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A linked active transport system for Na+ and K+ in a glial cell line
Brain Research, 104 (1976) 93-105
93
©Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A L I N K E D ACTIVE T R A N S P O R T SYSTEM F O R Na + A N D K + IN A G L I A L CELL LINE
G A R Y K U K E S , JEAN D E VELLIS* AND R A F A E L E L U L
Laboratory of Nuclear Medicine and Radiation Biology, Department of Anatomy, Mental Retardation Research Center and Brain Research Institute, School of Medicine, University of California, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted August llth, 1975)
SUMMARY
Ouabain (5 × l0 -4 M) induced a 6-fold increase in intracellular Na + and a 65 % loss of cellular K + in C6 glial cells which was accompanied by a 12 mV decrease in the resting membrane potential. Following ouabain washout intracellular ion concentrations and the membrane potential returned to control levels suggesting that C6 is capable of active Na + transport which is linked to uptake of K +. A portion of K + uptake under steady-state conditions is also active since K + influx was reduced 32 % by ouabain. Five m M cyanide significantly increased cell Na + and significantly decreased cell K + and the membrane potential. The similarity in the ratio of Na + gained/K + lost (ouabain 1.24, cyanide 1.41) suggests that the two agents inhibit the same ion transport system. Decreased temperature had the paradoxical effect of increasing intracellular K + while significantly decreasing both membrane potential and K + influx. Part of this effect may be due to the marked reduction in K + efflux at low temperature. At 6 °C cell loss of K + was much less than loss of K + with ouabain at 37 °C. The observation of a linked N a + - K + transport system in C6 cells confirms the hypothesis that coupled active N a + - K + exchange occurs in glial cells and suggests that ionic transport may regulate certain aspects of glial metabolism.
INTRODUCTION
Experimental observations of swelling of astrocytes and glial cells in vivo 6 and in culture 32 in the presence of ouabain suggest that these cells have the capacity to actively transport Na +. The drug in other systems specifically inhibits a N a + - K + * To whom requests for reprints should be addressed: Laboratory of Nuclear Medicine and Radiation Biology, 900 Veteran Avenue, Los Angeles, Calif., 90024, U.S.A.
94 ATPase so the resulting accumulation of intracellular Na + induces osmotic water inflow38. This mechanism could reasonably account for glial swelling since the enzyme is present in bulk glia separated from neurons 16, in glia hand dissected from brain 8, in subcutaneously grown astroglial nodules ~°, and in glial tumor cell lines 7. In the previous report 22, we have shown, using a glial cell line, that the permeability of the cells to Na ÷ is relatively high, and have suggested that active outward pumping must occur to keep the cell at the low steady-state Na + concentration. In the present study we investigate the capacity of C6 glial cells to actively transport Na + . MATERIALS AND METHODS
Electrophysiological recordings, flux measurements, and the determination of intracellular ion concentration and cell volume were carried out as previously described 22. Cells were grown under conditions previously reported 22. In experiments where the effect of drugs on the membrane potential was studied, the substance to be tested was added as a concentrated solution m a volume ljl0th that of the final solution. Ouabain (Stophanthidin G) was obtained from Calbiochem. Sodium cyanide and iodoacetate were reagent grade. Acetoxycycloheximide (5/zg/ml) was added to cells to determine whether ion transport could be sustained in the absence of protein synthesis. To test the degree of inhibition of protein synthesis by acetoxycycloheximide, the drug was given either 15 min prior to, or simultaneously with a pulse of 1 #Ci[14C]leucine (AmershamSearle). Cultures to which [~4C]leucine was given in the absence of acetoxycycloheximide served as control. After the incubation period, radioactive medium was poured off, cells were washed 3 times with 0.15 N saline and harvested with an equal volume of 10 ~ trichloroacetic acid (TCA) into conical tubes. The suspension was centrifuged at 4 °C for 15-20 min at 1500 rev./min, the supernatant was saved for determination of radioactivity and 5 % trichloroacetic acid was added to the pellet. The pellet was heated at 90 °C for 15 min to hydrolyze nucleic acids. After two washings with 5 % TCA and one wash in ethanol the pellet was then dissolved with 0.5 N N a O H in a water bath at 37 °C. Atiquots were taken for determination of radioactivity and protein. The samples were counted after the addition o f cabosil in 10 ml of a scintillation fluid consisting of a mixture of 870 ml toluene, 600 ml 100 % ethanol, 1000 ml paradioxane, 130 g naphthalene, and 130 ml Spectrafluor (AmershamSearle). Protein was measured by the method of Lowry using bovine serum albumin as a standard 24. Counts in the TCA precipitable fraction represent amino acid incorporated into protein. Those in the wash represent the free pool of amino acids. D N A content was measured by a modification of the method of Santen and Agranoff 33. After dissolution of cells in 3 ml of 0.5 N NaOH at 37 ~C for 1 h to hydrolyze RNA, an equal volume of ice-cold 70 % perchloric acid was added and the suspension centrifuged at 2000 rev./min for 15 min. The pellet containing DNA was heated (90 °C) in PCA to hydrolyze D N A for 15 min and after centrifugation the supernatant was saved. The pellet was washed and centrifuged twice more in I N
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Fig. 1. Effect of 5 × 10 -4 M ouabain on intracellular concentration of N a + and K +. Ouabain was added in fresh Ham's F10 plus 1 0 ~ fetal calf serum at t = 0. Filled symbols, in the presence of 5 x 10 -4 M ouabain (O, N a + ; A, K+); open symbols, control ( O , N a + ; A , K+). Curve through points (. . . . , N a + ; . . . . , K+). Each point is the mean of duplicate determinations.
PCA and the combined supernatant comprising the D N A fraction was read at 267 nm against appropriate blanks in a Cary scanning spectrophotometer. Aliquots were taken for protein determination to correct for extra absorbance from that source. RESULTS
Effect of ouabain on ionic concentration When cells were incubated in 5 x 10 -4 M ouabain, there was a rapid increase of intracellular N a + and a concomitant loss of cell K +. The maximum rate of concentration change (raM/liter cell water) occurred during the first 20 rain when K + fell from 133 to 83 m M and N a + increased from 16 to 78 mM. After this time period, K + loss and N a + accumulation were more gradual until a new steady-state level was reached by 90-100 rain with K + at 45 m M and N a + at 108 m M (Fig. 1). This re-
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Fig. 2. Effect of various concentrations of ouabain on intracellular N a + and K + at the end of 100 min incubation. Filled symbols, ouabain-treated (O, N a + ; , , K+); open symbols, control ( © , N a + ; A , K+). Curve through points ( , N a + ; . . . . , K+). Each point represents the mean of duplicates.
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Fig. 3. Intracellular concentration of Na + and K + following washout of ouabain. Cells were incubated in 5 x I0 -~ M ouabain for 100 min, washed rapidly 5 times and reincubated at time 0 in ouabain free medium plus 10% fetal calf serum. Open symbols (O, Na + ; z% K+). Curve through points ( , Na + : . . . . , K+). Each point represents the mean of duplicates.
presented a 65 ~ K ÷ loss a n d a 6-fold N a ÷ increase c o m p a r e d to controls. A f t e r rem a i n i n g fairly c o n s t a n t for several hours, K + b e g a n to increase slightly a n d N a b e g a n to fall, so b y the end o f a 12 h i n c u b a t i o n K ÷ was 52 m M a n d N a ÷ was 92 m M . A t o u a b a i n c o n c e n t r a t i o n s greater t h a n 10 -4 M, cells lost K ÷ a n d a c c u m u l a t e d N a ÷. Fig. 2 shows t h a t the loss o f K + at each c o n c e n t r a t i o n at the end o f 100 m i n i n c u b a t i o n was a c c o m p a n i e d by an a l m o s t e q u i m o l a r increase o f cell N a ÷. The finding t h a t t r a n s p o r t o f b o t h N a ÷ and K ÷ was h a l f m a x i m a l l y inhibited at 1.6 x 10 -4 M s t r o n g l y suggests t h a t the m o v e m e n t o f these ions is c o u p l e d . T h e increase in cell N a ÷ in all eases was a c c o m p a n i e d by cell swelling which at 10 -3 M o u a b a i n was 30 ~ .
K ÷ flux with ouabain I f K ÷ toss with o u a b a i n results f r o m the d r u g ' s c a p a c i t y to inhibit a N a ÷ - K ~ A T P a s e , K + influx w o u l d be d i m i n i s h e d to the extent that K + u p t a k e is actively med i a t e d by this enzyme. W i t h 5 x l0 -4 M o u a b a i n , K ÷ influx calculated f r o m u p t a k e o f 42K for 2 min was 8.9 :t: 0.2 (S.D.) p m o l e s / s q . c m . s e c (n - - 5), significantly less t h a n ( P < 0.005) the c o n t r o l level o f 13.0 :a: 1.7 (S.D.) p m o l e s / s q . c m . s e c (n - - l l L T h i s suggests t h a t a p o r t i o n o f K + u p t a k e is active.
