Choline For Acetylcholine Synthesis Research Articles

Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V max of choline uptake, but had no effect on K m for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.

648 Brain magnetic resonance spectroscopy (MRS) in Alzheimer's disease: Changes after treatment with xanomeline, an M1 selective cholinergic agonist

Objective: Higher than normal cellular levels of the phospholipid catabolic intermediate glycerophosphocholine have been found in postmortem brain tissue of persons with Alzheimer's disease. Proton magnetic resonance spectroscopy ('H-MRS) can detect a choline resonance that is largely due to glycerophosphocholine. The authors tested the hypothesis that treatment with xanomeline, an M 1 selective muscarinic cholinergic agonist, would be associated with a decrease in the 1 H-MRS choline resonance. Method: Patients with mild to moderate Alzheimer's disease received placebo or xanomeline for 6 months. 1 H-MRS spectra were collected at baseline and after treatment discontinuation for 12 patients, two taking placebo and 10 taking xanomeline at a dose of 25 mg t.i.d. (N=4), 50 mg t.i.d. (N=3), or 75 mg t.i.d. (N=3). Results: For the combined group of patients taking xanomeline, there was a significant decrease in the choline/creatine ratio from baseline to endpoint. Conclusions: Treatment of Alzheimer's disease with a cholinergic agonist is associated with a decrease in the MRS choline resonance. Xanomeline may reduce breakdown of cholinergic neuron membranes by reducing the cellular requirement for free choline for acetylcholine synthesis.

Synaptosomal phospholipase D potential role in providing choline for acetylcholine synthesis.

The phospholipase D of the rat brain synaptic membrane possesses the highest activity of this enzyme of any mammalian tissue examined. The synaptic phospholipase D activity is latent and barely detectable in the absence of 4 mM sodium oleate. Several other fatty acids were either less effective or ineffective as stimulators of activity compared to this monounsaturated fatty acid. The activity was decreased by hemicholinium-3, an inhibitor of choline uptake and slightly activated by neostigmine, an acetylcholinesterase inhibitor. Incubation of synaptosomes in the presence of sodium oleate and acetyl-coenzyme A resulted in the formation of a product chromatographing with acetylcholine. Acetylcholine formation was nearly undetectable in the absence of sodium oleate or acetyl-coenzyme A. These results implicate synaptosomal phospholipase D in releasing choline from phosphatidylcholine for acetylcholine formation.

Cytidine(5')diphosphocholine enhances the ability of haloperidol to increase dopamine metabolites in the striatum of the rat and to diminish stereotyped behavior induced by apomorphine

Experiments were performed to determine whether exogenous cytidine(5')diphosphocholine (CDP-choline) could modify release of dopamine in the striatum and behavior dependent on dopamine, perhaps by providing supplemental choline for synthesis of acetylcholine. Rats received water (control) or CDP-choline orally (100 mg/kg per day, for 5 days), either alone or before injection with haloperidol (1 mg/kg i.p.), apomorphine (0.15 mg/kg s.c.), or both. Stereotyped behavior was measured during the hour after administation of apomorphine; levels of the dopamine metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum were assessed at the end of this period (2 hr after administration of haloperidol). In rats receiving the CDP-choline, the stereotyped behavior observed after injection of apomorphine alone ( P < 0.01), or after haloperidol plus apomorphine ( P < 0.01), was attenuated. The pretreatment with CDP-choline also significantly increased levels of HVA (by 24%) and DOPAC (by 23%) in the striatum over appropriate controls in animals receiving haloperidol, or by 29 and 59% (averaging data for all time points), respectively, in animals receiving haloperidol plus apomorphine. One mechanism by which CDP-choline may affect behavior involves contributing choline to enhance synthesis of acetylcholine.

