Dual action of n-alcohols on neuronal nicotinic acetylcholine receptors.

Alcohol is known to modulate the activity of a variety of neuroreceptors and ion channels. Recently, neuronal nicotinic acetylcholine receptors (nnAChRs) have become a specific focus of study because not only are they potently modulated by alcohol but also they regulate the release of various transmitters, including gamma-aminobutyric acid (GABA) and dopamine, which play an important role in the behavioral effects of ethanol. Whereas the potency of normal alcohols (n-alcohols) to potentiate GABA(A) receptors and to inhibit N-methyl-D-aspartate receptors increases with carbon chain length, we have found that n-alcohols, depending on the carbon chain length, exert a dual action, potentiation and inhibition, on nnAChRs in primary cultured rat cortical neurons. The mechanism of dual action of n-alcohols on nnAChRs was further analyzed using human embryonic kidney cells expressing the alpha 4 beta 2 subunits. Shorter chain alcohols from methanol to n-propanol potentiated acetylcholine (ACh)-induced currents, whereas longer chain alcohols from n-pentanol to n-dodecanol inhibited the currents. n-Butanol either potentiated or inhibited the currents depending on the concentrations of ACh and butanol. The parameters for both potentiation (log EC(200)) and inhibition (log IC(50)) were linearly related to carbon number, albeit with different slopes. The slope for potentiation was -0.299, indicating a change in free energy change (Delta Delta G) of 405 cal/mol/methylene group, whereas the slope for inhibition was -0.584, indicating a Delta Delta G of 792 cal/mol. These results suggest that potentiating and inhibitory actions are exerted through two different binding sites. Ethanol decreased the potency of n-octanol to inhibit ACh currents, possibly resulting from an allosteric mechanism.

Mechanisms of alcohol-nicotine interactions: alcoholics versus smokers.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Bo Söderpalm. The presentations were (1) Nicotinic mechanisms and ethanol reinforcement: Behavioral and neurochemical studies, by Bo Söderpalm, M. Ericson, P. Olausson, and J. A. Engel; (2) Chronic nicotine and ethanol: Differential regulation in gene expression of nicotinic acetylcholine receptor subunits, by X. Zhang and A. Nordberg; (3) Nicotine-ethanol interactions at neuronal nicotinic acetylcholine receptors, by Toshio Narahashi, William Marszalec, and Gary L. Aistrup; (4) Relapse prevention in alcoholics by cigarette smoking? Treatment outcome in an observational study with acamprosate, by L.G. Schmidt, U. Kalouti, M. Smolka, and M. Soyka; and (5) Effect of nicotine on voluntary ethanol intake and development of alcohol dependence in male rats, by L. Hedlund and G. Wahlström.

Modulation of neuronal nicotinic acetylcholine receptors by halothane in rat cortical neurons.

Inhalational general anesthetics have recently been shown to inhibit neuronal nicotinic acetylcholine (ACh) receptors (nnAChRs) expressed in Xenopus laevis oocytes and in molluscan neurons. However, drug actions on these systems are not necessarily the same as those seen on native mammalian neurons. Thus, we analyzed the detailed mechanisms of action of halothane on nnAChRs using rat cortical neurons in long-term primary culture. Currents induced by applications of ACh via a U-tube system were recorded by the whole-cell, patch-clamp technique. ACh evoked two types of currents, alpha-bungarotoxin-sensitive, fast desensitizing (alpha 7-type) currents and alpha-bungarotoxin-insensitive, slowly desensitizing (alpha 4 beta 2-type) currents. Halothane suppressed alpha 4 beta 2-type currents more than alpha 7-type currents with IC(50) values of 105 and 552 microM, respectively. Halothane shifted the ACh dose-response curve for the alpha 4 beta 2-type currents in the direction of lower ACh concentrations and slowed its apparent rate of desensitization. The rate of recovery after washout from halothane block was much faster than the rate of recovery from ACh desensitization. Thus, the halothane block was not caused by receptor desensitization. Chlorisondamine, an irreversible open channel blocker for nnAChRs, caused a time-dependent block that was attenuated by halothane. These results could be accounted for by kinetic simulation based on a model in which halothane causes flickering block of open channels, as seen in muscle nAChRs. Halothane block of nnAChRs is deemed to play an important role in anesthesia via a direct action on the receptor and an indirect action to suppress transmitter release.

