Although Acetylcholine Research Articles

Multi-frequency electromagnetic radiation induces anxiety in mice via inflammation in the cerebral cortex.

Modern life is filled with radiofrequency electromagnetic radiation (RF-EMR) in various frequency bands, while the health risks are not clear. In this study, mice were whole-body exposed to 0.9/1.5/2.65GHz radiofrequency radiation at 4 W/kg for 2h per day for 4weeks to investigate the emotional effects. It was found that the mice showed anxiety but no severe depression. The ELISA results showed a significant decrease in amino acid neurotransmitters (GABA, DA, 5-HT), although acetylcholine (ACH) levels were not significantly altered. Furthermore, Western blot results showed that BDNF, TrkB, and CREB levels were increased in the cerebral cortex, while NF-κB levels were decreased. In addition, pro-inflammatory factors (IL-6, IL-1β, TNF-α) were significantly elevated, and anti-inflammatory factors (IL-4, IL-10) tended to decrease. In conclusion, multi-frequency electromagnetic radiation induces an inflammatory response through the CREB-BDNF-TrkB and NF-κB pathways in the cerebral cortex and causes a decrease in excitatory neurotransmitters, which ultimately causes anxiety in mice.

Multiple cholinergic receptor subtypes coordinate dual modulation of acetylcholine on anterior and posterior paraventricular thalamic neurons.

Paraventricular thalamus (PVT) plays important roles in the regulation of emotion and motivation through connecting many brain structures including the midbrain and the limbic system. Although acetylcholine (ACh) neurons of the midbrain were reported to send projections to PVT, little is known about how cholinergic signaling regulates PVT neurons. Here, we used both RNAscope and slice patch-clamp recordings to characterize cholinergic receptor expression and ACh modulation of PVT neurons in mice. We found ACh excited a majority of anterior PVT (aPVT) neurons but predominantly inhibited posterior PVT (pPVT) neurons. Compared to pPVT with more inhibitory M2 receptors, aPVT expressed higher levels of all excitatory receptor subtypes including nicotinic α4, α7, and muscarinic M1 and M3. The ACh-induced excitation was mimicked by nicotine and antagonized by selective blockers for α4β2 and α7 nicotinic ACh receptor (nAChR) subtypes as well as selective antagonists for M1 and M3 muscarinic ACh receptors (mAChR). The ACh-induced inhibition was attenuated by selective M2 and M4 mAChR receptor antagonists. Furthermore, we found ACh increased the frequency of excitatory postsynaptic currents (EPSCs) on a majority of aPVT neurons but decreased EPSC frequency on a larger number of pPVT neurons. In addition, ACh caused an acute increase followed by a lasting reduction in inhibitory postsynaptic currents (IPSCs) on PVT neurons of both subregions. Together, these data suggest that multiple AChR subtypes coordinate a differential modulation of ACh on aPVT and pPVT neurons.

Dual-Layered Nanomaterial-Based Molecular Pattering on Polymer Surface Biomimetic Impedimetric Sensing of a Bliss Molecule, Anandamide Neurotransmitter

In this endeavor, a novel electrochemical biosensor was designed using multiwall carbon nanotubes (MWCNTs)- and nickel nanoparticles (NiNPs)-embedded anandamide (AEA) imprinted polymer. The NiNPs so synthesized were mortared with MWCNTs and molecularly imprinted polymer (MIP), which enhanced sensitivity and selectivity of the developed sensor, respectively. The characterization methods of AEA-based MIP included X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and Brunauer–Emmett–Teller (BET) analysis, which supported the successful synthesis of the polymer. Electrochemical studies of fabricated sensor were performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy in potentiostatic mode (PEIS). In this first phase of AEA-specific sensor development, MWCNT/NiNP/MIP@SPE was found to successfully discriminate between different concentrations of AEA. The developed sensing platform demonstrated a 100 pM–1 nM linear range with a 0.01 nM detection limit (LOD), 0.0149 mA/pM sensitivity, and 50% stability within 4 months. The sensor demonstrated selectivity toward AEA: although acetylcholine (ACh) and dopamine acted as strong interfering components because of their chemical similarity, the spiked AEA samples demonstrated ∼90% recoveries. Hence, our results have passed the first step in AEA detection at home, although with a clinical setup, future advancement is still required.

Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors

Although acetylcholine (ACh ) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a ‘cholinergic (nicotinic) anti-inflammatory pathway’. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of α1, α4, α5, α7, α10, β1 and β3 subunits, down-regulated α6 and β2 subunits, and did not alter the relative quantity of α9 and β4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-α gene was mediated predominantly by the α-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes—by the mecamylamine-sensitive nAChRs. Both the Mec- and αBtx-sensitive nAChRs regulated expression of the IL-1β gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through α7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.

Structure and Function of the Nicotinic Arm of Acetylcholine Regulatory Axis in Human Leukemic T Cells

Although acetylcholine (ACh) is widely known as a neurotransmitter, it also functions as a local humoral factor translating environmental stimuli into alterations in T cell development and function. The cholinergic components present in neurons are expressed in T cells where they constitute an independent cholinergic system. Both non-immunologic and immunologic stimulations can alter expression and function of cholinergic elements in T cells. Recent studies have convincingly demonstrated regulation of immune system by auto/paracrine ACh, which provides a basis for development of new immunomodulatory therapies with nicotinic agonists. The purpose of our research is to integrate information about the structure and activity of the ACh regulatory axis with the phenotypic and functional alterations of T cells during their development and commitment. In this study, we used the Ach producing human leukemic T cell line CCRF-CEM (CEM) to investigate auto/paracrine mechanisms of T cell regulation through the nicotinic class of ACh receptors (nAChRs). The intact CEM expressed alpha3, alpha5, alpha6, alpha7, alpha 9, beta2 and beta4 nAChR subunits. Stimulation of CEM with 10 microg/ml of phytohemagglutinin (PHA) for 16 h upregulated expression of the alpha3, alpha5, alpha7, alpha9 and beta2 and downregulated that of alpha6 and beta4 subunits, indicating that TCR activation leads to overexpression of high Ca2+-permeable ACh-gated ion channels. Activation of alpha7- and alpha3 AChRs predominantly abrogated PHA-dependent upregulation of the pro-inflammatory cytokine TNF-alpha and IFN-gamma receptors, respectively, at the mRNA and protein levels. Signaling through alpha7 and alpha3 nAChRs also significantly (p<0.05) altered expression of the cell state regulators p21 and Bcl-2, respectively, suggesting that downregulation of inflammation via nAChRs includes effects on the T cell cycle progression and apoptosis. These findings indicate that constant stimulation of alpha7 and alpha3 nAChRs by endogenously released ACh controls T cell activation and that signaling downstream of distinct nAChR subtypes targets specific inflammatory and cell cycle genes. Learning the cholinergic pharmacology of inflammation should allow to regulate specific types of immune reactions by selectively activating or blocking the types of nAChRs expressed by the immune cells mediating specific immune reactions.

Self-vasodilating ability at the spastic site of patients with vasospastic angina: estimation by acetylcholine delayed phase.

Deficient nitric oxide (NO) release is thought to be the principal mechanism of coronary spasm, however, the precise mechanisms are unknown. Although acetylcholine (ACh) is used for provocation of coronary spasm, ACh is also used for the augmentation of blood flow and flow-mediated vasodilation is induced. We estimated the self-vasodilating ability (endothelial function) at the spastic site of coronary arteries in patients with vasospastic angina (VSA) during the provocation test of coronary spasm by ACh. This study included 93 patients with VSA and 77 patients with atypical chest pain (ACP). Intracoronary injection of ACh (20, 50, and 100 microg) was performed over 30 seconds and the coronary artery diameter of the spastic site was measured 3 to 4 minutes after ACh injection (delayed phase). The ability of dilation (AOD) was calculated as: ([diameter of delayed phase-baseline diameter]/[diameter after isosorbide dinitrate-baseline diameter]) x 100 (%). No significant difference was noted between the AOD in patients with ACP and VSA (28 +/- 36 vs 15 +/- 60%, respectively). The AOD values of 49% of patients with VSA were greater than the mean value of AOD of patients with ACP. At least almost half of the patients with VSA may have preserved self-vasodilating ability at the spastic site, and an abnormality other than endothelial dysfunction is involved in the mechanism of coronary spasm in these patients.

