Inhibition Of ACh Release Research Articles

Opposite effects of moderate heat stress and hyperthermia on cholinergic system of soil nematodes Caenorhabditis elegans and Caenorhabditis briggsae

Cholinergic system plays important role in all functions of organisms of free-living soil nematodes C. elegans and C. briggsae. Using pharmacological analysis we showed the existence of two opposite responses of nematodes cholinergic system to moderate and extreme heat stress. Short-term (15min) noxious heat (31–32°C) caused activation of cholinergic synaptic transmission in C. elegans and C. briggsae organisms by sensitization of nicotinic ACh receptors. In contrast, hyperthermia blocked cholinergic synaptic transmission by inhibition of ACh secretion by neurons. The resistance of behavior to extreme high temperature (36–37°C) was significantly higher in C. briggsae than in C. elegans, and thermostability of cholinergic transmission correlated with resistance of behavior to hyperthermia. Activation of cholinergic transmission by moderate heat stress can be the reason of movement speed increase in such adaptive behavior as noxious heat escape. Inhibition of ACh release is one of reasons for behavior failure caused by extreme high temperature since partial inhibition of ACh-esterase by aldicarb protected C. elegans and C. briggsae behavior against hyperthermia. Antagonist of mAChRs atropine almost completely prevented the rise in behavior thermotolerance caused by aldicarb. Pilocarpine, agonist of mAChRs, protected nematodes behavior against hyperthermia similarly with aldicarb. Therefore it is evident that it is the deficiency of mAChRs activity that is the reason for nematodes' behavior failure by hyperthermia.

Effects of κ- and μ-opioid agonists on cholinergic neurotransmission and contraction in isolated bovine trachealis

The effects of the selective μ-opioid agonist DAMGO and the selective κ-opioid agonist U-50488H on tritiated acetylcholine release ([3H]-ACh) and contractile responses to electrical stimulation (ES) were simultaneously determined in isolated bovine trachealis. The inhibitory effect of DAMGO 10−5M on [3H]-ACh release was not significantly different from the effect of the non-selective muscarinic agonist pilocarpine 10−5M, whereas the effect of U-50488H 10−5M was significantly greater. The effects of both opioids were not significantly different when muscles were pre- or co-incubated with the unselective muscarinic antagonist atropine 10−7M. Both DAMGO and U-50488H attenuated ES-induced contraction and this effect was significantly correlated with the inhibition of [3H]ACh-release (r2=0.8552). These data suggest that (1) opioids are important modulators of airway smooth muscle tone, (2) their effect is not altered by the activity of muscarinic autoregulation, and (3) their inhibitory effect of airway smooth muscle contraction can be almost totally explained by inhibition of ACh release.

Tuning adenosine A 1 and A 2A receptors activation mediates l-citrulline-induced inhibition of [ 3H]-acetylcholine release depending on nerve stimulation pattern

The influence of nerve stimulation pattern on transmitter release inhibition by l-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. l-Citrulline (10–470 μM), the NOS substrate l-arginine (10–470 μM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1–10 μM), concentration-dependently inhibited [ 3H]-acetylcholine ([ 3H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [ 3H]-ACh release inhibition by l-arginine (47 μM) and l-citrulline (470 μM), whereas the effect of SIN-1 (10 μM) remained unchanged. NOS inhibition with N ω-nitro- l-arginine (100 μM) prevented the effect of l-arginine, but not that of l-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-( p-nitrobenzyl)-6-thioinosine (10 μM), attenuated release inhibition by l-arginine and l-citrulline. With 5 Hz-trains, blockade of A 1 receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A 2A receptors with ZM241385 (10 nM), reduced the inhibitory action of l-arginine and l-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M 2 autoreceptors with AF-DX116 (10 nM) also attenuated the effects of l-arginine and l-citrulline with 50 Hz-bursts. l-Citrulline (470 μM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 μM), without significantly ( P > 0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived l-citrulline negatively modulates [ 3H]-ACh release by increasing adenosine outflow channelling to A 1 and A 2A receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A 1 receptors during 5 Hz-trains, inhibition of ACh release by l-citrulline at 50 Hz-bursts depends on the interplay between adenosine A 2A and muscarinic M 2 receptors.

Presynaptic serotonergic modulation of 5-HT and acetylcholine release in the hippocampus and the cortex of 5-HT 1B-receptor knockout mice

