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Hypertonic shrinking but not hypotonic swelling increases sodium concentration in rat brain synaptosomes
Brain Research Bulletin 73 (2007) 135–142
Hypertonic shrinking but not hypotonic swelling increases sodium concentration in rat brain synaptosomes Tatyana V. Waseem, Viktoriya A. Kolos, Liudmila P. Lapatsina 1 , Sergei V. Fedorovich ∗ Institute of Biophysics and Cell Engineering, Akademicheskaya Street, 27, Minsk 220072, Belarus Received 28 February 2007; accepted 1 March 2007 Available online 28 March 2007
Abstract Neurotransmitter release is dependent on both calcium and sodium influx. Hypotonic swelling and hypertonic shrinking of neurons evokes calcium-independent exocytosis of neurotransmitters into the synaptic cleft. To date, there are not too much data available on relationship between extracellular osmolarity and sodium concentration in presynaptic endings. In the present study we investigated the effects of hypotonic swelling and hypertonic shrinking on sodium levels, as measured using fluorescent dyes SBFI-AM and Sodium Green in rat brain synaptosomes. Reduction of incubation medium osmolarity from 310 to 230 mOsm did not raise the intrasynaptosomal sodium concentration. An increase of osmolarity from 310 to 810 mOsm is accompanied by a dose-dependent elevation of sodium concentration from 8.1 ± 0.5 to 46.5 ± 2.8 mM, respectively. This effect was insensitive to several channel inhibitors such as: tetrodotoxin, an inhibitor of voltage-gated sodium channels, bumetanide, an inhibitor of Na+ /K+ /2Cl− cotransport, gadolinium, an inhibitor of nonselective mechanosensitive channels, ruthenium red, an inhibitor of transient receptor potential channel and amiloride, an inhibitor of epithelial sodium channel/degenerin. Additionally, using the fluorescent dye BCECF-AM, we have shown that hypertonic shrinking caused a dose-dependent acidification of intrasynaptosomal cytosol, which suggests that the Na+ /H+ exchanger is not involved in the effect of increased osmolarity on cytosolic sodium levels. The increase in intrasynaptosomal sodium concentrations following increases in osmolarity is probably due to sodium influx through another sodium channels. © 2007 Elsevier Inc. All rights reserved. Keywords: Synaptosomes; Sodium; Hypertonic; Hypotonic; Sodium Green; SBFI
Neurotransmitter release depends on calcium and sodium influx. Hypotonic swelling and hypertonic shrinking of neuronal presynaptic endings evokes calcium-independent exocytosis with following neurotransmitter release. However, mechanism coupling changes in cell volume with neurotransmitter release is still unknown [15,38,47,52]. Neurotransmitter release caused by hypotonic swelling of presynaptic endings may simply be due to one or more of the following events: sodium influx through mechanosensitive channels; plasma membrane depolarization followed by influx of sodium and calcium through voltage-gated channels; activation of mechanosensitive anion channels or reversing of neurotransmitter transporters [47,48]. However, some other papers suggest that the relationship between neurotransmitter release and changes in cell volume might be more complex [1,32,52]. ∗
Corresponding author. Tel.: +375 172 84 2252; fax: +375 172 84 2359. E-mail address: [email protected] (S.V. Fedorovich). 1 Present address: Max-Delbr¨ uck-Centrum f¨ur Moleculare Medizin, Berlin, Germany. 0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.03.002
Firstly data concerning plasma membrane depolarization at hypotonic swelling of synaptosomes were contradictory: Tuz et al. [47] observed membrane depolarization following swelling, whereas Mongin et al. [32] found no changes in membrane potential. Secondly [47] concluded that hypotonicity-dependent exocytosis was dependent on extracellular sodium concentration, but did not quantify intrasynaptosomal sodium content. However, during hypotonic swelling, Aksentsev et al. [1] observed no changes 22 Na influx in synaptosomes. Finally results obtained with fluorescent styryl dyes suggest that intrasynaptosomal calcium regulates fusion pore dynamics during hypotonic swelling, rather than provoking exocytosis [52]. Taking together, these data suggest that the mechanism underlying neurotransmitter release at hypotonic swelling of presynaptic endings is not clear. Hypertonic shrinking is widely used as a model for estimation of the size of readily releasable pool of synaptic vesicles [36,38,49]. It is established that increasing of medium
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osmolarity leads to exocytosis without involving of calcium influx [25,38]. Neurotransmitter release induced by hypertonic shrinking similarly with Ca dependent exocytosis requires SNARE (soluble (N-ethylmaleimide-sensitive fusion protein) attachment receptor) proteins [2]. Tetanus toxin, which cleaves SNARE proteins, only partially inhibits glutamate release caused by hypertonic shrinking of synaptosomes [2]. These results suggest that under hypertonic conditions, neurotransmitters may be released in sodiumdependent manner directly from a cytosolic pool, as was shown in the presence of latrotoxin [29]. However the role of sodium in hypertonic induced neurotransmitter release remains unclear. Hypertonic shrinking activates many executive transport systems of regulatory volume increase (RVI) in most cell types [28]. Generally, this process involves sodium uptake. For instance, the Na+ /H+ exchanger is activated following increase of extracellular osmolarity in lymphocytes and erythrocytes [20,34], and hypertonic shrinking of synaptosomes leads to stimulation of Na+ /K+ /2Cl− cotransport [4]. Furthermore, osmotic shock can open many types of mechanosensitive channels, which are permeable for sodium [24,43]. To date, however, intrasynaptosomal sodium content during hypotonic swelling and hypertonic shrinking has yet to be quantified. Accurate quantification of intracellular sodium is important for development of drugs targeting neuronal sodium channels, as these drugs might be used to treat stroke, pain, migraine, some psychiatric diseases [6,9,19,55]. SBFI, a ratiometric dye, is usually used to measure sodium concentrations during changes in cell volume [3]. Unfortunately, this dye has some disadvantages, including a low signal/noise ratio and loading level and a requirement for equipment with two-wavelength excitation regime. To circumvent these problems, recently an indirect method of sodium measurements has been developed based on membrane potential changes during depolarizing sodium influx [26]. In our study, we measured sodium concentrations using Sodium Green, since it lacks the disadvantages of SBFI. Synaptosomes, isolated neuronal presynaptic endings, provide a useful model for investigation of neurotransmitter release mechanisms as they have the same intracellular signalization, ion transport, synaptic vesicles and synaptic proteins as intact endings [21,51,52]. Hypotonic swelling and hypertonic shrinking induces exocytosis and neurotransmitter release in synaptosomes [2,3,47,48,52,54]. In the present paper, we investigated cytsolic sodium concentration in rat brain synaptosomes at anisoosmotic conditions using fluorescent dyes (SBFI-AM, sodium-binding benzofuran isophtalate acetoxymethyl ester) [11,46] and Sodium Green [50,56]. We found that hypertonic shrinking but not hypotonic swelling leads to increase of intrasynaptosomal sodium content. 1. Materials and methods 1.1. Materials Sucrose was obtained from Fluka (Buchs, Switzerland). Pluronic acid F-127, monensin, gramicidin D, gadolinium chloride, veratrine, veratridine,
ruthenium red, amiloride, 2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM) were purchased from Sigma (St. Louis, MO, USA). Bumetanide and sodium-binding benzofuran isophtalate acetoxymethyl ester (SBFI-AM) were purchased from Calbiochem (La Jolla, CA, USA). Tetrodotoxin was from Sankyo Co. (Tokyo, Japan) and Sodium Green tetraacetate was obtained from Molecular Probes (Eugene, OR, USA).
