[3H]adenosine transport in rat dorsal brain stem using a crude synaptosomal preparation

~ Pergamon

0197-0186(94)E0048-X

Neurochem. Int. Vol. 25, No. 3, pp. 221-226, 1994 Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 01974)186/94 $7.00+0.00

[3H]ADENOSINE TRANSPORT IN RAT DORSAL BRAIN STEM USING A CRUDE SYNAPTOSOMAL PREPARATION ANDREW J. LAWRENCE*, MARGIE CASTILLO-MELlkNDEZ a n d BEVYN JARROTT Department of Pharmacology, Monash University, Wellington Road, Clayton, Victoria 3168, Australia (Received 11 February 1994 ; accepted 21 February 1994)

Abstract--The neuromodulator adenosine, has been shown to have the highest density of central uptake sites in the nucleus tractus solitarius in the dorsal brain stem. The nucleus tractus solitarius is involved in the central regulation of reflex cardiovascular activity suggesting that adenosine may also be involved in central cardiovascular control. Thus, the present study has characterized the transport of [3H]adenosine into rat dorsal brain stem synaptosomes. The process was found to be saturable and highly dependent on temperature. Furthermore, [3H]adenosine transport in rat dorsal brain stem synaptosomes was far more sensitive to the removal of sodium ions than has been previously reported for rat cortical synaptosomes. In addition transport was rapid, being linear for at least 30 s at 37°C, reaching equilibrium within 1 rain and had an apparent Kmvalue of 2.7 + 0.2 #M (n = 4) and a Vm~xvalue of 135.5 + 17.8 pmol/mg protein/min (n = 4). These kinetic parameters are within an order of magnitude of adenosine uptake processes found in rat cerebral cortical synaptosomes. Transport of [3H]adenosine was significantly inhibited by an excess of unlabelled adenosine (1 mM) and the adenosine uptake inhibitor S(4-nitrobenzyl)-6-thioinosine (100 /~M) while morphine (150 #M) and flurazepam (150 #M) were largely ineffective as inhibitors of the process, in contrast with previous findings in rat cortical synaptosomes. The present findings demonstrate the presence of a high affinity transport system for adenosine in the rat dorsal brain stem which appears to differ in some properties to uptake processes found in rat cortex.

The purine nucleoside adenosine has been shown to modulate neuronal function through a diversity of receptor-mediated mechanisms involving second messenger systems (Daly et al., 1983), transmembrane ion fluxes (Phillis and Wu, 1981) and neurotransmitter release (Fredholm and Hedqvist, 1980). Once released, the extracellular levels of endogenous adenosine are regulated primarily by transport systems (Wu and Phillis, 1984) by which adenosine is removed from the synaptic cleft, hence terminating the actions of this nucleoside (Gu and Geiger, 1992). A family of nucleoside transporters exist that have been categorized by a number of criteria: namely, their ability to translocate nucleosides across cell membranes by passive and facilitated equilibrative processes, or by sodium-dependent and concentrative transport systems; their relative affinities and selectivity for adenosine as substrate; and their sensitivity or resistance to the blocking effects of nitrobenzylthioinosine, dipyridamole and other *Author to whom all correspondence should be addressed. 221

nucleoside transport inhibitors (Geiger and Fyda, 1991). Adenosine uptake systems have been extensively examined using various tissue preparations (Bender et al., 1980). In central nervous system tissue, high-affinity transport systems have been demonstrated in rat cortical (Bender et aL, 1981) and in guinea pig neocortical synaptosomes (Barberis et al., 1981). These processes were found to be saturable and to reach completion within 60 s and have been shown to be sensitive to sodium ions, temperature and adenosine uptake inhibitors. Autoradiographic studies have demonstrated that, in the rat brain, the highest density of adenosine uptake sites occur in the nucleus tractus solitarius (NTS) in the dorsal brain stem (Bisserbe et al., 1984). The importance of the N T S as the major relay nucleus for input of baroreceptor afferents in central cardiovascular control has been established (Kumada et al., 1990). Neuronal cell groups in this nucleus are known to regulate reflex cardiovascular activity suggesting that adenosine could be involved in central cardiovascular function (Palkovits and Zaborszky,

