The Potassium Channel Blockers 4-Aminopyridine and Tetraethylammonium Increase the Spontaneous Basal Release of [H]5-Hydroxytryptamine in Rat Hippocampal Slices

Previous investigations have demonstrated that compounds capable of blocking presynaptic potassium channels can stimulate neurotransmitter release at both peripheral and central synapses. This study examined the in vitro effects of the “classical” potassium channel blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the spontaneous basal release of [H]5-hydroxytryptamine ([H]5-HT) from rat hippocampal slices using an automated superfusion apparatus. 4-AP and structural analogs increased the spontaneous basal release of [H]5-HT in a concentration-related manner. The rank order of potencies from the estimated EC50 values indicated that 3,4diaminopyridine (0.88 mM) ' 4-AP (1.2 mM) . 2-AP (89 mM) . 3-AP (100 mM) . pyridine (256 mM). TEA stimulated [H]5-HT release with an estimated EC50 value of 63 mM and was less efficacious than the pyridine congeners. The enhancement of release induced by 1 mM 4-AP was additive with 100 mM TEA and 5 mM veratridine but not with 3,4-diaminopyridine or KCl (25 and 50 mM). The release induced by 4-AP (0.3, 1 and 10 mM) and TEA (30, 100 and 300 mM) was significantly attenuated in a calcium-free buffer containing 1 mM ethylene glycolbis(b-aminoethyl ether N,N,N9,N9-tetraacetic acid. Tetrodotoxin (1 mM), a sodium channel blocker, was unable to block the response to 4-AP (1 mM) and TEA (100 mM). Notably, this concentration of tetrodotoxin reduced the stimulation of [H]5HT release produced by the sodium channel opener veratridine (5 mM). Taken together, the results demonstrate that potassium channel blockade can enhance the spontaneous basal release of [H]5-HT in rat hippocampal slices. These effects are at least partly dependent on extracellular calcium and do not appear to be mediated by modulating sodium channel function. A multiplicity of K channels are widely distributed in both peripheral nervous system and CNS tissue (Hille, 1992), where they regulate neuronal excitability and clearly modulate synaptic events governing neurotransmission. One important mechanism by which presynaptic K channels affect synaptic transmission is by controlling neurotransmitter release. Several hypotheses suggest that the blockade of K channels prolongs the action potential duration, which leads to an increased influx of extracellular Ca through voltagesensitive Ca channels and results in an enhanced release of neurotransmitter (Thesleff, 1980; Rudy, 1988). Aminopyridines and TEA have been used as standard reference compounds in a variety of studies involving the functions and properties of K channels (Glover, 1982; Rudy, 1988). These compounds have been classically employed as blockers of K efflux and conductances in a number of physiological preparations from both central and peripheral tissues (Rudy, 1988). Although there are differences in the selectivities of 4-AP and TEA for various K channels, these compounds share the ability to block presynaptic voltagedependent K channels and modulate the release of a variety of neurotransmitters. The facilitating effects of 4-AP on neurotransmitter release have been reported for norepinephrine (Hu and Fredholm, 1991), ACh (Tapia and Sitges, 1982; Dolezal and Tucek, 1983; Drukarch et al., 1989), dopamine (Boireau et al., 1991; Scheer and Lavoie, 1991), g-aminobutyric acid (Tapia et al., 1985) and glutamate (Tapia and Sitges, 1982; Tibbs et al., 1989). The stimulatory effects of 4-AP on ACh and dopamine release have been further corroborated in vivo using intrastriatal dialysis (Damsma et al., 1988; Dawson and Routledge, 1995). In addition, the K channel blocker TEA has been demonstrated to induce the release of ACh (Drukarch et al., 1989, norepinephrine (Hu et al., 1991) and dopamine (Boireau et al., 1991). The indirect modulation of ligand-gated K channel function by 5-HT through second-messenger coupling has been well documented (see Belardetti and Siegelbaum, 1988). In contrast, the ability of K channels to modulate the neuroReceived for publication October 18, 1996. ABBREVIATIONS: 4-AP, 4-aminopyridine; TMA, tetramethylammonium; 2-AP, 2-aminopyridine; 3-AP, 3-aminopyridine; 3,4-DAP, 3,4-diaminopyridine; EGTA, ethylene glycol-bis(b-aminoethyl ether) N,N,N9,N9-tetraacetic acid; TTX, tetrodotoxin; 5-HT, serotonin; K, potassium; KATP, ATP-sensitive potassium channels; Ca, calcium; Na, sodium; NMDA, N-methyl-D-aspartate; [H]5-HT, [H]5-hydroxytryptamine. 