1.2. Preparation of synaptosomes All animal experiments were carried out in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC). Purified synaptosomes were isolated from brain hemispheres of male Wistar rats (Minsk State Medical University, Minsk, Belarus, 12–16 weeks old, n = 45) in sucrose discontinuous gradient as described by Hajos [22]. Stock suspensions of synaptosomes (10 mg protein/ml) were prepared in medium A (132 NaCl, 5 KCl, 10 glucose, 1.3 MgCl2 , 1.2 NaH2 PO4 , 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, pH 7.4, 310 mOsm). Before the experiment, synaptosomes were preincubated for 10 min at 37 ◦ C, after that kept on ice and used within 3 h.
1.3. Measurement of intrasynaptosomal sodium concentration by fluorescent dye SBFI-AM Intrasynaptosomal sodium concentration was monitored by fluorescent dye SBFI-AM according Deri and Adam-Vizi [11]. Synaptosomes purification was carried out in medium A and then after additional washing in 10 volumes of medium B (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, 264 sucrose, pH 7.4, 310 mOsm) the pellet was resuspended in the same medium. The synaptosomal suspension was incubated in media containing of 10 M of SBFI-AM mixed with 20% of pluronic acid F127 for 30 min at 37 ◦ C. The suspension was then diluted 1:5 with medium B and incubated for a further 60 min at 37 ◦ C. Excess extracellular dye was removed by sedimentation and the final pellet resuspended in medium B. To investigate sodium concentration, 200 l of loaded synaptosomes was placed in a cuvette containing 2 ml of isotonic incubation medium A, to final protein concentration of approximately 0.6 mg/ml. Additional various reagents were then added directly to the cuvette. Fluorescence intensity was determined by measuring the ratio of light emitted at λem 510 nm to that passed through both excitation filters at λex 335/375 nm. Data were collected using ratio programme on spectrofluorimeter Cary Eclipse (“Varian”, USA) or SFL1211A (“Solar”, Belarus) at 37 ◦ C in constant stirring. An increase in the ratio of fluorescence intensity measured at 335/510 nm to the intensity at 375/510 nm indicated an increase in intracellular sodium content. Calibration curves describing the relationship between 335/375 ratio of fluorescence intensity and intracellular sodium levels were obtained for resulting quantification. In order to manipulate intrasynaptosomal sodium levels, synaptosomes were treated with both the Na+ /H+ ionophore monensin and the ionophore gramicidin D, which makes the plasma membrane permeable for monovalent cations. Assuming that under these conditions the cytosolic sodium concentration becomes the same as the concentration in extracellular medium. The appropriate amount of sodium chloride was equimolarly replaced by choline chloride in calibration samples. During the investigation of the effects of hypotonic swelling 860 l of hypotonic medium C (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, pH 7.4, 40 mOsm) was placed to cuvette containing 2 ml of synaptosomal suspension. This addition corresponds to lowering of osmolarity from 310 to 230 mOsm. The low sodium isotonic medium B was added in control experiments, to control for both dilution of the dye and demarcation of effect of lowering of sodium concentration from that of hypotonicity itself. To get resulted representative curve values of fluorescence intensity obtained in control experiments was extracted from values of fluorescence intensity obtained in experiments using hypotonic medium. Osmolarity of the incubation medium was increased by addition of 150–500 mM sucrose; an equal quantity of incubation medium A was added in control experiments. To get resulted representative curve values of fluorescence intensity obtained in control experiments was extracted from values of fluorescence intensity obtained in experiments using hypertonic medium.
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1.4. Determination of pHi by fluorescent dye BCECF-AM Intrasynaptosomal pH was monitored by fluorescent dye BCECF-AM (2 ,7 bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester) as described by Nachshen and Drapeau [33]. Synaptosomes were resuspended in medium A after additional washing in 10-volumes excess of the same medium. The suspension was first incubated in the presence of 10 M BCECF-AM for 15 min at 37 ◦ C, then diluted 1:1 with medium A and additionally incubated for 15 min at 37 ◦ C. Extracellular dye was washed out three times by sedimentation the final pellet was resuspended in medium A. In order to estimate intrasynaptosomal pH, 100 l of loaded synaptosomes were added to a cuvette containing 2 ml of isotonic incubation medium A resulting in a final protein concentration 1 mg/ml. Subsequent treatments were added directly to a cuvette. Fluorescence intensity was recorded at λex/em = 505/530 nm at 37 ◦ C in constant stirring. The suspension of synaptosomes was treated with the ionophores gramicidin D and monensin in order to establish a calibration curve. Aliquots of 2 l 0.5 M HCl were added to each sample for calibration, since this addition shifts pH of the suspension to 0.05 units. Manipulation of incubation medium osmolarity was performed as described above.
1.5. Investigation of intrasynaptosomal sodium content by fluorescent dye Sodium Green tetraacetate Synaptosomes purification was carried out in medium A and then after additional washing in 10 volumes of medium B (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, 264 sucrose, pH 7.4, 310 mOsm) the pellet was resuspended in the same medium. The synaptosomes suspension was incubated for 30 min at 37 ◦ C in presence of 10 M Sodium Green tetraacetate mixed with equal amount of 20% of pluronic acid F127. The suspension was then diluted 1:5 with medium B and incubated for an additional 60 min at 37 ◦ C. Extracellular dye was removed by sedimentation and the final pellet resuspended in medium B. To investigate sodium concentration, 200 l of loaded synaptosomes were added to cuvette containing 2 ml of incubation medium A given a final protein concentration 0.6 mg/ml. Subsequent treatments were added directly to a cuvette. Fluorescence intensity was recorded at λex/em = 512/550 nm on spectrofluorimeter SFL1211A at 37 ◦ C in constant stirring. An increase in intracellular sodium level was indicated by enhancement of fluorescence signal intensity. Alteration of incubation medium osmolarity was performed as described above.