Symposium overview: mechanism of action of nicotine on neuronal acetylcholine receptors, from molecule to behavior.

Nicotine has long been known to interact with nicotinic acetylcholine (ACh) receptors since Langley used it extensively to chart sympathetic ganglia a century ago. It has also been used as an effective insecticide. However, it was not until the 1990s that the significance of nicotine was increasingly recognized from the toxicological, pharmacological, and environmental points of view. This is partly because studies of neuronal nicotinic ACh receptors are rapidly emerging from orphan status, fueled by several lines of research. Since Alzheimer's disease is known to be associated with down-regulation of cholinergic activity in the brain, a variety of nicotine derivatives are being tested and developed for treatment of the disease. Public awareness of the adverse effects of nicotine has reached the highest level recently. Since insect resistance to insecticides is one of the most serious issues in the pest-control arena, it is an urgent requirement to develop new insecticides that act on target sites not shared by the existing insecticides. The neuronal nicotinic ACh receptor is one of them, and new nicotinoids are being developed. Thus, the time is ripe to discuss the mechanism of action of nicotine from a variety of angles, including the molecular, physiological, and behavioral points of view. This Symposium covered a wide area of nicotine studies: genetic, genomic, and functional aspects of nicotinic ACh receptors were studied, as related to anthelmintics and insecticides; interactions between ethanol and nicotine out the ACh receptor were analyzed, in an attempt to explain the well-known heavy drinker-heavy smoker correlation; the mechanisms that underlie the desensitization of ACh receptors were studied as related to nicotine action; selective pharmacological profiles of nicotine, and descriptions of some derivatives were described; and chronic nicotine infusion effects on memory were examined using animal models.

Basis of variable sensitivities of GABA(A) receptors to ethanol.

BACKGROUND: The GABA(A) system is believed to be one of the crucial target sites for ethanol. However, in the literature, data using various preparations yielded controversial conclusions regarding the ethanol potency to modulate the activity of GABA(A) receptors. We have previously shown that the potency of n-alcohols to potentiate GABA-induced currents is correlated with their carbon chain length. This correlation was further compared among four cell types in an attempt to explain the variable potencies of ethanol to potentiate GABA responses. METHODS: Whole-cell patch clamp experiments were performed to determine and compare the potencies of n-alcohols in potentiating GABA-induced currents in rat dorsal root ganglion (DRG) neurons, human embryonic kidney cells expressing the rat alpha1beta2gamma2S or alpha1beta2gamma2L subunits, and rat cortical neurons. RESULTS: The GABA(A) receptors of the four cell types tested were all sensitive to n-alcohols, albeit with different potencies and efficacies. The effective concentration to increase GABA-induced currents to 125% of control (EC125) was correlated with the carbon chain length of n-alcohols, but slopes for this relationship are different among DRG neurons, the alpha1beta2gamma2S, and alpha1beta2gamma2L subunits. Thus, the potencies of lower alcohols such as ethanol differed among these cell types although higher alcohols such as n-octanol were almost equally potent. In cortical neurons, however, the relationship was shifted in the direction of longer carbon chains, indicating that their sensitivity was lower than those of the other three cell types. The ethanol EC125 values as obtained by experiments or those by extrapolation (in parenthesis) from the EC125-carbon chain length relationship were: 169 (103) mM for DRG neurons, 501 (333) mM for the alpha1beta2gamma2L subunits, 781 (674) mM for the alpha1beta2gamma2S subunits, and (1897) mM for cortical neurons. CONCLUSIONS: It was concluded that the GABA(A) receptors of these four cell types were basically sensitive to n-alcohols including ethanol but the sensitivity curve was shifted to the lower side in the order of decreasing sensitivity of DRG neurons > alpha1beta2gamma2L > alpha1/beta2gamma2S > cortical neurons.

