Brain Research, 86 (1975) 499-503 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

499

Influence of potassium ions on accumulation and metabolism of ['4C]glucose by glial cells

RIFAAT D. SALEM, RICHARD HAMMERSCHLAG, HUMBERTO BRACHO AND RICHARD K. ORKAND* Department of Biology (R.D.S., H.B., R.K.O.), University of California at Los Angeles, Los Angeles, Calif. 90024 and Division of Neurosciences ( R.H.), City of Hope National Medical Center, Duarte, Calif. 91010 (U.S.A.)

(Accepted December 10th, 1974)

Impulse activity in neurons leads to an accumulation of potassium ions in the narrow intercellular spaces between neurons and gliaS, 13, and produces a depolarization of the glial membrane4,11,12,14. Thus, the level of potassium in the intercellular fluid is a function of impulse activity and could play a role in coordinating activity in neurons and glia 9. Recently, it has been demonstrated with the aid of fluorimetric techniques that increases in extracellular potassium, within the range that occurs during nervous activity, can affect the level of reduced pyridine nucleotides in isolated glial cells 1°. These results were observed in the optic nerve of Necturus maculosus, 2 months following surgical removal of the eye. After this time the axons have degenerated and the nerve consists solely of astrocytes that appear normal by cytological 1° and physiological a criteria. The present report describes additional potassium-induced metabolic alterations in 'all-glia nerves', as detected during uptake and metabolism of [14C]glucose in the presence of increased extracellular potassium. Eyeballs of Necturus maculosus were removed under cold anesthesia, and animals were maintained for 2 months to allow axon degeneration. Animals were then decapitated and the intracranial segment of optic nerve trunk (approximately 3 mm) was freed from capillaries and pia arachnoid and removed. Similar segments of nerve trunks were also removed from normal animals. Aliquots (1.5 #Ci) of [U-14C]glucose, 196 mCi/mmole (New England Nuclear Corp., Boston, Mass.) were dried in a vacuum centrifuge and redissolved in 15 #1 of normal or high K +, glucose-free Ringer solution to give a final glucose concentration o f 0.5 mM. Incubation solutions were transferred to 40 #1 wells drilled into a perspex block, and 1/tl aliquots were taken for determination of initial radioactivity. Matched numbers of nerve segments (2 or 3) were placed in each well and a glass cover slip was placed over the wells. After incubation at 20 °C for 5-60 min the nerves were rinsed * Present address • Department of Physiology and Pharmacology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pa. 19104, U.S.A.

500 TABLE I RATIOS OF TISSUERADIOACTIVITYIN OPTIC NERVESINCUBATEDIN HIGH FOTASSIUM-MEDIUM(15 m M K +) RELATIVETO NORMALMEDIUM(3 m M K +) Following incubation for 30 min at 20 °C, tissue was digested in 1 N NaOH. The digest was diluted to 0.5 ml, neutralized with HCI, and passed through 0.3 ml settled bed volumes of the cation-exchange resin AG-50-WX8 (Na+-form) packed in disposable pasteur pipets. Initial eluates, pooled with eluates from a 1 ml H20 wash of the resin, were designated : organic acid and neutral fraction. The amino acid fraction was eluted from the resin beds with 1 ml 3 M NHaOH. Each fraction was concentrated in a vacuum centrifuge and subjected to paper electrophoresis at pH 4.1 (0.2 M pyridine-acetate, 2.5 h/800 V) for separation of amino acids; pH 1.9 (0.5 M acetate-formate 3 h/800 V) for separation of organic acids; or thin layer chromatography on cellulose (BuOH-HOAc-H20, 3:1:1). Tissue radioactivity ratio All-glia nerves

Total radioactivity Amino acid fraction Organic acid and neutral fraction

1.71 -~ 0.13 (13)* 1.58 i 0.15 (6) 1.36 ~ 0.07 (6)

N o r m a l nerves

Total radioactivity

1.91 ± 0.20 (4)

* Values represent mean ± S.E.M.; number of experiments shown in parentheses. In each experiment 2 or 3 sets of nerves were compared. All ratios were significantly different from unity (P < 0.01).

