Crude Synaptosomal Membranes Research Articles

Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords.

The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer's disease or other neurodegenerative conditions.

Chronic nicotine and withdrawal affect glutamatergic but not nicotinic receptor expression in the mesocorticolimbic pathway in a region-specific manner

Tobacco addiction is a complex form of dependence process that leads high relapse rates in people seeking to stop smoking. Nicotine elicits its primary effects on neuronal nicotinic cholinergic receptors (nAChRs), alters brain reward systems, and induces long-term changes during chronic nicotine use and withdrawal.We analysed the effects of chronic nicotine treatment and withdrawal on the mesocorticolimbic pathway (a brain reward circuit in which addictive drugs induce widespread adaptations) by analysing the expression of nAChRs in the midbrain, striatum and prefrontal cortex (PFC) of mice receiving intravenous infusions of nicotine (4mg/kg/h) or saline (control) for 14 days and mice sacrified two hours, and one, four and 14 days after treatment withdrawal.We biochemically fractionated whole tissue homogenates in order to obtain crude synaptosomal membranes. Western blotting analyses of these membrane fractions, ligand binding and immunoprecipitation studies, showed that chronic nicotine up-regulates heteromeric β2* nAChRs in all three mesocorticolimbic areas, and that these receptors are rapidly removed from synapses upon the cessation of nicotine treatment. The extent of nicotine-induced nAChR up-regulation, and the time course of its reversal were comparable in all three areas.We also analysed the expression of glutamate receptor subunits (GluRs) and scaffold proteins, and found that it was altered in an area-specific manner during nicotine exposure and withdrawal.As the functional properties of GluRs are determined by their subunit composition, the observed changes in subunit expression may indicate alterations in the excitability of mesocorticolimbic circuitry, and this may underlie the long-term biochemical and behavioural effects of nicotine dependence.

Anxiolytic-like effects of compound zhi zhu xiang in rats.

The purpose of this study was to determine whether compound zhi zhu xiang (CZZX) exerts anxiolytic-like effects in rats. The animals were orally administered CZZX (0.75, 1.5, and 3 g/kg daily) for 10 days and tested in the elevated plus maze (EPM), Vogel conflict test (VCT), and open field. Repeated treatment with CZZX (3 g/kg/day, p.o.) significantly increased the percentage of both entries into and time spent on the open arms of the EPM compared with saline controls. In the VCT, repeated treatment with CZZX (1.5 and 3 g/kg/day, p.o.) significantly increased the number of punished licks. The drug did not change the total entries into the open arms of the EPM or interfere with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. In the open field, locomotion was not reduced, discarding the possible sedative effect of CZZX. In the binding assay, the binding of [3H] Ro 15-1788 (flumazenil) to the benzodiazepine binding site in washed crude synaptosomal membranes from rat cerebral cortex was affected by CZZX. These data indicate an anxiolytic-like profile of action for CZZX without sedative side effects, and this activity may be mediated by benzodiazepine binding site modulation at γ-aminobutyric acid-A receptors.

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

The anxiolytic-like effects of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) in mice

The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of 3H-flunitrazepam ( 3H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABA A receptors.

CaMKII-dependent Phosphorylation Regulates SAP97/NR2A Interaction

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

Age-related oxidative inactivation of Na +, K +-ATPase in rat brain crude synaptosomes

The study was undertaken to examine the status of Na +, K +-ATPase in aged rat brain and to verify if any alteration of this enzyme in aged brain could be related to an oxidative damage. The crude synaptosomes from rat brain were exposed in vitro to an oxidative stress in the form of a combination of Fe 2+ (100 μM) and ascorbate (2 mM) for up to 2 h when increased lipid peroxidation (nearly four-fold), extensive protein carbonyl formation and a marked decrease of Na +, K +-ATPase activity (approximately 88%) were observed. All these changes were prevented by the presence of a chain-breaking anti-oxidant, butylated hydroxytoluene (0.2 mM), in the incubation mixture. When the same crude synaptosomal membranes from the young (4–6 months) and aged (18–22 months) rat brains were analysed, a significant reduction of Na +, K +-ATPase activity (nearly 48%) along with significantly elevated levels of lipid peroxidation products and protein carbonyls could be detected in the aged animals in comparison to young ones. The latter data in combination with the results of in vitro experiments imply that the age-related decline of rat brain Na +, K +-ATPase activity is presumably the consequence of an enhanced oxidative damage in aging brain

