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.

Upregulation of surface alpha4beta2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine.

It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing alpha4beta2 receptors were chronically incubated (24-48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surface receptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nM) was the same as that needed to induce upregulation (9.9 nM). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nM), as predicted from cyclical desensitization models. (3) Functional desensitization of alpha3beta4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant alpha4beta2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of alpha4beta2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect the in vivo desensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.

Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C.

Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alpha4beta2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of alpha4beta2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tauf = 1.4 min (65% relative amplitude) and taus = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with taurec = 43 min. Recovery was largely eliminated after replacement of external Ca2+ with Ba2+ and slowed by calphostin C (taurec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (taurec = 14 min), a PKC activator, or by cyclosporin A (with taurec = 8 min), a phosphatase inhibitor. alpha4beta2 receptors containing a mutant alpha4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, alpha4beta2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the "permanent" inactivation of alpha4beta2 receptors observed after chronic nicotine treatment.

Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine.

The influence of alpha and beta subunits on the properties of nicotine-induced activation and desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes was examined. Receptors containing alpha4 subunits were more sensitive to activation by nicotine than alpha3-containing receptors. At low concentrations of nicotine, nAChRs containing beta2 subunits reached near-maximal desensitization more rapidly than beta4-containing receptors. The concentration of nicotine producing half-maximal desensitization was influenced by the particular alpha subunit expressed; similar to results for activation, alpha4-containing receptors were more sensitive to desensitizing levels of nicotine than alpha3-containing receptors. The alpha subunit also influenced the rate of recovery from desensitization; this rate was approximately inversely proportional to the apparent nicotine affinity for the desensitized state. The homomeric alpha7 receptor showed the lowest sensitivity to nicotine for both activation and desensitization; alpha7 nAChRs also demonstrated the fastest desensitization kinetics. These subunit-dependent properties remained in the presence of external calcium, although subtle, receptor subtype-specific effects on both the apparent affinities for activation and desensitization and the desensitization kinetics were noted. These data imply that the subunit composition of various nAChRs determines the degree to which receptors are desensitized and/or activated by tobacco-related levels of nicotine. The subtype-specific balance between receptor activation and desensitization should be considered important when the cellular and behavioral actions of nicotine are interpreted.