Control Of Acetylcholine Release Research Articles

Lung Edema and Mortality Induced by Intestinal Ischemia and Reperfusion Is Regulated by VAChT Levels in Female Mice

Acute lung injury induced by intestinal ischemia/reperfusion (I/R) is a relevant clinical condition. Acetylcholine (ACh) and the α7 nicotinic ACh receptor (nAChRα-7) are involved in the control of inflammation. Mice with reduced levels of the vesicular ACh transporter (VAChT), a protein responsible for controlling ACh release, were used to test the involvement of cholinergic signaling in lung inflammation due to intestinal I/R. Female mice with reduced levels of VAChT (VAChT-KDHOM) or wild-type littermate controls (WT) were submitted to intestinal I/R followed by 2 h of reperfusion. Mortality, vascular permeability, and recruitment of inflammatory cells into the lung were investigated. Parts of mice were submitted to ovariectomy (OVx) to study the effect of sex hormones or treated with PNU-282,987 (nAChRα-7 agonist). A total of 43.4% of VAChT-KDHOM-I/R mice died in the reperfusion period compared to 5.2% of WT I/R mice. The I/R increased lung inflammation in both genotypes. In VAChT-KDHOM mice, I/R increased vascular permeability and decreased the release of cytokines in the lung compared to WT I/R mice. Ovariectomy reduced lung inflammation and permeability compared to non-OVx, but it did not avoid mortality in VAChT-KDHOM-I/R mice. PNU treatment reduced lung permeability, increased the release of proinflammatory cytokines and the myeloperoxidase activity in the lungs, and prevented the increased mortality observed in VAChT-KDHOM mice. Cholinergic signaling is an important component of the lung protector response against intestinal I/R injury. Decreased cholinergic signaling seems to increase pulmonary edema and dysfunctional cytokine release that increased mortality, which can be prevented by increasing activation of nAChRα-7.

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

BackgroundVarious protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction.ResultsWe use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca2+, stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3′,5′-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion.ConclusionsTogether, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

Age-dependent contribution of P/Q- and R-type Ca2+ channels to neuromuscular transmission in lethargic mice.

β-Subunits of voltage-gated calcium channels (VGCCs) regulate assembly and membrane localization of the pore-forming α1-subunit and strongly influence channel function. β4-Subunits normally coassociate with α1A-subunits which comprise P/Q-type (Cav2.1) VGCCs. These control acetylcholine (ACh) release at adult mammalian neuromuscular junctions (NMJs). The naturally occurring lethargic (lh) mutation of the β4-subunit in mice causes loss of the α1-binding site, possibly affecting P/Q-type channel expression or function, and thereby ACh release. End-plate potentials and miniature end-plate potentials were recorded at hemidiaphragm NMJs of 5-7-week and 3-5-month-old lh and wild-type (wt) mice. Sensitivity to antagonists of P/Q- [ω-agatoxin IVA (ω-Aga-IVA)], L- (nimodipine), N- (ω-conotoxin GVIA), and R-type [C192H274N52O60S7 (SNX-482)] VGCCs was compared in juvenile and adult lh and wt mice. Quantal content (m) of adult, but not juvenile, lh mice was reduced compared to wt. ω-Aga-IVA (~60%) and SNX-482 (~ 45%) significantly reduced m in adult lh mice. Only Aga-IVA affected wt adults. In juvenile lh mice, ω-Aga-IVA and SNX-482 decreased m by >75% and ~20%, respectively. Neither ω-conotoxin GVIA nor nimodipine affected ACh release in any group. Immunolabeling revealed α1E and α1A, β1, and β3 staining at adult lh, but not wt NMJs. Therefore, in lh mice, when the β-subunit that normally coassociates with α1A to form P/Q channels is missing, P/Q-type channels partner with other β-subunits. However, overall participation of P/Q-type channels is reduced and compensated for by R-type channels. R-type VGCC participation is age-dependent, but is less effective than P/Q-type at sustaining NMJ function.