Ion movement after ouabain washout I n o r d e r to test the cell c a p a c i t y to r e a c e u m u l a t e K + a n d expel N a + after the n o r m a l c o n c e n t r a t i o n g r a d i e n t was reversed, cultures were i n c u b a t e d in 5 x t 0 -4 M o u a b a i n for 100 rain, w a s h e d r a p i d l y a n d the m e d i u m r e p l a c e d with fresh H F - 1 0 plus 10 ~ fetal c a l f serum. U n d e r these c o n d i t i o n s cells r a p i d l y lost N a ÷ a n d r e a c c u m u l a t e d K + . W i t h i n 10 m i n after o u a b a i n h a d been w a s h e d o u t i n t r a c e l l u l a r K ÷ h a d risen f r o m 40 to 160 m M cell water, while i n t r a c e l l u l a r N a + fell f r o m 120 m M cell w a t e r (Fig. 3). This d e m o n s t r a t e s clearly t h a t C6 h a s the c a p a c i t y to expel N a + in a m a n n e r which is linked to K + u p t a k e . Since this N a ÷ m o v e m e n t t a k e s place a g a i n s t a N a + conc e n t r a t i o n g r a d i e n t when the m e m b r a n e p o t e n t i a l is still negative (see Results-
97
Effect ofouabain on membrane potential) the transport is active. The rapid resumption of ion pumping, after ouabain is washed out, is taken to mean that the glycoside has a low affinity for the N a + - K + ATPase in C6. This is consistent with the finding that the complex formed between cardiac glycosides and Na+-K + ATPase of more ouabain sensitive species such as cow and dog has greater stability than the complex formed with the enzyme from rat, a ouabain insensitive species from which the C6 cell line is derivedL
Kinetics of Na + transport The rate constant for N a + loss after ouabain washout can be derived from the permeability equation: d(Na)i -
-
dt
kl(Nae) -- kz(Nal)
where Nae and Nai are the external and intracellular k2, the constants for influx and efflux are equal to volume, respectively. I f the permeability constants are if the final value for Nat can be found, then kl can be integration becomes: --kzt = In
(Nai) oo -- (Nai)t ( N a 0 oo -- (Nal)o
= k 2 --
Na + concentration and kl and K1 area/volume and K2 area/ not altered as Nai changes and eliminated and the solution on
In 2 t~
where (Na0o is the concentration at the beginning of ouabain washout, (Na0c~ the new steady-state following ouabain washout and t~ is the time required for half the Na + to be lost from the cells 's. F r o m 3 experiments where Na + was lost following ouabain washout, the value of the rate constant for efflux, kz, was 0.0116 i 0.004 sec-L This compared to a rate constant of 0.0256 sec -~ from experiments where effiux was determined by loss of 22Na from pre-loaded cells. The discrepancy could be due to the phenomenon in tracer flux experiments where exchange diffusion of internally labeled Na + for an external non-radioactive N a + gives an apparent flux and rate constant which is actually higher than the true value 38. That such a process takes place in C6 is strongly suggested by the observation reported previously z2 that N a + tracer efflux was 2.5 times larger than Na + influx measured by the non-radioactive method.
Effect of inhibition of protein synthesis on transport To test whether protein synthesis was required for ion pumping following ouabain washout, cells were exposed to 5/~g/ml acetoxycycloheximide (ACCh) and 5 x 10 -4 M ouabain for 100 min then washed and exposed to A C C h for the duration of the experiment. Preliminary experiments showed that a 15 rain preincubation with ACCh inhibited 98.5~o of [14C]leucine incorporated into the T C A precipitable fraction during a 15 min pulse while total protein/DNA decreased by only 1 0 ~ during a 6 h exposure to the inhibitor. Results showed that during the initial exposure to ouabain and in the period following ouabain washout there were no obvious differences in the ionic responses between ACCh-treated and ouabain control cultures (Fig. 4).
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Fig. 4. Effect of acetoxycycloheximide on the intracellular accumulation of Na + and loss of K + with ouabain and the ion movements following ouabain washout. Cells were incubated with 5 × 10 -4 M ouabain in the presence and abseaace of 5/tg/ml acetoxycloheximide. Panel A indicates K + concentration. Filled symbols, with A C C h (A), open symbols, without (A). At upward arrow (1') cells were washed free of ouabain and experimental cultures (A) were reincubated with 5/~g/ml ACCh. Panel B indicates Na + concentration: Filled symbols, ACCh ( 0 ) ; open symbols, without ((3). At downward arrow (~), cells were washed as in part A. Each point represents the mean of duplicates except panel B at t : 110 min which is a single determination. Curve through points with ACCh ( . . . . ), control ( --).
This indicates that the turnover rate of proteins involved in ion pumping is greater than the time of exposure to ACCh.
Effect of ouabain on the membrane potential The average membrane potential, measured within an hour after 10 -s M ouabain was given, declined in 3 experiments from the control value of --31 mV to -- 19 mV (Fig, 5A). Because we could not record from cells while changing the bathing solution it was difficult to determine whether there was an immediate depolarization with ouabain suggestive of an electrogenic pump 9. An experiment where two
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Fig. 5. Effect of ouabain and cyanide on membrane potentials in C6. A: (upper) membrane ~ i a l s recorded in 3 experiments in t ~ presence of 10 -3 M ouabain within 90 rain after addition:; Lower portion control at 37 °C. B: membrane potentials recorded in 4 experim©nts in t h ¢ i ~ of 5 cyanide within 90 min after the drug was added. Lower portion, control at 37 OC. Each rectangle represents the number of cells at the potential indicated by the interval on the abscissa.
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Fig. 6. Effect of ouabain on the time course of membrane potentials in C6. Membrane potentials between t -- 0 and t ~ 40 were recorded in Ham's F 10 plus 10 % fetal calf serum. At t 40 (denoted by f') fresh medium containing 5 × 10 -4 M ouabain was added and recordings were made for 160 min. Ouabain medium was then washed out (~,), fresh medium added and the cells were equilibrated for 9.5 h and recordings were again made. Each point represents a single membrane potential from a different cell in the presence (©) and absence (O) of ouabain. m e m b r a n e p o t e n t i a l s o f - - 4 5 m V a n d - - 4 0 mV were o b t a i n e d within 3 m i n after 5 × 10 -4 M o u a b a i n was a d d e d suggests t h a t any electrogenic c o m p o n e n t is p r o b a b l y insignificant, since these p o t e n t i a l s are n o t lower t h a n c o n t r o l p o t e n t i a l s r e c o r d e d i m m e d i a t e l y p r i o r to o u a b a i n a d d i t i o n (Fig. 6). A f t e r these two potentials were rec o r d e d , a p o t e n t i a l o f - - 2 8 mV was o b t a i n e d 14 m i n after o u a b a i n was a d d e d . D u r i n g the next 90 min no p o t e n t i a l greater t h a n - - 3 2 mV was seen. T h e most plausible e x p l a n a t i o n for the o u a b a i n - i n d u c e d d e p o l a r i z a t i o n is the loss o f cell K + and a c c u m u l a t i o n o f cell N a +. If the relative N a + / K + p e r m e a b i l i t y of 0.11 (see ref. 22) is n o t altered by o u a b a i n , the G o l d m a n equation, E = (61 log K0 + P N a / P K (Na0))/Ki + P N a / P K ( N a 0 predicts a d r o p from the c o n t r o l value o f - - 5 2 mV to --31 mV when the cells reach the steady-state c o n c e n t r a t i o n o f 109 m M N a + and 45 m M K + in 5 × 10 .4 M o u a b a i n . A l t h o u g h the m e a s u r e d potentials are lower t h a n the p r e d i c t e d values, the p o t e n t i a l decrease after 100 rain in 5 × 10 .4 M o u a b a i n to - - 2 6 mV ± 4.1 S.D. (n = 6) f r o m a p r e - o u a b a i n c o n t r o l of - - 4 3 mV ~ 6.6 S.D. (n = 4) is 17 mV c o m p a r e d to the p r e d i c t e d decrease o f 21 inV. A l s o in s u p p o r t o f this h y p o t h e s i s is the o b s e r v a t i o n t h a t 10 -4 M o u a b a i n which does n o t cause a n y s u b s t a n t i a l change in the ionic g r a d i e n t s h a d no effect on the m e m b r a n e potential. F u r t h e r m o r e , after o u a b a i n is washed out, the m e m b r a n e potential r e c o r d e d 9 h later r e t u r n e d to c o n t r o l levels. This is consistent with direct i o n measureTABLE I EFFECTS OF METABOLIC INHIBITORS ON CELLULAR
Na +
AND K + AFTER 1 0 0 MIN AT
37 °C
Values ± s.d. Inhibitor
Flasks (n)
ttg K+/mg protein
I~g Na+/mg protein
mM K+/liter mM Na+/liter cell H20 cell H20
1.5 mM CN1 mM1AA Control
4 4 4
38.0 ± 2.. 42.1 ~ 1.51 39.6 ± 1.30
5.08 ± 0.78 3.26±0.43 2.65 ± 0.37
152 ± 10 168 ± 6 158 ± 5
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Fig. 7. A: effect of 5 mMcyanide on intracellular Na ÷ and K + in C6. Filled symbols, 5 mM cyanide (0, Na + ; A, K+); open symbols, control (O, Na + ; •, K+). B: effect of 24 °C on Jntracellulat Na ÷ and K ÷. Filled symbols, 24 °C (0, Na + ; A, K +); open symbols, control at 37 °C. (O, Na + ; A, K+). In both A and B each point with bars representing one standard deviation, is the average of 3 determinations. Points without bars are the mean of duplicates. ments which show that cells maintain the control level steady-state ion concentrations following ouabain washout.