Expression of acetylcholine in lymphocytes and modulation of an independent lymphocytic cholinergic activity by immunological stimulation

Although acetylcholine (ACh) is classically thought of as a neurotransmitter in mammalian species, lymphocytes possess most of the components needed to constitute an independent non-neuronal cholinergic system. These include ACh itself, choline acetyltransferase (ChAT), highaffinity choline transporter, acetylcholinesterase, and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca2+ concentration and stimulates c-fos gene expression and nitric oxide synthesis. Which subpopulations of T lymphocytes are the source of blood ACh is not yet known, though expression of ChATmRNA was observed in human peripheral CD4+ (helper) but not in CD8+ (cytotoxic) T cells. Stimulation of lymphocytes with phytohemagglutinin, a T-cell activator, or with monoclonal antibodies that bind the CD7 and/or CD11a cell surface molecules activates lymphocytic cholinergic activity, as evidenced by increased synthesis and release of ACh and up-regulation of expression of ChATand M5 mAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.

The Ach-evoked Ca2+-activated K+ current in mouse mandibular secretory cells. Single channel studies.

Although acetylcholine (ACh) is able to activate voltage- and Ca2+-sensitive K+ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K+ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K+ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 micromol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb+ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K+ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K+ current is not carried substantially by BK channels and show that it is carried by a small-conductance K+ channel with quite different properties.

Distribution of efferent cholinergic terminals and alpha-bungarotoxin binding to putative nicotinic acetylcholine receptors in the human vestibular end-organs.

Although acetylcholine (ACh) has been identified as the primary neurotransmitter of the efferent vestibular system in most animals studied, no direct evidence exists that ACh is the efferent neurotransmitter of the human vestibular system. Choline acetyltransferase immunohistochemistry (ChATi), acetylcholinesterase (AChE) histochemistry, and alpha-bungarotoxin binding were used in human vestibular end-organs to address this question. ChATi and AChE activity was found in numerous bouton-type terminals contacting the basal area of type II vestibular hair cells and the afferent chalices surrounding type I hair cells; alpha-bungarotoxin binding suggested the presence of nicotinic acetylcholine receptors on type II vestibular hair cells and on the afferent chalices surrounding type I hair cells. This study provides evidence that the human efferent vestibular axons and terminals are cholinergic and that the receptors receiving this innervation may be nicotinic.

Alterations in muscarinic K+ channel response to acetylcholine and to G protein-mediated activation in atrial myocytes isolated from failing human hearts.

