Low Concentrations Of Acetylcholine Research Articles

The relation of neurohumors to autorhythmicity of the isolated ventricle of Strombus gigas (Gastropoda prosobranchia)

1. 1. Isolated ventricles of Strombus gigas may be made hypodynamic by long perfusion with sea water or by chilling. 2. 2. Acetylcholine will not restart ventricles made hypodynamic by either method. 3. 3. Low concentrations of acetylcholine do not have a significantly excitatory action on spontaneously beating isolated ventricles. 4. 4. 5-Hydroxytryptamine, which has an excitatory action on spontaneously beating isolated ventricles, will restart ventricles made hydrodynamic by either method but relatively high concentrations are required. 5. 5. It is concluded that neither 5-hydroxytryptamine nor acetylcholine has effects that suggest a local hormonal role in the heart of Strombus gigas.

Studies on chemical mechanisms of the action of neurotransmitters and hormones: II. Increased incorporation of 32P into phosphatides as a second, adaptive response to pancreozymin or acetylcholine in pigeon pancreas slices

The concentrations of pancreozymin or acetylcholine necessary to give hormone-responsive amylase extrusion, and those necessary to give hormone-responsive increases in 32P incorporation into phosphatides in pigeon pancreas slices, have been investigated. Low concentrations of pancreozymin or acetylcholine stimulated amylase extrusion with little or no effect on 32P incorporation into phosphatides. High concentrations of pancreozymin gave increased incorporation of 32P into phosphatides without increasing amylase extrusion above that observed with lower concentrations of hormone. Amylase extrusion in response to 10 −4 or 10 −6 m acetylcholine (plus eserine) was less than in response to lower concentrations of acetylcholine, but 32P incorporation into phosphatides was much higher with the higher concentrations of acetylcholine. These results provide further evidence that increased synthesis or turnover of phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in response to hormone is not a secondary consequence of zymogen granule extrusion (reverse pinocytosis). In view of the requirement for higher concentrations of hormone than those necessary to elicit amylase extrusion, it is suggested that the increased incorporation of 32P into phosphatides may represent a second, adaptive response to hormone—part of a gearing up of the cell to meet greater demands for the secretion of zymogens than it had previously been meeting.

Acetylcholine sensitivity of embryonic ventricular fibres.

IN adult hearts, only fibres of the sinoatrial and atrio-ventricular nodes are innervated. In adult hearts, innervated nodal fibres are very sensitive, while non-innervated fibres are relatively insensitive, to low concentrations of acetylcholine (ACh) and norepinephrine1,2. Similarly, in innervated skeletal muscle, the end plate membrane is much more sensitive to ACh than the rest of the membrane3. The presence of the nerve terminal is in fact responsible for the low ACh sensitivity of the remainder of the fibre membrane, as with denervation, all of the skeletal muscle membrane becomes quite sensitive to ACh (ref. 4). Formation of a new end plate on a denervated muscle fibre is followed by decreased ACh sensitivity of the rest of the fibre membrane5. Furthermore, Diamond and Miledi observed that, in rat embryo, formation of an end plate is followed by decreased ACh sensitivity of the remainder of the skeletal muscle fibre membrane6. The following experiments were carried out to determine whether innervation of nodal fibres in embryonic heart is accompanied by decreased ACh sensitivity in non-innervated ventricular muscle.

COMPARATIVE ACTIONS OF 1,10-PHENANTHROLINE AND QUINIDINE ON NORMAL AND FIBRILLATING ISOLATED ATRIA.

Summary1. A method of inducing fibrillation in isolated rabbit atria was explored. Atrial fibrillation can be readily produced and maintained under the conditions described by Holland and Burn(2). The method is improved here by the use of much lower concentration of acetylcholine. 2. 1,10-Phenanthroline is as effective as quinidine in stopping induced fibrillation in isolated rabbit atria. 3. 1,10-Phenanthroline, like quinidine, reduces the maximal following rate of isolated rabbit atria, but to a lesser extent. 4. The lowering of maximal following rate by 1,10-Phenanthroline and quinidine can be lessened by reduction of potassium in the medium. 5. At comparable concentrations, 1,10-Phenanthroline does not affect the intrinsic sinus rate of isolated rabbit atria, while quinidine reduces the intrinsic rate markedly. 6. 1,10-Phenanthroline, like quinidine, produces distinct reduction of the rate of depolarization and repolarization of the action potential of the rat atria.

