TAURINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— Uptake of [^3,5S]taurine by rat whole brain synaptosomes was studied at varying temperatures, under O₂, and N₂ atmospheres, during electrical stimulation and in the presence of dinitrophenol or variable taurine concentrations in the incubation medium. The morphology and purity of the synaptosomes was checked by electron microscopy. The respiration of the synaptosomes was linear for at least 90 min. The taurine uptake was energy- and temperature-dependent and significantly enhanced by electrical stimulation. The total uptake of taurine could be divided into three components, non-saturable influx and saturable high-affinity ( K _m= 46 μmol/l) and low-affinity ( K _m, = 6.3 mmol/l) transport systems. The efficacy of the high-affinity transport appears small in view of the postulated neurotrans-mitter role of taurine.     

CHOLINE UPTAKE BY CHOLINERGIC NEURON CELL SOMAS

The cellular compartments of ciliary ganglia take up choline by a single, saturable process with K_m=7.1 × 10^?5 M and V_max= 4.66 pmol/min per ganglion: Denervation of the ganglia and the resultant degeneration of nerve terminals caused no significant decrease of the rate of accumulation of choline by the ganglia. This indicates that the measured uptake is by the postganglionic ncurons and nonneural elements (NNE: glial and connective tissue cells) in the ganglia. This uptakc is not dependent on metabolic energy and is not affectcd by lowcring Na⁺ or raising K⁺ concentrations in the incubating mcdia but is depressed in the presence of ouabain and hemicholinium-3. The presence or Na⁺-dependent. rapidly saturable uptake in the preganglionic nerve terminals which is not detectablc kinetically is, however, inferred from a decrease in ACh synthesis in dcncrvatcd prcparations and a similar decrcasc in intact ganglia incubated in low Na+ solution.     

α-TOXIN BINDING PROTEINS IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA STUDIED BY IMMUNOCHEMICAL METHODS

—Crossed immunoelectrophoretic techniques were developed to study the efficiency of the various purification steps in the isolation of nicotinic acetylcholine receptor (nAChR) from Torpedo mormorata electric organ. A new α-neurotoxin binding assay based on immunoelectrophoresis is also presented. In crude extracts of Torpedo electric organ membranes one type of receptor molecule (M ñ; 300 , 000) was found; an earlier described higher molecular form was shown to be an artifact of affinity chromatography. Polyvalent antibodies against Torpedo electroplaque membranes, antibodies against purified membrane proteins and against Naja naja siamensisα-neurotoxin revealed four α-neurotoxin binding antigens (including nAChR). Two of these, nAChR and T₂, were specific for electroplaque membrane and showed partial immunoidentity but different biochemical and physical properties.     

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.     

Ca-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP₃) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP₃ mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP₃ mass levels under resting conditions. The IP₃ mass was neither modified by changes in external Ca²⁺ nor by a Ca²⁺-free medium containing EGTA. IP₃ mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K⁺ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca²⁺ emphasizing that Ca²⁺ entry, independently of the influx mechanism involved, leads to an IP₃ increase. The phospholipase C_β inhibitors U-73122 and U-73343 reduced K⁺-stimulated IP₃ levels while K⁺-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP₃ by agents that stimulate ACh release may suggest a possible link between IP₃ metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd     

CHARACTERISTICS OF D-GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of D-glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D-glucosamine uptake, after correcting for simple diffusion, obeys Michaelis-Menten kinetics. The apparent kinetic constants for the uptake process are K_m = 2.5 0.8 m m , V_max = 3.7 ± 1.2 nmol/mg protein/min. D-Glucose, D-mannose, 2-deoxy-D-glucose and 3-0-methyl-o-glucose are potent inhibitors of D-glucosamine uptake. 2-Deoxy-D-glucose and D-glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D-glucosamine uptake with apparent inhibitor constants ( K₁ ) of 7.0 × 10^-5, 2.3 × 10^-3 and 0.4 mM, respectively. The uptake is unaffected by Na⁺, Li⁺ and Mg²⁺, partially inhibited by NH₄⁺, Mn²⁺ and Ca²⁺, and slightly stimulated by PO₄-ions. D-Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D-glucosamine and 2-deoxy-D-glucose are about 4 times greater in 7-day-old than in the adult rat brains.     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

ON THE UPTAKE OF INOSITOL BY RAT BRAIN SYNAPTOSOMES

The uptake of inositol by rat brain synaptosomes occurs via an unsaturable process that even at substrate concentrations as low as 1 μM is unable to achieve a concentration gradient indicative of active transport. Dinitrophenol, ouabain and cytochalasin B did not affect uptake of the cyclitol. The data indicate that inositol uptake by rat synaptosomes occurs by diffusion or by a system with an affinity so low it is difficult to discern. The low capacity, saturable inositol uptake system observed in rabbit brain slices may reflect a species difference or uptake by elements of the slice other than neuronal membranes.     

