reduced cortical ecto-atpase activity in rat brains during prolonged status epilepticus induced by...

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Reduced Cortical Ecto-ATPase Activity in Rat Brains During

Prolonged Status Epilepticus Induced by Sequential Administration

of Lithium and Pilocarpine A6NES K. NA6u NANCY Y. WALTON

AND DAVID M. TRE1MAN

Department of Neurology, UCLA School of Medicine and VA West Los Angeles Medical Center, 10833 Le Conte Ave.,

Los Angeles, CA 90095-1769

Received February 14, 1996; Revised November 18, 1996; Accepted December 10, 1996

ABSTRACT

Considerable evidence indicates that ATP, acting intracellularly or as a neurotransmitter, can influence nerve cell physiology in a variety of ways. Defects in the functioning of ATP-metabolizing enzymes could therefore lead to disturbances in neurotransmission and creation of sustained neuronal discharges characteristic of status epilepticus. In this study we investigated synaptosomal ATPase changes in rat brains during lithium/pilocarpine-induced status epilepticus. After 2 h of continuous electroencephalographic spiking, both Mg 2+- and Ca2+-dependent ecto-ATPases were significantly decreased in freshly prepared synaptosomal preparations from the status rats. The intraceUulary acting Ca2*Mg2+-ATPase (Ca-pump) was also decreased, but no changes occurred in synaptosornal Na+K+-ATPase activi~ The difference between ecto-ATPase activities of the control and status rat brains was not affected by repeated freezing-thawing and lengthy storage. Possible involvement of reduced synaptosomal divalent cation-dependent ATPases in the pathophysiology of status epilepticus is discussed.

Index Entries: ecto-ATPase; synaptosomes; rat; lithium/pilocarpine; status epilepticus; subcellular brain fractions; Mg2+-depen - dent ATPase; Ca2+-dependent ATPase.

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

Molecular and Chemical Neuropathology 135 vol. 31, 1997

136 Nagy, Walton, and Treiman

Abbreviations: SE, status epilepticus; EEG, electroencephalogram; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis-(~ amino-ethyl ether) N,N'-tetraacetic acid; HEPES, N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid.

INTRODUCTION

Ecto-ATPase, the first member of the extracellular ATP-hydrolyzing cascade, was initially observed in nucleated avian red blood cells by Engelhardt (1957). Since then, ectonucleotide phosphatase enzymes have been found on the outer surface of many cells (Knowles et al., 1983; Plesner, 1994), including neuronal and glia cells (Stefanovic et al., 1976). The existence of an ecto-ATPase on the surface of intact nerve endings was predicted (Sorensen and Mahler, 1981; White, 1978) and was experimentally shown for the first time in chicken forebrain (Nagy et al., 1983). Later, ecto-ATPase activities were also described in synaptosomes from the electric organ of Torpedo Marmorata (Keller and Zim- mermann, 1983) and from various mammalian brains (Nagy et al., 1986; Gandhi and Ross, 1988).

Seizure activity causes perturbations in brain metabolism, including enzyme activities involved in ion transport and in phosphorylation of membrane proteins (Shin and McNamara, 1994; Buchhalter, 1993). Although seizure-related changes in brain Na+K§ have been reported, a survey of the literature (Lees, 1991; Grisar et al., 1992) reveals many discrepancies among observations, which are probably owing to variations in enzyme preparations, the brain regions studied, and the type of epilepsy model used. Fewer data are available on brain ecto- ATPases, but alterations in function of these enzymes have been reported in genetic models of epilepsy (Rosenblatt et al., 1976; Trams and Lauter, 1978) and in human temporal lobe epilepsy (Nagy et al., 1990).

We elected to study ecto-ATPase changes during prolonged seizure activity (lithium/pilocarpine-induced status epilepticus) because of the sug- gested association of these enzymes with the production of an endogenous neurosupressant, adenosine (Nagy, 1986). Seizure-related alterations in the mitochondrial ATP content and ATP-synthesizing enzyme (F1-ATPase) were also investigated in these experiments, and the results are published elsewhere (Walton et al., 1997). A preliminary report on ecto-ATPase changes in this status model has been presented (Nagy et al., 1995).

