Exposure to nicotine and sensitization of nicotine-induced behaviors.

Evidence for an important link between sensitization of midbrain dopamine (DA) neuron reactivity and enhanced self-administration of amphetamine and cocaine has been reported. To the extent that exposure to nicotine also sensitizes nucleus accumbens DA reactivity, it is likely that it will also impact subsequent drug taking. It is thus necessary to gain an understanding of the long-term effects of exposure to nicotine on nicotinic acetylcholine receptors (nAChRs), neuronal excitability and behavior. A review of the literature is presented in which different regimens of nicotine exposure are assessed for their effects on upregulation of nAChRs, induction of LTP in interconnected midbrain nuclei and development of long-lasting locomotor and DA sensitization. Exposure to nicotine upregulates nAChRs and nAChR currents and produces LTP of excitatory inputs to midbrain DA neurons. These effects appear in the hours to days following exposure. Exposure to nicotine also leads to long-lasting sensitization of nicotine's nucleus accumbens DA and locomotor activating effects. These effects appear days to weeks after drug exposure. A model is proposed in which nicotine exposure regimens that produce transient nAChR upregulation and LTP consequently produce long-lasting sensitization of midbrain DA neuron reactivity and nicotine-induced behaviors. These neuroadaptations are proposed to constitute critical components of the mechanisms underlying the initiation, maintenance and escalation of drug use.

Dorsal root ganglion neurons express multiple nicotinic acetylcholine receptor subtypes.

Although nicotinic agonists can modulate sensory transmission, particularly nociceptive signaling, remarkably little is known about the functional expression of nicotinic acetylcholine receptors (nAChRs) on primary sensory neurons. We have utilized molecular and electrophysiological techniques to characterize the functional diversity of nAChR expression on mammalian dorsal root ganglion (DRG) neurons. RT-PCR analysis of subunit mRNA in DRG tissue revealed the presence of nAChR subunits alpha2-7 and beta2-beta4. Using whole cell patch-clamp recording and rapid application of nicotinic agonists, four pharmacologically distinct categories of nicotinic responses were identified in cultured DRG neurons. Capacitance measurements were used to divide neurons into populations of large and small cells, and the prevalence of nicotinic responses was compared between groups. Category I (alpha7-like) responses were seen in 77% of large neurons and 32% of small neurons and were antagonized by 10 nM methyllycaconitine citrate (MLA) or or 50 nM alpha-bungarotoxin (alpha-BTX). Category II (alpha3beta4-like) responses were seen in 16% of large neurons and 9% of small neurons and were antagonized by 20 microM mecamylamine but not 10 nM MLA or 1 microM DHbetaE. Category II responses had a higher sensitivity to cytisine than nicotine. Two other types of responses were identified in a much smaller percentage of neurons and were classified as either category III (alpha4beta2-like) or category IV (subtype unknown) responses. Both the alpha7-like and alpha3beta4-like responses could be desensitized by prolonged applications of the analgesic epibatidine.

Expression of glycine receptors in rat sensory neurons vs. HEK293 cells yields different functional properties.

Many structure-function studies of the glycine receptor (GlyR), and other ligand-gated ion channels, use somatic cell lines or Xenopus oocytes as expression systems. Using a polyethylenimine-based technique, we transfected GlyR cDNA into primary cultures of rat dorsal root ganglion (DRG) neurons. We then compared the functional properties of wildtype and a mutant GlyR expressed in DRG neurons with HEK 293 cells. The glycine sensitivity of the wildtype GlyR was nearly identical for the two cell types. The mutant GlyR has an arginine for glutamine substitution at position 271 (R271Q), which results in low glycine sensitivity relative to wildtype receptors expressed in HEK cells. This point mutation is associated with startle disease (hyperekplexia) in humans. Mutant GlyR expression in DRG neurons resulted in a significantly lower glycine sensitivity than was seen in HEK cells. This supports the idea that neuron-specific post-translational modifications may be important for determining receptor function.

Long-term potentiation of excitatory inputs to brain reward areas by nicotine.

Nicotine reinforces smoking behavior by activating nicotinic acetylcholine receptors (nAChRs) in the midbrain dopaminergic (DA) reward centers, including the ventral tegmental area (VTA). Although nicotine induces prolonged excitation of the VTA in vivo, the nAChRs on the DA neurons desensitize in seconds. Here, we show that activation of nAChRs on presynaptic terminals in the VTA enhances glutamatergic inputs to DA neurons. Under conditions where the released glutamate can activate NMDA receptors, long-term potentiation (LTP) of the excitatory inputs is induced. Both the short- and the long-term effects of nicotine required activation of presynaptic alpha7 subunit-containing nAChRs. These results can explain the long-term excitation of brain reward areas induced by a brief nicotine exposure. They also show that nicotine alters synaptic function through mechanisms that are linked to learning and memory.

Cholinesterase inhibition by potato glycoalkaloids slows mivacurium metabolism.