222

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1977). T h e possibility that a d e n o s i n e plays a n e u r o m o d u l a t o r y role in central c a r d i o v a s c u l a r c o n t r o l areas is s u p p o r t e d by n e u r o p h a r m a c o l o g i c a l studies using n o r m o t e n s i v e rats in w h i c h m i c r o i n j e c t i o n o f a d e n o s i n e into the caudal N T S caused r e d u c t i o n s in b l o o d pressure a n d heart rate ( B a r r a c o el al., 1988, M o s q u d d a - G a r c i a et al., 19911. G i v e n the possible i n v o l v e m e n t o f a d e n o s i n e in the central c o n t r o l o f c a r d i o v a s c u l a r f u n c t i o n , the p r e s e n t investigation has a t t e m p t e d to c h a r a c t e r i z e the kinetic p a r a m e t e r s for [)H]adenosine t r a n s p o r t into rat dorsal brain stem s y n a p t o s o m e s a n d to investigate the effects o f various agents a n d e x p e r i m e n t a l c o n d i t i o n s on this t r a n s p o r t process.

EXPERIMENTAL PROCEDURES

Synaptosomal preparation Male Sprague-Dawley rats weighing between 200 250 g were decapitated and their brain stems removed and cut 3 mm rostral and 3 mm caudal of obex. This section of brain stem was then hemisected to obtain the dorsal portion (mean tissue wet weight 94_+ 2 mg, n = 16) which was homogenized in 5 ml of 0.32 M sucrose solution using an Ultra-Turrax homogenizer employing two x 10 s bursts. The homogenate was centrifuged at 1000 g for 10 rain at 4 C. Following centrifugation, the pellet was discarded and the remaining supernatant was centrifuged again at 12,000 g for 20 min at 4'C. The supernatant was then discarded and the pellet was resuspended in 50 vol (w/v) of Krebs/Hepes buffer of the following composition in m M : (NaCI, 110; glucose, 25; sucrose, 68.3 ; KCI, 5.3 ; CaCI> 1.8 ; MgSO~, 1.0 ; Hepes, 20 ; pH. 7.4 ; Gu and Gieger, 1992) and pre-incubated in a water bath at 37'C for 10 min before commencing the assay. Inhibition qf ['H] adenosine transioort The characteristics of the transport process were first studied under the following conditions: (1) the effects of N a free buffer (NaCI replaced with 110 mM choline chloride) ; (2) incubation at 4' C ; (3) an excess of unlabelled adenosine ( 1 raM) ; (4) morphine (150 ,uM) ; (5) flurazeparn (150 #M) ; (6) S(4-nitrobenzyl)-6-thioinosine (NBI, 100 #M) on [3H]adenosine transport into rat dorsal brain stem synaptosomes. Assays were performed in a final volume of 1000 /11 containing [3H]adenosine (final concentration 1 #M; specific activity 1 pCi/nmol), 100/,tl ofsynaptosomal suspension (75 100 #g of protein) and Krebs/Hepes buffer (pH 7.4) in the presence or absence of drugs. Following a 2 rain pre-incubation at 37 C, the reaction was initiated by addition of the crude synaptosomal preparation into the tubes and vortexing. The assay was allowed to continue for 60 s, after which the reaction was terminated by addition of ice-cold washing solution (260 mM sucrose, 2 mM CaCl2, 2 mM MgCl2 and 20 mM Tris HCl ; pH 7.5) and subsequent vacuum filtration through a Brandell cell harvester using GF/B filters. The particles retained on the filter were then washed twice with ice-cold washing solution. The radioactivity associated with the washed filter was then determined by scintillation counting offi-emission (efficiency- 50%).

N( t: Cl aZ

Time course O/ [:~tt]adeno.sim ' tran.V~Orl A time course tbr the process was obtained by incubating the crude synaptosomal preparation with ['H]adenosinc (final concentration I pM) for 10.20, 30, 40, 60, 90 and 120 s. Time courses were obtained with [~tt]adenosine alone to determine total purine transport, in the presence of unlabelled adenosine ( 1 inM) to define non-specitic transport. and in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (EHNA. 10/zM) and iodotubercidin (10 tiM) to determine total adenosine transport. The reaction was initiated as previously described and terminated b~ addition of ice-cold wash solution (5 ml) and subsequent individual vacuum filtration through a micro-glass fibre filter (GF/B) alter the appropriate incubation period. The filters were then washed twice ,~ith wash buffer ( - 5 roll, placed m a scintillation vial and counted as previously described hlilia/ rate o/[~tt]adenosine Iransporl Experiments were carried out using [~H]adenosine at final concentrations of 0.2. 0.4. 0.8, 1.0, 1.5 and 2.0 BM. The reaction was allowed to continue for 30 s after which the assay was terminated as described for the time course study.

