Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

REGULATION OF ACETYLCHOLINE SYNTHESIS: CONTROL OF CHOLINE TRANSPORT AND ACETYLATION IN SYNAPTOSOMES

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [²H₄]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [²H₄]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.     

Certain aspects of acetylcholine metabolism in teleost retina

Acetylcholine (ACh) synthesis and release from isolated superfused retina of the teleostEugerres plumieri has been studied under different physiological conditions. The retinas were superfused with Krebs-Ringer solutions containing [¹⁴C]choline and the extracellular space of 32% was determined by [³H]inulin. The retina accumulates choline (Ch) from the superfusion medium and this process is mediated by a high affinity transport system with aK _m of 1.82 M. The incorporated Ch is mainly utilized for the synthesis of ACh. The ACh content of the light-adapted retina is not significantly different from that of a dark-adapted one. However, the release of [¹⁴C]ACh from the light-adapted retina was 52% higher as compared to the release from the dark-adapted retina. Flicker stimulation induced a larger increase in ACh release, than from either light or dark adapted retina, proportional to flicker frequency. The results suggest that changes in ACh utilization were related to the function of cellular units responsible for light changes transduction rather than light detection.In partial fulfillment of a MSc degree.     

Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

Abstract: Using sequential incubations in media of different K⁺ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K⁺ and with 2 μM-[²H₉]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K⁺ solutions containing [²H₄]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K⁺ solution on the metabolism during a second low or high K⁺ incubation of a [²H₉]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [²H₉]Ch metabolism versus [²H₄]Ch metabolism. The most striking result we obtained was that [²H₉]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K⁺ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K⁺ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K⁺ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.     

GABA FLUXES IN PRESYNAPTIC NERVE ENDINGS FROM IMMATURE RATS

Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [³H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na⁺ and Ca²⁺ influx, K⁺ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri , 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [³H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm -OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [³H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca²⁺, unless the synaptosomes had been previously depleted of Na⁺ These data suggest that, although a Ca²⁺ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na⁺ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

Concomitant protein phosphorylation and endogenous acetylcholine release induced by nicotine: Dependency on neuronal nicotinic receptors and desensitization

Summary 1. Nicotine stimulated two Ca²⁺-dependent processes in rat frontal cortex synaptosomes: the phosphorylation of an 80-kDa protein band and the release of endogenous ACh.³ Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptosomal plasma membrane induced by the drug. Changes in the state of phosphorylation of the 80-kDa band (presumed to contain synapsin I) were correlated with changes in the release of ACh as follows, from 2 to 4.2. Blockade of predominant, nerve terminal P-type Ca²⁺ channels with -agatoxin-IVA, did not prevent nicotine from stimulating ACh release. In contrast, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylation induced by either nicotine or veratridine. Taken together, these data suggest that, upon nicotine stimulation, Ca²⁺ enters nerve terminals through two distinct pathways. The first, via Ca²⁺ channels, is necessary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotine-induced phosphorylation and release, is the neuronal nAChR itself.3. Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosphorylation. This shows that diminished release is associated to decreased phosphorylation of the 80-kDa protein band, most likely as a consequence of nicotine-promoted nAChR desensitization.4. Augmented ACh release and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in other neurons, increases intracellular Ca²⁺ (Cholewinskiet al., 1993), thus promoting desensitization of ACh release.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

Studies on a Possible Functional Coupling Between Presynaptic Acetylcholinesterase and High-Affinity Choline Uptake in the Rat Brain

The relationships between presynaptic acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 microM [N-methyl-3H]ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was 1/25 of the amount passing through the synaptosomal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

Dopamine Uptake by Rat Striatal Synaptosomes: Time-and Temperature-Dependent Decay and Protection by Dithiothreitol and Dopamine

The uptake of [³H]dopamine (DA) into rat striatal synaptosomes in the presence of a monoamine oxidase inhibitor was studied using a filtration technique. After a 10-min preincubation period, a fast initial uptake of [³H] DA was seen. Uptake reached a maximum after 4 min of incubation. If incubation was continued for more than 7 min, a gradual decrease in synaptosomal [³H]DA levels was found. Uptake was dependent on preincubation time; initial uptake velocity and maximal uptake decreased irreversibly with increasing preincubation periods. Moreover, the capacity of the synaptosomes to retain the [³H]DA during longer incubation times was progressively affected. The decrease in initial uptake activity was due to a decrease in the V_max of the transport system. Dithiothreitol (2.8 mM) protected synaptosomal uptake activity against deterioration at 37°C. Also, DA itself (10^-7M) stabilized the uptake mechanism if added to the suspension before preincubation was started. Since [³H]DA uptake observed after loading the synaptosomes with labeled DA was similar to the uptake seen if the synaptosomes were not previously loaded with DA, it was concluded that under these conditions synaptosomal DA is completely exchangeable with exogenous substrate. Prolonged storage of the synaptosomes at 0°C also resulted in a time-dependent decrease in uptake activity (t_1/2= 116 min). The addition of unlabeled DA or dithiothreitol to the suspension did not affect instability at 0°C.     