Effect of cyanide Since ouabain inhibits transport by blocking the pump's capacity to hydrotyze ATP, we looked at the effect of cyanide which limits ATP production at the mitochondrial level. At a concentration of 1.5 m M cyanide after 100 min, there was only a slight increase in cell Na + with no significant change in cell K + (Table I). With 5 m M cyanide there was a rapid increase in cell Na + and a concomitant fall in cell K ~ (Fig. 7A). During the first 20 min, Na ÷ increased from 20 to 68 m M while K ÷ fell from 133 to 99 mM. This represents a molar ratio of 1.41 Na ÷ gained/K + lost. Over the nest 40 min there was a reversal of this pattern and cells began to gain K ÷ and extrude Na + so that by the end of an hour, K + concentration had risen to 114 m M and Na + had fallen to 50 m M cell water. Iodoacetate, an inhibitor o f glycolysis, at 1 m M had little effect on the ionic concentration of Na + and K ÷ at the end of 100 min incubation (Table I). Membrane potentials recorded within an hour after 5 m M cyanide was added decreased significantly (P < 0.001) to 25 mV compared to the control of 36 mV. As in the case of ouabain this depolarization is most readily explained by the loss of cell K +.
Effect of temperature Decreased temperature reduces the active component of K + influx uptake in both squid axon 19 and mammalian skeletal muscle s without affecting K + efflux. Lowering the temperature in C6 produced a significant decrease (P < 0.O25), in K ÷ influx from the control value of 13.0 ± 1.7 S.D. pmoles/sq.em.sec (n = 11) at 37 °C to 10.9 ± 0.23 S.D, pmoles/sq.cm, sec (n = 5). If K + ettlux is not affected by lowered
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Fig. 8. C o m p a r i s o n of the effects of low t e m p e r a t u r e (6 °C), o u a b a i n at 6 °C, a n d o u a b a i n at 37 °C o n intracellular N a + a n d K + concentration. A : intracellular K + ( A , 6 °C; O , 6 °C plus 5 × 10 -4 M o u a b a i n ; O, 5 × 10 -4 M o u a b a i n at 37 °C). B: intracellular N a + (/k, 6 °C; O , 6 °C plus 5 x 10 -4 M o u a b a i n ; O, 5 × 10 -4 M o u a b a i n at 37 °C). E a c h point is the m e a n of duplicate determinations.
temperature, one would predict that the K + concentration in C6 at 24 °C would decrease over time. Contrary to this, we found that at the end of an incubation of 60 min. at 24 °C, intracellular K + was 162 raM, slightly higher than the control value of 154 raM. A possible explanation for this phenomenon is that passive outward K + movement in C6 is reduced at lower temperature. A finding which supports this hypothesis comes from experiments where ionic concentrations were studied in cells at 6 °C. Fig. 8 shows that in the first hour of incubation at 6 °C, cell K + decreased from 160 to 148 m M cell water, while Na + increased from 18 to 33 m M cell water. Because of the high temperature coefficient of Na +-K + ATPase s7, the enzyme is almost completely inhibited in other systems at 6 °C (see refs. 13 and 30). To test this in C6 we incubated cells at 6 °C with 5 × 10-4 M ouabain and found during the first hour that K + fell from 160 to 130 m M cell water, while cell Na + rose from 18 to 35 m M cell water so that the rate of concentration change of these ions was only slightly greater than at 6 °C without ouabain (Fig. 8). This suggests that the rate of
30-
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q0 -20 -30 -40 -50 60 MEMBRANEPOTENTIAl(mV) Fig. 9. Effects o f t e m p e r a t u r e o n t h e m e m b r a n e potential in C6. U p p e r portion, m e m b r a n e potentials at 24 °C. Lower portion, m e m b r a n e potentials at 37 °C. E a c h rectangle represents the n u m b e r o f cells at the potentials indicated by the interval on t h e abscissa.
102 ion transport at 6 °C mediated by a Na+-K + ATPase in C6 is low. When the loss of K* at low levels of ion pumping at 6 "~'Cis compared to K ~ loss at low levels of pumping with ouabain at 37 '~C (Fig. 8), it is apparent that outward K movement is greatly retarded at the lower temperature. Another observation which is consistent with a temperature-induced permeability change is that the membrane potential decreased to a significantly greater degree (P < 0.01) at 24 °C than the decline in potential predicted on the basis of temperature by the G o l d m a n equation (Fig. 9). The equation predicts a drop of 3.1 mV for a 13 °C temperature decrease while the actual membrane potential dropped by 6.3 mV to --29.7 inV. Although it is not apparent whether this involves a change in the ratio of PNa/PK, the depolarization is not induced by changes in the internal concentrations of K ÷ and Na ÷ as is the case with ouabain and cyanide. DISCUSSION
The movement of Na + out of the cell against both a concentration gradient and a negative transmembrane potential following ouabain washout clearly demonstrates that C6 is capable of active Na t transport. It is apparent from the reaccumulation of cell K + which takes place concurrently with the Na ÷ extrusion that in C6 glial cells as in other systems active Na t transport is linked to the uptake of K +,3,~z. A portion of the inward K + under steady-state conditions is also actively mediated since 5 .. 10 -4 M ouabain reduced K + influx by 32 ~ while decreasing external Na ÷ lowered it by 51 ~22. On the basis of the amounts of Na ~ gained and K ~ lost in experiments where ion transport was inhibited by ouabain and cyanide, an estimate of the ratio of N a : K transported by the p u m p can be made. During the first 20 min at 5 × 10 - 4 M ouabain cells lost 50 m M K ÷ and gained 62 m M Na ÷ giving a ratio of 1:24. This can be taken as the ratio of passive leak which would have to be balanced by active pumping in order to maintain the steady-state concentration if the pump were not inhibited ~9. Actually the ratio of active pumping may be slightly higher than this because the passive inward leak of Na ÷ and outward leak of K ÷ are probably retarded somewhat by the decreasing transmembrane gradient of these ions as the intracellular concentrations change with time. With 5 m M cyanide, the loss of K ~ and gain of Na ÷ over the first 20 min, 34 and 48 m M respectively, were slightly less than the concentration changes induced by ouabain. The flux ratio of 1.41. similar to that with ouabain, suggests that the two agents exert their inhibitory effect ultimately on the same transport system. Ouabain does so by blocking hydrolysis of A T P by the pump and cyanide by making high energy phosphate unavailable for transport processes. Interestingly, in mouse brain slices. the decrease in total A T P brought about by cyanide had to be much larger to effect a comparable increase in slice Na + and loss of K ÷ as that induced by ouabain z. In that study, blockage of ion transport suggested that intracellular K + loss was accompanied by an equimolar increase in N a t. In other systems, active flux ratios between 1 and 1.5 have been reported ~5.34.
