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.

Effect of potassium propionate on free and bound acetylcholine in frog muscle.

Frog sartorius muscles were homogenized under various conditions which allowed, by means of mass spectrometry, the measurement of total ACh, and different ACh compartments in the tissue: 'bound', 'free-1' and 'free-2' ACh. Bound ACh presumably corresponded to the vesicular compartment, and the free-1 and free-2 fractions to the cytoplasmic compartments of ACh. Stimulation of ACh release by La3+ ions for 60 min caused a decrease of both bound and free-2 ACh, but at 20 min bound ACh was reduced much more than free-2 ACh. Stimulation of ACh release by isotonic potassium propionate (KPr) solution for only 5 min caused a decrease of bound ACh, in contrast to free-1 and free-2 ACh which were not significantly changed. When muscles after 5 min stimulation in KPr were allowed to recover in normal Ringer, free-1 ACh did not change, but free-2 and bound ACh increased; after 180 min in Ringer bound ACh had recovered to control values. When ACh synthesis was prevented by hemicholinium-3 during recovery of the muscles in Ringer, bound ACh increased at the expense of free-2 ACh. In deuterium labeling experiments, in which the Ringer contained choline-d9, much more ACh-d9 was formed in stimulated than in unstimulated muscles. It appeared that almost all newly formed ACh was ACh-d9, since no significant synthesis of unlabeled ACh (ACh-d0) took place. Yet again, the amount of bound ACh-d0 significantly increased, apparently at the expense of preformed free-2 ACh-d0.(ABSTRACT TRUNCATED AT 250 WORDS)

Surplus acetylcholine and acetylcholine release in the rat diaphragm.

1. Skeletal muscles from rat, mouse and frog were incubated under different conditions and the amounts of acetylcholine (ACh) extractable from the tissue and released into the medium were determined by mass fragmentography. In some experiments measurements were made of the amounts of ACh ('bound' ACh) surviving in a muscle homogenate to which an excess of acetylcholinesterase had been added. In other experiments the membrane potentials, end-plate potentials (e.p.p.s), and miniature end-plate potentials (m.e.p.p.s) were studied. 2. During incubation in Ringer medium the ACh content of the rat hemidiaphragm usually did not change, but after inhibition of cholinesterase by soman the ACh content rose gradually from about 100 to 150 pmol to a plateau of about 400 pmol after 4 h. A similar formation of 'surplus ACh' after cholinesterase inhibition was found in the mouse diaphragm, but not in the frog sartorius muscle. 3. Surplus ACh accumulated predominantly in the end-plate region of the rat diaphragm. In muscles, 16-18 h after in vivo denervation, the capacity to form surplus ACh was decreased by more than 80%. 4. The amount of ACh diffusing from the resting hemidiaphragm into the incubation medium ('resting release') varied between 0.5 and 0.9 pmol min-1 in different experiments; it remained at the same level during accumulation of surplus ACh. It was reduced by more than 80% 16-18 h after denervation. 5. The amplitude of m.e.p.p.s and e.p.p.s did not increase while surplus ACh was accumulating. 6. Incubation of hemidiaphragms in Ringer solution containing [3H]choline caused the formation of [3H]ACh. Additional amounts of [3H]choline were incorporated into ACh when the nerve was stimulated for 60 min. However, incubation in the presence of soman (3,3-dimethyl-2-butylmethylphosphonofluoridate), in the absence of stimulation, did not cause an increase of the [3H]ACh content of the muscles. 7. From hemidiaphragms with active cholinesterase about 120 pmol ACh was lost after prolonged nerve stimulation or incubation with 50 mM-KCl in the presence of hemicholinium-3, and about 35 pmol remained in the tissue. In soman-treated muscles, containing surplus ACh, about as much ACh was released by nervous stimulation as from untreated hemidiaphragms, and much more ACh remained unreleased. 8. Transection of the muscle at both sides of the end-plate or incubation of intact muscles in the presence of 50 mM-KCl depolarized the muscle fibres to -35 and -31 mV, respectively. Surplus ACh was partially released by 50 mM-KCl, but not by muscle transection.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Acetylcholinesterase activity in intact and homogenized skeletal muscle of the frog.