CHARACTERIZATION OF HIGH AFFINITY CHOLINE UPTAKE BY TORPEDO CALIFORNICA T-SACS

Abstract— The high affinity choline uptake system present in T-sacs prepared from Torpedo californica electric organ was shown to be insensitive to external Ca²⁺ and to be absolutely dependent on external NaCl, with optimal uptake at approx 200 mM-NaCl. Both Na⁺ and Cl⁻ separately stimulate uptake. Uptake also exhibited an optimum at approx 10mM-external K⁺. Uptake was completely inhibited at 4°C. Approximately 50% of newly accumulated [³H]choline was released by depolarization of T-sacs regardless of the time or method of depolarization.     

DETERMINATION OF ACh CONCENTRATION IN TORPEDO SYNAPTOSOMES

Abstract— The concentrations of ACh and ATP of Torpedo electric organ synaptosomes were directly measured and found to be respectively 20.0 ± 6.4 mM and 3.1 ± 0.6 mM. The synaptosomal volume was estimated by a classical space marker technique using ¹⁴C inulin and tritiated water. After counting the synaptosomes in an haemocytometer (and knowing their volume), a mean diameter of 3.5 p was calculated. The use of these classical techniques was rendered possible because of the homogeneity of the fraction and the large size of Torpedo synaptosomes.     

COMPARATIVE STUDIES ON SYNAPTOSOMES: UPTAKE OF [N-Me-3H]CHOLINE BY SYNAPTOSOMES FROM SQUID OPTIC LOBES

Abstract— The uptake of [ N -Me-³H]choline into synaptosomes from squid optic lobes was studied using a Millipore filtration technique. When incubated in an artificial sea water medium at 26°C, but not at 0°C, the synaptosomes rapidly accumulated choline, most of which could be recovered as unchanged free choline. The accumulated choline was readily released by treatment of the synaptosomes with Triton X-100 or exposing them to hypo-osmotic conditions. The influx of choline increased with increasing concentrations of choline and could be resolved into saturable and non-saturable components. Kinetic analysis revealed the presence of two saturable components one of high affinity ( K _m about 2 μ m ) and one of lower affinity ( K _m 25 μ m ). The rate of choline uptake by these synaptosomes was considerably greater than by mammalian brain synaptosomes. Both high and low affinity systems were Na⁺-requiring and inhibited by hemicholinium no. 3, levorphanol and dextrorphan. NaCN, 2,4-dinitrophenol and ouabain also inhibited choline uptake, the high affinity system being particularly sensitive to these agents. It is suggested that the high affinity system is specific for cholinergic terminals.     

D-GALACTOSE TRANSPORT BY SYNAPTOSOMES ISOLATED FROM RAT BRAIN

Abstract— Synaptosomes prepared by differential and Ficoll density gradient centrifugation took up d -galactose by two saturable transport systems: one. a high affinity system with a K _m of 0-25 mn and V_max of 075 nmol/mg protein 3 min, the other, a low affinity system with a K_m of 47 mM and a V_max of 135 nmol/mg protein/3 min. The high affinity system was inhibited by 1-5 mM phlorizin but was unaffected by the absence of sodium ion or the presence of 1 mM ouabain. The low affinity system was unaffected by phlorizin or ouabain. Both systems were inhibited by high concentrations of glucose. 2-deoxyga-lactose. and inositol, and by 2.4-dinitrophcnol. Galactose was rapidly converted in synaptosomes to phos-phorylatcd intermediates and was more slowly oxidized to ¹⁴CO₂     

A ROLE FOR DIVALENT CATIONS IN THE UPTAKE OF NORADRENALINE BY SYNAPTOSOMES

–The effects of divalent cations on the initial rates of noradrenaline uptake by synaptosomes were determined using Millipore filtration to terminate the reaction. The removal of either Ca²⁺ or Mg²⁺ from the incubation medium had no effect on uptake, but when both Ca²⁺ and Mg²⁺ were removed, uptake was reduced. Uptake was also diminished when Ca²⁺ was absent and 1 mm -EGTA added to the medium. It appeared that Ca²⁺ was required for optimal uptake but that Mg²⁺ could partially substitute for Ca²⁺ in this regard. The reduction in the rate of uptake when both Ca²⁺⁰ and Mg²⁺ were absent could be rapidly and completely reversed by restoring Ca²⁺, Mg²⁺, or both Ca²⁺ and Mg²⁺ to the incubation medium. Of the divalent cations tested, Ca²⁺ and Mg²⁺, but not Mn²⁺, supported noradrenaline uptake. When the kinetics of uptake were examined, it was found that removing both Ca²⁺ and Mg²⁺ from the medium resulted in a reduction of the V_max for noradrenaline uptake. It is apparent from these results that, in addition to facilitating the release of noradrenaline from noradrenergic terminals, Ca²⁺ may also play a role in the uptake of noradrenaline by presynaptic nerve-endings in the CNS.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

