Strychnine: A Potent Competitive Antagonist of a-Bungarotoxin-Sensitive Nicotinic Acetylcholine Receptors In Rat Hippocampal Neurons

In our study, evidence is provided that strychnine, a competitive antagonist at glycine-gated Cl channels, is also a potent competitive antagonist at native a-7-containing, a-bungarotoxin-sensitive nicotinic acetylcholine receptor (nAChRs). To address the effects of strychnine on two types of nicotinic responses, the whole-cell mode of the patch-clamp technique was applied to rat hippocampal neurons in culture. Type IA and type II nicotinic currents evoked by acetylcholine (ACh) were inhibited by strychnine in a concentration-dependent manner with IC50s of 1.2 and 38 mM, respectively. Strychnine (2 mM) decreased the peak amplitude of the a-bungarotoxin-sensitive type IA current in a voltage-independent manner and prolonged the decay phase of this current. The concentration-response curve for ACh in evoking type IA current showed a parallel shift to the right in the presence of strychnine (2 mM); the EC50 for ACh was increased from 0.4 to 0.8 mM. These findings suggest that strychnine acts as a competitive antagonist of ACh at the a7 nAChRs that subserve type IA current. In contrast, the inhibition by strychnine of type II current was strongly voltage dependent, and the decay phase of this current was markedly accelerated by the toxin, suggesting an open-channel blockade by strychnine of the a4b2 nAChRs subserving type II currents. Preexposure of the neurons to strychnine enhanced its ability to decrease the peak amplitude of type II currents, indicating that the toxin may also act on a4b2 nAChR channels that are not open. It is concluded that strychnine is a potent competitive antagonist of ACh at neuronal a7 nAChRs and a noncompetitive antagonist at the a4b2 nAChR. Although strychnine (fig. 1) is a well-known competitive antagonist of glycine at glycine-gated Cl channels (Saitoh et al., 1994), its binding to glycine-resistant sites has been recognized for many years. For instance, numerous reports have provided evidence that strychnine can inhibit cholinergic transmission at the neuromuscular junction and in sympathetic ganglia (Lanari and Luco, 1939; Alving, 1961). A presynaptic nicotinic mechanism appears to account for the ability of the toxin (1–100 mM; Koelle and McKinstry, 1976) to block the release of ACh from sympathetic ganglia (Collier and Katz, 1970) and to block the release of catecholamines from adrenal medullary chromafin cells (10–30 mM; Kuijpers et al., 1994). However, it is unlikely that strychnine poisoning could be accounted for by these effects, because they are observed at concentrations that are considerably greater than those at which the alkaloid exerts its excitatory and depressant effects in the CNS (Franz, 1975). Nevertheless, findings that strychnine produces spiking in the cortex (Amato et al., 1969; Garcı́a Ramos et al., 1977), where glycine receptors are rare (Frostholm and Rotter, 1985), strongly support the notion that some of the toxic effects of this alkaloid can be accounted for by its action on receptors other than the glycine receptors in the CNS. In contrast to the previously noted low potency of strychnine at nonneuronal nAChRs, the toxin has been shown to have potent actions on certain subtypes of neuronal nAChRs. On the outer hair cells of the vestibular system, strychnine is even more potent than curare or a-BGT in antagonizing the Received for publication August 14, 1997. 1 This study was supported by United States Public Health Service Grants NS25296 and ES05730 and by PRONEX (Brazil). Some of the data have appeared previously in abstract form (Matsubayashi et al., 1996). ABBREVIATIONS: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; a-BGT, a-bungarotoxin; CNS, central nervous system; GABA, g-amino butyric acid; MEM, minimum essential medium; HEPES, (N-[2-hydroxyethyl]piperazine-N9-[2-ethane sulfonic acid]); DHbE, dihydro-berythroidine; ATP-RS, adenosine 59-trisphosphate-regenerating solution; EGTA, ethyleneglycol-bis-(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; t, decay-time constant; EC50, agonist concentration that evokes 50% of the maximal response; IC50, antagonist concentration that reduces by 50% the maximal response; nH, Hill coefficient; ANOVA, analysis of variance; Ki, apparent affinity of an antagonist for the receptor; Kd, apparent affinity of an agonist for the receptor. 