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1 Department of Pharmacology and Toxicology and 2 Department of Physiology Medical College of Virginia, Richmond, Virginia 23298-0524
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Tavalin, Steven J., Earl. F. Ellis, and Leslie S. Satin. Inhibition of the electrogenic Na pump underlies delayed depolarization of cortical neurons after mechanical injury or glutamate. J. Neurophysiol. 77: 632-638, 1997. We previously characterized the electrophysiological response of cortical neurons to a brief sublethal stretch-injury using an in vitro model of traumatic brain injury. This model revealed that cortical neurons undergo a stretch-induced delayed depolarization (SIDD) of their resting membrane potential (RMP) which is ~10 mV in magnitude. SIDD is dependent on N-methyl-D-aspartate (NMDA) receptor activation, neuronal firing, and extracellular calcium for its induction but not its maintenance. SIDD was maximal 1 h after the insult and required incubation at 37°C. The present study examined the mechanism mediating SIDD and its relation to glutamate receptor activation. The Na pump inhibitor ouabain was used to assess the contribution of the Na pump to the RMP of control and stretched neurons using whole cell patch-clamp techniques. The nitric oxide (NO) synthase inhibitor N
-nitro-L-arginine and a polyethylene glycol conjugate of superoxide dismutase were used to assess whether NO or superoxide anion, respectively, were involved in the induction of SIDD. Neurons were exposed to exogenous glutamate in the absence of cell stretch to determine whether glutamate alone can mimic SIDD. We report that SIDD is mediated by Na pump inhibition and is likely to result from reduced energy levels since the RMP of neurons dialyzed with a pipette solution containing 5 mM ATP were identical to controls. NO, but not superoxide anion, also may contribute to SIDD. A 3-min exposure to 10 µM glutamate produced a SIDD-like depolarization also associated with Na pump inhibition. The results suggest that Na pump inhibition secondary to alterations in cellular energetics underlies SIDD. Na pump inhibition due to glutamate exposure may contribute to traumatic brain injury or neurodegenerative diseases linked to glutamate receptor activation.
Two classes of injury contribute to the overall pathophysiology of traumatic brain injury (TBI). The primary injury results from the initial mechanical event itself while the secondary injury, of delayed onset and prolonged duration, results from a biochemical cascade triggered by the mechanical insult (Young 1987 Cell culture
Primary cultures were prepared from 1- to 2-day-old Zivic-Miller rats as previously described (Tavalin et al. 1995 Delivery of injury
Injury was delivered by use of a model 94A Cell Injury Controller (Commonwealth Biotechnology, Richmond, VA) as previously reported (Ellis et al. 1995 Electrophysiology
An Axopatch-1D (Axon Instruments, Forest City, CA) amplifier was used in tight-seal whole cell patch-clamp configuration (Hamil et al. 1981). Current- and voltage-clamp modes were used to measure the membrane potentials and currents of cultured cortical neurons. Patch pipettes (4-10 M Data acquisition and analysis
Data were acquired on a Macintosh Quadra 800 (Apple Computer, Cupertino, CA) using an Instrutech ITC-16 computer interface (Instrutech, Great Neck, NY) and Pulse Control (Herrington and Bookman, 1994 Na pump contribution to SIDD
Current-clamp recordings from stretched and control neurons revealed that the RMP of stretched neurons were significantly depolarized compared to control (
Dependence of SIDD on intracellular ATP
Various indices of cellular energetic status have been shown to decrease after TBI or excitotoxic insults (Ankarcrona et al. 1995
Contribution of nitric oxide and superoxide production to SIDD
NO and superoxide anion production have been implicated in glutamate excitotoxicity (Coyle and Puttfarcken 1993
Delayed depolarization of neuronal RMP by exogenous glutamate application
Because SIDD is blocked by glutamate receptor antagonists (Tavalin et al. 1995
The present study used an in vitro model of TBI (Ellis et al. 1995
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Widespread neuronal depolarization and elevations in extracellular potassium and glutamate occur immediately following the primary traumatic event in animals (Faden et al. 1989; Katayama et al. 1990; Nilsson et al. 1990; Takahashi et al. 1981) and are believed to couple the primary and secondary injuries. Activation of glutamate receptors, especially those of the N-methyl-D-aspartate (NMDA) subtype, after trauma has been implicated in the long-term pathophysiology of TBI, namely prolonged motor and memory impairments (Faden et al. 1989; Hayes et al. 1988, 1992). Although glutamate exposure is neurotoxic if glutamate is elevated sufficiently (Choi et al. 1987; Rothman et al. 1987), glutamate also has been implicated in the behavioral impairments due to TBI even in the absence of cell death (Hayes et al. 1992; Lyeth et al. 1990). Thus the mechanism by which glutamate receptor activation contributes to secondary injury and the pathophysiology of TBI remains unclear.
