Nicotinic Currents Research Articles

Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons.

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K+ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 microM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (IK(V)), IC50 63 microM; transient (IA) current, IC50 69 microM] and produced no detectable inhibition of the fast and slow Ca(2+)-activated K+ currents IC and IAHP or of a hyperpolarization-activated cation current (IQ/Ih) at 10-30 microM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl- currents with IC50 values of 7.6 and 26 microM respectively. It is concluded that linopirdine shows some 20-fold selectivity for M-channels among different K+ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.

Tacrine inhibits nicotinic secretory and current responses in adrenal chromaffin cells

Tacrine enhanced acetylcholine-induced catecholamine secretion with a concentration of up to 10 μM, but inhibited it at over 10 μM in perfused adrenal glands. Qualitatively the same result was obtaiend withphysostigmine. Both tacrine and physostigmine only inhibited the secretory responses to carbachol and/or nicotine in perfused glands and dispersed chromaffin cells. Acetylcholinesterase activity of adrenal homogenates was inhibited by tacrine and physostigmine in a concentration-dependent manner. In whole-cell patch-clamp experiments, tacrine and physostigmine caused reversible inhibition of nicotine-evoked inward currents with a dose range similar to that for the inhibitory action on the secretory response. These results suggest that the enhancing effect of tacrine and physostigmine on acetylcholine-induced catecholamine secretion results from the prevention of enzymatic hydrolysis of acetylcholine in adrenal glands and that the inhibitory effect is due to the inhibition of nicotinic receptor-mediated membrane currents in adrenal chromaffin cells.

Proadrenomedullin N-terminal 20 peptide (PAMP) reduces inward currents and Ca2+ rises induced by nicotine in bovine adrenal medullary cells.

It has been recently reported that proadrenomedullin N-terminal 20 peptide (PAMP), which is secreted with adrenomedullin and catecholamines from the adrenal medulla, inhibits catecholamine release stimulated with nicotine. In the present study, to elucidate anticholinergic mechanisms of PAMP we employed the whole-cell patch-clamp and the intracellular Ca2+ imaging techniques in cultured bovine adrenal medullary cells. PAMP inhibited nicotinic currents and [Ca2+]i rises induced by nicotine in a dose-dependent manner (10(-9)-10(6) M). These inhibitions were selective, since PAMP alone did not induce any ionic currents, moreover it did not affect voltage-dependent Ba2+ currents or high K+ (50 mM)-induced [Ca2+]i rises. The onset of the inhibitory effect of PAMP (10(-6) M) was very rapid and reached a steady-state level within 10 sec. The effect of PAMP (10(-6) M) lasted for about 10-15 min. Desensitization process of the nicotinic current fitted to a single exponential function with a time constant of 6.4 +/- 0.3 sec. When PAMP (10(-6) M) simultaneously added with nicotine (10(-5) M), the desensitization process was facilitated and fitted to two exponentials with time constants of 0.46 +/- 0.08 and 2.5 +/- 0.8 sec. From the present results, the inhibition by PAMP of nicotinic currents which was well associated with that of nicotine induced [Ca2+]i rises leads to the attenuation of catecholamine release probably, at least in part, due to the facilitation of the desensitization process of the nicotinic currents.

Melatonin modulates cholinergic transmission by blocking nicotinic channels in the guinea-pig submucous plexus

