Abstract
Tetraethylammonium (TEA), the quaternary ammonium ion and nonselective K+ channel blocker, is protective against neuronal apoptosis. We now tested two TEA analogs, tetrapentylammonium (TPeA) and tetrahexylammonium (THA), for their effects on apoptotic neuronal death and for their pharmacological profiles on membrane currents in cultured mouse cortical neurons. TPeA and THA (0.1–1.0 μM) attenuated staurosporine-induced caspase-3 activation and neuronal apoptosis. TPeA and THA blocked the outward delayed rectifier K+ (IK) current in concentration-dependent manners with IC50 values of 2.7 and 1.9 μM, respectively. IK was blocked by TPeA in a use-dependent manner, whereas THA blockedIK regardless of activation state of the channel. TPeA at 1 μM inhibited the high voltage-activated (HVA) Ca2+ current and the A-type K+ current (IA). TPeA (1–10 μM) also blocked the fast inactivating Na+ current. The ligand-gatedN-methyl-d-aspartate (NMDA) receptor current was not affected by up to 20 μM TPeA. THA at 1 μM showed inhibitory effects on IA, HVA Ca2+, and Na+ currents. THA (10 μM) suppressed NMDA currents. The data suggest that, as K+ channel blockers and apoptosis antagonists, TPeA and THA are much more potent than TEA; however, they have nonspecific actions on several voltage-gated or ligand-gated channels.
Apoptosis is a form of programmed cell death that occurs under physiological conditions as a mechanism of cell/tissue homeostasis (Kerr et al., 1972; Raff et al., 1993) and under certain pathological conditions (Thompson, 1995; Choi, 1996). A reduction in cell volume and activation of caspases are fundamental features of apoptosis (Kerr et al., 1972;Armstrong et al., 1997). Recent work from several groups suggests that changes in ionic content, primarily K+, play a pivotal role in the progression of apoptosis (Bortner and Cidlowski, 1999; Dallaporta et al., 1999; Orlov et al., 1999). In immune and neuronal cells, apoptotic insults trigger considerable intracellular K+ loss (Bortner et al., 1997; Yu et al., 1997,1999a), which may consequently cause caspase-3 activation and apoptosis (Hughes et al., 1997; Yu et al., 1999b). The excessive K+ efflux can be mediated by overactivation of voltage-gated K+ channels. The delayed rectifier K+ channel is up-regulated during certain stages of several apoptotic insults in cortical neurons (Yu et al., 1997,1998, 1999b), myeloblastic leukemia cells (Wang et al., 1999), and cholinergic septal cells (Colom et al., 1998). Tetraethylammonium (TEA) and other K+ channel blockers attenuate apoptotic cell death in cultured cortical neurons (Yu et al., 1997, 1999b; Yu and Choi, 1999), cholinergic septal cells (Colom et al., 1998), liver cells (Gantner et al., 1995), and in rat's cerebral cortex of transient focal ischemia (Choi et al., 1998).
TEA blocks K+ channels and apoptosis at millimolar concentrations (Yu et al., 1997). Dallaporta et al. (1999)recently showed that the TEA analog TPeA, at micromolar concentrations, blocked all features of apoptosis in thymocyte induced by dexamethasone, etoposide, γ-irradiation, or ceramide. TPeA, like TEA, inhibits several voltage-gated K+ channels from inside and outside of the membrane (Kirsch et al., 1991; Im and Quandt, 1992; Carl et al., 1993), including Ca2+-activated K+ channels (Langton et al., 1991; Carl et al., 1993), the inward-rectifier K+ channel (Spassova and Lu, 1993), and the ATP sensitive K+ channels (Davies et al., 1989). TPeA blocks Kv3.1 and Kv2.1 channels in a fast, reversible, and time-dependent manner. Unlike the voltage-dependent effects of TPeA and TEA from the cytoplasmic side of the membrane, the external binding of TPeA appears to be voltage independent (Carl et al., 1993; Jarolimek et al., 1995). TPeA may possess nonspecific effects on other ion channels. Intracellular-applied TPeA was shown to inhibit human cardiac Na+ channels expressed in a mammalian cell line (O'Leary and Horn, 1994). Intracellular-applied TPeA and other quaternary ammonium ions can block chloride channels in rat cortical neurons (Sanchez and Blatz, 1995). TPeA at high concentrations (>10–20 μM) reduced the contractions in smooth muscle, whereas TEA had no such effect; it was assumed that TPeA might have an inhibitory effect on nifedipine-sensitive Ca2+ channels (Kwok et al., 1998).
