HypoxiaÏischaemia (or anoxia) of the brain often occurs

during stroke and seizure, and cerebellar and hippocampal

neurons are especially vulnerable to such insults (Cervos-

Navarro & Diemer, 1991). An important event that occurs

early during hypoxiaÏischaemia is loss of ionic homeostasis

(for reviews see Hansen, 1985; Choi, 1988), which is

suggested to be closely linked to neuronal injury and brain

oedema. One major hypothesis as to the cause of neuronal

injury is that an intracellular Ca¥ (Cafl) overload results in

cytoskeletal perturbation, impaired mitochondrial function,

and the activation of proteases, endonucleases and

phospholipases (for review see Choi, 1988). On the basis of

in vivo and in vitro studies, it has also been suggested that

the influx of Na¤ and water contribute to neuronal swelling

and blebbing (Goldberg & Choi, 1993; Friedman & Haddad,

1994; Chidekel, Friedman & Haddad, 1997; Fung &

Haddad, 1997), since ischaemia induces a decrease in the

extracellular sodium concentration ([Na¤]ï) (Jiang et al.

1992), and the removal of extracellular sodium (Naº)

prevents ischaemia-induced morphological changes in

isolated hippocampal neurons (Friedman & Haddad, 1993).

It is therefore important that neurons maintain their intra-

cellular sodium and calcium concentrations within the

physiological range.

The mechanisms responsible for the Cafl overload seen using

the hypoxic or ischaemic model and metabolic inhibition are

controversial, but several possibilities have been suggested,

namely: (i) overactivation of voltage-sensitive Ca¥ channels

(Choi, 1988; Uematsu et al. 1991), (ii) overactivation of

NMDAÏnon-NMDA channels (Choi, 1988; Dubinsky &

Rothman, 1991; Uematsu et al. 1991; Goldberg & Choi,

1993), (iii) operation of the reverse mode of the Na¸—Cafi

exchanger (exchange of internal Na¤ for external Ca¥; Du et

al. 1997), (iv) inhibition of Ca¥-ATPase (Choi, 1988) and

(v) overproduction of reactive oxygen free radicals (for

review see Halliwell, 1992; Gunasekar et al. 1996). To

explain the hypoxiaÏischaemia-induced [Na¤]é increase,

two possible mechanisms have been proposed, involving

either TTX-sensitive Na¤ channels (Fung & Haddad, 1997)

or the Naº—Cafl exchanger (Chidekel et al. 1997).

Cerebellar granule cells form the largest population of

neurons in the brain and have important physiological

functions. However, the mechanisms of the metabolic

Journal of Physiology (1999), 515.1, pp.133—146 133

Early metabolic inhibition-induced intracellular sodium and

calcium increase in rat cerebellar granule cells

Wei-Hao Chen*‡, Kuan-Chou Chu*, Shyh-JongWu*, Jiahn-ChunWu†,

Hao_Ai Shui * and Mei-LinWu*

Institutes of *Physiology and †Anatomy, College of Medicine, National Taiwan University

and ‡Department of Internal Medicine, National Taiwan University Hospital, Taipei,

Taiwan, Republic of China

(Received 28 July 1998; accepted after revision 3 November 1998)

1. Possible mechanisms responsible for the increases in intracellular calcium ([Ca¥]é) and

sodium ([Na¤]é) levels seen during metabolic inhibition were investigated by continuous

[Ca¥]é and [Na¤]é measurement in cultured rat cerebellar granule cells. An initial small

mitochondrial Ca¥ release was seen, followed by a large influx of extracellular Ca¥. A large

influx of extracellular Na¤ was also seen.

2. The large [Ca¥]é increase was not due to opening of voltage-dependent or voltage-

independent calcium channels, activation of NMDAÏnon-NMDA channels, activation of the

Na¸—Cafi exchanger, or inability of plasmalemmal Ca¥-ATPase to extrude, or mitochondria

to take up, calcium.

3. The large [Na¤]é increase was not due to activation of the TTX-sensitive Na¤ channel, the

Na¸—Cafi exchanger, the Na¤—H¤ exchanger, or the Na¤—K¤—2Cl¦ cotransporter, or an

inability of Na¤—K¤-ATPase to extrude the intracellular sodium.

4. Phospholipase Aµ (PLAµ) activation may be involved in the large influx, since both were

completely inhibited by PLAµ inhibitors. Moreover, melittin (a PLAµ activator) or

lysophosphatidylcholine or arachidonic acid (both PLAµ activation products) caused similar

responses. Inhibition of PLAµ activity may help prevent the influx of these ions that may

result in serious brain injury and oedema during hypoxiaÏischaemia.

8560

Keywords: Metabolic inhibition, intracellular calcium, intracellular sodium

inhibition-induced [Ca¥]é changes in granule cells have not

been studied in detail, and there is no direct evidence for

[Na¤]é changes during such insult. By treating granule cells

with 5 mÒ CN¦-containing glucose-free medium to inhibit

both oxidative phosphorylation and glycolysis, we have

shown and characterized the changes in [Ca¥]é and [Na¤]é

during this process. Under these experimental conditions, a

small initial increase in [Ca¥]é is seen, probably as a result

of Ca¥ release from mitochondria, that is then followed by a

much larger influx of Ca¥and Na¤, possibly as a result of

phospholipase Aµ (PLAµ) activation. Reactive oxygen species

may also play a role in the process. Possible reasons for the

differences in results seen in this study and those involving

in vivo or brain slice studies are discussed.

METHODS

Solutions and chemicals

All test solutions were prepared in Hepes-buffered modified Tyrode

solution, containing (mÒ): 118 NaCl, 4·5 KCl, 1·0 MgClµ, 2·0 CaClµ,

11 glucose, 10 Hepes, adjusted to pH 7·4 with NaOH at 37°C

unless specified otherwise. When chemicals were added at

concentrations greater than 5 mÒ, the fraction of NaCl was reduced

accordingly to compensate the osmolarity. All chemicals were

purchased from Sigma. HOE694 and U_78517F were generous

gifts, respectively, from Dr H.-J. Lang (Hoechst Aktiengesellschaft,

Frankfurt Germany) and Dr E. J. Jacobsen (Medicinal Chemistry

Research Unit, Upjohn Laboratories, MI, USA).

Primary culture of cerebellar granule cells

Rat cerebellar granule cells were prepared and cultured essentially

as described previously (Gallo et al. 1982). In brief, 8-day-old

Wistar rats were killed by cervical dislocation and then

decapitated. The cerebella were removed and minced into 0·4 mm

cubes, and dissociated with 0·025% trypsin for 15 min at 37°C.

