High Affinity Uptake Process Research Articles

Effect of guanidinoethane sulfonate on taurine uptake by rat retina.

Guanidinoethane sulfonate (GES) markedly decreased 3H-taurine accumulated in the retina by the high-affinity uptake process. The effect of GES was dose-dependent. Analysis of the kinetics of GES effect revealed that it is a competitive inhibitor. The uptake of taurine by GES was less affected in rat cerebral cortex slices, where the inhibition by 1 mM GES was only 28%. Taurine accumulation by tissues of rats treated with GES (0.1% and 1% in the drinking water) was found to be particularly decreased in the retina, although accumulation by heart and liver was also affected by the higher dose. Taurine uptake by cerebellum and cerebral cortex slices was unaffected by GES. Treatment of rats with GES is known to produce an alteration in the structure and function of the retina, but apparently not in other organs. We discuss whether the marked effect of GES on taurine transport by the retina is related to the deleterious effect of the inhibitor in this organ.

Potassium activation of [3H]-choline accumulation by isolated sympathetic ganglia of the rat.

1 The effect of K-depolarization on the uptake of low and high concentrations of [3H]-choline by isolated superior sympathetic ganglia of the rat has been studied. 2 In unstimulated ganglia, the uptake of [3H]-choline (0.1 microM) ('high affinity uptake') was unaffected by denervation or by hemicholinium-3 (HC-3), suggesting uptake by structures other than cholinergic nerve terminals. 3 K-depolarization of the ganglia increased [3H]-choline accumulation by the high affinity uptake process but in contrast the 'low affinity' accumulation of [3H]-choline (100 microM) was decreased. 4 The K-activated, 'high affinity' component of choline uptake was highly sodium-dependent, inhibited by HC-3, and was abolished by denervation. 5 In incubation conditions designed to prevent transmitter release (Ca-free medium and high-Mg medium), the K-activated uptake of [3H]-choline was abolished. 6 It is concluded that in unstimulated ganglia, there is little choline uptake by nerve terminals. However, when the terminals are depolarized, choline uptake is increased by the activation of a sodium-dependent, HC-3-sensitive transport process. The activation of this uptake process is apparently associated with the release of acetylcholine from the terminals, or by changes in ionic fluxes, and not by the depolarization per se.

Effect of potassium depolarization and preganglionic nerve stimulation on the metabolism of [3H]-choline in rat isolated sympathetic ganglia.