Uptake of neurotransmitters and precursors by clonal lines of astrocytoma and neuroblastoma: III. Transport of choline

Clonal lines of glial, neuronal, and nonneural origin accumulate choline via a high-affinity carrier-mediated transport system withK m in the range of 10-14 μM. These cell lines also accumulate choline by a second system that is not saturable at 10 mM choline, and that may represent diffusion. The transport of choline in glial cells differs from that seen in neuronal cells with respect to its Na(+) requirement. The omission of Na(+) from the incubation medium reduces high-affinity choline transport in neuronal cells and enhances it in glial cells. Kinetic analysis of the data indicates that reversible cholinesterase inhibitors and hemicholinium-3 (HC-3) inhibit the high-affinity transport system for choline. On the other hand, the diffusional or low-affinity component of choline transport in either cell type appears to have no Na(+) requirement and is unaffected by either cholinesterase inhibitors or 10(-4) M HC-3. The neuronal-glial differences in the Na(+) requirement of choline transport may be related to the coupling of transport to choline metabolism, which differs in the two cell types. The presence of a high-affinity transport system for choline in clonal glial lines used as models of normal glia suggest that glia may modulate the availability of choline for acetylcholine synthesis at cholinergic synapses.

Choline Metabolism in Discrete Areas of the Brain: the Effect of Hemicholinium-3

When [Me-14C]choline is injected into the brain of the rat it is rapidly phosphorylated to give phosphorylcholine; subsequently the radioactivity appears in the phosphatidylcholine (Ansell & Spanner, 1968). The incorporation into discrete areas of the brain has now been investigated, together with the effect of injecting the labelled choline at different depths from the surface. The object of this was to study the effect of hemicholinium-3, which interferes with the transport and utilization of choline for acetylcholine synthesis. Adult female rat brains were dissected as described by Glowinski & Iversen (1966), and in Table 1 the specific activities of acetylcholinesterase (EC 3.1.1.7) and choline kinase (EC 2.7.1.32) and the concentrations of total phospholipid, phosphatidylcholine and phosphorylcholine are given. Acetylcholinesterase activity was unevenly distributed (cf. cerebellum and striatum) and paralleled acetylcholine distribution (Schmidt et al., 1972), but choline kinase activity, largely present in areas rich in neurons (McCaman, 1962), was, like the phosphorylcholine, evenly distributed (Table 1). When [Me-14C]choline (1 pCi; 0.016pmol) was injected into the region of the lateral ventricle there was an uneven distribution of radioactivity between the six regions of the brain. A distribution of the same order was found for [3H]noradrenaline by Carr & Moore (1969). Of the radioactivity recovered in the brain, 67 % was distributed between the hypothalamus and the midbrain, but there was less than 0.3 % in the striatum. It was therefore decided to vary the depth of injection to test whether this uneven distribution was merely a reflection of the site of injection or was, in fact, a preferential uptake. The choline was injected at the same point of entry as before but the distance from the surface varied from 1.5 to 4mm. After an exchange time of 0.5h the total radioactivity in the brain was slightly higher after the shallower injections than after those at 4mm. The distribution of radioactivity among the regions was very different from that found after the injections at 4mm. For example, after the injection at 1.5mm the percentage of the recovered activity in the hypothalamus was only 8 % compared with 29 % after that at 4mm. However, the cortex contained 35 % compared with 14% after the injection at 4mm. It is noteworthy that the percentage distribution in the midbrain was almost unaffected by the depth of injection. It had been found previously (Ansell & Spanner, 1968) that there was a very rapid phosphorylation of choline after intraventricular injection. This was borne out when the individual regions were examined, each region showing a high percentage of labelling in the phosphorylcholine after 0.5-4.0h of exchange. However, when the labelled choline was injected into the brain tissue above the ventricle there was a very considerable depression of the phosphorylation, i.e. to an average of 15 % after the injection at 1 Smm and of 19.5 % after the injection at 3.0mm compared with over 75 % after that at 4mm. The distribution of choline kinase activity when measured in vitro showed little variation from region to region (Table l), so that the reason for this discrepency has yet to be explained. The ratio of the specific radioactivity of phosphatidylcholine to that of phosphorylcholine in vivo was unaffected. The transfer of the labelled choline and its metabolites from the injected to the uninjected side of the brain was also examined after different depths of injection. The steep gradient found by Carr & Moore (1969) with [3H]noradrenaline and by Fibiger et al. (1972) with ~-[U-'~C]leucine was not so marked with [Me-14C]choline. The side of injection contained 70 % of the isotopic label compared with 30 % in the uninjected side after 1-4h of exchange. This ratio was unaffected by the depth of injection. The cortex, striatum and midbrain showed the highest gradient. The medulla and hypothalamus contained the same percentage of radioactivity on both sides, whereas the cerebellum