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Brain Research, 548 (1991) 222-227 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 000689939116578P

BRES 16578

Diazepam enhances intrasynaptosomal free calcium concentrations Joseph V. Martin, Donald R. Keir and Hsin-yi Lee Department of Biology, Rutgers University, Camden, N.J. 08102 (U.S.A.) (Accepted 4 December 1990) Key words: Benzodiazepine; Calcium ion; Diazepam; Fura-2; Fura-2/AM; Synaptosome

Synaptosomes prepared from brains of rats were incubated in different concentrations of diazepam under conditions designed to reduce the action of a reversed Na+/Ca2÷ exchanger. In synaptosomes depolarized in the presence of added Ca 2÷, doses of diazepam ranging from 0.1 to 100/~M were found to significantly enhance Ca 2÷ levels measured with the fluorescent dye fura-2, compared to control incubations without drug. Furthermore, doses of diazepam as low as 1/~M significantly increased the concentration of Ca 2+ in non-depolarized synaptosomes without added Ca 2+ in the medium. The effects of depolarization and diazepam treatment were synergistic in increasing the levels of intrasynaptosomal Ca2+ .

INTRODUCTION T h e early view of b e n z o d i a z e p i n e (BZ) r e c e p t o r function was derived from studies which focused almost exclusively on a single subtype of B Z r e c e p t o r (type I). This r e c e p t o r has a well-established association with a ~-aminobutyric acid ( G A B A A ) r e c e p t o r modulating the activity of a chloride channel 36'37. R e c e n t studies, however, have emphasized the existence of several structurally distinct subtypes of B Z receptors 19'25'3°-32. The functional h e t e r o g e n e i t y of the B Z receptors is reflected in a n u m b e r of presynaptic actions of B Z s that are not related to an action at a chloride channel zg. Several lines of evidence have shown that B Z s can affect the flux of calcium ions (Ca 2÷) into neural tissue 23"33. F o r e x a m p l e , m i c r o m o l a r concentrations of B Z facilitate the u p t a k e of 45Ca 2+ into depolarized s y n a p t o s o m e s 23'24"28 and enhance Ca2+-mediated K + conductance in electrophysiological experiments 3'4. In contrast, concentrations of B Z s in the hundreds of m i c r o m o l a r inhibit 45Ca2+ uptake to depolarized synaptosomes 16'17'35'4° and decrease Ca 2+ action potentials in myenteric neurons 6. These effects of B Z s are of potential pharmacological relevance, since behaviorally effective doses of B Z s give rise to m i c r o m o l a r concentrations of B Z s in the blood and brain of rats l°'H'15 and man 11. The effects of the drugs are p r e s u m a b l y d e p e n d e n t on the free concentration in the local environment of a suitable receptor, a measure which cannot be obtained experimentally. Hy-

d r o p h o b i c c o m p o u n d s such as d i a z e p a m are m o r e highly c o n c e n t r a t e d in brain than in b l o o d 1'1°, potentially increasing the local concentration of the drug 11. The lowest limit for free B Z available for binding at the r e c e p t o r would c o r r e s p o n d to the u n b o u n d B Z in serum. For d i a z e p a m , this concentration is in the range of 0 . 1 - 0 . 4 p M , or 8-fold less I than b l o o d concentrations of the B Z 11. We now provide evidence that the levels of free ionized Ca 2÷ can be significantly e n h a n c e d inside synaptosomes by concentrations of B Z s in the micromolar range or lower u n d e r conditions designed to minimize the contribution of a reversed N a ÷ / C a 2+ exchanger 5'41. In addition, synaptoplasmic levels of Ca z+ are dramatically enhanced by B Z s in the much higher concentration range known to inhibit 45Ca 2+ u p t a k e 1637"4°. A n earlier account of this w o r k was published elsewhere in a preliminary form 22. MATERIALS AND METHODS Male Sprague-Dawley rats (150-200 g) were obtained from Charles River Laboratories (Wilmington, MA) and housed in a facility with controlled temperature and humidity for one week before use. Fura-2, 4-Br A23187, and the hydrophobic acetoxymethyl ester of fura-2 (fura-2/AM) were obtained from Molecular Probes (Eugene, OR). Other drugs and reagents were purchased from Sigma (St. Louis, MO). Rats were decapitated, and the whole brain lacking cerebellum and brainstem was isolated. Synaptosomes were prepared at 0-4 °C by a rapid purification procedure which substantially reduces contamination of the preparation with cell debris and mitochondria 13. The brain tissue was homogenized in 10 vols. (w/v) of

Correspondence: J.V. Martin, Department of Biology, Rutgers University, Camden, NJ 08102, U.S.A.