We tested the antiapoptotic actions of TPeA and another long-chain quaternary ammonium ion tetrahexylammonium (THA) in cultured cortical neurons. To understand the mechanism of their antiapoptotic actions, we studied effects of TPeA and THA on the membrane currents carried by Ca2+, Na+, and K+ that are believed to be important in neuronal cell death. Our data suggest that TPeA and THA are neuroprotective against staurosporine-induced apoptosis; as potent K+ channel blockers, they also show strong inhibitory effects on several ion channels.
Materials and Methods
Cortical Cultures.
Mixed cortical cultures, containing neurons and a confluent glia bed, were prepared as described previously (Rose et al., 1993). Mice of 15 to 17 days gestation were anesthetized with halothane. Dissociated cortical cells were plated onto a previously established glial monolayer at a density of 3.5 to 4.0 hemispheres/10 ml in 35-mm culture dishes or 24-well plates (Falcon, Primaria, Lincoln Park, NJ), in Eagle's minimal essential medium (MEM, Earle's salts) supplemented with 20 mM glucose (final concentration = 25 mM), 5% fetal bovine serum, and 5% horse serum (HS). Medium was changed after 1 week to MEM containing 25 mM glucose and 10% HS, as well as 10 μM cytosine arabinoside to inhibit cell division. Subsequently, cultures were fed once weekly with MEM supplemented with 20 mM glucose. Cultures were kept in a 37°C, humidified incubator in a 5% CO2 atmosphere. All experiments were performed between 10 and 15 days in vitro. Glial cultures were prepared from dissociated neocortices of postnatal day 1 to 3 mice. Cells were plated at a density of 0.65 hemisphere/10 ml, in Eagle's MEM containing 25 mM glucose, 10% fetal bovine serum, 10% HS, and 10 ng/ml epidermal growth factor; confluent glial bed was formed in 1 to 2 weeks. Neuronal identity has been previously confirmed by Nissl staining and electrophysiological characterization, whereas the glial bed is immunoreactive for glial fibrillary acidic protein (Choi et al., 1987; Rose et al., 1993).
Electrophysiology.
Whole-cell voltage clamp was performed on cortical neuronal cultures in 35-mm dishes on the stage of an inverted microscope (Nikon, Tokyo, Japan) using an EPC-7 amplifier (List Electronics, Darmstadt, Germany); patch electrodes had tip resistance of 8 to 14 MΩ (fire-polished). Current was digitally sampled at 100 μs (10 kHz). The current signals were filtered by a 3-kHz, 3-pole Bessel filter. Current and voltage traces were displayed and stored on a Macintosh computer (Quadra 950; Apple Computer Corp., Cupertino, CA) using the data acquisition/analysis program package PULSE (HEKA Electronics, Lambrecht/Pfalz, Germany).
Voltage-gated K+, Ca2+currents, and NMDA-induced membrane current were recorded using solutions listed in Table 1. Experiments were performed at room temperature (21–22°C).
Neuronal Cell Death.
Neuronal cell death was assessed in 24-well plates by cell counts after staining with 0.4% trypan blue dye, and by measuring lactate dehydrogenase released into the bathing medium (Koh and Choi, 1987).
Caspase Activity Assay.
Caspase activity was measured as described previously by Armstrong et al. (1997). Briefly, cultures were washed three times with phosphate-buffered saline and lysed in 80 μl of buffer A (10 mM HEPES, 42 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, pH 7.4). Lysate (10 μl) was combined in a 96-well plate with 90 μl of buffer B (10 mM HEPES, 42 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, 10% sucrose, pH 7.4) containing fluorometric substrate (final concentration 30 μM) and incubated for 45 min at room temperature in the dark. Formation of fluorogenic product was determined in a Cytofluor fluorometric plate reader by measuring emission at 460 nm with 360-nm excitation. Caspase-3 like activity was defined asN-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin cleavage (Thornberry et al., 1997).
Cell Volume Assay.
For cell volume assay, the cell maximum cross-sectional area was measured using the MetaMorph imaging system (Universal Imaging Corporation, West Chester, PA) based on the assumption that cell soma swells and shrinks in a symmetrical manner as if it were a sphere. This assumption was validated in cortical cultures by measuring cell volume changes directly using optical sectioning techniques; and there was no difference between cell volume changes measured by optical sectioning and those calculated from cross-sectional area (Churchwell et al., 1996).