1 The effects of potassium depolarization and preganglionic nerve stimulation on the metabolism of [(3)H]-choline in the isolated superior sympathetic ganglion of the rat have been studied.2 When unstimulated (resting) ganglia were incubated for 10 min with a low concentration (0.1 muM) of [(3)H]-choline (high affinity uptake), approximately 75% of the accumulated radioactivity was present as [(3)H]-phosphorylcholine, 11% was [(3)H]-acetylcholine ([(3)H]-ACh) and the remainder was unchanged [(3)H]-choline.3 Depolarization of the ganglia with K (46 mM) before their incubation with [(3)H]-choline, increased [(3)H]-choline uptake by 70% and increased [(3)H]-ACh synthesis by more than 700%, so that [(3)H]-ACh represented almost 50% of the total radioactivity recovered. In contrast, the proportion of [(3)H]-phosphorylcholine fell to 36% of the total radioactivity recovered.4 The striking effect of K-depolarization on [(3)H]-ACh synthesis in ganglia occurred at a concentration of 30 mM or above, and the maximum effect was seen at 45-50 mM.5 Chronic denervation of the ganglia abolished all the effects of high-K on [(3)H]-choline metabolism. In resting ganglia, [(3)H]-ACh formation was reduced by over 80% but [(3)H]-phosphorylcholine synthesis and the level of unchanged [(3)H]-Ch were not affected by denervation.6 Exposure of the ganglia to low-Na or hemicholinium-3 (HC-3) greatly reduced [(3)H]-ACh synthesis in control resting ganglia and almost abolished the effects of high-K on [(3)H]-ACh synthesis.7 Prevention of transmitter release with high-Mg or low-Ca medium also prevented K-depolarization from stimulating [(3)H]-ACh synthesis.8 Preganglionic nerve stimulation had an effect on [(3)H]-choline metabolism similar to that of K-depolarization. Thus, at all the frequencies studied (1-30 Hz), [(3)H]-ACh synthesis was greatly increased and [(3)H]-phosphorylcholine was reduced, the maximum effects occurring at 3 Hz.9 When ganglia were incubated with a high concentration (100 muM) of [(3)H]-choline (low affinity uptake), a different pattern of metabolism was observed. Most of the radioactivity in resting ganglia was present as unchanged [(3)H]-choline (70%) with [(3)H]-phosphorylcholine and [(3)H]-ACh representing 23% and 6% of the total radioactivity respectively. K-depolarization decreased [(3)H]-choline uptake but increased the proportions of [(3)H]-phosphorylcholine and [(3)H]-ACh to 32% and 24% of the total radioactivity respectively.10 It is concluded that in unstimulated (resting) rat sympathetic ganglia most of the [(3)H]-choline transport and metabolism occurs in postsynaptic structures. However, depolarization of the presynaptic nerve terminals appears to trigger a sodium-dependent, HC-3 sensitive, high-affinity uptake process, and causes a dramatic increase in presynaptic [(3)H]-ACh synthesis together with a fall in postsynaptic [(3)H]-phosphorylcholine synthesis. These changes in choline metabolism cannot be due to the depolarization of the nerve terminals per se, because they were abolished by high-Mg or low-Ca, i.e. when transmitter release was prevented. Thus, the increase in ACh synthesis may be triggered by a fall in the intraterminal concentration of ACh or by the changes in Ca flux induced by depolarization. Our experiments do not provide evidence on these possible mechanisms.

Catecholamine transport in isolated lung parenchyma of pig.

1 Lung parenchyma strips of the pig incubated at 37 degrees C with [(3)H]-(-)-noradrenaline ([(3)H]-NA) or [(3)H]-(+/-)-isoprenaline ([(3)H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K(m)) for [(3)H]-NA and [(3)H]-Iso were 1.34 x 10(-6) M and 1.63 x 10(-6) M respectively, while apparent transport maxima (V(max)) were 4.86 and 1.63 x 10(-9) mol min(-1) g(-1) respectively.2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7 degrees C, pretreatment with ouabain (100 muM), phentolamine (15 muM) or phenoxybenzamine (80 muM). However, accumulation of radioactivity derived from ((3)H]-NA was inhibited selectively by cocaine (10 muM) and desipramine (1 muM), while normetanephrine (80 muM) and 3-O-methylisoprenaline (50 muM) caused much greater reductions in cellular radioactivity from [(3)H]-Iso than from ((3)H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.3 Cocaine (50 muM) which selectively reduced [(3)H]-NA transport, had no significant effect on the sensitivity (EC(50)) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 muM), also failed to alter the potency of NA, while normetanephrine (80 muM) caused a 2 fold decrease in potency.4 Phentolamine (15 muM), which reduced the cellular accumulation of radioactivity derived from [(3)H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 muM) and 3-O-methylisoprenaline (50 muM), which also depressed the accumulation of cellular radioactivity from [(3)H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of beta-adrenoceptor blockade. In sharp contrast, cortisol (80 muM) and U-0521 (60 muM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [(3)H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from alpha-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from beta-adrenoceptors mediating Iso-induced relaxation. However, beta-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

Characterization of high affinity dopamine uptake into the dopamine neurons of the hypothalamus