223 0.3 M sucrose and centrifuged at 1500 g for 10 min. The resulting pellet was resuspended in the initial volume of 0.3 M sucrose and again centrifuged at 1500 g for 10 min. The supernatants of the two initial centrifugations were pooled and centrifuged at 9000 g for 20 min. The pellet was resuspendend in 5.0 ml of 0.3 M sucrose and layered over 20 ml of 0.8 M sucrose. The tube was then centrifuged at 9000 g for 25 min, allowing an additional 5-min period each for gradual acceleration and deceleration of the rotor. The upper layer, along with a white band at the interface of the two layers, was discarded. The remaining supernatant layer was removed, diluted to 0.3 M sucrose, and centrifuged at 12 000 g for 20 min. The pellet was resuspendend in a buffered salt solution (BSS) specifically formulated for each experiment (see below) at a protein concentration 2° of 1.2-1.4 mg/ml. In the first experimental series, BSS containing Na ÷ ions (Na ÷ BSS, in raM: NaCI 136, KCI 5.6, MgCI2 1.3, glucose 11, Tris-HCl 20, pH 7.4) was used.In the second experimental series, to minimize the contribution of Ca 2÷ entry via reversed Na+/Ca 2÷ exchange, the synaptosomes were resuspended and maintained in a solution in which NaCI was replaced by isomolar choline chloride (choline BSS)5"38'39A1.The concentration of free Ca 2+ within the synaptosomes was determined by using fura-2/AM 12. Synaptosomes were incubated with 10/aM fura-2/AM at 37 °C for 45 min with shaking. The incubate was diluted with an additional 4 vols. of warm (37 °C) BSS and incubated at 37 °C for an additional 15 min to maximize hydrolysis of fura-2/AM to free dye by intrasynaptosomal esterases 34. The suspension was then centrifuged at 3200 g for 10 min, and the pellet was resuspended in fresh BSS in order to remove unbound dye from outside the synaptosomes. This centrifugation and resuspension procedure was

a measure of the maximal dye response in the presence of saturating levels of Ca 2+ (Figs. la and 2a). The concentration of Ca 2÷ within the synaptosomes was then calculated according to Grynkiewicz et al. 12, using a K d of 224 nM for fura-2 binding of Ca 2+. Data were analyzed by a two-way analysis of variance (ANOVA) with one repeated measure (time after depolarization in the presence of Ca 2÷) and one between-groups measure (dose of diazepam). Planned comparisons were made between individual drug treatment groups and the corresponding controls.

repeated, Dye-loaded synaptosomes (0.2 ml) were incubated with varying concentrations of diazepam or vehicle (10/al ethanol) in a final volume of 2 ml of the appropriate BSS at 0-4 °C for 45 min. Immediately prior to use, the synaptosomes were preincubated at 37 °C for 10 min. Fluorescence was measured at 500 nm in an Aminco-Bowman Model J4-8202 spectrofluorometer (Silver Spring, MD). The excitation wavelength alternated between 340 and 385 nm over time so that a ratio of measurements at the two wavelengths (R = F34o/F38s) could be made every 15 s. After a preliminary baseline measurement (Non-depolarized Condition), 0.1 ml of a solution (945 mM KC1 and 25.2 mM CaCI2) was added, bringing the final concentration of KCI to 45 mM and CaCI2 to 1.2 mM. Fluorescence measurements were then taken at 15-s intervals up to 2 min (Depolarized Condition). In all experiments, the fluorescent measurements of synaptosomes under different treatment conditions were made in a counterbalanced order, to minimize any effect of time after isolation of the synaptosomes. Comparison of replicates did not demonstrate a systematic variation over time in these experiments. Under these circumstances, most of the fluorescence was due to fura-2 within the synaptosomes. Quenching with added 40/aM Mn 2+ reduced the fluorescent response by 17% after 5 min, presumably by binding mainly to free fura-2 outside the synaptosomes. At higher concentrations Mn 2+ substantially inhibited the fluorescence measured, an effect which increased with time in a manner suggesting disruption of the synaptosomal membrane. Given the complex time- and dose-dependence of the effect of Mn 2÷ under our incubation conditions, we did not use Mn 2+ quenching as a correction factor for the present results. In a set of tubes processed along with the experimental samples, 1.8 ml of 0.5% sodium dodecylsulfate (SDS) in 4 mM ethyleneglycol-bis-(fl-aminoethyl ether) N,N'-tetraacetic acid (EGTA) was added to the synaptosomes in order to obtain a measure of the minimal dye response (RMu~)12 in the presence of very low Ca z+ (Figs. lc and 2c). To estimate the background fluorescence to be subtracted, a second set of parallel incubates included synaptosomes without dye. At the end of the 2-rain measurement of the effects of depolarization the non-quenching Ca 2+ ionophore 4-Br A231877 was added to each of the experimental samples at a final concentration of 5/aM. After 3 min, a final set of readings was made (RMAx) as

