BRAIN RESEARCH ELSEVIER
Brain Research 644 (1994) 1-6
Osmotic regulation of sodium pump in rat brain synaptosomes: the role of cytoplasmic sodium Sergei L. Aksentsev a, Alexander A. Mongin a, Sergei N. Orlov b,, Anatoli A. Rakovich a, Georgy V. Kaler a, Sergei V. Konev a a Institute ofPhotobiology, Belarus Academy of Sciences, Minsk 220733, Belarus b School of Biology, Moscow State University, Moscow 119899, Russia
(Accepted 4 January 1994)
Abstract The effect of hypoosmolality of incubation medium on the rat of ouabain-sensitive 86Rb+ transport in rat brain synaptosomes was studied. A decreased osmolality from 310 to 250 mOsm increased the rate of 86Rb+ uptake from 3.72 to 6.23 nmol/mg of protein min. To evaluate the involvement of cytoplasmic sodium in sodium pump stimulation inhibitors of ion channels and transport pathways able to increase [Na+]in were used. Tetrodotoxin (1 ~M), amiloride (0.5 mM) and verapamil (0.1 mM) had no influence on the osmotic response of the sodium pump. The decrease of sodium concentration in incubation medium to 15 mM, leading to a practical loss of its transmembrane gradient, did not abolish stimulation of pump. No increase in 22Na ÷ influx or intrasynaptosomal sodium content was registered at hypotonic conditions. It is suggested that osmotic regulation of Na+,K ÷ATPase is not connected with an increase of internal sodium through opening of sodium channels, or with activation of other membrane sodium-transporting systems. Key words: Sodium pump; Osmotic regulation; Synaptosome
I. Introduction At various pathological conditions accompanied by a decrease in concentration of electrolytes in the blood plasma, the brain cells are under significant osmotic stress. Thus, an acute hypo-natremia leads to edema of the brain tissue which may result in a potentially lethal organic damage of the brain [3,4,13]. In response to a decrease of electrolytes in plasma the brain is able to lower its osmolality through a loss of the osmotically active cations (sodium and potassium). Such a compensatory mechanism prevents the formation of an osmotic gradient between the brain and plasma and thereby the development of edema [3,4,23,24]. Recent studies have suggested that the sodium pump is involved in this compensatory mechanism. In particular, a decreased transport function of Na+,K+-ATPase in synaptosomes
* Corresponding author. Present address: Centre de recherche H6tel-Dieu de Montr6al, 3850 rue Saint-Urbain, Montr6al, Que., H2W 1T8, Canada. Fax: (1) (514) 843-2715. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00063-1
from female rats in comparison with that of males is well correlated with a higher death rate from brain edema in an acute experimental hyponatremia [13,14]. Similar data on a higher death rate in an acute symptomatic hyponatremia were observed in women of child-bearing age  and in female rabbits . These differences between the sexes may be explained by the fact that female sex hormones (estrogen and progesterone) inhibit Na+,K+-ATPase while the male hormone (testosterone) activates the enzyme [11,14,15,32, 34]. A pattern of electrolyte loss from rat brain during acute hypo-natremia may serve as indirect evidence of the sodium pump involvement in regulatory response to brain edema. In particular, within 1 hr after the beginning of hyposmotic stress (intra-peritoneal infusion of distilled water) there is a distinct decrease of Na + and C1- whereas brain K ÷ remains practically at a constant level . In order to reveal a role of the Na+,K+-ATPase in the protection of brain cells from the damaging effect of hyponatremia it is important to know whether the osmotically-dependent change of cell volume may in-
S.L. Aksentset et al./Brain Research 044 (1904) t t)