103 The finding that 1.5 m M cyanide and 1 m M iodoacetate did not affect ionic transport and that transport of Na + and K + apparently resumes in 5 m M cyanide after an initial inhibition may reflect a high rate of energy production in C6 cells. Concentrations of 10 m M sodium azide and 1 m M 2,4-dinitrophenol do not block uptake of the saturable, presumably energy requiring component of 7-aminobutyric acid uptake in C6 36. The 1 7 ~ drop in K ÷ influx when the temperature is dropped to 24 °C presumably reflects an inhibition of the active portion of the K + uptake which is also temperature sensitive in other systems. The paradoxical finding was that despite the decrease in K + influx, intracellular K + concentration was not reduced over time. From our finding of a markedly reduced loss of cell K + at 6 °C compared to that at 37 °C with ouabain, we postulated that decreased temperature in C6 significantly re:ards K + efflux. In this regard, it is interesting that although there is no difference in the amount of K + lost from cat heart muscle at 2-3 °C compared to ouabain at 27 °C zs, kidney cortical slices from hibernating mammals show significantly smaller K ÷ losses at 5-6 °C than with ouabain at higher temperatures 4°. Such a mechanism for preventing massive loss of K ÷ from glial cells following cold exposure may serve to protect the organism from the brain edema which accompanies exposure of brain cells to high levels of K ÷ (see refs. 4 and 23) and to prevent seizures elicited by local buildup of K ÷ (ref. 11). It has been postulated that the observed active uptake of K + in mammalian glial cells14, t6 is linked to outward Na + transportt7, 21. Such hypotheses have been based on finding significant N a + - K + ATPase activity in glial cell fractions8,16 and on the ouabain-induced swelling of glial cells in vivo 6 and in vitro 32. Our finding in C6 provides direct confirmation that K + uptake in glial cells is linked to outward active transport of Na +. One consequence o f a glial Na + pump, activated as in other systems by increased external K + (see reL 3) is that glial metabolism may be altered in response to nerve activity. As extracellular K + increases in the vicinity of active neurons, stimulation of the glial N a + p u m p could alter cellular ratios of A D P / A T P 39. Such stimulated glial Na + p u m p activity may explain the decrease in ATP 35 and in the increase in oxygen consumption 18 in glial cells exposed to high external K +. Although the mechanism is unknown, increases in external K +, comparable to those during neural activity, produce decreased levels of N A D H in pure glial optic nerve preparation of Necturus z7. A Na + p u m p in glia may also play a role in regulation of glial uptake of neurotransmitters. By increasing local Na + concentrations around synaptic terminals following nerve activity glial N a + pumping in concert with an electrogenic p u m p in neurons 31 may facilitate transport of G A B A by glial cells through stimulation of the Na+-dependent component of the G A B A uptake mechanism 2°,36. Evidence from the previous report suggests that the C6 cell line resembles normal gila with respect to permeability of Na +, K + and C1- and that its biophysical properties appear similal to those of normal tissue. Our present finding indicates C6 has retained the linked N a + - K + transport system strongly inferred in normal gila. T u m o r cell lines serve as useful models to the
104 extent they express particular characteristics of normal tissue. Although certain ionic properties in C6 are similar to those irt normal tissue, our present base for comparing normal and tumor glial cells is narrow. As more is learned about the function of glial cells and about fundamental processes of carcinogenesis, the base for comparison will broaden and C6, as a model for ionic mechanisms in glia, will be strengthened or weakened accordingly. In spite of the problems discussed in these papers, in addition to its present applicability to studies of ionic permeability, the C6 cell line appears to be a useful preparation for characterizing the coupled active Na+-K + transport system in glial cells. ACKNOWLEDGEMENTS
We wish to thank Ms. Ruth Cole and Diane Inglish for their expert help with tissue culture and Dr. H. Bracho for helpful comments in the preparation of the manuscript. The material in these two papers was included in a thesis submitted in partial fulfillment of the Degree of Doctor of Philosophy at the University of California, Los Angeles. Supported by USPHS Grants HD-04612, HD-05615 and CA 13538 and by Contract E(04-1)GEN-12 between E R D A and the University of California and NtH Pre-doctoral Fellowship 5-F01 G M 44694-03. REFERENCES 1 ALLEN, J. C., AND SCHWARTZ, A., A possible biochemical explanation for the insensitivity of the rat to cardiac glycosides, d. Pharmacol. exp. Ther., 168 (1969) 42-46. 2 BANAY-SCHWARTZ,M.. TELLER, n . N., GERGELY, A., AND LAJTHA, A., Tile effects of metabolic inhibitors on amino acid uptake and the levels of ATP, Na ÷ and K + in incubated slices of mouse brain, Brain Research, 71 (1974) 117-131. 3 BONTING, S. L., Sodium-potassium activated adenosinetriphosphatase and cation transport. In E. E. BITTAR (Ed.), Membranes and Ion Transport, Vol. l, Wiley-Interscience, London. 1970, pp. 257-363. 4 BOURKE, R. S., AND NELSON, K. M., Further studies on the K + dependent swelling of primate cerebral cortex in vivo: the enzymatic basis of the K + dependent transport of chloride, J. Neurochem., 19 (1972) 663-687. 5 CALKINS, E., TAYLOR, I. i J , AND HASTINGS, A. B., Potassium exchange in the isolated rat diaphragm; effect of anoxia and cold, Amer. J. PhysioL, 177 (1954) 211-218. 6 CONROG,J. L., GONATAS,N. K., AND FEIERMAN,J. R., Effect of intracerebral injection of ouabain on the fine structure of the rat cerebral cortex, Amer. d. Path., 51 (1967) 573-590. 7 COTMAN,C., FIERSCHMAN. H., AND TAYLOR. D,, Subcellular fractionation of cultured glial cells, J. NeurobioL, 2 (1971) 169-180. 8 CUMINS, J., AND HYD~N, H., Adenosine triphosphate levels and adenosine triphosphatases in neurons, glia and neuronal membranes of the vestibular nucleus, Biochim. Biophys. Acta (Amst.), 60 (1962) 271-283. 9 DE WEER, P., AND GEDULDIO, D.. Electrogenic sodium pump m squid giant axon, Science, 179 (1973) 1326-1328. 10 EMBREE, L. J., HESS, H. H., AND SHEIN, H. M., Sodium-potassium ATPase activity of normal and virally transformed hamster astroglia grown subcutaneously, Brain Research, 27 (1971) 422-425. 11 GLASER,G. H.. AND ZUCKERMAN,E. C., Potassium accumulation in extracellular spaces of brain as a possible cause of epileptogenic activity. In R. VIZIOLI (Ed.), Brain and Mind Problems. tl Pensiero Scientifico. Rome, 1968, pp. 309-329 12 GLYNN, I. M., Sodium and potassium movements in h u m a n red cells, J. PhysioL (Lond.), 134 (1956) 278-310.
105 13 GRUENER, N., AND AVI-DOR, Y., Temperature-dependence of activation and inhibition of ratbrain adenosine triphosphatase activated by sodium and potassium ions, Biochem. J., 100 (1966) 762-767. 14 HALJAMAE,H., AND HAMBERGER,A., Potassium uptake by both prepared neuronal and glial cells, J. Neurochem., 18 (1971) 1903-1912. 15 HARRIS, E. J., AND MAIZELS, M., The permeability of h u m a n erythrocytes to sodium, J. Physiol. (Lond.), 113 (1951) 506-524. 16 HENN, 17. A., HALJAMAE, H., AND HAMBERGER, A., Glial cell function: active control of extracellular K ÷ concentration, Brain Research, 43 (1972) 437443. 17 HERTZ, I., Possible role of neuroglia: a potassium mediated neuronal-neuroglial-neuronal impulse transmission system, Nature (Lond.), 206 (1965) 1091-1094. 18 HERTZ, I., Neurologlial localization of potassium and sodium effects on respiration in brain, J. Neurochem., 13 (1966) 1373-1387. 19 HODGKIN, A. L., AND KEYNES, R. D., Active transport of cations in giant axons from Sepia and Loligo, J. Physiol. (Lond.), 128 (1955) 28-60. 20 HUTCHISON, n . T., WERBACH, K., VANCE, C., AND HABER, B., Uptake of neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. 1. Transport of ~,-aminobutyric acid, Brain Research, 66 (1974) 265-274. 21 KIMELBERG,H. K., Active potassium transport and Na ÷ + K + ATPase activity in cultured glioma and neuroblastoma cells, J. Neurochem., 22 (1974) 971-976. 22 KUKES, G., ELUL, R., AND DE VELLIS, J., The ionic basis of the membrane potential in a rat glial cell line, Brain Research, O0 (1976) 00(~000. 23 LOD1N, Z., HARTMAN, J., KAGE, M. P., KORINKOVA, P., AND BOOHER, J., Potassium-induced hydration in cultured neural tissue, Neurobiology, 1 (1971) 69-85. 24 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. 25 McCONAGHEY, P. D., AND MAIZELS, M., Cation exchanges of lactose treated human red cells, J. Physiol. (Lond.), 162 (1962) 485-509. 26 MEDZEHRADSKY, F., NANDHASHRI, P. S., INDOYAGA-VARGAS,V., AND SELLINGER, O. Z., A comparison of ATPase activity of a glial cell fraction and the neuronal perikaryl fraction isolated in bulk from rat cerebral cortex, J. Neurochem., 18 (1971) 1599-1603. 27 ORKANO, P. M.. BRACHO, H. E., AND ORKAND, R., Glial metabolism alteration by potassium levels comparable to those during neural activity, Brain Research, 55 (1973) 467-471. 28 PAGE, E., GOERKE, R. J., AND STORM, S. R., Cat heart muscle in vitro. IV. Inhibition of transport in quiescent muscles, J. gen. Physiol., 47 (1964) 531-543. 29 POST, R. L., ALBRIGHT, C. D., AND DAYANI, K., Resolution of pump and leak components of sodium and potassium ion transport in human erythrocytes, J. gen. Physiol., 50 (1967) 1201-1220. 30 POST, R. L., SEN, A. K., AND ROSENTHAL, A. S., A phosphorylated intermediate in adenosine triphosphate dependent sodium and potassium transport across kidney, J. biol. Chem., 240 (1965) 1437-1445. 31 RANSOM, 13. R., AND GOLDRING, S., Slow hyperpolarization in cells presumed to be Alia in cerebral cortex of cat, J. Neurophysiol., 36 (1973) 879-893. 32 RENKAWEK, K., PALLADINI, G., AND IERADI, L., Morphology of glial cells in the presence of ouabain, Brain Research, 18 (1970) 363 367. 33 SANTEN, R. J., AND AGRANOFF, B. W., Studies on the estimation of deoxyribonucleic acid in rat brain, Biochim. biophys. Acta (Aust.), 72 (1963) 251-262. 34 SEN, A. K., AND POST, R. L., Stoichiometry and localization of adenosine triphosphate dependent sodium and potassium transport in the erythrocyte, J. biol. Chem., 239 (1964) 345-352. 35 SCHOUSBOE,A., BOOHER, J., AND HERTZ, |., Content of ATP in cultivated neurons and astrocytes exposed to balanced and potassium-rich media, J. Neurochem., 17 (1970) 1501-1504. 36 SCHRIER, B. K., AND THOMPSON, E. J., On the role of glial cells in the mammalian central nervous system. Uptake, excretion and metabolism of putative neuro-transmitters by cultured glial tumor cells, J. biol. Chem., 249 (1974) 1769-1780. 37 SKOU, J. C., Enzymatic basis for active transport o f N a + and K + across cell membrane, Physiol. Rev., 45 (1965) 596-615. 38 TOSTESON, D. C., AND HOEFMAN, J. F., Regulation of cell volume by active cation transport in high and low potassium sheep red cells, J. gen. Physiol., 44 (1960) 169-194. 39 WHITTAM, R., The Interdependence of Metabolism and Active Transport in Cellular Functions of Membrane Transport, Prentice-Hall, Englewood Cliffs, N.J., 1964, pp. 139 154. 40 WILLIS, J. S., Uptake of potassium at low temperatures in kidney cortex slices of hibernating mammals, Nature (Lond.), 204 0964) 691-693.
