- Email: [email protected]
Lactate production and release in cultured astrocytes
Neuroscience Letters, 86 (1988) 296 300 Elsevier Scientific Publishers Ireland Ltd.
296
NSL 05226
Lactate production and release in cultured astrocytes Wotfgang Walz and Srimathie Mukerji Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Sask. (Canada) (Received 5 October 1987; Revised version received 11 December 1987; Accepted 14 December 1987)
Key word~: Acidosis; Astrocyte; Cytotoxic brain swelling; Edema in the brain; Glial cell; Lactate Intracellular lactate content and release of lactate into the surrounding medium of mouse astrocytes in primary culture was measured using the lactate dehydrogenase method. During culturing the cellular content of astrocytes decreased from 400 to 200 nmol/mg protein. The total lactate released into the extracellular space, however, amounted to 75,000 nmot/mg within 98 h, corresponding to a lactate concentration of I0 mM in the cell culture dish. In another set of experiments, cytotoxic swelling was evoked by exposure of the cells to 60 mM K +, this situation caused a 40% increase in cellular volume and an increase in the KCI content of astrocytes. Within 3 h of a change to 60 mM K * the intracellular lactate content was increased by 100 nmol/mg (one third) and the lactate release in the extracellular space by about 2000 nmol/mg (twice as high as during exposure to 3 mM K+). However, due to the increased intracellular water content, the lactate concentration inside the cells remained unchanged. It is concluded that astrocytes produce substantial amounts of additional lactate during cytotoxic swelling. This lactate, however, is not increasing the intracellular osmolarity and most of the lactate is released into the extracellular space. Depending on the transmembrane transport mechanism it could have the capability to decrease the strong ion difference and contribute to acid shifts in the extracellular space.
Lactic acid production is one of the most favored theories in explaining cell damage of brain cells during anoxia, ischemia and hyperglycemia [2, 10]. Especially astrocytes, which contain more glycogen than neurons are suspected to produce lactate from these glycogen residues when the supply of oxygen and glucose is decreased [3]. During increased glucose supply in hyperglycemia substantial acid shifts are measured and lactate production is one of the prime suspects [11]. It is assumed from morphological studies [10] that as soon as the lactate concentration in the tissue during severe incomplete ischemia exceeds 16 raM, the tissue is irreversibly damaged. During focal ischemia where infarction can also occur lactate reaches higher levels [8]. In this study we investigate the lactate production and release into the surrounding medium of mouse astrocytes in primary cultures. An increase of extracellular K +
Correspondence: W. Walz, Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Sask., Canada S7N 0W0. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
297
to 60 mM evokes ion shifts and swelling of astrocytes, which resemble cytotoxic swelling [12-14]. This situation can be used as a simple model to study edema on a cellular basis. We investigated the lactate production and release of astrocytes in this experimental model system. In a first step we were interested in lactate production of astrocytes in the growth medium under normal culture conditions. Astrocytes were cultured as described by Hertz et al. [1] in minimal essential medium (MEM) growth medium. The growth medium contains 7.5 mM glucose. For the first week the horse serum concentration was 20%. Thereafter a 10% serum concentration was used. The cells were kept in 60 mm diameter culture dishes with 3 ml MEM medium. Twice a week the medium was exchanged with a fresh one ('feeding'). After 14 days the cultures are confluent monolayers and after 3 weeks they were usually used for physiological experiments. For measuring lactate