High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Modulation of synaptosomal high affinity choline transport.

Depolarization of synaptosomes produced by incubation in 35mMK⁺ Krebs Ringer phosphate buffer results in an increased Vmax and no change in K_T of the high affinity transport of [³H]-choline as determined upon re-incubation in normal K⁺ Krebs Ringer phosphate buffer. The high K⁺ induced increase in the uptake of choline appears to be independent of transmitter release. The K⁺ stimulated increase in the Vmax of the high affinity transport of choline is totally blocked by high, 11mM, Mg⁺². The proportion of choline converted to acetylcholine in synaptosomes previously depolarized is the same as those incubated in normal K⁺ Krebs Ringer; thus the absolute rate of acetylcholine synthesis in nerve terminals is increased as a result of prior depolarization.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Age- and Sex-Dependent Effects of Ethanol on Hippocampal Hemicholinium-3 Sensitive Choline Carriers During Postnatal Development of Rats

Vulnerability of hippocampal hemicholinium-3 (HC-3)-sensitive carriers to ethanol was evaluated in vitro during rat postnatal development. The high-affinity uptake of [³H]choline (HACU) and the specific binding of [³H]HC-3 were measured on synaptosomes from 7-, 14-, and 60-day- and 3-month-old male and female Wistar rats. Marked increases of basal (between 7 and 60 days of age) and of stimulated HACU levels via K⁺-depolarization (between 14 days and 3 months) but only a mild elevation in [³H]HC-3 binding (between 7 days and 3 months) associated with alterations in the binding site number were found. On the mature tissue, ethanol at high concentrations (5%) moderately inhibited the choline transport under basal conditions but totally eliminated depolarization effects. However, both age- and sex-dependent alterations in basal HACU mediated by high or low pharmacologically relevant alcohol concentrations (50–100 mM) were observed in the immature tissue. Namely, the dose- and incubation time–dependent inhibition of HACU associated with changes in the transport velocity was found in postnatal male but not female tissue. [³H]HC-3 binding site was not markedly sensitive to ethanol actions. Anisotropy measurements in the region of the hydrophilic heads of phospholipid bilayers and in the membrane hydrocarbon core indicated penetration of 100 mM ethanol to immature female but not male tissue. Our results suggest the noncompetitive binding of alcohol to choline carriers from immature male tissue and correspond with data reporting significant sexual dimorphism of postnatal hippocampal neurons. The direct effects of ethanol on male choline carriers can contribute to the inhibition of acetylcholine synthesis and to sex-dependent neurotoxic effects of alcohol applied in vivo during early and late postnatal period.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

Ethanol Alters Glutamate but Not Adenosine Uptake in Rat Astrocytes: Evidence for Protein Kinase C Involvement

Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [³H]glutamate uptake and inhibits [³H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [³H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [³H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [³H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [³H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [³H]glutamate uptake but not [³H]adenosine uptake by affecting PKC modulation of transporter activity.     

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

The effect of lidocaine on [³H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3·2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0·0005) or 55 per cent (n = 10; p < 0·0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [³H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until ³H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.     

Acetylation and phosphorylation of choline following high or low affinity uptake by rat cortical synaptosomes

Synaptosomal acetylcholine synthesis was found to be dependent on the presence of Na⁺-dependent HC-3 sensitive choline transport at low (5.5 mM) and high (35 mM) K⁺ concentrations. However, at 5, 20, and 100 M choline, choline phosphorylation was proportional to total choline uptake, in the presence or absence of high affinity transport. Only in the presence of eserine (50 M) did acetylcholine synthesis increase as the choline concentration was elevated from 20 M to 100 M, and this effect was observed at low and high K⁺ concentrations. Our results suggest that: 1) the synthesis of non-surplus synaptosomal ACh is dependent on high affinity choline transport; and 2) choline is equally likely to be phosphorylated after being taken up by low or high affinity transport.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

Stimulation of phosphatidylcholine biosynthesis by hemicholinium-3, a potent inhibitor of choline uptake in human leukemic monocyte-like U937 cells

The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [³H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [³H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [³H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.     

Assessment of Tracer 99mTc(V)-DMSA Uptake as a Measure of Tumor Cell Proliferation In Vitro

Purpose

To examine whether ^99mTc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation.

Methods

Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with ^99mTc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-³H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.

Results

In all cell lines rates of ^99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-³H]thymidine incorporation and expression of PCNA and Ki-67.

Conclusion

Rates of ^99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline.

The uptake and metabolism of [14C]choline in dissociated rat brain embryo cell cultures was examined as a function of the extracellular choline concentration. Choline uptake did not follow normal Michaelis-Menten kinetics, but rather exhibited two components with apparent Km of 0.016 mM and 0.96 mM. At low choline concentrations (high affinity uptake) most of the [14C]choline label was present in the phosphocholine fraction prior to the appearance of label in phospholipids. At high choline concentrations (low affinity uptake) a large proportion of the radioactivity was converted into acetylcholine. The dissimilarities between the formation of phosphocholine and acetylcholine as a function of choline concentration might be explained by the existence of two mutually independent enzymatic activities with different Km affinities for choline. Kinetic data augmented by double label studies, suggested that formation of choline phosphoglyceride proceeds entirely via a phosphocholine intermediate. Nearly all radioactivity in the lipid fraction is incorporated into choline phosphoglycerides. A higher turnover rate of choline incorporation into choline phosphoglycerides, accompanied by an increase in the levels of glycerophosphocholine, was observed in older cultures as compared to younger cultures. The metabolic implications of these findings in cultured brain cells in comparison with other in vitro systems are discussed.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

Effects of K+-Depolarization,Arachidonic Acid,Ethanol, and Aging on the High-Affinity Choline Transport in Rat Hippocampus

The Na⁺-dependent high-affinity choline uptake (HACU) transport and the [³H]hemicholinium-3 ([³H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3–6 months) and old (22 months) Wistar rats. In vitro effects of 100–300 M arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [³H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K⁺-depolarization effect on HACU and the decreased influence of AA on [³H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

A vinblastine sensitive high affinity choline uptake system

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 microM vinblastine, respectively. 2. In contrast, high affinity uptake of choline in the Limulus corpora pedunculata and abdominal ganglia, tissues in which a cholinergic function has been described, were unaffected. 3. In pulse-chase experiments, the cardiac ganglion was incubated in 0.1 microM [3H]choline for 60 min and then switched to an incubation medium containing 1 mM unlabelled choline for varying periods of time. 4. Under these conditions, a 3-fold increase of radiolabel above basal level was measured in the pellet fraction within 2 hr of post-labelling incubation. 5. Prior exposure of the ganglion to 500 microM vinblastine completely eliminated this increase of radioactivity in the pellet fraction. 6. Treatment of the radiolabelled pellet fraction with phospholipase C resulted in the solubilization of 72% of the radiolabel. 7. Ten (10) microM 5-hydroxytryptamine (5-HT), a concentration previously shown to inhibit spontaneous electrical activity within the cardiac ganglion, resulted in a 40% decrease in high affinity choline uptake in this tissue selectively. 8. These results are consistent with the view that a probable role of the Limulus cardiac ganglion HAChUS is the supply of choline subserving the synthesis of membrane phospholipid. 9. It is further speculated that this membrane phospholipid synthesis may be associated with synaptic vesicle turnover.