Tetanus toxin effect on the metabolism and release of acetylcholine in rat central nervous system

The effect of tetanus toxin in doses of 30 mcg/kg on the content, synthesis and release of acetylcholine, and on the activity of choline acetylase and acetylcholine esterase in the central nervous system of the rat was studied. The investigations were carried out after the appearance of tetanus. We found that the tetanus toxin: a) caused no changes in the acetylcholine content in the cerebral cortex and brain stem, and also in the cervical and lumbar parts of the spinal cord; b) stimulated acetylcholine synthesis in the brain stem and in the cervical and lumbar parts of the spinal cord but not in the cerebral cortex; c) activated choline acetylase; d) had no effect on acetylcholine esterase activity; e) released acetylcholine from the neurons in the brain stem and spinal cord. The release could not be inhibited by low concentration of potassium ions in the medium or increased with electrical stimulation.     

The resting release of acetylcholine by a retinal neuron

The cholinergic amacrine cells of the rabbit retina secrete acetylcholine by two mechanisms. One is activated by stimulation of the retina by light or depolarization of the amacrine cells by K+ ions. It requires the presence of extracellular Ca2+. The second is independent of extracellular Ca2+ and is unaffected by large depolarizations of the cells. It bears some similarity to the acetylcholine 'leakage' described at the neuromuscular junction. Although the Ca2+-independent mechanism accounts for about two thirds of the total acetylcholine release in the dark, the amount of acetylcholine released in this way is small compared with the release of acetylcholine triggered by stimulation of the retina with light. Its biological significance is unclear.     

Drug Effects on the Release of Endogenous Acetylcholine In Vivo: Measurement by Intracerebral Dialysis and Gas Chromatography–Mass Spectrometry

Intracerebral microdialysis was combined with a sensitive and specific gas chromatographic-mass spectrometric assay to measure the release of endogenous acetylcholine in the rat striatum in vivo. In rats anesthetized with urethane (1.2 g/kg i.p.), the levels of striatal acetylcholine dialyzed into a Ringer's perfusate were: (a) reliably measurable only in the presence of physostigmine; (b) stable at between 3 and 8 h of perfusion (30-75 pmol/20 min in the presence of 75 microM physostigmine); (c) reduced by calcium-free Ringer's solution, tetrodotoxin (0.1 microM), and vesamicol (1.0 microM); and (d) increased by elevated potassium (100 mM), atropine (3-300 microM), and haloperidol (0.75 mg/kg i.p.). In conscious unrestrained rats, the spontaneous release of striatal acetylcholine was not altered significantly following the administration of urethane. The changes in acetylcholine release observed in this study are consistent with the known actions of some drugs or ionic conditions on striatal cholinergic neurotransmission and are evident under the condition of urethane anesthesia. The present results demonstrate the sensitivity and suitability of this method for monitoring endogenous striatal acetylcholine release in vivo.     

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).     

Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants

Spontaneous and potassium-induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium-induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion-dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.     

Disulfide bonds in acetylcholine receptors of frog sympathetic ganglion neurons

Changes in responses of frog sympathetic ganglion neurons to perfusion with cholinomimetics were studied during modification of acetylcholine receptors by dithiothreitol and ferricyanide. Perfusion with dithiothreitol suppressed responses to carbachol, suberyldicholine, and 5-methylfurmethide, whereas subsequent perfusion with ferricyanide partly restored responses to suberyldicholine but suppressed responses to 5-methylfurmethide. Acetylcholine and tetramethylammonium, used as protectors, protected nicotinic and muscarinic receptors against the action of dithiothreitol, but acetylcholine was more effective than tetramethylammonium for nicotinic acetylcholine receptors. It is suggested that disulfide bonds, some of them located in the anionic centers of the receptors, are present in the recognition sites of acetylcholine receptors of the frog sympathetic ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 593–600, November–December, 1979.     