The study of hypothalamic dopamine (DA) neurons is complicated by the difficulty in distinguishing DA neurons from norepinephrine (NE) neurons and by the fact that they comprise only a small proportion of the catecholamine neuron population of the hypothalamus. We have studied DA uptake into nerve terminals of hypothalamic DA neurons using a synaptosomal preparation. Desmethylimipramine (DMI) was used to prevent uptake into synaptosomes from NE neurons, thus pharmacologically isolating dopaminergic from noradrenergic nerve terminals. This DMI-insensitive DA uptake in hypothalamus had all the properties of a high affinity uptake process; it was saturable, dependent on incubation time and incubation temperature, increased linearly with increasing amounts of tissue and was completely abolished by excess unlabeled DA. Also, it was completely abolished by benztropine, an inhibitor of amine uptake into DA neurons. We believe that DMI-insensitive DA uptake into hypothalamic synaptosomes represents uptake into DA nerve terminals. The DMI-insensitive DA accumulation in discrete areas of hypothalamus correlated well with the known prevalence of DA neurons relative to NE neurons in these areas: median eminence > median eminence-arcuate nucleus > mediobasal hypothalamus > whole hypothalamus. Comparison of the affinity constants for DA uptake into synaptosomes incubated without DMI revealed a 2-fold higher affinity constant for DA uptake in median eminence compared with striatum, but affinity constants in all the other hypothalamic regions examined (mediobasal hypothalamus, hypothalamus minus median eminence, whole hypothalamus) were identical to that of striatum. In contrast, comparison of the affinity constants for DA uptake in the presence of DMI revealed a 2–3-fold higher affinity constant for DA uptake in all these hypothalamic regions compared with striatum. It appears the tuberoinfundibular DA neurons and the other DA neurons of the hypothalamus have a high affinity uptake system for DA, although affinity for DA in all of these hypothalamic DA neurons appears to be 2–3-fold lower than that in striatal DA neurons. The data also suggest that the much larger mediobasal hypothalamus may serve as a model for studies of DA uptake into tuberoinfundibular DA neurons of the median eminence.

Uptake and metabolism of choline by the embryonic heart of the chick in vitro

1. 1. The uptake and metabolism of [ 14C]choline was investigated in isolated hearts from 4- to 14-day-old chick embryos. 2. 2. A high-affinity uptake system of choline was present in hearts from all ages of embryos examined: the K m values were 9–11 μM, while the V max values ranged from 5 (14-day ventricles) to 77 (4-day atria) pmol choline/mg protein/min. 3. 3. Hemicholinium-3, metabolic inhibitors, low temperature and low Na + concentrations reduced the high-affinity uptake process. 4. 4. The accumulated choline was converted mainly into phosphorylcholine in 4-day hearts, while the percentage of conversion into acetylcholine increased in older embryos.

Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte-macrophages

It is commonly assumed that the low density lipoprotein (LDL) degradation in extra-hepatic cells proceeds partly by way of the high-affinity LDL uptake process. In addition, it has been suggested that extra-hepatic LDL catabolism proceeds partly by way of preferential uptake and subsequent degradation of LDL by phagocytic scavenger cells. In order to study the effect of LDL and high density lipoprotein (HDL) on cholesterol metabolism in such scavenger cells, cultured human or pig monocytes were incubated with varying amounts of human or pig LDL, HDL and LDL/HDL mixtures, and the incorporation of [ 14C]acetate into cholesterol was measured. The addition of LDL suppressed endogenous cholesterol synthesis. In contrast, the addition of HDL had no effect on endogenous cholesterol synthesis. Incubation of monocytes with LDL/HDL mixtures reduced the total amount of de novo synthesized cholesterol to the same extent as incubation with LDL alone. Our results suggest that in cultured monocyte-macrophages HDL does not influence endogenous cholesterol synthesis, even in the presence of LDL.

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat.

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release of L-[3H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinity glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process for L-[3H]glutamate resulted in elevation of Vmax with no significant alteration in KT as compared to age-matched controls. Depolarization-induced release of L-[3H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.