Neuronally expressed Ras‐family GTPase Di‐Ras modulates synaptic activity in Caenorhabditis elegans

Ras-family GTPases regulate a wide variety of cellular functions including cell growth and differentiation. Di-Ras, which belongs to a distinct subfamily of Ras-family GTPases, is expressed predominantly in brain, but the role of Di-Ras in nervous systems remains totally unknown. Here, we report that the Caenorhabditis elegans Di-Ras homologue drn-1 is expressed specifically in neuronal cells and involved in synaptic function at neuromuscular junctions. Loss of function of drn-1 conferred resistance to the acetylcholinesterase inhibitor aldicarb and partially suppressed the aldicarb-hypersensitive phenotypes of heterotrimeric G-protein mutants, in which acetylcholine release is up-regulated. drn-1 mutants displayed no apparent defects in the axonal distribution of the membrane-bound second messenger diacylglycerol (DAG), which is a key stimulator of acetylcholine release. Finally, we have identified EPAC-1, a C.elegans Epac homologue, as a binding partner for DRN-1. Deletion mutants of epac-1 displayed an aldicarb-resistant phenotype as drn-1 mutants. Genetic analysis of drn-1 and epac-1 showed that they acted in the same pathway to control acetylcholine release. Furthermore, DRN-1 and EPAC-1 were co-immunoprecipitated. These findings suggest that DRN-1 may function cooperatively with EPAC-1 to modulate synaptic activity in C.elegans.

The Gα12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans

In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Galpha12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. We showed previously that RHO-1 stimulates ACh release by two separate pathways-one that requires UNC-13 and a second that does not. We show here that a non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons.

Acetylcholine Release at Neuromuscular Junctions of Adult Tottering Mice Is Controlled by N-(Cav2.2) and R-Type (Cav2.3) but Not L-Type (Cav1.2) Ca2+Channels

The mutation in the alpha(1A) subunit gene of the P/Q-type (Ca(v)2.1) Ca(2+) channel present in tottering (tg) mice causes ataxia and motor seizures that resemble absence epilepsy in humans. P/Q-type Ca(2+)channels are primarily involved in acetylcholine (ACh) release at mammalian neuromuscular junctions. Unmasking of L-type (Ca(v)1.1-1.2) Ca(2+) channels occurs in cerebellar Purkinje cells of tg mice. However, whether L-type Ca(2+) channels are also up-regulated at neuromuscular junctions of tg mice is unknown. We characterized thoroughly the pharmacological sensitivity of the Ca(2+) channels, which control ACh release at adult tg neuromuscular junctions. Block of N- and R-type (Ca(v)2.2-2.3), but not L-type Ca(2+) channels, significantly reduced quantal content of end-plate potentials in tg preparations. Neither resting nor KCl-evoked miniature end-plate potential frequency differed significantly between tg and wild type (WT). Immunolabeling of Ca(2+) channel subunits alpha(1A), alpha(1B), alpha(1C), and alpha(1E) revealed an apparent increase of alpha(1B), and alpha(1E) staining, at tg but not WT neuromuscular junctions. This presumably compensates for the deficit of P/Q-type Ca(2+)channels, which localized presynaptically at WT neuromuscular junctions. No alpha(1C) subunits juxtaposed with pre- or postsynaptic markers at either WT or tg neuromuscular junctions. Thus, in adult tg mice, immunocytochemical and electrophysiological data indicate that N- and R-type channels both assume control of ACh release at motor nerve terminals. Recruitment of alternate subtypes of Ca(2+) channels to control transmitter release seems to represent a commonly occurring method of neuronal plasticity. However, it is unclear which conditions underlie recruitment of Ca(v)2 as opposed to Ca(v)1-type Ca(2+) channels.

Ecto-AMP deaminase blunts the ATP-derived adenosine A2A receptor facilitation of acetylcholine release at rat motor nerve endings.

At synapses, ATP is released and metabolised through ecto-nucleotidases forming adenosine, which modulates neurotransmitter release through inhibitory A1 or facilitatory A2A receptors, according to the amounts of extracellular adenosine. Neuromuscular junctions possess an ecto-AMP deaminase that can dissociate extracellular ATP catabolism from adenosine formation. In this study we have investigated the pattern of ATP release and its conversion into adenosine, to probe the role of ecto-AMP deaminase in controlling acetylcholine release from rat phrenic nerve terminals. Nerve-evoked ATP release was 28 +/- 12 pmol (mg tissue)-1 at 1 Hz, 54 +/- 3 pmol (mg tissue)-1 at 5 Hz and disproportionally higher at 50 Hz (324 +/- 23 pmol (mg tissue)-1). Extracellular ATP (30 microM) was metabolised with a half time of 8 +/- 2 min, being converted into ADP then into AMP. AMP was either dephosphorylated into adenosine by ecto-5'-nucleotidase (inhibited by ATP and blocked by 200 microM alpha,beta-methylene ADP) or deaminated into IMP by ecto-AMP deaminase (inhibited by 200 microM deoxycoformycin, which increased adenosine formation). Dephosphorylation and deamination pathways also catabolised endogenously released adenine nucleotides, since the nerve-evoked extracellular AMP accumulation was increased by either alpha,beta-methylene ADP (200 microM) or deoxycoformycin (200 microM). In the presence of nitrobenzylthioinosine (30 microM) to inhibit adenosine transport, deoxycoformycin (200 microM) facilitated nerve-evoked [3H]acetylcholine release by 77 +/- 9 %, an effect prevented by the A2A receptor antagonist, ZM 241385 (10 nM). It is concluded that, while ecto-5'-nucleotidase is inhibited by released ATP, ecto-AMP deaminase activity transiently blunts adenosine formation, which would otherwise reach levels high enough to activate facilitatory A2A receptors on motor nerve terminals.