Binding affinity profile of betahistine and its metabolites for central histamine receptors of rodents

In order to clarify the interaction of betahistine (BH) and its metabolites [aminoethylpyridine (AEP) and hydroxyethylpyridine (HEP)] for receptors that mediate the physio-pharmacological activities of histamine, we performed in vitro competition binding studies to obtain their binding affinity profile for H1-, H2- and H3-histamine receptors prepared from rodent brains. Crude synaptosomal membranes were incubated in the absence (total binding) or presence of the unlabelled ligands used to saturate the specific binding, or with different concentrations of BH, AEP or HEP. Receptor binding methods were validated by running known standard drugs together with the test compounds. Like histamine, only BH interacted with H1-histamine receptors with comparable affinity (around 10−5M). BH and its metabolite AEP both interacted with the H3-histamine receptors, with μ M affinity. HEP still showed some affinity for the H3-receptors but with aKi only 1/50 that of the parent compound. Histamine showed 10−8M affinity for the H3-receptor sites and was the only ligand to interact with H2-histamine receptors, all the others giving affinities above the mM range. Hill coefficients (as slopes of the sigmoidal inhibition isotherms) were close to unity for BH against H1- and H3-binding sites and for AEP against H3-sites, indicating that these interactions take place in the absence of cooperativity. Histamine and HEP interacted with H1- and H3-receptors with a Hill coefficient less than unity for the former and higher than unity for the latter (presence of negative and positive cooperativity, respectively). The results suggest that BH and its metabolites may act as neurotransmitter modulators of the histaminergic system.

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin b 12 diet

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B 12 on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 μg vitamin B 12 per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B 12 ameliorates the second messenger-mediated injury.

Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [ 3 H ]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn 2+ ions and a pH optimum in the 7–8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a K m of 37.1±3.6 μM and a V max of 5.5 μmol min −1 l −1 per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726–735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AbetaPP) by the alpha-secretase pathway within the betaA4 domain to generate a soluble secreted N-terminal fragment (AbetaPPs). AbetaPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform beta1 and novel PKCepsilon from cytosol to membrane fractions, but there was no alteration in the proportion of AbetaPP associated with the Triton-soluble and -insoluble fractions. AbetaPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AbetaPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 microM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AbetaPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the alpha-secretase activity leading to the secretion of AbetaPPs can occur at the level of the presynaptic terminal.

Effect of sequence of administration on the pharmacokinetic interaction between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate or [3H]N-methylscopolamine in rats.

In rats the pharmacokinetic interactions between the anticholinergic drug biperiden and [3H]quinuclidinyl benzylate ([3H]QNB) or [3H]N-methylscopolamine ([3H]NMS) is affected by the sequence in which the drugs are administered. Drug concentrations in various tissues were determined after intravenous administration of [3H]QNB or [3H]NMS (325 ng kg(-1)). Biperiden (6.4 mg kg(-1)) was administered either 5 min before, concomitantly with or 20 min after injection of [3H]QNB or [3H]NMS. When biperiden was administered concomitantly with or before [3H]QNB, distribution of [3H]QNB among the regions of the brain and other tissues was reduced; at 4 h the ratio of the distribution of [3H]QNB for experimental animals to that for control animals ranged from 0.15 to 0.9. When biperiden was administered after [3H]QNB, the distribution of [3H]QNB in the brain and other tissues was significantly higher than for the other two treatments (P < 0.01). However, for [3H]NMS the sequence of administration had no effect on the distribution of the drug in the brain and other tissues except for the kidney. In-vitro, in crude synaptosomal membranes, the amount of [3H]QNB at 2 h relative to the control concentration at equilibrium was 87% when biperiden was added before [3H]QNB and 56% when biperiden was added after [3H]QNB. In both instances the concentration of [3H]NMS reached equilibrium within 30 min. These findings suggest that the difference between the rate constant of association and dissociation at the possible site of action gives rise to the effect of the sequence of administration on the pharmacokinetic interaction.

