Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl₂ but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.     

Solubilization of Membrane-Bound Acetyicholinesterase by a Phosphatidylinositol-Specific Phospholipase C

Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.     

Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy     

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G _M1 ³ species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to G_M2, G_M1, G_D1a, G_D1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that G_M1 is the major ganglioside species available for enzyme oxidation at the surface.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Large-scale preparation of synaptosomes from bovine brain using a zonal rotor technique

A zonal rotor technique for the preparation of synaptosomes in bulk from bovine brain frontal cortex based on an empirical transformation of a small-volume discontinuous surcrose density gradient arrangement is presented in detail. The procedure yields new information concerning synaptosomes prepared in sucrose gradients. Cerebroside analysis and electron microscopy show myelin contamination to be restricted to the leading, less dense edge of the synaptosomal profile, free mitochondria to the trailing, more dense edge. Exclusion of fringe areas yields a highly purified synaptosome preparation which entirely enters the next dense layer beyond the 0.81.2 M sucrose interface. This interface collects most of the oubain-sensitive (Na⁺, K⁺) adenosine triphosphatase activity. The purified synaptosomes display very high intrinsic sialidase activity and are rich in di-, tri-, and tetrasialogangliosides, the preferred substrates for the enzyme. Up to 90% of the cholinesterase activity in the zonal rotor synaptosome preparation is specific acetylcholinesterase.     

Synaptic vesicle acidification and exocytosis studied with acridine orange fluorescence in rat brain synaptosomes

The acidification of synaptic vesicles (SV) in rat brain synaptosomes was studied using acridine orange (AO) as a fluorescent probe. In synaptosomal suspensions the AO fluorescence was partially quenched, indicating the presence of an acidic compartment. In permeabilized synaptosomes, the quenching was augmented by MgATP and was sensitive to concanamycin A, a specific inhibitor of the V-type H⁺-ATPase known to be present in synaptic vesicles. Some ATP-dependent acidification was also observed without permeabilization, suggesting that a fraction of synaptosomes (ca. 15%) was unsealed, irrespective of the method used to prepare the synaptosomes (sucrose or Ficoll density gradient, sedimentation or flotation). Depolarization of synaptosomes with 30 mM KCl resulted in an immediate, albeit small, rise in AO fluorescence that was prevented by the removal of Ca²⁺ or by substituting NaCl for KCl. This response is consistent with depolarization-evoked release of the acidic contents of an exocytosis-competent pool of synaptic vesicles, representing ca. 5% of the total. No further AO release subsequent to the immediate phase was observed in depolarized synaptosomes, which indicates an extremely rapid reacidification. The results demonstrate that AO fluorescence is suitable for monitoring SV acidification within synaptosomes, and may be used to derive an independent estimate of the relative size of the immediately releasable SV pool. In addition, the use of AO might be advantageous for the assessment of synaptosomal integrity by comparing the ATP-dependent acidification in intact and permeabilized synaptosomes.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

Comparative studies on synaptosomes: Applicability of the rapid method for preparing synaptosomes to elasmobranch brain

A flotation technique for the rapid preparation of synaptosomes, originally developed for invertebrate nervous tissue, has now been successfully applied to that of an elasmobranch fish (Mustelis canis, dogfish). The technique involves submitting the supernatant, obtained after a homogenate has been centrifuged at low speed to remove nuclei and tissue debris to centrifugal fields of intermediate intensity (10⁶ g/min), appears to separate well-sealed synaptosomes from those less well sealed as judged by the criteria of osmotic shrinkage, enzyme occlusion, and choline uptake. The sealed synaptosomes do not equilibrate with the 0.8 M sucrose used as the homogenization medium and rise to form a coherent pellicle at the top of the tube. Due to the short (1–1/1/2 hr) preparation time, such synaptosomes may well prove useful in further metabolic studies.     

