High-affinity Uptake Mechanism Research Articles

Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer

ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high‐affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI‐Q system can import L‐Met, D‐Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI‐Q suggest that some substrates may be able to access the transporter through a solvent accessible tunnel in the cognate binding protein‐transporter complex. These studies suggest that the substrate delivery to MetNI transporter may be more flexible than other ABC transport systems.To test if the MetNI‐Q system transport system uses two mechanisms of substrate delivery, we designed a series of experiments to dissect the individual steps in the transport cycle. As the starting point, we have developed a fluorescent anisotropy assay to measure the binding affinity between the MetQ substrate‐binding protein and the MetNI transporter in the presence of different methionine derivatives. Our preliminary data show that MetNI binds to apo‐MetQ ~10 fold tighter than L‐Met liganded MetQ, and additionally may differ in the presence of D‐Met. This suggests that different methionine derivatives may utilize discrete transport models.

Genome mining and functional genomics for siderophore production in Aspergillus niger.

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.

Role of taurine as a possible transmitter in the thermoregulatory pathways of the rat.

Taurine (10 and 20 micrograms) injected unilaterally into the lateral ventricle of rats caused an increase in core temperature. Bilateral injection of taurine 2.5 and 5 micrograms into the preoptic region of the anterior hypothalamus induced a dose-related hyperthermia: higher doses (10 micrograms) caused hypothermia. Intrahypothalamically taurine-induced hyperthermia was blocked by prior injection of strychnine hydrochloride (5 and 15 micrograms); doses which alone had no effect on core temperature. Of the other inhibitory amino acids injected intrahypothalamically hypotaurine also induced a hyperthermia. GABA (10 micrograms) caused hypothermia; glycine (10 micrograms) had no effect. Potassium (50 mM) stimulated release of radioactivity from superfused slices of anterior hypothalamus prelabelled with [3H]taurine in a calcium-dependent manner. A high affinity uptake mechanism with a Km of 8.5 microM was demonstrated with [3H]taurine into slices of anterior hypothalamus. Taurine may have a neurotransmitter role in the anterior hypothalamus but whether the body temperature effects represent physiological or pharmacological events remains to be established.

Repeated Swim Impairs Serotonin Clearance via a Corticosterone-Sensitive Mechanism: Organic Cation Transporter 3, the Smoking Gun

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with increased extracellular serotonin (5-HT) in limbic brain regions. The mechanism through which this occurs remains unclear. One way could be via HPA axis-dependent impairment of serotonin transporter (SERT) function, the high-affinity uptake mechanism for 5-HT. Consistent with this idea, we found that 5-HT clearance rate in hippocampus was dramatically reduced in mice exposed to repeated swim, a stimulus known to activate the HPA axis. However, this phenomenon also occurred in mice lacking SERT, ruling out SERT as a mechanism. The organic cation transporter 3 (OCT3) is emerging as an important regulator of brain 5-HT. Moreover, corticosterone, which is released upon HPA axis activation, blocks 5-HT uptake by OCT3. Repeated swim produced a persistent elevation in plasma corticosterone, and, consistent with prolonged blockade by corticosterone, we found that OCT3 expression and function were reduced in these mice. Importantly, this effect of repeated swim to reduce 5-HT clearance rate was corticosterone dependent, as evidenced by its absence in adrenalectomized mice, in which plasma corticosterone levels were essentially undetectable. Behaviorally, mice subjected to repeated swim spent less time immobile in the tail suspension test than control mice, but responded similarly to SERT- and norepinephrine transporter-selective antidepressants. Together, these results show that reduced 5-HT clearance following HPA axis activation is likely mediated, at least in part, by the corticosterone-sensitive OCT3, and that drugs developed to selectively target OCT3 (unlike corticosterone) may be candidates for the development of novel antidepressant medications.