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sodium concentration. Veratridine has similar action. The effect of veratrine on sodium influx was blocked by tetrodotoxin (TTX), a selective inhibitor of voltage-gated sodium channels. These data are in agreement with the results from previous studies [11] and confirm that the fluorescent dye SBFI-AM can be used for accurate estimation of sodium levels in isolated neuronal presynaptic endings. 2.2. The effect of anisoosmotic conditions on sodium concentration in rat brain synaptosomes as estimated by SBFI-AM Fig. 1 shows that a decrease of medium osmolarity from 310 to 230 mOsm did not change the cytosolic sodium concentration. However, we found that a reduction of the sodium concentration while maintaining constant osmolarity in the extracellular medium results in a small increase in intrasynaptosomal sodium level. The reason of this paradox is unknown. Increase in incubation medium osmolarity caused a dosedependent increase in cytosolic sodium level (Fig. 1). Thus, direct measurements have shown that hypertonic shrinking, but not hypotonic swelling increases intrasynaptosomal sodium concentration. Previously we have shown that neither a reduction of osmolarity from 310 to 230 mOsm nor an increase to 810 mOsm affected lactate dehydrogenase release from synaptosomes [52,53]. More significant lowering of osmolarity than 230 mOsm can lead to lysis (data not shown). These results indicate that simple damage of plasma membrane is not sufficient to explain our data. In order to elucidate the mechanism by which hypertonic shrinking may affect the intrasynaptosomal sodium concentration, we treated the synaptosomal suspension with 500 nM TTX and 0.1 mM gadolinium, a nonselective inhibitor of mechanosensitive channels [27]. Neither inhibitor affected basal
1.6. Other methods Protein was assayed according to Lowry et al. [30] using bovine serum albumin as a standard. Data is presented as means ± S.E.M. where indicated, statistical significance was evaluated using two-tailed Student’s t-test.
2. Results 2.1. Regulation of sodium content in isotonic conditions We found that the cytosolic sodium concentration in rat brain synaptosomes in isotonic conditions was 8.1 ± 0.5 mM. This value is close to the results obtained in other laboratories using the same method [8,11,42]. A similar sodium concentration was reported in hippocampal neurons [39]. In order to confirm detection of increase in sodium levels, we treated the synaptosomes with veratrine which stabilizes voltage-gated sodium-channels in open state [5,11]. As expected, veratrine treatment increased intrasynaptosomal
Fig. 1. The effect of hypotonic swelling and hypertonic shrinking on sodium concentration in rat brain synaptosomes: (1) Isotonic medium. No addition was made. (2) Decreasing of osmolarity from 310 to 230 mOsm. (3) Decreasing of [NaCl]out from 132 to 92 mM, osmolarity 310 mOsm. (4) Increasing of osmolarity from 310 to 460 mOsm. (5) Increasing of osmolarity from 310 to 660 mOsm. (6) Increasing of osmolarity from 310 to 810 mOsm. Bars represent average sodium concentration during 5 min after additions. Data presented are mean values ± S.E.M. of 10 experiments. *P ≤ 0.05; **P ≤ 0.01 vs. control.
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Fig. 3. Hypertonic shrinking decreases intrasynaptosomal pH. Final sucrose concentration was 150; 350; 500 mM. Sucrose solutions were added where indicated. Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
Fig. 2. Gadolinium and TTX affects neither basal sodium content (A) nor hypertonicity-evoked sodium influx (B) as evoked by 500 mM sucrose in rat brain synaptosomes. Synaptosomes were preincubated 30 min with 100 M GdCl3 (1) or with 0.5 M TTX (2) after dye loading. Data are expressed as a fluorescence ratio. Bars represent average fluorescence ratio during 5 min after additions. One hundred percent level corresponds to fluorescence ratio in untreated synaptosomes. Data presented are mean values ± S.E.M. of five experiments.
sodium content nor sodium influx as evoked by 500 mM sucrose (Fig. 2) or 150 mM sucrose as well (data not shown). Previous studies have demonstrated the indicated concentrations of the inhibitors used are sufficient for channels blockade [11,12]. 2.3. The effect of hypertonic shrinking on intrasynaptosomal pH Sodium can potentially enter the cell during hypertonic shrinking via the epithelial sodium channel/degenerin [24,43] or via the Na+ /H+ exchanger [7]. Both transport systems are sensitive to inhibition by amiloride. Amiloride has autofluorescence with an excitation maximum of approximately 340–380 nm. Therefore, this compound and its derivatives might interfere with intracellular ion assays involving fluorescent dyes with similar excitation wavelengths [23,41,56]. Also it was shown that hippocampal neurons contain amiloride-insensitive Na+ /H+ exchanger [7].
If hypertonic shrinking stimulates Na+ /H+ exchange, we predicted a rise in intracellular pH accompanied by sodium influx. Therefore, we investigated the effect of increase of incubation medium osmolarity on intrasynaptosomal pH using fluorescent dye BCECF-AM. We showed that pHi in synaptosomes in basal conditions was less than incubation medium pH (Fig. 3), in agreement with observations reported by other laboratories [33,45]. Finally, we found that hypertonic shrinking caused dose-dependent acidification of synaptosomal cytoplasm but not alcalinization as would be expected under conditions of Na+ /H+ exchanger activation (Fig. 3). 2.4. Investigation of intrasynaptosomal sodium by fluorescent dye Sodium Green The amiloride-sensitive epithelial sodium channel/degenerin [24,43] is not the only route for sodium influx. Previous studies provide evidences for participation of bumetanide-sensitive Na+ –K+ –Cl cotransport [4] and ruthenium red-sensitive transient receptor potential (TRP) channels in sodium transport across cytoplasmic membrane [35]. Unfortunately, the compounds required for inhibition of these channels, bumetanide and ruthenium red, affect spectral properties of SBFI (data not shown); therefore an alternative method for measuring sodium levels is required. Intracellular sodium concentration can also be determined using Sodium Green [50,56]. Unlike SBFI, it is not ratiometric dye and its spectral properties allow it to be used with inhibitors with intrinsic fluorescent signal [56]. In order to determine whether Sodium Green could be used to reliably measure sodium levels, we treated synaptosomes with the sodium channel activator veratrine and sodium pump inhibitor ouabain. We found that both treatments caused an increase in [Na+ ]i , indicated by a rise in dye fluorescence intensity (Fig. 4A). Veratridine has similar effect (data not shown).