). Cortical neurons were stretched rapidly and reversibly in a controlled and quantifiable manner using an electronically controlled pulse of air to deform the silastic membrane substrate upon which cultures were plated. Electrophysiological recordings revealed that the resting membrane potential (RMP) of stretch-injured neurons was depolarized by ~10 mV. This depolarization required a 1-h incubation period after the mechanical insult and subsided by 24 h. We subsequently termed this phenomena stretch-induced delayed depolarization (SIDD). SIDD was dependent on the degree of cell stretch and required neuronal activity, extracellular calcium, and NMDA receptor activation for its induction but not for its maintenance. SIDD was not mediated by an increase in neuronal leak conductance and occurred in the absence of cell death (Tavalin et al. 1995). Because the induction of SIDD shows similarities to the induction of glutamate excitotoxicity (Choi 1987; Choi et al. 1988), the present study was undertaken to examine its mechanism and relation to glutamate action.
; Thomas 1972). The Na pump is coupled to cellular metabolic status as ~40% of the energy generated by respiration is used to maintain neuronal Na pump activity at rest (Astrup et al. 1981; Whittam 1962). Na pump activity contributes ~10 mV to neuronal RMP of sympathetic neurons (Jones 1989). Because TBI and glutamate excitotoxicity are associated with reductions in cellular energetic status (Ankarcrona et al. 1995; Faden et al 1989; Retz and Coyle 1982; Rothman et al. 1987), we hypothesized that Na pump inhibition may contribute to SIDD. The specific Na pump inhibitor ouabain was used to determine the contribution of the Na pump to the RMP of control and stretched neurons. We also voltage clamped neurons to measure directly ouabain-sensitive pump current.
) and is known to be associated with calcium-dependent nitric oxide (NO) and superoxide anion production, both of which may contribute to excitotoxic neuronal injury (Coyle and Puttfarcken 1993; Dawson et al. 1992). Thus we hypothesized that NO and superoxide anion may contribute to SIDD. The nitric oxide synthase inhibitor N-nitro-L-arginine (L-NOARG) was used to assess whether inhibition of NO production prevents SIDD. Similarly, the polyethylene glycol conjugate of superoxide dismutase (PEG-SOD) was used to determine whether degradation of superoxide is protective. Last, we determined whether exogenous glutamate was capable of eliciting a SIDD-like depolarization in the absence of mechanical perturbation to test the hypothesis that mechanisms similar to SIDD also contribute to excitotoxicity.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Briefly, neocortices were minced in saline and then trypsinized (0.125%) for 10 min at 37°C. Tissue was transferred to culture medium containing Dulbecco's modified essential medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (P/S). Cells were triturated and washed three times, counted, and plated at a density of 106 cells per well in six-well Flex Plates (Flexcell International, McKeesport, PA). The bottom of each well was a 25-mm diam and 2-mm thick silastic membrane. These silastic membranes were collagen-coated before plating cells. Plates were incubated at 37°C in a 95%-5% mixture of air-carbon dioxide, with 95% humidity. Culture medium was replaced with growth medium (DMEM, 30 mM glucose, P/S, and 5% horse serum) 2-3 days after plating and cultures were fed twice weekly. Experiments were carried out at 12-16 days in vitro at which time the glial cell layer was near confluency.