Lesioning of serotonergic afferents increases hippocampal ACh release and attenuates memory deficits produced by cholinergic lesions. Improved memory performance described in 5-HT 1B-knockout (KO) mice might thus be due to a weaker 5-HT 1B-mediated inhibitory influence of 5-HT on hippocampal ACh release. The selective delay-dependent impairment of working memory observed in these KO mice suggests, however, that cortical regions also participate in task performance, possibly via indirect influences of 5-HT on ACh release. To provide neuropharmacological support for these hypotheses we measured evoked ACh and 5-HT release in hippocampal and cortical slices of wild-type (WT) and 5-HT 1B KO mice. Superfused slices (preincubated with [ 3H]choline or [ 3H]5-HT) were electrically stimulated in the absence or presence of 5-HT 1B receptor ligands. In hippocampus and cortex, 5-HT 1B agonists decreased and antagonists increased 5-HT release in WT, but not in 5-HT 1B KO mice. In 5-HT 1B KO mice, 5-HT release was enhanced in both structures, while ACh release (in nCi) was reduced. ACh release was inhibited by 5-HT 1B agonists in hippocampal (not cortical) slices of WT but not of 5-HT 1B KO mice. Our data (i) confirm the absence of autoinhibition of 5-HT release in 5-HT 1B-KO mice, (ii) demonstrate a reduced release of ACh, and the absence of 5-HT 1B-receptor-mediated inhibition of ACh release, in the hippocampus and cortex of 5-HT 1B-KO mice, and (iii) are compatible with an indirect role of cortical ACh in the working memory impairment observed in these KO mice.

Short-term synaptic plasticity in rabbit pancreatic ganglia

The extrinsic innervation of the pancreas converges on a plexus of intrinsic pancreatic ganglia whose cholinergic neurons innervate acini, ducts, islets and blood vessels. Therefore, understanding ganglionic transmission is essential for understanding neural control of pancreatic secretion. Intracellular recordings of nicotinic fast excitatory postsynaptic potentials (fEPSPs) and action potentials (APs) were used to characterize and compare transmission in ganglia from the head/neck and body regions of the rabbit pancreas. Paired-pulse facilitation (PPF) or depression (PPD) of fEPSPs was observed in ganglia from both regions with PPF peaking and disappearing at shorter inter-stimulus intervals than PPD. PPF was most frequent in the head/neck (60%) and PPD (50%) in the body. Repetitive stimulation (10 Hz/5 s) evoked multiple forms of mid- and post-train plasticity. Facilitation during the first 1–2 s of train stimulation was reduced or reversed with continued stimulation due to development of synaptic depression and mid-train depression was of greater magnitude in the head/neck region. A brief (∼ 10 s) post-train augmentation was followed by a 1–2 min post-train depression that appeared to result from inhibition of ACh release. Regional differences in the frequency, magnitude, or duration of all forms of synaptic plasticity suggested regional differences in the extrinsic innervation patterns and possibly the function of pancreatic ganglia. In conclusion, rabbit pancreatic ganglia exhibit multiple forms of short-term synaptic plasticity that markedly alter the probability of postsynaptic firing, consistent with these ganglia being critical sites of synaptic integration and autonomic regulation of pancreatic secretion.

Multiple presynaptic and postsynaptic sites of inhibitory modulation by myomodulin at ARC neuromuscular junctions of Aplysia.

The functional activity of even simple cellular ensembles is often controlled by surprisingly complex networks of neuromodulators. One such network has been extensively studied in the accessory radula closer (ARC) neuromuscular system of Aplysia. The ARC muscle is innervated by two motor neurons, B15 and B16, which release modulatory peptide cotransmitters to shape ACh-mediated contractions of the muscle. Previous analysis has shown that key to the combinatorial ability of B15 and B16 to control multiple parameters of the contraction is an asymmetry in their peptide modulatory actions. B16, but not B15, releases myomodulin, which, among other actions, inhibits the contraction. Work in single ARC muscle fibers has identified a distinctive myomodulin-activated K current as a candidate postsynaptic mechanism of the inhibition. However, definitive evidence for this mechanism has been lacking. Here, working with the single fibers and then motor neuron-elicited excitatory junction potentials (EJPs) and contractions of the intact ARC muscle, we have confirmed two central predictions of the K-current hypothesis: the myomodulin inhibition of contraction is associated with a correspondingly large inhibition of the underlying depolarization, and the inhibition of both contraction and depolarization is blocked by 4-aminopyridine (4-AP), a potent and selective blocker of the myomodulin-activated K current. However, in the intact muscle, the experiments revealed a second, 4-AP-resistant component of myomodulin inhibition of both B15- and B16-elicited EJPs. This component resembles, and mutually occludes with, inhibition of the EJPs by another peptide modulator released from both B15 and B16, buccalin, which acts by a presynaptic mechanism, inhibition of ACh release from the motor neuron terminals. Direct measurements of peptide release showed that myomodulin also inhibits buccalin release from B15 terminals. At the level of contractions, nevertheless, the postsynaptic K-current mechanism is responsible for much of the myomodulin inhibition of peak contraction amplitude. The presynaptic mechanism, which is most evident during the initial build-up of the EJP waveform, underlies instead an increase of contraction latency.

Modulation of ACh release from airway cholinergic nerves in horses with recurrent airway obstruction.

