Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

Influence of tone on responses to acetylcholine in the rabbit pulmonary vascular bed

Pulmonary vascular responses to acetylcholine were compared under resting and high tone conditions of the intact-chest rabbit. Under resting tone conditions, intralobar injections of acetylcholine increased lobar arterial pressure in a dose-related manner. The pressor responses to acetylcholine under resting conditions were blocked by meclofenamate, indomethacin, atropine, and pirenzepine. When lobar vascular resistance was raised to a high steady level, low doses of acetylcholine decreased lobar arterial pressure, whereas higher doses elicited a biphasic response with the pressor component predominating at the highest dose studied. Under high tone conditions, only the pressor component of the response was blocked by meclofenamate or indomethacin, whereas pressor and depressor responses were blocked by atropine or the 600-micrograms/kg iv dose of pirenzepine. Pressor responses to acetylcholine under resting and high tone conditions were blocked by pirenzepine (50 micrograms/kg iv), whereas gallamine had no effect on responses to acetylcholine. The 50-micrograms/kg iv dose of pirenzepine had no effect on depressor responses or the depressor component of the response to acetylcholine. The present data support the concept that acetylcholine has significant cyclooxygenase-dependent pressor activity in the rabbit pulmonary vascular bed and suggest that this response is mediated by a muscarinic M1-type receptor. These data also show that, under high tone conditions, a vasodilator response or a vasodilator component of a biphasic response is unmasked. This response is not dependent on the release of cyclooxygenase products and is mediated by a muscarinic receptor that is neither of the M1- nor the M2-type.     

Nerve-Muscle Interaction In Vitro : Role of acetylcholine

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with α-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.     

Tone-dependent responses to acetylcholine in the feline pulmonary vascular bed

The effects of an increase in base-line tone on pulmonary vascular responses to acetylcholine were investigated in the pulmonary vascular bed of the intact-chest cat. Under conditions of controlled blood flow and constant left atrial pressure, intralobar injections of acetylcholine under low-tone base-line conditions increased lobar arterial pressure in a dose-related manner. When tone was increased moderately by alveolar hypoxia, acetylcholine elicited dose-dependent decreases in lobar arterial pressure, and at the highest dose studied, acetylcholine produced a biphasic response. When tone was raised to a high steady level with the prostaglandin analogue, U46619, acetylcholine elicited marked dose-related decreases in lobar arterial pressure. Atropine blocked both vasoconstrictor responses at low tone and vasodilator responses at high tone, whereas meclofenamate and BW 755C had no effect on responses to acetylcholine at low or high tone. The vasoconstrictor response at low tone was blocked by pirenzepine (20 and 50 micrograms/kg iv) but not gallamine (10 mg/kg iv). The vasodilator response at high tone was not blocked by pirenzepine (50 micrograms/kg iv) or gallamine or pancuronium (10 mg/kg iv). The present data support the concept that pulmonary vascular responses to acetylcholine are tone dependent and suggest that the vasoconstrictor response under low-tone conditions is mediated by a high-affinity muscarinic (M1)-type receptor. These data also suggest that vasodilator responses under high-tone conditions are mediated by muscarinic receptors that are neither M1 nor M2 low-affinity muscarinic-type receptor and that responses to acetylcholine are not dependent on the release of cyclooxygenase or lipoxygenase products.     

The Kinetics of Acetylcholine Turnover in a Resting Cholinergic Nerve Terminal and the Magnitude of the Cytoplasmic Compartment

Abstract: The magnitude of the cytosolic pool of acetylcholine in the cholinergic electromotor nerve terminals of Torpedo marmorata has been calculated to be 22 ± 3% of the total by comparing the isotopic ratio of acetylcholine with that of choline when slices of electric organ were incubated with 10 μM deuterated choline. The calculation is based on a two-compartment model that assumes the presence, in unstimulated tissue, of a vesicular pool of acetylcholine that does not exchange, under resting conditions, with a second cytosolic pool; the latter, by contrast, is subject to 'futile recycling' and comes into isotopic equilibrium with the tissue choline pool.     

ASPECTS OF ACETYLCHOLINE METABOLISM IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA

Abstract— —The synthesis of acetylcholine and its compartmentation were studied in the electric organ of Torpedo marmorata. When electric organ was homogenized in iso-osmotic NaCl-sucrose some 55 per cent of its acetylcholine content was lost unless very potent cholinesterase inhibitors were present. Slices of electric organ incubated in a suitable medium were found to synthesize radioactive-labelled acetylcholine from [ N-Me-³ H] choline. The specific activity of the labelled acetylcholine was higher in the trichloracetic acid extract of the organ slices than in an NaCl-sucrose homogenate. Acetylcholine-containing vesicles isolated from the NaCl-sucrose homogenate contained labelled acetylcholine with about the same specific activity as the parent homogenate. There was thus a fraction of acetylcholine in the incubated tissue of higher specific radioactivity that was lost when the tissue was homogenized. The acetylcholine-containing vesicles lose their acetylcholine when submitted to gel filtration under hypo-osmotic conditions. On standing at 5°C there were only small losses of acetylcholine from the vesicles but at 20°C the losses were substantial. Vesicles containing labelled acetylcholine were studied. On gel filtration under iso-osmotic conditions there was a considerable loss of labelled acetylcholine without a concomitant loss of bio-assayable acetylcholine. The pools of radioactive and bio-assayable acetylcholine are therefore not homogeneous in the vesicles as isolated.     