The dissociated cells were suspended in basal modified Eagle’s

medium containing 10% fetal calf serum, 25 mÒ KCl, 2 mÒ

glutamine, and 50 ìg ml¢ gentamicin, and consequently plated

onto poly-¬-lysine-coated 24 mm coverslips, and maintained in a

humidified 5% COµ incubator. Cytosine arabinoside (10 ìÒ) was

added 24 h after plating to kill and arrest the replication of the

non-neuronal cells, especially the astrocytes. The purity of the

granule cells is generally greater than 90% after 6—7 days in culture.

Determination of [Ca¥]é

The method for measuring intracellular [Ca¥]é levels was similar to

that used in our previous study (Wu et al. 1997). In brief, cells were

loaded for 60 min at room temperature (22—25°C) with 5 ìÒ

fura_2 AM (Molecular Probes), then a small group of cells (•5—10

cells for each experiment) were excited alternately with 340 and

380 nm wavelength light. The ratio of the emission at 510 nm with

the excitation wavelengths, respectively, of 340 and 380 nm was

calculated and converted to [Ca¥]é using the following equation

(Grynkiewicz et al. 1985):

[Ca¥]é = Kd (R − Rmin)Ï(Rmax − R) (Sf2ÏSb2),

where R is the ratio of the 510 nm fluorescence at 340 nm

excitation over that at 380 mm. Calibration constants are obtained

by adding 5 ìÒ ionomycin in solutions containing either 10 mÒ

Ca¥ (Rmax) or calcium-free solution containing 10 mÒ EGTA

(Rmin). A Kd of 224 nÒ was used (Grynkiewicz et al. 1985). Sf2ÏSb2

is the ratio of the 510 nm emissions at 380 nm excitation

determined at Rmin and Rmax, respectively. We tested whether the

Kd for fura_2 was altered when the intracellular pH (pHé) changed,

using an in vitro test (fura_2 free acid with different pH values,

from 6·0 to 8·0) and found that, in the pH range 6·3—8·0, the

340Ï380 ratio shows little change.

Determination and calibration of [Na¤]é

Granule cells were loaded with 5 ìÒ SBFI AM (Molecular Probes)

for 90 min at room temperature, then washed with the control

solution, and a small group of cells (•10 cells for each experiment)

were excited alternately with 340 and 380 nm wavelength light.

The ratio of the emission at 510 nm with the excitation

wavelengths, respectively, of 340 and 380 nm was calculated and

converted to a linear sodium scale by in vivo calibration. Since

SBFI has different spectral properties inside cells compared with

those in bulk solution and this may differ in different cell types

(Rose & Ransom, 1997), calibration of the dye signal within the cell

following each experiment allowed us to determine absolute values

for [Na¤]é. Calibration solutions were prepared using a combination

of high Na¤ solution (containing (mÒ): 115 sodium gluconate, 25

NaCl, 1 EGTA, 10 Hepes, 11 glucose) and high K¤ solution

(containing (mÒ): 115 potassium gluconate, 25 KCl, 1 EGTA, 10

Hepes, 11 glucose). The solutions contained the ionophores,

gramicidin D (2 ìÒ), monensin (40 ìÒ) and ouabain (1 mÒ), and

the pH was adjusted to 7·4 at 37°C. The mean apparent

dissociation constant (Kapp) at 37°C was 17·3 mÒ. The fluorescence

ratio was converted into the intracellular Na¤ concentration by the

following equation (Harootunian et al. 1989):

[Na¤]é = Kapp (R − RminÏRmax − R) (Sf2ÏSb2).

Statistics

All results were expressed as means ± s.e.m. for a given number of

experiments (n). Statistical difference was compared using Student’s

paired or unpaired t tests, and P < 0·05 was considered significant.

RESULTS

Metabolic inhibition induces an increase in [Ca¥]é

In rat cerebellar granule cells, the resting [Ca¥]é was found

to be 30 ± 10 nÒ (n = 56), similar to the value reported in

another study on granule cells (Courtney et al. 1990). As

shown in Fig. 1A, within 3·8 ± 0·1 min (n = 14) of the start

of perfusion, with inhibitors of both oxidative (5 mÒ KCN)

and glycolytic (glucose-free) metabolism (Allen & Orchard,

1986), [Ca¥]é began to increase, reaching levels of

250 ± 30 nÒ (n = 28) and 394 ± 28 nÒ (n = 32) at 10 and

15 min, respectively (Table 1). The [Ca¥]é level plateaued at

1188 ± 247 nÒ (n = 8) after 30 min perfusion (Table 1). In

all cases, following wash-off of the metabolic inhibitors,

[Ca¥]é returned almost to the resting level (Figs 1A and 8A).

For the convenience of comparison, the [Ca¥]é increases at

10 and 15 min of metabolic inhibition were measured in

subsequent studies.

Since intracellularly stored calcium can be easily washed out

by EGTA-containing solutions, Ca¥-free medium without

added EGTA was used to investigate the origin of the

[Ca¥]é increase. In calcium-free medium, metabolic

inhibition resulted in only a small initial increase in [Ca¥]é

(Fig. 1B and Table 1), but, when Cafi was restored to 2 mÒ

in the presence of metabolic inhibitors, the large increase in

W.-H. Chen and others J. Physiol. 515.1134

[Ca¥]é was again observed, suggesting that the initial small

increase is due to release from internal Ca¥ stores, whereas

the subsequent larger change is due to a massive influx of

extracellular Ca¥.

Two major internal calcium stores, the cytosolic endoplasmic

reticulum (ER) (i.e. IP×Ïryanodine-sensitive Ca¥ stores) and

mitochondria, could be involved in the initial Ca¥ release.

When cells were treated with a combination of cyclopiazonic

acid (CPA, 15 ìÒ) and ryanodine (10 ìÒ) under Ca¥-free

conditions (Fig. 1C), a small [Ca¥]é transient, probably due

to depletion of the ER stores, was seen in the absence of

metabolic inhibition. Complete depletion of the ER store by

Metabolic inhibition-evoked sodium and calcium increaseJ. Physiol. 515.1 135

Figure 1. Metabolic inhibition-induced [Ca¥]é increase

A, in the presence of 2 mÒ [Ca¥]ï. B, in Ca¥-free medium followed by addition of 2 mÒ [Ca¥]ï.