Prolein assay Protein content was determined by the Fotin-phenol procedure (Lowry et al., 1951), using bovine serum albumin standards (0.25 4.0 rag/rob. StatL~'tical anaO'si.s A paired Student's t-test was carried out for evaluation of the uptake characterization data. Statistical evaluation of the kinetic data (in the presence and absence of enzyme inhibitors) were carried out using one way analysis of variance (ANOVA) with a post-hoc Dunett's t-test. In all cases P < 0.05 was considered significant. Non-linear curve-fitting was performed with the program "Cricket Graph" on a Macintosh computer. Linearity of time courses were determined using least-squares linear regression. Materials [2-3H]adenosine was obtained from Amersham (U.K.). Adenosine and erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (EHNA) were from Sigma Chemical Co., (St Louis, MO, U.S.A.). lodotubercidin and S(4-nitrobenzyl)6-thioinosine (NBI) were from Research Biochemicals Incorporated (RBI), (U.S.A.). Morphine hydroehloride was from Glaxo (Australia) and flurazepam was from Roche (Australia). R ESU LTS

Transport characteristic.~ T h e t r a n s p o r t o f [3H]adenosine into a c r u d e p r e p a r a t i o n o f rat dorsal b r a i n stem s y n a p t o s o m e s was highly d e p e n d e n t o n t e m p e r a t u r e a n d s o d i u m ions (Table 1). S y n a p t o s o m e s i n c u b a t e d with [3H]ad, e n o s i n e (1 ~ M ) at 4~C for 60 s s h o w e d a significantly r e d u c e d t r a n s p o r t ( P < 0.001 ; n = 4) c o m p a r e d with t h o s e i n c u b a t e d at 3 7 C . F u r t h e r m o r e , the t r a n s p o r t o f [3H]adenosine in a m e d i u m in w h i c h N a C I h a d been replaced with choline chloride, was also significantly

[3H]Adenosine transport in dorsal brain stem Table 1. Inhibition of [~H]adenosine transport into rat dorsal brain stem synaptosomes Treatment Control 4°C Na ÷ free buffer Excess adenosine (1 mM) NBI (100 pM) Morphine (150 #M) Flurazepam (150 pM)

400

% Inhibition of control

n

-~ "~ 300 ~ "~

-83.8+1.8" 90.3+_3.2* 83.5 +_4.3* 25.0+6.1"* 7.7+_2.2** 5.3_+2.5

5 4 4 4 5 5 5

N ~ ea ~ 200 "~ "~ "~ °= ~ 100 "~ <

[3H]Adenosine (1 #M final concentration) was incubated for 60 s at 37C with dorsal brain stem synaptosomes (75-100 pg protein) under the various experimental conditions in an assay volume of 1000 /d. Values are mean+-SEM from n independent experiments, each performed in duplicate. A paired Student's t-test was carried out to determine the effects of the various treatments. *P < 0.01, **P < 0.05 significantly different from control.

reduced c o m p a r e d with the control ( P < 0.001; n = 4). In the presence of a 1000-fold m o l a r excess o f unlabelled adenosine (1 m M ) , the t r a n s p o r t of radiolabel was also inhibited significantly ( P < 0.001; n = 4). The adenosine u p t a k e blocker N B I (100 #M), partially inhibited [3H]adenosine transport (P=0.015; n=5) while m o r p h i n e (150 p M , P = 0.024; n = 5) a n d flurazepam (150 # M , P = 0.097 ; n = 5) h a d only a small effect as inhibitors of the t r a n s p o r t process (Table 1).

Time course of ~H]adenosine transport The t r a n s p o r t o f [3H]adenosine (1 p M ) into the rat dorsal brain stem s y n a p t o s o m a l suspension was linear for at least 30 s at 37°C (Fig. 1). T o determine non-specific or non-carrier mediated transport, the s y n a p t o s o m e s were i n c u b a t e d in the presence of unlabelled adenosine (1 m M ) a n d it can be seen in Fig. I(A), t h a t the non-specific t r a n s p o r t remained relatively c o n s t a n t t h r o u g h o u t a 120 s i n c u b a t i o n period. Binding o f [3H]adenosine to receptors a n d / o r transport sites as opposed to actual t r a n s p o r t represented 1 0 . 9 6 + 0 . 8 7 % of the total radioactivity added. The t r a n s p o r t of [3H]adenosine into rat dorsal b r a i n stem s y n a p t o s o m e s was also determined in the presence of inhibitors for adenosine deaminase (10/~M E H N A ) a n d adenosine kinase (10 p M iodotubercidin). U n d e r these conditions the process was not significantly different to t h a t in the absence of the enzyme inhibitors as seen in Fig. 1 (A). T h e net t r a n s p o r t of [3H]adenosine, o b t a i n e d by subtracting the non-specific u p t a k e from the total uptake, was rapid a n d reached equilibrium at approx. 60 s as illustrated in Fig. 1 (B). As for total transport, the net t r a n s p o r t of [3H]ad-

