Low Acetylcholine Research Articles

Relative importance of extracellular and intracellular Ca2+ for acetylcholine stimulation of insulin release in mouse islets.

Mouse islets were used to study whether mobilization of intracellular Ca2+ is sufficient to account for acetylcholine (ACh) amplification of glucose-induced insulin release. In the presence of 15 mM glucose, the acceleration of 45Ca efflux and insulin release by 1-100 microM ACh increased with the concentration of extracellular Ca2+ (0.25-2.5 mM). Low concentrations of the Ca2+-channel blockers D 600 (1 microM) or nifedipine (0.1 microM) partially inhibited glucose-induced insulin release and its amplification by ACh. At higher concentrations, D 600 (25 microM) or nifedipine (2 microM) practically abolished the ionic and secretory effects of 1 microM ACh. However, 100 microM ACh still caused a fast, large, but transient acceleration of 45Ca efflux, accompanied by a small, short-lived release of insulin. Similar results were obtained in a Ca2+-free medium, indicating that this peak of 45Ca efflux reflects Ca2+ mobilization. Addition of nifedipine or omission of Ca2+ during ACh stimulation rapidly and strongly inhibited 45Ca efflux and insulin release. Both glucose and ACh-induced 45Ca uptake were inhibited by D 600. Only high concentrations of ACh (100 microM) mobilize enough cellular Ca2+ to trigger a small and transient insulin release when Ca2+ influx is prevented or impossible. A continuous influx of Ca2+ is necessary for low ACh concentrations to increase release and for high concentrations to have a sustained effect. The amplification of release by the neurotransmitter results from a slight enhancement of Ca2+ influx associated with a marked increase in the effectiveness of incoming Ca2+ on the releasing machinery.

Kinetics of activation of acetylcholine receptors in a mouse muscle cell line under a range of acetylcholine concentrations

We studied, using the patch-clamp technique, the kinetics of single acetylcholine (ACh)-activated channels in a mouse muscle cell line. In the presence of high ACh concentrations we estimated the rate of channel isomerization into the open state (beta) from the dwell time between openings. Also, we obtained estimates for beta under low agonist concentrations by assuming a linear sequential model of channel activation and applying burst analysis. If the linear model is correct, then the two estimates of beta should agree since beta should be independent of ACh concentration. However, the estimates of beta obtained under low ACh concentrations were slower than those obtained independently under high ACh concentrations. The discrepancy in the estimates of beta suggests that the linear model is inadequate, but the discrepancy can be explained if open channels can close through two separate pathways. Two alternative kinetic models that can account for our data are discussed.

5-Hydroxytryptamine decreases the sensitivity of nicotinic acetylcholine receptor in bull-frog sympathetic ganglion cells.

The post-synaptic effects of 5-hydroxytryptamine (5-HT) were examined in neurones of bull-frog sympathetic ganglia with intracellular micro-electrode and voltage-clamp recording techniques. Atropine (1 microM) was used to block the muscarinic cholinoceptors. 5-HT reduced the amplitude of the fast excitatory post-synaptic potential (fast e.p.s.p.). 5-HT also reduced the mean amplitude of the miniature excitatory post-synaptic potentials (m.e.p.s.p.s) without affecting their frequency. Voltage-clamp studies showed that 5-HT decreased in a dose-dependent manner the amplitude of the acetylcholine (ACh) current produced by ionophoretic application of ACh to sympathetic neurones. The relationship between the log of the ACh dose, applied ionophoretically, and the peak ACh current (the dose-response curve) was examined in voltage-clamped neurones. 5-HT caused a parallel shift to the right of the dose-response curve for ACh. Analysis using a double reciprocal plot (Lineweaver-Burk plot) revealed that 5-HT increased the apparent dissociation constant (Km) of ACh for the receptor without changing the maximum ACh current (Vmax), suggesting a competitive antagonism. The relationship between the 5-HT dose and the magnitude of inhibition of the ACh current was obtained using two different amplitudes for the ACh response. The dose-response curve of 5-HT-induced inhibition using a relatively high amplitude ACh current, S1, was parallel with that for a relatively low amplitude ACh current, S2. The Dixon plot of these two curves yielded an apparent inhibition constant (Ki) of 42 microM. Both fast excitatory post-synaptic currents (fast e.p.s.c.s) and miniature excitatory post-synaptic currents (m.e.p.s.c.s) had single-exponential decay time courses. The time constants of fast e.p.s.c. decay (tau e) and m.e.p.s.c. decay (tau m) were not altered by 5-HT, suggesting that 5-HT does not change the kinetics of opening and closing of the ionic channel associated with the nicotinic receptor. 5-HT did not alter the reversal potential of the fast e.p.s.c. These results suggest that 5-HT decreases the sensitivity of the nicotinic receptor of sympathetic neurones, by interfering with ACh binding at the active site on the receptor-ionic-channel complex. 5-HT may physiologically inhibit cholinergic transmission as it is an endogenous substance which antagonizes the nicotinic receptor in post-ganglionic neurones of bull-frog sympathetic ganglia.