Evolving concepts in functional gastrointestinal disorders: promising directions for novel pharmaceutical treatments

In recent years there has been an increasing appreciation of the complexity of functional gastrointestinal disorders. These represent a spectrum of conditions which may affect any part of the gastrointestinal tract in which there appears to be dysregulation of visceral function and afferent sensation and a strong association with emotional factors and stress. There is a clear psychological dimension, with up to 60% of irritable bowel syndrome (IBS) patients reported to have psychological co-morbidities and altered pain perception is also common in comparison with control populations. The role of the enteric nervous system, the sensory pathways and the brain as well as the influence of the latter on sympathetic and parasympathetic outflow have likewise attracted increasing interest and have led to exciting new methods to study their complex interactions. The concept of low-grade inflammation, such as might occur after infection, acting as a trigger for neuromuscular dysfunction has also led to the broad integrative hypotheses that help to explain the biopsychosocial dimensions seen in functional gastrointestinal disease. The multi-component model places a major emphasis on neurogastroenterology and enteric and neuro-immune interactions where new approaches to pharmacotherapy lie. Drugs may affect motility, visceral sensation and other aspects of gut function such as secretion or absorption. More particularly, however, has been the search for and attempts to influence important mediators of these primary gut functions. Such targets include serotonin and selected 5-HT receptors, which are involved in gut motility, visceral sensation and other aspects of gut function, CCK receptors which are involved in the mediation of pain in the gut and nociception in the CNS, opioid receptors involved in pain in the brain, spinal cord and periphery, muscarinic M 3-receptors, substance P and neurokinin A and B receptors which are involved in motor adaptation and pain transmission in association with inflammation, gabba receptors involved in nociception and cannabinoid receptors which are involved in the control of acetyl choline release in the gut. With a better understanding of the structures and pathways involved in visceral perception and hyperalgesia, in the CNS, spinal cord and the gut and new pharmacological tools we will be better able to elucidate the neuropharmacology of visceral perception and its relationship to gut dysfunction. It is likely that there will be multiple therapeutic options based on the spectrum of abnormalities capable of causing the spectrum of symptoms of functional gastrointestinal disorders in any individual patient.

Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release.

Injections of inositol trisphosphate (IP3) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysia californica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca2+ release from various Ca2+ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP3-induced calcium release with heparin, abolished the effects of IP3, but not the effects of NAADP, suggesting the presence of an intracellular Ca2+ pool independent of those containing ryanodine receptors (RyR) or IP3 receptors. To reinforce electrophysiological observations, intracellular [Ca2+]i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca2+ concentration. RyR- and IP3-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP3-dependent, and NAADP-dependent Ca2+ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca2+ concentration that triggers transmitter release.

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype.

1. Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2. In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 microM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%). 3. Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 microM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM-1 microM), and enhanced by atropine (0.1-100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. 4. The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (-log EC50) orders at pre- and postjunctional level were: atropine > or = 4-diphenyl-acetoxy-N-piperidine (4-DAMP) > mamba toxin 3 (MT-3) > tripitramine > para-fluorohexahydrosiladiphenidol (pF-HHSiD) > or = methoctramine > or = pirenzepine > tripinamide, and atropine > or = 4-DAMP > pF-HHSiD >> pirenzepine = tripitramine > tripinamide > methoctramine >> MT-3, respectively. 5. The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

Tachykinins increase [ 3H]acetylcholine release in mouse striatum through multiple receptor subtypes

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar 9,Met(O 2) 11]substance P, [β-ala 8]neurokinin A 4–10 and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase–polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar 9,Met(O 2) 11]substance P, [β-ala 8]neurokinin A 4–10 and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 μM), a phospholipase C inhibitor, blocked [sar 9,Met(O 2) 11]substance P- and senktide-induced acetylcholine release, but had no effect on [β-ala 8]neurokinin A 4–10-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 μM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists ω-conotoxin GVIA (N type), nifedipine and nimodipine (L type), ω-conotoxin MVIIC and ω-agatoxin IVA (P/Q type), and Ni 2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.

