Introduction

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Aminopyridines, particularly 4-aminopyridine (4-AP), have been investigated as a means of beneficial symptomatic treatment in a variety of neurological conditions of demyelination diseases including multiple sclerosis, myasthenia gravis, and spinal cord injury (Murray and Newsom-Davis, 1981; Jones et al., 1983; Targ and Kocsis, 1985; Waxman, 1993; Segal and Brunnemann, 1997; Potter et al., 1998; Halter et al., 2000). 4-AP is a typical blocker of the voltage-gated, fast-inactivating A-type K⁺ channels or I_A channels (Mathie et al., 1998); the inhibition of I_A channels, specifically those located in the inter and para-nodal regions of axonal membranes, prolongs the duration of action potential and facilitates the propagation of action potentials in demyelinated nerve fibers (Bostock et al., 1981; Targ and Kocsis, 1985; Schauf, 1987; Kaji and Sumner, 1988; Blight, 1989; Shi and Blight, 1997). K⁺ channel blockers including 4-AP also increase Ca²⁺ influx, which can increase transmitter release in a wide range of neuronal types (Bowman and Savage, 1981; Glover, 1982; Soni and Kam, 1982; Hayes et al., 1994), beneficial for improving neurological conditions in diseases such as myasthenia gravis.

4-AP, on the other hand, is well known as an experimental convulsant for seizure induction (Spyker et al., 1980; Murray and Newsom-Davis, 1981; Yamaguchi and Rogawski, 1992; Pickett and Enns, 1996). Recent studies have shown that 4-AP, at commonly used concentrations, can cause apoptosis in hepatoblastoma cells (Kim et al., 2000) and malignant astrocytoma cell lines (Chin et al., 1997). Another classical K⁺ channel blocker tetraethylammonium (TEA) at high concentrations also shows toxic effects on cortical neurons (Yu et al., 1997). The mechanism of 4-AP- or TEA-induced neurotoxicity is unclear. A link to increases in intracellular free Ca²⁺ ([Ca²⁺]_i) was suggested for the pro-apoptotic effect of 4-AP (Kim et al., 2000). The role for Ca²⁺ in the induction of apoptosis, however, is controversial and complex. Increasing [Ca²⁺]_i may either induce or antagonize apoptosis (Dowd, 1995; Yu et al., 2001); furthermore, apoptosis may occur without alterations in [Ca²⁺]_i (Iseki et al., 1993; Beaver and Waring, 1994; Treves et al., 1994; Reynolds and Eastman, 1996; Ubol et al., 1996).

Emerging evidence now supports an ionic mechanism underlying apoptosis, associating with excessive K⁺ efflux and loss of intracellular K⁺ (Yu et al., 1997; Dallaporta et al., 1998; Hughes and Cidlowski, 1999). The pro-apoptotic K⁺ depletion can be mediated by K⁺-permeable ion channels (Yu et al., 1997, 1999) or by blocking the Na⁺,K⁺-ATPase (Xiao et al., 2002). In the latter case, a "hybrid death" of concomitant apoptosis and necrosis in same cells was associated with depletion of intracellular K⁺ and simultaneous accumulations of Ca²⁺ and Na⁺, respectively (Xiao et al., 2002). Supporting the contribution of over-activated K⁺ channels to apoptosis, K⁺ channel blockers such as TEA attenuate caspase activation and apoptotic death (Yu et al., 1997; Colom et al., 1998; Krick et al., 2001; Xiao et al., 2002).

As 4-AP and TEA may have potential clinical values in certain pathological conditions, understanding the mechanism of their adverse effects becomes necessary and important. Previous works showed that TEA was capable of blocking the Na⁺,K⁺-ATPase (Eckstein-Ludwig et al., 1998), thus the toxic effect of K⁺ channel blockers might be linked to a dysfunction of the Na⁺,K⁺-ATPase. In the present study, we tested the hypothesis that 4-AP and TEA may induce apoptosis or the hybrid death mediated by blocking the Na⁺,K⁺-ATPase.