; Tavalin et al. 1995). A single 50-ms pulse of compressed air was used to briefly deform the silastic membrane by 5.7 mm in all experiments. A 5.7-mm deformation is associated with a 31% stretch of the silastic membrane and adherent cells (Ellis et al. 1995). This level of injury is sublethal and reliably produces SIDD (Tavalin et al. 1995). Cells were stretched in normal external solution (see below). After the insult, cells were placed in a 95%-5% air-CO2 incubator at 37°C for various times. Cells then were washed three times with standard external solution. Drug solutions were made from concentrated stock solutions and diluted in external solution. L-NOARG (Fluka, Ronkonkoma, NY) and PEG-SOD (Sanofi-Winthrop, Collegeville, PA) were applied 30 min before injury and as above cells were washed three times with standard external solution after incubation.
) were pulled from borosilicate glass (WPI, Sarasota, FL) using a two-stage puller (Model 750B, David Kopf Instruments, Tujunga, CA). Neurons were visualized using an Olympus IMT-2 inverted microscope at ×225. Phase-bright pyramidal neurons were selected for experiments. Recordings of these adherent neurons were made in situ. Cells were perfused continuously (1-2 ml/min) during recordings with standard external solution, which consisted of (in mM) 11.1 glucose, 130 NaCl, 5 KCl, 3 CaCl2, 2 MgCl2, and 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES; pH 7.20). The standard internal solution consisted of (in mM) 135 KOH, 135 aspartic acid, 5 KCl, 2 NaCl, 2 MgCl2, 1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 0.2 CaCl2, and 10 HEPES (pH 7.20). For the ATP-containing pipette solution, MgATP (Sigma, St. Louis, MO) was added and MgCl2 was omitted. All recordings were made at room temperature (20°C). Some recordings were done in the presence of 1 µM tetrodotoxin (TTX; Sigma) to reduce spontaneous synaptic activity and neuronal firing.
) and Igor (Wavemetrics, Lake Oswego, OR) software. Currents and voltages were digitized and stored on VCR tape using an Instrutech VR-10-B digital data recorder. RMPs were determined within 1-3 min of establishing the whole cell configuration and were corrected for liquid junction potentials. Only cells showing stable resting potentials were included for analysis. No noticeable differences in sealing rates or successful establishment of the whole cell configuration were seen between control and stretched cells. Current-voltage (I-V) relationships were generated from a standard holding potential of 
60 mV using either test pulses (50-500 ms) or voltage ramps (10 mV/s). Input resistances at the 0 current potential (ZCP) were determined from linear regression of the I-V about the ZCP. RMPs measured in current clamp and ZCPs determined in voltage clamp were in excellent agreement. All data are expressed as the means ± SE.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
51.1 ± 2.2 mV; n = 14 vs. 63.4 ± 0.6 mV; n = 16, P < 0.01; Fig. 1A) as previously described (Tavalin et al. 1995). The contribution of the electrogenic Na pump to cortical neuron RMP was assessed by bath application of the pump blocker ouabain. Ouabain (1 mM) depolarized control cells to a significantly greater extent than stretched cells (8.7 ± 1.5 mV; n = 7 vs. 2.6 ± 0.9 mV; n = 9, P < 0.01; Fig. 1B), suggesting that the Na pump already was inhibited by stretch injury. Ouabain depolarization was maximal within ~5-7 min of application and was in most cases fully reversible (not shown). Although ouabain is effective at blocking Na pump activity at much lower concentrations (Brines and Robbins 1992), this concentration was used to produce a relatively rapid effect given the slow association kinetics of the drug (Brines and Robbins 1992). Thus 100 µM ouabain also depolarized control cells by 
7 mV but this took closer to 12 min (n = 2). Depolarization due to ouabain application often was accompanied by an increase in the frequency of excitatory postsynaptic potentials (EPSPs) and action potentials, presumably due to increased neurotransmitter release. However, it is unlikely that the ouabain depolarization was mediated by these excitatory events because ouabain depolarized neurons even in the presence of TTX (n = 2), which eliminated firing and large EPSPs. I-V relationships, obtained in voltage clamp, were quite variable but generally were found to be either linear over the range of voltages tested or showed inward rectification at potentials negative to 80 mV. No noticeable differences in the shape of I-V curves between stretched and control neurons were detected. The input resistance of stretched and control cells about the ZCP in voltage clamp was not significantly different(601 ± 62 M; n = 14 vs. 509 ± 71; n = 16, respectively). This indicates that the reduction of ouabain-induced depolarization observed in stretched neurons was not due to changes in pump current shunt conductance but in fact due to reduced pump current. Figure 1C shows that ouabain induced an inward pump current in voltage-clamped control neurons with no noticeable alteration in slope conductance. Figure 1D shows this pump current appears reduced in stretched cells. Na pump current, determined as the ouabain difference current at 60 mV, in stretched neurons was reduced significantly compared with controls (6.3 ± 1.3 pA; n = 9 vs. 22.1 ± 7.1; n = 7, P < 0.05). Taken together these results indicate that neuronal stretch-injury is associated with reduced Na pump activity.
