Increasing Concentrations Of Acetylcholine Research Articles

Factors affecting choline transport by the cat superior cervical ganglion during and following stimulation, and the relationship between choline uptake and acetylcholine synthesis

It has been shown previously that the accumulation of the choline analogues, homocholine and triethylcholine, by the cat superior cervical ganglion is enhanced during preganglionic nerve stimulation. In the present experiments, this enhanced accumulation was measured under various conditions in order to compare this phenomenon to the high affinity choline transport mechanism described for synaptosomes. Similar conditions were then used to determine the relationship between acetylcholine synthesis and the activation of choline transport in ganglia, the latter being inferred from the preceeding experiments. The enhanced accumulation of choline analogues by stimulated ganglia was found to be Na + -dependent, Cl − -independent, would accept Ba 2+ and Sr 2+ as substitutes for Ca 2+ , but was not apparent during chemical depolarization. Choline transport activity was increased without a proportional increase in acetylcholine synthesis when ganglia were stimulated during perfusion with a medium in which BaCl 2 replaced CaCl 2 ; hence, choline uptake and acetylcholine synthesis can be dissociated during stimulation. Acetylcholine synthesis was not impaired, although choline transport did not appear to be activated during exposure of ganglia to high [K + ] or veratridine; however, extracellular choline was utilized for the synthesis of acetylcholine during exposure of ganglia to high [K + ]. Accumulation of choline analogues was also enhanced during a period of rest that followed a period of conditioning stimulation; this phenomenon was found to be Na + -dependent, Cl − -independent and Ca 2+ -independent. The post-stimulation increase in choline transport activity was associated with a rebound increase in acetylcholine content and this was also eliminated by the substitution of Li + for Na + . The results of this study suggest that the relationship between choline uptake and acetylcholine synthesis might change following an alteration in functional state.

The effect of acetylcholine release on choline fluxes in isolated synaptic terminals.

As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca2+ and inhibited by high Mg2+ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 x 10(-8) cm . s-1 and to acetylcholine 1.8 x 10(-8) cm . s-1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol . min-1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo.

Evidence for noradrenergic mediation of the oxotremorine-induced increase in acetylcholine content in rat hippocampus

Evidence for noradrenergic mediation of the oxotremorine-induced increase in acetylcholine content in rat hippocampus

On the kinetics of the potassium channel activated by acetylcholine in the S-A node of the rabbit heart.

1. Voltage-clamp experiments were conducted on small specimens of rabbit sinoatrial node. In the same preparation the dose-response curve of the potassium current induced by application of different concentrations of acetylcholine (ACh), the time constant of relaxation and the current fluctuations were measured. From these measurements the apparent dissociation constant and the rate constants for the opening and closing of the ACh-activated potassium channel were estimated. 2. In the presence of neostigmine a measurable response was recorded at around 10(-8) M ACh, the saturation was reached at 10(-4) M, and the half saturation was attained at around 10(-6) M. 3. The time constant of relaxation at --35 mV decreased from 100 ms at 10(-8) M to 45 ms at 10(-4) M ACh. 4. The variance of the fluctuations of the ACh-activated current increased with increasing ACh concentration to a peak value of around 10(-5) M. 5. From the above 3 kinds of measurements, opening and closing rate constants of about 12s-1 and 10s-1, respectively, and a dissociation constant of 1.7 microM were calculated. 6. The Katz-Miledi model was considered to be appropriate to describe the reaction of ACh with the muscarinic receptor in the S-A node. 7. The current on ionophoretic application of ACh was computed using the rate constants and taking into account diffusion in the S-A node in which the density of receptors is low. The computed response had a similar time course to the recorded current.

Lack of involvement of dopaminergic and GABA neurones in the inhbitory effect of harmaline on the activity of striatal cholinergic neurones in the rat.