A variety of previous studies have demonstrated reduced diastolic potential and electrical activity in atrial specimens from patients with heart disease. Although K+ channels play a major role in determining resting membrane potential and repolarization of the action potential, little is known about the effects of preexisting heart disease on human atrial K+ channel activity. We characterized the inwardly rectifying K+ channel (IKI) and the muscarinic K+ channel [IK(ACh)] in atrial myocytes isolated from patients with heart failure (HF) and compared electrophysiological characteristics with those from donors (control) by the patch-clamp technique. Resting membrane potentials of isolated atrial myocytes from HF were more depolarized (-51.1 +/- 9.7 mV, mean +/- SD, n = 30 patients) than those from donors (-73.0 +/- 7.2 mV, n = 4 patients, P < .001). The action potential duration in HF was longer than that in donors. Although acetylcholine (ACh) shortened the action potential, reduced the overshoot, and hyperpolarized the atrial cell membrane in HF, these effects were attenuated compared with those observed in donors. The whole-cell membrane current slope conductance in HF was small, the reversal potential was more positive, and the sensitivity to ACh was less compared with donors. In single-channel recordings from cell-attached patches, IK1 channel conductance and gating characteristics were the same in HF and donor atria. When ACh was included in the pipette solution, IK(ACh) was activated in both groups. Single-channel slope conductance of IK(ACh) averaged 42 +/- 3 pS (n = 28) in HF and 44 +/- 2 pS (n = 4) in donors, and mean open lifetime was 1.3 +/- 0.3 milliseconds (n = 24) in HF and 1.5 +/- 0.4 milliseconds (n = 4) in donors. These values were virtually identical in the two groups (not significantly different, NS), although both single IK1 and IK(ACh) channel densities were less in HF. Channel open probability of IK(ACh) was also less in HF (4.0 +/- 1.2%, n = 24) than in donors (6.8 +/- 1.1%, n = 3, P < .01). The concentration of ACh at half-maximal activation was 0.11 mumol/L in HF and 0.03 mumol/L in donors. In excised inside-out patches, IK(ACh) from HF required higher concentrations of GTP and GTP gamma S to activate the channel compared with donors. These results suggest a reduced IK(ACh) channel sensitivity to M2 cholinergic receptor-linked G protein (Gi) in HF compared with donors. Atrial myocytes isolated from failing human hearts exhibited a lower resting membrane potential and reduced sensitivity to ACh compared with donor atria. Whole-cell and single-channel measurements suggest that these alterations are caused by reduced IK1 and IK(ACh) channel density and reduced IK(ACh) channel sensitivity to Gi-mediated channel activation in HF.

Chapter 30: Nerve growth factor affects the cholinergic neurochemistry and behavior of aged rats

The major central cholinergic systems include (1) the projection neurons of the medial septum and diagonal band of Broca (MS/DB) to the hippocampus; (2) the projection neurons of the nucleus basalis magnocellularis (NBM) to the amygdala and cerebral cortex; (3) the inter-neurons of the striatum. Although the basal forebrain projection neurons have been implicated in learning and memory, and the striatal inter-neurons in motor behaviors, it still is not clear how and to what extent the central cholinergic neurons are involved in specific behaviors. Similarly, relatively little is known about the cellular and molecular mechanisms that regulate animal behaviors mediated by the central cholinergic systems. Although acetylcholine (ACh) was the first neurotransmitter discovered in the central nervous system, it remains unclear how the synthesis, storage, and release of ACh are regulated, or how ACh neurochemistry might be altered by environmental stimuli or synaptic experience, both of which would impact on cholinergically mediated behaviors. The central cholinergic systems are particularly dysfunctional in Alzheimer's disease. Thus, our animal research on the regulation of central cholinergic transmission and behavior has focused on the deficits in the systems associated with senescence and the potential amelioration of age-related deficits by the administration of the neurotrophic protein, nerve growth factor (NGF). This chapter describes the current understanding of the regional regulation of ACh synthesis and release in young and old rats, and illustrates several correlations between age-related and NGF-induced alterations in cholinergic neurochemistry, electrophysiology, morphology, and behavior.

The effects of histamine and some related compounds on conditioned avoidance response in rats

When histamine (Hi) and other agonists were applied intraventricularly, Hi caused a dose-dependent inhibition of the avoidance response in rats; its ED50 was 3.60 μg. l-methylHi, l-methylimidazole acetic acid and imidazole acetic acid which are major metabolites of Hi produced no inhibitory effect even at 50 μg. H 1-agonists (2-methylHi and 2-thiazolylethylamine) also depressed the avoidance response; their dose-response lines run parallel to that of Hi. The depressant effects of H 2-agonists (4-methylHi and dimaprit) were relatively weak; their dose-response lines were not parallel to that of Hi. When antagonists were pretreated intravenously, Hi action was clearly antagonized by diphehydramine and pyrilamine, but not by cimetidine or ranitidine. Intraventricular injection of Hi mixed with cimetidine or ranitidine did not change the effect induced by Hi alone. The avoidance response was not affected by noradrenaline, dopamine or 5-hydroxytryptamine. Although acetylcholine (ACh) suppressed the avoidance response dose-dependently, its effect was much weaker than that of Hi. Pretreatment with cholinergic blocking drugs (atropine and scopolamine) antagonized ACh action but not Hi action. From these results, it is assumed that the inhibitory effect of Hi on the avoidance response is preferentially linked to the H 1-receptor. After intraventicular application of 3HHi, the highest radioactivity was determined in the hypothalamus.