0022-3565/98/2843-0904$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 3 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 284:904–913, 1998 904 at A PE T Jornals on N ovem er 7, 2016 jpet.asjournals.org D ow nladed from nicotinic actions of ACh (Bartolami et al., 1993; Erostegui et al., 1994; Guth et al., 1994). The anticholinergic effects of strychnine in the auditory system (Guth et al., 1994) have been observed at concentrations as low as 0.01 mM (Doi and Ohmori, 1993), and strychnine binds to the neuronal nAChRs of the outer hair cells with a Kd of approximately 35 nM (Lawoko et al., 1995). Of interest, in addition to the outer hair cell nAChRs, which are likely to be made up of the a9 subunit (Elgoyhen et al., 1994), homomeric a7 nAChRs, homomeric a8 nAChRs and homomeric a9 nAChRs ectopically expressed in Xenopus oocytes are highly sensitive to inhibition by toxicologically relevant concentrations of strychnine (Anand et al., 1993; Elgoyhen et al., 1994; Gerzanich et al., 1994; Peng et al., 1994). Studies directed at determining the ability of centrally acting compounds to interact with native neuronal nAChRs have been helpful in improving the understanding of the functions of these receptors in the CNS. For instance, studies of the interactions of epibatidine, a-conotoxin-ImI and amantadine with neuronal nAChRs have suggested that different subtypes of these receptors are likely to be involved in analgesia, convulsions and Parkinson’s disease. In fact, 1) epibatidine, a toxin isolated from the skin of the frog Epipedobates tricolor and known to cause analgesia when injected i.p. in mice (Badio and Daly, 1994), is a potent agonist at a4b2containing nAChRs in hippocampal neurons (Alkondon and Albuquerque, 1995); 2) a-conotoxin-ImI, a toxin isolated from the venom of Conus imperialis and shown to cause convulsions when injected intracerebroventricularly in mice and rats (Johnson et al., 1995), is a competitive antagonist selective for native a7-bearing nAChRs (Pereira et al., 1996) and 3) at therapeutically relevant concentrations, amantadine, a drug used to treat Parkinson’s disease, acts as a noncompetitive antagonist of ACh at a7-containing nAChRs in hippocampal neurons (Matsubayashi et al., 1997). Considering that, 1) homomeric a7 nAChRs heterologously expressed in Xenopus oocytes are sensitive to blockade by strychnine (Peng et al., 1994), 2) convulsions are among the well-documented toxic effects of strychnine, 3) hippocampal foci can be developed as a consequence of exposure to strychnine (Baker, 1965), and 4) a7-bearing nAChRs, which are expressed in the majority of the hippocampal neurons (Alkondon and Albuquerque, 1993), appear to be involved in convulsions, we decided to investigate the interactions of the toxin with the different subtypes of nAChRs (including the a7-bearing receptors) present on hippocampal neurons. As reported previously, hippocampal neurons can respond to nicotinic agonists with at least one of three pharmacologically and kinetically distinct types of nicotinic currents, namely types IA, II and III that arise from the activation of nAChRs composed of a7, a4b2, and a3b4 subunits, respectively (Alkondon and Albuquerque, 1993; Alkondon et al., 1994; Ishihara et al., 1995; Barbosa et al., 1996). Our study demonstrates that strychnine inhibits both a7 nAChR-mediated type IA currents and a4b2 nAChR-mediated type II currents recorded from rat hippocampal neurons. It is likely that some of the symptoms of strychnine-induced intoxication are associated with its ability to block a7 nAChRs in the CNS, because we provide evidence that the concentrations at which strychnine inhibits the activation of native a7 nAChRs present on hippocampal neurons are similar to those at which it interacts with glycine receptors in the