Chemicals.
The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD) was purchased from Enzyme Systems Products (Dublin, CA). TPeA, THA, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Statistics.
Changes were identified as significant ifP value in Student's t test was less than .05; multiple comparisons were done using one-way ANOVA test. Mean values were reported together with the S.E.M. unless otherwise specified.
Results
TPeA and THA Attenuated Staurosporine-Induced Neuronal Apoptosis.
Staurosporine, the nonselective protein kinase inhibitor, is a typical inducer of apoptosis (Bertrand et al., 1994;Koh et al., 1995); 0.2 μM staurosporine added into the culture medium induced about 20% cell body shrinkage and 50% of neuronal death in cortical cultures in 24 h (Fig. 1, A and B). Staurosporine stimulated caspase-3 like protease activation (Fig. 1D); consistently, the broad-spectrum caspase inhibitor Z-VAD (100 μM) blocked 87 ± 3% of the death (Fig. 1B). These caspase-mediated events confirmed the apoptotic nature of staurosporine toxicity. TPeA (0.1–1.0 μM) or THA (1.0 μM) coapplied with staurosporine blocked cell shrinkage, attenuated caspase activation, and reduced neuronal death (Fig. 1, A–D). High concentrations of TPeA alone (10 μM), however, showed toxic effects on cortical neurons (Fig. 1B). The protective effect by TPeA was persistent; significant attenuation of neuronal death remained 72 h after coapplication of 0.2 μM staurosporine and 1.0 μM TPeA (57 ± 3% protection; n = 12 cultures, P < .05 compared with staurosporine alone). The L-type Ca2+ channel antagonist nifedipine (2 μM;n = 4) and Na+ channel blocker tetrodotoxin (TTX) (1 μM; n = 4) had no significant protection against staurosporine-induced neuronal death (data not shown).
TPeA and THA Blocked Delayed Rectifier K+ Current in Use-Dependent and -Independent Manners.
Apoptotic cell body shrinkage is believed mainly to be due to excessive K+ efflux followed by water loss (Ojcius et al., 1991; Deckers et al., 1993; Duke et al., 1994; Beauvais et al., 1995;Bortner et al., 1997). The effects of TPeA and THA on staurosporine-induced cell body shrinkage suggested a blockage of K+ channel-mediated excessive K+ efflux. To characterize the K+ channel blocking activities, we studied effects of TPeA and THA on the major K+ currentsIK and IA in cortical neurons. Bath-applied TPeA and THA blockedIK at low micromolar concentrations. Measured by a voltage step of 300 ms from −70 to +40 mV applied every minute, TPeA and THA in 15 min suppressedIK steady-state current at IC50 of 2.7 ± 0.2 and 1.9 ± 0.2 μM, respectively (Fig. 2, A–C). The inhibitory effect of TPeA was partially reversible upon washing (Fig.2A). The effect of TPeA and THA was not dependent on the membrane potential; IK was blocked at positive and negative potentials after the membrane potential was transiently jumped to these levels (Fig. 2D). Alternatively, when the membrane potential was persistently held at −70 or −20 mV, theIK current activated by depolarizing pulses to +40 mV was blocked by 76 ± 7% (n = 5) and 67 ± 3% (n = 8), respectively (P= .19).
The mechanism of action for TPeA and THA was apparently different. TPeA blocked IK in a time- and use-dependent manner; IK activated by the depolarizing voltage pulse of one per minute (
TPeA preferably blocked the IK steady-state current, an action in agreement with an open channel blocker (Fig. 2A). To further confirm the use-dependent mechanism, no voltage step was applied during the first 5 min of incubation in 10 μM TPeA to keepIK channels in close state. After 5 min in TPeA, the first IK was 86 ± 4% of the control current (n = 6), whereas the current in cells subjected to
Effects of TPeA and THA on Ca2+ and Na+Currents.
TPeA and THA were potent antagonists at HVA Ca2+ currents; HVA currents were suppressed by 1 to 10 μM TPeA or THA in concentration-dependent manners (Fig.5). Up to 20 μM TPeA did not affect the fast inward Na+ current; however, 10 μM THA drastically blocked the Na+ current (Fig.6).
Effects of TPeA and THA on the NMDA Subtype of Glutamate Receptor Currents.