93
©Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A L I N K E D ACTIVE T R A N S P O R T SYSTEM F O R Na + A N D K + IN A G L I A L CELL LINE
G A R Y K U K E S , JEAN D E VELLIS* AND R A F A E L E L U L
Laboratory of Nuclear Medicine and Radiation Biology, Department of Anatomy, Mental Retardation Research Center and Brain Research Institute, School of Medicine, University of California, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted August llth, 1975)
SUMMARY
Ouabain (5 × l0 -4 M) induced a 6-fold increase in intracellular Na + and a 65 % loss of cellular K + in C6 glial cells which was accompanied by a 12 mV decrease in the resting membrane potential. Following ouabain washout intracellular ion concentrations and the membrane potential returned to control levels suggesting that C6 is capable of active Na + transport which is linked to uptake of K +. A portion of K + uptake under steady-state conditions is also active since K + influx was reduced 32 % by ouabain. Five m M cyanide significantly increased cell Na + and significantly decreased cell K + and the membrane potential. The similarity in the ratio of Na + gained/K + lost (ouabain 1.24, cyanide 1.41) suggests that the two agents inhibit the same ion transport system. Decreased temperature had the paradoxical effect of increasing intracellular K + while significantly decreasing both membrane potential and K + influx. Part of this effect may be due to the marked reduction in K + efflux at low temperature. At 6 °C cell loss of K + was much less than loss of K + with ouabain at 37 °C. The observation of a linked N a + - K + transport system in C6 cells confirms the hypothesis that coupled active N a + - K + exchange occurs in glial cells and suggests that ionic transport may regulate certain aspects of glial metabolism.
INTRODUCTION
Experimental observations of swelling of astrocytes and glial cells in vivo 6 and in culture 32 in the presence of ouabain suggest that these cells have the capacity to actively transport Na +. The drug in other systems specifically inhibits a N a + - K + * To whom requests for reprints should be addressed: Laboratory of Nuclear Medicine and Radiation Biology, 900 Veteran Avenue, Los Angeles, Calif., 90024, U.S.A.
94 ATPase so the resulting accumulation of intracellular Na + induces osmotic water inflow38. This mechanism could reasonably account for glial swelling since the enzyme is present in bulk glia separated from neurons 16, in glia hand dissected from brain 8, in subcutaneously grown astroglial nodules ~°, and in glial tumor cell lines 7. In the previous report 22, we have shown, using a glial cell line, that the permeability of the cells to Na ÷ is relatively high, and have suggested that active outward pumping must occur to keep the cell at the low steady-state Na + concentration. In the present study we investigate the capacity of C6 glial cells to actively transport Na + . MATERIALS AND METHODS
Electrophysiological recordings, flux measurements, and the determination of intracellular ion concentration and cell volume were carried out as previously described 22. Cells were grown under conditions previously reported 22. In experiments where the effect of drugs on the membrane potential was studied, the substance to be tested was added as a concentrated solution m a volume ljl0th that of the final solution. Ouabain (Stophanthidin G) was obtained from Calbiochem. Sodium cyanide and iodoacetate were reagent grade. Acetoxycycloheximide (5/zg/ml) was added to cells to determine whether ion transport could be sustained in the absence of protein synthesis. To test the degree of inhibition of protein synthesis by acetoxycycloheximide, the drug was given either 15 min prior to, or simultaneously with a pulse of 1 #Ci[14C]leucine (AmershamSearle). Cultures to which [~4C]leucine was given in the absence of acetoxycycloheximide served as control. After the incubation period, radioactive medium was poured off, cells were washed 3 times with 0.15 N saline and harvested with an equal volume of 10 ~ trichloroacetic acid (TCA) into conical tubes. The suspension was centrifuged at 4 °C for 15-20 min at 1500 rev./min, the supernatant was saved for determination of radioactivity and 5 % trichloroacetic acid was added to the pellet. The pellet was heated at 90 °C for 15 min to hydrolyze nucleic acids. After two washings with 5 % TCA and one wash in ethanol the pellet was then dissolved with 0.5 N N a O H in a water bath at 37 °C. Atiquots were taken for determination of radioactivity and protein. The samples were counted after the addition o f cabosil in 10 ml of a scintillation fluid consisting of a mixture of 870 ml toluene, 600 ml 100 % ethanol, 1000 ml paradioxane, 130 g naphthalene, and 130 ml Spectrafluor (AmershamSearle). Protein was measured by the method of Lowry using bovine serum albumin as a standard 24. Counts in the TCA precipitable fraction represent amino acid incorporated into protein. Those in the wash represent the free pool of amino acids. D N A content was measured by a modification of the method of Santen and Agranoff 33. After dissolution of cells in 3 ml of 0.5 N NaOH at 37 ~C for 1 h to hydrolyze RNA, an equal volume of ice-cold 70 % perchloric acid was added and the suspension centrifuged at 2000 rev./min for 15 min. The pellet containing DNA was heated (90 °C) in PCA to hydrolyze D N A for 15 min and after centrifugation the supernatant was saved. The pellet was washed and centrifuged twice more in I N
95
I 160Na +
~ 1202 E Z o 80l.--
k2
40-
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~< 0
30
60
9;
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7½0
MINUTES
Z
Fig. 1. Effect of 5 × 10 -4 M ouabain on intracellular concentration of N a + and K +. Ouabain was added in fresh Ham's F10 plus 1 0 ~ fetal calf serum at t = 0. Filled symbols, in the presence of 5 x 10 -4 M ouabain (O, N a + ; A, K+); open symbols, control ( O , N a + ; A , K+). Curve through points (. . . . , N a + ; . . . . , K+). Each point is the mean of duplicate determinations.
PCA and the combined supernatant comprising the D N A fraction was read at 267 nm against appropriate blanks in a Cary scanning spectrophotometer. Aliquots were taken for protein determination to correct for extra absorbance from that source. RESULTS
Effect of ouabain on ionic concentration When cells were incubated in 5 x 10 -4 M ouabain, there was a rapid increase of intracellular N a + and a concomitant loss of cell K +. The maximum rate of concentration change (raM/liter cell water) occurred during the first 20 rain when K + fell from 133 to 83 m M and N a + increased from 16 to 78 mM. After this time period, K + loss and N a + accumulation were more gradual until a new steady-state level was reached by 90-100 rain with K + at 45 m M and N a + at 108 m M (Fig. 1). This re-
I
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i
2x10-4
i
i
i
i
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i
i
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OUABAIN (M)
Fig. 2. Effect of various concentrations of ouabain on intracellular N a + and K + at the end of 100 min incubation. Filled symbols, ouabain-treated (O, N a + ; , , K+); open symbols, control ( © , N a + ; A , K+). Curve through points ( , N a + ; . . . . , K+). Each point represents the mean of duplicates.
96
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Na÷
u.l
u ~
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i
i
i
1
2
3
4
o 6
7
o 8
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Fig. 3. Intracellular concentration of Na + and K + following washout of ouabain. Cells were incubated in 5 x I0 -~ M ouabain for 100 min, washed rapidly 5 times and reincubated at time 0 in ouabain free medium plus 10% fetal calf serum. Open symbols (O, Na + ; z% K+). Curve through points ( , Na + : . . . . , K+). Each point represents the mean of duplicates.
presented a 65 ~ K ÷ loss a n d a 6-fold N a ÷ increase c o m p a r e d to controls. A f t e r rem a i n i n g fairly c o n s t a n t for several hours, K + b e g a n to increase slightly a n d N a b e g a n to fall, so b y the end o f a 12 h i n c u b a t i o n K ÷ was 52 m M a n d N a ÷ was 92 m M . A t o u a b a i n c o n c e n t r a t i o n s greater t h a n 10 -4 M, cells lost K ÷ a n d a c c u m u l a t e d N a ÷. Fig. 2 shows t h a t the loss o f K + at each c o n c e n t r a t i o n at the end o f 100 m i n i n c u b a t i o n was a c c o m p a n i e d by an a l m o s t e q u i m o l a r increase o f cell N a ÷. The finding t h a t t r a n s p o r t o f b o t h N a ÷ and K ÷ was h a l f m a x i m a l l y inhibited at 1.6 x 10 -4 M s t r o n g l y suggests t h a t the m o v e m e n t o f these ions is c o u p l e d . T h e increase in cell N a ÷ in all eases was a c c o m p a n i e d by cell swelling which at 10 -3 M o u a b a i n was 30 ~ .
K ÷ flux with ouabain I f K ÷ toss with o u a b a i n results f r o m the d r u g ' s c a p a c i t y to inhibit a N a ÷ - K ~ A T P a s e , K + influx w o u l d be d i m i n i s h e d to the extent that K + u p t a k e is actively med i a t e d by this enzyme. W i t h 5 x l0 -4 M o u a b a i n , K ÷ influx calculated f r o m u p t a k e o f 42K for 2 min was 8.9 :t: 0.2 (S.D.) p m o l e s / s q . c m . s e c (n - - 5), significantly less t h a n ( P < 0.005) the c o n t r o l level o f 13.0 :a: 1.7 (S.D.) p m o l e s / s q . c m . s e c (n - - l l L T h i s suggests t h a t a p o r t i o n o f K + u p t a k e is active.
