Rat Brain Synaptosomal Plasma Membranes Research Articles

Effect of acute stress on NTPDase and 5′-nucleotidase activities in brain synaptosomes in different stages of development

The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto-nucleotidase activities at specific stages of postnatal development (15-, 30-, 60- and 90-day-old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72h post-stress. At 1h after stress NTPDase and ecto-5′-nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short-term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long-term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72h post-stress. In adult rats only ATP hydrolysis was decreased after 24h, indicating that ecto-ATPase was affected by stress. Ecto-5′-nucleotidase hydrolysing activity was decreased within 24h in adult rats, while in 15- and 30-day old rats it decreased 72h post-stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto-5′-nucleotidase was detected. Our results highlight the developmental-dependence of brain ecto-nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time-dependent ecto-nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats.

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‐β1a or the III‐β1a 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‐β1a 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.

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.

Modulation of rat brain synaptosomal plasma membrane achieved by atracurium and its metabolite laudanosine.

Atracurium besylate and laudanosine cause excitement and seizures when introduced into the central nervous system of laboratory animals. We examined the modulation of lipid-protein interaction in the lipid environment of rat brain synaptosomal plasma membrane (SPM)-bound enzymes as a possible mechanism leading to these effects. The effect of various concentrations of atracurium besylate and laudanosine, or of varying duration of SPM, on the activity of Na+/K+-stimulated ATPase, Mg2+-stimulated ATPase and 5'-nucleotidase were assessed. The modulation of lipid protein interaction by laudanosine was estimated on the basis of the temperature dependence and cooperative behaviour of Na+/K+-stimulated ATPase. The effect of atracurium besylate or laudanosine on Na+/K+-stimulated ATPase activity was biphasic. Maximal enzyme stimulation appeared at 10(-4) M atracurium besylate or 10(-8) M laudanosine, and at 30 min of pre-incubation with both drugs. Arrhenius plots of Na+/K+-stimulated ATPase showed a transition temperature of 23.0 +/- 1.2 degrees C in control SPM and shifted to 16.5 +/- 0.9 degrees C (p < 0.01) in SPM treated with 10(-8) M laudanosine. The Hill coefficients for the allosteric inhibition of Na+/K+-stimulated ATPase by fluoride decreased from 1.99 +/- 0.22 in controls to 1.06 +/- 0.11 (p < 0.001) in the presence of 10(-8) M laudanosine. Our results suggest that laudanosine, one of the principal metabolites of atracurium besylate, affects nerve cell function in rats through the perturbation of the membrane lipid structure accompanied by SPM-bound enzyme dysfunction.

Modulation of synaptosomal plasma membrane-bound enzyme activity through the perturbation of plasma membrane lipid structure by bupivacaine.

We investigated modulations of lipid dynamics and lipid-protein interactions of rat brain synaptosomal plasma membrane (SPM) as one of the possible mechanisms by which the local anesthetic bupivacaine (BPV) has an adverse effect on nerve cell function, with SPM-bound enzyme activity used as a functional probe. The kinetics of BPV impact on the activity of the endoenzymes Ca2+/Mg2+-stimulated ATPase and Na+/K+-stimulated ATPase and the active concentrations of the drug were relevant to those that produce biphasic systemic toxicity. Arrhenius plots of these enzymes showed a transition temperature of 26.6 +/- 1.8 degrees C and 24.5 +/- 1.2 degrees C (mean +/- SD), respectively, in control SPM, which shifted to 17.1 +/- 0.95 degrees C (P < 0.01) and 18.2 +/- 0.85 degrees C (P < 0.05) in SPM treated with 10(-5) M BPV. The Hill coefficients for the allosteric inhibition of Ca2+/Mg2+-stimulated ATPase by Na+ and Na+/K+-stimulated ATPase by fluoride decreased from 1.73 +/- 0.20 and 1.95 +/- 0.25, respectively, in controls to 0.92 +/- 0.09 (P < 0.001) and 1.09 +/- 0.11 (P < 0.001) in the presence of 10(-5) M BPV. The fluidity perturbation in the microenvironment of the ectoenzyme acetylcholinesterase was observed only at 5 x 10(-3) M BPV, as confirmed by the disparity in transition temperature between the controls (22.3 +/- 1.2 degrees C) and the BPV-treated SPM (17.5 +/- 0.8 degrees C, P < 0.01) and that in the Hill coefficient in the two groups: 2.15 +/- 0.24 and 0.97 +/- 0.12 (P < 0.001), respectively. We propose that under physiological conditions, the neutral and protonated forms of local anesthetics can affect nerve cell function through the asymmetric perturbation of the membrane lipid structure, accompanied by synaptosomal plasma membrane-bound enzyme dysfunction.