Intrastriatal kainic acid elicits functional supersensitivity of muscarinic receptors regulating dopamine release

Acetylcholine enhanced in a concentration-dependent way the K⁺ (15 mM)-evoked release of [³H]dopamine from synaptosomes isolated from rat corpus striatum and prelabeled with the radioactive catecholamine. The concentration-effect curve of ACh obtained in presence of 1.2 mM Ca²⁺ was progressively shifted to the left when [Ca²⁺] was lowered to 0.4 and to 0.2 mM. Intrastriatal injections of kainic acid reduced (70%) the uptake of [³H]choline in synaptosomes prepared 8 days after the lesion but did not affect significantly the uptake of [³H]dopamine. Also the release of [³H]dopamine evoked by K⁺ was minimally affected by kainic acid treatment. In contrast, acetylcholine (tested in presence of 1.2 or 0.2 mM Ca²⁺) was much more effective in enhancing [³H]dopamine release in synaptosomes from kainic acid-lesioned than from unlesioned striata. The results suggest that muscarinic receptors located on dopamine nerve terminals undergo supersensitivity following intrastriatal kainic acid injection.     

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

The association of [³H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)–1]^–1. Arthenius-type plots of [(r_o/r)–1]^–1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10^–10–10^–4 M). These effects were reversed by naloxone (10^–4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.     

EFFECT OF VINBLASTINE AND COLCHICINE ON UPTAKE AND RELEASE OF PUTATIVE TRANSMITTERS BY SYNAPTOSOMES AND ON BRAIN ACTOMYOSIN-LIKE PROTEIN

Abstract— The effects of several inhibitors, including vinblastine and colchicine, on the accumulation of a number of putative transmitters by a rat brain synaptosomal preparation and their subsequent release by excess K⁺ was examined. In addition, the effect of the alkaloids on the ATPase activity of the actomyosin-like protein, neurostenin, isolated from the synaptosomal preparation, was studied. The uptakes of radioactive glutamate, GABA, dopamine and norepinephrine were energy-dependent, as evidenced by their susceptibility to 0.01 mM carbonyl cyanide m-chlorophenylhydrazone (Cl-CCP), 01 mM ouabain and temperature. The active accumulations of GABA, dopamine and norepinephrine were also greatly inhibited by 1 mM6-hydroxydopamine (6-OHDA), 01 mM mersalyl, 0.05–0.25mM vinblastine and 0.1–1.0 mM colchicine. Vinblastine was approximately 10-fold more potent (K₁, ?0.1 mM) than colchicine as an inhibitor. The release of actively accumulated dopamine or norepinephrine by excess K⁺ (increasing the [K⁺] from 5 to 30 mM) was inhibited somewhat when vinblastine was present during the entire incubation period. If the synaptosomes were preloaded with the radioactive compounds prior to addition of vinblastine, there was no discernible effect on the relative amount of material released by excess K⁺. However, the addition of inhibitor under the latter conditions caused a leakage of radioactivity into the medium even without excess K⁺ being present. Glutamate accumulation was somewhat different from that of GABA, dopamine or norepinephrine. Although it required energy for uptake, 6-OHDA, mersalyl, vinblastine or colchicine were not inhibitory. Studies of the oxidative metabolism of glutamate and GABA by this synaptosomal preparation indicated that the mechanisms of inhibition by vinblastine was not attributable to a metabolic effect. Both vinblastine and colchicine inhibited the Mg²⁺-stimulated, but not the Ca²⁺-activated ATPase of neurostenin. This effect was probably attributable to an interaction of the vinblastine with the neurin moiety of this actomyosin-like protein. We suggest that the inhibitory phenomena exhibited by vinblastine and colchicine in this synaptosomal preparation arose from the effect of these alkaloids on the neurin associated with the synaptic membrane.     

Effects of taurine on calcium binding and accumulation in rabbit hippocampal and cortical synaptosomes

The effect of taurine on Ca²⁺ binding and uptake was studied with rabbit brain cortical and hippocampal synaptosomes. Taurine (25 mM) increased by 25% the high affinity ⁴⁵Ca²⁺ binding in the cortical fraction and by 55% in hippocampal synaptosomes but had no effect on low affinity Ca²⁺ binding. Taurine decreased significantly the fluorescence of the chlorotetracycline-hydrophobic Ca²⁺ chelate probe in both synaptosomal fractions which suggests a shift of bound Ca²⁺ from the hydrophobic to the hydrophilic part of the membranes. The uptake of ⁴⁵Ca²⁺ by rabbit brain synaptosomes, when measured in control and 65 mK K⁺-containing media, was not influenced by taurine. However, taurine inhibited significantly the ⁴⁵Ca²⁺ uptake in synaptosomes incubated in media containing moderately increased K⁺ concentrations (14 and 20 mM K⁺). The effects of taurine are discussed in conjunction with its stabilizing effect on excitable membranes.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.