Neuronal nicotinic acetylcholine receptors: a new target site of ethanol.

Whereas a variety of neuroreceptors and ion channels have been demonstrated to be affected by ethanol including GABAA receptors, NMDA receptors, non-NMDA glutamate receptors, 5-HT3 receptors and voltage-gated calcium channels, neuronal nicotinic acetylcholine receptors (nnAChRs) have recently emerged as a new target site of ethanol. The nnAChRs are different from the muscle type nicotinic AChRs with respect to their molecular architecture and pharmacology. This article briefly reviews the structure, distribution and function of nnAChRs for which a considerable amount of information has been rapidly accumulated during the past 5-10 years. The potent and unique action of ethanol on nnAChRs has been unveiled only during the past few years. Most recent developments along this line of ethanol action are discussed in this paper.

Ethanol-nicotine interactions at alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in rat cortical neurons.

Numerous studies have indicated a correlation between ethanol intake and cigarette smoking in heavy drinkers. We have studied the underlying pharmacological basis of this relationship using cultured rat cortical neurons. These neurons express nicotinic receptors having characteristics similar to those described for the alpha4beta2 subunit combination. In the presence of alpha-bungarotoxin both acetylcholine (ACh) and nicotine evoked currents with respective EC50 values of 4.3 and 3.4 microM. The maximal nicotine-activated response, however, was only 56% that of the maximal ACh current. It was previously shown that 10 to 100 mM of ethanol potentiated ACh-mediated currents in these neurons. We demonstrate that 100 mM ethanol similarly potentiates currents evoked by 300 nM (40%) and 1 microM nicotine 61%). This suggests that an ethanol-induced potentiation of nicotinic currents may enhance the acute positive reinforcement associated with nicotine and could increase tobacco use during heavy ethanol intake. However, further experimentation indicated that the continuous perfusion of 30, 100, or 300 nM nicotine desensitizes ACh-evoked currents by 38, 54, and 62%, respectively, with little direct receptor-channel activation. The residual ACh currents of nicotine-desensitized receptor channels were potentiated by 100 mM ethanol to nearly the extent as were the undesensitized control responses. We propose that the opposing effect of ethanol on nicotine-induced desensitization could also explain the increased tobacco use observed with excessive drinking. Thus, ethanol has a dual effect regarding nicotine. It enhances acute nicotine-mediated receptor activation, although opposing the net effect of nicotine-induced receptor channel desensitization.

Ethanol modulation of nicotinic acetylcholine receptor currents in cultured cortical neurons.

Ethanol, at physiologically relevant concentrations, significantly enhanced high-affinity neuronal nicotinic acetylcholine receptor (NnAChR) currents insensitive to alpha-bungarotoxin (alpha-BuTX-ICs) in cultured rat cortical neurons in a fast and reversible manner, as determined by standard whole-cell patch-clamp recording techniques. The enhancement was (mean +/- S.D.) 7.7 +/- 5% to 192 +/- 52% upon coapplication of 3 to 300 mM ethanol with 1 to 3 microM ACh. No plateau for this ethanol-induced enhancement of alpha-BuTX-ICs was reached. The maximal alpha-BuTX-IC evoked by very high concentrations of ACh also was increased upon coapplication of ethanol. In contrast, ethanol weakly inhibited low-affinity NnAChR currents sensitive to alpha-BuTX (alpha-BuTX-SCs) (5 +/- 4% to 29 +/- 6% inhibition by 10 to 300 mM ethanol at 300 to 1000 microM ACh). This neuronal preparation also enabled comparison of ethanol action on NnAChRs with its action on N-methyl-D-aspartate receptor currents and gamma-aminobutyric acid receptor currents within the same neurons. Ethanol (100 mM) was more potent at enhancing NnAChR alpha-BuTX-ICs (61 +/- 9% enhancement) than it was at enhancing gamma-aminobutyric acid receptor current (3 +/- 3% enhancement-not statistically significant) or at inhibiting N-methyl-D-aspartate receptor currents (approximately 35 +/- 7% inhibition). Thus, NnAChRs, particularly those insensitive to alpha-BuTX, may be sensitive conduits through which ethanol can mediate some of its actions in the brain.

