Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

Subcellular Distribution of Mammalian Tachykinins in Rat Basal Ganglia

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.     

Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain

We have made a monoclonal antibody which specifically recognizes smg p25A among many ras p21/ras p21-like GTP-binding proteins thus far purified from bovine brain membranes. By use of this antibody, we have investigated the localization and subcellular distribution of smg p25A in rat brain by light and electron microscopic immunocytochemistry and by immunoblotting. By light microscopic immunocytochemistry, specific immunoreactivity is widely distributed, most abundant in neuropil, weak in neuronal somata, and absent from white matter. By electron microscopic immunocytochemistry, intense labeling is demonstrated on most of the synapses and concentrated in the presynaptic area where synaptic vesicles are observed. Presynaptic plasma membranes are weakly labeled but mitochondria, postsynaptic plasma membranes, and postsynaptic densities are unlabeled. In subcellular fractionation analysis of cerebrum, about one-fifth of smg p25A is found in the soluble cytosol fraction and the rest is found in the particulate fraction. About half of the particulate-bound smg p25A is recovered in the P2 fraction containing synaptosomes, mitochondria, and myelin, among which a major portion of smg p25A is recovered in the synaptosomal fraction. In the synaptosomal fraction, smg p25A is concentrated about 8-fold in the fraction containing synaptic vesicles and about 3-fold in the fraction containing synaptic plasma membranes compared with the original homogenate. smg p25A is present at a low level in the fraction containing synaptosomal soluble substances but almost absent from the fractions containing intrasynaptosomal mitochondria or post-synaptic densities. These results suggest that smg p25A plays important roles in the regulation of synaptic functions such as exo-endocytotic recycling of synaptic vesicles during neurotransmitter release.     

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Localization of vasopressin in synaptic vesicles of extra-hypothalamic rat brain

The subcellular localization of vasopressin (VP) from extra-hypothalamic areas of rat brain was investigated by measuring its distribution (a) along a continuous sucrose gradient; (b) during the preparation of isolated nerve endings (synaptosomes) and (c) during the preparation of synaptic vesicles.Quite large amounts of vasopressin are isolated in the same fractions as mitochondria, as well as synaptosomes. Osmotic rupture of membrane bound organelles in the homogenate results in the vasopressin being measured largely in the fraction containing synaptic vesicles. These results would suggest that vasopressin could be released by nerve terminals which is consistent with the hypothesis that it may have a neurotransmitter/neuromodulator function in the CNS.     

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.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Distribution of proteins in different subcellular fractions of rat brain studied by two-dimensional gel electrophoresis

The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.     

Different Subcellular Localization of Muscarinic and Serotonin (S2) Receptors in Human, Dog, and Rat Brain

Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain.     

Synaptic junctional glycoconjugates from chick brain

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

Isolation of synaptic junctional complexes of high structural integrity from rat brain

A new method has been developed for isolating synaptic junctional complexes (SJC) of high structural integrity. The major step in the isolation involves homogenization of a synaptosomal membrane (SM) fraction in a biphasic system consisting of Freon 113 and an aqueous phase containing 0.2% Triton X-100. Well-preserved SJCs, along with membrane vesicles, were recovered in the aqueous phase after low-speed centrifugation of the homogenate. The membranes were subsequently separated from the SJCs by centrifugation on a discontinuous sucrose density gradient. The purity and identity of subcellular fractions were monitored by thin sectioning electron microscopy, using specific and nonspecific staining methods. From the electron microscope studies we conclude that SJCs and their components occupy about 65% of the area covered by structures in this fraction. The assay of enzyme activities indicates that homogenization in Triton-Freon and subsequent steps of the isolation procedure affect the activities of Na, K-ATPase, cytochrome oxidase, and acid phosphatase to different extents, but do not cause total inactivation. Electrophoresis of the SJC-enriched fraction on sodium dodecyl sulfate-polyacrylamide gels has demonstrated that a polypeptide which co-migrates with tubulin is the major component in this fraction, and that a polypeptide co-migrating with actin is also present.     

γ-Glutamylaminotransferase and Transglutaminase in Subcellular Fractions of Rat Cortex and in Cultured Astrocytes

The subcellular distribution of gamma-glutamylamino-transferase (EC 2.3.2.2) and transglutaminase (EC 2.3.2.1) has been investigated in rat brain tissue fractionated by a centrifugation and sedimentation technique. Neither of these enzymes was enriched in the synaptosomal fraction. Comparing the in vitro grown astrocytes with synaptosomes, we find that both of these enzymes may possibly be more important in the glial element of the synaptic region. gamma-Glutamylaminotransferase is most abundant in capillaries, confirming previous reports.     

Brain Ca2+/Mg(2+)-ATPase activity and seasonal adaptation of the Djungarian dwarf hamster Phodopus sungorus.

1. Of three sets of Djungarian dwarf hamster, two groups were raised during winter under greatly differing circumstances. One winter group was raised within a climate controlled cage in which the ambient temperature was maintained at 22 degrees C and whereby conditions of light vs darkness were maintained in a constant 12 hr cycle. The second winter group was raised out of doors whereby the hamsters were subjected to prevailing seasonal environmental conditions. A third group was studied under summer conditions, as well. Ca(2+)-, Mg(2+)- and (Ca2+/Mg2+)-ATPase activity was analysed in cellular (= total homogenate) and subcellular fractions (P1-, synaptosomal fraction, synaptic membranes) from cortex, cerebellum and basal brain. 2. The data obtained indicate similar ATPase activity in the cortical homogenates of the winter indoor and summer hamsters. 3. Winter outdoor animals experiencing normal torpidity, however, exhibited reduced ATPase activity by about 50%. 4. Cortical subcellular fractions yielded different results: both the winter and the summer groups showed high ATPase activity in the synaptosomal and synaptic membrane fractions. 5. In the total cerebellar homogenate, the hamsters raised under summer and winter conditions showed the greatest enzyme activity, although less activity was seen in the subcellular fractions. 6. The ATPase activity in the basal brain was found to be nearly identical in all three hamster groups.     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity.

We have purified cholinergic synaptic vesicles from the electric organs of two related marine elasmobranchs, Torpedo californica and Narcine brasiliensis, to a specific activity higher than had previously been obtained. We have demonstrated the homogeneity of the vesicles by biophysical criteria. The purification scheme consisted of differential centrifugation, flotation equilibrium in sucrose density gradients, and permeation chromatography on glass bead columns of average pore size 3000 A. Our criteria for purity were that bound acetylcholine, bound nucleotide triphosphate, protein, and lipid--phosphorus behave identically when vesicles were analyzed by procedures which depend on vesicle size, density, and charge. Contaminants were not detected when vesicles were fractionated by preparative and analytical sedimentation, by preparative equilibrium sedimentation using glycerol density gradients, or by electrophoresis in Ficoll density gradients. Pure synaptic vesicles, which have been purified 290-fold from the initial homogenate, contain per mg of protein: 8 mumol of acetylcholine, 3 mumol of ATP, and 7 mumol of lipid phosphorus. These procedures may be of general value in the purification of membrane vesicles.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.