R. L. Polak

Acetylcholine metabolism and choline uptake in cortical slices.

Abstract— The uptake of [14C]choline was studied in cortical slices from rat brain after their incubation in a Krebs‐Henseleit medium containing either 4.7 mm‐KCl (low K), 25 mm‐KCl (high K) or 25 mm‐KCl without calcium (Ca free, high K). With 0.84 μm‐[14C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P2 than in fractions P1 and S2. With 17 and 50μm‐[14C]choline in the medium greater amounts of [14C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P2 followed Michaelis‐Menten kinetics with Km values of 2.1 and 2.3 μm after incubation in low and high K medium, respectively. Hemicholinium‐3 only slightly inhibited choline uptake from a medium with 0.84μm‐[14C]choline, but it abolished the extra‐uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium‐3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8‐fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high‐affinity uptake system, of which the activity is geared to the rate of ACh synthesis.

An analysis of acetylcholine in frog muscle by mass fragmentography

Extracts of frog sartorius muscles were assayed for their acetylcholine (ACh) content by means of pyrolysis-gas chromatography/mass spectrometry. Freshly dissected whole muscles contained 43 ± 3.1 (22) pmol ACh, and apart from ACh, no other related ester was detected. The ACh content varied among different animals, but was relatively independent of muscle mass. In denervated muscles the ACh content began to decrease after a delay of two days and, by the eighth day of denervation, reached a steady value of about 26% of the control. Muscles were divided into endplate free segments and segments containing endplates. ACh was localized predominantly in the endplate segment, but a small amount was found in the endplate free region. The endplate segments of denervated muscles contained ACh at the same low concentration as ACh in non-endplate segments. The ACh concentration in non-endplate segments was not affected by denervation. During incubation in the presence of diisopropylfluorophosphate muscles released 2.1 pmol/h into the medium. During the incubation the ACh content of the muscles remained constant. It is concluded that there is about 12 pmol extraneural ACh in sartorius muscle, and that about 30 pmol is in the nerve terminals. If it is assumed that one half of the neural ACh is contained in the synaptic vesicles of motor nerve terminals, then each vesicle would contain, on the average, some 8 x 103 molecules of ACh.

Electrophysiological and chemical determination of acetylcholine release at the frog neuromuscular junction.

1. Mass fragmentography was used to measure the tissue content and release of acetylcholine (ACh) by frog sartorius muscles, which had been previously treated with an irreversible cholinesterase inhibitor. The frequency of miniature end‐plate currents (m.e.p.c.s) was also measured. 2. Exposure of muscles for 15 min to 2 mM‐LaCl3 resulted in a large release of ACh which subsided to low levels after 1 h. About 4 h later treatment with 50 mM‐KCl, or with the calcium ionophore A 23187, or with a second dose of LaCl3, all failed to augment ACh release, notwithstanding the fact that the ACh content of La3+‐treated muscles was about the same as that of controls. 3. Hypertonic NaCl or raised KCl concentrations were used to increase m.e.p.c.s and this also increased ACh release; it was estimated that each quantum corresponded to the release of 12 000 molecules of ACh. 4. ACh release by nerve stimulation was greatly potentiated by 10 mM‐tetraethylammonium chloride, and this enabled the ACh released by ten, and even single, stimuli to be detected; it was calculated from the ACh released and the quantal content that each quantum contained on the average 13 000 molecules. 5. ACh released by nerve stimulation at 0.2/s in the absence of tetraethylammonium was about half that expected on the basis of previous estimates of quantal content; it was increased about two‐fold by alpha‐bungarotoxin. 6. It is concluded that chemical and electrical stimulation of the nerve evoked quantal ACh release, without influencing non‐quantal ACh leakage. The results are consistent with the view that ACh quanta are derived from synaptic vesicles. They also show that resting ACh release is not due to leakage of ACh ions along an electrochemical gradient in the membrane.

The effect of lanthanum ions on acetylcholine in frog muscle.

