The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake

Abstract: The Na⁺ and K⁺ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na⁺ (290 MM) and low internal K⁺ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na⁺, 96 mM; K⁺, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na⁺ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na⁺ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.     

Subcellular localization and ionic properties of acetyl-coenzyme A synthetase in the electric organ of Torpedo marmorata.

Acetyl-CoA synthetase activity was shown to be present in pure cholinergic synaptosomes from electric organ of Torpedo marmorata. After osmotic disruption of synaptosomes a substantial part of the activity was recovered in the soluble fraction. The effects of varying pH and increasing K+ concentrations on the synaptosomal enzyme activity were shown to differ from those observed with the mitochondrial enzyme. Whereas this latter enzyme showed optimal activity above pH 8.5, and a maximal activation in the presence of 120 mM-K+, the synaptosomal enzyme exhibited an optimal activity at pH 7.9 and a moderate K+ stimulatory effect with an optimal concentration of 30 mM.     

Neurotransmitter release from viable purely cholinergic Torpedo synaptosomes

Viable synaptosomes from the electric organ of Torpedo have been prepared and partially purified. The synaptosomes contain about 100 fold more acetylcholine (Ach) than do mammalian synaptosomes, synaptic vesicles and mitochondria. The Torpedo synaptosomes release Ach by K depolarization in the presence of Ca ions, and manifest an ionophore-mediated Ca-dependent Ach release. These results demonstrate that the synaptosomes contain the neurosecretion apparatus in a functional viable state. Since this preparation uniquely contains only one neurotransmission system (cholinergic), it is most suitable for structural and functional investigations of neuro transmission.     

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.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

In Vitro Evidence for Increased Facilitation of Striatal Acetylcholine Release via Pre- and Postsynaptic NMDA Receptotors in Hemiparkinsonian Rats

Abstract : The NMDA-evoked acetylcholine release from striatal slices and synaptosomes was investigated in rats subjected to unilateral injection of 6-hydroxydopamine into the substantia nigra. In slices prepared from the striatum contralateral to the lesion, the NMDA-evoked endogenous acetylcholine release was not significant at 10 μ M NMDA and maximal at 100 μ M NMDA (124 ± 19%). Conversely, in slices taken from the dopamine-depleted striatum, NMDA was effective even at 10 μ M (41 ± 4%), and at 100 μ M (196 ± 24%) efficacy was nearly doubled. In synaptosomes prepared from the contralateral striatum, NMDA maximally stimulated 20 m M KCl-induced endogenous acetylcholine release at 1 μ M (66 ± 5.1%), with lower concentrations (0.01-0.1 μ M ) being ineffective. Conversely, in synaptosomes prepared from the dopamine-depleted striatum, NMDA maximally enhanced the K⁺-evoked acetylcholine release at 0.1 μ M (118 ± 12.4%). Concentration-response curves of NMDA-evoked acetylcholine release in sham-operated rats could be superimposed on those observed in the contralateral striatum of the 6-hydroxydopamine-lesioned animals. The present data support the view of an increased glutamatergic regulation of striatal acetylcholine release via pre- and postsynaptic NMDA receptors during Parkinson's disease.     

Na(+)-Ca2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata.

Synaptic plasma membranes obtained by hypo-osmotic treatment of purified Torpedo ocellata synaptosomes, contain an electrogenic Na(+)-Ca2+ exchange system. The dependence of the initial reaction rate on [Ca2+] reveals a single binding site for Ca2+ with an average apparent Km of 13.66 (S.D. = 12.07) microM [Ca2+] and maximal reaction velocity of Vmax = 11.33 (S.D. = 5.93) nmol/mg protein per s. The dependence of the initial rate of the Na+ gradient dependent Ca2+ influx on the internal [Na+] exhibits a sigmoidal curve which reaches half-maximal reaction rate at 170.8 (S.D. = 19.9) mM [Na+]. Addition of ATP gamma S does not change the K0.5 to Na+. The average Hill coefficient is 3.09 (S.D. = 0.86) indicating that 3-4 Na+ ions are exchanged for each Ca2+. Na+ gradient dependent Ca2+ uptake in Torpedo SPMs takes place also in the absence of K+ suggesting that K+ co-transport is not obligatory. The temperature dependence of the initial and steady-state rates of Na+ gradient dependent Ca2+ influx reveal that maximal reaction velocities of the Torpedo exchanger are attained between 15 and 20 degrees C. The energy of activation between 0 and 20 degrees C is 20,826 cal/mol. In comparison, rat brain synaptic plasma membrane Na(+)-Ca2+ exchanger reaches maximal reaction rates between 30 and 40 degrees C. Reconstitution of Torpedo or rat brain Na(+)-Ca2+ exchangers into a membrane composed of either Torpedo or brain phospholipids, does not alter the temperature dependence of the native Torpedo or rat brain Na(+)-Ca2+ exchangers; inspite of considerable differences in the composition of the fatty acyl chains that are esterified to brain and Torpedo phospholipid head groups and differences in membrane fluidity that were detected. An ATP-dependent Ca2+ pump, which is insensitive to FCCP, is also present in the same synaptic membrane.     

Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes.

Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.     

Presynaptic Glycine Receptors Influence Plasma Membrane Potential and Glutamate Release

Glycine is a classical inhibitory neurotransmitter however presynaptic glycine receptors have rather depolarizing action. Reasons for latter phenomenon are unknown. In the present paper we have investigated how glycine influences cytosolic chloride level monitored by fluorescent dye SPQ, membrane potential monitored by fluorescent dye DiSC3(5) and [¹⁴C]-glutamate release in synaptosomes. We estimated that cytosolic chloride concentration in synaptosomes was about 52 ± 1 mM. Glycine (1 mM) induced chloride efflux and caused slow plasma membrane depolarization. Chloride efflux was almost completely blocked by 100 μM strychnine whilst glycine-induced depolarization was only partially. We also showed that 1 mM glycine induced [¹⁴C]-glutamate release via a strychnine-insensitive pathway. Hence we have concluded that glycine was able to induce two independent effects in synaptosomes: (1) Chloride efflux with following depolarization. This efflux was sensitive to strychnine and thereby is probably conducted through glycine-gated ion channels. (2) Glutamate release seems to be mediated by glycine transporters.     

ADP-Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals

Abstract: Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [³²P]NAD⁺ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of M_r= 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcel-lular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with M_r= 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nu-cleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

Large-Scale Purification of Torpedo Electric Organ Synaptosomes

Abstract: A procedure for the large-scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium. Two hundred grams of electric organ can be fractionated in a single run, allowing biochemical studies on presynaptic membrane constituents.     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

Cholinergic Vesicle Specific Proteoglycan: Stability in Isolated Vesicles and in Synaptosomes During Induced Transmitter Release

Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co-release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P-40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co-release with acetylcholine or ATP.     

Attenuation of glyceraldehyde-3-phosphate dehydrogenase activity and ATP levels in rat brain synaptosomes by acrylamide

The effect of neurotoxin acrylamide (AC) on energy metabolism has been studied in a purified preparation of the synaptosomes. The synaptosomes were prepared by the flotation technique in a discontinuous Ficoll/sucrose gradient. The purity of the synaptosomes was checked by electron microscopy and by assaying the activity of marker enzymes. By these criterias, free mitochondrial contamination in the synaptosomes was found to be >2%. Incubation of the synaptosomes with different concentrations of AC (2.5, 5.0, and 10mM) produced a concentration-dependent inhibition (15, 35, and 60%, respectively) of glyceraldehyde-3-phosphate dehydrogenase activity. Acrylamide also produced a time-dependent decrease of ATP concentrations in the synaptosomes; about 25% loss of ATP was seen within 1h, while about 60% ATP was lost after 120 min incubation with 10 mM AC. The effect of known inhibitors of glycolysis-iodoacetic acid (IAA), and of oxidative phophorylation-rotenone and antimycin A, was also studied on ATP synthesis by the synaptosomes. IAA was found to be the most potent inhibitor of ATP synthesis, while both rotenone and antimycin A were equally effective in blocking ATP synthesis in the synaptosomes. These studies show that the synaptosome might be used as a suitablein vitro model to study the effect of neurotoxin such as AC on neuronal energy metabolism.Special issue dedicated to Dr. Sidney Ochs.     

Immunohistochemical localization of cholinergic nerve terminals

Summary Most of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.     

PLASMA VASOPRESSIN LEVELS AND WATER CONSERVATION IN FASTING, POSTWEANED NORTHERN ELEPHANT SEAL PUPS (MIROUNGA ANGUSTIROSTRIS)

Northern elephant seal pups ( Mirounga angustirostris ) use several physiological mechanisms to conserve water during their 8- 12-wk postweaning fast. Urine was analyzed from five animals and plasma from four of those animals was sampled serially throughout the postweaning period to quantify changes in urine concentrating ability and electrolyte homeostasis at various stages of the fast (Early = 1-4 wk, Mid = 4-7 wk, Late = 7-10 wk). Plasma osmolarities stayed relatively constant at 301.6 f 13.1 mOsm/kg. Urine osmolarities ranged from 1,053.5 ± 78.6 mOsm/kg (Early), to 1,585.0 ± 136.7 mOsm/kg after eight weeks, dropping to 1,214.3 ± 349.6 mOsm/kg (Late). Urine concentrations for Na⁺ and K⁺ early in the fast were 55.0 ± 14.6 mM and 180.8 ± 48.6 mM, respectively, declining to 8.2 ± 3.3 mM and 91.4 ± 29.0 mM later. Plasma vasopressin concentrations ranged from 34.8 ± 18.2 pg/ml (Early) to 4.8 ± 1.3 pg/ml (Late). The pups appear to conserve their body water by producing a concentrated urine, thus reducing urinary water loss. The significance of the antidiuretic role of vasopressin in the conservation of body water in these pups could not be conclusively determined from the results.