	Materials and Methods

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Neocortical Cultures. Mixed cortical cultures (containing neurons and a confluent glia bed) were prepared as described previously (Rose et al., 1993). Briefly, neocortices were obtained at 15- to 17-days gestation from fetal mice; they were dissociated and plated onto a poly-D-lysine- and laminin-coated base (near-pure neuronal culture) or a previously established glial monolayer (mixed culture) at a density of 0.35 to 0.40 hemispheres/ml in 24- or 96-well plates or 35-mm dishes (Falcon Primaria; BD Biosciences, Franklin Lakes, NJ) depending on experimental requests. Cultures were maintained in Eagle's minimal essential medium (MEM, Earle's salts) supplemented with 20 mM glucose, 5% fetal bovine serum, and 5% horse serum (HS). Medium was changed after 1 week to MEM containing 20 mM glucose and 10% HS, as well as cytosine arabinoside (10 µM) to inhibit cell division. Glial cultures used for mixed cultures were prepared from dissociated neocortices of postnatal days 1 to 3 mice. Glial cells were plated at a density of 0.06 hemispheres/ml in Eagle's MEM containing 20 mM glucose, 10% fetal bovine serum, 10% HS, and 10 ng/ml epidermal growth factor; a confluent glial bed was formed in 1 to 2 weeks.

Electrophysiological Recordings of Na⁺,K⁺-Pump Current. The 35-mm culture dish containing cortical neurons was placed on the stage of an inverted microscope, membrane currents were recorded by whole-cell configuration using an EPC-9 amplifier (List Electronic, Darmstadt, Germany). Recording electrodes of 8 to 10 M (fire-polished) were pulled from Corning Kovar Sealing 7052 glass pipettes (PG52151-4, WPI Instruments, Waltham, MA) by a Flaming-Brown micropipette puller (P-80/PC, Sutter Instrument Co., Novato, CA). Current and voltage signals were displayed on a computer monitor and collected by a data acquisition/analysis program PULSE (HEKE, Lambrect, Germany). Currents were digitally sampled at 0.33 kHz and filtered at 3 Hz by a 3-pole Bessel filter.

Assessment of Cell Death. Neuronal cell death was assessed in 24-well plates by measuring lactate dehydrogenase (LDH) released into the bathing medium (MEM + 20 mM glucose and 30 mM NaHCO₃) using a multiple-plate reader (Molecular Devices Corp., Sunnyvale, CA). Validation of the LDH method for measuring apoptotic death has been performed before (Gottron et al., 1997). Neuronal loss is expressed as the percentage of LDH released in each experimental condition normalized to negative (sham wash) and positive (complete neuronal death induced by 24-h exposure to 300 µM NMDA) controls.

Electron Microscopy. Cultures in 35-mm dishes were fixed in glutaraldehyde (1% glutaraldehyde, 0.1 M sodium cacodylate buffer, pH 7.4) for 30 min at 4°C, washed with 0.1 M sodium cacodylate buffer, and postfixed in 1.25% osmium tetroxide for 30 min. Cells were then stained en bloc in 4% aqueous uranyl acetate for 1 h, dehydrated through a graded ethanol series, embedded in Poly/Bed 812 resin (Polysciences Inc., Warrington, PA), and polymerized in a 60°C oven overnight. Thin sections (62 nm) were cut on a Reichert Ultracut Ultramicrotome (Mager Scientific, Dexter, MI), mounted on 150-mesh copper grids, and poststained in uranyl acetate and Reynold's lead citrate (Electron Microscopy Sciences, Fort Washington, PA). Sections were photographed using a transmission electronic microscope (Zeiss 902; LEO Electronic, Thornwood, NY).

Chemicals. 4-Aminopyridine, tetraethylammonium chloride, gadolinium chloride, and strophanthidin were purchased from Sigma-Aldrich (St. Louis, MO). The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) was obtained from Enzyme Systems Products (Dublin, CA).

Statistics. Student's two-tailed t test was used for comparison of two experimental groups; multiple comparisons were done using one-way analysis of variance test followed by Tukey's test for multiple pairwise tests. Changes were identified as significant if the P value was less than 0.05. Mean values were reported together with the standard error of mean (S.E.M.).