Analysis of monoamine accumulations in the neuronal tissues of Mytilus edulis (bivalvia)—II: Pharmacological alteration of pedal ganglia monoamine uptake

1. Lineweaver-Burk plots revealed the presence of two uptake mechanisms for serotonin, dopamine and norepinephrine. The high affinity uptake processes were found to have K m 1 values of 0.281, 2.68 and 3.17 × 10 −6 M and V max 1 values of 0.154, 1.048 and 0.165 nmol/g/min for serotonin, dopamine and norepinerphine, respectively. 2. At low biogenic amine concentrations uptake 1 contributes more than uptake 2 to the total amount of amine accumulated, as determined by the Michaelis-Menten equation, whereas at higher concentrations the reverse is true. 3. 3-Chlorimiprimine was the most potent and select agent found for inhibiting serotonin uptake (ID 50 value 0.5 × 10 −6 M). The same is true of benztropine for inhibiting dopamine uptake (ID 50 value 0.2 × 10 −6 M). 4. DALA ( D-ala 2-met-enkephalinamide) and morphine were found to be inhibitors of monoamine uptake within the concentration range 100–140 μM. 5. The high affinity uptake mechanisms were found to be highly sensitive to Na + omissions from the incubation medium. 6. The monoamines were found to affect each other's uptake. These results suggest the existence of intraganglionic regulatory mechanisms involving neurotransmitter interrelationships.

Characterization of separated cell types from developing rat cerebellum. Transport of [3H]GABA by preparations enriched in Purkinje cells and astrocytes.

The characteristics of [3H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [3H]GABA. In comparison with astrocytes, the Vmax of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10(6) cells) and the apparent Kt twofold greater (2 vs. 1 microM). In contrast to these cell types, uptake was very low in granule cell-enriched preparations. cis-1,3-aminocyclohexane carboxylic acid was a potent inhibitor of [3H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for beta-alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [3H]GABA transport was Na+ dependent in both cell classes. However, veratridine and ouabain selectively blocked [3H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [3H]GABA transport systems and the underlying metabolic processes.

Kinetics of [ 3H]choline uptake in abdominal ganglia of Limulus polyphemus

The uptake of [ 3H]choline by isolated ganglia of the ventral nerve cord of Limulus potyphemus was studied. The uptake process was linear for 90 min and was concentration dependent. Kinetic analysis suggested the existence of a high affinity uptake process ( K t 14 μM and V max 0.19 pmole/mg/min) and a low affinity uptake process ( K t 14 μM and V max 0.89 pmole/mg/min). The high affinity uptake system showed a greater dependence on sodium ions and was more sensitive to inhibition by hemicholinium-3. Neither uptake system was greatly influenced by the absence of calcium or potassium. It is suggested that this system may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.

Subcellular distribution of [ 3H]choline, choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase activities in rabbit retina

In order to obtain further information on the retinal cholinergic system, the uptake and subcellular distribution of [ 3H]choline by the rabbit retina in vitro was examined. Low (6·5 × 10 −8 m) concentrations of choline were used in order to study the high affinity uptake system which appears to be exclusively associated with cholinergic neurotransmitter function in the mature CNS. The subcellular distribution of enzymes of the retinal cholinergic system (choline acetyl-transferase, ChAT; acetylcholinesterase, AChE; and butyrylcholinesterase, BuChE) were also studied and compared with the subcellular distribution of [ 3H]choline. Rabbit retinae rapidly accumulated radioactivity from the external medium and after 45 min incubation, tissue/medium ratios of approximately six were obtained. A preliminary investigation demonstrated that the uptake was temperature dependent and was reduced by about 50% in the absence of sodium ions or the presence of hemicholinium (H.C.3.) (0·1 m m) in the incubation medium. Primary subcellular fractionation of retinae incubated with [ 3H]choline indicated that accumulation of radioactivity by the crude mitochondrial fraction (P2) was reduced if sodium was absent from the incubations. Since the P2 fraction from rabbit retina contains synaptosomes, this result may be indicative of a sodium-dependent high-affinity accumulation of exogenous choline by retinal cholinergic synaptosomes. On continuous, sucrose density gradients more than 80% of the P2 radioactivity appeared to be localized in a population of osmotically-sensitive particles which had a median equilibrium density equivalent to 1·25 m-sucrose. Furthermore, over 85% of the ChAT activity on such gradients was present in particles with the same equilibrium density as those accumulating choline. The primary subcellular distribution of AChE and BuChE showed that both enzymes were present in the total particulate fractions (P1 and P2) although BuChE was a relatively more soluble enzyme than AChE. Both enzymes had a broader distribution than ChAT on density gradients. The likely localization of AChE and BuChE in synaptosome membrane fragments and glial particles respectively may thus point to the ubiquitous nature of such particles on sucrose gradients. The considerable activity of the cholinergic enzymes and the large proportion of [ 3H]choline accumulated by the crude nuclear fraction (P1) may be due to the presence of the large photoreceptor synaptosomes present in this fraction, but more likely to the presence of unbroken tissue fragments. It is concluded that the rabbit retina possesses a sodium-dependent, high affinity uptake process for choline. This process is present in particles which may prove to be retinal cholinergic nerve endings.