C, depletion of endoplasmic reticulum (ER) calcium stores using 15 ìÒ CPA and 10 ìÒ ryanodine in Ca¥-

free medium. D, in Ca¥-free medium in the presence of the mitochondrial uncoupler carbonyl cyanide

m_chlorophenyl-hydrazone (CCCP, 100 ìÒ). The metabolic inhibitors used were 5 mÒ KCN (CN¦) in

glucose-free (Glc-free) medium. All experiments were performed in Hepes-buffered solution (pH 7·4) at

37°C.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 1. Metabolic inhibition (MI) induces an initial small [Ca¥]é increase, followed by a marked

Ca¥ influx

––––––––––––––––––––––––––––––––––––––––––––––

[Ca¥]é (nÒ)

––––––––––––––––

Treatment 2 mÒ [Ca¥]ï 0 Cafi

––––––––––––––––––––––––––––––––––––––––––––––

Time after MI

10 min 250 ± 30 (28) 49 ± 6 (10)

15 min 394 ± 28 (32) –

30 min 1188 ± 247 (8) –

CPA + Rya – 15 ± 3 (8)

CPA + Rya + MI – 44 ± 5†

CCCP 138 ± 9 (8) 105 ± 8 (8)

CCCP + MI 961 ± 155* 99 ± 9‡

––––––––––––––––––––––––––––––––––––––––––––––

The first three rows show [Ca¥]é at different times (10—30 min) after metabolic inhibition (glucose-free

solution containing 5 mÒ CN¦). CPA, cyclopiazonic acid (15 ìÒ); Rya, ryanodine (10 ìÒ). The results are

expressed as means ± s.e.m. The numbers in parentheses indicate the number of experiments (n).

*P < 0·05, Student’s unpaired t test, compared with control group (250 ± 30 nÒ, n = 28); †P > 0·05,

Student’s unpaired t test, compared with control group (49 ± 6 nÒ, n = 10); ‡P > 0·05, Student’s paired t

test, compared with its own control (paired rows).

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

these two blockers in granule cells was confirmed by the lack

of further Ca¥ release (data not shown) on addition of

0·5 mÒ carbachol (an agonist of the IP×-sensitive calcium

store in granule cells) in the presence of 10 ìÒ ryanodine

(depletion of the ryanodine-sensitive store) and 15 ìÒ CPA

(Irving et al. 1992). In the presence of ryanodine and CPA,

the initial small [Ca¥]é rise induced by metabolic inhibition

was still seen (Fig. 1C and Table 1), therefore suggesting

that the IP×ÏER store does not play a major role in this

process. In contrast, under the same Ca¥-free conditions,

this initial small peak was no longer seen in the presence of

100 ìÒ carbonyl cyanide m_chlorophenyl-hydrazone (CCCP)

(Fig. 1D and Table 1), a proton ionophore that collapses the

negative mitochondrial membrane potential, and results in

mitochondrial calcium release and inhibition of calcium

uptake in granule cells (Budd & Nicholls, 1996). This

indicates that it is the mitochondria that are responsible for

the initial Ca¥ release seen during early metabolic

inhibition.

Lack of involvement of Ca¥ and NMDAÏnon-NMDA

channels, the plasmalemmal Na¸—Cafi exchanger and

Ca¥-ATPase in the large increase in [Ca¥]é during

metabolic inhibition

Anoxia induces marked depolarization of the membrane

potential in hippocampal slices (Fung & Haddad, 1997), and

thus the Ca¥ influx seen during hypoxiaÏischaemia could

possibly be due to the opening of voltage-dependent Ca¥

channels. Granule cells possess L_ and N-type, but not TÏP-

type, Ca¥ channels (Pearson et al. 1995). However, in our

experiments, 10 ìÒ nifedipine (an L-type Ca¥ channel

W.-H. Chen and others J. Physiol. 515.1136

Figure 2. NMDA and non-NMDA receptors are not involved in the metabolic inhibition-induced

[Ca¥]é influx

Calcium influx in the presence of the NMDA receptor antagonist MK_801 (10 ìÒ) (A), the non-NMDA

receptor antagonist DNQX (6 ìÒ) (B) and after pretreatment with both inhibitors (C).

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 2. Effects of various treatments on MI-induced [Ca¥ ]é

increase

–––––––––––––––––––––––––––––

Treatment Ä[Ca¥]é (nÒ)

–––––––––––––––––––––––––––––

MI (10 min) 250 ± 30 (28)

Nifedipine 190 ± 30 (4)

ù-Conotoxin 200 ± 40 (4)

SK&F 96365 200 ± 45 (4)

Gadolinium 235 ± 70 (4)

Flufenamic acid 235 ± 37 (4)

MK_801 268 ± 14 (4)

DNQX 248 ± 18 (5)

MK_801 + DNQX 262 ± 11 (4)

Na¤ free 191 ± 27 (4)

MI (10 min) 218 ± 46 (4)

K¤ free 11 ± 3 *

MI (10 min) 196 ± 25 (4)

pHï 8·5 548 ± 21 *

MI (10 min) 188 ± 10 (4)

Eosin B 312 ± 21 *

–––––––––––––––––––––––––––––

*P < 0·05, Student’s paired t test, compared with its own control

for the last three experiments (paired rows). The rest of the

experiments were compared using Student’s unpaired t test with

the control [Ca¥]é value after the first 10 min of metabolic

inhibition (MI) (250 ± 30 nÒ, see Table 1); no significant difference

was found. n values given in parentheses.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

blocker) or 1 ìÒ ù-conotoxin GVIA (an N-type Ca¥

channel blocker) had no effect on the Ca¥ influx after

10 min perfusion with metabolic inhibitors (Table 2).

The possible involvement of the two other calcium-

permeable channels was also tested, namely the calcium

release-activated calcium (CRAC) channel (present in

granule cells; Simpson et al. 1995) and the non-selective

cation channel. Neither the CRAC channel blocker SK&F

96365 (30 ìÒ) nor the two non-selective cation channel

blockers flufenamic acid or gadolinium (both 100 ìÒ) had

any effect on the metabolic inhibition-induced increase in

[Ca¥]é (Table 2).

As NMDAÏnon-NMDA channels are involved in hypoxia/

ischaemia-induced Ca¥ increase in hippocampal and

cortical neurons (Dubinsky & Rothman, 1991; Uematsu et

al. 1991), this possibility was investigated in granule cells.

When NMDA receptors were activated by 100 ìÒ NMDA in

Mg¥-free solution containing 10 ìÒ glycine, the resultant

[Ca¥]é increase could be blocked by 10 ìÒ MK_801,

whereas the metabolic inhibition-induced [Ca¥]é increase

was not (Fig. 2A and Table 2). Similarly, the [Ca¥]é increase

induced by the non-NMDA agonist kainic acid (100 ìÒ) was

blocked by 6 ìÒ DNQX, but the metabolic inhibition-

induced [Ca¥]é increase was not (Fig. 2B and Table 2).