Pharmacological approaches to study of choline- and GABA-receptor sta tes in neuronal membranes after low doses irradiation

As a result of the study of both the acetylcholine (10(-10) M; 10(-6) M) and gamma-aminobutyric acid (10(-9) M; 10(-5) M) effects on active K+ transport in rat brain cortex slices using any selective antagonists of cholinoreceptors (ChR) and GABA-receptors (GABA-R) it had been shown that after whole-body 25 cGy irradiation (1.75 mGy/min) metabotropic muscarinic ChR and GABAB-R were involved into processes of neurotransmitter modulation, whereas under the normal conditions they were mediated via ionotropic nicotinic ChR and GABAA-R. Observed changes were supposed to be an adaptive reaction. Perhaps, postirradiation structure-functional disorders in receptors were pointed as one of the reasons leading to essential changes in the interneuronal metabolic communication processes in CNS.     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

Acetylcholine-ATP binding by direct membrane electrode measurement.

Controversy concerning acetylcholine-ATP interaction and the possible role of such binding for acetylcholine storage in synaptic vesicles has been resolved by direct binding measurements using an acetylcholine selective membrane electrode. At pH 7.4, acetylcholine was found to bind ATP^?4 with a 1:1 stoichiometry and a thermodynamic formation constant of 175M^?1. The interaction of acetylcholine with HATP^?3 and MgATP^?2 was found to be much weaker with formation constants of approximately 20M^?1 and 25M^?1, respectively. The data indicate that ATP binding could not account for more than 20% of acetylcholine storage under the conditions known to exist in synaptic vesicles.     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Exchangeability of radioactive acetylcholine with the bound acetylcholine of synaptosomes and synaptic vesicles

1. The exchangeability with added radioactive acetylcholine of the acetylcholine in isolated presynaptic nerve terminals (synaptosomes) and isolated synaptic vesicles was studied by a Sephadex-column method. 2. A substantial proportion of the synaptosomal acetylcholine is exchangeable with added radioactive acetylcholine. It is liberated by hypo-osmotic shock and ultrasonic treatment, and behaves as though it occupies the cytoplasmic compartment of synaptosomes. 3. Methods of isolating vesicles from hypo-osmotically ruptured synaptosomes in optimum yield are discussed. 4. The acetylcholine of synaptic vesicles isolated on a sucrose density gradient is released by hypo-osmotic conditions, suggesting that it is enclosed by a semi-permeable membrane; however, it is not easily released by ultrasonic treatment. 5. Added radioactive acetylcholine does not exchange with vesicular acetylcholine under a variety of different conditions. These include addition of ATP and Mg(2+), and pre-loading of the synaptosome with radioactive acetylcholine before hypo-osmotic rupture. This failure to exchange is discussed in terms of the possible storage mechanism of vesicular acetylcholine.     

AN ISOTOPIC MICROMETHOD FOR THE MEASUREMENT OF CHOLINESTERASE ACTIVITY IN INDIVIDUAL CELLS

—A microisotopic method for measuring acetylcholinesterase activity in isolated cells is described. The assay employs [¹⁴C]acetylcholine and can measure 7 × 10^-12 moles of acetylcholine hydrolysed/hr in 50-150 samples per experiment. The method described has been applied to the measurement of cholinesterase activity in individual sympathetic ganglion cells of the cat. It has been shown that under standard conditions the substrate has complete access to the enzymatic site.     

Protection of Ischaemic Synaptosomes from Calcium Overload by Addition of Exogenous Lactate

In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic- and ischaemic-type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic-type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic-type conditions. Evidence is presented to suggest that the effect of L-lactate was due to the lactate moiety itself rather than the associated acidosis.     

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel.

The reversible acetylcholine esterase inhibitor (-)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorata electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maelicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable-albeit only from other than the transmitter binding site(s).     

Early effect of glucose and oxygen deprivation on the spontaneous acetylcholine release from the myenteric plexus of the guinea pig ileum

The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself.     

The development of desensitization during rhythmic activity of the synapse and in the course of generating a single signal

The possibility of desensitization (DS) development under the rhythmic activity and during the generation of the single response to acetylcholine was studied in the frog sartorius muscle by voltage-clamp technique. It was revealed, that under conditions promoting DS (hyperpolarization, muscle warming, rise of calcium concentration as well as treatment by the DS--potentiating agent--proadifen) decrease of the postsynaptic membrane sensitivity to the acetylcholine can develop after few multiquantal end-plate currents--and when acetylcholinesterase is inhibited--during the response to a single quantum of acetylcholine.     