TEA and its derivatives may have inhibitory effects on NMDA receptor channels (Wright et al., 1991). Extracellularly applied 10 μM (n = 3) or 20 μM (n = 5) TPeA did not show significant effect on NMDA currents recorded at −70 or +40 mV (Fig. 7, A and B). THA, on the other hand, suppressed NMDA current at 10 μM (n = 3) (Fig. 7B), so THA, although a potent K+ channel blocker, is less selective than TPeA.
Discussion
The present study shows that, as K+ channel blockers, TPeA and THA are 1000-fold stronger than TEA in central neurons. Consistently, TPeA and THA attenuate neuronal apoptosis at 0.1 to 1.0 μM, concentrations that inhibit IKcurrents and are anticipated to prevent the extra K+ efflux triggered by an apoptotic insult (Yu et al., 1997). In addition, TPeA and THA are potent blockers atIA, HVA Ca2+, and Na+ currents at similar or higher concentrations; on the other hand, the ligand-gated NMDA receptor channel was only affected by THA.
Although K+ channel blockers such as TEA attenuate apoptotic cell death, the mechanism of action is under debate. It was proposed that the protective effect by K+ channel blockers is due to an increase in [Ca2+]i as a result of activation of voltage-activated Ca2+ channels (Colom et al., 1998). A contribution from a Ca2+channel-mediated [Ca2+]iincrease, however, was excluded in the protective effects of TEA and high K+ medium in cortical neurons, based on the fact that complete blockages of HVA Ca2+ currents and [Ca2+]i increases did not eliminate the TEA- or high K+-induced protection (Yu et al., 1997, 1999b). In the present study, TPeA and THA, as potent K+ and Ca2+channel blockers, attenuated staurosporine-induced caspase activation and neuronal apoptosis. These results further support the idea that an increase in [Ca2+]i may not contribute to the protective effect of K+channel blockers in cortical neurons. In addition, our data are consistent with a recent report that TPeA does not have an inhibitory effect on NMDA receptors (Sobolevsky et al., 1999). Although TPeA and THA block the fast inward Na+ current, it may not be the mechanism of protection because TTX showed no protection against apoptotic death; instead, TTX slightly increased the staurosporine-induced death (20% increase; n = 4,P = .052), perhaps due to consequently diminished activity of Na+/K+-ATPase and reduced K+ uptake.
As derivatives of TEA, TPeA and THA displayed distinctive mechanisms of block on IK channels. The block ofIK by TPeA was strongly use dependent, suggesting its binding site was only accessible when the channels were in open state. Effect of TPeA on IK was time dependent, and this may partly reflect a possible act on the putative internal quaternary ammonium site because of its higher lipid solubility than TEA (Snyders and Yeola, 1995). In contrast to TPeA, block of IK by THA did not require preopening of IK channels, probably suggesting a different biding site for THA located outside of the pore region. Activation of IK channels, however, did accelerate the blocking effect of THA, implying that THA might also act on the TPeA site or a second binding site for THA. Bath-applied TPeA and THA blocked IK in a voltage-independent manner, suggesting that the extracellular binding sites for TPeA and THA were conceivably not deeply inside of the channel pore region, consistent with the larger sizes of these compounds.
This study demonstrates that although TPeA and THA are potent K+ channel blockers, their ion channel selectivity is limited and the intricate blocking of several channels may explain the toxic effects at higher concentrations. More selective channel blockers specifically targeting atIK channels will be needed as useful tools for blocking proapoptotic excessive K+ efflux and apoptotic death.
Footnotes
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Send reprint requests to: Shan Ping Yu, Department of Neurology, Box 8111, School of Medicine, Washington University, St. Louis, MO 63110. E-mail: yus{at}neuro.wustl.edu
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↵1 This work was supported by research grants from American Heart Association (9950207N), National Science Foundation (IBN-9817151), and National Institutes of Health (3257ADRC16).
- Abbreviations:
- TEA
- tetraethylammonium
- TPeA
- tetrapentylammonium
- THA
- tetrahexylammonium
- MEM
- minimal essential medium
- HS
- horse serum
- Z-VAD
- Z-Val-Ala-Asp(OMe)-fluoromethyl ketone
- TTX
- tetrodotoxin
- HVA
- high-voltage-activated
- NMDA
- N-methyl-d-aspartate
- Received March 29, 2000.
- Accepted June 29, 2000.
- The American Society for Pharmacology and Experimental Therapeutics