Early life LiCl-pilocarpine-induced status epilepticus reduces acutely hippocampal glutamate uptake and Na+/K+ ATPase activity

Status epilepticus-induced hippocampal neuronal loss is mainly associated with excitotoxicity induced by increased levels of extracellular glutamate which is normally neutralized by high-affinity uptake mechanism. The energy source for the glutamate uptake is the electrochemical Na+ gradient maintained by Na+/K+ ATPase pump. In this study, we investigated the effect of early-life-induced status epilepticus on hippocampal Na+/K+ ATPase activity and glutamate uptake. Rat pups 15days old were injected i.p. with LiCl (3mEq/kg) 12–18h prior to s.c. pilocarpine administration (60mg/kg). Hippocampal Na+/K+ ATPase activity and glutamate uptake were evaluated 1.5, 12 and 24h after SE induction. LiCl-pilocarpine-induced SE decreased Na+/K+ ATPase activity and glutamate uptake by 42 and 38%, respectively, 1.5h after SE induction. However, 12 and 24h after SE induction the pump activity and glutamate uptake returned to control levels. SE early in life increased hippocampal number of degenerating neurons in the CA1 subfield and dentate gyrus 24h after SE induction. In conclusion, SE induced early in life causes short-term disruption in hippocampal Na+/K+ ATPase activity and glutamate uptake, which may be related to neuronal death found in CA1 subfield.

Methane oxidation in temperate soils: effects of inorganic N

Additions of inorganic nitrogen (N) to an oak soil with significant potential for methane (CH 4) oxidation resulted in differential reduction in CH 4 oxidation capacity depending on N species added. Nitrate, rather than nitrite or ammonium, proved to be the strongest inhibitor of CH 4 oxidation in oak soil. Both high (CH 4 at 10 μl l −1) and low (CH 4 at 5 ml l −1) affinity CH 4 oxidation in oak soil was completely inhibited at a nitrate concentration similar to that present in an alder soil from the same experimental site. The alder soil showed no capacity for low affinity CH 4 oxidation. A ‘low nitrate’ forest soil (oak) showed high affinity, low capacity CH 4 oxidation upto around 1 ml l −1 CH 4, above which both high and low affinity CH 4 oxidation became apparent following a lag phase, indicating either an induced high affinity uptake mechanism or the existence of distinct low affinity and high affinity methanotroph populations. High affinity CH 4 oxidation became saturated at CH 4 concentrations >500 μl l −1, while low affinity CH 4 oxidation became saturated at ∼30 ml l −1 CH 4. In a ‘high nitrate’ forest soil (alder), CH 4 oxidation appeared to be due to high affinity CH 4 oxidation only and became undetectable at CH 4 concentrations >5 ml l −1.

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system.

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.

The Transport of Choline

ABSTRACTCholine has many physiological functions throughout the body that are dependent on its available local supply. However, since choline is a charged hydrophilic cation, transport mechanisms are required for it to cross biological membranes. Choline transport is required for cellular membrane construction and is the rate-limiting step for acetylcholine production. Transport mechanisms include: (1) sodium-dependent high-affinity uptake mechanism in synaptosomes, (2) sodium-independent low-affinity mechanism on cellular membranes, and (3) unique choline uptake mechanisms (e.g., blood–brain barrier choline transport). A comprehensive overview of choline transport studies is provided. This review article examines landmark and current choline transport studies, molecular mapping, and molecular identification of these carriers. Information regarding the choline-binding site is presented by reviewing choline structural analog (hemicholinium-3 and 15, and other nitrogen/methyl-hydroxyl compounds) inhibition studies. Choline transport in Alzheimer's disease, brain ischemic events, and aging is also discussed. Emphasis throughout the article is placed on targeting the choline transporter in disease and use of this carrier as a drug delivery vector.