Ion movement after ouabain washout I n o r d e r to test the cell c a p a c i t y to r e a c e u m u l a t e K + a n d expel N a + after the n o r m a l c o n c e n t r a t i o n g r a d i e n t was reversed, cultures were i n c u b a t e d in 5 x t 0 -4 M o u a b a i n for 100 rain, w a s h e d r a p i d l y a n d the m e d i u m r e p l a c e d with fresh H F - 1 0 plus 10 ~ fetal c a l f serum. U n d e r these c o n d i t i o n s cells r a p i d l y lost N a ÷ a n d r e a c c u m u l a t e d K + . W i t h i n 10 m i n after o u a b a i n h a d been w a s h e d o u t i n t r a c e l l u l a r K ÷ h a d risen f r o m 40 to 160 m M cell water, while i n t r a c e l l u l a r N a + fell f r o m 120 m M cell w a t e r (Fig. 3). This d e m o n s t r a t e s clearly t h a t C6 h a s the c a p a c i t y to expel N a + in a m a n n e r which is linked to K + u p t a k e . Since this N a ÷ m o v e m e n t t a k e s place a g a i n s t a N a + conc e n t r a t i o n g r a d i e n t when the m e m b r a n e p o t e n t i a l is still negative (see Results-
97
Effect ofouabain on membrane potential) the transport is active. The rapid resumption of ion pumping, after ouabain is washed out, is taken to mean that the glycoside has a low affinity for the N a + - K + ATPase in C6. This is consistent with the finding that the complex formed between cardiac glycosides and Na+-K + ATPase of more ouabain sensitive species such as cow and dog has greater stability than the complex formed with the enzyme from rat, a ouabain insensitive species from which the C6 cell line is derivedL
Kinetics of Na + transport The rate constant for N a + loss after ouabain washout can be derived from the permeability equation: d(Na)i -
-
dt
kl(Nae) -- kz(Nal)
where Nae and Nai are the external and intracellular k2, the constants for influx and efflux are equal to volume, respectively. I f the permeability constants are if the final value for Nat can be found, then kl can be integration becomes: --kzt = In
(Nai) oo -- (Nai)t ( N a 0 oo -- (Nal)o
= k 2 --
Na + concentration and kl and K1 area/volume and K2 area/ not altered as Nai changes and eliminated and the solution on
In 2 t~
where (Na0o is the concentration at the beginning of ouabain washout, (Na0c~ the new steady-state following ouabain washout and t~ is the time required for half the Na + to be lost from the cells 's. F r o m 3 experiments where Na + was lost following ouabain washout, the value of the rate constant for efflux, kz, was 0.0116 i 0.004 sec-L This compared to a rate constant of 0.0256 sec -~ from experiments where effiux was determined by loss of 22Na from pre-loaded cells. The discrepancy could be due to the phenomenon in tracer flux experiments where exchange diffusion of internally labeled Na + for an external non-radioactive N a + gives an apparent flux and rate constant which is actually higher than the true value 38. That such a process takes place in C6 is strongly suggested by the observation reported previously z2 that N a + tracer efflux was 2.5 times larger than Na + influx measured by the non-radioactive method.
Effect of inhibition of protein synthesis on transport To test whether protein synthesis was required for ion pumping following ouabain washout, cells were exposed to 5/~g/ml acetoxycycloheximide (ACCh) and 5 x 10 -4 M ouabain for 100 min then washed and exposed to A C C h for the duration of the experiment. Preliminary experiments showed that a 15 rain preincubation with ACCh inhibited 98.5~o of [14C]leucine incorporated into the T C A precipitable fraction during a 15 min pulse while total protein/DNA decreased by only 1 0 ~ during a 6 h exposure to the inhibitor. Results showed that during the initial exposure to ouabain and in the period following ouabain washout there were no obvious differences in the ionic responses between ACCh-treated and ouabain control cultures (Fig. 4).
98
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120
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120
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MINUTES
Fig. 4. Effect of acetoxycycloheximide on the intracellular accumulation of Na + and loss of K + with ouabain and the ion movements following ouabain washout. Cells were incubated with 5 × 10 -4 M ouabain in the presence and abseaace of 5/tg/ml acetoxycloheximide. Panel A indicates K + concentration. Filled symbols, with A C C h (A), open symbols, without (A). At upward arrow (1') cells were washed free of ouabain and experimental cultures (A) were reincubated with 5/~g/ml ACCh. Panel B indicates Na + concentration: Filled symbols, ACCh ( 0 ) ; open symbols, without ((3). At downward arrow (~), cells were washed as in part A. Each point represents the mean of duplicates except panel B at t : 110 min which is a single determination. Curve through points with ACCh ( . . . . ), control ( --).
This indicates that the turnover rate of proteins involved in ion pumping is greater than the time of exposure to ACCh.
Effect of ouabain on the membrane potential The average membrane potential, measured within an hour after 10 -s M ouabain was given, declined in 3 experiments from the control value of --31 mV to -- 19 mV (Fig, 5A). Because we could not record from cells while changing the bathing solution it was difficult to determine whether there was an immediate depolarization with ouabain suggestive of an electrogenic pump 9. An experiment where two
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Fig. 5. Effect of ouabain and cyanide on membrane potentials in C6. A: (upper) membrane ~ i a l s recorded in 3 experiments in t ~ presence of 10 -3 M ouabain within 90 rain after addition:; Lower portion control at 37 °C. B: membrane potentials recorded in 4 experim©nts in t h ¢ i ~ of 5 cyanide within 90 min after the drug was added. Lower portion, control at 37 OC. Each rectangle represents the number of cells at the potential indicated by the interval on the abscissa.
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Fig. 6. Effect of ouabain on the time course of membrane potentials in C6. Membrane potentials between t -- 0 and t ~ 40 were recorded in Ham's F 10 plus 10 % fetal calf serum. At t 40 (denoted by f') fresh medium containing 5 × 10 -4 M ouabain was added and recordings were made for 160 min. Ouabain medium was then washed out (~,), fresh medium added and the cells were equilibrated for 9.5 h and recordings were again made. Each point represents a single membrane potential from a different cell in the presence (©) and absence (O) of ouabain. m e m b r a n e p o t e n t i a l s o f - - 4 5 m V a n d - - 4 0 mV were o b t a i n e d within 3 m i n after 5 × 10 -4 M o u a b a i n was a d d e d suggests t h a t any electrogenic c o m p o n e n t is p r o b a b l y insignificant, since these p o t e n t i a l s are n o t lower t h a n c o n t r o l p o t e n t i a l s r e c o r d e d i m m e d i a t e l y p r i o r to o u a b a i n a d d i t i o n (Fig. 6). A f t e r these two potentials were rec o r d e d , a p o t e n t i a l o f - - 2 8 mV was o b t a i n e d 14 m i n after o u a b a i n was a d d e d . D u r i n g the next 90 min no p o t e n t i a l greater t h a n - - 3 2 mV was seen. T h e most plausible e x p l a n a t i o n for the o u a b a i n - i n d u c e d d e p o l a r i z a t i o n is the loss o f cell K + and a c c u m u l a t i o n o f cell N a +. If the relative N a + / K + p e r m e a b i l i t y of 0.11 (see ref. 22) is n o t altered by o u a b a i n , the G o l d m a n equation, E = (61 log K0 + P N a / P K (Na0))/Ki + P N a / P K ( N a 0 predicts a d r o p from the c o n t r o l value o f - - 5 2 mV to --31 mV when the cells reach the steady-state c o n c e n t r a t i o n o f 109 m M N a + and 45 m M K + in 5 × 10 .4 M o u a b a i n . A l t h o u g h the m e a s u r e d potentials are lower t h a n the p r e d i c t e d values, the p o t e n t i a l decrease after 100 rain in 5 × 10 .4 M o u a b a i n to - - 2 6 mV ± 4.1 S.D. (n = 6) f r o m a p r e - o u a b a i n c o n t r o l of - - 4 3 mV ~ 6.6 S.D. (n = 4) is 17 mV c o m p a r e d to the p r e d i c t e d decrease o f 21 inV. A l s o in s u p p o r t o f this h y p o t h e s i s is the o b s e r v a t i o n t h a t 10 -4 M o u a b a i n which does n o t cause a n y s u b s t a n t i a l change in the ionic g r a d i e n t s h a d no effect on the m e m b r a n e potential. F u r t h e r m o r e , after o u a b a i n is washed out, the m e m b r a n e potential r e c o r d e d 9 h later r e t u r n e d to c o n t r o l levels. This is consistent with direct i o n measureTABLE I EFFECTS OF METABOLIC INHIBITORS ON CELLULAR
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Values ± s.d. Inhibitor
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Fig. 7. A: effect of 5 mMcyanide on intracellular Na ÷ and K + in C6. Filled symbols, 5 mM cyanide (0, Na + ; A, K+); open symbols, control (O, Na + ; •, K+). B: effect of 24 °C on Jntracellulat Na ÷ and K ÷. Filled symbols, 24 °C (0, Na + ; A, K +); open symbols, control at 37 °C. (O, Na + ; A, K+). In both A and B each point with bars representing one standard deviation, is the average of 3 determinations. Points without bars are the mean of duplicates. ments which show that cells maintain the control level steady-state ion concentrations following ouabain washout.