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FIG. 1.
Effect of stretch on RMP and ouabain sensitivity of cultured cortical neurons. A: current-clamp recordings revealed that neurons subjected to 5.7-mm silastic membrane deformation were depolarized significantly compared with controls as previously described (Tavalin et al. 1995 ). *P < 0.01. Numbers in parentheses represents number of cells. B: ouabain (1 mM) depolarized control cells in current clamp to a significantly greater extent than stretched cells, suggesting that Na pump already was inhibited in stretched cells. *P < 0.01. C: I-V relationship for a typical voltage-clamped control cell (
). Ouabain (
) induced an inward Na pump current in control cells without an alteration in slope conductance. There appeared to be no marked voltage dependence to Na pump current. D: I-V relationship for a typical voltage-clamped stretched cell (). I-V relationship in presence of ouabain (). Ouabain-sensitive current was reduced in this cell suggesting that stretch-induced delayed depolarization (SIDD) is mediated by inhibition of Na pump current.
; Faden et al. 1989; Rothman et al. 1987). To test whether Na pump inhibition was due to a reduction in cellular energy levels, ATP was supplied to the cell via the patch pipette. When ATP (5 mM) was included in the pipette solution, the RMPs of control and stretched neurons were no longer significantly different (60.9 ± 1.1 mV;n = 16 vs. 61.2 ± 1.0 mV; n = 17, respectively; Fig. 2A). Whole cell dialysis with 5 mM ATP had no effect on the RMP of control cells, suggesting that cellular energy levels were compromised only in stretched cells. In addition, ouabain depolarized stretched and control neurons to the same extent (8.3 ± 2.2 mV; n = 6 vs. 8.0 ± 1.8 mV; n = 6; Fig. 2B) when ATP was included in the pipette solution, implying that ATP restored the Na pump of stretched cells. The input resistances of stretched and control neurons under these conditions were not significantly different (525 ± 53 M; n = 16 vs. 481 ± 49; n = 17), suggesting that the restoration of the ouabain-induced depolarization was by recovery of Na pump current rather than by changes in input resistance. In voltage clamp, ouabain was found to induce an inward current in control neurons dialyzed with 5 mM ATP without alteration of membrane slope conductance (Fig. 2C). In stretched neurons supplied with ATP, ouabain induced an inward current (Fig. 2D) much like that of controls. Na pump current was not significantly different in control and stretched neurons (26.1 ± 9.1 pA; n = 6 vs. 20.0 ± 7.0 pA; n = 6, respectively), indicating that the Na pump current of stretched cells could be recovered by pipette ATP. These results indicate that Na pump inhibition underlies SIDD and that pump inhibition is likely via a reduction in cellular energy levels.
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FIG. 2.