for 30-40 sec through several 100 #1 pools of non-radioactive Ringer solution, transferred to microtubes containing 50 #1 1 N NaOH, and incubated for 30 min at 50 °C to dissolve the tissue. Total tissue radioactivity was determined in 3 #1 aliquots of the NaOH digest, with 10 ml Bray's fluid 2 used as scintillant. The remaining NaOH digest was neutralized with HCI, and separated into amino acid and organic acid fractions by ion exchange chromatography by methods detailed in the legend to Table I. Ringer solution for nerve incubation had the following composition (mM): NaCI (110); KCI (3); CaCI2 (2); Tris-maleate (5); pH 7.4. Solutions with increased K + concentrations were prepared by substituting NaCI by KC1. Initial experiments carried out to determine the time-course of uptake of [14C]glucose into the all-glia nerves showed that total radioactivity in tissue exposed to 15 m M K+-medium was approximately 70 ~ greater than that in tissue incubated in normal medium (Table I). Experiments using normal optic nerves (axons and glia) showed a similar stimulation of [14C]glucose uptake by high K + (Table I). The increased uptake of [14C]glucose by the all-glia nerves was abolished when the nerves were disrupted by immersing them in distilled water at 60 °C for 10 min. The distribution of radioactivity in Krebs' cycle intermediates (organic acids) and in amino acids was examined to determine if high K ÷ levels might also be affecting the metabolism of [~4C]glucose taken up into the all-glia tissue. Total radioactivity in the organic acid and neutral fraction prepared from tissue exposed to high K+-medium was significantly increased over radioactivity in tissue incubated in normal medium (Table I). However, no selective effects by high K ÷ on organic acid metabolism were revealed by comparing percentages of radioactivity in individual components of this

501 50c~

40-

..1-1

~

.~ 20"

~.~ ~g

[•

10-

radioactivity ratio high k+:normal

unknown

succinate

1.62' (0.16)

1.42 ~" (0.24)

~ citrate

1.51 ~ (0.08)

, Z , I,n

a-keto glutarate 1.19 ~ (0.09)

oxalacetate 1.40 ~' (0.25)

I I

I I~"1

pyruvate I.:59 ,e" (0.36)

Fig. 1. Radioactive organic acid components of all glia optic nerves following incubation (30 min, 20 °C) with lt4C]glucose in normal (open columns), and 15 mMK ÷ (hatched columns) media. Bars show percentage means of 6 experiments; S.E.M. indicated on each bar. See legend, Table I for conditions of electrophoretic separation. The unidentified band migrated between the origin and succinate. Values below abscissa represent mean ratios of radioactivity for each component, between the 2 incubation conditions; S.E.M. shown within parentheses, t P < 0.05 when ratio was tested for significance of difference from unity, o Ratio not significantly different from unity.

fraction following electrophoretic separation. Approximately 60 70 of the radioactivity in tissue incubated in either normal or high K+-medium was accounted for by 2 bands with mobilities similar to citrate and succinate, and by a third unidentified band migrating between the origin and succinate. Bands corresponding to a-ketoglutarate, pyruvate and oxaloacetate accounted for another 5 % of the total radioactivity (Fig. 1). The radioactivity ratios for each component (counts/min in high K+-medium - counts/min in normal medium) were all within a similar range, and only citrate and the unknown component showed ratios significantly different from unity (Fig. 1, below abscissa). Total radioactivity i n the amino acid fraction of tissue exposed to 15 m M K +medium also increased relative to that in tissue incubated in normal medium (Table 1). Four main bands of radioactivity were obtained when this fraction was subjectcd to electrophoresis at p H 4.1: (i) 'neutral' amino acids (comprising greater than 90 % glutamine and alanine as determined by thin-layer chromatography); (ii) glutamate; (iii) aspartate; and (iv) a band migrating more anionic than aspartate. Wl'~en percentages of total amino acid radioactivity in each band were compared for tissues exposed to normal and high K+-medium the neutral band and glutamate showed slight decreases, while the unknown band showed a significant increase (Fig. 2). Comparisons of the radioactivity ratios showed that all 4 bands obtained from tissue exposed to high K +medium reflected stimulation of metabolism. But, unlike the effect on organic acids, the K + effect on the amino acid components occurred to markedly differing extents,

502 50-

.E

40J

~. 30-6o 2o-

L

10

L./1 radioactivity ratio high k + : normal

J

J

J

J

J

J

J

..-"1

J

neutralS

glutamate

asportate

unknown

1.26 ° (0.24)

1.42 $ (0. 1.5)

1.96 ¢ (0.2.5)

2.85 ~ (0.83)

Fig. 2. Radioactive amino acid components of all-glia optic nerves. For incubation conditions, details of electrophoresis, and explanation of symbols, see legends to Table I and Fig. 1. The unidentified band migrated as more anionic than aspartate. Radioactivity ratios shown below abscissa are explained in legend to Fig. 1. t P < 0.05.