Effect of cyanide Since ouabain inhibits transport by blocking the pump's capacity to hydrotyze ATP, we looked at the effect of cyanide which limits ATP production at the mitochondrial level. At a concentration of 1.5 m M cyanide after 100 min, there was only a slight increase in cell Na + with no significant change in cell K + (Table I). With 5 m M cyanide there was a rapid increase in cell Na + and a concomitant fall in cell K ~ (Fig. 7A). During the first 20 min, Na ÷ increased from 20 to 68 m M while K ÷ fell from 133 to 99 mM. This represents a molar ratio of 1.41 Na ÷ gained/K + lost. Over the nest 40 min there was a reversal of this pattern and cells began to gain K ÷ and extrude Na + so that by the end of an hour, K + concentration had risen to 114 m M and Na + had fallen to 50 m M cell water. Iodoacetate, an inhibitor o f glycolysis, at 1 m M had little effect on the ionic concentration of Na + and K ÷ at the end of 100 min incubation (Table I). Membrane potentials recorded within an hour after 5 m M cyanide was added decreased significantly (P < 0.001) to 25 mV compared to the control of 36 mV. As in the case of ouabain this depolarization is most readily explained by the loss of cell K +.
Effect of temperature Decreased temperature reduces the active component of K + influx uptake in both squid axon 19 and mammalian skeletal muscle s without affecting K + efflux. Lowering the temperature in C6 produced a significant decrease (P < 0.O25), in K ÷ influx from the control value of 13.0 ± 1.7 S.D. pmoles/sq.em.sec (n = 11) at 37 °C to 10.9 ± 0.23 S.D, pmoles/sq.cm, sec (n = 5). If K + ettlux is not affected by lowered
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Fig. 8. C o m p a r i s o n of the effects of low t e m p e r a t u r e (6 °C), o u a b a i n at 6 °C, a n d o u a b a i n at 37 °C o n intracellular N a + a n d K + concentration. A : intracellular K + ( A , 6 °C; O , 6 °C plus 5 × 10 -4 M o u a b a i n ; O, 5 × 10 -4 M o u a b a i n at 37 °C). B: intracellular N a + (/k, 6 °C; O , 6 °C plus 5 x 10 -4 M o u a b a i n ; O, 5 × 10 -4 M o u a b a i n at 37 °C). E a c h point is the m e a n of duplicate determinations.
temperature, one would predict that the K + concentration in C6 at 24 °C would decrease over time. Contrary to this, we found that at the end of an incubation of 60 min. at 24 °C, intracellular K + was 162 raM, slightly higher than the control value of 154 raM. A possible explanation for this phenomenon is that passive outward K + movement in C6 is reduced at lower temperature. A finding which supports this hypothesis comes from experiments where ionic concentrations were studied in cells at 6 °C. Fig. 8 shows that in the first hour of incubation at 6 °C, cell K + decreased from 160 to 148 m M cell water, while Na + increased from 18 to 33 m M cell water. Because of the high temperature coefficient of Na +-K + ATPase s7, the enzyme is almost completely inhibited in other systems at 6 °C (see refs. 13 and 30). To test this in C6 we incubated cells at 6 °C with 5 × 10-4 M ouabain and found during the first hour that K + fell from 160 to 130 m M cell water, while cell Na + rose from 18 to 35 m M cell water so that the rate of concentration change of these ions was only slightly greater than at 6 °C without ouabain (Fig. 8). This suggests that the rate of
30-
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102 ion transport at 6 °C mediated by a Na+-K + ATPase in C6 is low. When the loss of K* at low levels of ion pumping at 6 "~'Cis compared to K ~ loss at low levels of pumping with ouabain at 37 '~C (Fig. 8), it is apparent that outward K movement is greatly retarded at the lower temperature. Another observation which is consistent with a temperature-induced permeability change is that the membrane potential decreased to a significantly greater degree (P < 0.01) at 24 °C than the decline in potential predicted on the basis of temperature by the G o l d m a n equation (Fig. 9). The equation predicts a drop of 3.1 mV for a 13 °C temperature decrease while the actual membrane potential dropped by 6.3 mV to --29.7 inV. Although it is not apparent whether this involves a change in the ratio of PNa/PK, the depolarization is not induced by changes in the internal concentrations of K ÷ and Na ÷ as is the case with ouabain and cyanide. DISCUSSION
The movement of Na + out of the cell against both a concentration gradient and a negative transmembrane potential following ouabain washout clearly demonstrates that C6 is capable of active Na t transport. It is apparent from the reaccumulation of cell K + which takes place concurrently with the Na ÷ extrusion that in C6 glial cells as in other systems active Na t transport is linked to the uptake of K +,3,~z. A portion of the inward K + under steady-state conditions is also actively mediated since 5 .. 10 -4 M ouabain reduced K + influx by 32 ~ while decreasing external Na ÷ lowered it by 51 ~22. On the basis of the amounts of Na ~ gained and K ~ lost in experiments where ion transport was inhibited by ouabain and cyanide, an estimate of the ratio of N a : K transported by the p u m p can be made. During the first 20 min at 5 × 10 - 4 M ouabain cells lost 50 m M K ÷ and gained 62 m M Na ÷ giving a ratio of 1:24. This can be taken as the ratio of passive leak which would have to be balanced by active pumping in order to maintain the steady-state concentration if the pump were not inhibited ~9. Actually the ratio of active pumping may be slightly higher than this because the passive inward leak of Na ÷ and outward leak of K ÷ are probably retarded somewhat by the decreasing transmembrane gradient of these ions as the intracellular concentrations change with time. With 5 m M cyanide, the loss of K ~ and gain of Na ÷ over the first 20 min, 34 and 48 m M respectively, were slightly less than the concentration changes induced by ouabain. The flux ratio of 1.41. similar to that with ouabain, suggests that the two agents exert their inhibitory effect ultimately on the same transport system. Ouabain does so by blocking hydrolysis of A T P by the pump and cyanide by making high energy phosphate unavailable for transport processes. Interestingly, in mouse brain slices. the decrease in total A T P brought about by cyanide had to be much larger to effect a comparable increase in slice Na + and loss of K ÷ as that induced by ouabain z. In that study, blockage of ion transport suggested that intracellular K + loss was accompanied by an equimolar increase in N a t. In other systems, active flux ratios between 1 and 1.5 have been reported ~5.34.
103 The finding that 1.5 m M cyanide and 1 m M iodoacetate did not affect ionic transport and that transport of Na + and K + apparently resumes in 5 m M cyanide after an initial inhibition may reflect a high rate of energy production in C6 cells. Concentrations of 10 m M sodium azide and 1 m M 2,4-dinitrophenol do not block uptake of the saturable, presumably energy requiring component of 7-aminobutyric acid uptake in C6 36. The 1 7 ~ drop in K ÷ influx when the temperature is dropped to 24 °C presumably reflects an inhibition of the active portion of the K + uptake which is also temperature sensitive in other systems. The paradoxical finding was that despite the decrease in K + influx, intracellular K + concentration was not reduced over time. From our finding of a markedly reduced loss of cell K + at 6 °C compared to that at 37 °C with ouabain, we postulated that decreased temperature in C6 significantly re:ards K + efflux. In this regard, it is interesting that although there is no difference in the amount of K + lost from cat heart muscle at 2-3 °C compared to ouabain at 27 °C zs, kidney cortical slices from hibernating mammals show significantly smaller K ÷ losses at 5-6 °C than with ouabain at higher temperatures 4°. Such a mechanism for preventing massive loss of K ÷ from glial cells following cold exposure may serve to protect the organism from the brain edema which accompanies exposure of brain cells to high levels of K ÷ (see refs. 4 and 23) and to prevent seizures elicited by local buildup of K ÷ (ref. 11). It has been postulated that the observed active uptake of K + in mammalian glial cells14, t6 is linked to outward Na + transportt7, 21. Such hypotheses have been based on finding significant N a + - K + ATPase activity in glial cell fractions8,16 and on the ouabain-induced swelling of glial cells in vivo 6 and in vitro 32. Our finding in C6 provides direct confirmation that K + uptake in glial cells is linked to outward active transport of Na +. One consequence o f a glial Na + pump, activated as in other systems by increased external K + (see reL 3) is that glial metabolism may be altered in response to nerve activity. As extracellular K + increases in the vicinity of active neurons, stimulation of the glial N a + p u m p could alter cellular ratios of A D P / A T P 39. Such stimulated glial Na + p u m p activity may explain the decrease in ATP 35 and in the increase in oxygen consumption 18 in glial cells exposed to high external K +. Although the mechanism is unknown, increases in external K +, comparable to those during neural activity, produce decreased levels of N A D H in pure glial optic nerve preparation of Necturus z7. A Na + p u m p in glia may also play a role in regulation of glial uptake of neurotransmitters. By increasing local Na + concentrations around synaptic terminals following nerve activity glial N a + pumping in concert with an electrogenic p u m p in neurons 31 may facilitate transport of G A B A by glial cells through stimulation of the Na+-dependent component of the G A B A uptake mechanism 2°,36. Evidence from the previous report suggests that the C6 cell line resembles normal gila with respect to permeability of Na +, K + and C1- and that its biophysical properties appear similal to those of normal tissue. Our present finding indicates C6 has retained the linked N a + - K + transport system strongly inferred in normal gila. T u m o r cell lines serve as useful models to the