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Fig. 4. Investigation of intrasynaptosomal sodium level by Sodium Green fluorescent dye. (A) Veratrine increases intrasynaptosomal sodium levels (Ver). Pre-treatment of synaptosomes with TTX prevents the effect of veratrine (TTX + Ver). Ouabain has the similar effect as veratrine on sodium levels (O). Veratrine (0.25 mg/ml), tetrodotoxin (500 nM) + veratrine (0.25 mg/ml) and ouabain (1 mM) were added where indicated. (B) The effect of hypotonic swelling and hypertonic shrinking on sodium levels as indicated by Sodium Green fluorescence. Additions of different media were made where indicated. Results of control experiments (Ic) were extracted from results of experiments on investigation of hypertonic shrinking and hypotonic swelling (Ie). Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
Preincubation of synaptosomes with tetrodotoxin abolished the effect of veratrine in sodium influx. Next, we confirmed that hypertonic shrinking but not hypotonic swelling, increases sodium concentration in rat brain synaptosomes (Fig. 4B). Thus, Sodium Green can be used to accurately determine intrasynaptosomal sodium concentrations and to investigate osmolarity induced sodium transport in synaptosomes. Furthermore, this dye has a better signal/noise ratio than SBFI. Amiloride, bumetanide and ruthenium red do not affect sodium increase during sucrose-induced hypertonic shrinkage (Fig. 5). Also all tested compounds did not affect increase in sodium level caused by 150 mM sucrose application (data not shown). Concentration of amiloride, bumetanide and ruthenium red was sufficient for complete blocking of investigated transport systems [4,24,35,43].
Fig. 5. The effect of ion transport inhibitors on hypertonicity-evoked increase of sodium levels, as measured by rise of Sodium Green fluorescence in rat brain synaptosomes. Synaptosomes were treated with 1 mM amiloride (Amil) (panel A); 100 M bumetanide (Bum) (panel B) or 50 M ruthenium red (RR) (panel C) 30 min. Con: non-treated synaptosomes. Results of control experiments (Ic) were extracted from results of experiments on investigation of hypertonic shrinking (Ie). The osmolarity was increased to 810 mOsm where indicated. Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
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3. Discussion In the present study, we have demonstrated that changes in osmolarity modulate sodium content in neuronal presynaptic endings. Hypotonic swelling did not increase sodium levels in synaptosomal cytosol (Fig. 1) despite previous work suggested swelling might activate voltage-gated and mechanosensitive sodium channels [47]. Increase of synaptosomal volume when isolated presynaptic endings were placed in 240 mOsm incubation medium was confirmed by Babila et al. [4]. Our preparation has at least voltage-gated sodium channels because sodium concentration increase was evoked by veratrine and it was blocked by TTX (Fig. 4A). These data suggest that neurotransmitter release induced by hypotonic swelling of synaptosomes [17,47,48,52,54] is therefore unlikely to be mediated by sodium influx through voltage-gated or mechanosensitive channels. Tuz and colleagues [47,48] showed that both complete removal of sodium from incubation medium and treatment with lanthanides can partially inhibit neurotransmitter release in synaptosomes induced by reduction in incubation medium osmolarity. Unfortunately, no direct measurement of sodium concentration was performed in these reports. Furthermore, sodium withdrawal might affect many different ion gradients required for normal housekeeping of presynaptic endings. Lanthanides have been shown to have many targets beyond mechanosensitive channels [12,14,18]. Finally, we showed that osmotic stimulation of the sodium pump by hypotonic swelling did not significantly change sodium content in synaptosomes. Our results are in agreement with data of Aksentsev et al. [1], when used 22 Na to determine sodium influx. By contrast, we found that hypertonic shrinking led to a rapid dose-dependent increase in cytosolic sodium concentration (Figs. 1 and 4B). The effect develops within several seconds and reaches the value 46.5 ± 2.8 mM. Our data suggest that Na+ /H+ exchange is not activated in synaptosomes following enhances in osmolarity. Moreover, we did not find evidence for involvement of transient receptor potential channels, voltage-gated channels, Na+ , K+ , Cl− cotransport, or epithelial sodium channel/degenerin, as sodium influx was not affected by TTX, Gd3+ , bumetanide, amiloride or ruthenium red (Figs. 2 and 5). Some of these compounds can influence not only sodium transport in synaptosomes, but, for instance, neurotransmitters release itself [40,41,44]. Used methods permit direct measurement of intrasynaptosomal sodium and allow it to exclude some possible side effects. Our observation that hypertonic shrinking is accompanied by dose-dependent acidification (Fig. 3) provides further evidence that Na+ /H+ exchange in synaptosomes is not activated by an increase of osmolarity. Taken together, our experiments therefore suggest a mechanism other than the basic transport system might be involved in regulating sodium influx during hypertonic shrinking in rat brain synaptosomes. Two such mechanisms are: activation of sodium channels unknown origin or sodium concentrating upon hypertonic shrinking due to reduction in cytosolic volume. Ashton and
Ushkaryov [3] describe that, in synaptosomes, increasing incubation medium osmolarity by 100 and 500 mOsm leads to a decrease in cytosolic volume of 50% and 85%, respectively. These values approximately correspond to observation that in our experiments sodium content increases in two and eight times above basal level. However, it is unlikely that this effect is due to a reduction in the volume of the shrunk presynaptic endings. Firstly, if changes in cytosolic volume affect sodium concentration, then we would expect to see a decrease in sodium concentration when the size of the nerve endings is increased. As shown in Figs. 1 and 4, hypotonic swelling had no effect on sodium levels. Secondly, Ashton and Ushkaryov [3] discovered that calcium increase following hypertonic shrinking occurs due to release from intracellular stores, not from simple mechanical concentration of calcium ions. Finally, rises in intrasynaptosomal sodium levels have been shown to activate sodium pump [13]. Therefore, we would expect cytosolic Na+ levels to be lower than the levels we observed, unless hypertonic shrinking activates sodium channels. Taken together, our experiments do not completely discard possibility of cytoplasmic volume reduction as the cause of increase of [Na]i , but we rather suggest activation of sodium channels in presynaptic endings. Although we were unable to identify these channels using a pharmacological approach, we cannot conclusively rule out involvement of some members of the epithelial sodium channel/degenerin family that are not sensitive to amiloride [24,31] and might underlie the nature of the phenomenon described. Unfortunately, lack of specific inhibitors prevents direct investigation of the contribution of sodium increase to neurotransmitter release following hypertonic shrinking. Recent studies have demonstrated that sodium may have several roles in the synapse; an increase in sodium concentration can directly affect SNARE proteins [10] or lead to calcium release from mitochondria through Na+ /Ca2+ exchanger followed by exocytosis [37,48]. Sodium influx might also affect non-vesicular neurotransmitter release, since Ashton et al. [2] found that hypertonic-induced neurotransmitter release in synaptosomes was only partially blocked by SNARE-cleaving clostridia toxins. Our data suggest that such cytosolic neurotransmitter efflux might be driven by reversal of neurotransmitter transporters after sodium influx [16,29]. Therefore, increases in presynaptic sodium concentrations may be involved in regulation of both vesicular and nonvesicular neurotransmitter release. Acknowledgments This work was supported by CAEN-ISN (Committee in Aid of Neurochemistry of International Society of Neurochemistry) and The Physiological Society on “Centres of excellence” scheme. Initial experiments were supported by The Wellcome Trust (grant 069417/Z/02/Z). We thank Dr. A. Pooler for improvement of English.