being slight for the neutral amino acids, and pronounced for glutamate, aspartate, and the unknown component (Fig. 2, below abscissa). To assess whether the observed effects of high K+-medium might be due in any degree to the lowered Na + levels 6, 2 experiments were carried out in which all-glia tissues were incubated in [14C]glucose in normal medium, and in a medium with normal K +, 95 m M Na +, and sucrose added to maintain isotonicity. No differences were observed either in total tissue radioactivity or in radioactivity of amino acid or organic acid fractions, between the samples incubated in normal and in low Na+-media. The present studies, utilizing a pure glia optic nerve preparation, demonstrate metabolic changes in these cells in response to increased extracellular K +-at a concentration similar to that reached in intercellular clefts during normal neuronal activity. The main effect of the high K+-medium was to increase the tissue accumulation of radioactivity from [14C]glucose relative to that in tissue exposed to normal medium. Since this effect occurred to a similar magnitude in all-gtia nerve segments as in normal nerve segments containing axons and glia, it appears that the increased K + m a y be selectively affecting glial cells. The observed tissue radioactivity is likely to be predominantly in intracellular compartments: electrophysiological studies have determined that the half-time of washout of K + from extracellular compartments in N e c t u rus optic nerve is 6 sec (Bracho et al., unpublished observations) so that washing the tissue after the incubation period in isotope-free medium for 5 half-times should have removed 90-95 % of extracellular radioactivity. The extensive flow of radioactivity

503 through citric acid cycle intermediates to amino acids more directly indicates an intracellular accumulation of [14C]glucose. An additional effect of increased extracellular K + is suggested by the analyses of radioactivity in the amino acid components; following electrophoresis an unknown compound (that is likely an anionic amino compound) showed a marked increase in radioactivity to an extent not seen in the other amino acid components. The increased radioactivity in the organic acids in high K+-medium appeared to be largely secondary to the enhanced accumulation of [14C]glucose since the increase was similar for all the components analyzed. These observations of increased accumulation and metabolism of [14C]glucose in astrocytes in response to K+-enriched external medium are in accord with several indices of K+-induced metabolic alterations described in preparations of mammalian glial cells: the potassium-induced decrease in levels of ATP 15 and increase in oxygen consumption 1,7,s, will need to be studied concomitantly with pyridine nucleotide levels 1° and glucose accumulation, to further clarify the metabolic as well as the functional role of K + as a possible mediator of neuron-glia interaction. This work was supported by U.S. Public Health Service Research Grants NS08346 (R.K.O.) and NS-09885 (R.H.), and by PHS Training Grant NS-05670 (H.B.).

1 ALEKSIDZE,N. G., AND BLOMSTRAND,C., Influence of potassium ions on the respiration of the neuron and the neuroglia of the lateral vestibular nucleus of the rabbit, Proc. Acad. Sci. U.S.S.R., Biochem. Set., 186 (1969) 140-141. 2 BRAY,G. A., A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter, Analyt. Biochem., 1 (1960) 279-285. 3 COHEN, i . W., The contribution by glial cells to surface recordings from the optic nerve of an amphibian, J. Physiol. (Lond.), 210 (1970) 565-580. 4 DENNIS,i . J., AND GERSCHENFELD,H. i . , Some physiological properties of identified mammalian neuroglial cells, J. Physiol. (Lond.), 203 (1969) 211-222. 5 FRANKENHAEUSER,B., AND HODGKIN, A. L., The after-effects of impulses in the giant nerve fibres of Loligo, J. Physiol. (Lond.), 131 (1956) 341-376. 6 FRIEDE,R. L., The enzymatic response of astrocytes to various ions in vitro, J. Cell Biol., 20 (1964) 5-15. 7 HERTZ, L., Neuroglial localization of potassium and sodium effects on respiration in brain, J. Neuroehem., 13 (1966) 1373-1387. 8 HERTZ, L., DITTMANN,L., AND MANDEL,P., K ÷ induced stimulation of oxygen uptake in cultured cerebral glial cells, Brain Research, 60 (1973) 517-520. 9 KUrFLER, S. W., Neuroglial cells: physiological properties and a potassium-mediated effect of neuronal activity on the glial membrane potential, EEOC.roy. Soc. B, 168 (1967) 1-21. 10 ORKAND, P. M., BRACHO, H., AND ORKAND, R. K., Glial metabolism: alteration by potassium levels comparable to those during neural activity, Brain Research, 55 (1973) 467-471. 11 ORKAND, R. K., NICHOLS,J. G., AND KUEELER,S. W., Effect of nerve impulses on the membrane potential of gliai cells in the central nervous system of amphibia, J. Neurophysiol., 29 (1966) 788-806. 12 PAPE, L. G., AND KATZMAN, R., Response of glia in cat sensorimotor cortex to increased extracellular potassium, Brain Research, 38 (1972) 71-92. 13 PRINCE, D. A., Lux, H. D., AND NEHER, E., Measurement of extracellular potassium activity in cat cortex, Brain Research, 50 (1973) 489-495. 14 RANSOM,B. R., AND GOLDRING, S., Ionic determinants of membrane potential of cells presumed to be glia in cerebral cortex of cat, J. Neurophysiol., 36 (1973) 855-868. 15 SCHOUSBOE,A., BOOHER,J., AND HERTZ, L., Content of ATP in cultivated neurons and astrocytes exposed to balanced and potassium-rich media, J. Neurochem., 17 (1970) 1501-1504.