KINETICS OF ADENOSINE UPTAKE INTO ASTROCYTES

Abstract— Kinetics for uptake of adenosine, a putative inhibitory transmitter, were measured in normal, i.e. non-transformed, astrocytes in cultures obtained from the dissociated, cortex-enriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with a rather intense (V_max 0.36nmol/min per mg protein) high affinity ( K _m 3.4 μ m ) uptake following Michaelis-Menten kinetics and inhibited by 100 μ m -papaverine. The V_max was about two times higher than that reported in the literature for brain slices suggesting that a considerable part of the adenosine uptake in brain slices occurs into glial cells. Such an accumulation of adenosine into normal astrocytes may play a major role in nucleoside and nucleotide metabolism in the brain and help in regulating the extracellular adenosine concentration.     

EFFECTS OF ETHANOL ON THE ACTIVITY OF ADENOSINE TRIPHOSPHATASE AND ACETYLCHOLINESTERASE IN SYNAPTOSOMES ISOLATED FROM GUINEA-PIG BRAIN

Nerve ending fractions from guinea-pig cerebral cortex contained more than one-half of the Na-K ATPase activity present in the original homogenate. Ethanol at concentrations ranging from 0·043 to 2·57 m inhibited the Na-K ATPase to a significantly greater extent than the Mg-activated ATPase or AChE. The inhibition of membrane-bound Na-K ATPase by ethanol was of the non-competetive type and the activity of Na-K ATPase was increasingly inhibited by alcohols of increasingly longer chain length. The ability of various alcohols to inhibit membrane-bound Na-K ATPase activity was correlated with their lipid solubility.     

CATION UPTAKE BY INTACT NUCLEI FROM RAT CEREBRAL CORTEX

A method is described for determining the uptake of cations by intact nuclei from rat cerebral cortex. The divalent cations of magnesium, manganese and calcium were found to be concentrated in the nuclear pellet to levels well above the initial concentration of the medium, whereas little uptake of either potassium or sodium was observed. The binding of divalent cations to the nuclei will produce a cationic environment quite different from that of the external medium. Such localized differences may play a role in the control of nuclear activities.     

l-[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of [³⁵S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent K _m values of 0.14 and 1.4 m m . The low K _m system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.     

BREAKDOWN OF CREATINE PHOSPHATE AND ATP IN NERVE TERMINALS AND ELECTROPLAQUES OF THE TORPEDO ELECTRIC ORGAN: COMPARISON WITH THE ELECTRICAL ENERGY DISSIPATED

Abstract— The electrical work performed by the electric organ of Torpedo was compared with the energy provided by the net breakdown of ATP and creatine phosphate (CrP). The electrical work was calculated for single impulses and for repetitive stimulations. The content in CrP and ATP was measured at different times in the course of stimulation and during the period of recovery. The chemical expenditure due to activity of the nerve terminals was distinguished from the total expenditure by the use of curare which interrupts synaptic transmission but does not interfere to any great extent with the release of acetylcholine. In the presence of curare the breakdown of phosphagen started only after more than 1 min of stimulation; it represented the loss of about 20-25% of the initial store. In untreated tissue the breakdown of CrP and ATP occurred in two phases and continued within the first minute after the end of the stimulation; as much as 77% of the phosphagen content was utilized under these conditions. The recovery of ATP and CrP was completed only 3-5 h after stimulation, a long time after the restoration of the physical capabilities of the tissue. The electrical energy dissipated during activity was smaller than the chemical energy provided by the net breakdown of phosphagens. This suggests that only a fraction of the chemical energy is utilized directly to compensate for the physical work accomplished, i.e. for the restoration of the ionic electromotive force. The electric organ also requires chemical energy for other purposes, particularly in the nerve endings where the presynaptic machinery seems to utilize an important fraction of the high energy phosphates stored in the tissue.     

SCORPION TOXIN-INDUCED CATECHOLAMINE RELEASE FROM SYNAPTOSOMES

Abstract— A toxin purified from crude venom of the scorpion L. quinquestriatus releases [3H]norepinephrine from synaptosomes prepared from rat brain. The toxin-induced release is dependent on duration of exposure and concentration of toxin in the medium. The absence of calcium in the medium diminishes toxin-induced release but does not abolish it. Toxin-induced release is diminished by tetrodotoxin or, to a lesser extent, by desmethylimipramine. Since the released tritium is present predominantly as norepinephrine, it appears that toxin-induced release is similar to that produced by veratradine or tyramine and is distinct from reserpine induced release.