Melatonin, a hormone produced and released by the pineal gland, is also synthesized by cells of the gastrointestinal wall, where it might be a local regulator of gut functions. In this study, we investigated the possible role of melatonin as a modulator of the enteric nervous system. Intracellular recordings were made in neurons of the submucosal plexus from the guinea-pig ileum to measure the melatonin effects on their electrophysiological properties. Melatonin did not alter the membrane potential, the membrane resistance and the noradrenergic inhibitory postsynaptic potentials. However, melatonin (30–3000 μM) reversibly decreased the amplitude of nicotinic excitatory postynaptic potentials (EPSPs) in a concentration-dependent manner (IC 50 = 247 μM). These actions of melatonin were not modified by the presence of idazoxan and atropine indicating that they are not mediated by endogenous release of acetylcholine, noradrenaline, or by direct activation of α 2-adrenoceptors or muscarinic receptors. The superfusion of melatonin also blcoked the nicotinic depolarizations induced by locally applied acetylcholine, indicating that at least part of its effects are postsynaptic. In voltage-clamp experiments, using the whole-cell configuration, melatonin also inhibited the nicotinic inward currents induced by acetylcholine ( I ACh) in a concentration-dependent manner (IC 50 = 257 μM). Melatonin decreased the maximal I ACh but did not affect the potency of acetylcholine to induce this current, indicating a noncompetitive antagonism. This effect was voltage-dependent. Our observations indicate that melatonin inhibits the fast EPSPs by directly and specifically blocking the nicotinic channels. The relative high concentrations of melatonin required to produce such an effect rules this out as one of its humoral actions. Such an effect, however, might be of physiological significance close to the cells that release melatonin in the gastrointestinal wall or in other organs.

The role of nicotinic receptors and calcium channels in mipafox induced inhibition of catecholamine release in bovine chromaffin cells

Depolarization induced catecholamine release from chromaffin cells was decreased 28% by N,N′-diisopropyl diamido-phosphorofluoridate (mipafox), an organophosphorus compound (OP) causing neurotoxic effects, while secretion stimulated by nicotinic agonist was inhibited 65%. The reversibility of this effect and the fact that calcium-dependent secretion from digitonin-permeabilized cells was unaffected by mipafox suggest that this compound affects the ionic currents implicated in catecholamine release. Patch-clamp experiments showed that the activity of voltage-dependent calcium channels (VDCC) was inhibited 35% by mipafox being this effect reversible whereas only minor effects were detected on Na + and K + currents. Finally, we studied the effect of mipafox on nicotinic ionic currents in chromaffin cells. In this case, the OP was able to cause reversible inhibition reaching maximal effects of 50–60%. In conclusion, nicotinic receptors and VDCC should be considered as potential targets in order to understand the neurotoxicity of these chemicals.

Activation and cooperative multi-ion block of single nicotinic-acetylcholine channel currents of Ascaris muscle by the tetrahydropyrimidine anthelmintic, morantel.

1. We have investigated activation and block, by the tetrahydropyrimidine anthelmintic, morantel, of nicotinic-acetylcholine receptor (AChR) currents in membrane vesicles isolated from somatic muscle cells of the nematode parasite Ascaris suum. Standard single-channel recording techniques were employed. Morantel in the pipette (6 nM to 600 microM), activated single nicotinic AChR currents. 2. Kinetic properties of the main-conductance state of morantel-activated currents were investigated in detail throughout the concentration range, 0.6 microM to 600 microM. Open-time distributions were best fitted by a single exponential. Mean open-times were slightly voltage-dependent, increasing from 0.9 ms at +75 mV to 1.74 ms at -75 mV in the presence of 0.6 microM morantel. At low concentrations, closed-time distributions were best fitted by the sum of two or three exponential components. 3. As the concentration of morantel was increased (100-600 microM), fast-flickering open channel-block was observed at positive potentials, even though morantel, a cation, was only present at the extracellular surface of the membrane. The block rate was dependent on morantel concentration and both block rate and duration of block increased as the potential became less positive. A simple channel-block mechanism did not explain properties of this block. 4. At negative potentials, as the morantel concentration increased, a complex block was observed. With increases in morantel concentration two additional gap components appeared in closed-time distributions: one was short with a duration (approximately 13 ms) independent of morantel concentration; the other was long with a duration that increased with morantel concentration (up to many minutes). In combination, these two components produced a marked reduction in probability of channel opening (Po) with increasing morantel concentration. The relationship between the degrees of block and morantel concentration had a Hill coefficient of 1.6, suggesting the involvement of at least two blocking molecules. The data were analysed by use of a simple sequential double block kinetic model.

Vasoactive intestinal polypeptide modulation of nicotinic ACh receptor channels in rat intracardiac neurones.