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Hypertonic shrinking but not hypotonic swelling increases sodium concentration in rat brain synaptosomes Tatyana V. Waseem, Viktoriya A. Kolos, Liudmila P. Lapatsina 1 , Sergei V. Fedorovich ∗ Institute of Biophysics and Cell Engineering, Akademicheskaya Street, 27, Minsk 220072, Belarus Received 28 February 2007; accepted 1 March 2007 Available online 28 March 2007
Abstract Neurotransmitter release is dependent on both calcium and sodium influx. Hypotonic swelling and hypertonic shrinking of neurons evokes calcium-independent exocytosis of neurotransmitters into the synaptic cleft. To date, there are not too much data available on relationship between extracellular osmolarity and sodium concentration in presynaptic endings. In the present study we investigated the effects of hypotonic swelling and hypertonic shrinking on sodium levels, as measured using fluorescent dyes SBFI-AM and Sodium Green in rat brain synaptosomes. Reduction of incubation medium osmolarity from 310 to 230 mOsm did not raise the intrasynaptosomal sodium concentration. An increase of osmolarity from 310 to 810 mOsm is accompanied by a dose-dependent elevation of sodium concentration from 8.1 ± 0.5 to 46.5 ± 2.8 mM, respectively. This effect was insensitive to several channel inhibitors such as: tetrodotoxin, an inhibitor of voltage-gated sodium channels, bumetanide, an inhibitor of Na+ /K+ /2Cl− cotransport, gadolinium, an inhibitor of nonselective mechanosensitive channels, ruthenium red, an inhibitor of transient receptor potential channel and amiloride, an inhibitor of epithelial sodium channel/degenerin. Additionally, using the fluorescent dye BCECF-AM, we have shown that hypertonic shrinking caused a dose-dependent acidification of intrasynaptosomal cytosol, which suggests that the Na+ /H+ exchanger is not involved in the effect of increased osmolarity on cytosolic sodium levels. The increase in intrasynaptosomal sodium concentrations following increases in osmolarity is probably due to sodium influx through another sodium channels. © 2007 Elsevier Inc. All rights reserved. Keywords: Synaptosomes; Sodium; Hypertonic; Hypotonic; Sodium Green; SBFI
Neurotransmitter release depends on calcium and sodium influx. Hypotonic swelling and hypertonic shrinking of neuronal presynaptic endings evokes calcium-independent exocytosis with following neurotransmitter release. However, mechanism coupling changes in cell volume with neurotransmitter release is still unknown [15,38,47,52]. Neurotransmitter release caused by hypotonic swelling of presynaptic endings may simply be due to one or more of the following events: sodium influx through mechanosensitive channels; plasma membrane depolarization followed by influx of sodium and calcium through voltage-gated channels; activation of mechanosensitive anion channels or reversing of neurotransmitter transporters [47,48]. However, some other papers suggest that the relationship between neurotransmitter release and changes in cell volume might be more complex [1,32,52]. ∗
Corresponding author. Tel.: +375 172 84 2252; fax: +375 172 84 2359. E-mail address: [email protected] (S.V. Fedorovich). 1 Present address: Max-Delbr¨ uck-Centrum f¨ur Moleculare Medizin, Berlin, Germany. 0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.03.002
Firstly data concerning plasma membrane depolarization at hypotonic swelling of synaptosomes were contradictory: Tuz et al. [47] observed membrane depolarization following swelling, whereas Mongin et al. [32] found no changes in membrane potential. Secondly [47] concluded that hypotonicity-dependent exocytosis was dependent on extracellular sodium concentration, but did not quantify intrasynaptosomal sodium content. However, during hypotonic swelling, Aksentsev et al. [1] observed no changes 22 Na influx in synaptosomes. Finally results obtained with fluorescent styryl dyes suggest that intrasynaptosomal calcium regulates fusion pore dynamics during hypotonic swelling, rather than provoking exocytosis [52]. Taking together, these data suggest that the mechanism underlying neurotransmitter release at hypotonic swelling of presynaptic endings is not clear. Hypertonic shrinking is widely used as a model for estimation of the size of readily releasable pool of synaptic vesicles [36,38,49]. It is established that increasing of medium
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osmolarity leads to exocytosis without involving of calcium influx [25,38]. Neurotransmitter release induced by hypertonic shrinking similarly with Ca dependent exocytosis requires SNARE (soluble (N-ethylmaleimide-sensitive fusion protein) attachment receptor) proteins [2]. Tetanus toxin, which cleaves SNARE proteins, only partially inhibits glutamate release caused by hypertonic shrinking of synaptosomes [2]. These results suggest that under hypertonic conditions, neurotransmitters may be released in sodiumdependent manner directly from a cytosolic pool, as was shown in the presence of latrotoxin [29]. However the role of sodium in hypertonic induced neurotransmitter release remains unclear. Hypertonic shrinking activates many executive transport systems of regulatory volume increase (RVI) in most cell types [28]. Generally, this process involves sodium uptake. For instance, the Na+ /H+ exchanger is activated following increase of extracellular osmolarity in lymphocytes and erythrocytes [20,34], and hypertonic shrinking of synaptosomes leads to stimulation of Na+ /K+ /2Cl− cotransport [4]. Furthermore, osmotic shock can open many types of mechanosensitive channels, which are permeable for sodium [24,43]. To date, however, intrasynaptosomal sodium content during hypotonic swelling and hypertonic shrinking has yet to be quantified. Accurate quantification of intracellular sodium is important for development of drugs targeting neuronal sodium channels, as these drugs might be used to treat stroke, pain, migraine, some psychiatric diseases [6,9,19,55]. SBFI, a ratiometric dye, is usually used to measure sodium concentrations during changes in cell volume [3]. Unfortunately, this dye has some disadvantages, including a low signal/noise ratio and loading level and a requirement for equipment with two-wavelength excitation regime. To circumvent these problems, recently an indirect method of sodium measurements has been developed based on membrane potential changes during depolarizing sodium influx [26]. In our study, we measured sodium concentrations using Sodium Green, since it lacks the disadvantages of SBFI. Synaptosomes, isolated neuronal presynaptic endings, provide a useful model for investigation of neurotransmitter release mechanisms as they have the same intracellular signalization, ion transport, synaptic vesicles and synaptic proteins as intact endings [21,51,52]. Hypotonic swelling and hypertonic shrinking induces exocytosis and neurotransmitter release in synaptosomes [2,3,47,48,52,54]. In the present paper, we investigated cytsolic sodium concentration in rat brain synaptosomes at anisoosmotic conditions using fluorescent dyes (SBFI-AM, sodium-binding benzofuran isophtalate acetoxymethyl ester) [11,46] and Sodium Green [50,56]. We found that hypertonic shrinking but not hypotonic swelling leads to increase of intrasynaptosomal sodium content. 1. Materials and methods 1.1. Materials Sucrose was obtained from Fluka (Buchs, Switzerland). Pluronic acid F-127, monensin, gramicidin D, gadolinium chloride, veratrine, veratridine,
ruthenium red, amiloride, 2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM) were purchased from Sigma (St. Louis, MO, USA). Bumetanide and sodium-binding benzofuran isophtalate acetoxymethyl ester (SBFI-AM) were purchased from Calbiochem (La Jolla, CA, USA). Tetrodotoxin was from Sankyo Co. (Tokyo, Japan) and Sodium Green tetraacetate was obtained from Molecular Probes (Eugene, OR, USA).