b e l o w ) . (In s e p a r a t e c o n t r o l e x p e r i m e n t s , a d d i t i o n o f 55

RESULTS In c o n t r o l s y n a p t o s o m e s w h i c h h a d b e e n s u s p e n d e d in B S S c o n t a i n i n g N a +, d e p o l a r i z a t i o n w i t h e l e v a t e d K + in t h e p r e s e n c e o f C a 2÷ d o u b l e d t h e c o n c e n t r a t i o n o f s y n a p t o p l a s m i c C a 2+ by 15 s o f i n c u b a t i o n (Figs. 3 and 4). T h i s stage o f t h e r e s p o n s e to i n c r e a s e d K + c o n c e n t r a t i o n clearly c o r r e s p o n d s to t h e w e l l - d o c u m e n t e d fastp h a s e C a 2+ c h a n n e l o f p r e s y n a p t i c t e r m i n a l s T M . A m u c h less d r a m a t i c a d d i t i o n a l i n c r e a s e ( 1 4 % ) in C a 2+ c o n c e n t r a t i o n was m e a s u r e d b e t w e e n 15 s a n d 120 s a f t e r d e p o l a r i z a t i o n . T h e s e c o n d a r y ' s l o w - p h a s e ' C a 2+ inc r e a s e 26'41 is p r o b a b l y r e l a t e d to u p t a k e o f C a 2+ t h r o u g h a r e v e r s e d N a + / C a 2+ e x c h a n g e r 2'5"21'27'38'39'41 (see

m M KCI in t h e a b s e n c e o f a d d e d C a 2+ d e c r e a s e d i n t r a s y n a p t o s o m a l C a z+ c o n c e n t r a t i o n by 1 5 % . I s o t o n i c c h o l i n e c h l o r i d e a l o n e (55 m M ) i n c r e a s e d i n t r a s y n a p t o s o m a l C a 2+ by 7 %

, w h e r e a s C a 2÷ a l o n e (1.2 m M )

i n c r e a s e d i n t r a s y n a p t o s o m a l C a 2+ by 2 0 % . ) T h e effects of d e p o l a r i z a t i o n in t h e p r e s e n c e o f C a 2+ a r e r e f l e c t e d in a highly significant effect in t h e A N O V A (df = 8,296; F = 308.15, P < 10-6). M o r e i m p o r t a n t l y , t h e d o s e o f d i a z e p a m also was a highly significant f a c t o r in t h e s e statistical a n a l y s e s (df =

~ "-

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300 350 400 451 Exeltation Wavnlength (nm) Fig. 1. Fluorescence excitation spectra of synaptosomes loaded with fura-2 in buffered salt solution (BSS) containing Na ÷, measured at 500 nm. a, for determination of RMAX, the maximal dye response in the presence of saturating Ca 2÷, a spectrum was taken 3 rain after addition of 5 /aM 4-Br A23187; b, control synaptosomes were incubated in non-depolarizing conditions (dotted line); c, for determination of RMu~, 1.8 ml of 0.5% SDS in 4 mM EGTA was added to 0.2 ml of synaptosomes in order to obtain a measure of the minimal dye response in the presence of very low Ca 2+.

