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.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

ISOLATION OF [3H]ACETYLCHOLINE POOLS BY SUBCELLULAR FRACTIONATION OF CEREBRAL CORTEX SLICES INCUBATED WITH [3H]CHOLINE

Abstract— Subcellular fractions were isolated from tissue incubated in [³H]choline with or without the addition of 33 mM-KCl. Radioactive and bioassayable ACh were measured in the synaptosomes, synaptosomal cytoplasm and in the vesicles. After incubation with KCI the vesicles, as isolated, contained ACh of a lower specific activity than the cytoplasmic ACh. Therefore the vesicle fraction as isolated does not represent the source of the high specific activity ACh released upon K⁺ stimulation. However the vesicle fraction is heterogeneous. Most of the bioassayable ACh but little of the radioactive ACh in the vesicles passed through iso-osmotic Sephadex columns. These results raise the question of the existence of vesicles which contain highly radioactive ACh but which lose it during their isolation by current methods. Different possible forms of heterogeneity are discussed.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

SYNTHESIS OF RADIOACTIVE ACETYLCHOLINE FROM [3H]CHOLINE AND ITS RELEASE FROM CEREBRAL CORTEX SLICES IN VITRO

Abstract— Guinea pig cerebral cortex slices were incubated for 60 min in a medium containing [³H]choline with or without the addition of 33 mM-KCl for the last 30 min. KC1 caused the release into the medium of large amounts of both bioassayable and radioactive ACh, while at the same time their concentrations in the tissue decreased. The specific activity (d.p.m./pmol) of the ACh released by KC1 was greater than that released in control incubations, indicating that it comes from a newly synthesized, more radioactive store. The amounts of [³H]choline, [³H]ACh and the specific activity of tissue acetylcholine reached a plateau in the tissue 30 min after the addition of isotope. However isotopic equilibrium was not achieved because the specific activity of the ACh released, with or without KC1 in the subsequent 30 min, was less than the specific activity of the ACh remaining in the tissue. This implies the existence of a pool of ACh in the tissue which is turning over very slowly or is being synthesized from a less radioactive pool of choline. This pool of ACh does not contribute substantially to that released by KC1. Levorphanol at 10⁻³ M, as well as the analgesically inactive stereoisomer, dextrorphan, blocked the KCl-stimulated release of both bioassayable and radioactive ACh. These drugs demonstrate the coupling of synthesis and release of ACh in cerebral cortex slices.     

IN VITRO EXPERIMENTS ON THE METABOLISM, ACCUMULATION AND RELEASE OF 5-HT IN THE NERVOUS SYSTEM OF THE SNAIL HELIX POMATIA

Abstract— The accumulation, metabolism and stimulated-induced release of 5-HT in the nervous system of the snail was studied. When nervous tissue was incubated at 24°C in a medium containing [¹⁴C]5-HT or [³H]tryptophan, tissue: medium ratios of about 25:1 and 4:1 respectively were obtained after 45 min incubation. The process responsible for [¹⁴C]5-HT accumulation showed properties of an active transport system: it was temperature sensitive and was greatly inhibited by dinitrophenol and ouabain. Furthermore, the accumulation process was inhibited by imipramine and desipramine. Of a number of analogues of indole, N-acetyl-5-HT and 5-hydroxytryptophan were the most potent in the inhibition of the accumulation of [¹⁴C]5-HT. The presence of a large molar excess of amino acids had little effect. A small amount (less than 14 per cent) of the accumulated [¹⁴C]5-HT was metabolized to form 5-hydroxyindole acetic acid, even after long periods (2 h) of incubation. The accumulated [³H]tryptophan was metabolized to form 5-hydroxytryptophan and 5-HT; the content of formed [³H]5-HT increased with incubation time whilst the [³H]5-hydroxytryptophan remained more or less constant. The presence of p-chlorophenylalanine in the incubation medium did not interfere with the accumulation of [³H]tryptophan, though it inhibited the formation of [³H]5-hydroxytryptophan and to a greater extent [³H]5-HT. A rapid efflux of the accumulated [¹⁴C]5-HT from snail nervous tissue was observed on electrical stimulation. Slower release resulted when the Ca²⁺ ion content of the incubation medium was replaced by Mg²⁺ ions. There is also a slight efflux of radioactive substances following electrical stimulation in tissues previously incubated in [³H]tryptophan. Most of this radioactivity was attributed to the formed [³H]5-HT. The data support the idea that 5-HT is a transmitter-substance in the snail Helix pomatia, and that re-uptake of the substance is a method of inactivating the released amine.     

