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.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

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 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.     

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.     

PROPERTIES OF THE UPTAKE AND RELEASE OF GLUTAMIC ACID BY SYNAPTOSOMES FROM RAT CEREBRAL CORTEX

Abstract– (1) The uptake and release of glutamic acid by guinea-pig cerebral cortex slices and rat synaptosomal fractions were studied, comparing the naturally occurring l - and non-natural d -isomers. Negligible metabolism of d -glutamic acid was observed in the slices. (2) Whereas in the cerebral slices the accumulation of glutamic acid was almost the same for the two isomers, d -glutamic acid was accumulated into the synaptosomal fraction at a markedly lower rate than was the L-isomer. (3) The uptake systems for d -isomer into the slices and synaptosomal fraction were found to be of single component, in contrast with the two component systems, high and low affinity components, for the uptake of l -glutamic acid. The apparent K_m values for the uptake of d -glutamic acid into the slices and synaptosomal fraction were comparable with those reported for the low affinity components for l -isomer. The uptake systems for d -glutamic acid were dependent on the presence of Na⁺ ions in the medium, like those for l -glutamic acid and GABA. (4) The evoked release of radioactive preloaded d -glutamic acid was observed both from the slices and synaptosomal fraction following stimulation by high K⁺ ions in the medium. From these observations, it is evident that the evoked release of an amino acid by depolarization in vitro is not necessarily accompanied by a high affinity uptake process. (5) The uptake of l -glutamic acid, expecially into the synaptosomal fraction, was highly resistant to ouabain. On the other hand, the uptake rate of d -glutamic acid and GABA into the synaptosomal fraction was inhibited by varying concentrations of ouabain in accordance with the inhibition for brain Na-K ATPase. (6) The uptake of l -glutamic acid into subfractions of the P₂ fraction was studied in relation to the distribution of the ‘synaptosomal marker enzymes’. An attempt to correlate the activities of enzymes of glutamic acid metabolism with the uptake of l -glutamic acid into the synaptosomal fraction from various parts of brain was unsuccessful. The high affinity uptake of l -glutamic acid was found to be very active in the synaptosomal fraction from any part of brain examined.     

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.     

EVIDENCE FOR THE HETEROGENEITY OF L-2,4-DIAMINOBUTYRIC ACID UPTAKE IN RAT CORTEX

The uptake of L-[³H]DABA by rat cerebral cortex slices was studied. Analysis of the kinetic data obtained provides evidence that DABA entry is mediated by both high and low affinity carriers. When cortical slices were incubated in the presence of equimolar [³H]DABA and [¹⁴C]GABA the ratio of entry of the two radionuclides was found to depend upon the loading concentration. The specificity of the uptake of 1 μM and 1 mM-L-DABA was examined: GABA and DABA were relatively potent inhibitors of 1 μM-DABA uptake whereas an equal concentration of histidine did not produce significant inhibition. In contrast, DABA and histidine were markedly more potent as inhibitors of 1 mM-DABA uptake than was GABA. It is concluded from these experiments that L-DABA is transported into cortical slices by a carrier which has high affinities for both DABA and GABA and by a second lower affinity carrier which prefers DABA as a substrate to GABA. On the basis of a comparison of the effects of inhibitors on [³H]DABA and [³H]GABA uptake it is estimated that approx 26% of DABA uptake at 1 μM does not occur by the high affinity carrier whereas at 1 mM-DABA this proportion rises to 62–67%.     

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.     

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.     

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.     

METABOLITES OF [3H]ACETATE BOUND TO SYNAPTIC VESICLES ISOLATED FROM RAT CEREBRAL CORTEX

Abstract— Synaptic vesicles were isolated from rat cerebral cortex after an intraventricular injection of [³H]acetate. The labelled substances bound to the synaptic vesicles were released by exposure to acid, separated from the vesicle membranes by Sephadex column chromatography and identified by thin-layer chromatography and thin-layer electrophoresis. The three major peaks of radioactivity were glutamate, glutamine and gamma-aminobutyric acid. Their presence in synaptic vesicles is consistent with the concept of an integration of energy metabolism, membrane regulation and synaptic transmission.     

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.     

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.     

THE UPTAKE OF γ-[3H]AMINOBUTYRIC ACID BY SLICES FROM VARIOUS REGIONS OF RAT BRAIN AND THE EFFECT OF LITHIUM

Abstract— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [³H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity ( K_m = 1.2 ± 10^-5 M) while the other has a lower affinity ( K_m = 4 ± 10^-4 M). Studies at low GABA concentration (5 ± 10^-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation.
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.     

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

Abstract— Radioactive acetylcholine ([¹⁴C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[¹⁴C]ACh medium, apparently by a passive diffusion process at concentrations > 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm -[¹⁴C]ACh and about constant, 27%, for the range 5-50mm -[¹⁴C]ACh. All its ACh is released on homogenization with 0.1 n -perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [¹⁴C]ACh. Deprivation in the incubation media of glucose or Na⁺ or Ca²⁺. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na⁺, Ca²⁺ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na⁺ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

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.     

THE RELEASE IN VIVO OF [3H]ACETYLCHOLINE FROM CAT CAUDATE NUCLEUS AND CEREBRAL CORTEX BY ATROPINE, PENTYLENETETRAZOL, K+-DEPOLARIZATION AND ELECTRICAL STIMULATION

Abstract— In the present experiments, the resting and stimulus evoked release of newly synthesized [³H]acetylcholine from the caudate nucleus, the cerebral cortex and the cerebellar cortex into the perfusate of the push-pull cannula was studied in the unanesthetized, midpontine, pretrigeminally transected cat following infusion at the push-pull site of [³H]choline. Separation of the metabolites in the perfusate revealed that after 20 min, approximately 20% of the recovered radioactivity in the sample was in a lipid fraction, about 10% was found to be phosphorylcholine and around 3% was observed to be incorporated into acetylcholine. The rest of the recovered radioactivity remained as choline. Electrical stimulation applied directly to the caudate nucleus, local potassium depolarization, atropine and pentylenetetrazol were all observed to result in a significant and stimulus dependent increase in the levels of [³H]acetyIchoIine, but not [³H]choline or [¹⁴C]urea in the effluent of the push-pull cannula located in the caudate nucleus. A similar release of newly synthesized [³H]acetylcholine was observed following atropine and potassium stimulation in the cerebral but not the cerebellar cortex. The specificity of this evoked increase in the levels of [³H]acetylchoiine is substantiated by obtaining the release with stimuli having different modes of action, by the absence of stimulus evoked changes in the levels of other water-soluble elements found in the perfusate and by the absence of an observable release of [³H]acetylcholine in perfusion experiments involving the cerebellum, a tissue not thought to have strong cholinergic innervation. The percentage increases in release of [³H] acetylcholine over baseline levels evoked by the various methods closely corresponded to those reported in the literature for authentic acetylcholine. This was taken to suggest that the neuronal pools containing endogenous acetylcholine and those containing newly synthesized acetylcholine, if not identical, were disposed to behave in the same manner following the activation of the synapse.