Ethanol modulation of excitatory and inhibitory synaptic interactions in cultured cortical neurons.

The effects of ethanol on spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were studied in a culture of embryonic rat cortical neurons. In these experiments, EPSCs and IPSCs were recorded concurrently as inward and outward currents, respectively. These spontaneous currents were dominated by a slow (<1 Hz) repetitive pattern of prolonged N-methyl D-aspartate (NMDA)-EPSCs and co-occurring IPSCs when Mg2+ was left out of the perfusate. A 3- to 5-min bath perfusion of 100 mM ethanol reduced the average integrated EPSC by 65%, while simultaneously potentiating IPSCs by about 3-fold. EPSC frequency was also reduced by about one-third. NMDA-mediated EPSCs were inhibited more than non-NMDA currents. A perfusion of 30 mM ethanol was less effective and probably represents a threshold concentration for these effects. The ethanol inhibition of currents evoked by directly applied glutamate or NMDA to these cells was much less than that observed for spontaneous EPSCs. Currents evoked by exogenous gamma-aminobutyric acid (GABA) application were never potentiated by ethanol. When spontaneous NMDA-EPSCs were blocked with an NMDA antagonist, ethanol no longer potentiated the IPSCs. However, benzodiazepine treatment increased these IPSCs 2-fold. In other experiments, spontaneous IPSCs were blocked by a GABA(A) antagonist. Here, the EPSCs occurred as groups of repetitive bursts. Ethanol decreased the total number of EPSCs per burst but did not decrease their overall amplitude, as in the control recordings. Thus, the way in which ethanol affects concurrently recorded spontaneous EPSCs and IPSCs appears different from the way in which it affects isolated GABA- and NMDA-evoked currents. In addition, the antagonist studies show that concurrently activated NMDA and GABA channels each tend to limit the responses of the other. Thus, the overall effect of ethanol on spontaneous activity may result, in part, by a modification of this synaptic interaction.

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 microM was very potent in accelerating the desensitization of currents induced by acetylcholine. (5) The ethanol modulation was subunit- and state-dependent, occurring in the alpha3beta4 combination but only weakly in the alpha3beta2 combination. (6) In contrast, halothane at 430 microM (approximately 1 MAC) potently suppressed ACh-induced currents in the alpha3beta2 subunit combination.

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 microM, 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.

Ion channel properties of a protein complex with characteristics of a glutamate/N-methyl-D-aspartate receptor.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Subconductance-state currents generated by imidacloprid at the nicotinic acetylcholine receptor in PC 12 cells.

Nitromethylene heterocyclic insecticides are known to act on the nicotinic acetylcholine receptor-channel complex. The effects of the nitromethylene heterocycle imidacloprid on the nicotinic acetylcholine receptor of clonal rat pheochromocytoma (PC 12) cells were studied using the single-channel patch clamp technique. Imidacloprid generated single-channel currents of multiple conductance states in PC12 cells. Both acetylcholine and imidacloprid induced single-channel currents of main conductance and subconductance states with conductances of 33.3 and 9.4 pS by acetylcholine and 30.4 and 9.8 pS by imidacloprid. However, the main conductance currents were generated predominantly by acetylcholine, whereas the subconductance currents were generated predominantly by imidacloprid. Partial block of acetylcholine-induced currents by imidacloprid may be explained on the basis of the single-channel behavior.