ACTIVE UPTAKE OF [3H]5-HT BY SYNAPTIC VESICLES FROM RAT BRAIN

The question of whether synaptic vesicles accumulate [³H]5-HT by an active process was investigated in a mixed population of vesiclcs from whole rat brain. The temperature dependence and the effect of metabolic inhibitors were studied in synaptosomal suspensions and vesicular fractions. Arrhenius plots for synaptosomes differed from those for vesicles as did the temperature coefficients for these two fractions. For synaptosomes the Q₁₀ was 7 and for vesicles 1.6. However, if ATP was added to the incubation, the temperature dependence of vesicular amine accumulation became manifest; the Arrhenius plot resembled that of synaptosomes and the Q₁₀ was greater than 20 indicating strong temperature dependence. In the presence of ATP, vesicular uptake was stimulated approx 8-fold. Ouabain, dinitrophenol and NEM inhibited synaptosomal uptake but failed to affect [³H]5-HT accumulation by vesicles in the absence of ATP. When ATP was added, vesicular uptake was also blocked by NEM but was unaffected by either ouabain or DNP. Total observed uptake consisted of two components, one ATP-dependent and one nonsaturable and ATP-independent. The active process had a K_m= 1.25 × 10^?7 M and could be completely blocked by either 10^?3 M or 10^?7 M-reserpine. Active vesicular [³H]5-HT uptake was magnesium dependent and was inhibited by sodium and potassium. Cation effects on uptake were specific and could not be accounted for by either changes in osmotic pressure or ionic strength. It was concluded that synaptic vesicles from whole rat brain accumulate [³H]5-HT by an active process.     

EFFECTS OF AMINO-OXYACETIC ACID ON [3H]GABA UPTAKE BY RAT BRAIN SLICES

Abstract— The effect of amino-oxyacetic acid on the uptake of [³H]GABA by rat brain slices was studied. When added simultaneously with [³H]GABA, amino-oxyacetic acid had no significant effect on [³H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [³H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10^--⁶ M) had no effect on [³H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [³H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [³H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [³H]GABA uptake.     

THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

STUDIES OF THE UPTAKE AND RELEASE OF [3H]β-ALANINE BY FROG SPINAL SLICES

Abstract— [³H]β-Alanine was accumulated by frog spinal cord slices by two transport components with estimated K_m values of 31 M ('high-affinity') and 11 HIM ('low affinity') respectively. The high affinity uptake exhibited sodium ion and energy dependence, temperature sensitivity, had a very low V_max (10.4 nmol/g/min) compared to GABA and glycine, was competitively inhibited by GABA (K_t 2 M), and was significantly reduced by the presence of glycine and of taurine in the incubating medium.
When slices preloaded with [³H]β-alanine were superfused with medium containing depolarizing concentrations of potassium ions, there was a small, but consistent, increase in [³H]β-alanine efflux: 1.4 times prestimulation rates in 40 mM potassium. When the superfusate was altered by omission of calcium and addition of concentrations of magnesium (10 mm), manganese (1 mM), and cobalt (1 mM) ions sufficient to block reflex transmission in the isolated in vitro frog cord, the potassium-evoked release was not blocked. Release was decreased by lanthanum ions (1 mM). Release of [³H]GABA and [³H]glycine in parallel experiments was inhibited by magnesium, manganese, cobalt and lanthanum. Veratridine significantly increased the release of [³H]GABA and [³H]glycine but not of [³H]β-alanine.
These observations demonstrate the non-specificity of β-alanine uptake and the unconventional nature of the calcium-dependence of β-alanine release and therefore do not lend support to the hypothesis that β-alanine functions as a neurotransmitter in frog spinal cord.     

THE EFFECT OF DELTA-AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF [3H]GABA IN RAT BRAIN SYNAPTOSOMES

§-Aminolaevulinic acid (§-ALA) is an omega amino acid which can be considered as an analogue of γ-aminobutyric acid (GABA). We have examined the effect of §-ALA on [³H]GABA uptake and release in the synaptosome fraction of rat cerebral cortex and report: (1) High concentrations of §-ALA (0.75-5 mM) stimulated [³H]GABA release very markedly, the stimulation with 1mM and 5mM-§-ALA exceeding the maximum obtainable with unlabelled GABA; (2) Low concentrations of §-ALA (0.1-0.5 mM) produced little stimulation of [³H]GABA efflux, less than that produced by similar concentrations of unlabelled GABA; (3) 0.1 mM-§-ALA reduced the stimulation of [³H]GABA efflux elicited by 55 mM-K⁺ and the combination of 1 mM-§-ALA and 55mM-K⁺ produced a lower stimulation of efflux than 1 mM-§-ALA alone; (4) §-ALA inhibits [³H]GABA uptake in a linearly competitive fashion and inhibition is maximal at 0.5 mM-§-ALA. These results are discussed in relation to the neuronal high affinity GABA transport mechanism and inhibition of the synaptosomal Na⁺ and K⁺ -dependent ATPase. It is also postulated that §-ALA increases the chloride conductance of the synaptosomal membrane, possibly by acting on presynaptic GABA receptors.     

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.