fluence the sodium pump activity. Such an approach seems even more justified because in a number of cells of various origin rates of ion fluxes can be increased by changes in osmolality of external medium. This volume-dependent regulation of ion transport underlies processes of regulatory volume decrease (RVD) after swelling in hyposmotic medium and regulatory volume increase (RVI) upon hyperosmotic shrinkage [12,17, 20,27,31 ]. Here we report that swelling of rat brain synaptosomes results in stimulation of ouabain-sensitive 86Rb+ uptake and this effect is not connected with an increase in intracellular concentration of Na +
2. Materials and methods Reagents. Tetrodotoxin was purchased from Sankyo Co. (Japan). Ouabain, veratrine, amiloride, verapamil, furosemide were from Sigma Chem. Co. (USA), choline chloride, sucrose - Serva (Germany), glucose, Tris and all salts - B D H (UK), 86RbCI and 22NaCI - Isotope (Russia). Bumetanide was a gift of Prof. J. D u h m (University of Munich, Germany). Preparation procedures. Synaptosomes were isolated from brain hemispheres of 12- to 16-week-old male white rats by the method of Hajos  and stock suspensions (15.0 mg p r o t e i n / m l ) were prepared in m e d i u m A consisting of (in raM): 132 NaC1, 5 KC1, 1.3 MgCl2, 1.2 NaH2PO4, 10 glucose, 20 Tris-HCl (pH 7.4, 37°C). W h e n osmolality was varied the stock suspension was diluted with m e d i u m B where NaCI was lowered to 92 m M and sucrose was added to concentration 72 raM. Transport measurements. The rates of K + (86Rb+) uptake were determined in m e d i u m A or in media in which concentration of NaCI was decreased to 92 m M and osmolality varied by addition of sucrose. Synaptosome suspensions in medium A or B were preincubated for 7 min at 37°C and then 100/zl aliquots were dispensed into the tubes containing 900 /~1 of respective medium at 37°C and additionally 1 /xCi of S6RbCI. To determine the rates of 22Na+ uptake aliquots (100 /zl) of synaptosomal suspension in m e d i u m A were introduced into 900/zl of prewarmed medium C with osmolality 250 m O s m containing (in mM): eholine chloride - 9 2 , KCI - 5 , MgC12 - 1.3, N a H 2 P O 4 - 1.2, glucose - 10, Tris-HC1 - 2 0 (pH 7.4) or into the same m e d i u m containing additionally 72 m M sucrose (osmolality 310 mOsm). Both media contained 1 /~Ci/ml 22NaCI. After 3 min of incubation at 37°C in a shaker the reactions were terminated by a rapid vacuum filtration through the filters G F / F ( W h a t m a n , UK) with two washings by 7.5 ml of cooled m e d i u m containing (in mM): 140 NaCI, 5 KC1, 20 Tris-HCl buffer pH 7.4, 4°C. T h e filters were dried in the air (at 70°C) for 1 h and placed into scintillation liquid. Radioactivity of samples was m e a s u r e d in scintillation counter Mark-III (Netherlands) using program 10. The 86Rb+ and 22Na+ uptake values were corrected for nonspecific adsorption on filters and synaptosomes evaluated as uptake at zero time of incubation under conditions described above. The rates of S6Rb+ and 22Na+ uptake were calculated as V = A / a c t where A is radioactivity (cpm) of a sample containing c m g of protein, a is the specific radioactivity of 86Rb+ or 22Na+ related to the contents of K ÷ or Na ÷ in incubation medium ( c p m / m m o l ) , t is the incubation time (3 min). T h e rate of ouabain-sensitive K ÷ (S6Rb ÷) uptake was calculated as a difference between the total uptake and that in the presence of 0.2 m M ouabain. The measurement of sodium content in synaptosomes. A content of cytoplasmic sodium was determined by flame photometry essentially
as in  with minor modifications. Briefly. synaplosome suspensiorl was incubated as in the ion transport measurements under isosmotie and hyposmotic conditions. T h e n it was filtered through filters G F / ( ' (Whatman, UK) with two washings by 7.5 ml of cooled medium containing 260 m M sucrose, 2 m M MgCI 2 and 10 mM Tris-HC1 (pit 7.4, 4°C). The filters were placed into 2 ml of lysis medium consisting of 7 m M LiNO 3 for 1 h at room temperature and aliqu(~ts (l,5 rot) were taken for sodium measurements on a llame [lholomelclF L A P H O 4 (Carl Zeiss Yena, Germany). The content of sodium i~).s was expressed as m e a n values per mg of protein. Other methods. Protein was assayed according to Lowry at al. i22] using bovine serum albumin as a standard.