Enzymatic hydrolysis of acetylcholine (ACh) was determined in intact frog sartorius muscles or their homogenates. The Vmax was 29 nmol min-1 in intact muscles and 46 nmol min-1 per muscle in homogenates, and the Km was 6 and 0.2 mM, respectively. The muscle was divided into small segments, which were homogenized; the junctional cholinesterase (ChE) accounted for 60% of total enzyme activity. At low substrate concentrations the rate of hydrolysis was up to 30 times higher in homogenates than in intact muscles. This difference was greatly reduced at very high substrate concentrations. It appears that most of the ChE in intact muscle is 'occluded' to external ACh, mainly because the ChE at the edges of the synaptic cleft prevents the ACh from reaching the enzyme situated further inwards, which consequently does not contribute to its hydrolysis; homogenization makes all synaptic ChE accessible to added ACh. Incubation of sartorius muscles with collagenase caused an 80% decrease in ChE activity (determined in homogenates) of end-plate-containing parts which became similar to that in end-plate-free parts on which collagenase had little effect. Histochemistry showed that the tendon-muscle junction contained folds which were stained intensively for ChE. Diethyldimethylpyrophosphonate , neostigmine, eserine, and di-isopropyl fluorophosphonate inhibited ChE activity in this order of potency. The I50 values (i.e. the concentrations of the drugs which caused a 50% inhibition) were about 5 times higher in intact than in homogenized tissue. Neostigmine, 0.15 and 0.4 microM, increased the time constant of miniature end-plate currents 1.3- and 1.8-fold, and slowed down ChE activity of muscle homogenates by 1.4 and 2.1 times, respectively, without significantly affecting ACh hydrolysis by intact muscles. This indicates that synaptic ChE is not present in large excess. It is concluded that ChE activity measured in homogenates presents a better picture of in situ ChE activity than that measured in whole muscles especially for evaluating the effect of ChE inhibitors. A mathematical model for ChE-hindered diffusion of ACh is presented in an Appendix.

Potassium propionate causes preferential loss of 'bound' acetylcholine in frog muscle.

Bound and free acetylcholine (ACh) were measured in frog sartorius muscles by mass fragmentography. Upon incubation of the muscles for 5 min with potassium propionate, which stimulated the release of ACh, there was a 2-fold reduction of bound ACh. In contrast, the amount of free ACh remained unchanged. After 65 min recovery from stimulation in normal Ringer solution containing deuterium-labelled choline, free ACh was labelled to a higher degree than bound ACh. The results are in agreement with the idea that ACh is synthesized in the cytoplasmic compartment of the motor nerve terminal, and subsequently transferred to the vesicles from which it is released upon stimulation.

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.

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.

Eaton-Lambert syndrome: acetylcholine and choline acetyltransferase in skeletal muscle.

In biopsied intercostal muscle from six patients with Eaton-Lambert syndrome, we measured acetylcholine content and release and choline acetyltransferase. Both the spontaneous and the KCl-evoked release of acetylcholine were abnormally low. On the other hand, the acetylcholine content and the level of choline acetyltransferase activity were within the range of values earlier found in healthy human intercostal muscle. These results are consistent with the view that the defect in this syndrome lies not in the synthesis or storage of the transmitter but in the mechanism of release itself.

Giant miniature endplate potentials induced by 4-aminoquinoline.