Na+ Dependence of Tyrosine Transport Across the Synaptosomal Membrane Reflects Changes in the Morphology of Synaptosomes

The Na+ dependence of tyrosine uptake into rat brain synaptosomes and synaptosomal plasma membrane vesicles (SPMV) was examined in the present study. At low tyrosine concentrations, the isoosmotic substitution of Na+ by sucrose in the incubation medium led to an increase of tyrosine uptake in synaptosomes and to a decrease in SPMV. The removal of extracellular Ca2+ and Mg2+ and addition of isoosmotic sucrose completely prevented the augmented tyrosine uptake in Na+-free incubated synaptosomes. Morphological differences were found at the electron-microscopic level when synaptosomes were incubated in Na+-free and Na+-containing media. The internal volume measured for synaptosomes incubated in a Na+-free medium was almost half of that obtained in a Na+-containing medium, in good agreement with the observations made with the electron microscope. Also, the omission of Ca2+ and Mg2+ resulted in a specific swelling of only the synaptosomes incubated in Na+-free medium. When synaptosomes and SPMV were preloaded with several neutral amino acids, the tyrosine uptake rate was greatly increased, indicating fully operational exchange mechanisms for these amino acids. We propose that the enhancement of high-affinity synaptosomal tyrosine uptake observed in Na+-free medium is a consequence of a specific shrinkage of the synaptosomes and a parallel increase of the exchange rate with endogenous neutral amino acids.     

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Effect of Aging on Tyrosine Hydroxylase Protein Content and the Relative Number of Dopamine Nerve Terminals in Human Caudate

This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15-63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.     

Separation of synaptic junctional complexes from thin layers of rabbit cerebral cortex

Synaptic junctional fractions were separated from rabbit brain by procedures based on combining the methods of Cotman and Taylor [4], Orosz et al. [16, 17] and Lisman et al. [13]. Thin layers of cerebral cortices were homogenized to obtain a crude mitochondrial-synaptosomal fraction. The sedimentation rates of mitochondria and mitochondria containing synaptosomes were increased by raising the density of mitochondria with an insoluble dense formazan deposit inside mitochondria after iodo-nitrotetrazolium treatment. The synaptic plasma membrane fraction isolated by this method contained no mitochondrial contamination. After Triton X-100 treatment the insoluble residues of the detergent were centrifuged through discontinuous sucrose gradients. A great enrichment of morphologically identifiable intact synaptic junctions was observed in some of the obtained interface layers.     

Caffeine-sensitive calcium stores in presynaptic nerve endings: a physiological role?

Ca2+-sensitive minielectrodes and the fluorescent cytosolic calcium probes, quin2 and fura2, were used to study some aspects of calcium homeostasis in intact and permeabilized synaptosomes from whole rat brain. Experiments in permeabilized synaptosomes revealed the existence of a vesicular, non-mitochondrial, ATP-dependent calcium uptake system with a vanadate sensitivity similar to that of brain microsomes, or endoplasmic reticulum-type calcium sequestering organelles. By using the fluorescent probes it was possible to show that caffeine mobilizes calcium from these internal stores in intact synaptosomes. Our results indicate a role of the caffeine sensitive calcium stores in the buffering of calcium loads elicited by plasma membrane depolarization.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Isolation of metabolically distinct synaptosomes on Percoll gradients

Synaptosomes were prepared from whole rat brain by six different methods based on gradients of sucrose, Ficoll or Percoll. In these, the synthesis and calcium-specific release of amino acids were assessed by two different procedures. Preparations based on sucrose showed the least calcium-specific release, followed by Ficoll-derived synaptosomes. As previously described, Percoll gave two separate populations of synaptosomes, both very active in terms of release of aspartate, glutamate, and GABA. The data involving release and synthesis were not identical, but did agree in the following: in low-density synaptosomes, haloperidol blocked both the release and synthesis of glutamate, but was without effect in the heavier populatin. 2-chloroadenosine and 2-oxoglutarate affected both release and synthesis only in the high-density population. Dopamine blocked aspartate release and synthesis only in the high-density population. These results suggest that haloperidol interferes with glutamate release and synthesis via a mechanism which may not involve adenosine, serotonin, or dopamine.