Effects on reinforced and nonreinforced responses of depressed and of elevated brain acetylcholine levels

Abstract Two experiments are reported which were designed to study effects on reinforced and nonreinforced responses of manipulating the cholinergic neurotransmitter system both by depressing and by elevating brain acetylcholine (ACh) levels. Atropine sulphate, a cholinergic antagonist, and diisopropyl fluorophosphate, an indirect agonist, were used to produce the respective changes. The relatively simple operant response of running with food reinforcement in a straight alley was chosen as the behaviour to be studied in order to minimize the array of possible competing responses which in earlier studies had been shown to confound interpretation of the influence of differential reinforcement on behavioural effects of cholinergic agents. Although not always statistically significant, low ACh levels produced depression and high ACh levels enhancement in all responses studied. Effects of the cholinergic antagonist were relatively greater on the most highly reinforced response only when the competing response ...

Acetylcholine receptor channel properties during development of Xenopus muscle cells in culture.

Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch-clamp technique. At all stages of differentiation two different unitary-event amplitudes were observed, corresponding to high-gamma (single-channel conductance) (64 pS) and low-gamma (46 pS) channel types. No change in conductance occurred for either channel type during the 6-day in vitro period examined. At resting membrane potential (-85 mV) the low-gamma channel exhibited a mean open time of approximately 2 ms which, on the average, was 2-3-fold longer than that measured for the high-gamma channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development. In recordings with low ACh concentration (0.1-0.25 microM) both high-gamma and low-gamma channel types exhibited non-stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop-out' rate was faster for the low-gamma channel. A developmental increase in the proportion of high-gamma events occurred between day 1 (16%) and day 5 (56%) in culture, paralleling the time-dependent changes in the channel kinetics based on ACh-activated membrane noise. We conclude that the development of non-junctional muscle membrane is associated with increased expression of high-gamma channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.

Properties of non-junctional acetylcholine receptor channels on innervated muscle of Xenopus laevis.

Patch-clamp recordings of current through acetylcholine-activated channels were made from non-junctional membrane of innervated myotomal muscle from Xenopus laevis. Two classes of acetylcholine (ACh) receptor channels were identified on the basis of current amplitudes. Both amplitude classes exhibited current-voltage relations which deviated from linearity as the extrapolated reversal potential was approached (-5 to -12 mV). Over the range of greatest linearity the conductances of the two classes were 64 and 44 pS. Both event classes were blocked by alpha-bungarotoxin. At the normal resting membrane potential (approximately -95 mV) the larger conductance channel (gamma) exhibited an apparent mean channel open time of less than 1 ms, compared to approximately 2 ms for the smaller gamma class. The apparent open time was voltage-dependent, changing e-fold with a 63 mV hyperpolarization for the high gamma channel and 93 mV hyperpolarization for the low gamma channel. At low ACh concentrations (0.1-0.3 microM) both amplitude classes exhibited bursts of successive openings separated by brief closures of less than 0.5 ms. Bursts were separated by longer closed intervals of 1 to greater than 100 ms. Closed interval histograms revealed corresponding populations of brief and long closures, indicating that at least two kinetic processes are required to describe the distribution of closed intervals. In the absence of exogenous ACh, channels were observed in an occasional patch which showed a conductance and extrapolated reversal potential similar to ACh-activated channels. In such patches the event frequency could occasionally be altered by adjusting the negative pressure applied to the patch. The two main conductance classes of ACh activated channels were observed to coexist in most patches. However, the most frequent event observed in non-junctional membrane of innervated muscle corresponded to the high gamma class. In this respect, the non-junctional ACh receptors bore a greater similarity to junctional ACh receptors than to non-junctional receptors reported for denervated muscle.