Moreover, pretreatment with both blockers had no effect on

the subsequent metabolic inhibition-induced [Ca¥]é increase

(Fig. 2C and Table 2), strongly suggesting that, under the

present experimental conditions, neither NMDA nor non-

NMDA receptors are involved in the metabolic inhibition-

induced [Ca¥]é increase.

Metabolic inhibition-evoked sodium and calcium increaseJ. Physiol. 515.1 137

Figure 3. Role of the Na¸—Cafi exchanger in the metabolic inhibition-induced [Ca¥]é increase

Intracellular sodium (A and C) and calcium (B and D) measurements. A, under metabolic inhibition (Glc-

free + CN¦), a significant [Na¤]é increase was seen which was reversed upon changing to Na¤-free medium

(all external Na¤ replaced with N-methyl-ª_glucamine). B, [Ca¥]é was increased with metabolic inhibition

in Na¤-free medium. C, inhibition of Na¤—K¤-ATPase with either strophenthidin (100 ìÒ) or K¤-free

medium increased [Na¤]é. D, [Ca¥]é was higher under metabolic inhibition than in K¤-free medium.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 3. Effects of PLAµ inhibitors or metal ions on MI-,

melittin- or fatty acid-induced [Ca¥]é increases

–––––––––––––––––––––––––––––

Treatment Ä[Ca¥]é (nÒ)–––––––––––––––––––––––––––––

MI (10 min) 203 ± 32 (4)

+ Mepacrine 20 ± 2 *

MI (10 min) 168 ± 28 (4)

+ Antiflammin_1 14 ± 3 *

Melittin 229 ± 23 (5)

+ Ni¥ 124 ± 21*

Melittin 207 ± 15 (5)

+ Co¥ 72 ± 11 *

MI (10 min) 340 ± 43 (7)

+ Ni¥ 32 ± 9*

MI (10 min) 163 ± 21 (4)

+ Co¥ 14 ± 5*

LPC 430 ± 36 (5)

+ Ni¥ 184 ± 24*

LPC 512 ± 73 (4)

+ Co¥ 85 ± 18 *

AA 265 ± 30 (5)

+ Ni¥ 127 ± 17*

AA 194 ± 30 (4)

+ Co¥ 93 ± 8*

–––––––––––––––––––––––––––––

Values represent Ä[Ca¥]é after 10 min of metabolic inhibition.

*P < 0·05, Student’s paired t test compared with its own control.

+ indicates the presence of inhibitors during the various

treatments. AA, arachidonic acid; LPC, lysophosphatidylcholine.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Ischaemia has been demonstrated to cause depolarization and

activation of the TTX-sensitive Na¤ channel in hippocampal

slices (Fung & Haddad, 1997), and the subsequent increase

in [Na¤]é can lead to an increase in [Ca¥]é through

activation of the Na¸—Cafi exchanger (Goldberg & Choi,

1993). This possibility was examined in the following

experiments. In granule cells, the resting [Na¤]é, measured

using SBFI AM, was 7·8 ± 0·6 mÒ (n = 70). Within

3·2 ± 0·2 min (n = 19) of the onset of metabolic inhibition,

[Na¤]é started to increase, reaching a level of 30·0 ± 1·5 mÒ

(n = 12) at 15 min (Fig. 3A); this effect was reversed by

replacement of all external sodium ions with N-methyl-

ª_glucamine (NMDG) (Fig. 3A). Comparison of the results

seen in Na¤-containing (Fig. 1A) and Na¤-free medium

(Fig. 3A) shows that the increase in [Na¤]é, but not that in

[Ca¥]é (Fig. 3B), was inhibited under Na¤-free conditions

(Table 2), suggesting that the Na¸—Cafi exchanger may not

be involved. The above results are also summarized in

Table 5.

In order to raise Na¸ to a level similar to that seen under

conditions of metabolic inhibition and determine whether

this [Na¤]é could induce an increase in [Ca¥]é, the Na¤—K¤-

ATPase was inhibited using K¤-free medium; the resulting

increase in [Na¤]é is shown in Fig. 3C. However, the [Ca¥]é

increase induced by K¤-free medium was much less than

that seen during metabolic inhibition (Fig. 3D and Table 2),

again suggesting that the Na¸—Cafi exchanger was not

involved. Interestingly, a commonly used Na¤—K¤-ATPase

blocker, strophenthidin (100 ìÒ), did not induce an increase

in [Na¤]é comparable to that produced by K¤-free medium

(Fig. 3C); the reason for this is unknown, but it may be due

to K¤-free medium hyperpolarizing the membrane potential,

resulting in a greater sodium influx. Other possibilities

cannot be ruled out completely.

Since intracellular ATP levels can be depleted during brain

ischaemia (for review see Blaustein, 1988), we tested whether

the metabolic inhibition-induced [Ca¥]é increase was due to

inhibition of plasma membrane Ca¥-ATPase. Before testing

this possibility, we conducted a set of control experiments

consisting of two successive treatments with 5 mÒ CN¦-

containing glucose-free medium, which resulted in similar

calcium responses (236 ± 14 and 231 ± 19 nÒ, P > 0·05,

Student’s paired t test, n = 4).

Ca¥-ATPase is a Cafl—Hº exchanger and, in granule cells,

is blocked at a pHï of 8·5 (reduced [H¤]ï) (Khodorov et al.

1995). When Ca¥-ATPase was blocked at pHï 8·5, the

metabolic inhibition-induced [Ca¥]é increase was significantly

higher than at pHï 7·4 (Fig. 4A and Table 2). A similar

result was obtained using another potent Ca¥-ATPase

blocker, Eosin B (20 ìÒ, Gatto & Milanick, 1993) (Fig. 4B

and Table 2). The above results therefore suggest that Ca¥-

ATPase is still operating during early metabolic inhibition.

In neurons, mitochondria have a high capacity to take up

the excess Ca¥ during calcium overload (Gunter et al. 1994;

Kiedrowski & Costa, 1995; Budd & Nicholls, 1996; Wang &

W.-H. Chen and others J. Physiol. 515.1138

Figure 4. Role of calcium extrusion mechanisms in the metabolic inhibition-induced [Ca¥]é

increase

When Ca¥-ATPase was blocked either by pHï 8·5 (A) or Eosin B (20 ìÒ, B), the metabolic inhibition-

induced [Ca¥]é increase is larger. The mitochondrial blocker CCCP (100 ìÒ) released stored calcium in the

mitochondria both during (C) and after (D) metabolic inhibition. In the buffer system for pHï 8·5 medium,

10 mÒ bicine replaced 10 mÒ Hepes.