	Results

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Suppression of Na⁺,K⁺-Pump Currents by 4-AP and TEA. 4-AP is regarded as a classical A-type K⁺ channel blocker and a blocker for certain delayed rectifier channels (Mathie et al., 1998). In cortical neurons it selectively inhibited I_A current triggered by voltage pulses from 110 mV to 10 mV, with an IC₅₀ of 0.7 mM (n = 5 to 9 cells; the holding potential between pulses was 70 mV). To measure the tonic Na⁺,K⁺-pump activity, an inward membrane current, I_pump, was triggered by brief application of the selective inhibitor strophanthidin (500 µM) in the presence of voltage-gated channel blockers (Fig. 1). Bath-applied 4-AP at low millimolar concentrations markedly suppressed I_pump recorded at 70 mV (IC₅₀ = 1.2 mM) (Fig. 1). The pan K⁺ channel blocker TEA also inhibited I_pump with an IC₅₀ of 5.2 mM at 70 mV (n = 8). Elevating extracellular K⁺ stimulates the Na⁺,K⁺-pump activity and generates an outward current. This outward I_pump associated with activation of the Na⁺,K⁺-pump was blocked by bath-applied 4-AP with an IC₅₀ of 4.2 mM. Similar to a previous report on TEA (Eckstein-Ludwig et al., 1998), the 4-AP effect was voltage-dependent; stronger I_pump inhibition was achieved at depolarized membrane potentials (Fig. 2). 4-AP blocked the Na⁺,K⁺-pump activity only at an extracellular site; intracellular application of 5 mM 4-AP exhibited no effect on I_pump (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Block of Na+,K+-pump currents by 4-AP. Na+,K+-pump currents were recorded in cultured cortical neurons using whole-cell recordings in the presence of K+, Na+, and Ca2+ channel blockers. A, the inward current associated with the tonic pump activity was induced by local application of the Na+,K+-ATPase inhibitor strophanthidin (500 µM). In the presence of bath-applied 5 mM 4-AP, much smaller pump current was recorded in the same cell. B, elevated extracellular K+ triggered an outward membrane current resulting from an enhanced pump activity; this outward pump current was markedly inhibited by 500 µM strophanthidin or 5 mM 4-AP. C, dose-response relationship of 4-AP effect on the Na+,K+-pump. The dose-response curve was fitted by the two exponential equation, yielding an IC50 of 1.17 mM on the pump current recorded at the holding potential of 70 mV (n = 10 for each concentration point).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Characterization of 4-AP effects on Na+,K+-pump currents. A, the inhibitory effect of 4-AP on the Na+,K+-pump activity was voltage-dependent; at depolarized membrane potentials 5 mM 4-AP induced greater suppression of the pump current, n = 7-10. B, 4-AP affected the Na+,K+-pump activity only from an extracellular site. Bath application of 4-AP (5 mM) blocked about 50% of the outward pump current, whereas 4-AP (5 mM) in the internal solution caused little effect (n = 8 for each column; 5- to 10-min application). Control currents were recorded before the 4-AP application in same cells. C, as a drug application control, 1 mM 4-AP included in the intracellular solution drastically suppressed IA current 5 to 10 min after establishing the whole-cell configuration. *, significantly different (P < 0.05) from controls.

Hybrid Neuronal Death Induced by 4-AP and TEA. 4-AP at low millimolar concentrations exhibited dose-dependent toxicity to cortical neurons; significant cell death occurred after a 24-h incubation in 5 mM 4-AP and after a 48-h incubation in 0.1 to 10 mM 4-AP (Fig. 3). The EC₅₀ of 4-AP toxic effect was 4.1 mM at 48 h (n = 8 cultures). TEA also showed time- and concentration-dependent toxic effect on cortical neurons at relatively higher concentrations (10 mM) (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Neuronal death induced by 4-AP and TEA. Cortical neuronal death was assessed by LDH release 24 to 48 h after adding 4-AP and expressed as the percentage of full damage induced by excessive activation of NMDA receptors (300 µM plus 10 µM glycine). A, dose-response relationship for 4-AP toxicity; the EC50 for the 4-AP effect calculated from an exponential curve fitting was 5.7 mM at 24 h and 4.1 mM at 48 h. B, the 4-AP-induced neuronal death was partly attenuated by the caspase inhibitor Z-VAD-FMK or by the membrane-permeable Ca2+ chelator BAPTA-AM (10 µM) co-applied with 4-AP. Combined application of Z-VAD-FMK plus BAPTA-AM resulted in additional neuroprotection. The residue death may be partly due to the BAPTA toxicity and undefined mechanism. C, the classical K+ channel blocker TEA showed neurotoxicity at higher concentrations and after longer exposure; significant cell death was observed after exposure to 10 and 20 mM TEA for 48 h. Z-VAD-FMK (100 µM) significantly reduced TEA-induced cell death. n  12 cultures for each group. *, significant difference from 4-AP or TEA alone (P < 0.05).