3. Results and discussion
As shown in Fig. 1 86Rb+ uptake in the absence of inhibitors (the total uptake) reaches its maximum level after the 10th min of incubation while ouabain-sensitive uptake becomes saturated by the 5th min. The rate of transport was evaluated as an average value of 86Rb+ uptake for 3 min of incubation. The decrease of external sodium from 132 mM to 92 mM while isotonicity is maintained by addition of sucrose results in reduction of the rate of ouabain-sensitive uptake from 7-10 to 3.5-4.5 nmol K÷/mg protein min (data not shown). Osmolality change from 320 (92 mM NaC1 + 72 mM sucrose) to 250 mOsm (92mM NaCI without sucrose) leads to an increase in the rate of ouabain-sensitive transport (6.43 _+0.27 vs. 3.78 _+ 0.20 nmol K+/mg protein min) (Fig. 2, Table 1). This may be connected with activation of the Na+,K +ATPase due to the increment of [Na+]in induced by sodium influx or with the direct effect of hypotonic swelling on the sodium pump. The following pathways of sodium influx into synaptosomes are known presently: voltage-dependent sodium channels [1,9], Na +/Ca 2+ exchange [10,19], Na + / H + exchange [ 18,25 ]
8eRb* uptake, nmol/mg of protein eo
t, rain Fig. 1. Kinetics of S6Rb+ uptake in rat brain synaptG~,omes in incubation medium A. 1, total uptake; 2, uptake in the presence of 0.2 m M ouabain; 3, ouabain-sensitive component of the uptake.
S.L. Aksentsev et al. / Brain Research 644 (1994) 1-6
80RI~ uptake, nmol/mg of protein, min
Oemolelity, mOem Fig. 2. Effect of osmolality of incubation m e d i u m on the rate of S6Rb+ uptake in synaptosomes. The concentration of [Na ÷ ]o is 92 mM. 1, total uptake; 2, uptake in the presence of 0.2 m M ouabain; 3, ouabain-sensitive c o m p o n e n t of the uptake. Data are m e a n s -+ S.E. of 3 experiments performed in quadruplicates.
and Na+,K÷,2CI - cotransport . Therefore we tested the effect of specific inhibitors of sodium channels and sodium transport systems on the volume-dependent activation of the sodium pump. Blockage of sodium channels by tetrodotoxin added in the media with osmolalities 310 and 230 mOsm had no influence on the sodium pump stimulation (Table 1). It should be noted, however, that in the presence of tetrodotoxin the total and ouabain-sensitive 86Rb+ uptake are slightly decreased. The data of Table 1 were obtained upon incubation of synaptosomes in the medium B containing 92 mM NaC1. At 132 mM NaC1 (medium A) the effect of tetrodotoxin is more distinct Table 1 Effects of inhibitors of various ion-transporting systems on the rate of ouabain-sensitive 86Rb+ uptake in rat brain synaptosomes Inhibotor
Control 1 ~M TTX 0.5 m M amiloride 0.1 m M verapamil 25 ~ M bumetanide + 0.5 m M furosemide
Ouabain-sensitive 86Rb + uptake n m o l / m g of protein min
Number of experi-
310 m O s m a 250 m O s m b
3.78 + 0.20 2.64+0.44 4.12_+0.57 3.90+0.42 4.87 _+0.43
4 2 2 1 3
6.42+ 0.27 5.54+0.54 7.49+0.66 7.12+0.31 5.48 + 0.48
** * * *
Data are m e a n s + S.E. a Composition of m e d i u m 310 m O s m (in mM): NaCI 92, KCI 5, MgCI 2 1.3, N a H 2 P O 4 1.2, glucose 10, sucrose 72, Tris-HCl 20 (pH 7.4). b Composition of m e d i u m 250 m O s m (in mM): NaCI 92, KCI 5, MgCI 2 1.3, N a H 2 P O 4 1.2, glucose 10, sucrose 18, Tris-HC1 20 (pH 7.4). * P < 0.05; ** P < 0.01. *** Each experiment are m e a n s of four replicates.