Microinjection of kainic acid into the rat hippocampus

The effects of unilateral injection of kainic acid into the rate hippocampus have been examined in terms of morphologic, neurochemical and behavioral sequelae. Infusion of 10 nmoles if kainate causes a rapid and complete degeneration of neuronal perikarya in the entire hippocampal formation followed by gliosis and atrophy of the region. This unilateral neuronal loss is accompanied by a 50% decrease in the specific activity of the biochemical markers for GABAergic neurons including glutamic acid decarboxylase, endogenous GABA and synaptosomal uptake of [ 3H]GABA. The extrinsic hippocampal cholinergic and noradrenergic afferents also exhibit significant alteration. Although the specific activity of choline acetyltransferase is unaffected and the specific activity of tyrosine hydroxylase is significantly increased in the injected hippocampus, the synaptosomal high affinity uptake process for [ 3H]choline and [ 3H]norepinephrine are significantly reduced at 10 days after injection. Whereas the level of endogenous acetylcholine is elevated in the lesioned hippocampus at 2 days after injection, the level of endogenous norepinephrine is reduced. For several hours after intrahippocampal injections of 5 nmoles or more of kainate, rats exhibit epileptiform behavior. Intrahippocampal injection of kainate may be a useful rodent model for temporal lobe seizure disorders.

The uptake, metabolism and release of homocholine: studies with rat brain synaptosomes and cat superior cervical ganglion.

Abstract—The accumulation of [3H]homocholine (3‐trimethylamino‐propan‐1‐01) by isolated synaptosomes prepared from rat brain was resolved kinetically into a high (KT= 3.0 μM) and a low (KT= 14.5 μM) affinity system. Although homocholine was not acetylated by solubilized choline acetyltransferase, 64% of the homocholine accumulated by intact synaptosomes via the high affinity uptake process was acetylated. Homocholine was also acetylated in the superior cervical ganglion of the cat, and the amount of acetylhomocholine formed was increased (12‐fold) by preganglionic nerve stimulation. In ganglia, acetylhomocholine was available for release by preganglionic nerve impulses, and its release was Ca2+‐dependent, It is concluded that homocholine can form a cholinergic false transmitter, and that the substrate specificity of choline acetyltransferase in vitro might be different from that in situ.

The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat

The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat was established owing to the combined use of microdissection techniques and biochemical microassays. The density of 5-HT terminals in various areas of both structures was quantified first by measuring 5-HT levels in microdiscs of frozen tissue. Since the high affinity uptake process for 5-HT appeared undamaged in isotonic homogenates of previously frozen (−5 °C) tissues, it was possible to confirm the findings obtained with the measurement of 5-HT levels by also determining 5-HT uptake activity in these microdiscs. However, in the rat neostriatum, but not in the cat caudate nucleus, [ 3H]5-HT even at a very low extracellular concentration ( 4.4 × 10 −8 M ) was taken up not only by serotoninergic terminals but also to a significant extent by dopaminergic terminals. In presence of benztropine, this second component was suppressed and [ 3H]5-HT uptake activity could then be considered as a specific marker of serotoninergic terminals also in the neostriatum of the rat. In both species, 5-HT terminals were mainly localized in the ventrocaudal area of the structure. In this area, 5-HT levels were among the highest values found in the brain (17 ng/mg protein). The density of 5-HT terminals decreased progressively from the caudal to the rostral planes of the neostriatum in rats or the caudate nucleus in cats. The poorest area, i.e. the dorsorostral zone, contained about 4 times less 5-HT than the ventrocaudal zone of the structure. Electrolytic lesion of the dorsalis (B7) and centralis superior (B8) raphe nuclei during early life resulted in a large decrease of 5-HT levels (−90%) in various parts of the neostriatum of adult rats. The present findings might be of interest to further analyze the role of serotoninergic neurons in extrapyramidal functions.