1. The effects of vasoactive intestinal polypeptide (VIP) on isolated parasympathetic neurones of rat intracardiac ganglia were examined under voltage clamp using dialysed and perforated patch whole-cell and excised outside-out membrane patch recording configurations. 2. VIP reversibly potentiated nicotinic ACh-evoked whole-cell currents, with half-maximal potentiation (EC50) obtained with 260 pM VIP. However, VIP had no effect on muscarinic ACh-evoked currents, ATP-evoked currents, or depolarization-activated ionic currents in these neurones. 3. VIP-induced potentiation of nicotinic ACh-evoked whole-cell currents was observed following cell dialysis, and was inhibited reversibly by bath application of the VIP receptor-binding inhibitor L-8-K (5 microM) or the neuronal nicotinic receptor antagonist mecamylamine (3 microM). 4. The signal transduction pathway mediating VIP-induced potentiation of nicotinic ACh-evoked currents involves a guanine nucleotide-binding protein (G-protein) but not cyclic AMP. Intracellular application of 100 microM GDP-beta-S, or pre-incubation of neurones with pertussis toxin, inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. 5. In outside-out membrane patches, co-application of ACh (4 microM) and VIP (4 nM) decreased the duration of closings between bursts and clusters of bursts of ACh single-channel activity relative to control (4 microM, ACh alone). VIP, however, did not alter single ACh receptor channel current amplitude, duration of closings and openings within a burst, or mean burst duration. 6. VIP-induced modification of nicotinic ACh receptor channel kinetics results in an increase in the open-channel probability which is sufficient to account for the VIP-mediated potentiation of nicotinic ACh-evoked whole-cell currents. 7. The potentiation of nicotinic ACh-evoked currents by VIP is likely to account for the altered neuronal activity observed in the mammalian intracardiac ganglia in vivo and consequent changes in heart rate and cardiac contractility.

Nicotinic Receptors of Different Species Exhibit Differential Sensitivities to Nitromethylene and Organophosphate Insecticides

The effects of nitromethylene heterocycle (NMH) and organophosphate (OP) insecticides were studied on nicotinic acetylcholine receptors (nAChR) in cultured cells of different species origin, in order to examine the selectivity of these compounds at the level of the target sites. In mouse muscle BC3H1, mouse NIE-115 and human SH-SY5Y neuroblastoma, and locust thoracic ganglion cells, the neurotransmitter, acetylcholine (ACh), induces a similar transient inward current. Dependent on the cell type, the six NMHs acted as agonists and/or antagonists on nAChR. Distinct agonistic effects of NMHs on nAChR are observed on insect neurons only. Further, NMHs potently block nicotinic responses in insects, while mammalian cells are only moderately affected. In all cases, the neuronal type nAChR was more sensitive to blocking than the endplate type nAChR in mammalian cells.Parathion and paraoxon at micromolar concentrations inhibit ACh-induced nicotinic inward currents. The insecticide, parathion, is a 50-fold more potent inhibitor than its acetyl-cholinesterase-inhibiting metabolite, paraoxon. Moderate differences in sensitivity to the blocking action of the OPs appear to exist among cells of different species. The results demonstrate that the experimental approach of fundamental electrophysiology and the use of cell lines are relevant tools for investigating the species specificity of neurotoxic compounds.

Muscarinic receptor activation modulates ligand-gated ion channels in an insect motoneuron via changes in intracellular calcium

An isolated ganglion preparation has been used to make recordings from an identified cockroach neuron in situ. We have shown that the magnitude of conductance increase of ligand-gated ion channels produced by agonists can be modulated by activation of muscarinic ACh receptors (mAChRs); currents through nicotinic ACh receptors (nAChRs) are suppressed, while GABA currents can be either enhanced or suppressed. mAChR activation causes a rise in [Ca 2+ ] 1 measured using the fluorescent Ca 2+ indicator fluo-3. This rise in [Ca 2+ ] 1 appears to trigger modulation of nicotinic ACh and GABA currents, since the effects of mAChR activation are mimicked by brief increases in [Ca 2+ ] 1 produced by flash photolysis of the Ca 2+ chelator nitr-5. These modulatory effects outlast the rise in [Ca2+]j evoked by mAChR activation or photolytic Ca 2+ release by 5—10 min. The prolongation by Li+ of the modulatory effects mediated by mAChRs suggests a possible involvement of the phosphoinositol pathway. These results indicate that mAChRs acting via intracellular messengers can exert a powerful modulatory influence upon nACh or GABA gated ion channels of neurons in which these receptors are co-localized.