1. Frog sartorius muscles were treated with an irreversible cholinesterase inhibitor and then incubated in Ringer with 2 mM‐LaCl3. The amounts of ACh in the tissue and medium were assayed by mass fragmentography, miniature end‐plate potentials (min. e.p.p.s) were recorded and the end‐plate was investigated by electron microscopy. 2. Addition of La3+ caused in normal, but not in denervated, muscles a discharge of both min. e.p.p.s and chemically detectable ACh. After 30 min both min. e.p.p.s and ACh release decreased. Between 4 and 5 hr after the addition of La3+ min. e.p.p.s had practically ceased and the rate of ACh release was almost back to that in the absence of La3+. 3. La3+ caused a 50% reduction in the ACh content of the tissue within the first 30 min; thereafter ACh gradually increased to 110% by 5 hr. At this time synaptic vesicles were practically absent in most terminals. The ACh was predominantly located in the end‐plate regions of the muscles, before as well as after the incubation with La3+. ACh in end‐plate free parts of the muscles was unchanged by La3+. 4. Hemicholinium‐3 inhibited the synthesis of ACh in the muscles, but it had almost no influence on La3+‐induced ACh release. 5. From these and other results, it is concluded that the ACh released by La3+ originates exclusively from the nerve terminals, that most likely this ACh is released via exocytosis from synaptic vesicles, and that the synthesis of ACh following the release of ACh takes place in the nerve terminals. The results further indicate that in freshly excised muscle the greater part (80‐90%) of the ACh contained in the nerve terminals is located in the vesicles.

Free and bound acetylcholine in frog muscle.

1. Frog sartorius muscles were divided into end‐plate containing (e.p.) and end‐plate‐free (non‐e.p.) segments or homogenized in Ringer solution at 0 degrees C in the presence or absence of added acetylcholinesterase from electric eel. ACh was extracted from the tissue or from the homogenates and measured by mass fragmentography. 2. The concentration of ACh in non‐e.p. segments was about six times lower than that in e.p. segments. 3. Homogenization of muscles in Ringer caused the hydrolysis of a small fraction (‘free‐1’) of total ACh; addition of extra acetylcholinesterase caused hydrolysis of another, greater, fraction (‘free‐2’ ACh). The esterase‐resistant (‘bound’) ACh was stable at 0 degrees C up to 15 min of incubation. 4. Denervation for 15 days, which caused the disappearance of the nerve terminals, did not influence ACh in non‐e.p. segments, but reduced total and bound ACh by about 75%, and free‐2 ACh by 90%. 5. Treatment with La3+ ions, which caused the disappearance of synaptic vesicles, did not influence total ACh, but reduced bound ACh by 75%, whereas free‐1 and free‐2 ACh were increased. 6. Electrical stimulation of the nerve at 5 sec‐1 or incubation with 50 mM‐KCl did not affect ACh in the non‐e.p. segments, but reduced by roughly 60% total, bound, and free ACh. 7. It is concluded that about 75% of bound ACh derives from synaptic vesicles, corresponding to 11,000 molecules per vesicle, and 25% from non‐neural ACh; that free‐1 and free‐2 ACh derive mainly from the nerve terminal cytoplasm, although they may be contaminated by vesicular ACh.

Neural and Non-Neural Acetylcholine in the Rat Diaphragm

The compartmentation of acetylcholine (ACh) and of choline acetyltransferase in the rat diaphragm was analysed by measuring their contents in muscle segments containing endplates (e. p.) and endplate-free segments (non-e. p.) at different times following section of the phrenic nerve. In addition ACh release was determined before and after denervation. Freshly dissected hemidiaphragms contained about 125 pmol of ACh; more than 90% of this was localized in the e. p. portion. Between 10 and 18 h after denervation the ACh content of the e. p. portion decreased by 80% and its ACh concentration became approximately equal to that in the non-e. p. region, whose ACh content did not change. Spontaneous release of ACh was reduced by denervation and ACh release evoked by 50 mM KCl was practically abolished. Choline acetyltransferase activity in freshly dissected preparations was about 30 nmol of ACh per gram per hour, Km 0.5 mM. About 65% of the enzyme disappeared in the first 24 h and the remaining 35% between 24 and 50 h after denervation. A different enzyme capable of ACh synthesis was found in the muscle fibres; its activity did not decrease after denervation. It is concluded that about 70% of the ACh in the diaphragm is contained in the motor nerve terminals, about 10% in the intramuscular nerve fibres and the remainder in the muscle fibres, and that about 65% of choline acetyltransferase is in the motor terminals and 35% in the nerve fibres.

Acetylcholine in intercostal muscle from myasthenia gravis patients and in rat diaphragm after blockade of acetylcholine receptors.

Publisher Summary This chapter discusses whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. It also investigates rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin, or by immunization with AChR purified from Torpedo electroplax. ACh was extracted from the intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients. Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. It was found that tetrodotoxin depressed the amount of ACh released into the medium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from the animals that had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms.