content the cells were washed with salt solution (containing the same ion and glucose composition as growth medium) for 3 times (lasting about 10 s). The cells were scraped into salt solution, the contents of 4-5 culture dishes were pooled together. Aliquots of 200/~1 of the cell suspension were taken out and added to 400/ll of ice-cold 8% perchloric acid. The solution was then centrifuged, the pellet was assayed for protein contents with the method of Lowry et al. [7] and the supernatant used for lactate determination. For this determination the Sigma technique was used, which uses excess nicotinamide adenine dinucleotide ( N A D ) and lactate dehydrogenase which will form pyruvate. Newly formed pyruvate was trapped with hydrazine. Thus, N A D H formation becomes a measure of lactate originally present. This formation was measured spectrophotometrically with a Spectronic 601 instruA
B 5O0
30,000
6
~
6
tu
400
lO
25,0000 ul
20,000
,
~
~ ~ ~5
15,000
10,000
J
J
z 0
300
~oo
5
~ ~
E ~c
100
D J
5,000
w 0
0 E
,
10 TIME AFTER
i
,
,
,
,
50 FEEDING
I
100
(hrll)
I
I
I
J
0
10
20
30
'~ × u~
AGE (days)
Fig. 1. A: lactate accumulation in the growth medium of 21-day-old astrocytes after feeding (at time 0). The lactate content is given as per culture dish, which contains 3 ml medium and approximately 0.4 mg protein (n - I3 16, S.E.M. are given). B: accumulation of lactate during the culturing of astrocytes in primary culture. The lactate content was measured at different ages (3 29 days) 48 h after the last feeding. The lactate content of the cells (nmol/mg; filled circles) and the lactate concentration (mM; unfilled circles) of the growth medium in the culture dish was measured separately approximately 48 h after the last feeding ( n - 3 6, S.E.M. are given).
E ~
298 ment. For measuring lactate in the fluid surrounding the cells 200/A samples of the fluid in the cell cultures were taken out o f the cell cultures before the washing procedures and assayed in the same way. Fig. IA shows the results of a first experimental series to measure lactate released by 3-week-old astrocytes within the 4-90 h following feeding. As can be seen lactate is consistently released from the astrocytes within the first day after feeding at about a rate of 1000 nmol/mg.h. This is the same magnitude of release as found by other groups [6, 9]. The final plateau value reached after 3 days is about 30,000 nmol per culture dish or 75,000 nmol/mg. This corresponds to a concentration of 10 mM of lactate in a culture that was not fed for 3--4 days. Fig. I B shows the results of a second experimental series undertaken to measure cellular content and extracellular release during development in culture. The samples were taken out approximately 48 h after feeding. The cellular content of lactate actually declines from about 400 nmol/mg after 22 days. The lactate concentration in the growth medium increases continuously from about 1.5 m M to about 8 mM at day 14 and stays then relatively stable. At 22 days the concentration of 7.5 mM corresponds to a lactate production of 68,000 nmol/mg which is released into the growth medium, whereas the cellular content is 157 nmol/mg. This means during the growth in culture lactate is almost exclusively released into the surrounding growth medium till it reaches a concentration of about 8 raM, where the lactate content stabilizes. This might be due to the use of lactate as energy fuel [ 15]. Quite obviously astrocytes have the tendency to shuttle almost all of their newly synthesized lactate across the membrane into the extracellular space. They are able to tolerate concentrations of at least 10 mM. It is now of considerable interest to investigate the lactate production and release of astrocytes in situations where they show cytotoxic swelling. It would be important to determine if in this situation (1) intracellular lactate accumulation increases the osmolarity of astrocytes to contribute to the increased water uptake, and if (2) lactate is released by astrocytes during cytotoxic swelling and the lactate concentration o f the extracellular space increases and leads to an acid shift of the neuronal microenvironment. Recently, we developed an experimental system [13, 14] by which the astrocytes are exposed from their normal 3 m M K ÷ concentration to 60 m M K ÷ (leaving the sum of the Na ÷ and K ÷ concentration constant by reducing the Na ÷ concentration). The cells swell within 2 min and double their K ÷ and triple their CI- contents. In anoxia, ischemia and hypoglycemia an increase of extracellular K ÷ between 40 and 80 m M accompanies cytotoxic brain edema. We therefore think that this phenomenon evoked by high external K ÷ is a relatively simple model for cytotoxic events in vivo. We exposed astrocytes for various times (2 min to 3 h) to salt solutions containing either 3 or 60 mM and analyzed the cellular lactate content and the lactate release into the surrounding salt solution. Fig. 2A shows the lactate content of the cells. At 3 mM K ÷ there is a slight increase from around 150 nmol/mg to 220 nmol/mg in 3 h solution. At 60 mM K ÷ the values increase between 15 and 45 min to 260 nmol/mg and stay elevated at about 320 nmol/mg at 3 h. From the measured volume of the astrocytes in these experimental situations, one can calculate the approximate intracellular lactate concentration. Despite the small increase in lactate content at 60 mM the lactate concentration is
299 A
400
-
300
-
200
-
6 0 mM
× E E
LU
0 tO
100-
,<
0
i 0
,< .J
5FO
i 100
I 150
(minutes)
TIME
5000t
60 mM ×
(~
4000-
E E
v
w
J
3000/
/
z
/
/
/
/
/
2000-
1000-
7
I
0
510
I
100 rIME
1
;
0
(minutes)
Fig. 2. A: time course of changes in the cellular content of lactate (nmol/mg) after cells were washed 3 times with 3 mM K + salt solution and then exposed to either 3 or 60 mM K + salt solution for up to 3 h (n = 4 6, S.E.M. are given). B: time course of lactate accumulation into the salt solution (in nmol/mg) during exposure to either 3 or 60 mM K + salt solution. Experiments were the same as in A.
virtually unchanged, due to the v o l u m e increase at 60 raM: a lactate concentration o f approximately 35 m M can be calculated for 3 and 60 m M K + [12]. Fig. 2B shows the a c c u m u l a t i o n o f lactate outside the cells in the salt solution. As can be seen the m a x i m a l release o f 2000 n m o l / m g within about 2 h in 3 m M salt solution is about doubled in salt solution c o n t a i n i n g 60 m M K +. These experiments together show clearly that 60 m M K + evokes an increased K + production, but almost all o f it is released into the extracellular fluid. A remaining problem is why the lactate production in 3 and 60 m M K + is not different after 15 min o f exposure to salt solution when all o f the ion m o v e m e n t s and most o f the water m o v e m e n t s causing cytotoxic swelling like p h e n o m e n a are already terminated (see ref. ! 3). The results demonstrate that the increased lactate production is a delayed p h e n o m e n o n . The initial entry of KCI is probably under D o n n a n type forces and it is therefore not energy dependent
300 a n d thus a n initial increased energy p r o d u c t i o n is n o t obligatory [14]. Therefore, lactate p r o d u c t i o n p r e s u m a b l y reflects increased cell m e t a b o l i s m secondary to K ÷ entry, m e m b r a n e d e p o l a r i z a t i o n a n d swelling. Clearly it can be said that lactate changes alone are likely to provide insufficient osmotic drive to result in swelling. D e p e n d i n g o n the t r a n s m e m b r a n e m e c h a n i s m o f lactate release into the ECS, released lactate could possibly change the strong ion difference a n d c o n t r i b u t e to extracellular acidification [4, 5]. W . W . is a scholar of the Medical Research C o u n c i l o f C a n a d a . O p e r a t i n g funds were provided by the M R C o f C a n a d a . 