Neuronal Nicotinic Acetylcholine Receptor Channels Research Articles

Is postoperative delirium a relevant outcome?

potentially confounding neurological dysfunctions. In their study with 100 postoperative patients without a known risk factor of POD, the incidence of POD diagnosed using CAM was 28 %. Their result suggested that the incidence of POD was common in patients after major surgery even without known risk factors. Major surgery might evoke a strong stress response. Recent studies provided the hypothesis that it may introduce postoperative delirium or cognitive dysfunction [9, 10]. As the authors have pointed out, prior studies have commonly been conducted in postoperative patients with known risk factors; their study is relevant and novel. Another finding was a significant association of the use of thiopental with the risk of POD compared with propofol. Thiopental, a barbiturate class of drug, has effects on the gamma-aminobutyric acid (GABA) receptor [11] and propofol is also considered to act through potentiation of GABA receptor activity. Although their finding on thiopental may be skewed by the bias of patient characteristics and conditions which may cause anesthesiologists to choose thiopental rather than propofol, the potential action of thiopental on non-GABA-ergic ligand-gated ion channels, including the neuronal nicotinic acetylcholine receptor channels, may contribute to its association [12]. Although there are potential hypotheses for the etiology and pathophysiology of POD including cholinergic inhabitation, serotonin deficiency, dopamine activation [13], GABA activity [14], and melatonin activity [15], they are not fully understood [16]. This is one of the reasons why there are still just a few strategies for preventing POD [17]; the use of dexmedetomidine in post-cardiac surgery patients [18], multicomponent interventions, and antipsychotics. In this regard, further study should be warranted to develop a detailed understanding and preventive strategy for these postoperative complications. Delirium is characterized by the acute onset of mental status or a fluctuating course and inattention and disorganized thinking or an altered level of consciousness [1], which is common in critically ill patients [2]. Recently, the diagnosis of delirium is recommended by using the Confusion Assessment Method for the intensive care unit (CAM-ICU) [3]. Postoperative delirium (POD) was defined as so because it occurs postoperatively. Although operation, anesthetics, and analgesics can contribute to POD, it is not just associated with emergence from anesthesia, but often occurs between postoperative days 1 and 3. Although POD is commonly recovered from within a short period of time [4], patients with POD are more likely to die or develop dementia and require institutionalization [5]. A number of risk factors are reported for POD, including older age, preoperative cognitive or functional impairment, decreased postoperative hemoglobin, markedly abnormal sodium potassium and glucose, the presence of alcohol abuse, noncardiac thoracic surgery, aortic aneurysm surgery, history of delirium, preoperative use of narcotics, low postoperative oxygen saturation, history of cerebrovascular disease, and untreated pain [6, 7]. In a recent prospective observational study conducted by Saporito and Sturini [8], they excluded patients with any known preexisting predisposing factor for POD; types of surgery, preoperative dementia, sensory deficits, requirement of psychotropic drugs, and alcoholism etc., and other

Voltage‐dependent modulation of the neuronal nicotinic acetylcholine receptor‐channel by cartap

Nicotinic acetylcholine receptor-channels (AChR) are believed to be the target site of cartap. In order to clarify the mechanisms of the cartap interaction with nicotinic AChRs, single-channel patch clamp experiments were performed using rat clonal phaeochromocytoma (PC12) cells. Cartap increased the short closures occurring during single-channel openings induced by acetylcholine (ACh). Thus, when co-applied with acetylcholine, cartap markedly shortened the open time associated with bursts. These effects were voltage-dependent, being more prominent at negative voltages. These results strongly suggest that cartap acts as an open channel blocker at the neuronal nicotinic AChR. © 1999 Society of Chemical Industry

Voltage-dependent modulation of the neuronal nicotinic acetylcholine receptor-channel by cartap

Nicotinic acetylcholine receptor-channels (AChR) are believed to be the target site of cartap. In order to clarify the mechanisms of the cartap interaction with nicotinic AChRs, single-channel patch clamp experiments were performed using rat clonal phaeochromocytoma (PC12) cells. Cartap increased the short closures occurring during single-channel openings induced by acetylcholine (ACh). Thus, when co-applied with acetylcholine, cartap markedly shortened the open time associated with bursts. These effects were voltage-dependent, being more prominent at negative voltages. These results strongly suggest that cartap acts as an open channel blocker at the neuronal nicotinic AChR.