104 extent they express particular characteristics of normal tissue. Although certain ionic properties in C6 are similar to those irt normal tissue, our present base for comparing normal and tumor glial cells is narrow. As more is learned about the function of glial cells and about fundamental processes of carcinogenesis, the base for comparison will broaden and C6, as a model for ionic mechanisms in glia, will be strengthened or weakened accordingly. In spite of the problems discussed in these papers, in addition to its present applicability to studies of ionic permeability, the C6 cell line appears to be a useful preparation for characterizing the coupled active Na+-K + transport system in glial cells. ACKNOWLEDGEMENTS
We wish to thank Ms. Ruth Cole and Diane Inglish for their expert help with tissue culture and Dr. H. Bracho for helpful comments in the preparation of the manuscript. The material in these two papers was included in a thesis submitted in partial fulfillment of the Degree of Doctor of Philosophy at the University of California, Los Angeles. Supported by USPHS Grants HD-04612, HD-05615 and CA 13538 and by Contract E(04-1)GEN-12 between E R D A and the University of California and NtH Pre-doctoral Fellowship 5-F01 G M 44694-03. REFERENCES 1 ALLEN, J. C., AND SCHWARTZ, A., A possible biochemical explanation for the insensitivity of the rat to cardiac glycosides, d. Pharmacol. exp. Ther., 168 (1969) 42-46. 2 BANAY-SCHWARTZ,M.. TELLER, n . N., GERGELY, A., AND LAJTHA, A., Tile effects of metabolic inhibitors on amino acid uptake and the levels of ATP, Na ÷ and K + in incubated slices of mouse brain, Brain Research, 71 (1974) 117-131. 3 BONTING, S. L., Sodium-potassium activated adenosinetriphosphatase and cation transport. In E. E. BITTAR (Ed.), Membranes and Ion Transport, Vol. l, Wiley-Interscience, London. 1970, pp. 257-363. 4 BOURKE, R. S., AND NELSON, K. M., Further studies on the K + dependent swelling of primate cerebral cortex in vivo: the enzymatic basis of the K + dependent transport of chloride, J. Neurochem., 19 (1972) 663-687. 5 CALKINS, E., TAYLOR, I. i J , AND HASTINGS, A. B., Potassium exchange in the isolated rat diaphragm; effect of anoxia and cold, Amer. J. PhysioL, 177 (1954) 211-218. 6 CONROG,J. L., GONATAS,N. K., AND FEIERMAN,J. R., Effect of intracerebral injection of ouabain on the fine structure of the rat cerebral cortex, Amer. d. Path., 51 (1967) 573-590. 7 COTMAN,C., FIERSCHMAN. H., AND TAYLOR. D,, Subcellular fractionation of cultured glial cells, J. NeurobioL, 2 (1971) 169-180. 8 CUMINS, J., AND HYD~N, H., Adenosine triphosphate levels and adenosine triphosphatases in neurons, glia and neuronal membranes of the vestibular nucleus, Biochim. Biophys. Acta (Amst.), 60 (1962) 271-283. 9 DE WEER, P., AND GEDULDIO, D.. Electrogenic sodium pump m squid giant axon, Science, 179 (1973) 1326-1328. 10 EMBREE, L. J., HESS, H. H., AND SHEIN, H. M., Sodium-potassium ATPase activity of normal and virally transformed hamster astroglia grown subcutaneously, Brain Research, 27 (1971) 422-425. 11 GLASER,G. H.. AND ZUCKERMAN,E. C., Potassium accumulation in extracellular spaces of brain as a possible cause of epileptogenic activity. In R. VIZIOLI (Ed.), Brain and Mind Problems. tl Pensiero Scientifico. Rome, 1968, pp. 309-329 12 GLYNN, I. M., Sodium and potassium movements in h u m a n red cells, J. PhysioL (Lond.), 134 (1956) 278-310.
105 13 GRUENER, N., AND AVI-DOR, Y., Temperature-dependence of activation and inhibition of ratbrain adenosine triphosphatase activated by sodium and potassium ions, Biochem. J., 100 (1966) 762-767. 14 HALJAMAE,H., AND HAMBERGER,A., Potassium uptake by both prepared neuronal and glial cells, J. Neurochem., 18 (1971) 1903-1912. 15 HARRIS, E. J., AND MAIZELS, M., The permeability of h u m a n erythrocytes to sodium, J. Physiol. (Lond.), 113 (1951) 506-524. 16 HENN, 17. A., HALJAMAE, H., AND HAMBERGER, A., Glial cell function: active control of extracellular K ÷ concentration, Brain Research, 43 (1972) 437443. 17 HERTZ, I., Possible role of neuroglia: a potassium mediated neuronal-neuroglial-neuronal impulse transmission system, Nature (Lond.), 206 (1965) 1091-1094. 18 HERTZ, I., Neurologlial localization of potassium and sodium effects on respiration in brain, J. Neurochem., 13 (1966) 1373-1387. 19 HODGKIN, A. L., AND KEYNES, R. D., Active transport of cations in giant axons from Sepia and Loligo, J. Physiol. (Lond.), 128 (1955) 28-60. 20 HUTCHISON, n . T., WERBACH, K., VANCE, C., AND HABER, B., Uptake of neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. 1. Transport of ~,-aminobutyric acid, Brain Research, 66 (1974) 265-274. 21 KIMELBERG,H. K., Active potassium transport and Na ÷ + K + ATPase activity in cultured glioma and neuroblastoma cells, J. Neurochem., 22 (1974) 971-976. 22 KUKES, G., ELUL, R., AND DE VELLIS, J., The ionic basis of the membrane potential in a rat glial cell line, Brain Research, O0 (1976) 00(~000. 23 LOD1N, Z., HARTMAN, J., KAGE, M. P., KORINKOVA, P., AND BOOHER, J., Potassium-induced hydration in cultured neural tissue, Neurobiology, 1 (1971) 69-85. 24 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. 25 McCONAGHEY, P. D., AND MAIZELS, M., Cation exchanges of lactose treated human red cells, J. Physiol. (Lond.), 162 (1962) 485-509. 26 MEDZEHRADSKY, F., NANDHASHRI, P. S., INDOYAGA-VARGAS,V., AND SELLINGER, O. Z., A comparison of ATPase activity of a glial cell fraction and the neuronal perikaryl fraction isolated in bulk from rat cerebral cortex, J. Neurochem., 18 (1971) 1599-1603. 27 ORKANO, P. M.. BRACHO, H. E., AND ORKAND, R., Glial metabolism alteration by potassium levels comparable to those during neural activity, Brain Research, 55 (1973) 467-471. 28 PAGE, E., GOERKE, R. J., AND STORM, S. R., Cat heart muscle in vitro. IV. Inhibition of transport in quiescent muscles, J. gen. Physiol., 47 (1964) 531-543. 29 POST, R. L., ALBRIGHT, C. D., AND DAYANI, K., Resolution of pump and leak components of sodium and potassium ion transport in human erythrocytes, J. gen. Physiol., 50 (1967) 1201-1220. 30 POST, R. L., SEN, A. K., AND ROSENTHAL, A. S., A phosphorylated intermediate in adenosine triphosphate dependent sodium and potassium transport across kidney, J. biol. Chem., 240 (1965) 1437-1445. 31 RANSOM, 13. R., AND GOLDRING, S., Slow hyperpolarization in cells presumed to be Alia in cerebral cortex of cat, J. Neurophysiol., 36 (1973) 879-893. 32 RENKAWEK, K., PALLADINI, G., AND IERADI, L., Morphology of glial cells in the presence of ouabain, Brain Research, 18 (1970) 363 367. 33 SANTEN, R. J., AND AGRANOFF, B. W., Studies on the estimation of deoxyribonucleic acid in rat brain, Biochim. biophys. Acta (Aust.), 72 (1963) 251-262. 34 SEN, A. K., AND POST, R. L., Stoichiometry and localization of adenosine triphosphate dependent sodium and potassium transport in the erythrocyte, J. biol. Chem., 239 (1964) 345-352. 35 SCHOUSBOE,A., BOOHER, J., AND HERTZ, |., Content of ATP in cultivated neurons and astrocytes exposed to balanced and potassium-rich media, J. Neurochem., 17 (1970) 1501-1504. 36 SCHRIER, B. K., AND THOMPSON, E. J., On the role of glial cells in the mammalian central nervous system. Uptake, excretion and metabolism of putative neuro-transmitters by cultured glial tumor cells, J. biol. Chem., 249 (1974) 1769-1780. 37 SKOU, J. C., Enzymatic basis for active transport o f N a + and K + across cell membrane, Physiol. Rev., 45 (1965) 596-615. 38 TOSTESON, D. C., AND HOEFMAN, J. F., Regulation of cell volume by active cation transport in high and low potassium sheep red cells, J. gen. Physiol., 44 (1960) 169-194. 39 WHITTAM, R., The Interdependence of Metabolism and Active Transport in Cellular Functions of Membrane Transport, Prentice-Hall, Englewood Cliffs, N.J., 1964, pp. 139 154. 40 WILLIS, J. S., Uptake of potassium at low temperatures in kidney cortex slices of hibernating mammals, Nature (Lond.), 204 0964) 691-693.