Alpha-toxin binding to acetylcholine receptor alpha 179-191 peptides: intrinsic fluorescence studies

Interactions between two alpha-toxins and the synthetic peptides alpha 179-191 from both calf and human acetylcholine receptor alpha-subunit sequences have been studied by measurements of quenching of intrinsic fluorescence after toxin addition. Dissociation constants of approx. 5 x 10(-8) M for binding of calf peptide by both alpha-cobratoxin and erabutoxin a have been estimated. The binding of alpha-cobratoxin to calf peptide, which leads to marked quenching of fluorescence intensity, is inhibited by a 10(4) molar excess of acetylcholine. The human alpha 179-191 peptide binds to alpha-cobratoxin, but not, under comparable conditions, to erabutoxin a.     

Autoradiographic identification of acetylcholine in the rabbit retina

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.     

The functions of acetylcholine in the rabbit retina

Rabbit retinas were incubated in vitro under conditions known to maintain their physiological function. The acetylcholine stores of the cholinergic amacrine cells were labelled by incubation in the presence of [3H]choline. The tissue was then mounted in a fast-flow superfusion chamber, and the release of [3H]acetylcholine under various conditions was measured by liquid cation exchange or high-voltage electrophoresis. When the retina was stimulated by flashing light, the rate of appearance of radioactive acetylcholine in the superfusate increased, with a latency shorter than the resolution of the system. The rate of release of acetylcholine remained elevated as long as the light was flashing, and returned rapidly to baseline when the light was extinguished. A one minute stimulation with steady light caused a burst of acetylcholine release following stimulus onset and a second, smaller, burst following stimulus cessation. In the presence of 2-amino-4-phosphonobutyrate (APB), an agent known to eliminate selectively the transmission of ON responses to the proximal retina, steady light caused acetylcholine release only at stimulus cessation. Other retinas were labelled with [3H]choline, then incubated for 10-80 min in the presence of flashing light (to promote acetylcholine release) and either control medium or medium containing 100 micron APB (to prevent release from cells activated by stimulus onset). These retinas were quick-frozen, freeze-dried and radioautographed on dry emulsion. In retinas incubated under control conditions [3H]acetylcholine was initially present within two bands within the inner plexiform layer. The two bands became fainter together as the tissue's [3H]acetylcholine was released. APB selectively retarded the depletion of [3H]acetylcholine from the band nearest the ganglion cell layer. We conclude that the displaced cholinergic amacrine cells release acetylcholine at the transient when light appears, and the conventionally placed cholinergic amacrine cells release acetylcholine at the transient when light is extinguished. The retinal ganglion cells that receive a light-driven cholinergic input are distinguished from those that do not by a great sensitivity to slow stimulus motion. It is proposed that the dense plexus of cholinergic dendrites and the transient nature of acetylcholine release combine to create the local subunit that enables detection of motion within regions smaller than those ganglion cells' receptive fields.     

LES COMPARTIMENTS D''ACETYLCHOLINE DE L''ORGANE ELECTRIQUE DE LA TORPILLE ET LEURS MODIFICATIONS PAR LA STIMULATION

Abstract— Changes in ‘free’ and ‘bound’ acetylcholine before and after stimulation have been investigated in vivo and in slices of electric organ of Torpedo marmorata incubated or superfused with physiological saline solutions. Spontaneous miniature end-plate potentials could be recorded and on electrical stimulation discharges of up to 30 V could be elicited. The electrical response fell off rapidly on repetitive stimulation. ‘Bound’ acetylcholine is that which relhains after the tissue has been homogenized since any ‘free’ acetylcholine is hydrolysed by the esterases when the tissue is disrupted. ‘Free’ acetylcholine can therefore be determined as the difference between the total acetylcholine found when the tissue is extracted with trichloroacetic acid and that which remains when the tissue is homogenized. Most of the ‘bound’ acetylcholine is present in synaptic vesicles. Stimulation of the tissue until the electrical response had fallen was accompanied by a drop in the level of ‘free’ acetylcholine. Lowered calcium and increased magnesium concentrations in the medium caused a decrease in the electrical response to stimulation and a decrease in the fall of ‘free’ acetylcholine. In other experiments, a decrease of both compartments was noticed at the end of the stimulation period. However the drop in ‘bound’ acetylcholine could also be elicited after the ‘free’ had fallen, by continuing the stimulation. When anticholinesterases were put in the medium, acetylcholine released on stimulation could be collected. On pre-incubation of the slice with [¹⁴C]choline, the acetylcholine stores became labelled. The specific radioactivity of the ‘free’ acetylcholine fluctuated on serial stimulations, whereas the specific radioactivity of the ‘bound’ acetylcholine remained stable under these experimental conditions. It is concluded that the ‘free’ compartment of acetylcholine is the most immediately available for release on stimulation.