Effect of inclusion of 5 mM ATP within internal pipette solution on SIDD. A: resting membrane potentials (RMPs) of control and stretched neurons were no longer significantly different when ATP was included in pipette (compare with Fig. 1A). B: ouabain depolarized control and stretched neurons to same extent when ATP was in pipette solution, suggesting that ATP relieved inhibition of Na pump seen in stretched cells dialyzed without ATP (compare with Fig. 1B). C: I-V relationship of control neurons was not affected by whole cell dialysis with pipette solution containing 5 mM ATP ( ). I-V obtained in ouabain () shows that ouabain induced an inward current without alteration in slope conductance suggesting that Na pump current was not modified by presence of high ATP in pipette solution (compare with Fig. 1C). D: I-V relationship of a representative stretched cell dialyzed with 5 mM ATP (). Zero current potential was similar to that of control cells. Enhanced sensitivity to ouabain () shows that ATP restored an inward pump current that was absent in stretched cells without pipette ATP (compare with Fig. 1D). These results suggest that inclusion of ATP within the patch pipette restored Na pump current and that Na pump current underlies SIDD.
; Dawson et al. 1992). Because both of these species have been reported to inhibit mitochondrial function, we hypothesized they might be a metabolic link between the activation of glutamate receptors following stretch and Na pump inhibition. To test whether either of these species are involved in SIDD, neurons were treated with either L-NOARG (1 mM) to block NO synthesis or PEG-SOD (100 U) to degrade superoxide. As shown in Fig. 3, stretched cells treated with L-NOARG were no longer significantly depolarized compared with controls (54.9 ± 2.3 mV; n = 10 vs. 60.2 ± 0.8 mV; n = 9), although they remained somewhat depolarized. This suggests that L-NOARG may have been modestly protective. Stretched cells treated with PEG-SOD, however, remained significantly depolarized compared to controls (53.7 ± 2.1 mV; n = 9 vs. 63.4 ± 1.9 mV; n = 8, P < 0.01). These results suggest that NO may contribute weakly to SIDD, whereas superoxide anion is unlikely to be involved in SIDD.
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FIG. 3.
Contribution of nitric oxide (NO) and superoxide to SIDD. Pretreatment with NO synthase inhibitor N -nitro-L-arginine (L-NOARG; 1 mM) was used to test whether NO production was involved in induction of SIDD. SIDD was attenuated partially, suggesting that NO may contribute to SIDD. Pretreatment with a polyethylene glycol conjugate of superoxide dismutase (PEG-SOD; 100 U) was used to assess involvement of superoxide in SIDD. Treatment with PEG-SOD did not offer any protection, suggesting that superoxide is unlikely to be involved in SIDD. *P < 0.01.
), it was of interest to determine whether application of exogenous glutamate to neurons in the absence of stretch could mimic SIDD. Thus cortical neurons were exposed to 10 µM glutamate for 3 min at room temperature. This dose of glutamate was used because it has been reported to be sublethal (Choi et al. 1987; Coulter et al. 1992; Wang et al. 1994) and SIDD is also not associated with cell death (Tavalin et al. 1995). Neurons exposedto glutamate and then incubated for 1 h were found to bedepolarized significantly compared with control neurons(54.7 ± 2.7 mV; n = 7 vs. 61.7 ± 1.0 mV; n = 7, P < 0.05; Fig. 4). In addition, the input resistance of glutamate-treated neurons was not significantly different from controls (559 ± 70 M; n = 7 vs. 603 ± 63 M; n = 7), as is also the case for SIDD. Additionally, neurons that remained at room temperature after glutamate exposure were not depolarized (62.0 ± 1.1 mV; n = 6; Fig. 4), suggesting that incubation is required for this glutamate-induced persistent depolarization much like incubation is required for SIDD (Tavalin et al. 1995). These results suggest that glutamate produced a delayed depolarization by a mechanism similar to SIDD. This was further tested by applying ouabain to glutamate-treated neurons. Ouabain depolarized glutamate-treated neurons by 1.7 ± 0.9 mV (n = 3) similar to the blunted ouabain depolarization seen with stretched cells (Fig. 1A). These results suggest that glutamate-induced delayed depolarization, like SIDD, was mediated by Na pump inhibition.