MATERIALS AND METHODS

Materials

Enzyme substrates and Percoll were purchased from Sigma (St. Louis, MO). All other reagents were of analytical grade.

Molecular and Chemical Neuropathology Vol. 31, 1997

Ecto-ATPase Changes in Status Epilepticus 137

Animal Preparation and Experimental Protocol Status epilepticus (SE) was induced in adult, male Sprague-Dawley

rats by sequential administration of lithium and pilocarpine according to a protocol described elsewhere (Walton and Treiman, 1988). Briefly, rats were prepared surgically with four epidural recording electrodes 1 wk prior to experimentation. On the day prior to assay, rats were injected intraperitoneally with 3.3 mmol/kg lithium chloride. SE was induced by ip injection of 40 mg/kg pilocarpine HC1 20-22 h after the lithium chloride injection. EEG was monitored continuously throughout the episode of SE; status animals were sacrificed under anesthesia (ketamine/ xylazine, 87/13 mg/kg) after 2 h of continuous spiking on EEG. Brains were immediately removed and kept in ice-cold sucrose medium until subcellular fractionation.

In pilot experiments, we have established that neither the surgical procedure nor drug treatment without seizure onset affected the enzyme activities studied here. Therefore, a matching naive rat was used as control and was anesthetized and decapitated at the same time as each SE rat. Brain tissue preparation and enzyme assays were carried out simultaneously for each matching pair of rats (six pairs total).

Preparation of Intact Synaptosomes and Synaptosomal Plasma Membranes from Rat Brains

Brain cortex samples (average 0.63 g) were homogenized in cold, isoosmotic sucrose/buffer medium (0.32 M sucrose; 5 mM HEPES, pH 7.5; 0.1 mM EDTA) in a Teflon-glass homogenizer at approximately 800 rpm. After removing the nuclear and cell debris, synaptosomes were isolated from the crude mitochondrial pellet (P2) by using sucrose/Percoll (7.5, 10, and 20%) discontinuous density gradients (Nagy et al., 1984). After centrifugation at 15,000g for 20 rain, synaptosomes were collected from the 10/20% Percoll interface and stored on ice until further analysis. Part of the synaptosomal fractions were disrupted by freeze-thawing once in a hypo-osmotic medium, in order to expose the intracellular active-sites of the Na§ +-, and Ca2*Mg2+-ATPase enzymes. Synaptic plasma membrane fractions were prepared from control rat brain tissues according to Cottman and Matthews (1971), except that synaptosomes were separated on Percoll gradients. Synaptic plasma membrane preparations were used in our in vitro drug experiments.

Enzyme Assays Ecto-Mg2+-ATPase activity of intact synaptosomes was determined

in an incubation medium (total volume 0.25 mL) containing 50 mM HEPES buffer (pH 7.4), 1.5 mM MgCI~, and 0.5 mM EGTA. Sodium



azide (5 raM) and ouabain (2 mM) were added to the reaction mixtures to inhibit any possible mitochondrial Ft-ATPase and Na+K+-ATPase activity, respectively. Twenty-five microliters of synaptosomal preparation (0.4-0.5 mg /mL protein) were added to the reaction medium and preincubated for 2 min at 37~ The enzyme reactions were initiated by the addition of 1 mM ATP (vanadate-free), and were allowed to pro- ceed for 1-2 min, at 37~ in a shaking water bath. The reactions were terminated by the addition of 5% trichloroacetic acid, chilled on ice, and centrifuged at 2500g x 15 min. Samples of the supernatants (0.1 mL) were taken for assay of the released inorganic phosphate (Pi) by the method of Lanzetta et al. (1979) using malachite green as a color reagent and KH2PO4 as a reference standard. The assay of ecto-Ca 2+- ATPase was conducted in essentially the same way as the Mg2+-ATPase, except that 1.5 mM CaCl~ replaced MgCI2, and 0.1 mM EDTA was used instead of EGTA in the Ca2+-ATPase reactions. The Na+K+-ATPase activity was taken as the difference of the ATPase activity measured in a complete ecto-Mg2+-ATPase incubation medium, plus 125 mM NaC1 and 5 mM KC1 in the absence and presence of 2 mM ouabain. To deter- mine the Ca2+Mg2+-ATPase (Ca-pump) activity, 0.15 mM CaC12 was also included in the reaction medium used for Mg2+-ATPase assay. These two latter enzyme activities were measured in disrupted synaptosomal fractions.

