. PMO-IV has a similar location but is less abundant in the vestibular nuclei of the sensory brain stem and in the motor nucleus. PMO-V in contrast is poorly present in most brain areas compared to the other iso-enzymes, apart of the Purkinje layer of the cerebellar cortex. Finally PMO-VI is mainly present in the oriens layer and in the stratum radiatum of the hippocampus formation, in the supraoptic and lateral magnocellular nucleus of the hypothalamus, in the mesencephalic trigeminal nucleus, in the ventral auditory nucleus and in the facial nucleus of the brain stem as well as in red nucleus of the reticular formation and in the Purkinje layer of the cerebellar cortex. These data show that the iso-enzymes are located in specific brain nuclei. The significance of the results in respect to the yet very poorly defined function of PMO's is discussed.