In experiments on the isolated extensor digitorum longus muscle of the rat it was shown that 4-aminoquinoline (125-250 micro M) altered the amplitude distribution of spontaneous miniature endplate potentials to include a large portion of giant miniature endplate potentials with slow rise and decay times. Similar, slow-rising giant miniature endplate potentials were induced by the drug at neuromuscular junctions with regenerating nerve terminals, i.e. in a condition where spontaneous as well as evoked transmitter release is depressed. The appearance of giant miniature endplate potentials was not correlated with inhibition of cholinesterase since neostigmine (3 micro M) failed to induce such potentials. Nerve impulse evoked endplate potentials of amplitudes similar to the spontaneous giant miniature endplate potentials had a faster and more uniform rise time. The results suggest that 4-amino-quinoline, by a direct action on the nerve terminal, causes the release of larger than normal quanta of acetylcholine. Quantitative assays of acetylcholine released before and in the presence of 4-aminoquinoline gave similar values showing that the amounts of acetylcholine which give rise to the giant miniature potentials contribute little to the total amount of acetylcholine liberated.

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 KC1 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.

Choline acetyltransferase in skeletal muscle from patients with myasthenia gravis.

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.

Congenital myasthenia: end-plate acetylcholine receptors and electrophysiology in five cases.

The nature of the defect in congenital myasthenia was investigated in biopsy specimens of intercostal muscle from 5 male patients whose symptoms presented between birth and 2 years of age. Miniature end-plate potentials were reduced in amplitude in all 5 patients. The number of acetylcholine receptors as determined by alpha-bungarotoxin binding was normal in case 1 and reduced in cases, 2, 4, and 5. The shape of the end-plates as shown by autoradiography and cholinesterase staining was normal in case 1 and elongated in cases 2, 4, and 5. In cases 3, alpha-bungarotoxin binding was slowly reversible, and there were some muscle fibers with multiple end-plate regions. The acetylcholine content of the muscle was normal in all 5 cases. None of the patients had serum antibody to human acetylcholine receptor as measured by immunoprecipitation or inhibition of alpha-bungarotoxin binding. We conclude that congenital myasthenia is a heterogeneous condition of nonimmune etiology in which both presynaptic and postsynaptic defects can be found.

Acetylcholine content and release in denervated or botulinum poisoned rat skeletal muscle.

1. The acetylcholine (ACh) content and spontaneous and evoked release of ACh in rat extensor digitorum longus (EDL) muscles were determined by pyrolysis-mass fragmentography. The determinations were made on muscles paralysed by local application of botulinum toxin (BoTx) type A, on unpoisoned muscles, surgically denervated or reinnervated muscles.2. The ACh content of unpoisoned control muscles was nearly uniform between animals and varied in the experimental series between 36 and 50 pmol. BoTx failed to affect the ACh content after 2 d of poisoning and caused a slight increase in content after 8 d. Surgical denervation reduced the ACh content within 24 h to less than 10% of innervated muscles and upon reinnervation the ACh content was restored. Following cholinesterase inhibition the ACh content of innervated and denervated muscles increased somewhat, about equally with time.3. Spontaneous release of ACh varied in normal innervated muscles between 40 and 100 fmol/min. In the presence of 25 mm-KCl the rate of release increased about fourfold. In BoTx poisoned muscles spontaneous release was reduced by up to 60% of control and high potassium failed to accelerate the release at 2 d after poisoning and caused only a small increase at 8 d. Denervated muscles released ACh at a rate which was less than 20% of control and it was not accelerated by high potassium.4. The results show that more than 90% of total ACh in the innervated EDL muscle is present in the nerve and its terminals. The remaining ACh is apparently formed and stored in the muscle tissue. BoTx caused a larger reduction in ACh release than can be accounted for by assuming a selective blockade of quantal release of transmitter. It suggests that BoTx has an inhibitory effect also on non-quantal ACh release.

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.

Acetylcholine synthesizing enzymes in frog skeletal muscle.

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (Vmax, 2nmol . g-1 . h-1, Km for choline, approx. 50 microM). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4-(1-naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine-synthesizing activity of a different enzyme not saturated by 10 mM-choline. The non-specific enzyme was present at and away from the endplate area, and it was not affected by denervation.