Thayer, 1996). The following experiment was performed to

test whether this process was blocked during early metabolic

inhibition, thus resulting in the marked [Ca¥]é increase.

When 100 ìÒ CCCP was added during the upstroke of the

calcium increase (Fig. 4C, arrowhead), a further transient

[Ca¥]é increase (867 ± 156 nÒ, n = 4), presumably due to

mitochondrial release (Kiedrowski & Costa, 1995; Budd &

Nicholls, 1996), was seen, and [Ca¥]é returned to the original

level when CCCP was removed (Fig. 4C). Furthermore, a

rapid rise in [Ca¥]é (722 ± 100 nÒ, n = 4; Fig. 4D) was seen

when 100 ìÒ CCCP was added during the recovery period

from metabolic inhibition. This suggests that mitochondrial

calcium uptake was at least partially functioning under the

experimental conditions.

Role of PLAµ and membrane lipids in the large

metabolic inhibition-induced [Ca¥]é increase

PLAµ activity in rat neocortical neurons is increased during

brain ischaemia (Umemura et al. 1992). When this possibility

was tested in granule cells, the marked Ca¥ influx was

completely and reversibly blocked by either of two potent

PLAµ inhibitors, mepacrine (50 ìÒ, Fig. 5A and Table 3)

and antiflammin_1 (200 nÒ, Fig. 5B and Table 3) (L�offler et

al. 1985; Lloret & Moreno, 1992). It was also blocked by

divalent cations, Ni¥ (Fig. 5C and Table 3) and Co¥

(Fig. 5D and Table 3). Melittin (50 nÒ), a potent PLAµ

activator (Choi et al. 1992; Clapp et al. 1995), induced a

marked increase in the [Ca¥]é, which was similarly blocked

by these divalent cations (Fig. 5E and F and Table 3).

In the brain, PLAµ can be activated when the [Ca¥]é is

increased to the range 0·01—1 ìÒ (Farooqui et al. 1997). Since

the initial small Ca¥ increase was due to mitochondrial Ca¥

release, the question then arose as to whether the calcium

response was initiated by the small mitochondrial calcium

release, which then activated PLAµ and caused the large

Ca¥ influx. However, this possibility seems unlikely, since,

following 10—15 min pretreatment with 100 ìÒ CCCP in

the absence or presence (Fig. 6A, arrowhead) of 2 mÒ

[Ca¥]ï, the [Ca¥]é was changed little (Table 1), and the

Metabolic inhibition-evoked sodium and calcium increaseJ. Physiol. 515.1 139

Figure 5. Role of PLAµ in the metabolic inhibition-induced [Ca¥]é increase

Effect of two PLAµ inhibitors, mepacrine (50 ìÒ, A) and antiflammin_1 (200 nÒ, B). Ni¥ (C) and Co¥ (D)

(both at 1 mÒ) markedly inhibit the calcium influx induced by metabolic inhibition. The melittin (50 nÒ, a

potent PLAµ activator)-induced calcium influx can be also inhibited by Ni¥ (E) and Co¥ (F) (both 1 mÒ).

subsequent metabolic inhibition-induced Ca¥ influx was

even greater (Fig. 6B, second arrowhead, 961 ± 155 nÒ,

n = 4) than that seen in the absence of CCCP (250 ± 30 nÒ,

n = 28, Fig. 1A and Table 1). This suggests that there is no

direct coupling between the initial mitochondrial calcium

release and the PLAµ activation-mediated calcium influx.

PLAµ activation results in the release of free fatty acids,

including arachidonic acid (AA), and lysophospholipids,

including lysophosphatidylcholine (LPC), and both induce

calcium overload during cardiac ischaemia (Jones et al.

1989; Donck et al. 1992). The effect of AA or LPC on the

metabolic inhibition-induced [Ca¥]é increase was tested.

LPC (3 ìÒ) caused a marked increase in the [Ca¥]é in Cafi-

containing solution similar to that seen during metabolic

inhibition, and this effect was blocked by either 1 mÒ Ni¥

(Fig. 7A and Table 3) or Co¥ (Fig. 7B and Table 3). A

similar result was seen using AA (10 ìÒ, Fig. 7C and D and

Table 3). Inhibitors of cyclo-oxygenase (10 ìÒ indomethacin),

lipo-oxygenase (10 ìÒ MK_886 + 10 ìÒ baicalein) or

cytochrome P450 (10 ìÒ econazole) did not affect the

metabolic inhibition-induced calcium increase (data not

shown), indicating that AA metabolites are probably not

involved.

W.-H. Chen and others J. Physiol. 515.1140

Figure 6. Role of the mitochondria in the calcium response

A, a small [Ca¥]é increase, induced by CCCP addition (Ca¥-free medium), is followed by a similar change

in [Ca¥]é (arrowhead) in the presence of 2 mÒ [Ca¥]ï. B, after CCCP pretreatment (first arrowhead), 5 mÒ

CN¦ in glucose-free medium induced a fast calcium influx (second arrowhead) in 2 mÒ [Ca¥]ï-containing

medium. The concentration of CCCP was 100 ìÒ.

Figure 7. Role of the membrane degradation products LPC and AA, in the metabolic inhibition-

induced [Ca¥]é increase

Ni¥ (1 mÒ) inhibited the LPC (3 ìÒ, A)- and AA (10 ìÒ, C)-induced [Ca¥]é increase. Co¥ (1 mÒ) also

inhibited LPC (3 ìÒ, B)-and AA (10 ìÒ, D)-induced [Ca¥]é increase.

Role of free radicals in metabolic inhibition-induced

[Ca¥]é increase

In cerebellar granule cells, addition of CN¦ has been shown

to activate NMDA channels and simultaneously generate

reactive oxygen species (ROS), which can be removed by

superoxide dismutase (SOD) and ¬_NAME (a nitric oxide

synthase inhibitor) (Gunasekar et al. 1996). Moreover,

stimulation of the NMDA receptorÏchannel results in NMDA-

dependent Oµ¦· production and neurotoxicity in granule

cells (Lafon-Cazal et al. 1993). Since ROS overproduction

can cause an increase in [Ca¥]é in other cells (Dubinsky &

Rothman, 1991; Uematsu et al. 1991), we tested whether

overproduction of ROSÏNO contributed to the metabolic

inhibition-induced Ca¥ influx seen in granule cells.