The action of 5-hydroxytryptamine and acetylcholine on the rectum of the venus clam, Mercenaria mercenaria

1. 1. An isolated preparation of the rectum of Mercenaria mercenaria has been developed and tested with a variety of componds/ 2. 2. 5-Hydroxytryptamine excites the rectum. Low doses often induce rhythmical activity; higher concentrations inhibit the beat while increasing tone.5-Hydroxytryptamine is mimicked by tryptamine and lysergic acid diethylamide and antagonized by methylsergide. Benzoquinonium chloride, d-tubocurarine and morphine, the latter to a slight extent, antagonize the tone increase while augmenting the beat induced by 5-hydroxytryptamine. Atropine potentiates the tone increase; its effect on beat is related to its tonotropic effect. 3. 3. Acetylcholine produces both a phasic and a tonic response. The first is a transient tension increase. The tonic effect is bimodal; low concentrations diminish, and high concentrations augment, rectal tone. The tone, amplitude and frequency of recta stimulated to beat by 5-hydroxytryptamine are decreased by low concentrations of acetylcholine, while large doses increase tone and obliterate the beat. Low doses of benzoquinonium (10 −5 M) augment the acetylcholine tone increase; higher doses are usually antagonistic, depending also upon the acetylcholine concentration. Atropine (5 × 10 −5) is a more effective antagonist than an equimolar dose of benzoquinonium. The effect of acetylcholine apparently depends on the rectal tension prior to the dose. 4. 4. 5-Hydroxytryptamine appears to stimulate cholinergic nerves as well as directly exciting the rectal musculature. Multiple sites of action of acetylcholine are suggested.

Evidence supporting cholinergic transmission at the lateral geniculate body of the cat

The neurons in the dorsal nucleus of the lateral geniculate body in encéphale isolé preparations of cats have been activated by orthodromic stimulation of the contralateral optic nerve and by antidromic stimulation of the optic radiation. The evoked geniculate response was recorded by extracellular microelectrodes. Afferent volleys limited to the large fibres the optic tract evoked a diphasic positive-negative presynaptic wave followed after a brief delay of 0·3 msec by a negative postsynaptic wave. Repetitive firing, consisting of several negative waves of small amplitude following the postsynaptic component, was frequently observed in the control preparation. The effects of drugs administered by intracarotid injection were evaluated by noting the amplitude, latency, duration, and refractory period of the geniculate response. Post-tetanic potentiation was studied in control and drug-treated preparations. The postsynaptic component was readily depressed by DHE, depressed to a lesser extent by atropine, and unaffected by d-tubocurarine; it was enhanced by low concentrations of acetylcholine and by physostigmine. Higher concentrations of acetylcholine had a depressant effect. Tetanic stimulation of the optic nerve relieved the depression imposed by DHE and by atropine. It depressed the postsynaptic wave after the wave had been enhanced by low doses of acetylcholine. The presynaptic spike, the antidromic response, and the refractory period remained unaltered after drug administration. 5-Hydroxytryptamine produced a depression of the postsynaptic component which was not relieved by tetanic stimulation. The data evidenced a close resemblance between the pharmacological responses of Renshaw cells and of geniculate neurons and indicate that at least one of the mechanisms subserving transmission in the lateral geniculate nucleus is cholinergic in nature.