Protein Half-Lives of Two Subunits of an NMDA Receptor-like Complex, the 71-kDa Glutamate-Binding and the 80-kDa CPP-Binding Protein

We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997,Biochem. Biophys. Res. Commun.237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (± 2.4) and 18 (± 1.2) hours for CBP and GBP, respectively.

Effect of Verapamil on Changes in Brain Protein Phosphorylation after Oral Administration of Tri-O-cresyl Phosphate to Hens

The endogenous phosphorylation of specific brain proteins in white Leghorn laying hens treated with tri-o-cresyl phosphate (TOCP) with and without verapamil, a calcium channel blocker, which has been shown to ameliorate clinical signs and nervous lesions associated with organophosphate-induced delayed neuropathy (OPIDN), was determined. Verapamil was given im at the dose of 7 mg/kg/day for 4 days beginning 1 day before administration of TOCP (750 mg/kg, po). Phosphorylation of crude synaptosomal (P2) membrane proteins was assayedin vitrousing [γ-32P]ATP as a phosphate donor. Following resolution of brain membrane proteins by SDS-PAGE, specific protein phosphorylation was detected by autoradiography and quantified by densitometry TOCP caused, 21 days after dosing, an enhancement of the phosphorylation of membrane proteins with apparent molecular weights of 40, 55, and 60 kDa by as much as 253, 322, and 609%, respectively. Verapamil administered to TOCP-treated hens abolished the enhancement and even caused some inhibition. This indicates that calcium channel blocker may contribute to the amelioration of OPIDN by preventing the enhancement of phosphorylation of brain membrane proteins.

Proline-glutamate interactions in the CNS

1. 1. Crude synaptosomes (P 2) and synaptosomal membranes were prepared from normal C57/B110 mouse brains and Wistar rats respectively. 2. 2. [ 3H]Pro binding to mouse brain synaptic membranes was examined in the presence of competitive NMDA antagonist, MK-801, or HA-966. Conversely, the effects of l-proline on [ 3H]MK-801 binding were also probed. The effects of l-proline on glutamatemedicated [Ca +2], levels were tested. 3. 3. The authors could not detect any effect of proline on glutamate-mediated [CA +2] i levels using FURA-2 in synaptosomes or neuroblastoma cells. 4. 4. NMDA competitive antagonists, AP-7, CPP, and CGS 19755 inhibit [ 3H]Pro binding to mouse brain synaptic membranes. 5. 5. MK-801, a NMDA channel blocker, also inhibits [ 3]Pro binding, but 200 mM praline is incapable of inhibiting [ 3H] MK-801 binding. 6. 6. HA-966, a glycine site partial agonist inhibits [ 3]Pro binding. Praline has modest effects on [ 3H]glycine binding.

Membrane receptors for estrogen, progesterone, and testosterone in the rat brain: fantasy or reality.

1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes. 2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17 beta-E-6-[125I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimated Kd of about 3 +/- 0.7 nM (X +/- SE; n = 3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with a Kd of 34 +/- 7 nM and, a Bmax of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (Kd = 1-2 nM) and one low affinity (Kd = 62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[125I]BSA indicate that the estimated Kd of 30 +/- 2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement with Kd values computed from Scatchard-derived data using the LIGAND algorithm. 3. 17 beta-E-6-[125I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[125I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22. 4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase. 5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the "reality of membrane steroid receptors."