0022-3565/97/2821-0262$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 282:262–270, 1997 262 at A PE T Jornals on Jne 5, 2017 jpet.asjournals.org D ow nladed from chemical characteristics of 5-HT neurotransmission has received little attention. Anden and Leander (1979) reported that 4-AP administered peripherally did not change the turnover of 5-HT or dopamine but markedly accelerated that of norepinephrine in the brain and spinal cord. In contrast, recent data from Pei et al. (1995) using in vivo microdialysis demonstrated that 4-AP and TEA, tested at single concentrations and perfused directly into the hippocampus, enhanced 5-HT efflux in this brain region. Discrepancies between the two studies may be explained on the basis of routes of administration and differences in technical methods used to quantitate the levels of neurotransmitters. The present study extends the previous findings involving K channel blockers and neurotransmitter release by examining the ability of 4-AP, pyridine analogs and TEA to enhance the spontaneous basal release of [H]5-HT from rat hippocampal slices. In contrast to the previous study by Pei et al. (1995), experiments were designed to determine the full concentration-effect relationships for these agents and to examine the additivity of neurochemical responses between K channel blockers and chemical depolarizing agents such as veratridine and KCl. Finally, investigations examining the biochemical mechanism by which these agents facilitated the spontaneous release of [H]5-HT were determined by studying the Ca and Na dependence of these effects. Materials and Methods Animals. Male albino Sprague-Dawley rats (Charles River, Kingston, NY) were group-housed on a 12-hr light/12-hr dark lighting cycle under standard laboratory conditions. Animals were acclimated for a period of at least 7 days before experimentation and weighed between 300 and 400 g. Materials. The following drugs and chemicals were used in this study: [H]5-HT (specific activity 5 97–99 Ci/mmol) (Amersham Corporation, Arlington Hts., Il), 4-AP, 3,4-DAP, 3-AP, 2-AP, pyridine and glybenclamide (Sigma Chemical Co., St. Louis, MO), TTX, veratridine, charybdotoxin, TEA and quinine (RBI, Natick, MA) and Apamin (Latoxan, Rosans, France). Fluoxetine was kindly provided by Lilly-Laboratories (Indianapolis, IN). All other reagents utilized were of the highest chemical grade and purity. Neurotransmitter release. The rats were sacrificed by decapitation, and the brains were rapidly removed and placed on ice for dissection. The hippocampus was removed and washed in ice-cold Krebs buffer. Hippocampal tissue was subsequently chopped into slices (0.25 mm by 0.25 mm) using a McIlwain tissue chopper and suspended in a volume of oxygenated (95% O2/CO2) Krebs buffer (pH 7.4; 37°C). The composition of the buffer was (mM): NaKH2PO4, 1.2; NaCl, 118; KCl, 4.8; glucose, 10; CaCl2, 1.3; MgSO4, 1.2; NaHCO3, 25; ascorbic acid, 0.1; pargyline, 0.128. The slices were subsequently incubated with 100 nM [H]5-HT at 37°C for 60 min with occasional agitation. After incubation with [H]5-HT, the slices were gently triturated and 200-ml aliquots of the preparation were loaded into the chambers of a Brandel (Gaithersburg, MD) superfusion apparatus that were enclosed by polyethylene filter discs. During the remainder of the experiment, the tissue preparation was superfused with Krebs buffer containing 10 mM fluoxetine to prevent the reuptake of 5-HT. After loading of the tissue into the apparatus, each chamber was continuously superfused with buffer for 45 min at a flow rate of 0.6 ml/min to remove excess radioactivity that was not incorporated into the tissue preparation. The flow rate was reduced to 0.3 ml/min during the remainder of the experiment, which was determined in preliminary studies to produce the optimal conditions for a stable base line, adequate oxygenation and fraction collection volumes. After the washout period, eighteen 5-min fractions were collected at a superfusion flow rate of 0.3 ml/min. Spontaneous release was recorded at base-line levels during the initial three collection fractions. Drug or appropriate vehicle was added during a 15-min period that began after the collection of the base-line fractions. Experiments designed to test the effects of K-induced depolarization and release were performed by substituting an equimolar concentration of K for Na. Studies investigating the Ca-dependent effects of [H]5-HT release were performed by substituting 1 mM EGTA for CaCl2. Compound or vehicle was subsequently washed out of the system by returning to standard buffer superfusion for the remainder of the experiment. Upon completion of the experiment, the total amount of radioactivity for each fraction, as well as that remaining in the slices and filters, was counted using standard scintillation techniques. Filters and slices were solubilized in 1 ml of NCS Solubilizer (0.6 N; Amersham) before the addition of Ready Organic scintillation cocktail (Beckman, Fullerton, CA). Calculation of fractional release of [H]5-HT. The release of [H]5-HT was expressed as a percentage of the labeled transmitter present in the superfusion chamber at the time of collection such