Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

Characteristics of 3H-substance P binding sites in rat brain membranes

Binding characteristics of 3H-Substance P (SP) were studied with rat brain membranes using a method applied to peripheral tissues by Lee and Snyder [15]. This method was well applicable to central nervous system (CNS) tissues. The results in the present study indicate that specific 3H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of 3H-SP. Scatchard analysis of specific binding data reveals a single class of binding sites with a high affinity (Kd = 0.30 nM) and a low density (Bmax = 27.7 fmol/mg protein) in rat brain membranes. A Hill plot of the displacement curve of 3H-SP with unlabelled SP showed no indication for cooperativity (nH = 0.83). The relative potencies of binding of various SP fragments at 3H-SP binding sites were fairly parallel to the length of the C-terminal fragments. Neurotransmitters not structurally related to SP produced no effect on 3H-SP binding even when used at micromolar concentrations.     

High-Affinity Uptake of Spermine by Slices of Rat Cerebral Cortex

Abstract: The accumulation of the polyamine spermine into 0.1-mm prisms of rat cerebral cortex has been investigated at both 37°C and at 4°C. Kinetic analysis of the temperature-sensitive portion of uptake indicates two high-aftinity saturable components together with an unsaturable component at high concentrations. The 'very high'– affinity saturable system ( K _m= 3.8 nM) was temperature- and sodium-dependent, and significantly reduced by metabolic inhibitors, findings that are consistent with an active transport system for spermine into brain tissue. The 'high'– affinity saturable component ( K _m= 0.44 μM) was sodium-dependent and inhibited by ouabain, but only partially susceptible to inhibition by 2,4-dinitrophenol and sodium cyanide. The significance of these results with respect to the function of spermine in the central nervous system is discussed.     

Nicotinic acetylcholine receptor of mammalian brain: naja toxin binding to subcellular fractions of rat brain

Abstract— Subcellular fractions of rat brain cortex bound 6.8 ± 0.4 fmol of ¹²⁵I-labeled α-naja toxin per milligram of protein. Toxin binding was saturable and specific e.g. nicotinic agonist and antagonist blockable. Regions in rat brain varied in saturable, specific toxin binding with hippocampus, olfactory area and corpus striatum displaying greatest toxin binding. We conclude that ¹²⁵I-α-naja toxin is a suitable probe for nicotinic cholinergic receptor in brain.     

A Charge-Deficient Analogue of Spermine with Chelating Properties

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu²⁺ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 303–311.Original Russian Text Copyright © 2005 by Khomutov, Grigorenko, Skuridin, Demin, Vepsalainen, Casero, Woster.     

Studies on detergent released choline acetyltransferase from membrane fractions of rat and human brain

The relationship between soluble and membrane choline acetyltransferase (ChAT) was studied. Differential solubilization of rat and human brain yielded ChAT in the soluble and membrane fractions. The addition of 1% Triton X-100 to membrane fractions resulted in a release of ChAT. A comparable release of lactate dehydrogenase was also observed. The Triton released ChAT and soluble ChAT from rat and human brain were efficiently purified by immuno-affinity chromatography. A single molecular weight of 68,000 was observed for both forms of rat and human brain ChAT. Epitope maps produced from both forms of human brain ChAT were identical. It is concluded that Triton release ChAT is identical to soluble ChAT and simply represents occluded soluble ChAT.     

Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [(3)H]MK-801 binding site were delineated along with the enhancement of [(3)H]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.     

Effects of D-Tubocurarine on the binding of met-enkephalin to synaptosomal fractions of rat brain

D-tubocurarine has been found to inhibit the binding of ³H-met-enkephalin to “synaptosomal mitochondrial” fractions of rat whole brain in Tris-HCl buffered media with an ID₅₀ of 65 μM. Lineweaver-Burk curves indicated competitive inhibition and Hill plots revealed the existence of two binding mechanisms, both of which showed negative cooperativity.     

5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes

The interaction of 5-aminolevulinic acid (ALA) with GABA_A receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [³H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1–10 mM) significantly inhibited the binding of [³H]muscimol (12 nM), with a similar potency in rat and human membranes (IC₅₀ = 199 vs. 228 M, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [³H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.     