Dopamine D 1 receptor-mediated control of striatal acetylcholine release by endogenous dopamine

The role of dopamine D 1 and D 2 receptors in the control of acetylcholine release in the dorsal striatum by endogenous dopamine was investigated by monitoring with microdialysis the effect of the separate or combined administration of the dopamine D 1 receptor antagonist, SCH 39166 {(−)- trans-6,7,7 a,8,9,13 b-exahydro-3-chloro-2-hydroxy- N-methyl-5 H-benzo-[ d]-naphto-[2,1 b]-azepine hydrochloride} (50 μg/kg subcutaneous (s.c.)), of the dopamine D 2/D 3 receptor agonist, quinpirole ( trans-(−)-4 aR,4 a,5,6,7,8,8 a,9-octahydro-5-propyl-1 H-pyrazolo-(3,4- g)-quinoline hydrochloride) (5 and 10 μg/kg s.c.), and of the D 3 receptor selective agonist, PD 128,907 [ S(+)-(4 aR,10 bR)-3,4,4 a,10 b-tetrahydro-4-propyl-2 H,5 H-[1]benzopyrano-[4,3- b]-1,4-oxazin-9-ol hydrochloride] (50 μg/kg s.c.), on in vivo dopamine and acetylcholine release. Microdialysis was performed with a Ringer containing low concentrations (0.01 μM) of the acetylcholinesterase inhibitor, neostigmine. Quinpirole (10 μg/kg s.c.) decreased striatal dopamine and acetylcholine release. Administration of PD 128,907 (50 μg/kg) decreased dopamine but failed to affect acetylcholine release. SCH 39166 (50 μg/kg s.c.) stimulated dopamine release and reduced acetylcholine release. Pretreatment with quinpirole reduced (5 μg/kg s.c.) or completely prevented (10 μg/kg s.c.) the stimulation of dopamine release elicited by SCH 39166 (50 μg/kg s.c.); on the other hand, pretreatment with quinpirole (5 and 10 μg/kg) potentiated the reduction of striatal acetylcholine release induced by SCH 39166 (50 μg/kg s.c.). Similarly, pretreatment with PD 128,907 (50 μg/kg) which prevented the increase of dopamine release induced by SCH 39166 (50 μg/kg), potentiated the reduction of striatal acetylcholine transmission elicited by SCH 39166. Thus, pretreatment with low doses of quinpirole or PD 128,907 influences in opposite manner the effect of SCH 39166 on striatal dopamine and acetylcholine release, counteracting the increase of dopamine release and potentiating the decrease in acetylcholine release. These results provide further evidence for the existence of a tonic stimulatory input of endogenous dopamine on striatal acetylcholine transmission mediated by dopamine D 1 receptors.

Calcium channels controlling acetylcholine release in the guinea-pig isolated anterior pelvic ganglion: an electropharmacological study

An electropharmacological analysis of the type(s) of calcium channel controlling neurotransmitter release in preganglionic sympathetic nerve terminals in the guinea-pig anterior pelvic ganglion has been carried out. Conventional intracellular recording techniques were used to record excitatory postsynaptic potentials as a measure of neurotransmitter release. Excitatory postsynaptic potentials were abolished by hexamethonium (30–100 μM) and are therefore mediated by acetylcholine acting at nicotinic receptors. In studies of more than 150 cells, the N-type calcium channel blocker ω-conotoxin GVIA (100–300 nM) failed to block the initiation of the nerve impulse by the excitatory postsynaptic potential. In single-cell studies, ω-conotoxin GVIA (1 μM) sometimes altered the configuration of the excitatory postsynaptic potential/cell body nerve action potential complex, but on only one occasion was the excitatory postsynaptic potential reduced below the threshold required to initiate the action potential. Nifedipine (10 μM), ω-agatoxin IVA (100 nM) and ω-conotoxin MVIIC (300 nM), applied alone or in combination with ω-conotoxin GVIA (300 nM), were also ineffective. However, excitatory postsynaptic potentials evoked by trains of stimuli (0.1–0.5 Hz) were markedly reduced or abolished by the non-specific calcium channel blocker ω-grammotoxin SIA (300 nM). When trains of stimuli were delivered at higher frequencies (4 Hz), the block induced by ω-grammotoxin SIA could be overcome, and excitatory postsynaptic potentials were able to initiate action potentials even when ω-conotoxin GVIA, ω-agatoxin IVA and ω-conotoxin MVIIC were also present. The calcium channel(s) controlling acetylcholine release was (were) blocked by low concentrations of cadmium ions (30 μM) at all stimulation frequencies studied (0.1–50 Hz). Thus, the dominant calcium channels controlling acetylcholine release in sympathetic ganglia are not the L, N, P or Q types. At low frequencies of stimulation, ω-grammotoxin SIA-sensitive calcium channels play a dominant role in acetylcholine release, but at higher stimulation frequencies yet another pharmacologically distinct calcium channel (or subtype) supports neurotransmitter release.