1 Hertz, L., Juurlink, B.H.J., Szuchet, S. and Walz, W., Cell and tissue culture. In A.A. Boulton and G.B. Baker (Eds.), Neuromethods, Vol. I, Humana, Clifton, 1985, pp. 117-167. 2 Kimelberg, H.K. and Ransom, B.R., Physiological and pathological aspects of astrocytic swelling. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 3, Academic, Orlando, 1986, pp. 129-166. 3 Klatzo, I., Pathophysiologic aspects of cerebral ischemia. In D.W. Tower (Ed.), The Nervous System, Vol. 1, Raven, New York, 1975, pp. 313-322. 4 Kraig, R.P., Ferreira-Filho, C.R. and Nicholson, C., Alkaline and acid transients in the cerebellar microenvironment, J. Neurophysiol., 49 (1983) 831-849. 5 Kraig, R.P., Pulsinelli, W.A. and Plum, F., Hydrogen ion buffering during complete brain ischemia, Brain Res., 342 (1985) 281--290. 6 Lopes-Cardozo, M., Larsson, O.M. and Schousboe, A., Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures and neurons from mouse cerebral cortex, J. Neurochem., 46 (1986) 773-778. 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 8 Norenberg, M.D., Mozes, L.W., Gregorios, J.B. and Norenberg, L.O., Effects of lactic acid on astrocytes in primary cultures, J. Neuropathol. Exp. Neuro., 46 (1987) 154-166. 9 Pauwels, P.J., Opperdoes, F.R. and Trouet, A., Effects of antimycin, glucose deprivation, and serum on cultures of neurons, astrocytes, and neuroblastoma cells, J. Neurochem., 44 (t985) 143-148. 10 Plum, F., What causes infarction in ischemic brain?, Neurology, 33 (1983) 222-233. 11 Siesjo, B.K. and Wieloch, T., Cerebral metabolism in ischemia: neurochemical basis for therapy, Br. J. Anaesthesiol., 57 (1985) 47~i2. 12 Walz, W., Swellingand potassium uptake in cultured astrocytes, Can. J. Physiol. Pharmacol., 65 (1987) 1051-1057. 13 Walz, W., The role of potassium in cytotoxic brain edema. In M.D. Norenberg (Ed.), Biochemical Pathology of Astrocytes, Liss, New York, in press. 14 Walz, W. and Mukerji, S., KC1 movements during potassium-induced cytotoxic swelling of cultured astrocytes, Exp. Neurol., 99 (1988) 17- 29. 15 Yu, A.C.H. and Hertz, L., Metabolic sources of energy in astrocytes. In L. Hertz et al. (Eds.), Glutamate, Glutamate and GABA in the Central Nervous System, Liss, New York, 1983, pp: 431-438.
296
NSL 05226
Lactate production and release in cultured astrocytes Wotfgang Walz and Srimathie Mukerji Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Sask. (Canada) (Received 5 October 1987; Revised version received 11 December 1987; Accepted 14 December 1987)
Key word~: Acidosis; Astrocyte; Cytotoxic brain swelling; Edema in the brain; Glial cell; Lactate Intracellular lactate content and release of lactate into the surrounding medium of mouse astrocytes in primary culture was measured using the lactate dehydrogenase method. During culturing the cellular content of astrocytes decreased from 400 to 200 nmol/mg protein. The total lactate released into the extracellular space, however, amounted to 75,000 nmot/mg within 98 h, corresponding to a lactate concentration of I0 mM in the cell culture dish. In another set of experiments, cytotoxic swelling was evoked by exposure of the cells to 60 mM K +, this situation caused a 40% increase in cellular volume and an increase in the KCI content of astrocytes. Within 3 h of a change to 60 mM K * the intracellular lactate content was increased by 100 nmol/mg (one third) and the lactate release in the extracellular space by about 2000 nmol/mg (twice as high as during exposure to 3 mM K+). However, due to the increased intracellular water content, the lactate concentration inside the cells remained unchanged. It is concluded that astrocytes produce substantial amounts of additional lactate during cytotoxic swelling. This lactate, however, is not increasing the intracellular osmolarity and most of the lactate is released into the extracellular space. Depending on the transmembrane transport mechanism it could have the capability to decrease the strong ion difference and contribute to acid shifts in the extracellular space.