Choline Acetyltransferase in Skeletal Muscle from Patients with Myasthenia Gravis

Abstract— Acetylcholine synthesis in homogenates of human intercostal muscle was measured by a radiochemical method. Choline acetyltransferase activity in control muscle was about 20 nmol.g−1.h−1. The enzyme was found only in the endplate area of the muscle. At high substrate concentrations its activity was overshadowed by the acetylcholine synthesizing activity of a different enzyme not saturated by 10 mm‐choline. The nonspecific enzyme was present at and away from the endplate area. Choline acetyltransferase in parasternal samples of intercostal muscle from myasthenia gravis patients was about 2.5 times higher than in samples, taken from a more lateral location, of control patients, but the Km for choline was not altered (0.24 mm). It is suggested that in myasthenia gravis the shortage of acetylcholine receptors is partially compensated for by increased synthesis, storage, and release of the transmitter.

Effect of chloride ions on giant miniature end‐plate potentials at the frog neuromuscular junction.

1. Frog sartorius muscles were incubated in Ringer solutions with a raised K+ concentration (high K+) and then allowed to recover in medium with a normal K+ concentration. During the recovery period miniature end‐plate potentials (m.e.p.p.s) were recorded with intracellular electrodes. In addition, the acetylcholine (ACh) released from muscles in the presence of high K+ was measured by a mass spectrometric method. 2. Incubation in a high‐K+ medium induced the appearance of giant miniature end‐plate potentials (g.m.e.p.p.s). However, if the Cl‐ of the medium was substituted by propionate, very few g.m.e.p.p.s were observed. This was due to the absence of Cl‐ and not to the presence of propionate. The frequency of g.m.e.p.p.s was also greatly depressed when the Cl‐ concentration was lowered from 120 to 60 mM. 3. The amount of ACh released into a high‐K+ medium was the same, regardless of whether Cl‐ or propionate was the anion. 4. When Cl‐ was replaced by NO3‐ or Br‐, incubation in high‐K+ Ringer solution induced the appearance of g.m.e.p.p.s. However, SO4(2‐), like propionate, was unable to substitute for Cl‐ in this respect. 5. The frequency of g.m.e.p.p.s was correlated with that of m.e.p.p.s during the recovery period. However, when the K+ concentration was raised to 17 mM the frequency of m.e.p.p.s greatly increased, whereas that of the g.m.e.p.p.s did not change significantly. 6. G.m.e.p.p.s disappeared in the presence of curare, but persisted in the presence of tetrodotoxin or in a Ca2+‐lacking medium. However, g.m.e.p.p.s failed to appear when the medium had lacked Ca2+ during the stimulation. 7. It is tentatively concluded that g.m.e.p.p.s are associated with Cl‐‐dependent processes which occur after stimulation of transmitter release, and which are linked with the endocytotic retrieval of presynaptic membrane.

The effect of anions on bound acetylcholine in frog sartorius muscle.

1. Frog sartorius muscles were treated with an irreversible cholinesterase inhibitor and then incubated in isotonic potassium propionate solution (isotonic KPr). Total and bound, presumably vesicular, acetylcholine (ACh) in the tissue and ACh in the medium were assayed by mass fragmentography, miniature end‐plate potentials (MEPPs) were recorded and the end‐plates were investigated by electron microscopy. 2. Incubation in isotonic KPr for 30 min stimulated ACh release and concomitantly decreased total and bound ACh. Nerve stimulation for 30 min by trains of impulses (0.1 s trains of 100 Hz, 1 train s‐1) in normal‐potassium propionate‐containing solution had the same effects. 3. When the tissue was incubated in normal‐K+ Ringer solution for 3 h, following chemical or electric stimulation, bound ACh recovered to about 75% of the initial value, provided that Cl‐ ions were present in the medium. In the presence of propionate instead of Cl‐ ions almost no recovery of bound ACh took place. There was also recovery of bound ACh in the presence of either NO3‐ or gluconate ions. In NO3‐ it was the same as in Cl‐, but in gluconate it was less than found in Cl‐ ‐containing medium. 4. Recovery of total ACh, in contrast to bound ACh, took place even in the presence of propionate ions, showing that extracellular Cl‐ is not required for the synthesis of ACh. 5. In terminals recovered in normal Ringer solution, many synaptic vesicles were found, but terminals ‘recovered’ in propionate solution were depleted of vesicles. 6. From these and other results it is concluded that the recycling of synaptic vesicles normally requires the presence of extracellular chloride.