Direct Functional Effects of Parathion and Paraoxon on Neuronal Nicotinic and Muscarinic M3 Acetylcholine Receptors

The direct effects of two organophosphates, parathion and paraoxon, on nicotinic and muscarinic acetylcholine receptors in cells from different species were investigated, by using the whole-cell voltage clamp technique. The effects of neuronal type nicotinic receptors in mouse N1E-115, human SH-SY5Y and locust thoracic ganglion cells, and on human muscarinic M3 receptors transfected in Chinese hamster ovary (CHO) cells, were analysed. Parathion and paraoxon inhibit acetylcholine-induced nicotinic inward currents at concentrations in the micromblar range. Surprisingly, parathion is a more potent inhibitor than its active acetyl-cholinesterase-inhibiting metabolite, paraoxon. Moderate differences in sensitivity to the blocking action of the organophosphates appear to exist between cells from different species. Both parathion (100nM) and paraoxon (1μM) induce ion currents in M3-transfected CHO cells. They appear to act as agonists on the human muscarinic M3 receptor, affecting the receptors in a different manner to that of acetylcholine.

Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.

Potentiation and inhibition of subtypes of neuronal nicotinic acetylcholine receptors by Pb 2+

Effects of inorganic lead (Pb 2+) on defined subtypes of neuronal nicotinic acetylcholine receptors have been investigated. Voltage clamp experiments have been performed on Xenopus oocytes expressing α3β2, α3β4 and α4β2 neuronal nicotinic acetylcholine receptor subunit combinations. In oocytes expressing the α3β2 subunit combination Pb 2+ enhances the peak amplitude of nicotinic acetylcholine receptor-mediated inward currents evoked by superfusion with 100 μM acetylcholine. At concentrations of 1–250 μM, Pb 2+ potentiates α3β2 receptor-mediated inward current concentration dependently by a factor of 1.1–11.0. Inward currents evoked by low (3 μM) and high (1 mM) concentrations of acetylcholine are potentiated to a similar extent. Conversely, in oocytes expressing the α3β4 subunit combination Pb 2+ inhibits the nicotinic receptor-mediated inward currents evoked with μM acetylcholine. Inhibitory effects are observed in the concentration range of 1 nM–100 μM Pb 2+, but the degree of inhibition varies between oocytes. A similar inhibition of the α4β2 nicotinic receptor-mediated inward current by Pb 2+ indicates that α as well as β subunits are involved in the potentiating and inhibitory effects. Possible reasons for the variation in the inhibitory effects of Pb 2+ on α3β4 and α4β4 nicotinic receptor-mediated inward currents have been investigated and are discussed. The divalent cations Ca 2+ and Mg 2+ potentiate both α3β2 and α3β4 nicotinic receptor-mediated inward currents. The distinct modulation of receptor function by Pb 2+ and by Ca 2+ and Mg 2+ and the dependence of the modulatory effect of Pb 2+ on subunit composition suggest that Pb 2+ interacts with multiple sites on the α and β subunits of neuronal nicotinic acetylcholine receptors.

Alpha 2-Adrenoreceptor-mediated inhibition of acetylcholine-induced noradrenaline release from rat sympathetic neurons: an action at voltage-gated Ca2+ channels.

[3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in 3H outflow which was half-maximal at about 300 microM and within 5 s. The overflow induced by 10 s exposure to 300 micro A acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 microM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow irrespective of the stimulus. The selective alpha2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximately 25% maximal inhibition) than electrically induced overflow ( > or = 45% maximal inhibition). These inhibitory effects were antagonized by the alpha2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 microM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 microM). UK 14,304 had no effect when 3H overflow was evoked by acetylcholine in the presence of 300 microM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of teh patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 microM) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 microM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 microM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. alpha2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and effects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha2-adrenoceptor.

Acetylcholine receptor desensitization induced by nicotine in rat medial habenula neurons.