Ester-splitting activity of the electroplax

1.1. The hydrolysis of acetylcholine, dl-acetyl-β-methylcholine, ethyl chloroacetate, triacetin and butyrylcholine by intact and homogenized isolated single cells and by the connective tissue between electroplax has been tested. Some connective tissue remains attached to the electroplax; a correction for this extracellular esterase was therefore applied to evaluate esterase activity of the electroplax proper.2.2. The electroplax enzyme displays the characteristics of acetylcholinesterase. The connective tissue enzyme has neither the characteristics of acetylcholinesterase, human-serum cholinesterase, aliesterase nor arylesterase. In some respects, however, it resembles the cholinesterase of certain piscine plasmas.3.3. Hydrolysis of low concentrations of acetylcholine by intact cells is mainly by acetylcholinesterase of the electroplax, whereas high concentrations of acetylcholine are mainly hydrolyzed by the connective tissue esterase. Both enzymes are inhibited by physostigmine. Ethyl chloroacetate is hydrolyzed by at least 2 enzymes in the electroplax, only one of which is inhibited by physostigmine.4.4. There is a strong permeability barrier to the penetration of acetylcholine to the interior enzyme of the intact electroplax. This barrier is less strong for dl-acetyl-β-methylcholine, ethyl chloroacetate, triacetin and butyrylcholine.5.5. Concentrations of physostigmine which block electrical activity markedly depress acetylcholine hydrolysis. Tetracaine even in much higher concentrations than required to block electrical activity only slightly inhibits acetylcholine hydrolysis. Inhibition by neostigmine is intermediate between that of tetracaine and physostigmine.

Pharmacological Observations on the Desheathed Last Abdominal Ganglion of the Cockroach1

The unstabilizing action of very low concentrations of anticholinesterase agents on the cereal nerve-giant fiber synapses in the last abdominal ganglion of the roach, Periplaneta americana (L.) (Roeder, Kennedy and Samson, 1947; Roeder, 1948 and Roeder and Kennedy, 1955) as well as identification of acetylcholine in insect nervous tissue (Lewis, 1953; Augustinsson and Grahn, 1954;) suggest that acetylcholine is involved in synaptic transmission in insects. However, although low concentrations of acetylcholine have been shown to increase the rhythmic contractions of the roach heart (Krijgsman and Krijgsman, 1950) and intestine (Kooistra, 1950), even very high concentrations (1.0%) of acetylcholine had no consistent effects when applied directly to the last ganglion (Roeder, 1948). Tobias, Kollros and Savit (1946) found that between 7 and 10 gms. acetylcholine must be injected per kg. of roach to produce convulsions and death. Demonstration by Hoyle (1952, 1953) that the connective tissue sheath which envelops the insect nervous system restricts the diffusion of potassium ions lent support to a hypothesis that externally applied or injected acetylcholine may fail to reach the interior of the ganglion in effective quantities rapidly enough to stimulate. Recent studies (Twarog and Roeder, 1956) indicate that the sheath surrounding the abdominal cord of the roach is indeed a barrier to the diffusion of acetylcholine as well as sodium and potassium ions. In connection with these last studies, a method was developed for the simple and effective desheathing of ganglia and connectives of the roach abdominal cord.

CHEMICAL TRANSMISSION AND ADAPTATION

Acetylcholine and factor I appear to be transmitter substances of excitatory and inhibitory regulatory nerve fibers supplying the sensory neurons of stretch receptor organs of the crayfish. Sudden application of a low concentration of acetylcholine causes the impulse frequency to jump to a peak value. But immediately the frequency falls again and gradually reaches a steady state which is not far above the previous frequency level. If the acetylcholine is now withdrawn there follows a silent period after which the frequency returns to its original level. The time course of these events is identical with that of adaptations to sudden increase or decrease of stretch. Factor I in sufficiently low concentrations causes an immediate fall in impulse frequency (silent period) which is followed by a return to a value near the previous frequency level. Withdrawal of factor I is followed by excitation and again return of the frequency to the rate measured before the application of factor I. The time course of these phenomena is identical with that of adaptations to sudden decrease and increase of stretch. It is suggested that adaptation may be a property not only of sensory neurons but of neurons in general and that even central neurons may be considered as receptor neurons inasmuch as they respond to chemically transmitted excitatory and inhibitory stimuli.