(Fig. 3). The reason for tetrodotoxin-induced inhibition of the sodium pump is presumably a decrease of [Na+]in due to the blockage of sodium channels some of which are remaining open even at nondepolarizing conditions. Another pathway of sodium influx, the N a + / H ÷ exchange was inhibited with amiloride (0.5 mM) which like tetrodotoxin failed to change the swelling-induced stimulation of the sodium pump (Table 1). In order to evaluate a contribution of Na+/Ca 2÷ exchange system we used 0.1 mM verapamil. At this concentration verapamil inhibits not only potential-dependent calcium channels but also Na-dependent 45Ca2+ influx into synaptosomes mediated by Na+/Ca 2÷ exchanger . It follows from Table 1 that verapamil did not prevent activation of sodium pump in hypotonic medium. Thus, sodium channels, N a ÷ / H ÷ and Na+/Ca 2+ exchange systems are not involved in volume regulation of sodium pump. Bumetanide, a specific inhibitor of Na÷,K+,2C1cotransport (25 /~M) in combination with furosemide (0.5 mM)which inhibits both Na÷,K÷,2CI- and K÷,CIcotransport [6,12,27] decreased significantly the activation of pump by swelling (Table 1). However, these inhibitors for reasons unknown increased by about 30% the ouabain-sensitive 86Rb+ uptake in isotonic medium (at 92 mM Na÷). The last fact raises doubts about the actual specificity of bumetanide and furosemide and complicates an interpretation of the response of S6Rb+ uptake to these drugs under hypotonic conditions. To resolve such a problem we made additional experiments in which the sodium concentration in incubation medium was reduced to 15 mM. With that aim 100/~1 aliquots of synaptosome suspen-
86Rb*uptake, nmol/mg of protein, min I 15
Fig. 3. Effect of 1 /xM tetrodotoxin on S6Rb+ uptake in rat synaptosomes in incubation medium A. 1 and 3, total uptake; 2 and 4, ouabain-sensitive uptake; 3 and 4, uptake in the presence of 1 /xM tetrodotoxin. Data are m e a n s + S.E. of 2 experiments performed in quadruplicates. * P < 0.05.
S.L. Aksentser et a l . / B r a i n Research 644 (1994) / n
Na*(~2Na*) uptake, nmol/mg, min
86Rb*uptake, nmol/mg of protein, min i 2
1 2 310 mOsm
3 4 2 3 0 mOsm
Fig. 4. Hypotonic stimulation of 86Rb+ uptake in rat brain synaptosomes in the medium with reduced concentration of sodium ([Na + ]o = 15 mM). Aliquots (100/zl) of synaptosomal suspension in medium A were introduced into 900/xl of prewarmed medium with osmolality 230 mOsm containing (in mM): choline chloride 92, KC1 5, MgCI: 1.3, NaH2PO 4 1.2, glucose 10, Tris-HCl 20 (pH 7.4) or into the same medium containing additionally 72 mM sucrose (osmolality 310 mOsm). The rate of S6Rb+ transport was measured as described in Materials and Methods. Data are means _+S.E. of 2 experiments performed in quadruplicates. * P < 0.01.
sion in m e d i u m A w e r e i n t r o d u c e d into 9 0 0 / z l s a m p l e s o f m e d i u m C k e p t at 37°C. U n d e r t h e s e c o n d i t i o n s a t r a n s m e m b r a n e s o d i u m g r a d i e n t is p r a c t i c a l l y abolished. H o w e v e r , j u d g i n g by t h e results o f Fig. 4, a relative v a l u e o f t h e s o d i u m p u m p s t i m u l a t i o n is the s a m e as in m e d i a c o n t a i n i n g 92 m M N a ÷. Since a d r a s t i c r e d u c t i o n o f e x t e r n a l s o d i u m m u s t inhibit s o d i u m influx via Na+,K+,2C1 - c o t r a n s p o r t this fact is a s t r o n g a r g u m e n t a g a i n s t i n v o l v e m e n t o f N a + , K ÷, 2C1- c o t r a n s p o r t in activation o f N a + , K + - A T P a s e by h y p o t o n i c stress. F u r t h e r , we h a d m e a s u r e d directly N a ÷ influx a n d s o d i u m c o n t e n t in s y n a p t o s o m e s in isotonic a n d h y p o t o n i c m e d i a . A t 15 m M of e x t e r n a l s o d i u m t h e influx o f 22Na+ (Fig. 5) is essentially t h e s a m e at 310 a n d 230 m O s m . Fig. 5 shows also t h a t in the b o t h m e d i a t h e o p e n i n g o f s o d i u m c h a n n e