When the cells were continuously perfused with 5 mÒ CN¦-

containing glucose-free medium (•30 min), a marked

increase was seen in the [Ca¥]é, which slowly plateaued at

1188 ± 247 nÒ (n = 8) (Fig. 8A and Tables 1 and 4). In the

presence of SOD + ¬_NAME (Fig. 8B), the metabolic

inhibition-induced [Ca¥]é increase was not significantly

different, possibly in part due to variable calcium peaks in

this set of experiments (Table 4). However, using either

U_78517 F (20 ìÒ, a potent lipid peroxidation inhibitor and

intracellular Oµ¦· scavenger, Fig. 8C) (Hall et al. 1991) or

N_(2-mercaptopropionyl)-glycine (N-MPG) (an OH· scavenger,

Fig. 8D) (Bolli et al. 1989), the calcium rise in the upstroke

was halted and the peak [Ca¥]é levels were significantly

lower (Table 4). The above results therefore suggest that free

radicals may play a role in the metabolic inhibition-induced

calcium increase.

Role of the Naº—Cafl exchanger, Na¤—H¤ exchanger,

Na¤—K¤—2Cl¦ cotransporter, voltage-gated Na¤

channels and PLAµ in metabolic inhibition-induced

[Na¤]é increase

It is possible that the large Na¤ influx seen in granule cells

during early metabolic inhibition could occur via the

Naº—Cafl exchanger as a result of the large [Ca¥]é increase;

however, the magnitude of the metabolic inhibition-induced

increase in [Na¤]é seen in calcium-free medium (Fig. 9A and

Table 5) was essentially similar to that seen in calcium-

containing medium (Fig. 3A and Table 5). A further

possibility was that metabolic inhibition induced a decrease

in the pHé (Wu & Vaughan-Jones, 1994) and consequently

stimulated the Na¤—H¤ exchanger to produce an increase in

[Na¤]é. However, addition of 60 ìÒ HOE 694 (a Na¤—H¤

exchanger blocker) had no effect on the metabolic inhibition-

induced [Na¤]é increase (Fig. 9B and Table 5). Furthermore,

Metabolic inhibition-evoked sodium and calcium increaseJ. Physiol. 515.1 141

Figure 8. Effect of antioxidants on the metabolic inhibition-induced [Ca¥]é increase

A, control, showing response to metabolic inhibitors (•30 min). B—D, response in the presence of anti-

oxidants. The concentrations of ¬_NAME, superoxide dismutase (SOD), U_78517F and N-MPG were

300 ìÒ, 100 units ml¢, 20 ìÒ and 10 mÒ, respectively.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 4. Effects of metal ions on the AA- or LPC-induced

[Ca¥]é increase

–––––––––––––––––––––––––––––

Treatment Ä[Ca¥]é (nÒ)

–––––––––––––––––––––––––––––

MI (30 min) 1188 ± 247 (8)

¬_NAME + SOD 1309 ± 428 (4)

U_78517F 241 ± 37† (4)

N-MPG 302 ± 32† (4)

–––––––––––––––––––––––––––––

†P < 0·05, Students’s unpaired t test compared with the control

value of 1188 ± 247 nÒ.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

the Na¤—K¤—2Cl¦ cotransporter or the voltage-gated

sodium channel could not account for the Naº influx, since

neither 10 ìÒ bumetanide (a co-transporter blocker) (Table 5,

with bumetanide autofluorescence corrected) nor 1 ìÒ TTX

(Fig. 9C and Table 5) had any effect. Interestingly, 50 ìÒ

mepacrine completely and reversibly inhibited the Naº

influx (Fig. 9D and Table 5, with mepacrine autofluorescence

corrected). Moreover, Ni¥ blocked both the metabolic

inhibition- and melittin-induced Naº influxes (Fig. 9E and F

and Table 5).

DISCUSSION

In cultured rat cerebellar granular cells, metabolic inhibition

induces an increase in the [Ca¥]é, which can be divided into

two stages, an initial small increase due to calcium release

from the mitochondria, and a subsequent large increase due

to calcium influx, probably involving PLAµ activation, as

does the observed large influx of Naº. These results,

obtained from cultured cells, differ from the findings from

previous in vivo or brain slice studies. These discrepancies

are discussed below.

Role of the mitochondria in the [Ca¥]é response seen

during metabolic inhibition

Recent studies have shown that the mitochondria play an

important role in Ca¥ buffering during glutamate-induced

Ca¥ overload in granule cells (Kiedrowski & Costa, 1995;

Budd & Nicholls, 1996). In the present study, when

mitochondrial calcium uptake was blocked by CCCP

(Kiedrowski & Costa, 1995; Budd & Nicholls, 1996) in the

presence of 2 mÒ [Ca¥]ï (Fig. 6A and B, first arrowheads),

the [Ca¥]é was higher (•130 nÒ, Table 1) than that seen in

the absence of CCCP (i.e. basal state, •30 nÒ), suggesting

that mitochondria may play an important role in regulating

the basal [Ca¥]é. Moreover, metabolic inhibition caused a

greater increase in [Ca¥]é when mitochondrial calcium

uptake was blocked (Fig. 6B, second arrow), providing

further evidence that mitochondria have a high capacity to

take up the overloaded calcium, as reported by other

investigators (Kiedrowski & Costa, 1995; Budd & Nicholls,

1996).

W.-H. Chen and others J. Physiol. 515.1142

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 5. Influx of extracellular Na¤ under various conditions

–––––––––––––––––––––––––––––

Treatment [Na¤]é

or Ä[Na¤]éÏÄt

–––––––––––––––––––––––––––––

Basal 7·8 ± 0·6 mÒ (70)

MI 30·0 ± 1·5 mÒ (12)

Ca¥-free 36·7 ± 2·0 mÒ (4)

HOE 694 29·7 ± 7·2 mÒ (4)

Bumetanide 27·5 ± 1·2 mÒ (4)

Mepacrine −1·4 ± 0·5 mÒ (4)

MI + Ni¥ 2·0 ± 0·7 mÒ (4)

Melittin + Ni¥ 10·9 ± 3·1 mÒ (4)

MI 1·8 ± 0·6 mÒ min¢

MI + TTX 1·7 ± 0·6 mÒ min¢

–––––––––––––––––––––––––––––

The values show the [Na¤]é increase after 15 min of metabolic

inhibition except for the basal value. †P < 0·05, Student’s unpaired

t test, compared with the control value (30·0 ± 1·5 mÒ, n = 12).

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Figure 9. Possible mechanisms involved in the [Na¤]é increase during metabolic inhibition

A, metabolic inhibition increased [Na¤]é under Ca¥-free conditions. In the presence of Ca¥ (2 mÒ) the

metabolic inhibition-induced [Na¤]é increase was not inhibited either by addition of the Na¤—H¤ exchange

blocker HOE 694 (60 ìÒ, B), or the Na¤ channel blocker TTX (1 ìÒ, C). However, the [Na¤]é increase was

inhibited by the PLAµ blocker mepacrine (50 ìÒ, D), and Ni¥ (1 mÒ, E). Addition of Ni¥ also inhibited

the effect of the PLAµ stimulator melittin on [Na¤]é (50 nÒ, F).