View larger version (140K): [in this window] [in a new window]   Fig. 4.   Ultrastructural alterations of hybrid injury induced by 4-AP and TEA. Apoptotic and necrotic morphological alterations were examined by electron microscopy. A, normal control cells showed large nuclei, intact and distinguishable cellular organelles, and the intact membranes. B, after 15 h of treatment in the medium containing 5 mM 4-AP, many cells showed shrunken nuclei of apoptotic change, but meanwhile the vacuolization of swollen cytoplasm and disruption of the organelles were signs of necrotic damage. C, about 30 h after onset of 4-AP treatment, numerous cells showed even more condensed nuclei and chromatin clamps (arrow), typical of apoptosis; on the other hand, the chaotic disruption of the cytoplasm was consistent with necrosis. D, the apoptotic nuclei condensation was largely prevented by the caspase blocker Z-VAD-FMK (100 µM), whereas cytoplasm and the plasma membrane still underwent necrotic changes (30-h 4-AP plus Z-VAD-FMK). E, the 4-AP-induced hybrid nature was not affected by co-applied MK-801 (1 µM). F, a representative cell after a 30-h incubation in 20 mM TEA; the changes in nucleus and cytoplasm suggested apoptosis and necrosis, respectively, in the same cell. Magnification, 4000×.

	Discussion

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This study provides new insight into the 4-AP pharmacology and neurotoxicity. We showed that, in addition to being a classical K⁺ channel blocker, 4-AP is a potent antagonist of the Na⁺,K⁺-ATPase. The latter property is likely responsible for the induction of a hybrid death of cortical neurons, consistent with our previous demonstration that the hybrid death was induced by failure of the Na⁺,K⁺-ATPase (Xiao et al., 2002 and see below). The additional protection gained from co-applied Z-VAD-FMK and BAPTA-AM supports the mixed nature of 4-AP toxicity; it is not clear why the residual cell death was not sensitive to caspase inhibition and Ca²⁺ buffering. Caspase-independent apoptosis and/or BAPTA toxicity (Fig. 3) may play a role in this observation.

Inhibiting K⁺ channels by TEA or 4-AP are protective against apoptosis in several cell types (Yu et al., 1997; Colom et al., 1998; Dallaporta et al., 1999; Wang et al., 1999, 2000; Krick et al., 2001); on the other hand, 4-AP is toxic in malignant astrocytoma cell lines and HepG2 human hepatoblastoma cells (Chin et al., 1997; Kim et al., 2000). Our data suggest that blocking the Na⁺,K⁺-ATPase, but not the K⁺ channels, is likely the primary mechanism underlying the toxic effect of 4-AP. Supporting this notion, both the 4-AP block of Na⁺,K⁺-ATPase and 4-AP toxicity are similarly concentration-dependent; more importantly, ouabain-induced hybrid death was attenuated but not exaggerated by blocking K⁺ channels or reducing K⁺ efflux (Xiao et al., 2002). Additional evidence can be found in the study where 4-AP inhibits outward K⁺ currents and cell proliferation with similar efficacy in malignant astrocytoma U87 and A172 cells; however, 4-AP induces apoptosis only in U87 cells but not in A172 cells (Chin et al., 1997). It will be interesting and important to know whether this discrepancy is results from different effects of 4-AP on the Na⁺,K⁺-ATPase in these cells.

In spite of a long research history on 4-AP, the inhibitory effect of 4-AP on the Na⁺,K⁺-pump activity has never been recognized before. This oversight is probably due to the fact that blocking K⁺ channels and blocking the Na⁺,K⁺-pump result in similar consequences, including membrane depolarization and increases in intracellular Ca²⁺. In this regard, it is possible that some previously observed effects induced by 4-AP are in fact at least partly a result of dysfunction of the Na⁺,K⁺-pump. For example, 4-AP at 1 mM suppressed axonal conductance accompanied by marked membrane depolarization (Shi and Blight, 1997), which can be partly explained by an inhibitory effect on the Na⁺,K⁺-pump. In addition, an enhanced membrane depolarization and disruption of ion homeostasis are likely important contributors to the convulsant side effects of 4-AP.

The K⁺ channel blocker TEA can directly block the Na⁺,K⁺-ATPase in a voltage-dependent manner (Eckstein-Ludwig et al., 1998). 4-AP is structurally unrelated to TEA but shows even stronger inhibition on the Na⁺,K⁺-ATPase. Similar to TEA, we confirmed that the 4-AP effect is voltage-dependent, with high inhibition at more depolarized membrane potentials. Like TEA, 4-AP blocks the Na⁺,K⁺-pump only at an extracellular site. It remains to be defined whether 4-AP blocks the pump via a direct competitive mechanism like TEA.

The block of Na⁺,K⁺-pump and induction of hybrid neuronal death by 4-AP and TEA suggest that more selective K⁺ channel blockers without the adverse action on the Na⁺,K⁺-pump will be needed for therapeutic uses. The more selective compounds may avoid or reduce the side effects associated with membrane depolarization and disruption of ion homeostasis therefore preclude induction of the hybrid cell injury.