Lactic acid production is one of the most favored theories in explaining cell damage of brain cells during anoxia, ischemia and hyperglycemia [2, 10]. Especially astrocytes, which contain more glycogen than neurons are suspected to produce lactate from these glycogen residues when the supply of oxygen and glucose is decreased [3]. During increased glucose supply in hyperglycemia substantial acid shifts are measured and lactate production is one of the prime suspects [11]. It is assumed from morphological studies [10] that as soon as the lactate concentration in the tissue during severe incomplete ischemia exceeds 16 raM, the tissue is irreversibly damaged. During focal ischemia where infarction can also occur lactate reaches higher levels [8]. In this study we investigate the lactate production and release into the surrounding medium of mouse astrocytes in primary cultures. An increase of extracellular K +
Correspondence: W. Walz, Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Sask., Canada S7N 0W0. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
297
to 60 mM evokes ion shifts and swelling of astrocytes, which resemble cytotoxic swelling [12-14]. This situation can be used as a simple model to study edema on a cellular basis. We investigated the lactate production and release of astrocytes in this experimental model system. In a first step we were interested in lactate production of astrocytes in the growth medium under normal culture conditions. Astrocytes were cultured as described by Hertz et al. [1] in minimal essential medium (MEM) growth medium. The growth medium contains 7.5 mM glucose. For the first week the horse serum concentration was 20%. Thereafter a 10% serum concentration was used. The cells were kept in 60 mm diameter culture dishes with 3 ml MEM medium. Twice a week the medium was exchanged with a fresh one ('feeding'). After 14 days the cultures are confluent monolayers and after 3 weeks they were usually used for physiological experiments. For measuring lactate content the cells were washed with salt solution (containing the same ion and glucose composition as growth medium) for 3 times (lasting about 10 s). The cells were scraped into salt solution, the contents of 4-5 culture dishes were pooled together. Aliquots of 200/~1 of the cell suspension were taken out and added to 400/ll of ice-cold 8% perchloric acid. The solution was then centrifuged, the pellet was assayed for protein contents with the method of Lowry et al. [7] and the supernatant used for lactate determination. For this determination the Sigma technique was used, which uses excess nicotinamide adenine dinucleotide ( N A D ) and lactate dehydrogenase which will form pyruvate. Newly formed pyruvate was trapped with hydrazine. Thus, N A D H formation becomes a measure of lactate originally present. This formation was measured spectrophotometrically with a Spectronic 601 instruA
B 5O0
30,000
6
~
6
tu
400
lO
25,0000 ul
20,000
,
~
~ ~ ~5
15,000
10,000
J
J
z 0
300
~oo
5
~ ~
E ~c
100
D J
5,000
w 0
0 E
,
10 TIME AFTER
i
,
,
,
,
50 FEEDING
I
100
(hrll)
I
I
I
J
0
10
20
30
'~ × u~
AGE (days)
Fig. 1. A: lactate accumulation in the growth medium of 21-day-old astrocytes after feeding (at time 0). The lactate content is given as per culture dish, which contains 3 ml medium and approximately 0.4 mg protein (n - I3 16, S.E.M. are given). B: accumulation of lactate during the culturing of astrocytes in primary culture. The lactate content was measured at different ages (3 29 days) 48 h after the last feeding. The lactate content of the cells (nmol/mg; filled circles) and the lactate concentration (mM; unfilled circles) of the growth medium in the culture dish was measured separately approximately 48 h after the last feeding ( n - 3 6, S.E.M. are given).