1. The activation and desensitization properties of nicotinic acetylcholine receptor (nAChR) channels were examined in acutely isolated medial habenula (MHb) neurons using whole cell patch-clamp recordings. nAChR-mediated currents were evoked by applying known concentrations of nicotinic agonists using rapid solution exchange techniques. 2. At a membrane potential of -60 mV, nAChR currents were observed above a concentration of approximately 100 mM nicotine. The peak current amplitude at low doses of agonist was proportional to the square of the concentration of nicotine, indicating that at least two molecules of agonist were required for channel opening. The concentration of nicotine required for half-maximal nAChR activation was estimated as 77 microM from a complete concentration-response curve. 3. During the continuous activation (2-5 s) of nAChRs by high concentrations of nicotine (300 microM), the current desensitized rapidly and extensively. The desensitization phase was described by the sum of two exponentials, with time constants of 210 and 1,435 ms. The fast component comprised 74% of the desensitizing phase of the current. Recovery from desensitization induced by 2- s applications of 300 microM nicotine was also fast and could be reasonably well described by a single exponential with a time constant of approximately 800 ms. Both the time courses of desensitization and recovery from desensitization were slightly slower at positive membrane potentials. 4. Incubation of neurons with low concentrations of nicotine (100 nM-10 microM) caused a slowly developing but pronounced desensitization of the nAChRs. In these cases desensitization was assessed from the reduction in the amplitude of the peak nicotinic current induced by repetitively applied pulses of a higher test concentration of agonist. A 5-min continuous exposure to 1 microM nicotine reduced the amplitude of the acetylcholine (30 microM, 1 s) test response to < 30% of its control value. As with higher concentrations of nicotine, the onset of the desensitization induced by 1 microM nicotine was biexponential, with fast and slow time constants of 15 s and 1.74 min, respectively. Recovery from the desensitization induced by these longer applications of nicotine was much slower than that observed with the brief pulses of high concentrations of nicotine. The concentration required for half-maximal desensitization after a 5-min incubation was approximately 300 nM. 5. Peak nAChR currents were approximately 85% smaller at +40 mV compared with -40 mV. The receptors that do not open at positive potentials desensitize almost as well as they would at negative potentials after channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

ω-Conotoxin GVIA blocks nicotine-induced catecholamine secretion by blocking the nicotinic receptor-activated inward currents in bovine chromaffin cells

We have studied the contribution of N-type voltage-dependent Ca 2+ channels to both norepinephrine and epinephrine secretion from bovine chromaffin cells induced by high K + or nicotine using ω-conotoxin GVIA, a selective blocker of N-type voltage-dependent Ca 2+ channels. We found that high K + (75 mM) induced catecholamine secretion was not affected by exposure of bovine chromaffin cells to ω-conotoxin GVIA (1 μM). However, nicotine-induced both norepinephrine and epinephrine secretion were similarly blocked (about 25%) by ω-conotoxin GVIA (1 μM). This effect could be explained by a potent (about 80%) and reversible blockade of the inward current induced by nicotine receptor activation in bovine chromaffin cells. The results indicate that besides the blockade of N-type voltage-dependent channels, ω-conotoxin GVIA is a potent and reversible blocker of the nicotinic receptor-induced currents in chromaffin cells.

Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurones of Lymnaea stagnalis

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were studied under two-electrode voltage-clamp. Neuronal nicotinic acetylcholine receptor currents were studied at low acetylcholine (ACh) concentrations (< or = 200 nM). At these levels, control currents were non-desensitizing and proportional to the square of the ACh concentration. 2. IC50 concentrations were determined for the steady-state inhibition of the ACh-activated current by 31 general anaesthetics plus the non-anaesthetic alcohol n-tridecanol. The general anaesthetics included inhalational agents, n-alcohols, n-alkane-(alpha,omega)-diols, cycloalcohols and an n-alkane. 3. Anaesthetic inhibition was independent of voltage and consistent with two anaesthetic-binding sites on the receptor. 4. IC50 concentrations for inhibiting the neuronal nicotinic ACh receptor correlated well (r = 0.97) with EC50 concentrations for general anaesthesia. The maximum deviation from the line of identity was less than fourfold. The inhalational agents tended to be more potent as inhibitors of the ACh receptor than as general anaesthetics, while the alcohols and diols were less potent. 5. The inhibition of the ACh-induced current by the homologous series of n-alcohols exhibited a cutoff at the same position (just after dodecanol) as found for the induction of general anaesthesia in tadpoles. 6. Polarity profile maps of the anaesthetic-binding sites on the neuronal nicotinic ACh receptor were calculated from IC50 concentrations for the homologous series of n-alcohols and n-alkane-(alpha,omega)-diols. They reveal amphiphilic sites with apolar regions capable of accommodating the hydrocarbon chains of n-alcohols as large as decanol. A striking resemblance was found to profiles previously calculated from data for tadpole general anaesthesia.