Harmaline (10 mg/kg i.p. 45 min) increased acetylcholine (ACh) levels in the rat striatum but did not affect the ACh content of the parietal cortex, the hippocampus or certain limbic nuclei. This change in striatal ACh levels may reflect a decreased utilization of the transmitter. The selective effect of harmaline on striatal cholinergic neurones is probably mediated by interneuronal processes. Various hypotheses were examined: 1. The harmaline-induced increase in striatal ACh levels was not abolished after destruction of the nigrostriatal dopaminergic neurones. Harmaline (10−5 M or 10−4 M) and its metabolites (harmalol, harmine) also failed to modify the activity of the striatal dopamine-sensitive adenylate cyclase. This excludes any involvement of dopaminergic mechanisms in the action of harmaline on striatal cholinergic neurones. 2. The harmaline-induced increase in striatal ACh levels could not be prevented by diazepam (5 mg/kg i.p. 65 min). In contrast the increase in ACh content elicited by picrotoxin (25 mg/kg i.p. 60 min) was abolished by diazepam. Furthermore, the harmaline-and picrotoxin-induced rises in striatal ACh levels were additive. These results suggest that GABA mechanisms are not involved in the effect of harmaline on striatal cholinergic neurones. 3. Parachlorophenylalanine pretreatment (48 and 24 h, 300 mg/kg i.p.) which reduced striatal serotonin (5-HT) levels by 95%, failed to affect ACh levels in the striatum. The inhibitor of 5-HT synthesis did not prevent the harmaline-induced rise in ACh levels. Harmaline (10−5 M or 10−4 M), harmine and harmalol, did not stimulate the 5-HT sensitive adenylate cyclase in the colliculi of newborn rats. It is thus unlikely that harmaline reduces the activity of striatal cholinergic neurones by its effects on 5-HT transmission. Moreover, other monoamine oxidase inhibitors (pargyline and tranylcypromine) were also ineffective on cholinergic neurones.

Effects of physostigmine and electrical stimulation on the acetylcholine content of the guinea-pig ileum

Incubation with physostigmine (7.7 μM) caused an approximately 2 fold increase in the acetylcholine content of the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum. This effect was due mainly to an increase in ‘free’ acetylcholine, which was directly assayable in either the homogenate after removal of cell debris or the supernatant fraction (100,000 g for 60 min) after subcellular fractionation. Acetylcholine output during stimulation at 0.017, 0.1 or 1 Hz was maintained for 60 min at a rate 2–4 times greater than the non-stimulated output; there was no change in content. At 10 Hz, output was high at the start of stimulation and then decreased continuously; there was a proportionate loss of mainly ‘free’ acetylcholine from the tissue. Mn 2+, hexamethonium, morphine and noradrenaline, which depressed acetylcholine output during stimulation at 0.1 Hz, had no effect on the acetylcholine content nor did they affect the increase in acetylcholine content during incubation with physostigmine.

Interactions of acetylcholine and epinephrine on the dynamics of insulin release in vitro.

An in vitro system for perifusion of rat pancreatic islets has been utilized to define the effects of epinephrine on acetylcholine-induced insulin release over varying concentrations of the two agents. Perifusion of islets with epinephrine before challenge with acetycholine produced marked enhancement of both phases of cholinergically induced insulin release; enhancement of the first phase being more marked with increase in acetylcholine concentration and the converse being observed with the second phase. Perifusion of islets with epinephrine during stimulation with acetylcholine produced inhibition of insulin release, an effect dependent upon the concentration of epinephrine and of acetylcholine. There was an order of difference in the acetycholine concentration needed to overcome significant epinephrine-mediated inhibition of the first phase of insulin release (5 X 10(-4) mug/ml) and that needed to overcome inhibition of the second phase (5 X 10(-3) mug/ml). Comparison of the effects of various concentrations of epinephrine on glucose- and acetyl-choline-induced insulin release revealed that epinephrine was a less potent inhibitor of the first phase of acetylcholine-induced insulin release than of the first phase of glucose-induced insulin release. These data provide some insight into the potential interactions between cholinergic and adrenergic autonomic systems in modifying insulin release.

Increase in striatal acetylcholine by picrotoxin in the rat: Evidence for a gabergic-dopaminergic-cholinergic link

Picrotoxin, 2 mg/kg i.p., a GABA receptor blocking agent, increased rat striatal acetylcholine content by approximately 70% without altering the levels of this amine in the cerebral hemispheres, mesencephalon, diencephalon, hippocampus and cerebellum. Striatal choline levels were concomitantly decreased by about 25%. This dose of picrotoxin also increased striatal homovanillic acid levels by about 30%, an effect which was not antagonized by pretreatment with the dopamine receptor stimulating agent, piribedil. Picrotoxin did not affect striatal choline-O-acetyltransferase or cholinesterase activity after in vitro incubation. The action of picrotoxin on striatal acetylcholine levels was partially antagonized by pimozide and completely blocked by alpha-methyl-para-tyrosine pretreatment while the intraventricular injection of 6-hydroxydopamine was without effect. Convulsions were not prevented by any of these treatments. The results are interpreted as follows: picrotoxin released dopamine through disinhibition of the dopaminergic neurons as a result of blockade of gabergic receptors. The increased dopaminergic activity inhibited cholinergic neurons and lead to an increase in acetylcholine content. The data thus provide evidence for a possible gabergic (inhibitory)—dopaminergic (inhibitory)-cholinergic link terminating in the striatum.

