Planarians require an intact brain to behaviorally react to cocaine, but not to react to nicotine.

Planarians possess a rudimentary brain with many features in common with vertebrate brains. They also display a remarkable capacity for tissue regeneration including the complete regeneration of the nervous system. Using the induction of planarian seizure-like movements (pSLMs) as a behavioral endpoint, we demonstrate that an intact nervous system is necessary for this organism to react to cocaine exposure, but not necessary to react to nicotine administration. Decapitated planarians (Girardia tigrina) display pSLMs indistinguishable from intact worms when exposed to nicotine, but cocaine-induced pSLMs are reduced by about 95% upon decapitation. Decapitated worms recover their normal sensitivity to cocaine within 5 days after head amputation. In worms where half of the brain was removed or partially dissected, the expression of cocaine-induced pSLMs was reduced by approximately 75%. Similar amputations at the level of the tail did not show a significant decrease to cocaine exposure. To the best of our knowledge, our work is the first report that explores how regenerating planarians react to the exposure of cocaine.

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction.

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

Characterization of cembranoid interaction with the nicotinic acetylcholine receptor.

The class of diterpenoids with a 14-carbon cembrane ring, the cembranoids, includes both competitive and noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR). All 20 coelenterate-derived cembranoids studied in this report inhibited [piperidyl-3,4-3H]-phencyclidine ([3H]-PCP) binding to its high-affinity site on the electric organ AChR, with IC50s ranging from 0.9 microM for methylpseudoplexaurate to 372 microM for lophotoxin. Inhibition was complete with all cembranoids but lophotoxin and most Hill coefficients were close to 1. Methylpseudoplexaurate and [3H]-PCP binding was competitive. Methylpseudoplexaurate and the fourth most potent cembranoid, eunicin, competed with each other for [3H]-PCP displacement, indicating that there exist one or more cembranoid sites on the AChR. Cembranoid affinity for the AChR correlated with hydrophobicity, but was also dependent on other features. Methylpseudoplexaurate and n-octanol also competed with each other for [3H]-PCP displacement, indicating that the cembranoid site is linked to the n-octanol site on the AChR. Unlike lophotoxin, the five cembranoids tested did not inhibit [125I]Tyr54-alpha-bungarotoxin binding to the AChR agonist sites. All seven cembranoids tested on oocyte-expressed electric organ AChR reversibly blocked acetylcholine-induced currents, although the inhibitor concentration curves were shallow and the inhibition was incomplete.

The 9-arginine residue of alpha-conotoxin GI is responsible for its selective high affinity for the alphagamma agonist site on the electric organ acetylcholine receptor.

The two agonist-binding domains of the electric organ nicotinic acetylcholine receptor are located at the alphagamma and alphadelta subunit interfaces. alpha-Conotoxins GI and MI are competitive antagonists of this receptor and, like d-tubocurarine, bind to the alphagamma site with much higher affinity than to the alphadelta site. In the present study, alpha-conotoxin SIA also displayed strong affinity for the alphagamma site but no measurable affinity for the alphadelta site, thus showing even greater site-selectivity. In contrast, alpha-conotoxin SI does not distinguish between the two agonist sites, although its sequence differs from that of GI at only three positions: GI, ECCNPACGRHYSC; SI, ICCNPACGPKYSC. Analogues of SI and GI modified at these three positions were studied to identify the determinants of GI's alphagamma selectivity. Substituting arginine for proline at position 9 produced peptides which displayed "GI-like" selectivity for the alphagamma site. Conversely, substituting proline for arginine at position 9 resulted in "SI-like" nonselective inhibitors. An SI analogue having alanine in place of proline 9 did not distinguish between the two agonist sites and displayed about the same affinity as SI, indicating the importance of the arginyl cation. Interchanging the residues at position 1 or at position 10 influenced the affinity for the receptor but did not measurably change peptide selectivity. Therefore, of the three sequence differences in SI and GI, the variation at position 9, proline and arginine, respectively, is sufficient to account for GI's selective high-affinity binding to the alphagamma site on the electric organ acetylcholine receptor.

Differential effects of dimethyl sulfoxide on nicotinic acetylcholine receptors from mouse muscle and Torpedo electrocytes.

The present study examined the effects of 0.1% dimethyl sulfoxide (DMSO) on nicotinic acetylcholine receptors (nAChR) from mouse muscle and Torpedo californica electrocytes. Receptors were expressed in Xenopus laevis oocytes and studied with voltage-clamp. When applied simultaneously with acetylcholine, DMSO did not inhibit current amplitude of either receptor. Preincubation with DMSO for 1 min reduced current amplitude by approximately 50% from oocytes expressing electrocyte receptor. Preincubation did not affect the muscle receptor. With electric organ membranes, 0.1% DMSO did not block either [alpha-(125)I]bungarotoxin binding to the nAChR agonist site or [3H]phencyclidine binding to its high affinity site on resting or desensitized receptor. These data suggest that DMSO might be affecting the electrocyte receptor through a second messenger system.

The alpha-conotoxins GI and MI distinguish between the nicotinic acetylcholine receptor agonist sites while SI does not.

The alpha-conotoxins are paralytic peptide toxins from Indo-Pacific cone snails. This paper presents a detailed analysis of how alpha-conotoxins inhibit [125I]-alpha-bungarotoxin (125I-BTX) equilibrium binding to the acetylcholine receptor (AChR) from electric organ of Torpedo californica and Torpedo nobiliana. All three alpha-conotoxins studied, SI, GI, and MI, completely inhibited 125I-BTX binding with the same order of potency in both species (MI approximately GI > SI approximately d-tubocurarine). BTX-concentration curves showed that this inhibition is competitive. However, while SI appeared to bind to a homogeneous population of sites, both GI and MI displayed curare-like heterogeneous binding. Studies using partially-blocked AChR demonstrated that both GI and MI display different affinities toward the two agonist sites, much like small curariform antagonists do. The high-affinity site for these two alpha-conotoxins is also the high-affinity d-tubocurarine site, which is believed to be located at the alpha gamma-subunit interface. The high-affinity binding of MI and GI was of the same order of magnitude as that of d-tubocurarine; however, their affinity for the other agonist site was somewhat greater than that of dTC, resulting in less site selectivity. Despite being homologous to GI and MI, SI did not distinguish between the two sites. A possible molecular basis for this difference is presented.