Lactate dehydrogenase was used as a cytoplasmic marker and was quantified by the method of Jolmson (1960).

Protein Assay The technique of Bradford (1976) was used for protein determina-

tions because residual Percoll present in our subcellular brain fractions did not interfere with this assay (Terland et at., 1979). However, membrane proteins had to be solubi!ized prior to determination, which we achieved by pretreating all samples with 0.2% (end concentration) sodium deoxycholate for 15 min at room temperature. Sodium deoxycholate at this concentration did not interfere with the colorimetric protein measurement.

Data Analysis

Enzyme activity changes in the epileptic brain samples were compared with those of the matched control brain samples. Significance of these changes was then estimated using Student's t-test or analysis of variance procedures. Individual comparisons were made using multiple t-tests, with Dunnett's correction for probability. Effect of in vitro drug concentrations on synaptosomal plasma membrane ecto-ATPases was evaluated by one-way analysis of variance (ANOVA). Data are given as mean + SEM, unless indicated otherwise.



RESULTS

By definition, plasma membrane-bound ecto-ATPases have their active site facing the extracellular space where they utilize ATP released from cells. It was therefore important to ascertain that the subcellular particles used for this enzyme activity assay were intact. The low activity of lactate dehydrogenase, a well-accepted marker for leaky plasma membranes (Johnson and Whittaker, 1963), indicated that synaptosomal preparations from both control and epileptic brains were well-preserved (Table 1). The proportion of damaged particles in our synaptosomal preparations (11%) was comparable to that of previous works (for review, see Nagy et al., 1984), regardless of whether the brain tissue samples were from control or SE rats. In order to ensure that the divalent cation- dependent ATPase values reflected ecto-ATPase activities in spite of the presence of these limited number of broken particles, specific inhibitors of intracellular ATPases (ouabain for Na~K+-ATPase, sodium azide for F1- ATPase, and EGTA for Ca-pump-ATPase) were always included in the reaction media.

Synaptosomal ATPase activities were determined from the cerebral cortex of six control rats and six SE rats, which were sacrificed after 120 min of continuous EEG spiking induced by lithium/pilocarpine treatment. In each experiment, we simultaneously measured and compared enzyme activities in one control and one status animal. As presented in Table 2, both ecto-Mg2+-ATPase and ecto-Ca2§ activities were statistically significantly decreased in synaptosomes from the status rat brains when compared to the corresponding ecto-ATPase values of control rat brains (t = 7.806, p < 0.01 for ecto-Mg2+-ATPase; t = 5.931, p < 0.01 for ecto-Ca2~-ATPase). Of the two measured intracellular ATPases, only the Ca2+Mg2+-ATPase (Ca-pump) showed a significant SE-related decrease in enzyme activity (t = 5.736, p < 0.05). The extent of decrease was simi- lar to that found for the two ecto-ATPases (see Table 2). Under the same conditions, there was no change in Na+K§

We have tested the direct effect of lithium and pilocarpine on ecto- ATPase activities using control rat brain synaptosomal plasma membranes as an enzyme source. ATPase activity of the purified membrane preparations were comparable to that of the intact synaptosomes, since activity of the non-ecto type ATPase was suppressed by the addition of specific inhibitors (1 mM ouabain for Na ~K+-ATPase; 1 mM sodium azide for mitochondrial F1-ATPase) or by omitting the necessary substrates from the assay medium (Ca-pump ATPase). Analysis of variance revealed no statistically significant change in either Mg2+-ATPase or Ca2§ activities when lithium chloride, pilocarpine, or both drugs were added at concentrations ranging from 0.5 ~tM to 1.0 mM (Table 3).