1.2. Preparation of synaptosomes All animal experiments were carried out in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC). Purified synaptosomes were isolated from brain hemispheres of male Wistar rats (Minsk State Medical University, Minsk, Belarus, 12–16 weeks old, n = 45) in sucrose discontinuous gradient as described by Hajos [22]. Stock suspensions of synaptosomes (10 mg protein/ml) were prepared in medium A (132 NaCl, 5 KCl, 10 glucose, 1.3 MgCl2 , 1.2 NaH2 PO4 , 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, pH 7.4, 310 mOsm). Before the experiment, synaptosomes were preincubated for 10 min at 37 ◦ C, after that kept on ice and used within 3 h.
1.3. Measurement of intrasynaptosomal sodium concentration by fluorescent dye SBFI-AM Intrasynaptosomal sodium concentration was monitored by fluorescent dye SBFI-AM according Deri and Adam-Vizi [11]. Synaptosomes purification was carried out in medium A and then after additional washing in 10 volumes of medium B (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, 264 sucrose, pH 7.4, 310 mOsm) the pellet was resuspended in the same medium. The synaptosomal suspension was incubated in media containing of 10 M of SBFI-AM mixed with 20% of pluronic acid F127 for 30 min at 37 ◦ C. The suspension was then diluted 1:5 with medium B and incubated for a further 60 min at 37 ◦ C. Excess extracellular dye was removed by sedimentation and the final pellet resuspended in medium B. To investigate sodium concentration, 200 l of loaded synaptosomes was placed in a cuvette containing 2 ml of isotonic incubation medium A, to final protein concentration of approximately 0.6 mg/ml. Additional various reagents were then added directly to the cuvette. Fluorescence intensity was determined by measuring the ratio of light emitted at λem 510 nm to that passed through both excitation filters at λex 335/375 nm. Data were collected using ratio programme on spectrofluorimeter Cary Eclipse (“Varian”, USA) or SFL1211A (“Solar”, Belarus) at 37 ◦ C in constant stirring. An increase in the ratio of fluorescence intensity measured at 335/510 nm to the intensity at 375/510 nm indicated an increase in intracellular sodium content. Calibration curves describing the relationship between 335/375 ratio of fluorescence intensity and intracellular sodium levels were obtained for resulting quantification. In order to manipulate intrasynaptosomal sodium levels, synaptosomes were treated with both the Na+ /H+ ionophore monensin and the ionophore gramicidin D, which makes the plasma membrane permeable for monovalent cations. Assuming that under these conditions the cytosolic sodium concentration becomes the same as the concentration in extracellular medium. The appropriate amount of sodium chloride was equimolarly replaced by choline chloride in calibration samples. During the investigation of the effects of hypotonic swelling 860 l of hypotonic medium C (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, pH 7.4, 40 mOsm) was placed to cuvette containing 2 ml of synaptosomal suspension. This addition corresponds to lowering of osmolarity from 310 to 230 mOsm. The low sodium isotonic medium B was added in control experiments, to control for both dilution of the dye and demarcation of effect of lowering of sodium concentration from that of hypotonicity itself. To get resulted representative curve values of fluorescence intensity obtained in control experiments was extracted from values of fluorescence intensity obtained in experiments using hypotonic medium. Osmolarity of the incubation medium was increased by addition of 150–500 mM sucrose; an equal quantity of incubation medium A was added in control experiments. To get resulted representative curve values of fluorescence intensity obtained in control experiments was extracted from values of fluorescence intensity obtained in experiments using hypertonic medium.
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1.4. Determination of pHi by fluorescent dye BCECF-AM Intrasynaptosomal pH was monitored by fluorescent dye BCECF-AM (2 ,7 bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester) as described by Nachshen and Drapeau [33]. Synaptosomes were resuspended in medium A after additional washing in 10-volumes excess of the same medium. The suspension was first incubated in the presence of 10 M BCECF-AM for 15 min at 37 ◦ C, then diluted 1:1 with medium A and additionally incubated for 15 min at 37 ◦ C. Extracellular dye was washed out three times by sedimentation the final pellet was resuspended in medium A. In order to estimate intrasynaptosomal pH, 100 l of loaded synaptosomes were added to a cuvette containing 2 ml of isotonic incubation medium A resulting in a final protein concentration 1 mg/ml. Subsequent treatments were added directly to a cuvette. Fluorescence intensity was recorded at λex/em = 505/530 nm at 37 ◦ C in constant stirring. The suspension of synaptosomes was treated with the ionophores gramicidin D and monensin in order to establish a calibration curve. Aliquots of 2 l 0.5 M HCl were added to each sample for calibration, since this addition shifts pH of the suspension to 0.05 units. Manipulation of incubation medium osmolarity was performed as described above.
1.5. Investigation of intrasynaptosomal sodium content by fluorescent dye Sodium Green tetraacetate Synaptosomes purification was carried out in medium A and then after additional washing in 10 volumes of medium B (5 KCl, 1.3 MgCl2 , 1.2 NaH2 PO4 , 10 glucose, 2.0 CaCl2 , 15 HEPES, 5 Tris-aminometan, 264 sucrose, pH 7.4, 310 mOsm) the pellet was resuspended in the same medium. The synaptosomes suspension was incubated for 30 min at 37 ◦ C in presence of 10 M Sodium Green tetraacetate mixed with equal amount of 20% of pluronic acid F127. The suspension was then diluted 1:5 with medium B and incubated for an additional 60 min at 37 ◦ C. Extracellular dye was removed by sedimentation and the final pellet resuspended in medium B. To investigate sodium concentration, 200 l of loaded synaptosomes were added to cuvette containing 2 ml of incubation medium A given a final protein concentration 0.6 mg/ml. Subsequent treatments were added directly to a cuvette. Fluorescence intensity was recorded at λex/em = 512/550 nm on spectrofluorimeter SFL1211A at 37 ◦ C in constant stirring. An increase in intracellular sodium level was indicated by enhancement of fluorescence signal intensity. Alteration of incubation medium osmolarity was performed as described above.