The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 µ M [3H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (125I seeds and a clinical 60Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with 125I, 5 µM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous 125I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a 60Co source; and (4) 5 µM hemicholinium significantly depressed the accumulation of tritium in both the control and the 125I-irradiated groups, and there was no longer a difference between 125I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed. [Neurol Res 2001; 23: 669-675]

Phosphorus uptake and translocation in salt-stressed melon plants

Summary Melon seedlings ( Cucumis melo L. cv.Galia) were grown hydroponically to study the effect of salinity (80 mmol/LNaCl) on phosphate (Pi) uptake and translocation at two levels of Pi (25 μmol/L and 1 mmol/L). Net uptake rates of Pi were determined by depletionof the medium and by plant content. Salinity decreased Pi uptake at low Pi (high affinity uptake mechanism), 25 μmol/L, although no specific competitive inhibition of Pi uptake by Cl − was observed. When plants were grown with high Pi (1 mmol/L), the uptake of Pi through the low affinity system was increased by 80 mmol/L NaCl. Salinity also reduced the phosphorus flux, as Pi, through the xylem. It is hypothesised that high levels of NaCl decrease the mobility of Pi stored in vacuoles, and as a result, inhibit export from this storage compartment to other parts of the plant.

Salt and Water Relations, and Nitrogen Excretion, in the Amphibious African Freshwater Crab Potamonautes Warreni in Water and in Air

ABSTRACT Mechanisms of salt and water conservation, and nitrogen excretion, were investigated in the freshwater amphibious crab Potamonautes warreni from the High Veld of South Africa. Adaptations to fresh water were assessed as pre-adaptations to air-breathing, and nitrogen excretion was examined as a potential constraint to terrestrial excursions. P. warreni was able to regulate water and salt loss in water up to 40 % sea water, but not in 80 % sea water. The water permeability of P. warreni was low and, since 97 % of the haemolymph filtrate was reabsorbed in the antennal organ, urinary water loss was minimal (0.7 μl g−1 h−1). The minimum equilibrium [Na] of P. warreni was low (0.116 mmol l−1), as were the rates of both Na loss (0.22 μmol g−1 h−1) and Ca loss (0.29 μmol g−1 h−1). The low loss rates were due to urinary salt conservation of approximately 90 % or better and to low permeability , and were compensated for by a high-affinity uptake mechanism (Jmax=0.76 μmol g−1 h−1 and Km=0.18 mmol l−1). Acclimating P. warreni to low Na concentrations increased maximum net Na uptake rate to 1.77 μmol g−1 h−1. Nitrogen excretion in P. warreni was almost 100 % ammoniotelic, and there was no accumulation of haemolymph or urinary ammonia or urea when in air. P. warreni was unable to excrete ammonia to air, but in water the rate of excretion was nearly 70 μmol kg−1 h−1. Crabs in amphibious conditions showed pulses of elevated NH3 excretion (350 μmol kg−1 h−1) when subsequently submerged, while for crabs breathing air for 3 days this pulse reached 4.9 mmol kg−1 h−1. Air-breathing P. warreni with artificially irrigated branchial chambers excreted double the amount of NH3via the gills compared with crabs from amphibious conditions. Water and salt conservation form useful pre-adaptations to terrestrial forays. While the relatively low water loss extends the duration of emersion, P. warreni is required to return briefly to water to excrete stored nitrogenous waste. The nature of the store remains to be determined.

Foetal ventral mesencephalic cell suspension grafts to the 6-hydroxydopamine-lesioned rat reduce the rate of dopamine uptake in the contralateral striatum

The present study employed fast cyclic voltammetry, at carbon-fibre microelectrodes, to monitor and compare the rate of dopamine uptake in the rat striatum contralateral to (a) the 6-hydroxydopamine (6-OHDA)-lesioned/grafted striatum and (b) the 6-OHDA-lesioned/sham grafted striatum. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted striatum of unilaterally 6-OHDA-lesioned rats. Six weeks after grafting, animals with functional, mature grafts were monitored for dopamine elimination in the contralateral striatum following electrical stimulation of the median forebrain bundle, before and after treatment with the dopamine uptake inhibitor GBR 12909. Compared to animals with sham grafts, amphetamine-amplified rotational behaviour was significantly reduced in animals with grafts of foetal ventral mesencephalic tissue. Fast cyclic voltammeric measurements followed by evaluation with the aid of a kinetic model revealed that in grafted animals, the rate of dopamine uptake via the high affinity uptake mechanism, following treatment with GBR 12909, was significantly reduced when compared to sham grafted animals.