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FIG. 4.
Effect of exogenous glutamate on RMP of control cortical neurons. Cells were exposed to 10 µM glutamate for 3 min at room temperature. Cells then either remained at room temperature or were incubated for 1 h at 37°C in an air-CO2 incubator. Neurons that were not incubated after glutamate exposure had RMPs similar to controls. In contrast, neurons incubated for 1 h after glutamate were depolarized significantly compared with controls. These results suggest that exogenous glutamate alone can mimic SIDD with respect to extent of depolarization and absolute requirement for incubation. *P < 0.05.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
) to understand the mechanism mediating SIDD, a delayed electrical response to stretch injury we reported recently (Tavalin et al. 1995). Whole cell patch-clamp recordings indicated that the Na pump was inhibited in stretched neurons. This inhibition was likely due to reduced cellular energy levels because the RMPs of stretched neurons were depolarized when ATP was not included in the pipette solution but were restored to control levels by 5 mM ATP. Restoration of control RMPs in stretched neurons by ATP occurred in parallel with restoration of Na pump current, suggesting that Na pump inhibition underlies SIDD. This is consistent with our previous observation that SIDD was not associated with altered membrane conductance (Tavalin et al. 1995).
) and neuronal firing (Thomas 1972), both of which are required for SIDD (Tavalin et al. 1995). However, it is possible that intracellular ATP concentrations remained unchanged after stretch but that ADP concentration increased. Although increased intracellular ADP would reduce the ATP/ADP ratio and thus pump activity, other actions of ATP can not be excluded. However, we believe our metabolic hypothesis is reasonable given what is known about SIDD and previously reported effects of glutamate on neurons. Because SIDD requires calcium for its induction (Tavalin et al. 1995), calcium-dependent uncoupling of mitochondrial oxidative phosphorylation, which may be involved in excitotoxicity, also might contribute to SIDD. Because a large fraction of mitochondrial respiration fuels the Na pump even at rest (Astrup et al. 1981), mitochondrial disruption thus would lead to Na pump inhibition. Mitochondria play an important role in buffering glutamate-induced calcium entry (White and Reynolds 1995), and thus excessive calcium loads would likely disrupt oxidative phosphorylation. For example, the glutamate receptor agonist kainate has been reported to produce a calcium-dependent depolarization of mitochondrial membrane potential, which is known to compromise ATP synthesis (Bindokas and Miller 1995). Acidification of hippocampal neurons after glutamate exposure also has been attributed to calcium-dependent uncoupling of mitochondrial respiration (Wang et al. 1994). Although the specific mechanisms involved in reducing cellular energy levels with regards to SIDD remain unknown, the hypothesis that altered cellular metabolism underlies SIDD is consistent with reduced bioenergetic status previously linked to TBI through decreases in the ratio of phosphocreatine to inorganic phosphate (Faden et al. 1989) and excitotoxicity via decreases in cellular ATP levels (Retz and Coyle 1982; Rothman et al 1987). Additionally, the time course of SIDD (Tavalin et al. 1995) strongly resembles that for changes in energy charge following exposure of cerebellar granule cells to glutamate (Ankarcrona et al. 1995).
). We found that interfering with NO production appeared to attenuate SIDD but did not fully prevent its induction. How NO production may contribute to SIDD, however, remains unclear. Although it has been reported that NO inhibits mitochondrial electron transport (Dawson et al. 1992), which would be expected to decrease ATP synthesis, inhibition of NO synthesis also has been reported to enhance the Na pump activity in central neurons (Nathanson et al. 1995). Because the Na pump is important in terminating glutamate-induced depolarizations (Fukuda and Prince 1992), enhancement of Na pump activity may more effectively terminate glutamate action after stretch and enhance neuronal repolarization.
). To test whether superoxide contributes to SIDD, PEG-SOD was used to enhance superoxide degradation. Although PEG-SOD did not prevent SIDD, we cannot exclude the possibility that other ROS contribute. The lack of an involvement of superoxide, however, may be consistent with our previous observation that SIDD is not associated with cell death (Tavalin et al. 1995).