224

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Fig. 2. Fluorescence excitation spectra of synaptosomes loaded with fura-2 in buffered salt solution lacking Na ÷ (choline BSS), measured at 500 nm. a, for determination of RraAX, the maximal dye response in the presence of saturating Ca 2÷, a spectrum was taken 3 min after addition of 5 /~M 4-Br A23187; b, control synaptosomes were incubated in non-depolarizing conditions (dotted line); c, for determination of RMxN, 1.8 ml of 0.5% SDS in 4 mM EGTA was added to 0.2 ml of synaptosomes in order to obtain a measure of the minimal dye response in the presence of very low Ca 2÷.

Fig. 4. Concentration dependence of effects of diazepam on depolarized synaptosomes in BSS containing Na ÷. The measurements were made at 15 s after the addition of 0.1 ml of a solution (945 mM KC1 and 25.2 mM CaCI2) bringing the final concentration of KCI to 45 mM and CaCI 2 to 1.2 mM. Results are presented as mean + S.E.M. for the combined data of two separate experiments including a total of 11-12 replicates at each point. Asterisk indicates significance of planned comparisons between that individual group and the appropriate control group incubated without drug (*P < 0.01).

4,37; F = 11.01, P < 10-6). The effect of diazepam was only apparent at the highest dose, 100/~M (Figs. 3 and 4). Planned comparisons showed significant differences (P < 0.05) only for the 100 /~M dose group (compared to control incubations without drug) in the non-depolarized synaptosomes (Fig. 3) and in synaptosomes depolarized in the presence of Ca 2+ (Fig. 4). A tendency toward a decrease in Ca 2÷ concentration in synaptosomes treated with 0 . 1 - 1 0 / z M diazepam was noted (Figs. 3 and 4). However, this apparent difference was not statistically significant in planned comparisons to control incubations without drug. The interaction term of the A N O V A was significant (df = 32,296; F = 5.56, P < 10-6), possibly

reflecting a lesser effect of the drug on non-depolarized synaptosomes (Fig. 3) than after depolarization in the presence of Ca 2+ (Fig. 4). In synaptosomes incubated in the presence of Na + ions, 100/zM diazepam substantially increased free Ca 2+ concentrations in non-depolarized synaptosomes, an effect which appeared to be synergistic with the effects of depolarization in the presence of Ca 2+. Lower doses of diazepam were without effect under these incubation conditions. One possible mechanism by which diazepam could alter cytosol levels of Ca 2+ would be through an effect on the Na÷/Ca 2+ exchanger which acts to remove Ca 2÷ from the cytoplasm under normal physiological condi-

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Fig. 3. Concentration dependence of effects of diazepam on non-depolarized synaptosomes in BSS containing Na + . Results are presented as mean _+ standard error of the mean (S.E.M.) for the combined data of two separate experiments including a total of 11-12 replicates at each point. Asterisk indicates significance of planned comparisons between that individual group and the appropriate control group incubated without drug (*P < 0.01).

0

- Control O. 1 1 10 1 O0 Concentration of Diazepam 04M)

Fig. 5. Concentration dependence of effects of diazepam on non-depolarized synaptosomes in choline BSS lacking Na ÷ . Results are presented as mean + S.E.M. for the combined data of two separate experiments including a total of 11-12 replicates at each point. Asterisks indicate significance of planned comparisons between that individual group and the appropriate control group incubated without drug (*P < 0.01).

225 so0 ~" ,S = 700 + 400

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-Control 0.1 1 10 100 Concentrotion of Oiazepam O~M)

Fig. 6. Concentration dependence of effects of diazepam on depolarized synaptosomes in choline BSS lacking Na÷. The m e a surements were made at 15 s after the addition of 0.1 ml of a solution (945 mM KCi and 25.2 mM CaCI2) bringing the final concentration of KCI to 45 mM and CaCI2 to 1.2 mM. Results are presented as mean + S.E.M. for the combined data of two separate experiments including a total of 11-12 replicates at each point. Asterisks indicate significance of planned comparisons between t h a t individual group and the appropriate control group incubated without drug (*P < 0.01; **P < 0.05).