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sodium concentration. Veratridine has similar action. The effect of veratrine on sodium influx was blocked by tetrodotoxin (TTX), a selective inhibitor of voltage-gated sodium channels. These data are in agreement with the results from previous studies [11] and confirm that the fluorescent dye SBFI-AM can be used for accurate estimation of sodium levels in isolated neuronal presynaptic endings. 2.2. The effect of anisoosmotic conditions on sodium concentration in rat brain synaptosomes as estimated by SBFI-AM Fig. 1 shows that a decrease of medium osmolarity from 310 to 230 mOsm did not change the cytosolic sodium concentration. However, we found that a reduction of the sodium concentration while maintaining constant osmolarity in the extracellular medium results in a small increase in intrasynaptosomal sodium level. The reason of this paradox is unknown. Increase in incubation medium osmolarity caused a dosedependent increase in cytosolic sodium level (Fig. 1). Thus, direct measurements have shown that hypertonic shrinking, but not hypotonic swelling increases intrasynaptosomal sodium concentration. Previously we have shown that neither a reduction of osmolarity from 310 to 230 mOsm nor an increase to 810 mOsm affected lactate dehydrogenase release from synaptosomes [52,53]. More significant lowering of osmolarity than 230 mOsm can lead to lysis (data not shown). These results indicate that simple damage of plasma membrane is not sufficient to explain our data. In order to elucidate the mechanism by which hypertonic shrinking may affect the intrasynaptosomal sodium concentration, we treated the synaptosomal suspension with 500 nM TTX and 0.1 mM gadolinium, a nonselective inhibitor of mechanosensitive channels [27]. Neither inhibitor affected basal
1.6. Other methods Protein was assayed according to Lowry et al. [30] using bovine serum albumin as a standard. Data is presented as means ± S.E.M. where indicated, statistical significance was evaluated using two-tailed Student’s t-test.
2. Results 2.1. Regulation of sodium content in isotonic conditions We found that the cytosolic sodium concentration in rat brain synaptosomes in isotonic conditions was 8.1 ± 0.5 mM. This value is close to the results obtained in other laboratories using the same method [8,11,42]. A similar sodium concentration was reported in hippocampal neurons [39]. In order to confirm detection of increase in sodium levels, we treated the synaptosomes with veratrine which stabilizes voltage-gated sodium-channels in open state [5,11]. As expected, veratrine treatment increased intrasynaptosomal
Fig. 1. The effect of hypotonic swelling and hypertonic shrinking on sodium concentration in rat brain synaptosomes: (1) Isotonic medium. No addition was made. (2) Decreasing of osmolarity from 310 to 230 mOsm. (3) Decreasing of [NaCl]out from 132 to 92 mM, osmolarity 310 mOsm. (4) Increasing of osmolarity from 310 to 460 mOsm. (5) Increasing of osmolarity from 310 to 660 mOsm. (6) Increasing of osmolarity from 310 to 810 mOsm. Bars represent average sodium concentration during 5 min after additions. Data presented are mean values ± S.E.M. of 10 experiments. *P ≤ 0.05; **P ≤ 0.01 vs. control.
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Fig. 3. Hypertonic shrinking decreases intrasynaptosomal pH. Final sucrose concentration was 150; 350; 500 mM. Sucrose solutions were added where indicated. Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
Fig. 2. Gadolinium and TTX affects neither basal sodium content (A) nor hypertonicity-evoked sodium influx (B) as evoked by 500 mM sucrose in rat brain synaptosomes. Synaptosomes were preincubated 30 min with 100 M GdCl3 (1) or with 0.5 M TTX (2) after dye loading. Data are expressed as a fluorescence ratio. Bars represent average fluorescence ratio during 5 min after additions. One hundred percent level corresponds to fluorescence ratio in untreated synaptosomes. Data presented are mean values ± S.E.M. of five experiments.
sodium content nor sodium influx as evoked by 500 mM sucrose (Fig. 2) or 150 mM sucrose as well (data not shown). Previous studies have demonstrated the indicated concentrations of the inhibitors used are sufficient for channels blockade [11,12]. 2.3. The effect of hypertonic shrinking on intrasynaptosomal pH Sodium can potentially enter the cell during hypertonic shrinking via the epithelial sodium channel/degenerin [24,43] or via the Na+ /H+ exchanger [7]. Both transport systems are sensitive to inhibition by amiloride. Amiloride has autofluorescence with an excitation maximum of approximately 340–380 nm. Therefore, this compound and its derivatives might interfere with intracellular ion assays involving fluorescent dyes with similar excitation wavelengths [23,41,56]. Also it was shown that hippocampal neurons contain amiloride-insensitive Na+ /H+ exchanger [7].
If hypertonic shrinking stimulates Na+ /H+ exchange, we predicted a rise in intracellular pH accompanied by sodium influx. Therefore, we investigated the effect of increase of incubation medium osmolarity on intrasynaptosomal pH using fluorescent dye BCECF-AM. We showed that pHi in synaptosomes in basal conditions was less than incubation medium pH (Fig. 3), in agreement with observations reported by other laboratories [33,45]. Finally, we found that hypertonic shrinking caused dose-dependent acidification of synaptosomal cytoplasm but not alcalinization as would be expected under conditions of Na+ /H+ exchanger activation (Fig. 3). 2.4. Investigation of intrasynaptosomal sodium by fluorescent dye Sodium Green The amiloride-sensitive epithelial sodium channel/degenerin [24,43] is not the only route for sodium influx. Previous studies provide evidences for participation of bumetanide-sensitive Na+ –K+ –Cl cotransport [4] and ruthenium red-sensitive transient receptor potential (TRP) channels in sodium transport across cytoplasmic membrane [35]. Unfortunately, the compounds required for inhibition of these channels, bumetanide and ruthenium red, affect spectral properties of SBFI (data not shown); therefore an alternative method for measuring sodium levels is required. Intracellular sodium concentration can also be determined using Sodium Green [50,56]. Unlike SBFI, it is not ratiometric dye and its spectral properties allow it to be used with inhibitors with intrinsic fluorescent signal [56]. In order to determine whether Sodium Green could be used to reliably measure sodium levels, we treated synaptosomes with the sodium channel activator veratrine and sodium pump inhibitor ouabain. We found that both treatments caused an increase in [Na+ ]i , indicated by a rise in dye fluorescence intensity (Fig. 4A). Veratridine has similar effect (data not shown).