To the best of our knowledge, the present study is the first

to show that calcium release from mitochondria, but not

from the ryanodineÏIP×-sensitive stores (ER), is responsible

for the initial increase in [Ca¥]é during metabolic inhibition

in neurons (Fig. 1C and D). This finding raised the interesting

possibility of the initial small Ca¥ release resulting in PLAµ

activation, thus generating the subsequent large Ca¥ influx.

However, this seems unlikely, since pretreatment with CCCP

alone, which, as mentioned above, caused little change in

[Ca¥]é, did not result in the large rapid Ca¥ influx seen

during metabolic inhibition (compare Figs 1A, 6A and 6B).

We therefore suggest that the initial mitochondrial calcium

release is not coupled to the subsequent large calcium influx

seen during metabolic inhibition. This also suggests that

neither mitochondrial depolarization nor inhibition of

mitochondrial ATP synthesis by CN¦ can account for the

large calcium influx.

Another interesting point is that mitochondrial calcium

uptake seems to be at least partially functioning after short-

term metabolic inhibition, as shown by the fact that it was

still possible for CCCP to induce a large calcium release from

the mitochondria both during (Fig. 4C) and after (Fig. 4D)

metabolic inhibition. The larger CCCP-induced calcium

release seen in Ca¥-containing medium compared with

Ca¥-free medium (Fig. 1D) is probably due to greater

calcium influx, resulting in greater mitochondrial uptake,

during metabolic inhibition. In the carotid body, CN¦

depolarizes the mitochondrial potential, resulting in the

release of stored calcium (Duchen & Biscoe, 1992). However,

we found that the mitochondria are still able to take up the

influxed calcium after CN¦ treatment (Fig. 4C), which may

depolarize the mitochondrial potential in granule cells. One

possible explanation is that CN¦ causes less depolarization

than that produced by CCCP, which we cannot accurately

measure using rhodamine_123. Other possibilities cannot be

ruled out completely.

Role of PLAµ and membrane phospholipids in the

large influxes of Ca¥ and Na¤

[Ca¥]é increase. During early ischaemia (within 15 min),

both phospholipase C (PLC) and PLAµ are activated in the

rat neocortex (Umemura et al. 1992). In rat hepatocytes,

PLAµ can also be activated by metabolic inhibition (Sakaida

et al. 1992). The present study shows that two potent PLAµ

blockers, mepacrine and antiflammin_1, were able to

completely block the large Ca¥ influx (Fig. 5A and B).

Moreover, melittin, a potent activator of endogenous PLAµ

(Choi et al. 1992), mimicked the effect of metabolic

inhibition (Fig. 5E and F). These results therefore strongly

suggest that PLAµ, which is probably stimulated during

hypoxiaÏischaemia, is involved in the large Ca¥ influx seen

during metabolic inhibition. However, the possibility that

PLC activation also contributes to the calcium influx cannot

be entirely excluded.

Brain ischaemia-induced PLAµ activation can result in the

accumulation of free fatty acids produced by membrane

phospholipid degradation. Of these, AA produces the

greatest increase (within 5 min) and the levels of AA are

found to remain high even following cessation of brain

ischaemia in many in vivo animal studies (Abe et al. 1987;

Umemura et al. 1992). The role of lysophospholipids in the

metabolic inhibition-induced calcium increase in neurons,

however, is less clear. We suggest that activation of PLAµ

results in the accumulation of LPC and AA, which then

contribute to the large Ca¥ influx, since addition of either

of these products produced a calcium response similar to

that induced by metabolic inhibition. Downstream AA

metabolites are probably not important, as cyclo-oxygenase,

lipo-oxygenase or cytochrome P450 inhibitors did not affect

the metabolic inhibition-induced calcium increase. In addition

to LPC and AA, other fatty acids or lysophospholipids may

also contribute to the Ca¥ influx.

Role of ROS. In cerebellar granule neurons, generation of

Oµ¦· is significantly increased following NMDA activation

(Lafon-Cazal et al. 1993). Moreover, CN¦ has been shown to

activate NMDA channels and simultaneously generate

reactive oxygen species (ROS) in granule cells (Gunasekar et

al. 1996). However, none of these studies examined possible

changes in [Ca¥]é during the generation of ROSÏNO. Our

results suggest that intracellular production of Oµ¦· and OH·

may play a role in the metabolic inhibition-induced [Ca¥]é

increase, since either U_78517F (a potent Oµ¦· scavenger

and inhibitor of lipid peroxidation) or N-MPG (an intra-

cellular OH· scavenger) significantly inhibited the large

calcium influx (Table 4). However, addition of SOD (a

scavenger of extracellular Oµ¦·) and ¬_NAME (an NO ·

scavenger) had little effect (Table 4). Since AA and LPC

induced the calcium influx similar to that seen during

metabolic inhibition, this suggests that lipid peroxidation-

induced ROS overproduction (possibly intracellular Oµ¦· and

OH·) (Easton & Fraser, 1998) is also involved in the large

calcium influx seen during metabolic inhibition.

[Na¤]é increase. In brain slices, anoxia induces a decrease in

the extracellular Na¤ levels, and removal of Naº prevents

anoxia-induced morphological changes in hypoglossal

neurons (Jiang et al. 1992). The possibility that anoxia-

induced depolarization results in a TTX-sensitive Na¤ influx

in hippocampal slices has been suggested (Fung & Haddad,

1997), since TTX significantly attenuates both the

depolarization rate and the rate of input resistance decline.

Another study in cultured neocortical neurons reports that

the Naº—Cafl exchanger plays a more important role than

the TTX-sensitive Na¤ channel, as amiloride (a non-specific

blocker of the Naº—Cafl exchanger), rather than TTX,

prevents the anoxia-induced morphological changes

(Chidekel et al. 1997).

In this study, we found the [Na¤]é increased to •30 mÒ

after 15 min metabolic inhibition (Fig. 3A). The involvement

of the Naº—Cafl exchanger, Na¤—H¤ exchanger,

Na¤—K¤—2Cl¦ cotransporter and TTX-sensitive Na¤

channel (Fig. 9 and Table 5) was ruled out. However, PLAµ

Metabolic inhibition-evoked sodium and calcium increaseJ. Physiol. 515.1 143

activation appears to be involved, since mepacrine completely

and reversibly blocked the Na¤ response (Fig. 9D), and

melittin mimicked the effect of metabolic inhibition on the

[Na¤]é (Fig. 9F). These facts, together with the similar time

course (•3 min) of the onsets of the [Ca¥]é and [Na¤]é

increases, suggest that the Ca¥ and Na¤ influxes probably

occur via LPCÏAA-activated non-selective cation or leak

channels. Further investigations are required to clarify this

issue.