tions 2'21'27. Incubation of synaptosomes in BSS containing Na ÷ at concentrations approximating those found in extracellular fluid results in the accumulation of excess Na ÷ in the cytoplasms '39'41. When intracellular Na + is elevated, the Na+/Ca 2÷ exchanger can act in a reversed mode to take up Ca 2+ from outside of the cell8'27. The effects of such a reversed Na+/Ca 2+ exchanger can be minimized by replacement of NaCI with choline chloride in the incubation solution 5 ' 38 ' 39 '41 . Thus, we examined the effects of diazepam on the level of free Ca 2÷ in synaptosomes maintained in choline BSS (Figs. 5 and 6). These synaptosomes generally showed lower concentrations of Ca 2+ under both non-depolarized and depolarized conditions compared to synaptosomes maintained in Na ÷ BSS (Figs. 3 and 4). Depolarization of synaptosomes in the presence of Ca 2÷ in choline BSS caused an increase of 47% in levels of synaptoplasmic Ca 2÷ (Figs. 5 and 6), a proportionately smaller effect than that seen in the incubation with Na ÷ BSS (Figs. 3 and 4). However, the effect of depolarization in the presence of Ca 2+ remained significant in the A N O V A (dr = 8,416; F = 411.42, P < 10-6). Against this lowered background level of Ca 2+, the effect of diazepam was more readily apparent in choline BSS (Fig. 5 and 6) than in Na ÷ BSS (Figs. 3 and 4). The between-groups factor of concentration of diazepam was again highly statistically significant (dr = 4,52; F = 74.12, P < 10-6). Planned comparisons at 15 s after depolarization showed significant differences (P < 0.05) in intracellular Ca/+ levels in synaptosomes treated with doses of diazepam as low as 0.1 a M as separately compared to the control incubation without drug (Fig. 6). Planned comparisons also showed

significant differences (P < 0.01) for the 1,10 and 100aM dose groups (each compared to the control incubation without drug) in the non-depolarized synaptosomes (Fig. 5), and at 15 s after depolarization in the presence of Ca 2+ (Fig. 6). As in the experiment using Na ÷ BSS, the A N O V A also indicated a significant interaction of drug treatment group by time after depolarization (df = 32,416; F = 34.36, P < 10-6). This interaction suggests a synergism between the effects of depolarization in the presence of Ca 2+ and dose of BZ in enhancing free Ca 2+ levels in synaptosomes.

DISCUSSION The present experimental methods and results differ from those of earlier research in several important ways. The current work examines a measure of total synaptoplasmic free Ca 2+ concentration, whereas previous research measured fluxes of Ca 2+ using 4SCa2+ as a tracer 16"17'24'2s'4°. In addition, previous work used widely differing dose ranges of BZs; some investigators demonstrated stimulatory effects of low (e.g. 1 aM) doses of BZs 24'2s and others reported inhibitory effects of much higher (e.g. 100 aM) doses 16'17'40. In our experiments using choline BSS, we were able to greatly enhance the effects of diazepam on synaptosomal Ca 2+ levels, apparently by reducing a background Ca 2+ uptake attributable to the action of a reversed Na+/Ca 2+ exchanger 8'27'41. The stimulatory effects of low micromolar doses of BZs on uptake of 45Ca2+ to depolarized synaptosomes24' 28 are consistent with our present findings of an increase of intrasynaptosomal Ca 2÷ levels due to these doses of diazepam. Our results further extend earlier findings by demonstrating a significant stimulatory effect of diazepam on Ca 2+ levels in non-depolarized synaptosomes in choline BSS. Furthermore, a significant effect of 0.1 a M diazepam was demonstrated in the depolarized synaptosomes. These changes in Ca 2+ levels are clearly within the range affecting exquisitely sensitive neural processes such as release of neurotransmitter 18 and enhancement of Ca2+-mediated K + conductance 3'4. The inhibitory effects of higher doses of BZs (e.g. 100 aM) on uptake of Ca/+ to depolarized synaptosomes 16' 17,35,40 and other neural tissues 6'14 initially appear at variance with the present findings. However, since many types of Ca 2÷ channels are inactivated by elevated intracellular Ca 2+ levels 9, the possibility arises that the dramatic increase in synaptoplasmic Ca 2÷ in 100 a M diazepam serves to block influx of further Ca 2÷. Resolution of the origin of the observed increase in free Ca 2÷ (i.e. intraceUular or extracellular sources) and the relationship of this finding to effects on Ca 2+ flux or currents awaits further investigation.

226

Acknowledgements. This study was supported by grants from the Rutgers University Research Council, Busch Fund, and NIH (NS23200).

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