E ~
298 ment. For measuring lactate in the fluid surrounding the cells 200/A samples of the fluid in the cell cultures were taken out o f the cell cultures before the washing procedures and assayed in the same way. Fig. IA shows the results of a first experimental series to measure lactate released by 3-week-old astrocytes within the 4-90 h following feeding. As can be seen lactate is consistently released from the astrocytes within the first day after feeding at about a rate of 1000 nmol/mg.h. This is the same magnitude of release as found by other groups [6, 9]. The final plateau value reached after 3 days is about 30,000 nmol per culture dish or 75,000 nmol/mg. This corresponds to a concentration of 10 mM of lactate in a culture that was not fed for 3--4 days. Fig. I B shows the results of a second experimental series undertaken to measure cellular content and extracellular release during development in culture. The samples were taken out approximately 48 h after feeding. The cellular content of lactate actually declines from about 400 nmol/mg after 22 days. The lactate concentration in the growth medium increases continuously from about 1.5 m M to about 8 mM at day 14 and stays then relatively stable. At 22 days the concentration of 7.5 mM corresponds to a lactate production of 68,000 nmol/mg which is released into the growth medium, whereas the cellular content is 157 nmol/mg. This means during the growth in culture lactate is almost exclusively released into the surrounding growth medium till it reaches a concentration of about 8 raM, where the lactate content stabilizes. This might be due to the use of lactate as energy fuel [ 15]. Quite obviously astrocytes have the tendency to shuttle almost all of their newly synthesized lactate across the membrane into the extracellular space. They are able to tolerate concentrations of at least 10 mM. It is now of considerable interest to investigate the lactate production and release of astrocytes in situations where they show cytotoxic swelling. It would be important to determine if in this situation (1) intracellular lactate accumulation increases the osmolarity of astrocytes to contribute to the increased water uptake, and if (2) lactate is released by astrocytes during cytotoxic swelling and the lactate concentration o f the extracellular space increases and leads to an acid shift of the neuronal microenvironment. Recently, we developed an experimental system [13, 14] by which the astrocytes are exposed from their normal 3 m M K ÷ concentration to 60 m M K ÷ (leaving the sum of the Na ÷ and K ÷ concentration constant by reducing the Na ÷ concentration). The cells swell within 2 min and double their K ÷ and triple their CI- contents. In anoxia, ischemia and hypoglycemia an increase of extracellular K ÷ between 40 and 80 m M accompanies cytotoxic brain edema. We therefore think that this phenomenon evoked by high external K ÷ is a relatively simple model for cytotoxic events in vivo. We exposed astrocytes for various times (2 min to 3 h) to salt solutions containing either 3 or 60 mM and analyzed the cellular lactate content and the lactate release into the surrounding salt solution. Fig. 2A shows the lactate content of the cells. At 3 mM K ÷ there is a slight increase from around 150 nmol/mg to 220 nmol/mg in 3 h solution. At 60 mM K ÷ the values increase between 15 and 45 min to 260 nmol/mg and stay elevated at about 320 nmol/mg at 3 h. From the measured volume of the astrocytes in these experimental situations, one can calculate the approximate intracellular lactate concentration. Despite the small increase in lactate content at 60 mM the lactate concentration is
299 A
400
-
300
-
200
-
6 0 mM
× E E
LU
0 tO
100-
,<
0
i 0
,< .J
5FO
i 100
I 150
(minutes)
TIME
5000t
60 mM ×
(~
4000-
E E
v
w
J
3000/
/
z
/
/
/
/
/
2000-
1000-
7
I
0
510
I
100 rIME
1
;
0
(minutes)
Fig. 2. A: time course of changes in the cellular content of lactate (nmol/mg) after cells were washed 3 times with 3 mM K + salt solution and then exposed to either 3 or 60 mM K + salt solution for up to 3 h (n = 4 6, S.E.M. are given). B: time course of lactate accumulation into the salt solution (in nmol/mg) during exposure to either 3 or 60 mM K + salt solution. Experiments were the same as in A.