Induction of choline acetyltransferase mRNA in human mononuclear leukocytes stimulated by phytohemagglutinin, a T-cell activator

The induction of mRNA for choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis was investigated in human mononuclear leukocytes (MNL) stimulated by phytohemagglutinin (PHA), a T-cell activator, using the reverse transcription-polymerase chain reaction. Stimulation of MNL by PHA induced the expression of ChAT mRNA, and potentiated ACh synthesis. ChAT mRNA induction required more time than the induction of interleukin-2 mRNA. Expression of the gene encoding the vesicular ACh transporter, which mediates ACh transport in cholinergic neurons, was not observed in PHA-stimulated MNL, suggesting that the mechanisms controlling ACh release from T-lymphocytes differ from those in cholinergic neurons. These findings demonstrate that activation of T-lymphocytes up-regulates ACh synthesis in the blood, and suggest that ACh plays an important role as a neuroimmunomodulator besides its role as a neurotransmitter.

Differential Localization of Voltage-Dependent Calcium Channel α1Subunits at the Human and Rat Neuromuscular Junction

Neurotransmitter release is regulated by voltage-dependent calcium channels (VDCCs) at synapses throughout the nervous system. At the neuromuscular junction (NMJ) electrophysiological and pharmacological studies have identified a major role for P- and/or Q-type VDCCs in controlling acetylcholine release from the nerve terminal. Additional studies have suggested that N-type channels may be involved in neuromuscular transmission. VDCCs consist of pore-forming alpha1 and regulatory beta subunits. In this report, using fluorescence immunocytochemistry, we provide evidence that immunoreactivity to alpha1A, alpha1B, and alpha1E subunits is present at both rat and human adult NMJs. Using control and denervated rat preparations, we have been able to establish that the subunit thought to correspond to P/Q-type channels, alpha1A, is localized presynaptically in discrete puncta that may represent motor nerve terminals. We also demonstrate for the first time that alpha1A and alpha1B (which corresponds to N-type channels) may be localized in axon-associated Schwann cells and, further, that the alpha1B subunit may be present in perisynaptic Schwann cells. In addition, the alpha1E subunit (which may correspond to R/T-type channels) seems to be localized postsynaptically in the muscle fiber membrane and concentrated at the NMJ. The possibility that all three VDCCs at the NMJ are potential targets for circulating autoantibodies in amyotrophic lateral sclerosis is discussed.

Opioid receptor-mediated control of acetylcholine release in human neocortex tissue.

The effects of various opioid receptor agonists and antagonists on evoked acetylcholine release were studied in slices of human neocortex prelabelled with [3H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K+ (20 mM). The delta-opioid receptor agonist DPDPE and the kappa-opioid receptor agonist U50488 reduced the evoked [3H]-overflow (acetylcholine release) in a concentration-dependent fashion; the delta-opioid receptor antagonist naltrindole and the kappa-opioid receptor antagonist norbinaltorphimine, respectively, antagonized these effects. Application of the mu-opioid receptor agonist DAGO also resulted in an inhibition of acetylcholine release; however, both delta- and kappa-opioid receptor antagonists were able to block this effect. The mu-opioid receptor agonists morphine and (+)-nortilidine had no effect. These results indicate that acetylcholine release in human neocortex is inhibited through delta- and kappa-opioid receptors, but not through mu-opioid receptors. Acetylcholine release was significantly increased by the delta-opioid receptor antagonist naltrindole in the presence of a mixture of peptidase inhibitors providing evidence for a delta-opioid receptor-mediated inhibition of acetylcholine release by endogenous enkephalin. K(+)-evoked acetylcholine release in the presence of TTX was inhibited by U50488, but not by DPDPE, suggesting the presence of kappa-opioid receptors on cholinergic terminals and the localization of delta-receptors on cortical interneurons. Therefore, the potent effect of DPDPE on acetylcholine release is likely to be indirect, by modulation of intrinsic cortical neurons. These interneurons probably do not use GABA as neurotransmitter since both GABAA and GABAB receptor agonists (muscimol and baclofen, respectively) were without effect on acetylcholine release.