Human placental cholinergic system: Occurrence, distribution and variation with gestational age of acetylcholine in human placenta

Although acetylcholine (ACh)-like activity was demonstrated in human placental extracts by a number of investigators, substances responsible for this activity were not identified. We found by gas Chromatographic techniques that the major component of the ACh-like activity of the term placenta was ACh (112 ± 7 nmoles/g of wet tissue). These results were confirmed by the separation of ACh from other quaternary ammonium compounds by column chromalography using Amberlite CG-50 resin. The placenta could be stored at 4 for a number of days without significant loss of ACh. Freezing and thawing of the placenta destroyed ACh. This indicates that ACh is bound within membranes. There were high concentrations of ACh in all segments of the placenta. The ACh concentrations in the concentric segments next to the periphery and the umbilical cord were lower than ACh concentrations in other segments. The concentrations of ACh in floating villi and the basal plate, which included the anchoring villi, were about 322 and 210% respectively, of that of the chorionic plate. There was variation in ACh content with gestational age: the highest concentration was found at about 22 weeks (wk) of gestation (nmoles/g at 9–12 wk, 129; 13–16 wk, 342 ± 31; 17–20 wk, 317 ± 32; 21–24 wk, 723 ± 63; 25–28 wk, 231; 29–32 wk, 249; 33–36 wk, 153 ± 15; 37–40 wk, 105 ± 7; and 41–44 wk, 88 ± 5). These observations associate ACh with syncytiotrophoblast. Choline acetyltransferase (ChA) has a similar pattern of variation with gestational age. The placental cholinergic system (as indicated by ChA ACh) was fully formed at the early fetal period of histogenesis and functional maturation, during which the fetus exhibits the fastest rate of growth.

EFFECTS OF DICHLOROISOPROTERENOL AND PHENOXY BENZAMINE ON THE TRANSMEMBRANE POTENTIALS OF THE ISOLATED RABBIT’S HEART

The inhibitory effects of α-receptor blocking agents such as dibenamine, yohimbine and chlorpromazine on the transmembrane potentials in the atria isolated from intact and reserpinized rabbits were reported previously (1-3). Although acetylcholine (ACh) as well as adrenaline and noradrenaline could restart the atrial action potential which had been ceased by the application of the adrenolytics, the restarting effect of ACh showed considerable differences from that of catecholamines. It was described by several investigators that positive inotropic and chronotropic responses of catecholamines in the mammalian heart were not prevented by α-receptor blocking agents (4) but were antagonized by dichloroisoproterenol (DCI), a β-receptor blocking agent (5, 6). In the isolated heart preparations, the stimulating effect of ACh was blocked by the pretreatment with DCI or by the full reserpinization of the animals (7-10). These results proposed the assumption that the stimulating effect of ACh derived from endogenously released catecholamine. However, as already confirmed in the previous report (2), the evidence that the addition of ACh could restart the action potential which had been ceased by the pretreatment of α-receptor blocking agents even in the reserpinized rabbit was against the afcre-mentioned assumption. In order to elucidate whether the stimulating effect of ACh derives from endogenously released catecholamine or not, the effects of ACh and catecholamine on the transmembrane potentials of the heart should be observed in relation to β-receptor blocking effects of DCI. The present experiments have been designed to compare the effects of DCI and phenoxybenzamine, an α-receptor blocking agent, on the atrial and ventricular transmembrane potentials in the isolated rabbit's heart, and to elucidate the correlation between the actions of DCI and ACh or catecholamines. Further, the effects of DCI and phenoxybenzamine on the conduction time and the effective refractory period of the ventricle have been comparatively studied.