Modulation of Synaptosomal Plasma Membrane-Bound Enzyme Activity Through the Perturbation of Plasma Membrane Lipid Structure by Bupivacaine

We investigated modulations of lipid dynamics and lipid-protein interactions of rat brain synaptosomal plasma membrane (SPM) as one of the possible mechanisms by which the local anesthetic bupivacaine (BPV) has an adverse effect on nerve cell function, with SPM-bound enzyme activity used as a functional probe. The kinetics of BPV impact on the activity of the endoenzymes Ca2+/Mg2+-stimulated ATPase and Na+/K+-stimulated ATPase and the active concentrations of the drug were relevant to those that produce biphasic systemic toxicity. Arrhenius plots of these enzymes showed a transition temperature of 26.6 +/- 1.8[degree sign]C and 24.5 +/- 1.2[degree sign]C (mean +/- SD), respectively, in control SPM, which shifted to 17.1 +/- 0.95[degree sign]C (P < 0.01) and 18.2 +/- 0.85[degree sign]C (P < 0.05) in SPM treated with 10-5 M BPV. The Hill coefficients for the allosteric inhibition of Ca2+/Mg2+-stimulated ATPase by Na+ and Na+/K+-stimulated ATPase by fluoride decreased from 1.73 +/- 0.20 and 1.95 +/- 0.25, respectively, in controls to 0.92 +/- 0.09 (P < 0.001) and 1.09 +/- 0.11 (P < 0.001) in the presence of 10-5 M BPV. The fluidity perturbation in the microenvironment of the ectoenzyme acetylcholinesterase was observed only at 5 x 10-3 M BPV, as confirmed by the disparity in transition temperature between the controls (22.3 +/- 1.2[degree sign]C) and the BPV-treated SPM (17.5 +/- 0.8[degree sign]C, P < 0.01) and that in the Hill coefficient in the two groups: 2.15 +/- 0.24 and 0.97 +/- 0.12 (P < 0.001), respectively. Implications: We propose that under physiological conditions, the neutral and protonated forms of local anesthetics can affect nerve cell function through the asymmetric perturbation of the membrane lipid structure, accompanied by synaptosomal plasma membrane-bound enzyme dysfunction. (Anesth Analg 1997;85:1337-43)

High affinity binding of 3H-verapamil to rat brain synaptic membrane is antagonized by pyrethroid insecticides.

Deltamethrin is a powerful neuroactive agent causing increased neurotransmitter release at the synapse. To understand the cause for such an action of this pyrethroid insecticide, we have studied its binding characteristics to the calcium channel by utilizing 3H-verapamil as a specific ligand. It was found that deltamethrin is capable of competing at the same binding site as 3H-verapamil. Also, it was found that nonlabeled verapamil could reduce the binding of 3H-deltamethrin (tested at 1 nM) to its binding site. The EC50 value of verapamil to displace 50% of 3H-deltamethrin was in the order of 0.3 microM, which coincides with the approximate KD value for this ligand to bind with calcium channels in the rat brain synaptosomal plasma membranes.

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.

Phosphatidic acid is a specific activator of phosphatidylinositol-4-phosphate kinase.

The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

1. 1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd). 2. 2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr. 3. 3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation. 4. 4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [( r 0/ r) — 1] −1 at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

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 degrees in the presence or in absence of 20 microM 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 microM Ca2+. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 nM); higher concentrations (up to 40 nM) 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 [(ro/r)-1]-1. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the membrane at 22.7 +/- 1.2 degrees was perturbed by calmodulin such that the temperature was reduced to 16.3 +/- 0.9 degrees and 15.5 +/- 0.8 degrees in the absence or in the presence of 20 microM Ca2+. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited break points at 26.7 +/- 1.4 degrees and 22.3 +/- 1.0 degrees in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3 +/- 0.9 degrees in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1 +/- 0.9 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

A Radioimmunoassay for the Phosphoprotein B‐50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.

Iminoglycine transport system in synaptosomes and its interaction with enkephalins.

Evidence is presented which suggests that proline, pipecolic acid, and glycine are accumulated by a common transport system in rat brain cortical synaptosomes and synaptosomal plasma membrane vesicles. This system is Na+ dependent and appears to be similar to the iminoglycine transport system present in renal tubules and in renal brush border membranes. The opioid pentapeptides Leu- and Met-enkephalin specifically inhibit the uptake of these three imino/amino acids, presumably by interaction with a nonopioid receptor, since the inhibition is not affected by the opiate antagonist naloxone and occurs with des-tyrosyl enkephalins as well as with the intact pentapeptides.