In order to assess the stability of the observed ecto-ATPase activity changes, aliquots of each synaptosomal preparation were frozen and



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Ecto-ATPase Changes in Status EpilepUcus 141

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Molecular and Chemical Neuropalhology Vol. 31, 1997


Table 3 In Vitro Effect of Lithium and Pilocarpine on fiynaptosoma| Ecto-ATPases

Percentage activity change b

Drug added a MgATPase CaATPase

Lithium chloride Pilocarpine hydrochloride Lithium chloride plus

pilocarpine hydrochloride

3.1 _+ 1.29 -1.8 _+ 0.55 1.6 _+ 0.92 -0.8 + 0.48

-0.3 + 0.63 0.1 + 0.69

'~Concentrations are from 0.5 laM to 1 mM. bActivity changes were calculated as percentage of control activities (465.2 + 2.62 for

MgATPase and 332.5 _+ 2.21 for CaATPase; n = 30). p for all calculated F values was greater than 0.05.

stored for various lengths of time. We repeated the enzyme measure- ments in the same fractions after 1 d and after 1 mo of storage at -20~ As a result of the freeze-thawing process, the majority of synaptosomes were damaged in both control and status brain fractions. However, the presence of specific inhibitors and enzyme assay conditions prevented intracellular ATPases from intervening in the ecto-ATPase assays. Results from these studies are shown in Fig. 1. Two-way ANOVA, using experimental group (status vs control) as one factor and duration of storage as the other factor, revealed that only the group factor was significantly related to the measured enzyme activities (F = 12.445, p < 0.01 for ecto- Mg2+-ATPase; F = 9.332, p < 0.01 for ecto-Ca2§ Neither the duration of storage nor the interaction of storage duration and group caused significant alteration in the enzyme activities.

DISCUSSION

During the last few years, convincing evidence has shown that ATP is stored within vesicles and coreleased with various neurotransmitters upon depolarization of the nerve endings (Stone, 1981; Gordon, 1986; Westfall et al., 1990). When released into neural junctions, ATP can fulfill multiple roles either by modulat ing the release and function of other neurotransmitters (Phillis and Wu, 1981; Gordon, 1986; Lindgren and Smith, 1987; Terrian et al., 1989; White, 1988) or by acting itself as a neurotransmitter in the peripheral nervous system (Stone, 1981). Recent studies suggest that ATP can also function as a fast excitatory neurotransmitter in the central nervous system (Edwards et al., 1992; Evans et al., 1992; Silinsky et al., 1992). Al though there is some controversy in the literature (Chaudry et al., 1985), the widely held belief is that phospho- rylated nucleosides cannot be taken up by intact cells (i.e., the excitatory action of ATP on purinergic receptors cannot be terminate unless first being hydrolyzed to adenosine by a series of ATP-metabolizing enzymes

blolecular and Chemical I'~europathology Vol. 31, 1997

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Fig. 1. Effect of freeze-thawing and storage on synaptosomal ecto-ATPase changes observed in rats with lithium/pilocarpine-induced status epilepticus. Synaptosomal preparations from control ( ~ ) and status epilepticus ([]) rat brains were assayed for ecto-MgATPase (A) and ecto-CaATPase (B) activities freshly, after 1 d storage at -20~ (freeze-thawed once), and after 1 mo storage at -20~ (freeze-thawed twice). Ecto-ATPase activities were measured in the presence of inhibitors of intracellular ATPases as described in the Methods sec- tion. Each bar shows the mean enzyme activity expressed as nmol Pi /min/mg protein, with an error bar indicating the standard error, n = 6.

(ecto-ATPase, ecto-ADPase, adenylate kinase and 5'-nucleotidase; Zim- mermann et al., 1979; Nagy, 1986). Adenosine itself is a powerful inhibitor of neurotransmitter release (Stone, 1981; Pedata, 1986; Richard- son et al., 1987; Williams, 1990), but its production is controlled by the overall hydrolysis rate of extracellular ATP. This is supported by the



observation that ATP can exert a "feed forward" inhibition on ADPase and 5'-nucleotidase, the latter two members of the extracellular ATP- hydrolyzing chain (Kluge et al., 1975; and James and Richardson, 1993). Through prolonging the presence of the excitatory transmitter, ATP, and delaying the production of the inhibitory modulator, adenosine, in the synaptic cleft, defective synaptosomal ecto-ATPases could increase seizure Susceptibility in the brain.