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 Km 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.

Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain

Ontogenic development of central cholinergic neurons in rat brain was examined by measuring the activity of choline acetyltransferase (CAT), concentration of acetylcholine (ACh) after focused microwave irradiation, the activity of the high affinity uptake process for choline and the apparent muscarinic receptor as quantified by specific binding of [ 3H]3-quinuclidinyl benzilate (QNB). For whole brain, the specific activity of CAT increases from 1 to 8% of adult between 15 days gestation and 7 days postpartum and then increases linearly to 83% by 4 weeks postpartum. The concentration of ACh is 22% of adult at 15 days gestation, rises to 29% by birth and attains adult levels by 4 weeks postpartum. The developmental rise in specific binding of [ 3H]QNB is intermediate between CAT and ACh with 10% of adult concentration of receptor at birth and a linear increase to 90% by 4 weeks postpartum. The development of the uptake of [ 3H]choline parallels that of CAT. In all regions of the neonatal rat brain, the relative level (% adult) of ACh is higher than [ 3H]QNB binding, which is higher than CAT. The neonatal medulla-pons has higher levels of [ 3H]QNB binding and activity of CAT (% adult) and develops more rapidly than the parietal cortex and corpus striatum; the hypothalamus and midbrain-thalamus exhibit intermediate rates of development.

The uptake of [3H] choline by snail (Helix pomatia) nervous tissue in vitro.

Abstract— When suboesophageal ganglia of the snail Helix comalia were incubated at 25°C in a medium containing [3H]choline, tissue: medium ratios of about 14:1 were obtained after 20 min incubation, and only 15°, of the accumulated choline was metabolized to form [3H]acetylcholine. The uptake of [3H]choline showed saturation kinetics and was dependent upon temperature and sodium ions. Kinetic analysis suggested the existence of a high affinity uptake process (Km= 1.7 μM, Vmax= 0.21 nmol/g/min) and a low affinity process (Km= 100 μM, Vmax= 1.2 nmol/g/min). The high affinity uptake differed from the low affinity system in that it was sensitive to various metabolic inhibitors and was competitively inhibited by low concentrations of hemicholinium‐ and acetylcholine. Neither uptake system was greatly influenced by the absence of calcium, potassium or magnesium ions or by the presence of low concentrations of 5‐HT, dopamine. tetrabenazine, chlorpromazine, decamethonium, nalaxone or imipramine. The high affinity uptake process may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.

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.

An evaluation of l-glutamate as the transmitter released from optic nerve terminals of the pigeon

The possibility was investigated that L-glutamic acid is the excitatory transmitter released from the optic nerve terminals of the pigeon optic tectum. (1) Superficial layers of the tectum contained high levels of endogenous glutamate and accumulated L-[3H]glutamate by a high affinity uptake process. (2) Subcellular and autoradiographic studies indicated that 10-30% of the exogenously accumulated L-[3H]glutamate was localized within synaptosomes, and that 11-15% of the synaptosomes had been labelled. (3) The glutamate-accumulating synaptosomes sedimented to the same isopycnic density as pinched-off optic nerve terminals. (4) GABA-and noradrenaline-accumulating synaptosomes were also associated with this subcellular population. (5) Retinal ablation did not change endogenous glutamate concentrations or the high affinity uptake of glutamate. The results are discussed in relation to a possible role for L-glutamate as the 'optic nerve transmitter' and in the context of previous evidence implicating glutamate as an excitatory transmitter.