BACKGROUND: The duration of action for many pharmaceutical agents is dependent on their breakdown by endogenous hydrolytic enzymes. Dietary factors that interact with these enzyme systems may alter drug efficacy and time course. Cholinesterases such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze and inactivate several anesthetic drugs, including cocaine, heroin, esmolol, local ester anesthetics, and neuromuscular blocking drugs. Natural glycoalkaloid toxins produced by plants of the family Solanaceae, which includes potatoes and tomatoes, inhibit both AChE and BuChE. Here the authors assess the extent to which two solanaceous glycoalkaloids (SGAs), alpha-solanine and alpha-chaconine, can alter the effects of neuromuscular blocking drugs and cholinesterase inhibitors in vivo and in vitro. METHODS: Inhibition of purified human AChE and BuChE by SGAs, neuromuscular blocking drugs, and cholinesterase inhibitors was assessed by an in vitro colorimetric cholinesterase assay. In vivo experiments were carried out using anesthetized rabbits to test whether SGAs affect recovery from mivacurium-induced paralysis. RESULTS: SGAs inhibited human BuChE at concentrations similar to those found in serum of individuals who have eaten a standard serving of potatoes. Coapplication of SGAs (30-100 nm) with neuromuscular blocking drugs and cholinesterase inhibitors produced additive cholinesterase inhibition. SGA administration to anesthetized rabbits inhibited serum cholinesterase activity and mivacurium hydrolysis. In addition, SGA prolonged the time needed for recovery from mivacurium-induced paralysis (149 +/- 12% of control; n = 12). CONCLUSIONS: These findings support the hypothesis that inhibition of endogenous enzyme systems by dietary factors can influence anesthetic drug metabolism and duration of action. Diet may contribute to the wide variation in recovery time from neuromuscular blockade seen in normal, healthy individuals.

Target-specific control of nicotinic receptor expression at developing interneuronal synapses in chick.

Neuronal differentiation and development of synaptic specializations are strongly influenced by cellular interactions. We compared the effects of interaction with distinct autonomic targets on the molecular and biophysical differentiation of 'upstream' neuron-neuron synapses. Contact with cardiac tissue induced expression of nicotinic receptor channels (nAChRs) distinct from those induced by renal tissue in presynaptic autonomic neurons. The kinetics of cholinergic currents at interneuronal synapses are dictated by the peripheral target contacted. Analysis of the nAChR channel subtypes and subunits in individual neurons demonstrated that the profile of transmitter receptors expressed at mature neuron-neuron synapses develops from the convergent influences of input-derived (anterograde) and target-specific (retrograde) signals.

Molecular diversity of neuronal nicotinic acetylcholine receptors.

The potent behavioral and cognitive effects of nicotine highlight the physiological importance of nicotinic acetylcholine receptors (nAChRs). These receptors are part of the superfamily of neurotransmitter-gated ion channels that are responsible for rapid intercellular communication. Molecular cloning of the protein subunits that make up these receptors has led to greater understanding of the pharmacology and physiology of nAChRs. This review outlines our current understanding of the molecular constituents of these receptors and some of the recent studies of the structural determinants of receptors function.

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

Mechanism of extracellular Ca2+ receptor-stimulated hormone release from sheep thyroid parafollicular cells.

1. Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5-HT) in response to increased external Ca2+. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled receptor for external Ca2+ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release. 2. Whole-cell recordings in the permeabilized-patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+]0) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca2+]0 activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near -25 mV indicating poor ion selectivity. 3. The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd3+ > Ba2+ > Ca2+ > > Mg2+. The insensitivity of these cells to high external Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4. Elevation of [Ca2+]0 also stimulates increases in intracellular Ca2+ concentration ([Ca2+]i) and this effect is largely inhibited by the Ca2+ channel blocker nimodipine, indicating that L-type voltage-gated Ca2+ channels contribute to the response to elevated [Ca2+]0. 5. Elevated [Ca2+]0 induces an inward current under conditions where the only permeant external cation is Ca2+, indicating that influx via the cation conductance is another source of the increases in [Ca2+]i. 6. Extracellular Ca2+ stimulates 5-HT release with an EC50 of 1.5 mM. Nimodipine blocks 90% of the Ca2+0-induced 5-HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca2+ channels in CaR-induced hormone secretion. Although earlier studies indicate that high [Ca2+]0 induces release of Ca2+ from intracellular stores, thapsigargin-induced depletion of these stores did not affect secretion from these cells, indicating that Ca2+ influx is necessary and sufficient for the Ca2+0-induced 5-HT secretion. 7. Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca2+0-induced 5-HT release by 50% while phorobol ester-induced 5-HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway. 8. We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 had no effect on the response to elevated [Ca2+]0. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca(2+)-induced 5-HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca2+]0.

Natural inhibitors of cholinesterases: implications for adverse drug reactions.