Voltage-dependent modulation of the neuronal nicotinic acetylcholine receptor-channel by cartap

Voltage-dependent modulation of the neuronal nicotinic acetylcholine receptor-channel by cartap

Modulation of the neuronal nicotinic acetylcholine receptor-channel by the nootropic drug nefiracetam

The effects of nefiracetam (DM-9384) on the neuronal nicotinic acetylcholine (ACh) receptor-channel were studied by the whole-cell patch clamp technique using PC12 cells. Nefiracetam had a dual effect on ACh-induced currents: it augmented the currents induced by low concentrations (10–30 μM) of ACh and suppressed those induced by high concentrations (100–1000 μM) of ACh. These effects were reversible after washing with drug-free solution. The stimulating effect of nefiracetam was clearly observed at a concentration of 10 μM, and slight increases in currents were detected even at 0.1 μM or 1 μM. Nefiracetam at 100 μM suppressed the currents induced by a low concentration (10 μM) of ACh. The rate of desensitization of ACh-induced current was greatly accelerated by nefiracetam, and this effect could not be reversed by washing with drug-free solution. When added to the internal pipette solution, the protein kinase A inhibitor KT 5720 (0.6 μM), but not the protein kinase C inhibitor calphostin C (0.5 μM), abolished the nefiracetam stimulation of the ACh receptor. Pre-incubation of cells with 200 ng/ml pertussis toxin for 24 h also abolished the nefiracetam action. Thus, the nefiracetam modulation of the neuronal nicotinic ACh receptor-channel is exerted via G proteins and protein kinase A. The stimulation of the ACh receptor may be directly related to the cognitive enhancing action of nefiracetam.

Effects of Nitenpyram on the Neuronal Nicotinic Acetylcholine Receptor-Channel in Rat Phaeochromocytoma PC12 Cells

Nitenpyram, (E)-N-(6-chloro-3-pyridylmethyl)-N-ethyl-N'-methyl-2-nitro-vinylidenediamine, is known to act on the nicotinic acetylcholine receptor-channel (AChR). We examined the effects of nitenpyram on the neuronal type of the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using a patch clamp technique to clarify the mechanisms of action at the single-channel level. Nitenpyram itself induced single-channel currents mediated by the neuronal nicotinic AChR. Co-application of ACh and nitenpyram opened the channels exhibiting the two conductance states. The proportion of main conductance levels was decreased and that of subconductance levels was increased in the presence of nitenpyram. The open time and closed time distribution, but not for the burst duration, for the main conductance state currents with co-application of ACh and nitenpyram were similar to those of ACh-induced current. These changes of single-channel kinetics result in a decrease in probability of openings of main conductance state currents explaining the effects of nitenpyram on the neuronal nicotinic AChR. The effects of nitenpyram on the acetylcholine-induced current are deemed directly responsible for the toxic actions in animals.

Ion channel modulation as the basis for general anesthesia

(1) Modulation of the function of the GABA A and neuronal nicotinic acetylcholine receptor channels caused by general anesthetics and modulation of the GABA A receptor-channel by halothane, enflurane, isoflurane, and n-octanol was channel state-dependent. (3) Halothane modulation of the GABA A receptor was independent of subunits, but n-octanol modulation was subunit-dependent. (4) Ethanol at 30–100 μM was very potent in accelerating the desensitization of currents induced by acetylcholine. (5) The ethanol modulation was subunit- and state-dependent, occurring in the α3 β4 combination but only weakly in the α3 β2 combination. (6) In contrast, halothane at 430 μM (∼1 MAC) potently suppressed ACh-induced currents in the α3 β2 subunit combination.

Dimensions of neuronal nicotinic acetylcholine receptor channel as estimated from the analysis of the channel-blocking effects.

Acetylcholine-induced membrane currents and excitatory postsynaptic currents (EPSCs) were recorded from the neurons of rat superior cervical ganglion (SCG) using the whole-cell patch clamp and the two-electrode voltage clamp techniques, correspondingly. The EPSC decay was bi-exponential, with fast and slow components characterized by time constants 5.5 +/- 0.5 msec and 20.4 +/- 1.2 msec (mean +/- SEM; n = 23), respectively. Blocking of these currents by a series of newly synthesized bis-cationic ammonium compounds, the pentamethonium and pentaethonium derivatives, was analyzed. Blocking effects were due to a block of nicotinic acetylcholine receptor (nAChR) open channel, with mean blocker binding rate constants for the fast component three to five times higher than those for the slow component. Dimensions of a nAChR ionic channel were deduced from a relationship between blocking activity of the compounds and the size of the projections of their three-dimensional molecular models on the neuronal membrane plane. The results suggest that there are two populations of nAChRs in rat SCG neurons; while these polulations differ in the rate constants of the binding by the blocker to their open channels, they exhibit similar channel diameter, 11.8 A, at the level at which the blockers bind to the channel.