) and glutamate levels are elevated after TBI (Faden et al. 1989; Katayama et al. 1990; Nilsson et al. 1990), it was important to determine whether glutamate alone could mimic SIDD in the absence of mechanical stretch. It is known that toxic glutamate exposure produces terminal neuronal depolarization and disruption of input resistance (Coulter et al. 1992). However, the glutamate treatment used in our present study was much more moderate in terms of concentration and duration of exposure than that which is used to cause cell death (Choi et al. 1987; Coulter 1992). Neurons exposed to our glutamate protocol without a subsequent incubation period had normal RMPs, consistent with the observation that neuronal RMPs return to baseline values after sublethal glutamate exposures (Coulter et al. 1992). However, our finding that incubation is required for glutamate to mimic SIDD may be related to the observation that sustained calcium entry or repeated glutamate exposures at 37°C inhibit the Na pump of hippocampal CA1 neurons (Fukuda and Prince 1992). Additionally, the time course of Na pump inhibition by calcium entry or glutamate exposures (Fukuda and Prince 1992) resembled the time course for SIDD induction (Tavalin et al. 1995). Our finding that glutamate inhibits the Na pump in a delayed manner thus appears consistent with these earlier studies. Our results further suggest that Na pump suppression after sustained calcium entry (Fukuda and Prince 1992) also may be due to reduced cellular energy levels. Although the results of the glutamate exposure experiments indicate that glutamate exposure alone is sufficient to mimic SIDD, mechanically induced alterations in glutamate receptors also may contribute to SIDD (Zhang et al. 1996).
). We favor the possibility, however, that prolonged incubation (
15-60 min) at 37°C allows a [Ca2+]i-dependent intracellular cascade to occur that starts with the activation of NMDA receptors and involves a mitochondria-mediated alteration in ATP/ADP with the concomitant inhibition of the electrogenic Na pump. It seems reasonable to us that such a biochemical cascade might contain at least one temperature-dependent step.
; Thompson and Prince 1986) and with development (Fukuda and Prince 1992; Haglund et al. 1985). This suggests that SIDD amplitude, which is in part a function of pump current amplitude, would be enhanced in older neurons and at warmer temperatures. However, neuronal RMP has been reported to be relatively unaltered by changes in temperature (Thompson et al. 1985) or postnatal developmental stage (McCormick and Prince 1987), whereas membrane input resistance decreases with increased temperature (Thompson et al. 1985) and postnatal age (Fukuda and Prince 1992; McCormick and Prince). These findings suggest that the electrogenic Na pump contribution to RMP and consequently SIDD actually may be similar at various temperatures or at different stages of postnatal development.
; Brines et al. 1995; Novelli et al. 1988). Our linkage of SIDD to Na pump inhibition might explain why sublethal TBI, which is associated with increased glutamate, increases neuronal vulnerability to delayed secondary insults like ischemia (Jenkins et al. 1989). Additionally, because the Na pump plays an important role in restoration of ionic gradients and cell repolarization after tetanic stimulation (Thomas 1972) or brief glutamate applications (Fukuda and Prince 1992), pump inhibition may alter membrane excitability and neuronal information processing. Such alterations could contribute to the behavioral deficits that occur in the wake of TBI.
; Lees 1991).
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ACKNOWLEDGEMENTS |
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We thank J. McKinney, K. Willoughby, and S. Liang for their technical expertise and for preparing neuronal cultures.
S. Tavalin was supported by National Institute of Drug Abuse Training Grant DA-07027, and E. Ellis was supported by a Jacob Javits Neuroscience Investigator Award (NS-27214). In addition, support from a Center of Excellence Grant-in-Aid from the Commonwealth of Virginia to J. Povlishock is gratefully acknowledged.
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FOOTNOTES |
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Address for reprint requests: L. S. Satin, Dept. of Pharmacology and Toxicology, Medical College of Virginia, Richmond, VA 23298-0524.
Received 24 May 1996; accepted in final form 20 September 1996.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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