Acetylcholine-induced responses in the salivary gland cells ofHelisoma trivolvis

This study describes the actions of acetylcholine (ACh) on the salivary gland cells of Helisoma. Perfusion of the salivary gland cells with ACh produces a long-lasting depolarization accompanied by an increase in the input conductance of the gland cells. The depolarization is often followed by a long-lasting hyperpolarization. Carbamylcholine, tetramethylammonium, and choline also produce depolarizing responses. Nicotine and pilocarpine produce only a small depolarization in the gland cells. The following cholinergic antagonists are effective in blocking the gland-cell response to ACh: tetraethylammonium, atropine, hexamethonium, d-tubocurarine, and strychnine. A new preparation, the "isolated acinus," was utilized to obtain the reversal potential of the ACh response. The mean reversal potential in 10 preparations was -7 +/- 8 mV. The depolarizing phase of the response is dependent on the presence of both external calcium and external sodium ions. The long-lasting hyperpolarization is produced by the activity of an electrogenic sodium-potassium pump. The properties of the acetylcholine receptors on the salivary gland cells of Helisoma are compared with those described in other gastropod preparations.     

Aging Decreases the Sensitivity of Rat Cortical Synaptosomes to Calcium Ionophore-Induced Acetylcholine Release

The capacity of calcium ions to trigger acetylcholine release was studied in cerebral cortical synaptosomes from adult (6-month-old) and senescent (24-month-old) rats, using a calcium ionophore, A23187, that bypasses voltage-sensitive calcium channels. The potency but not the efficacy of the A23187 was reduced with respect to releasing acetylcholine (ACh) in the aged animals. There was no age-related difference in the synthesis of ACh or potency of the ionophore with respect to increasing 45calcium uptake. These results suggest that aging reduces the sensitivity of cerebral cortical nerve terminals to calcium-triggered ACh-release.     

Effects of Physostigmine and Some Nitric Oxide-Cyclic GMP-Related Compounds on Muscarinic Receptor-Mediated Autoinhibition of Hippocampal Acetylcholine Release

Abstract: We have investigated the effects of (a) the cholinesterase inhibitor physostigmine and (b) drugs that are known to change intracellular cyclic GMP levels on the autoinhibition of acetylcholine release from rat hippocampal slices. Autoinhibition was triggered by submaximal electrical stimulation in both the absence and presence of physostigmine. The results obtained indicate that an unusual increase in the extracellular acetylcholine content, such as that induced by cholinesterase inhibition, is not essential for autoinhibition triggering. Dibutyryl cyclic GMP reduced significantly the stimulation-evoked acetylcholine release in the presence, but not in the absence, of atropine. Neither sodium nitroprusside nor glyceryl trinitrate exerted a dibutyryl cyclic GMP-like effect. N ^G-Nitro-L-arginine did not lessen the autoinhibition. These results indicate that an increase in the intracellular cyclic GMP level reduces acetylcholine release, and that the muscarinic receptor stimulation-nitric oxide synthesis-(soluble) guanylyl cyclase activation pathway is not involved in the cholinergic autoinhibition process.     

Suppression of In Vivo Neostriatal Acetylcholine Release by Vesamicol: Evidence for a Functional Role of Vesamicol Receptors in Brain

Experiments examined the effects of peripheral and central administration of the vesicular acetylcholine transport blocker vesamicol (AH5183) on the content, synthesis, and release of acetylcholine in the rat brain in vivo. In time course studies, a single intraperitoneal dose of DL-vesamicol (5 mg/kg) rapidly and reversibly (within 2 h) doubled the content of acetylcholine in the striatum and hippocampus, without affecting choline levels or the rate of transmitter synthesis. In microdialysis experiments, the same peripheral dose of drug produced a reversible 55% reduction in endogenous striatal acetylcholine release. A similar inhibitory effect was produced by direct intrastriatal perfusion with vesamicol. Moreover, this effect of vesamicol was (a) concentration-dependent and saturable (EC50 = 68 nM), (b) rapidly reversible, (c) stereospecific for the L-isomer, and (d) poorly mimicked by a vesamicol analog with lower plasma membrane permeability. This profile of effects is consistent with an interaction with a specific vesamicol receptor as defined by previous in vitro binding studies. These results support a functional role for vesamicol receptors in modulating central cholinergic transmission in vivo.     