Cartap Modulation of the Nicotinic Acetylcholine Receptor-Channel of Clonal Rat Phaeochromocytoma (PC12) Cells

Cartap, the synthetic analog of nereistoxin, is known to act on the nicotinic acetylcholine receptor-channel (AChR). Effects of cartap on the neuronal nicotinic AChR of clonal rat phaeochromocytoma (PC 12) cells were studied using a single-channel patch clamp method. When cartap was co-applied with acetylcholine, the single-channel openings were shortened and occurred in bursts separated by brief closures or gaps. The current amplitudes in the presence of cartap were virtually identical to those of currents induced by ACh only. Mean open time and burst duration were significantly decreased in the presence of cartap in a dose-dependent manner. These changes of single-channel kinetics result in a significant decrease in total charge carried through the open AChR explaining the suppressive effect of cartap on nicotinic AChR. Results suggest that cartap could act as an open channel blocker at the nicotinic AChR.

Modulation of the Neuronal Nicotinic Acetylcholine Receptor Channel by Imidacloprid and Cartap

Imidacloprid, a nitromethylene heterocycle insecticide, and cartap, a synthetic analog of nereistoxin, are known to act on the nicotinic acetylcholine receptor channel. The effects of imidacloprid and cartap on the nicotinic acetylcholine receptor channel of clonal rat phaeochromocytoma cells were studied using the single-channel patch-clamp technique. Acetylcholine at a concentration of 10 μM induced currents predominantly in the main conductance state, whereas imidacloprid at 10 μM induced currents predominantly in the subconductance state. No current was evoked by cartap at concentrations ranging from 0.1 to 300 μM. Coapplication of acetylcholine and imidacloprid generated currents in both main conductance state and subconductance state. Coapplication of acetylcholine and cartap induced the main conductance state currents, forming bursts. The distributions of open time and burst duration of currents induced by acetylcholine, imidacloprid, acetylcholine plus imidacloprid, and acetylcholine plus cartap showed multiexponential components. Cartap, when coapplied with acetylcholine, markedly shortened the open time reflecting the appearance of bursts. These results indicate that imidacloprid and cartap modulate the nicotinic acetylcholine receptor channel with different mechanisms of action. Imidacloprid acts as a partial agonist generating predominantly subconductance state currents, whereas cartap acts as an open channel blocker at the nicotinic acetylcholine receptor channel.

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.

Functional properties of neuronal nicotinic acetylcholine receptor channels expressed in transfected human cells.

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat alpha 3 beta 4, chick alpha 3 beta 2, alpha 4 beta 2, alpha 7 and alpha 8), expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of alpha 8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19-23 and 32-45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric alpha 7 and alpha 8 nAChRs (6-7 ms) than for heteromeric nAChRs (1-3 ms), with the exception of alpha 3 beta 4 nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (alpha 3 beta 4 and alpha 3 beta 2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChR channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.

Potent modulation of neuronal nicotinic acetylcholine receptor-channel by ethanol

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 μM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.

Potent modulation of neuronal nicotinic acetylcholine receptor-channel by ethanol

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC 50 of 88.5 μM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.

Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.

Diversity in primary structure and function of neuronal nicotinic acetylcholine receptor channels

Neuronal nicotinic acetylcholine receptors are oligomeric protein complexes whose component subunits are each encoded by a family of homologous genes. The current challenge is to determine the functional contributions of the related subunits to the receptor-linked ion channels they compose and to uncover the physiological impact of the distinct channel classes expressed in vivo. In the past year, new approaches to the analysis of these receptors have yielded important insights into their stoichiometry, pharmacology and functional properties.

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.

Voltage-dependent block by magnesium of neuronal nicotinic acetylcholine receptor channels in rat phaeochromocytoma cells.