Acetylcholine turnover and aggression in related three strains of mice

Abstract 1. 1. This study compares brain levels of acetylcholine (ACh) and choline, and ACh brain turnover in 3 inbred strains of mice, CF-1, C57B1/6 By and Balb-c-By; these parameters were measured in control (aggregated) mice as well as following two paradigms, mild footshock and isolation, applied alone or in combination. 2. 2. Enzymic radioassay method was used to measure brain ACh and choline; ACh turnover was evaluated by two computation methods. 3. 3. Aggregated Balb-c-By mice exhibited low ACh and choline levels as compared to those of the mice of the two other strains. ACh levels of C57B1/6 By and CF-1 mice did not differ significantly; choline levels of CF-1 mice were almost twofold higher than those of C57B1/6 By mice. 4. 4. ACh turnover of aggregated CF-1 mice was about twice as high as that of C57B1/6 By and more than twice higher than that of Balb-c-By mice. 5. 5. The effects of shock, isolation, or shock combined with isolation on the brain levels of ACh and choline of mice of the 3 strains were inconsistent and, frequently absent. 6. 6. ACh turnover was significantly decreased in the brains of CF-1 by the two paradigms, whether employed alone or in combination; there was no additive effect when footshock was combined with isolation. 7. 7. ACh turnover did not change consistently after the two paradigms, employed alone or in combination, in the case of C57B1/6 By and Balb-c-By mice; in some cases, ACh turnover was increased. 8. 8. Footshock proved to be a mild inducer of aggression as compared to isolation in the 3 mice strains; footshock combined with isolation proved to synergize with respect to aggression in the 3 strains. Whether exposed to footshock and isolation alone or to the combination of these procedures, CF-1 mice were significantly more aggressive than Balb-c-By and C57B1/6 By mice. 9. 9. It is emphasized that consistent facilitatory effect of the two paradigms on aggression as well as additive effect on aggression resulting from combining footshock and isolation should be contrasted with the inconsistent effects of the two paradigms, applied alone or in isolation, on ACh turnover; altogether, the data did not indicate that aggression is related to increased brain ACh turnover.

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.

Factors affecting action of acetylcholine on transmembrane flux of K in isolated rabbit atria.

The effects of temperature, cocaine, anoxia, acetylcholine (ACh), K, Na and Ca on influx and efflux of K in isolated rabbit atria have been studied. Increasing the K concentration in the medium (K0) increases both efflux and influx of K; having a much greater effect on the latter. Both fluxes were estimated to be equal at a K0 of 6.0–6.5 mm. ACh increases both influx and efflux. An increase of K0 hardly modifies the effects of ACh in efflux, but greatly enhances its effect on influx. In the presence of ACh both fluxes were estimated to be equal at a K0 of 4.0–4.5 mm. Studies on the influence of temperature, cocaine and anoxia suggest that at K0's less than 5.4 the two fluxes are passive in nature. At K0's above this, influx appears to have an active component. Evidence is presented which suggest that at low K0's ACh affects only passive movements of K, whereas at higher K0's the agent stimulates active transport. A decrease of Na in the medium to one-fifth depresses efflux and influx about 50% when the Ca concentration is maintained at a normal level. A decrease of Ca blocks the effect of low Na. These findings are interpreted to mean that Ca either increases passive movements of K or determines the degree of coupling between Na and K fluxes.