PURPOSE: Acetylcholinesterase and butyrylcholinesterase are two closely related enzymes important in the metabolism of acetylcholine and anaesthetic drugs, including succinylcholine, mivacurium, and cocaine. The solanaceous glycoalkaloids (SGAs) are naturally occurring steroids in potatoes and related plants that inhibit both acetylcholinesterase and butyrylcholinesterase. There are many clinical examples of direct SGA toxicity due to cholinesterase inhibition. The aim of this study was to review the hypotheses that (1) SGAs may be the evolutionary driving force for atypical butyrylcholinesterase alleles and that (2) SGAs may adversely influence the actions of anaesthetic drugs that are metabolized by acetylcholinesterase and butyrylcholinesterase. SOURCE: The information was obtained by Medline search and consultation with experts in the study of SGAs and cholinesterases. PRINCIPAL FINDINGS: The SGAs inhibit both acetylcholinesterase and butyrylcholinesterase in numerous in vitro and in vivo experiments. Although accurate assays of SGA levels are difficult, published data indicate human serum SGA concentrations at least ten-fold lower than required to inhibit acetylcholinesterase and butyrylcholinesterase in vitro. However, we review evidence that suggests the dietary ingestion of SGAs can initiate a cholinergic syndrome in humans. This syndrome appears to occur at SGA levels lower than those which interfere with anaesthetic drug catabolism. The world distribution of solanaceous plants parallels the distribution of atypical alleles of butyrylcholinesterase and may explain the genetic diversity of the butyrylcholinesterase gene. CONCLUSION: Correlative evidence suggests that dietary SGAs may be the driving force for atypical butyrylcholinesterase alleles. In addition, SGAs may influence the metabolism of anaesthetic drugs and this hypothesis warrants experimental investigation.

Presynaptic ionotropic receptors.

Recent studies have provided new insights into the role of presynaptic ligand-gated ion channels in modifying synaptic transmission. Along with a growing list of different types of presynaptic ionotropic receptors and the cell types that express them, there have been advances in characterizing the molecular components of the receptors as well as the signaling processes that link receptor activation to changes in neurotransmitter release. Perhaps most striking is the recent convergence of data from biochemical, molecular and electrophysiological studies, implicating presynaptic ionotropic receptors in the effects of psychoactive and addictive drugs.

Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.

The behavioral and cognitive effects of nicotine suggest that nicotinic acetylcholine receptors (nAChRs) participate in central nervous system (CNS) function. Although nAChR subunit messenger RNA (mRNA) and nicotine binding sites are common in the brain, there is little evidence for synapses mediated by nAChRs in the CNS. To test whether, CNS nAChRs might modify rather than mediate transmission, the regulation of excitatory synaptic transmission by these receptors was examined. Nanomolar concentrations of nicotine enhanced both glutamatergic and cholinergic synaptic transmission by activation of presynaptic nAChRs that increased presynaptic [Ca2]i. Pharmacological and subunit deletion experiments reveal that these presynaptic nAChRs include the alpha 7 subunit. These findings reveal that CNS nAChRs enhance fast excitatory transmission, providing a likely mechanism for the complex behavioral effects of nicotine.

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells.

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

Bradykinin modulates the electrophysiology of cultured rat sensory neurons through a pertussis toxin-insensitive G protein.

Application of the nonapeptide bradykinin (BK) to rat sensory neurons in culture elicits two types of electrical responses in a subpopulation of the cells: (i) an excitatory inward cation current (I(BK)), and (ii) an inhibition of voltage-activated calcium currents (I(Ca)). Ion replacement experiments and current-voltage measurements indicate that I(BK) shows little selectivity for monovalent cations. We investigated the role of GTP-binding proteins in the BK signal transduction pathway. Internal dialysis of neurons with the inhibitory guanine nucleotide analogue, GDPbetaS, decreased the percentage of cells responding and the magnitude of the responses. This finding lead us to propose that a G protein is involved in this transduction pathway. We have attempted to identify the G protein subtype involved using pretreatment with the bacterial toxins pertussis toxin (PTX) and cholera toxin (CTX). PTX potentiated I(BK) slightly, but had no effect on the inhibition of I(Ca) by BK. In contrast, PTX pretreatment blocked the effects of norepinephrine and neuropeptide Y on I(Ca), confirming that the PTX was capable of interfering with appropriate G protein signaling in these neurons. Pretreatment with CTX had no effect on the BK-induced inward current response. Therefore we propose that the BK signal transduction pathway utilizes a G protein that is not part of the G(i), G(o), G(t), or G(s) families.

Species-specific expression of cholecystokinin messenger RNA in rodent dorsal root ganglia.

The expression of cholecystokinin (CCK) messenger RNA (mRNA) was examined in dorsal root ganglia of rat and guinea pig using in situ hybridization histochemistry and RNA (Northern) blot hybridization with synthetic oligodeoxyribonucleotide (oligomer) probes. In guinea pig, CCK mRNA was detected in small and medium-sized neuronal perikarya comprising approximately 10-15% of the total dorsal root ganglia cell population. In contrast, in neurons of rat dorsal root ganglia, CCK mRNA was not detectable. Northern blot analyses revealed a single CCK mRNA species of expected size (0.8 kb) in guinea pig, but not rat, dorsal root ganglia. A 0.8 kb CCK mRNA was, however, detected in cortex of both rat and guinea pig. These data suggest that CCK is normally not synthesized in neurons of rat dorsal root ganglia and that there are species differences in CCK gene expression in mammalian sensory ganglia.