Clozapine inhibition of methenkephalin binding to synaptosome-enriched fractions of rat whole brain and hippocampus

Clozapine has been found to inhibit the binding of [³H]met-enkephalin to synaptosome-enriched fractions prepared from rat whole brain and hippocampus in a Tris-buffered media without added ions. The IC₅₀ value for hippocampus is 28.5 M. The inhibition constant for whole brain (41.0 M) was found to be slightly larger than the corresponding value for chlorpromazine, which was measured under similar experimental conditions.     

O -·2增强谷氨酸与其受体的结合力及EBSELEN的保护作用

用放射配体测定受体法研究了黄嘌呤(X)/黄嘌呤氧化酶(XO)体系产生的超氧阴离子自由基(O -·2)对［3H］DL-谷氨酸与大鼠大脑皮层突触膜谷氨酸受体结合的影响,结果表明O -·2明显增强谷氨酸与其受体的结合力,此作用能被2-苯基-1,2-苯并异硒唑-3(2H)酮(EBSELEN)(1 μmol/L)所抑制.     

增强谷氨酸与其受体的结合力及EBSELEN的保护作用

用放射配体测定受体法研究了黄嘌呤 (X) /黄嘌呤氧化酶 (XO)体系产生的超氧阴离子自由基 (O·2 )对 [3H]DL 谷氨酸与大鼠大脑皮层突触膜谷氨酸受体结合的影响 ,结果表明O·2 明显增强谷氨酸与其受体的结合力 ,此作用能被 2 苯基 1 ,2 苯并异硒唑 3 ( 2H)酮 (EBSELEN) ( 1 μmol/L)所抑制     

Cerebral Polyamine Metabolism: Inhibition of Spermidine Biosynthesis by Dicyclohexylamine

Since a specific inhibition of cerebral spermidine (Spd) synthase activity by alicyclic amines was preliminarily observed in vitro, we examined the in vivo inhibitory effectiveness of dicyclohexylamine (DCHA) on Spd biosynthesis in 21-day-old rat brain. For this purpose a previously reported HPLC procedure (Porta et al., 1981a) was modified to analyze the cerebral levels of DCHA at the time of polyamine determinations. The intraperitoneally injected DCHA was shown to cross the blood-brain barrier easily, reaching high levels in the cerebral tissue (approximately 750 nmol/g brain) within 1 h of its administration. The effect of the drug on the polyamine metabolism resulted in a significant depletion of Spd biosynthesis from the sixth hour after the treatment and in an earlier and prolonged increase of the putrescine (Pt) steady-state levels. Conversely, the spermine (Spm) endogenous pools remained unchanged throughout the 24-h post-DCHA period. Moreover, following the intracerebral administration of [1,4-14C]Pt, significantly lower specific radioactivity (s.r.a.) values for labeled Pt and Spd were recorded in the brains of DCHA-treated animals. Conversely, after intracerebral [14C]Spd injection, the s.r.a. of newly formed [14C]Spm remained unchanged, confirming the specificity of the DCHA effect on the Spd biosynthesis.     

γ-Aminobutyric Acid Metabolism and Behavioral Effects After Intraventricular Injection of Spermine in Chicks

Effects of intraventricularly injected spermine on behavior and electrocortical activity and gamma-aminobutyric acid (GABA) metabolism after a single dose of 1.13 mumol/animal were studied. Decrease in locomotor activity, sedation or sleep, and electrocortical synchronization that lasted approximately 2 h were observed. In addition spermine caused a significant increase in GABA content in diencephalon and brainstem, 30 min after administration. Concomitantly a significant increase of glutamate decarboxylase (GAD) activity was observed in cerebral hemispheres, diencephalon, and brainstem. Reduction in gamma-aminobutyrate: alpha-oxoglutarate amino-transferase (GABA-T) levels occurred in the diencephalon along with a significant increase of GABA-T in the brainstem. The present results demonstrate that spermine has the capacity to affect GABA metabolism and are in favor of the suggestion that endogenous polyamines may modulate GABAergic mechanisms.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.