Occurrence and characterization of a low-affinity choline uptake mechanism in the internal tissues of the cestode Hymenolepis diminuta

[3H]Choline was taken up by tissue slices of the cestode Hymenolepis diminuta by sodium-dependent and sodium-independent mechanisms. The sodium-dependent uptake was saturable, against an apparent concentration gradient, and by analysis of the kinetics of uptake could be delineated into a high-affinity choline uptake (HAChU) mechanism (Kt of approximately 2.0 microM; Vmax of approximately 0.15 pmol/mg wet weight tissue/min), and a low-affinity choline uptake (LAChU) mechanism (Kt of approximately 20.0 microM and a Vmax of approximately 2.0 pmol/mg wet weight tissue/min). Unlike the HAChU system, the LAChU system was unaffected by potassium. Furthermore, the responses of the LAChU and the HAChU transporters to pharmacological agents were distinctive, indicating that these transporters are two separate entities. Moreover, the entry or exit of choline via the LACh transporter was dependent upon the direction of the sodium gradient. Furthermore, the majority of released radiolabel from preloaded [3H]choline was associated with choline and was via a proteinaceous transporter. The present study is the first to provide direct evidence for a partial chemosmotic coupling of a LAChU system to the sodium electrochemical gradient.

Colocalization of glycine in substance P-amacrine cells of the larval tiger salamander retina.

The present study was performed as part of a systematic examination of glycine's coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of glycine high-affinity uptake to examine whether tiger salamander substance P-amacrine cells express these glycine markers. Double-label analyses revealed two populations of substance P-amacrine cells that express glycine immunoreactivity and glycine high-affinity uptake. The large majority of double-labeled cells were situated in the innermost cell row of the inner nuclear layer, while a smaller number were located in the inner nuclear layer in the second cell row distal to the inner plexiform layer. Double-label immunocytochemistry revealed that these double-labeled cells accounted for 91.7% of substance P-immunoreactive amacrine cells. A slightly lower percentage (90.1%) of substance P-amacrine cells were found to exhibit a glycine high-affinity uptake mechanism. Substance P-amacrine cells that did not co-label for markers of glycine activity were situated in the innermost cell row of the inner nuclear layer. Substance P-immunoreactive displaced amacrine cells were not observed to co-label for either glycine immunoreactivity or glycine high-affinity uptake. The present study reveals that the large majority of substance P-amacrine cells in the larval tiger salamander retina co-express markers of glycine activity. This finding suggests a functional diversity in the population of tiger salamander substance P-amacrine cells relative to their coexisting relationship with a major inhibitory neurotransmitter.

Stereospecificity of High-and Low-Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.

Characterization of a high-affinity choline uptake mechanism in the cestode Hymenolepis diminuta

Radiolabelled choline was taken up by tissue slices of the cestode Hymenolepis diminuta by a sodium-dependent and a sodium-independent mechanism. The sodium-dependent uptake was saturable, against a concentration gradient, displayed structural specificity, and was inhibited, in part, by hemicholinium-3. Kinetic analysis of the sodium-dependent choline uptake showed an apparent Kt = 2.0 microM and a Vmax = 0.146 pmol.mg-1 wet weight tissue.min-1, which is consistent with a high-affinity choline uptake (HAChU) mechanism. The rate of uptake or release of choline depended on the magnitude and direction of the sodium gradient, was diminished by high- or low-potassium, but was not chloride or sulphate dependent. A homoexchange mechanism for HAChU was not demonstrated. Evidence was obtained to suggest that HAChU or release of endogenous ACh is regulated by autoreceptors. The choline taken up by the HAChU mechanism was but slowly converted to ACh and other products.

Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurons.

We have measured [3H]dopamine ([3H]DA) uptake and tyrosine hydroxylase-immunopositive immunostaining in cells acutely dissociated from the embryonic ventral mesencephalon (MSC). DA and its metabolites as well as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) activities were determined in homogenates taken from the MSC and striatum (STR). In the embryonic ventral MSC measurable DA and tyrosine hydroxylase (TH) immunostaining were present as early as embryonic day (E) 12.5. At E14 the number of TH+ neurons was about 50% of the values at E18. In the MSC, DA concentration increased sharply at E16 and reached a plateau before birth that was 10-fold lower than adult values. In the STR, DA was first detected at E16, suggesting that DA fibers reach the STR at this embryonic stage. High-affinity DA uptake appeared in the MSC only at E16, concomitantly with the arrival of DA fibers in the STR, increased sharply between E16 and E18, and reached a plateau before birth. This uptake mechanism was not selective for catecholamine uptake inhibitors. Thus, DA synthesis in the MSC preceded the onset of high-affinity uptake mechanism, which could be correlated to the beginning of striatal DA innervation. Measurable MAO and COMT activities were detected as early as E13 (MSC) and E15 (STR), but not DA metabolites, which appeared later. We conclude that the high-affinity DA uptake mechanism in MSC DA neurons develops coincident with the arrival of DA fibers to the STR. The sharp increase of DA uptake between E16 and E18 is due only in part to an increase in the number of TH+ cells. These results support the hypothesis that in vivo the target STR neurons regulate the maturation of MSC DA cells.

Memory-improving action of glucose: indirect evidence for a facilitation of hippocampal acetylcholine synthesis

The effect of a 3 g/kg glucose injection on the velocity of the sodium-dependent high-affinity choline uptake mechanism in the hippocampus was both measured in quiet control mice and in mice immediately after training in an operant bar pressing task. Glucose did not significantly change high-affinity choline uptake in resting animals. High-affinity choline uptake in the hippocampus was increased by training in the operant bar pressing task. Glucose significantly reduced the amplitude of the increase in high-affinity choline uptake observed in the trained animals. Similarly, a 3 g/kg glucose injection also attenuated the increase in high-affinity choline uptake observed in animals injected with 1 mg/kg scopolamine. Finally, a 3 g/kg glucose injection significantly attenuated the amnesia produced by a post-training 1 mg/kg scopolamine injection in mice trained for an operant bar pressing task. These results provide additional evidence for an action of glucose on hippocampal cholinergic activity under conditions of high acetylcholine demand. This action may be mediated via an increase in acetyl coenzyme A availability, one of the precursors of acetylcholine. This facilitative effect of glucose on hippocampal acetylcholine synthesis may constitute the physiological basis for its facilitative action on memory and its attenuation of scopolamine amnesia.

Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine

A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.

Colocalization of NGF receptor immunoreactivity and [ 3H]GABA uptake activity in developing rat septum/diagonal band neurons in vitro

Neurons in the septum (SEP) and vertical limb of the diagonal band (vDB) of 6-day-old rats were dissociated and cultured for 3 days. Cells with high affinity uptake mechanism for γ-aminobutyric acid (GABA) were identified autoradiographically using 16 nM [ 3H]GABA. Cells that expressed nerve growth factor (NGF) receptor immunoreactivity were identified immunocytochemically using MC192, a monoclonal antibody against NGF receptor. Double labeling experiments combining [ 3H]GABA uptake with immunostaining for NGF receptor showed that 46% of the NGF receptor-immunoreactive cells accumulated [ 3H]GABA. The result was discussed in relation to a possible involvement of the developing septal GABAergic neurons in NGF-induced neuronal survival.