To evaluate the functional status of neuronal alpha2-adrenoceptors (ARs) and beta2-ARs on ACh release in horses with recurrent airway obstruction (RAO), we examined the effects of the physiological agonists epinephrine (Epi) and norepinephrine (NE) and the beta2-agonists RR- and RR/SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the beta2-agonist, increases ACh release from airways of control horses only after the autoinhibitory muscarinic receptors are blocked by atropine, we also tested the hypothesis that if there is an M2-receptor dysfunction in equine RAO, SS-formoterol should increase ACh release even in the absence of atropine. ACh release was evoked by electrical field stimulation and measured by HPLC. Epi and NE caused less inhibition of ACh release in horses with RAO than in control horses. At the catecholamine concentration achieved during exercise (10(-7) M), the inhibition induced by Epi and NE was 10.8 +/- 13.2 and 3.4 +/- 6.8%, respectively, in equine RAO versus 41.0 +/- 6.4 and 27.1 +/- 5.6%, respectively, in control horses. RR- and RR/SS-formoterol (10(-8) to 10(-5) M) increased ACh release to a similar magnitude as that in control horses. These results indicate that neuronal beta2-ARs are functioning; however, the alpha2-ARs are dysfunctional in the airways of horses with RAO in response to circulating catecholamines. SS-formoterol (10(-8) to 10(-5) M) facilitated ACh release in horses with RAO even in the absence of atropine. Addition of atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone. The magnitude of augmentation in horses with RAO in the absence of atropine was similar to that in control horses in the presence of atropine. The latter observations could be explained by neuronal muscarinic-autoreceptor dysfunction in equine RAO.

Lambert-Eaton syndrome antibodies inhibit acetylcholine release and P/Q-type Ca2+ channels in electric ray nerve endings.

1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.

New Generic Approach to the Treatment of Organophosphate Poisoning: Adenosine Receptor Mediated Inhibition of Ach-Release

Current treatment of acute organophosphate (OP) poisoning includes a combined administration of a cholinesterase reactivator (oxime), a muscarinic receptor antagonist (atropine) and an anticonvulsant (diazepam). This treatment is not adequate since it does not prevent neuronal brain damage and incapacitation. Here, as in a recent review' it is stated that other therapeutic approaches may improve protection.Former studies on the “direct effects” of oximes led to the conclusion that drug-induced inhibition of acetylcholine (ACh)-release shortly (I min) after the acute OP-intoxication, could prevent and counteract convulsions and improve survival. in general, the accumulation of ACh in the synaptic cleft is considered to be responsible for the symptoms that ultimately lead to death. Therefore, prevention or suppression of this excessive accumulation of ACh could be a generic approach to antagonize OP-poisoning. Preliminary evidence for this concept has been put forward 1,2.Evaluation of drugs that would be able to prevent and counteract ACh accumulation, led to the conclusion that adenosine receptor agonists could be promising candidates.Pilot experiments demonstrated that intramuscular administration of the adenosine receptor agonists NEC A (5′-N-ethylcarboxamido-adenosine) or CPA (N6-cyclopentyl adenosine) 1 min following a subcutaneous soman poisoning (1.5 -2LD50) in rats, resulted in (I) prevention or postponement of chewing, salivation, convulsive activity, and respiratory distress (cholinergic symptoms), (2) improvement of survival rate (24 h), (3) a low level of extracellular brain ACh, as opposed to high levels of extracellular brain ACh in untreated animals.It is concluded that (1) adenosine agonists protect acutely soman-poisoned rats without the need of additional treatment with atropine, oxime or diazepam, (2) prevention of ACh accumulation in this way may be a new generic approach in the treatment of OP-poisoning.

Effect of subtype-specific Ca(2+)-antagonists and Ca(2+)-free media on the field stimulation-evoked release of ATP and [3H]acetylcholine from rat habenula slices.

The involvement of different subtypes of voltage-sensitive (Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACH were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca(2+)-channel blocker omega-conotoxin GVIA (omega-CgTX, 0.01-1 microM) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 microM) and the inorganic Ca(2+)-channel blocker Ca2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca(2+)-free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 microM), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca(2+)-free media. Tetrodotoxin (TTX, 1 microM), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [CA2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.

Alpha-adrenergic and muscarinic cholinergic inhibition of ACh release in guinea pig trachea: role of neuronal K+ channels

Our goals were to establish that an alpha 2-adrenergic agonist (clonidine) inhibits ACh release from airway nerve endings and to test effects of iberiotoxin (IBTX), an inhibitor of fast-conductance, Ca(2+)-activated K+ channels on alpha 2-adrenergic and muscarinic attenuation of ACh release. Guinea pig tracheas were mounted between electrodes in buffer containing indomethacin and neostigmine, and high-performance liquid chromatography with electrochemical detection was used to measure ACh release during electrical field stimulation. Clonidine inhibited ACh release in a concentration-dependent fashion [maximum reduction: 48 +/- 3%; 50% inhibitory constant (IC50): 0.1 microM], and idazoxan, alpha 2-adrenergic antagonist, reversed the effect. However, IBTX failed to alter clonidine-induced attenuation of ACh release. In contrast, IBTX did cause an increase in tracheal tension. In addition, IBTX failed to reverse any of the potent autoinhibitory effects of endogenous ACh. Our results confirm the presence of inhibitory alpha 2-adrenergic receptors. However, activation of IBTX-sensitive K+ channels does not appear necessary for either alpha 2-adrenergic or muscarinic cholinergic inhibition of ACh release.