ATP-evoked membrane current in guinea pig adrenal chromaffin cells

Adenosine 5′-triphosphate (ATP) evoked an inward current in whole-cell voltage-clamped adrenal chromaffin cells of the guinea pig. The reversal potential ( E rev) of ATP-evoked current was about 0 mV in normal external solution and was shifted towards negative potentials by substituting Tris + or sucrose, but not Ca 2+, for the extracellular Na +. This current was mediated by the activation of non-selective cation channels and had some different properties from nicotinic current. It is suggested that these channels may function as a part of the ATP-induced Ca 2+ influx pathway in guinea pig chromaffin cells.

Natriuretic peptides inhibit nicotine-induced whole-cell currents and catecholamine secretion in bovine chromaffin cells: evidence for the involvement of the atrial natriuretic factor R2 receptors.

There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R1C but not the ANF-R1A receptor subtype. Evidence for the existence of ANF-R2 receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R2-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R2 receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R2 receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that the effect of CNP is predominantly mediated by the ANF-R2 and not the ANF-R1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCl-induced secretions were affected by natriuretic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

I 1-imidazoline receptor modulating substances influence ouabain-induced cardiac arrhythmia

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC50=366 μM); the K+-evoked release of catecholamines was unaffected. Clonidine (100 μM) and moxonidine (100 μM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at −80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents (IACh) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast (τon=31 ms); the recovery of the current after washout of agmatine also occurred very rapidly (τoff=39 ms). Efaroxan (10 μM) did not affect the inhibition of IACh elicited by 1 mM agmatine. IACh was blocked 90% by 100 μM clonidine and 50% by 100 μM moxonidine. The concentration–response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 μM agmatine. The blockade of IACh caused by agmatine (100 μM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block IACh. At 1 mM, agmatine blocked INa by 23%, IBa by 14%, IK(Ca) by 16%, and IK(VD) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.

Species-specific Effects of Nitromethylene Heterocycle Insecticides on Nicotinic Receptors in Locust Neurons and Mouse N1E-115 and BC3H1 Cells

The effects of nitromethylene heterocycle (NMH) insecticides on subtypes of nicotinic acetylcholine (nACh) receptors were investigated in locust thoracic ganglion neurons, mouse N1E-115 neuroblastoma cells, and mouse BC3H1 muscle cells by using electrophysiological techniques. In locust neurons, all of the six NMH insecticides tested induced transient inward currents resembling nicotinic ACh-induced inward currents, while, in the continued presence of the NMH compounds, the ACh-induced inward current was blocked. The amplitude of the inward current and the blocking effects of the NMH insecticides were enhanced by concentrations between 0.1 and 10μM. Cross-desensitisation with the ACh-induced inward current confirmed that the NMH-induced inward current was governed by the activation of nACh receptors. Mammalian endplate type nACh receptors in BC3H1 cells and mammalian neuronal type nACh receptors in N1E-115 cells were much less sensitive to the NMH insecticides than the locust neuronal nACh receptors. At a concentration of 10μM, which blocked 80–100% of the ACh-induced inward current in locust neurons, NMH insecticides only partially blocked the ACh-induced inward currents mediated by the two subtypes of mammalian nACh receptors. NMH insecticides also failed to induce significant agonist effects in the mammalian cells at this concentration.The results provide a possible explanation for the selectively greater toxicity of NMH insecticides to insects than to vertebrates, at the level of nACh receptor subtypes and, hence, demonstrate that this in vitro approach is valuable for the investigation of species-specific interactions of compounds at their target site.