1. The effects of Mg2+ on the single-channel conductance of neuronal nicotinic acetylcholine receptors were examined using receptors expressed by the rat phaeochromocytoma cell line, PC12. PC12 cells express at least three conductance classes of channels that are activated by acetylcholine, the largest conductance class being the most prevalent. This receptor channel is blocked by intracellular and extracellular Mg2+. 2. The effects of Mg2+ are asymmetrical; at a given concentration, internal Mg2+ is more effective at blocking outward currents than external Mg2+ is at blocking inward currents. Receptor channels are blocked at concentrations of Mg2+ that are low compared to the concentration of the main permeant cation, Na+, and the block is voltage dependent. 3. The block by Mg2+ is not complete as Mg2+ can permeate the channel. With 80 mM-extracellular Mg2+ (no extracellular Na+), the channel has an inward slope conductance of 2.9 pS. 4. The block by extracellular Mg2+ can be described by a one site, two barrier model for the channel which includes a negative surface charge on the external surface of the membrane. The parameters of the model place the binding site for Mg2+ at 52% of the membrane field from the outside with an apparent dissociation constant of 14 mM. However, the same parameters cannot describe the block by intracellular Mg2+. The deviations from the model suggest that the receptor channel may have more than one binding site for Mg2+.

Nicotinic receptor-elicited sodium flux in rat pheochromocytoma PC12 cells: Effects of agonists, antagonists, and noncompetitive blockers

Nicotinic agonists stimulate 22Na flux in rat pheochromocytoma PC12 cells. The stimulatory effect of carbamylcholine is maximal at 1 mM, while the stimulatory effect of nicotine and anatoxin maximize at the same level at 100 microM and 10 microM, respectively. The tertiary amines arecolone and isoarecolone have no effect on flux at 100 microM, while the methiodides at 100 microM stimulate flux to an extent similar to 1 mM carbamylcholine. Dihydro and alcohol analogues of isoarecolone methiodide have markedly smaller effects on flux. A preincubation for 2 to 20 min with carbamylcholine (2 mM), nicotine (300 microM), anatoxin (30 microM) or isoarecolone methiodide (100 microM) causes marked desensitization to a subsequent carbamylcholine-elicited stimulation of flux. d-Tubocurarine, mecamylamine, hexamethonium, and chlorisondamine inhibit carbamylcholine-elicited flux with IC50 values of 1.0, 0.8, 43, and 0.020 microM, respectively. Atropine has no effect at 1 microM, but reduces the response to carbamylcholine by 50% at 8.6 microM, presumably as a noncompetitive blocker. Other noncompetitive blockers of nicotinic acetylcholine-receptors, such as histrionicotoxins, gephyrotoxin, pumiliotoxin C, phencyclidine, bupivacaine and piperocaine, inhibit carbamylcholine-elicited stimulation of 22Na flux with IC50 values from 0.3 to 1.8 microM. In contrast to d-tubocurarine, which inhibits carbamylcholine-elicited desensitization, and mecamylamine, which has no apparent effect on desensitization, chlorisondamine and certain noncompetitive blockers appear to enhance desensitization. The effects of agonists, antagonists and noncompetitive blockers at the neuronal nicotinic acetylcholine receptor-channel of PC12 cells are compared to their effects on binding of [125I]alpha-bungarotoxin to agonist-recognition sites and of [3H]perhydrohistrionicotoxin to noncompetitive blocker sites of the nicotinic acetylcholine receptor-channel of electric ray (Torpedo) electroplax membranes. There are marked differences in relative potencies for the two types of nicotinic acetylcholine receptor-channel.

Changes in the conductance of the neuronal nicotinic acetylcholine receptor channel induced by magnesium

We have investigated the effect of magnesium on the single-channel conductance of neuronal nicotinic acetylcholine receptors (nAChRs) in nerve growth-factor treated rat pheochromocytoma (PC12) cells. The patch-clamp technique was used to record single-channel currents from cell attached and excised, outside-out patches in the presence of various internal and external Mg2+ concentrations. Mg2+ reduced the single-channel conductance in a concentration-dependent manner with an IC50 of 9.2 mM for external Mg2+ (inward conductance) and 0.69 mM for external Mg2+ (outward conductance). Both estimated and measured conductances for divalent cation-free CsCl solutions were around 60 pS. We also find that divalent cations are not involved in the inward rectification of whole-cell ACh-induced currents in these cells. Our results imply that the amino acids screened by divalent cations sense electric fields only weakly and are presumably outside the lipid bilayer. They also suggest that the density and the number of charges (or both) differ on either side of the ion pore.