Determination of Endogenous Acetylcholine Release in Freely Moving Rats by Transstriatal Dialysis Coupled to a Radioenzymatic Assay: Effect of Drugs

The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 microliter min-1. The perfusates were collected at 10-min intervals. In the presence of 1 and 10 microM physostigmine, acetylcholine release was 4.5 +/- 0.02 and 7.3 +/- 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+-stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium-3 (20 micrograms intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of approximately 63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.     

Clinical Concentrations of Volatile Anesthetics Reduce Depolarization-Evoked Release of [3H]Norepinephrine, but Not [3H]Acetylcholine, from Rat Cerebral Cortex

We compared the potencies of halothane, enflurane, and methoxyflurane in producing unconsciousness in vivo and in inhibiting the release of [3H]norepinephrine and [3H]acetylcholine in vitro. Rats were anesthetized with various concentrations of each anesthetic, and responsiveness was determined by a hemostat tail pinch. Slices of cerebral cortex were equilibrated with similar concentrations of each agent in vitro, and potassium-evoked release of [3H]norepinephrine and [3H]acetylcholine was determined. For both studies, brain concentrations of the anesthetics were determined by heptane extraction and gas chromatography. Using this method, we found that brain concentrations of all three agents which caused unconsciousness in vivo also reduced depolarization-evoked release of [3H]norepinephrine by approximately 30% in vitro. The release of [3H]acetylcholine was unaffected by similar concentrations of these anesthetics. Such selective interference with stimulus-secretion coupling in central noradrenergic, and possibly other, neurons might contribute to the depressant actions of volatile anesthetics. The differential effects on norepinephrine and acetylcholine release also suggest differences in the mechanisms by which these two transmitters are released.     

Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus

Intracellular recordings have been made from salivary gland cells of the pond snail Planorbis corneus. Gland cells produced a dose-dependent biphasic response to the bath application of acetylcholine (ACh), an initial depolarization being followed by a hyperpolarization. Nicotine and the nicotinic agonist tetramethylammonium had an excitatory action on the gland cells. The muscarinic agonists acetyl-beta-methyl choline and arecoline were also stimulants, but muscarine, bethanechol and pilocarpine produced no response from gland cells at 10(-3) M. A number of cholinergic antagonists, including atropine, hexamethonium and curare, effectively blocked the response to ACh. The depolarizing phase of the ACh response resulted from an increased membrane permeability to Na+ ions, though the participation of other ionic species cannot be ruled out. The hyperpolarizing phase of the ACh response was produced by the activity of an electrogenic Na+/K+ pump.     

Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors.

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.     

The measurement of nanogram amounts of acetylcholine in tissues by pyrolysis gas chromatography

Abstract— A method previously described for measuring ACh in biological effluents has been simplified and extended for use with tissues. The tissue is homogenized in acetonitrile containing propionylcholine as the internal standard and after centrifugation the acetonitrile is removed by shaking with toluene. To the aqueous solution is added a solution of KI-I₂ to precipitate the quaternary compounds. The precipitate is dissolved in aqueous acetonitrile and then drawn through a small column of ion-exchange resin to convert the periodides of the quaternary compounds to chlorides which are then simultaneously pyrolysed and gas chromatographed. On the column the pyrolytic product of choline has a slower retention time than that of acetylcholine; under these circumstances the choline present in tissues does not obscure the measurement of acetylcholine. Specificity was demonstrated by several procedures including mass spectroscopy. The method can measure 25 ng (171 pmoles) of acetylcholine in extracts of brain, simply, and with high reproducibility. With the usual gas chromatograph, 16 samples can be run in a working day. The content of acetylcholine in rat brain was 26.4 nmol/g or almost precisely the values found with other gas chromatographic methods. The pyrolytic method was shown to be applicable to the detection of biologically interesting substances other than choline esters, including betaine, carnitine and the non- quaternary compound, ?-aminobutyric acid, which is readily converted to a volatile compound (probably its methyl ester) when pyrolysed in the presence of tetramethylammonium hydroxide. Of additional general interest is the demonstration of the advantages of acetonitrile as a solvent for extracting water-soluble compounds from tissues.     