Surgical anaesthesia with pentobarbital prevents the effect of local SCH 23390 on rat striatal acetylcholine release in a strain-dependent manner

This study was performed in order to clarify existing discrepancies about the ability of the D1 antagonist SCH 23390 to reduce striatal acetyicholine (ACh) release after intrastriatal application by reverse dialysis. The possibility that negative findings were related to the use of pentobarbital rather than Equithesin as surgical anaesthesia for implanting microdialysis probes, and of Wistar rather than Sprague-Dawley rats, was tested. SCH 23390, applied by reverse dialysis at the concentration of 24µM, although able to reduce dialysate ACh in male Wistar rats implanted under Equithesin anaesthesia, failed to do so in rats of the same strain implanted 24h or 3 days earlier under pentobarbital anaesthesia. In male Sprague-Dawley rats, local SCH 23390 (24µM) reduced striatal dialysate ACh, both in rats implanted under Equithesin as well as in rats implanted under pentobarbital anaesthesia. Systemic SCH 23390 (0.3mg/kg s.c.) reduced dialysate A Ch both in Wistar and in Sprague-Dawley rats implanted under pentobarbital anaesthesia, but was more effective in the Sprague-Dawley strain. These observations, although consistent with a striatal localization of D1-receptors controlling ACh release, can be explained as being the result of a strain-dependent barbiturate-induced inactivation of D1-mediated control of ACh transmission, which is potentiated by the local changes induced by microdialysis probe implant.

Down-regulation of non-L-, non-N-type (Q-like) Ca 2+ channels by Lambert-Eaton myasthenic syndrome (LEMS) antibodies in rat insulinoma RINm5F cells

The action exerted on non-L-, non-N-type (Q-like) Ca 2+ channels by immunoglobulins G (IgGs) obtained from two patients with Lambert-Eaton myasthenic syndrome (LEMS) was investigated in the rat insulinoma RINm5F cell line. LEMS IgGs reduced by 30–36% the whole-cell Ba 2+ currents through Q-like Ca 2+ channels at +10 mV without significantly modifying their voltage dependence and activation kinetics. Single- and multiple-channel recordings in cell-attached and outside-out patches of cells treated with LEMS IgGs showed no significant changes of the channel elementary properties but rather a decreased number of active channels per patch. This suggests that Q-like current depression by LEMS autoantibodies is mostly due to a down-regulation of functioning Ca 2+ channels. In agreement with previous observations, LEMS IgGs also reduced by 20–33% the dihydropyridine-sensitive (L-type) Bat+ current. The suggested down-regulation of Q-like channels by LEMS IgGs in RINm5F cells may have a functional correlation with the depressive action of LEMS autoantibodies on the PIQ-type Ca 2+ channels controlling acetylcholine release from mammalian neuromuscular junctions.

Regional distribution and ontogeny of 5-HT 4 binding sites in rat brain

The tritiated specific antagonist [ 3H]GR 113808 was used to study the brain regional distribution of 5-HT 4 binding sites in rat during development. In adult brain, high densities of binding sites were found in the limbic system (islands of Calleja, olfactory tubercle, fundus striati, ventral pallidum, septum, hippocampus and amygdala) and in the striato-nigro-tectal pathway. Only low densities of binding sites were found in the prenatal brain, except in the brainstem. Marked density increases were observed during the second and third postnatal week in most forebrain regions. This ontogenic pattern indicates that 5-HT 4 receptors probably exert a minor role in developmental processes, while, in the adult, the expression of 5-HT 4 receptors in limbic areas strongly suggests a role for these receptors in emotional functions. The synchronous appearance of 5-HT 4 receptors and cholinergic markers also indicates that these receptors might control acetylcholine release. This hypothesis has been confirmed by functional evidence and further supports the view that 5-HT 4 drugs could improve cognitive functions in diseases with impaired cholinergic transmission (e.g., Alzheimer's disease)