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Incubation time (s) Fig. 1. Time course of [3H]adenosine (1 #M) transport into rat dorsal brain stem synaptosomal suspensions. Data points are mean + SEM from four independent assays, each performed in duplicate. (A) Total [3H]adenosine (1 ~M) transport in the absence (open circles) or presence (closed circles) of EHNA and iodotubercidin (10 pM each). Closed triangles represent non-specific transport defined in the presence of a 1000-fold molar excess of unlabelled adenosine. (B) Net transport of [3H]adenosine (1 pM) into rat dorsal brain stem synaptosomes, obtained by subtracting non-specific from total shown in A. enosine, over the time points studied, did not differ in either the absence or presence of enzyme inhibitors.

Transport kinetics The rate o f [3H]adenosine t r a n s p o r t was determined over a 30 s i n c u b a t i o n time, as the process was still linear at this time point, with [3H]adenosine in concentrations ranging from 0.2-2/aM. Direct linear plots (Eisenthal a n d C o r n i s h - B o w d e n , 1974) constructed from these data revealed a high affinity t r a n s p o r t system h a v i n g a dissociation c o n s t a n t (Km) of 2.7 + 0 . 2 # M (range 2.30-3.05 # M , n = 4) a n d a maximal rate (Vmax) of 135.5+ 17.8 p m o l / m g p r o t e i n / m i n (range 112-182 p m o l / m g protein/min, n - 4 ) . Figure 2 shows the kinetic data represented by a L i n e w e a v e r Burk plot.

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l/S [.ttM]q Fig. 2. Lineweaver-Burk plot of [3H]adenosine(0.2-2.0/~M) transport initial rates (pmol/mg protein/30 s) in rat dorsal brain stem synaptosomal suspensions.Data points are mean±SEM from four independent assays, each performed in duplicate. DISCUSSION

In the rat, the highest density of central adenosine uptake sites has been observed in the NTS and these sites are thought to be related to some of the central cardiovascular effects of adenosine (Bisserbe et al., 1984). In the present study, the transport of [3H]adenosine into rat dorsal brain stem synaptosomes was found to be dependent on temperature and sodium ions suggesting more than a simple passive diffusion process. These results are consistent with findings by Bender and colleagues (1981) who demonstrated a transport process for [~H]adenosine in rat cortical synaptosomes which was temperature dependent. However, in cortical synaptosomes, removal of Na + ions caused only a 35% inhibition of [3H]adenosine transport compared to over 90% inhibition obtained in the present study. This observation suggests that purine transport in dorsal brain stem synaptosomes has a greater active component compared to cortical synaptosomes. The possibility that the difference between previous studies in cortical synaptosomes is due to glial contamination in the present system is unlikely. Ohkubo and coworkers (1991) described a transport system for adenosine in cultured glial cells from rat hippocampus that was dependent on temperature but indepedent of sodium ions. In guinea pig neocortical synaptosomes a transport system for [3H]adenosine has also been reported which was inhibited by 61% on the removal of Na + ions and proceeded 10 times faster at 37~:C compared to 0~C (Barberis et al., 1981). NBI and related compounds have been shown to be highly potent inhibitors of adenosine uptake in erythrocytes (Jarvis and Young, 1980). However, in