Role of NMDAÏnon-NMDA channels, voltage-

sensitive or insensitive Ca¥ channels, the Na¸—Cafi

exchanger and Ca¥-ATPase in the large [Ca¥]é

increase in metabolic inhibition

Activation of NMDAÏnon-NMDA channels during hypoxia/

anoxia can result in an increased [Ca¥]é in hippocampal

neurons (Dubinsky & Rothman, 1991) and the cerebral

cortex (Uematsu et al. 1991). However, in cultured granule

cells, neither of these channels is involved in the calcium

increase seen following metabolic inhibition (Fig. 2 and

Table 2). Voltage-dependent and -independent Ca¥ channels

also seem not to be involved, since the specific blockers of

these channels had no inhibitory effect (Table 2). In the

present study, Ni¥, a known blocker of voltage-dependent

Ca¥ channels, non-selective leak channels (see Nilius et al.

1993 for review) and the Naº—Cafl exchanger (Du et al.

1997), was able to inhibit the metabolic inhibition-, LPC-,

AA- or melittin-induced Ca¥ increase (Figs 5 and 7 and

Table 2). On the basis of the above data, we speculate that

the blocking effect of the metal ions on the Ca¥ÏNa¤

response in granule cells is probably via non-selective cation

or leak channels; this point requires further investigation.

Na¸ exchange for Cafi, with the resultant Ca¥ overload,

was one possible explanation for the metabolic inhibition-

induced [Ca¥]é increase, since a marked [Na¤]é increase was

indeed observed. However, this possibility can be discounted,

since the use of Na¤-free medium (to block the Na¸—Cafi

exchanger, Fig. 3B) or K¤-free medium (to induce high

[Na¤]é, Fig. 3D) did not affect Ca¥ influx.

Another possibility was inhibition of the plasmalemmal

Ca¥-ATPase. This is also unlikely, since, in the presence of

pHï 8·5 or Eosin B (Fig. 4A and B and Table 2), the Ca¥

influx was augmented, suggesting that Ca¥-ATPase is still

active during early metabolic inhibition. The possibility that

the increase in [Ca¥]é, seen in Fig. 4A, was due to

extracellular alkalinization (pHï 8·5) and an increased

conductance of the calcium channels is also unlikely, since

specific calcium channel blockers had no effect on the

calcium influx (Table 2). It is intriguing to note that granule

cell Ca¥-ATPase was not inhibited during early metabolic

inhibition (ATPé, •0·35 mÒ, Ekholm et al. 1992); this is

probably because the Km of ATP for Ca¥-ATPase is low

(•0·03 mÒ, Caroni & Carafoli, 1980). However, another

possibility is that Ca¥-ATPase in granule cells may be not

so important for calcium extrusion, since the rate of calcium

recovery during wash-off of the metabolic inhibitors was

similar either in the presence or absence of Ca¥-ATPase

blockers (Fig. 4A and B). Therefore, other calcium extrusion

mechanisms (e.g. the mitochondria) may be more important

than Ca¥-ATPase during recovery from Ca¥ load.

Comparison between the mechanisms suggested in the

present work and those suggested by in vivo or brain

slice studies

In vivo studies and those involving brain slices have

demonstrated that the extracellular ion composition is

greatly altered during hypoxiaÏischaemia of the brain. For

example, a rapid depolarization of the membrane potential,

an increase in the concentration of extracellular K¤

(50—80 mÒ) and decreases in pHï and POµ have been observed

within 5—10 min of the onset of brain hypoxiaÏischaemia

(reviewed in Hansen, 1985). Many mechanisms have

therefore been suggested (see Introduction) to explain the

changes in [Ca¥]é and [Na¤]é seen in the brain during such

insult. In addition to these mechanisms, activation of

NMDAÏnon-NMDA channels, resulting from accumulation

of glutamate in the extracellular space, has recently been

demonstrated (for review see Choi, 1988; Uematsu et al.

1991).

The present study, using cultured cells, shows that PLAµ

activation plays an important role in the increases in [Ca¥]é

and [Na¤]é seen during metabolic inhibition. The conditions

are not entirely consistent with those in in vivo and slice

studies, as the extracellular ion composition is constant and

totally oxygen-free conditions are not achieved. This may

also explain the lack of involvement of the NMDAÏnon-

NMDA, voltage-dependent Ca¥ channels or involvement of

oxygen free radicals during the metabolic inhibition.

Moreover, the cultured neurons used may have very different

properties from cells in the intact brain, e.g. neonatal

(cultured) cells tolerate anoxia longer than adult cells, and

receptor or channel densities may change during the

culturing process. Because of these differences, the

possibility that activation of PLAµ may also be involved in

hypoxia/ischaemia-induced [Ca¥]éÏ[Na¤]é changes should be

investigated in vivo or in slices.

In summary, the present study shows that, during

metabolic inhibition, there is an initial small calcium release

from mitochondria, followed by a large influx of extracellular

Ca¥ and Na¤, the latter probably being mediated by

products, including fatty acids and free radicals, formed on

PLAµ activation. We have shown that the calcium extruding

mechanisms in granule cells appear to be at least partially

functioning during early metabolic inhibition. Complete

recovery to the resting [Ca¥]é seen following wash-off of

the metabolic inhibitors (Figs 1A and 8A) therefore

indicates that PLAµ can be reversibly activated by short-

term metabolic inhibition; this point requires further

investigation. The mechanism suggested in this study as an

explanation for the metabolic inhibition-induced Cafi and

Naº influxes differs from those suggested in in vivo and

slices studies on neurons; this may be due to the different

W.-H. Chen and others J. Physiol. 515.1144

working conditions used. However, as granule cells are the

most abundant neurons in the brain, the findings in the

present study may be important in the understanding of

the mechanisms involved in calcium and sodium overload

under metabolic inhibition. Inhibition of PLAµ activity

might have implications in preventing serious brain injury

and oedema during hypoxiaÏischaemia.

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Acknowledgements

The authors would like to thank the National Science Council for

financial support (NSC 87-2314-B002-190).

Corresponding author

M.-L. Wu: Institute of Physiology, College of Medicine, National

Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan,

Republic of China.

Email: mlw@ha.mc.ntu.edu.tw

W.-H. Chen and others J. Physiol. 515.1146