Fluorescent probes for opioid receptors

Naloxone, naltrexone and oxymorphone were labeled in position 6 with fluorescein by coupling their hydrazone analogs with fluorescein isothiocyanate. Naloxone was also coupled in a similar way with rhodamine-B. The compounds thus obtained were: “6-FN”, [1-(N)-fluoresceinyl naloxone thiosemicarbazone]; “6-FNX”, [1-(N)-fluoresceinyl naltrexone thiosemicarbazone]; “6-FO”, [1-(N)-fluoresceinyl oxymorphone thiosemicarbazone]; “6-RN”, [1-(N)-tetramethylrhodaminyl-B naloxone thiosemicarbazone]. These compounds were tested for opioid receptor binding in rat brain synaptosomal plasma membranes and for biological activity on the guinea pig ileum. All compounds retained activity, showing some changes in affinity and binding profile compared to their parent compounds. “6-FN”, for example, showed decreased (∼ 10x) affinity compared to naloxone in opiate displacement analysis but retained potency in displacement of opioid peptides. Antagonist activity of “6-FN” in the guinea pig ileum bioassay was about ten-fold less than that of naloxone.

Alteration of synaptosomal plasma membrane cholesterol content: membrane physical properties and cation transport proteins.

The level of nerve membrane cholesterol was altered by in vitro incubation of rat brain synaptosomal plasma membrane with liposomes having varying cholesterol contents. The normal plasma membrane cholesterol/phospholipid ratio of 0.3-0.4 (mol/mol) could be decreased by about one-half or increased more than 100%. Fluorescence polarization measurements were made using the probe 1,6-diphenyl-1,3,5-hexatriene. At temperatures below 35 percent C, lowering membrane cholesterol led to increased apparent microviscosity, while raising cholesterol content produced little change. However, at 45 percent C a continuous direct relationship existed between experimental membrane cholesterol/phospholipid ratio (ranging from 0.18 to 0.73) and apparent microviscosity. Under standard liposome-synaptosomal plasma membrane exchange conditions, 80% of the initial specific [(3)H]saxitoxin binding activity to the voltage-dependent sodium channel and at least 95% of the (Mg2+,K+)-p-nitrophenylphosphatase activity were preserved. Our results indicate that neither the characteristics of toxin binding nor the kinetics of this enzyme activity is dependent upon membrane cholesterol content.

Immunohistochemical localization of a phosphoprotein (B-50) isolated from rat brain synaptosomal plasma membranes

The endogenous phosphorylation in vitro of at least 5 protein bands of rat brain synaptosomal membranes (SPM) is inhibited by the behaviorally active peptide ACTH1-24. One of these proteins, the phosphoprotein band B-50, was isolated from rat brain SPM by SDS-slab gel electrophoresis. An antiserum to B-50 was raised in rabbits. The presence of antibodies to B-50 in the antiserum was demonstrated by immunoperoxidase staining of cryostat sections of a polyacrylamide gel containing the antigen. The production of antibodies was monitored by an indirect immunofluorescence technique using cryostat sections of quick-frozen rat cerebellum. Immunofluorescent staining of the molecular and granular layers was observed, whereas the white matter and the perikarya and Purkinje cells of the cerebellum were not stained. With the use of the peroxidase-antiperoxidase (PAP) method, the immunohistochemical localization of the antigen in the molecular and granular layer of the cerebellum was confirmed. Regions rich in neuropil, like the CA1, CA2, CA3, CA4 and dentate gyrus showed an intense immunostaining. Thus, in agreement with the synaptic origin of B-50, the antiserum reacted with tissue components present in brian regions in synaptic contacts.

ACTH-induced excessive grooming in the rat

ACTH-induced excessive grooming in the rat

ACTH‐SENSITIVE PROTEIN KINASE FROM RAT BRAIN MEMBRANES

Abstract—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B‐50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X‐100 in 75 mM‐KCl solubilized 15% of the total B‐50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH1–24. The rate of endogenous phosphorylation of protein band B‐50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B‐50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE‐cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH‐sensitive B‐50 protein kinase.

ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated(32)P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH1-24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10(-10) to 10(-4) M. In a number of protein bands, a biphasic effect of ACTH1-24 was observed: in concentrations of 10(-4)-10(-5) M, a reduced amount of(32)P was found; in concentrations of 10(-6)-10(-7) M, hardly any effect could be detected, whereas consistently at concentrations around 10(-8) M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH1-24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.