Rosenblatt et al. (1976) reported substantially decreased Ca2+-ATPase and slightly decreased Mg2+-ATPase activities in brain homogenates of audiogenic seizure-prone DBA/2N mice. Glial cells of these seizure- prone mice were also shown to have ecto-ATPase deficiency (Trams and Lauter, 1978). These investigators hypothesized that the prolonged presence of extracellular ATP, caused by insufficient hydrolysis, could lead to increased tissue excitability. We have shown that synaptosomal ecto- ATPase activities are decreased in actively spiking regions (documented during surgery) of temporal cortex tissue from humans with temporal lobe epilepsy (Nagy et al., 1990). We postulated that this diminished ecto- ATPase activity is the result of a prolonged hyperexcitable state of the epileptic cortical tissue and that the diminished synaptosomal ecto- ATPase activity could limit the rate of adenosine response that was observed immediately after convulsions (During and Spencer, 1992). Cor- relation between altered brain ecto-ATPase activity and seizure develop- ment has been earlier reported from other laboratories as well. For example, a major synaptosomal protein (67 kDa) has been shown to be significantly reduced in brains of EL mice as a result of repeated convulsions (Yamagami et al., 1987). The 67-kDa molecule was not identified by Yamagami et al. at the time of their study. However, a synaptosomal ecto-ATP diphosphohydrolase with a molecular weight of 60-70 kDa since has been described in rat brains (Battastini et al., 1995). A significant correlation was established by Palayoor and Seyfried (1984) between reduced brain ecto-Ca2+-ATPase activity and genetically inherited seizure susceptibility in DBA/2J (D2) inbred strains and B6 x D2 (BXD) recom- binant inbred strains of audiogenic mice. Later, the same group (Allen and Seyfried, 1994) did not find significant association between expres- sion of an Ntp gene (encoding the Ca 2§ or Mg2+-stimulated hydrolysis of nucteoside triphosphates) and susceptibility of DBA/2J (D2) mice to audiogenic seizures. Since this latter research has been carried out with brain microsomes, and identity between the microsomal and plasma membrane ecto-ATPases has not yet been established, this discrepancy between their earlier and recent findings might be explained by possible differences between these two types of ecto-ATPases.

In our experiments, we found that prolonged seizure activity (status epilepticus) induced by lithium/pilocarpine administration also resulted in reduced synaptosomal ecto-ATPase activitj6 Simultaneously, activity of the intrasynaptosomal Ca2§ (calcium-pump) was also decreased. The diminished function of these three ATPases was not the result of gen-



eral degradation of enzyme activities because marker enzyme (total lactate dehydrogenase) and Na+K+-ATPase activities in the same fraction were indistinguishable from those of the control rats. The fact that these ecto- ATPase changes could be preserved during lengthy storage and repeated freeze-thawing implies that permanent alteration probably occurred at the protein level. In an earlier study; we found that sequential lithium and pilocarpine injections did not cause a reduction in synaptosomal ecto-ATPase activities if occurrence of seizures was prevented by simultaneous injection of the anticonvulsant diazepam (Nagy et al., 1995a). This finding, in addition the fact that in vitro convulsive drug treatment did not affect the synaptosomal ecto-ATPase activity, suggests that sustained seizure activity caused the observed enzyme deterioration. Diminished function of the ecto- ATPases could, in turn, further increase seizure severity: the ecto-ATPases, by limiting the availability of the endogenous neurosupressant adenosine; and the calcium pump, by delaying the removal of excess calcium, which accumulates during seizures. Clinical observations suggest that the longer SE is allowed to continue in humans, the more refractory to treatment it becomes. Rats with prolonged lithium/pilocarpine-induced generalized, convulsive SE also have been shown to be extremely refractory to thera- peutic interventions (Morrisett et al., 1987; Walton and Treiman, 1988; Wal- ton and Treiman, 1991). SE in this model can always be controlled with diazepam administered at the onset of the status episode, but becomes completely refractory if status is allowed to continue until the EEG displays continuous, high-amplitude, rapid spiking (Walton and Treiman, 1988). It may be that seizure-induced reduction in brain ecto-ATPase activity plays a role in the transition from the treatment-responsive to the refractory stage of SE. This question should be the subject of further investigation.

ACKNOWLEDGMENTS

This study was supported by VA Medical Research Funds. The authors thank Esperanza Esteban, Tasha McMahon, and Adam Wong for skillful technical assistance.

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reduced cortical ecto-atpase activity in rat brains during prolonged status epilepticus induced by...

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