Modification of the characteristics of dopamine transporter in brain regions and spinal cord of morphine tolerant and abstinent rats

The specific binding of [ 3H]GBR 12935 to crude synaptosomal membranes of brain regions and spinal cord of morphine tolerant and abstinent rats was investigated. Male Sprague-Dawley rats were implanted with 6 morphine pellets each containing 75 mg of morphine base during a 7-day period. Placebo pellet implanted rats served as controls. Rats sacrificed without removal of the pellet were considered tolerant whereas those from which pellets were removed 16 hr prior to sacrificing were labeled abstinent. The binding of [ 3H]GBR 12935 was initially determined at a 1 nM concentration in all brain regions and spinal cord, which was followed by the determination of B max and K d values in the corpus striatum, a highly enriched region for the dopamine transporter. In morphine tolerant rats, the binding of [ 3H]GBR 12935 was increased in the hypothalamus (182%) but was decreased in the corpus striatum (34%) and spinal cord (30%). The decrease in binding in the corpus striatum was due to an increase in the K d value of [ 3H]GBR 12935. However, during morphine withdrawal, the binding of [ 3H]GBR 12935 was still higher in the hypothalamus (255%) but was decreased in the hippocampus (53%). Thus, chronic administration of morphine results in changes in the dopamine transporter function in selected brain regions and the spinal cord, and these changes are dependent upon whether or not the animals are undergoing the abstinence syndrome.

Antioxidant defense systems in the brains of type II diabetic mice.

The specific activities of superoxide dismutase, catalase, and glutathione S-transferase (mu subtype) were significantly lower in the brains of mice with type II diabetes than in the brains of control mice. On the other hand, the specific activity of glutathione peroxidase was unaltered. The concentration of vitamin E, but not that of total glutathione and ascorbate, was increased in the brains of the type II diabetic mice. The relative amount of polyunsaturated fatty acids (as determined with soybean lipoxygenase) was increased in whole brains and crude synaptosomal membranes of the type II diabetic mice. Endogenous levels of thiobarbituric acid-positive material were decreased in both whole brain homogenates and crude synaptosomal membranes of the db/db mice. Susceptibility of lipids within whole brain homogenates and crude synaptosomal membranes of mice with type II diabetes to peroxidation with iron/ascorbate was also markedly decreased compared with that of controls. Vitamin E is known to quench lipid peroxidation. Therefore, decreased lipid peroxidation in the type II mouse brain may be due to increased vitamin E content.

Lipid metabolism and membrane composition are altered in the brains of type II diabetic mice.

CBL/57 strain db/db mice exhibit type II (noninsulin-dependent) diabetes. The affected mice are markedly hyperinsulinemic, hyperglycemic, and hypercholesterolemic, and their serum K+ levels are decreased. The brains of the diabetic mice are significantly smaller than those of their lean, control littermates, but the protein concentration is normal. The low brain weight is accompanied by a loss of major fatty acid components within the whole brain, nerve endings, and mitochondrial membranes. Cholesterol levels are low in whole brain but are not significantly different from normal in the synaptosomal membranes. The phospholipid concentration is significantly decreased in whole brain homogenates, crude synaptosomal membranes, and crude mitochondrial membranes of the diabetic mice. In addition, the specific activities of membrane-bound synaptosomal acetylcholinesterase, Na+,K(+)-ATPase, and Mg(2+)-ATPase are decreased in crude synaptosomal membranes of the diabetic mice. The specific activities of carnitine palmitoyltransferase I and carnitine acetyltransferase are significantly increased in the crude mitochondrial fraction isolated from the brains of the type II diabetic mice, whereas the specific activity of pyruvate dehydrogenase complex is decreased. The specific activities of two other mitochondrial enzymes--monoamine oxidase B and citrate synthase--and a cytosolic enzyme--lactate dehydrogenase--are unaltered. The ability to synthesize cyclic AMP is markedly decreased in the brains of the diabetic mice. The concentrations of carnitine and of the amino acids, glutamate, aspartate, glutamine, and serine are unaltered, whereas glycine levels are significantly elevated in the brains of the db/db mice. The data suggest that in vivo the brains of the diabetic mice exhibit a decreased capacity for glucose oxidation and increased capacity for fatty acid oxidation. This hypothesis is supported by the finding that cerebral mitochondria isolated from the db/db mice oxidize [1-14C]palmitate to 14CO2 at a rate almost twice that of control mitochondria. The present findings emphasize the potentially serious alteration of brain metabolism in uncontrolled type II diabetes.

Production and characterization of an antiserum against the 5-HT 1B receptor for immunocytochemical localization

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7–8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1±3.6 μM and a Vmax of 5.5 μmol min−1 l−1 per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726–735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.