Marine Invertebrate Preparations useful in the Bioassay of Acetylcholine and 5-Hydroxytryptamine

CERTAIN isolated muscle and heart preparations of marine invertebrates are highly sensitive to acetylcholine, to 5-hydroxytryptamine (enteramine, serotonin), or to both of these substances. Bacq1 was the first and Ambache and Sawaya2 among the more recent to demonstrate the sensitivity to acetylcholine of muscle strips from holothurians (Echinodermata). Inhibition of heart-beat of the bivalve mollusc, Venus mercenaria, was first used by Welsh3 for the quantitative estimation of acetylcholine in tissue extracts. Recently, the same heart has been found to respond by increases in amplitude and frequency to low concentrations of 5-hydroxytryptamine4. Earlier, it had been reported5,6 that certain molluscan hearts had a low threshold to 5-hydroxytryptamine or tissue extracts containing this substance. At the Marine Biological Laboratory, during the summer of 1953, the need arose for the quantitative estimation of acetylcholine and 5-hydroxytryptamine in certain tissues. Several species of echinoderms and molluscs from the Plymouth region were secured, and isolated muscle and heart preparations were screened for their suitability in bioassay of these substances. Available holothurians were Holothuria nigra, Cucumaria normani, C. elongata, and a single specimen of Thyone sp. Pieces or entire lengths of the longitudinal muscle bands were dissected free from the inner body wall and set up in the manner commonly used for muscle strips during recording of contractions. In the case of C. normani the peristomal (pharyngeal) retractor muscles were also tried. While all these preparations contracted in low concentrations of acetylcholine, especially after eserinization, the difficulties that were encountered by earlier workers1,7 who had used other species of holothurians were obvious. Chief among these were spontaneous contractions, tendency to contract and remain contracted after eserine, and sensitivity to mechanical stimulation after eserine. This last was especially marked in the muscles from the single specimen of Thyone which, otherwise, were most promising since they were not spontaneously active.

Effect of calcium chloride on choline esterase.

THE effect of salts on choline esterase activity has been studied by a number of investigators1–13. The results may be summarized as follows : (1) True choline esterase : salts with univalent or divalent cations inhibit at low concentrations of acetylcholine, whereas these salts activate at high concentrations of acetylcholine ; the optimum substrate concentration is shifted to higher values, though this was not found by Augustinsson10. (2) Pseudo-choline esterase : salts with univalent cations inhibit, whereas divalent cations activate. In most experiments, hitherto, acetylcholine only was used as substrate, and choline esterase activities were determined manometrically, thus introducing a possible complicating effect of bicarbonate and other buffers present in the system. An effect of the salts (especially at high concentrations) on the bicarbonate – carbon dioxide equilibrium cannot be excluded.

Plasma Cholinesterase in Male and Female Rats

Beveridge and Lucas1 found that the serum cholinesterase activity of mature female rats was three to five times greater than that of mature males or immature females. Similar differences in the cholinesterase activity of rat liver due to sex were also reported by Zeller and co-workers2. These reports were made before Mendel and Rudney3 had established that there are two cholinesterases in the body—one a specific or true cholinesterase, exerting its maximum activity at low concentrations of acetylcholine (about 3 mgm. per cent), and the other a non-specific or pseudo-cholinesterase, capable of hydrolysing other esters besides those of choline and having its maximum effect at high concentrations of acetylcholine (above 300 mgm. percent). Beveridge and Lucas1 and Zeller2 had estimated rat cholinesterase at high concentrations of acetylcholine which would mask the activity of the specific enzyme and thus represent mainly the activity of the pseudocholinesterase.

A Nerve-Muscle Preparation From the Snail

ABSTRACT A nerve-muscle preparation from the snail and its method of removal from the animal are described. Further description is given of the apparatus used for obtaining records of its activity. The nerve appears to contain fibres which fall into two groups according to their diameters. It is suggested that the thin fibres are derived from the thick ones. No physiological evidence of double innervation has been found. There is clear evidence of neuro-muscular facilitation. The muscle contracts in response to low concentrations of acetylcholine.