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Fig. 4. Investigation of intrasynaptosomal sodium level by Sodium Green fluorescent dye. (A) Veratrine increases intrasynaptosomal sodium levels (Ver). Pre-treatment of synaptosomes with TTX prevents the effect of veratrine (TTX + Ver). Ouabain has the similar effect as veratrine on sodium levels (O). Veratrine (0.25 mg/ml), tetrodotoxin (500 nM) + veratrine (0.25 mg/ml) and ouabain (1 mM) were added where indicated. (B) The effect of hypotonic swelling and hypertonic shrinking on sodium levels as indicated by Sodium Green fluorescence. Additions of different media were made where indicated. Results of control experiments (Ic) were extracted from results of experiments on investigation of hypertonic shrinking and hypotonic swelling (Ie). Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
Preincubation of synaptosomes with tetrodotoxin abolished the effect of veratrine in sodium influx. Next, we confirmed that hypertonic shrinking but not hypotonic swelling, increases sodium concentration in rat brain synaptosomes (Fig. 4B). Thus, Sodium Green can be used to accurately determine intrasynaptosomal sodium concentrations and to investigate osmolarity induced sodium transport in synaptosomes. Furthermore, this dye has a better signal/noise ratio than SBFI. Amiloride, bumetanide and ruthenium red do not affect sodium increase during sucrose-induced hypertonic shrinkage (Fig. 5). Also all tested compounds did not affect increase in sodium level caused by 150 mM sucrose application (data not shown). Concentration of amiloride, bumetanide and ruthenium red was sufficient for complete blocking of investigated transport systems [4,24,35,43].
Fig. 5. The effect of ion transport inhibitors on hypertonicity-evoked increase of sodium levels, as measured by rise of Sodium Green fluorescence in rat brain synaptosomes. Synaptosomes were treated with 1 mM amiloride (Amil) (panel A); 100 M bumetanide (Bum) (panel B) or 50 M ruthenium red (RR) (panel C) 30 min. Con: non-treated synaptosomes. Results of control experiments (Ic) were extracted from results of experiments on investigation of hypertonic shrinking (Ie). The osmolarity was increased to 810 mOsm where indicated. Experiments run in parallel under the different experimental conditions tested. Curves represent three to five independent experiments.
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3. Discussion In the present study, we have demonstrated that changes in osmolarity modulate sodium content in neuronal presynaptic endings. Hypotonic swelling did not increase sodium levels in synaptosomal cytosol (Fig. 1) despite previous work suggested swelling might activate voltage-gated and mechanosensitive sodium channels [47]. Increase of synaptosomal volume when isolated presynaptic endings were placed in 240 mOsm incubation medium was confirmed by Babila et al. [4]. Our preparation has at least voltage-gated sodium channels because sodium concentration increase was evoked by veratrine and it was blocked by TTX (Fig. 4A). These data suggest that neurotransmitter release induced by hypotonic swelling of synaptosomes [17,47,48,52,54] is therefore unlikely to be mediated by sodium influx through voltage-gated or mechanosensitive channels. Tuz and colleagues [47,48] showed that both complete removal of sodium from incubation medium and treatment with lanthanides can partially inhibit neurotransmitter release in synaptosomes induced by reduction in incubation medium osmolarity. Unfortunately, no direct measurement of sodium concentration was performed in these reports. Furthermore, sodium withdrawal might affect many different ion gradients required for normal housekeeping of presynaptic endings. Lanthanides have been shown to have many targets beyond mechanosensitive channels [12,14,18]. Finally, we showed that osmotic stimulation of the sodium pump by hypotonic swelling did not significantly change sodium content in synaptosomes. Our results are in agreement with data of Aksentsev et al. [1], when used 22 Na to determine sodium influx. By contrast, we found that hypertonic shrinking led to a rapid dose-dependent increase in cytosolic sodium concentration (Figs. 1 and 4B). The effect develops within several seconds and reaches the value 46.5 ± 2.8 mM. Our data suggest that Na+ /H+ exchange is not activated in synaptosomes following enhances in osmolarity. Moreover, we did not find evidence for involvement of transient receptor potential channels, voltage-gated channels, Na+ , K+ , Cl− cotransport, or epithelial sodium channel/degenerin, as sodium influx was not affected by TTX, Gd3+ , bumetanide, amiloride or ruthenium red (Figs. 2 and 5). Some of these compounds can influence not only sodium transport in synaptosomes, but, for instance, neurotransmitters release itself [40,41,44]. Used methods permit direct measurement of intrasynaptosomal sodium and allow it to exclude some possible side effects. Our observation that hypertonic shrinking is accompanied by dose-dependent acidification (Fig. 3) provides further evidence that Na+ /H+ exchange in synaptosomes is not activated by an increase of osmolarity. Taken together, our experiments therefore suggest a mechanism other than the basic transport system might be involved in regulating sodium influx during hypertonic shrinking in rat brain synaptosomes. Two such mechanisms are: activation of sodium channels unknown origin or sodium concentrating upon hypertonic shrinking due to reduction in cytosolic volume. Ashton and
Ushkaryov [3] describe that, in synaptosomes, increasing incubation medium osmolarity by 100 and 500 mOsm leads to a decrease in cytosolic volume of 50% and 85%, respectively. These values approximately correspond to observation that in our experiments sodium content increases in two and eight times above basal level. However, it is unlikely that this effect is due to a reduction in the volume of the shrunk presynaptic endings. Firstly, if changes in cytosolic volume affect sodium concentration, then we would expect to see a decrease in sodium concentration when the size of the nerve endings is increased. As shown in Figs. 1 and 4, hypotonic swelling had no effect on sodium levels. Secondly, Ashton and Ushkaryov [3] discovered that calcium increase following hypertonic shrinking occurs due to release from intracellular stores, not from simple mechanical concentration of calcium ions. Finally, rises in intrasynaptosomal sodium levels have been shown to activate sodium pump [13]. Therefore, we would expect cytosolic Na+ levels to be lower than the levels we observed, unless hypertonic shrinking activates sodium channels. Taken together, our experiments do not completely discard possibility of cytoplasmic volume reduction as the cause of increase of [Na]i , but we rather suggest activation of sodium channels in presynaptic endings. Although we were unable to identify these channels using a pharmacological approach, we cannot conclusively rule out involvement of some members of the epithelial sodium channel/degenerin family that are not sensitive to amiloride [24,31] and might underlie the nature of the phenomenon described. Unfortunately, lack of specific inhibitors prevents direct investigation of the contribution of sodium increase to neurotransmitter release following hypertonic shrinking. Recent studies have demonstrated that sodium may have several roles in the synapse; an increase in sodium concentration can directly affect SNARE proteins [10] or lead to calcium release from mitochondria through Na+ /Ca2+ exchanger followed by exocytosis [37,48]. Sodium influx might also affect non-vesicular neurotransmitter release, since Ashton et al. [2] found that hypertonic-induced neurotransmitter release in synaptosomes was only partially blocked by SNARE-cleaving clostridia toxins. Our data suggest that such cytosolic neurotransmitter efflux might be driven by reversal of neurotransmitter transporters after sodium influx [16,29]. Therefore, increases in presynaptic sodium concentrations may be involved in regulation of both vesicular and nonvesicular neurotransmitter release. Acknowledgments This work was supported by CAEN-ISN (Committee in Aid of Neurochemistry of International Society of Neurochemistry) and The Physiological Society on “Centres of excellence” scheme. Initial experiments were supported by The Wellcome Trust (grant 069417/Z/02/Z). We thank Dr. A. Pooler for improvement of English.
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