l s with v e r a t r i n e results in a c o n s i d e r a b l e i n c r e a s e o f influx which was c o m p l e t e l y b l o c k e d by t e t r o d o t o x i n . T h e latter data demonstrate that our experimental conditions a r e s u i t a b l e for d e t e c t i o n o f any physiological c h a n g e in s o d i u m influx. S i m i l a r results w e r e o b t a i n e d in m e a s u r e m e n t s o f t h e i n t r a s y n a p t o s o m a l s o d i u m cont e n t at 92 m M o f e x t e r n a l N a ÷ (Fig. 6). A g a i n we f a i l e d to n o t i c e any effect of h y p o t o n i c i t y while d e p o l a r i z a t i o n with v e r a t r i n e c a u s e d a significant i n c r e m e n t o f N a ÷ which was p r e v e n t e d by t e t r o d o t o x i n . T h u s it m a y b e c o n c l u d e d t h a t s t i m u l a t i o n o f t h e p u m p is a d i r e c t c o n s e q u e n c e o f s y n a p t o s o m e swelling a n d is n o t m e d i a t e d by e l e v a t e d activity o f s o m e o t h e r t r a n s p o r t
Fig. 5. Na + (~2Na+) influx in rat brain synaptosomes in the media with different osmolalities ([Na+]o = 15 mM). Experiments were performed as described in Fig. 4. 1, control; 2, in the presence of 0.5 /~M TTX; 3, in the presence of veratrine (50 #g/ml); 4, TTX+ veratrine. Data are means± S.E. of two experiments performed in quadruplicates. * P < 0.001
system c a p a b l e o f i n c r e a s i n g t h e c y t o p l a s m i c s o d i u m c o n c e n t r a t i o n . T h e s e results a r e in a g o o d a g r e e m e n t with conclusions of V e n o s a  on [ N a + ] i n - i n d e p e n d e n t h y p o o s m o t i c s t i m u l a t i o n o f s o d i u m p u m p in frog muscle. E a r l i e r a s w e l l i n g - i n d u c e d s t i m u l a t i o n o f N a + , K +A T P a s e was r e g i s t e r e d in g i a n t snail n e u r o n s a t the high c o n t e n t o f i n t e r n a l s o d i u m as an o b l i g a t o r y c o n d i tion . R e c e n t l y we o b s e r v e d a h y p o t o n i c a c t i v a t i o n o f t h e s o d i u m p u m p in c u l t u r e d C6 g l i o m a cells ( d a t a in press). In this c o n n e c t i o n it s e e m s p l a u s i b l e that
JJmol Na+/mg of protein 140 I 120
80 60 40 20
Fig. 6. The content of sodium in rat brain synaptosomes in the media with various tonicity ([Na+]o = 92 mM). Experiments were performed as described in Materials and Methods: 1, control; 2, in the presence of 0.5/zM TTX; 3, in the presence of veratrine (50 p.g/ml); 4, TTX+veratrine. Data are means+_S.E. of two experiments performed in quadruplicates. * P < 0.05.
S.L. Aksentsev et al. / Brain Research 644 (1994) 1-6
activation under cell swelling is a peculiar feature of the sodium pump in the excitable tissue. All the cells studied so far responded to hyposmotic swelling by an increase of K ÷ efflux via K÷,CI - cotransport, K + / H+-exchange or stretch-activated K channels [8,12,20, 28,29,31]. For nervous cells the activation of similar systems could be disadvantageous since the decrease of internal K ÷ would reduce resting potential and decrease excitability. Activation of sodium pump would be a compensatory mechanism of such a loss of K ÷. On the other hand, uncompensated transfer of ions by the sodium pump (3 Na ÷ outward in exchange for 2 K ÷ inward) makes it possible in principle to decrease an osmolar gradient between the brain and plasma and thereby to prevent the cell swelling without K ÷ loss. The involvement of the Na÷,K+-ATPase in regulatory volume decrease (RVD) was suggested for rat astrocytes  and lymphoma ceils . However, the efficiency of Na+,K+-ATPase in the RVD process (approximately two-fold activation) is much smaller in comparison with that of the K + transport systems mentioned above. Synaptosomes posses significant volume regulatory decrease capability . A relative contribution of various ion pathways in this reaction remains to be seen.
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