Stress-Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor

The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (gamma-aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of delta-aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas delta-aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicuculline and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexamethonium and d-tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, beta-alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and delta-aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.     

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo

One of the hallmarks of type 2 diabetes is that pancreatic β cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic β cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M₃ muscarinic acetylcholine receptor subtype in pancreatic β cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M₃ receptors in pancreatic β cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that β cell M₃ muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.     

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.     

Histaminergic Modulation of Hippocampal Acetylcholine Release In Vivo

Abstract: In order to elucidate the modulatory role of the histaminergic neural system in the cholinergic neural system, the acetylcholine release from the CA1-CA3 region in the hippocampus of anesthetized rats was studied by an in vivo microdialysis method coupled with HPLC-electrochemical detection. The mean value for the basal acetylcholine release was 0.98 β 0.04 pmol/20 min. The acetylcholine release was increased to 172% of the basal level when an electrical stimulation at 200 μA was applied to the tuberomammillary nucleus. An administration of α-fluoromethylhistidine (100 mg/kg i.p.) blocked the electrically evoked release of histamine both from the septal-diagonal band complex and the hippocampus, and abolished the electrically evoked release of acetylcholine from the hippocampus. Zolantidine (5 mg/kg i.p.) attenuated the increase in the electrically stimulated acetylcholine release, but pyrilamine (5 mg/kg i.p.) did not attenuate the increase in the acetylcholine release. These drugs showed no significant effect on the basal acetylcholine release. An administration of ( R )-α-methylhistamine (5 mg/kg i.p.) caused a decrease in the acetylcholine release to 48.7% of the basal level, whereas thioperamide (5 mg/kg i.p.) caused an increase in the acetylcholine release 60 min after the injection. These results suggest that the histaminergic system may contribute to the modulation of the activity of the septohippocampal cholinergic system, mainly through H₂ receptprs.     

Uptake of Some Quaternary Ammonium Ions by Human Erythrocytes

In many biophysical studies on erythrocytes some quaternary ammonium ions are used as replacements for Na⁺ and K⁺ of the physiological solutions. The object of this work was to study the possible uptake of quaternary ammonium ions by erythrocytes. Uptake of C¹⁴–choline chloride and C¹⁴–tetramethylammonium chloride by human erythrocytes was proved. It was shown that the compounds were neither incorporated into phospholipids of the cell nor converted to any other metabolites. Studies of uptake as a function of time, at several external concentrations of choline and tetramethylammonium, showed that within the first 4 hours uptake was a linear function of time regardless of the external concentration of the quaternary ammonium ions. The effects of various external concentrations of choline and tetramethylammonium ions on the rate of uptake by the cells were studied. The results showed the presence of two distinct mechanisms for the uptake of choline: one, a facilitated uptake mechanism which becomes saturated at low external concentrations of the ion; the other, a simple diffusion mechanism in which the rate of uptake is proportional to concentration. For the facilitated part of the uptake the external choline concentration at which half-maximum rate was obtained was found to be 0.02 mm. Although the kinetic studies with tetramethylammonium ion were not as extensive as those with choline, they did suggest the presence of similar mechanisms for the uptake of both ions. Tetramethylammonium and tetraethylammonium ions were shown to be competitive inhibitors of the facilitated choline uptake.