Inhibition by oxotremorine of acetylcholine resting release from guinea pig-ileum longitudinal muscle strips.

1. Longitudinal muscle strips of the guinea-pig ileum were incubated in Tyrode solution containing either DFP or physostigmine as cholinesterase inhibitor. After a 90 min preincubation period the acetylcholine resting release into the medium was determined. Acetylcholine was estimated by gas chromatography. 2. The resting release was 0.39 nmol/g times min irrespective of the cholinesterase inhibitor used. In the presence of hexamethonium, or after omission of external calcium, the resting release fell by 50 and 55 per cent, respectively. 3. Oxotremorine (10-5 and 10-4M) significantly inhibited the resting release of acetylcholine by 25 and 33 per cent, respectively. The inhibitory effect of oxotremorine was completely reversed by atropine (3 times 10-7 M). Oxotremorine did not reduce the spontaneous release of acetylcholine that occurred either in the presence of hexamethonium or in the absence of external calcium. 4. The acetylcholine content of the muscle strips was approximately doubled during the preincubation with a cholinesterase inhibitor. The subsequent incubation with oxotremorine did not lead to a further increase in the endogenous acetylcholine content. However, incubation of the muscle strips with oxotremorine in the absence of a cholinesterase inhibitor led to a rise in the endogenous acetycholine concentration. In in vivo experiments, oxotremorine also caused an increase in the acetylcholine content of the muscle strips. The possibility is discussed that the rise in the acetylcholine concentration following the administration of oxotremorine is a consequence of the decreased release. 5. It is concluded that oxotremorine inhibits the resting release of acetylcholine by activation of neuronal muscarinic receptors. The inhibitory effect of exotremorine is linked to that fraction of the acetylcholine resting release that is calcium-dependent and that arises from propagated activity in cholinergic neurones. The results are consistent with the hypothesis of a feed-back control of acetylcholine release mediated by inhibitory muscarinic receptors.

Release of acetylcholine in cochlear nucleus upon stimulation of the crossed olivocochlear bundle

It has been established that the crossed olivocochlear bundle endings (COCB) in the cochlea release acetylcholine (ACh). According to Dale's principle, any single neuron will release the same transmitter at all its terminals. Therefore, the dorsal and ventral cochlear nuclei were perfused with a physiological salt solution and samples were collected during and in the absense of COCB stimulation to determine whether stimulation would or would not cause ACh release. Stimulation of the COCB caused a statistically meaningful increase in ACh content of the dorsal cochlear nucleus perfusates. The Ach determinations of the ventral cochlear nucleus perfusates were confounded by the presence of an unknown (but not uninteresting) agent which opposed ACh on the bioassay object. Although evidence was adduced indicating release of ACh coincidental with COCB stimulation, it cannot be said with certainty to come from COCB collaterals. Therefore, the method employed cannot be said to have asked the most incisive experimental question. Nonetheless, the research can be said to have produced provocative results and some interesting new hypotheses.

Rapid transport of acetylcholine in rat sciatic nerve proximal and distal to a lesion.