virtually unchanged, due to the v o l u m e increase at 60 raM: a lactate concentration o f approximately 35 m M can be calculated for 3 and 60 m M K + [12]. Fig. 2B shows the a c c u m u l a t i o n o f lactate outside the cells in the salt solution. As can be seen the m a x i m a l release o f 2000 n m o l / m g within about 2 h in 3 m M salt solution is about doubled in salt solution c o n t a i n i n g 60 m M K +. These experiments together show clearly that 60 m M K + evokes an increased K + production, but almost all o f it is released into the extracellular fluid. A remaining problem is why the lactate production in 3 and 60 m M K + is not different after 15 min o f exposure to salt solution when all o f the ion m o v e m e n t s and most o f the water m o v e m e n t s causing cytotoxic swelling like p h e n o m e n a are already terminated (see ref. ! 3). The results demonstrate that the increased lactate production is a delayed p h e n o m e n o n . The initial entry of KCI is probably under D o n n a n type forces and it is therefore not energy dependent
300 a n d thus a n initial increased energy p r o d u c t i o n is n o t obligatory [14]. Therefore, lactate p r o d u c t i o n p r e s u m a b l y reflects increased cell m e t a b o l i s m secondary to K ÷ entry, m e m b r a n e d e p o l a r i z a t i o n a n d swelling. Clearly it can be said that lactate changes alone are likely to provide insufficient osmotic drive to result in swelling. D e p e n d i n g o n the t r a n s m e m b r a n e m e c h a n i s m o f lactate release into the ECS, released lactate could possibly change the strong ion difference a n d c o n t r i b u t e to extracellular acidification [4, 5]. W . W . is a scholar of the Medical Research C o u n c i l o f C a n a d a . O p e r a t i n g funds were provided by the M R C o f C a n a d a . 1 Hertz, L., Juurlink, B.H.J., Szuchet, S. and Walz, W., Cell and tissue culture. In A.A. Boulton and G.B. Baker (Eds.), Neuromethods, Vol. I, Humana, Clifton, 1985, pp. 117-167. 2 Kimelberg, H.K. and Ransom, B.R., Physiological and pathological aspects of astrocytic swelling. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. 3, Academic, Orlando, 1986, pp. 129-166. 3 Klatzo, I., Pathophysiologic aspects of cerebral ischemia. In D.W. Tower (Ed.), The Nervous System, Vol. 1, Raven, New York, 1975, pp. 313-322. 4 Kraig, R.P., Ferreira-Filho, C.R. and Nicholson, C., Alkaline and acid transients in the cerebellar microenvironment, J. Neurophysiol., 49 (1983) 831-849. 5 Kraig, R.P., Pulsinelli, W.A. and Plum, F., Hydrogen ion buffering during complete brain ischemia, Brain Res., 342 (1985) 281--290. 6 Lopes-Cardozo, M., Larsson, O.M. and Schousboe, A., Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures and neurons from mouse cerebral cortex, J. Neurochem., 46 (1986) 773-778. 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 8 Norenberg, M.D., Mozes, L.W., Gregorios, J.B. and Norenberg, L.O., Effects of lactic acid on astrocytes in primary cultures, J. Neuropathol. Exp. Neuro., 46 (1987) 154-166. 9 Pauwels, P.J., Opperdoes, F.R. and Trouet, A., Effects of antimycin, glucose deprivation, and serum on cultures of neurons, astrocytes, and neuroblastoma cells, J. Neurochem., 44 (t985) 143-148. 10 Plum, F., What causes infarction in ischemic brain?, Neurology, 33 (1983) 222-233. 11 Siesjo, B.K. and Wieloch, T., Cerebral metabolism in ischemia: neurochemical basis for therapy, Br. J. Anaesthesiol., 57 (1985) 47~i2. 12 Walz, W., Swellingand potassium uptake in cultured astrocytes, Can. J. Physiol. Pharmacol., 65 (1987) 1051-1057. 13 Walz, W., The role of potassium in cytotoxic brain edema. In M.D. Norenberg (Ed.), Biochemical Pathology of Astrocytes, Liss, New York, in press. 14 Walz, W. and Mukerji, S., KC1 movements during potassium-induced cytotoxic swelling of cultured astrocytes, Exp. Neurol., 99 (1988) 17- 29. 15 Yu, A.C.H. and Hertz, L., Metabolic sources of energy in astrocytes. In L. Hertz et al. (Eds.), Glutamate, Glutamate and GABA in the Central Nervous System, Liss, New York, 1983, pp: 431-438.