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rat dorsal brain stem synaptosomes, NBI (100 tLM) inhibited [~H]adenosine transport by only 25%, whereas 50% inhibition (at a concentration o1"50 i t M ) has been observed in cultured rat hippocampal neurons (Ohkubo el al.. 1991 ). In the latter study. [~H]adenosine transport in cultured hippocampal glial cells was more sensitive to blockadc by NBI (> 75% inhibition at a concentration of 50 t t M ) than the neuronal process, suggesting the relative lack of effect of NB! in the dorsal brain stem preparation is not due to contamination by functional gliasomes. Since completion of the present study, recent experimental evidence has demonstrated that NBI (0.1 10 ttM) was ineffective in inhibiting the uptake of adenosine into rat spinal cord synaptosomes while dipyridamole (0.01-10 I,M) was highly effective, suggesting the involvement of heterogeneous carrier proteins in adenosine transport in the spinal cord (Sweeney et al., 1993). This could account for the low inhibition by NBI observed in the crude preparation of dorsal brain stem synaptosomes in the present study. Concentration response assays with NBI will help to determine whether the inhibitor can further block [~H]adenosine transport in the dorsal brain stem. Benzodiazepines (Phillis et al., 1980a, Bender e/al., 1981) and morphine (Phillis et al., 1980b, Bender et al., 1981) have been shown to inhibit adenosine transport in rat cortical synaptosomes due to blockade of the reuptake system. Therefore, the effects of morphine and flurazepam on the transport process in rat dorsal brain stem synaptosomes were also investigated : however, these drugs were lbund to be largely ineffective in this brain region. These observations are in contrast with tindings in rat cortical synaptosomes in which transport of [~H]adenosine was inhibited by more than 50"/0 in the presence of either morphine (150 #M) or flurazepam (100 I t M ) (Bender et al., 1981). This differential sensitivity could be due to adenosine subserving different roles in the cortex and in the NTS, In cortex, adenosine acts predominantly as an inhibitory neuromodulator whereas in the NTS it causes both inhibition and excitation of neurons (Barraco et al., 1991). Furthermore, pain and anxiety have a high degree of cortical involvement. In addition, it has been suggested that interactions between adenosine and benzodiazepines exist due to similarities in the localization of adenosine uptake sites and benzodiazepine receptors in the brain (Bisserbe et al., 1984). Both sites have been shown to be present in high density in brain structures such as the pyriform cortex, central gray, substantia nigra, the dorsal tegmentum and other brain regions (Young and Kuhar, 1980): however, benzodiazepine receptors

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[3H]Adenosine transport in dorsal brain stem have been found to be present only in very low densities in the pars commissuralis of the N T S (Young and Kuhar, 1980). It appears, therefore, that the inhibitory action of benzodiazepines on adenosine uptake are directly related to specific brain areas. Experiments were carried out to determine the time course for [3H]adenosine transport into dorsal brain stem synaptosomes. The process was found to be linear for at least 30 s, and also rapid, reaching equilibrium at approximately 60 s. These findings are in agreement with studies in rat cortical synaptosomes (Bender et al., 1981) and in human erythrocytes (Oliver and Paterson, 1971) where a rapid component in which transport reaches completion within 1 min has been demonstrated. Previous studies on the transport of [3H]adenosine into cortical synaptosomes have reported that between 47% (post-mortem human, G u et al., 1993) and over 70% (rat, Bender et al., 1981) of the [3H]adenosine taken up by the synaptosomes remains unchanged after 30 60 s in the absence of enzyme inhibitors for adenosine deaminase and adenosine kinase. Due to these findings, the experiment was repeated in the presence of E H N A (adenosine deaminase inhibitor) and iodotubercidin (adenosine kinase inhibitor). However statistical evaluation of the data revealed no significant differences between the process in the absence or presence of enzyme inhibitors, suggesting that adenosine was the main purine taken up by this process over the time course studied. In addition, the present data indicate that unlike cortical preparations, metabolism of [3H]adenosine did not appear to be occurring in rat dorsal brain stem synaptosomes at the time points used in these experiments. It is possible, however, that metabolism may be an important feature influencing transport at earlier times between 0-10 s. Finally, the rate of [3H]adenosine transport into dorsal brain stem synaptosomes was investigated using 30 s as an incubation period, since the process was found to be still linear at that time point. Transport of [3H]adenosine began to show signs of saturation at a concentration of approx. 2/~M. Similarly, in rat cortical synaptosomes, [3H]adenosine transport has been shown to reach saturation at approx. 1.4/~M (Bender et al., 1981). Kinetic analysis of these data revealed a high affinity transport carrier having a Vrnax of 135.5 pmol/mg protein /min and a Km value of 2.7 #M. These results are consistent with findings in guinea pig neocortical synaptosomes (Barberis et al., 1981), in rat cerebral cortical synaptosomes (Gu and Geiger, 1992) and in cultured rat hippocampal neurons (Ohkubo et al., 1991). Additional studies utilizing a wider range of [3H]adenosine concentrations

are currently underway to determine if a low affinity component of nucleoside transport exists in purified dorsal brain stem synaptosomes, as in synaptosomal preparations from other brain regions. In conclusion, these findings suggest that, in the rat dorsal brain stem, a rapid and high-affinity transport carrier for adenosine is present which appears to differ in several properties to that found in rat cortex. Whether adenosine uptake in this brain region is related to central cardiovascular control remains to be elucidated. Acknowledyements--This work was supported by a grant

from the NH and MRC of Australia. AJL is the recipient of a Wellcome Trust research travel grant.

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