1. Changes in distribution of acetylcholine (ACh) along the length of the sciatic nerve of the rat have been studied up to 24 hours after axotomy produced usually by crushing but sometimes by cutting or ligating the nerves. The ACh was extracted with trichloracetic acid from 5 mm lengths of nerve cut relative to the lesion and estimated by bio-assay techniques. 2. Axotomy usually produced an increase in the ACh content in the 5 mm on either side of the lesion within 1–2 minutes of operation. Subsequently there was a continued marked increase in ACh content in the 5 mmproximal to the lesion up to nearly three times the control level by 12 hours, with no further increase by 24 hours. In the 5 mmdistal to the lesion there was a further slight increase up to 60% above control by 6 hours with a subsequent decline to about the control level by 24 hours. In the more distal parts of the nerve (i.e. 5–20 mm distal to the lesion) there was a decline in ACh content of about 20% by 6–12 hours after operation. The three types of axotomy produced similar changes. 3. The initial small increase of ACh on both sides of the lesion was probably due to local synthesis of ACh, as previously described by other authors. It is suggested that the marked proximal increase in ACh was due to interruption of a proximo-distal transport of ACh and that the small decline in the more distal parts of the nerve was due to continued transport of ACh out of that segment of the nerve following operation; the size of this decline has been taken as an estimate of the proportion of axonal ACh which is rapidly transportable (20% of the total). The rate of transport of this fraction of the axonal ACh has been estimated as about 5 mm/hour. The rest of the axonal ACh is thought to be either stationary or moving slowly with bulk proximo-distal movement of the axoplasm.

Effect of amantadine on cerebral acetyl-choline release and content in the guinea pig

The effect of amantadine on cortical acetylcholine release and on cerebral acetyl-choline content in guinea pigs was examined in comparison with amphetamine. Both drugs stimulated gross behaviour and increased cortical acetylcholine release in conscious freely-moving animals. The effects of amantadine were still evident in guinea pigs pretreated with reserpine or α-methyl- p-tyrosine, but were abolished by the combined inhibition of catecholamine synthesis and granular uptake. On the contrary, α-methyl- p-tyrosine pretreatment alone, prevented amphetamine effects both on behaviour and on acetylcholine release. Amantadine increased acetylcholine content in the caudate nucleus, α-methyl- p-tyrosine showed the opposite effect, while amphetamine was ineffective. The discussion is based on the hypothesis that the activation of the cerebral adrenergic neurons is associated with a cholinergic counter-action. The conclusion is reached that the role of dopamine in determining this counter-action is predominant.

Duration of diazinon induced changes in the brain acetylcholine of rats

The duration of in vivo increase in the brain acetylcholine of rats after administration of Diazinon has been determined. It was found that Diazinon increased acetylcholine content in the brain for 72 hours, which is suggestive of a brief in vivo activity of the compound in rats. The results are discussed in connection with the prolonged activity of the compound in plants in which it is reported to undergo activation.

Effect of alpha-methyl dopa on acetylcholine content of rat brain, heart and intestine.

RESERPINE, guanethidine and syrosingopine, which are potent hypotensive drugs, have been shown to produce side effects suggestive of increased parasympathetic activity. Some of these effects, such as bradycardia and diarrhoea, are attributable to an increased acetylcholine content of heart and intestine1, 2.

Effect of chlorpromazine on tissue metabolism in vitro.

IT has been pointed out that central inhibition is connected with an increase of acetylcholine content in brain tissue. On the other hand it has also been shown that some central depressants reduce the oxygen uptake of normal respiring brain tissue in vitro 1 or in brain slices which were stimulated electrically2.

Acetylcholine release at parasympathetic synapses.

Summary.The submaxillary gland was perfused with eserinized plasma and the perfusate tested for acetylcholine on cat's blood pressure. Small amounts of acetylcholine could always be detected in the venous outflow; during stimulation of the chorda tympani there was an increase in acetylcholine content. When the perfusion fluid contained curare (Intocostrin 10‐4), stimulation of the chorda was also accompanied by an increase in acetylcholine content of the perfusate but only to about a third or a fourth of the value obtained on stimulation without curare. The average output of acetylcholine in ten experiments on cats (two minutes stimulation, 20 shocks/sec.) was 0.144 μg without and 0.050 μg with curare. It is suggested that the acetylcholine obtained in the latter case originated from the parasympathetic nerve endings in the submaxillary ganglion, since it is very unlikely that with this concentration of curare transmission occurs across the ganglion even in the presence of eserine. This assumption is further supported by pharmacological evidence and by the fact that the chorda tympani contains acetylcholine in relatively high concentration.This work was originally planned in discussions with Professor F. C. MACINTOSH, when one of us (N.E.) had the advantage of working with him at the National Institute for Medical Kesearch in London. The authors are greatly indebted to him for these discussions and for his kindness in looking over the manuscript.The investigation has been aided by a grant to one of us (N.E.) from the Swedish Medical Eesearch Council.