Pharmacological Comparison of Transient and Persistent [H]Dopamine Release from Mouse Striatal Synaptosomes and Response to Chronic L-Nicotine Treatment

L-Nicotine stimulates a biphasic release of [H]dopamine from mouse striatal synaptosomes which does not persist after agonist is removed. Approximately 80% of the initial release is transient and disappears with a half-time of less than 1 min; the other 20% persists for several minutes (t1/2, 5–10 min). Both the transient and persistent phases were investigated by 10-min exposures to agonists with an in vitro perfusion technique. A series of nicotinic agonists and antagonists were used to determine the pharmacological relationship of the two phases. Parameters measured included EC50 and Vmax values and desensitization rates for both phases for agonists, Ki values for antagonists and Ki values for low concentrations of agonists. The results are consistent with both phases being mediated by a single type of receptor. In addition, the effects of chronic nicotine treatment on transient and persistent [H]DA release were measured. For both phases, release was decreased approximately 15% by chronic infusion of 4.0 mg/kg/hr L-nicotine. Correlation of the results with inactivation of a portion of the receptors rather than a reversible desensitization is discussed. Nicotine-evoked [H]DA release from striatal synaptosomes is mediated by a receptor exhibiting nicotinic pharmacology (Rapier et al., 1988, 1990; Grady et al., 1992, 1994; Rowell and Hillebrand, 1994; El-Bizri and Clarke, 1994; Lippiello et al., 1995) and is largely dependent upon calcium influx through voltage-gated calcium channels (Soliakov et al., 1995; Turner et al., 1993). NIC-stimulated [H]DA release from rodent striatal synaptosomes is biphasic with one component being a transient response with micromolar affinity for L-NIC and substantial initial release, and the other, a persistent response of low nanomolar affinity for L-NIC but low initial release (Grady et al., 1994). The persistent response does not require previous transient response; i.e., it is not a residual or secondary effect of high concentrations of L-NIC (Rowell, 1995; Grady et al., 1994). The persistent release is dependent on external calcium and is blocked by DHbE, which indicates that it is a nicotinic response requiring calcium influx (Rowell, 1995). It is well established that nAChRs exist in multiple forms with somewhat different pharmacology depending on the subunit composition (McGehee and Role, 1995; Sargent, 1993). Clear evidence of different pharmacology for agonists and antagonists has been demonstrated for different combinations of subtypes expressed in Xenopus oocytes (Luetje and Patrick, 1991; Luetje et al., 1993; Cachelin and Jaggi, 1991; Gross et al., 1991; Cachelin and Rust, 1994; Gerzanich et al., 1995; McGehee and Role, 1995). Electrophysiological measurements of agonist and antagonist potency for nAChRs in rat medial habenula, interpeduncular nucleus, superior cervical ganglion and hippocampus have shown that signals can be differentiated on the basis of pharmacology (Mulle and Changeux, 1990; Mulle et al., 1991; Covernton et al., 1994; Alkondon and Albuquerque, 1993, 1995). These potency variations are thought to reflect differences in subtype composition. Functional responses of nAChRs measured by ion flux and neurotransmitter release from brain slices, cell lines and synaptosomes also differ pharmacologically (Wonnacott et al., 1995; Lukas, 1993; Wong et al., 1995; Sacaan et al., 1995, 1996; Gopalakrishnan et al., 1996; Clarke and Reuben, 1996). For a subtype to form functional presynaptic receptors on dopaminergic terminals in striatum, the message should be detectable in substantia nigra. Message for alpha-3, alpha-4, alpha-5, beta-2 and beta-3 has been shown to exist in subReceived for publication September 30, 1996. 1 This work was supported by grants DA-03194 and DA-00197 from the National Institute on Drug Abuse. ABBREVIATIONS: ACh, acetylcholine; ATR, atropine; ATX, anatoxin-a; CARB, carbachol; CYT, cytisine; DA, dopamine; DEC, decamethonium; DFP, diisopropyl fluorophosphate; DHbE, dihydro-b-erythroidine; EPI, (1)-epibatidine; HEPES, N-[2-hydroxyethyl]-piperazine-N9-[2-ethanesulfonate]; HEX, hexamethonium; MEC, mecamylamine; MeCARB, methylcarbachol; MLA, methyllycaconitine; nAChR, neuronal nicotinic acetylcholine receptor; nBTX, neuronal bungarotoxin; NIC, nicotine; dTC, d-tubocurarine; TMA, tetramethylammonium. 0022-3565/97/2821-0032$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:32–43, 1997 32 at A PE T Jornals on A ril 8, 2017 jpet.asjournals.org D ow nladed from stantia nigra of mice (Marks et al., 1992; Marks, M. J., unpublished results); therefore, it is possible that the transient and persistent phases of [H]DA release from mouse striatal synaptosomes are the result of stimulation of two different subtypes of nAChR. In this case, the persistent and transient responses may express different pharmacologies. Alternatively, the two responses could be mediated by a single receptor which exists in two states. Such a two-state desensitizing model was originally proposed by Katz and Thesleff (1957) (see also Ochoa et al., 1989; Changeux, 1990), and subsequently this two-state model was shown to adequately explain the binding properties of L-[H]NIC to rat brain membrane (Lippiello et al., 1987). Support for the single-receptor hypothesis comes from the slow onset of the persistent response (Rowell, 1995), and the ability of low concentrations of L-NIC to antagonize the transient response evoked by higher concentrations of the agonist (Grady et al., 1994; Lippiello et al., 1995). To determine whether it is likely that the two phases of [H]DA release are the result of stimulation of two different receptor populations or kinetic manifestations of one receptor subtype, the pharmacology of the transient and persistent phases of agonist-induced [H]DA release were compared.