Adenosine Transport by Rat and Guinea Pig Synaptosomes: Basis for Differential Sensitivity to Transport Inhibitors

Adenosine transport by rat and guinea pig synaptosomes was studied to establish the basis for the marked differences in the potency of some transport inhibitors in these species. An analysis of transport kinetics in the presence and absence of nitrobenzylthioinosine (NBTI) using synaptosomes derived from several areas of rat and guinea pig brain indicated that at least three systems contributed to adenosine uptake, the Km values of which were approximately 0.4, 3, and 15 microM in both species. In both species, the system with the Km of 3 microM was potently (IC50 of approximately 0.3 nM) and selectively inhibited by NBTI. This NBTI-sensitive system accounted for a greater proportion of the total uptake in the guinea pig than in the rat and was inhibited by dipyridamole, mioflazine, and related compounds more potently in the guinea pig. Preliminary experiments with other species indicate that adenosine transport in the mouse is similar to that in the rat, whereas in the dog and rabbit, it is more like that in the guinea pig. In the rat, none of the systems appeared to require Na+, but the two systems possessing the higher affinities for adenosine were inhibited by veratridine- and K(+)-induced depolarization. The transport systems were active over a broad pH range, with maximal activity between pH 6.5 and 7.0. Our results are consistent with the possibility that adenosine transport systems may be differentiated into uptake and release systems.     

Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Abstract: Transport of GABA by a high-affinity transport system ( K _m≃ 10⁻⁵ M) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [³H]DABA by auto-radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive [³H]GABA and [³H]DABA transport. For each, transport requires external sodium, is abolished by ionophores that dissipate the Na⁺ gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor ( K _i≃ 0.6 MM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles.     

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC-fluorescence detection. Both basal and KCl-stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 +/- 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 +/- 83 pmol/mg protein/5 s. Ethanol caused a dose-dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol-stimulated release does not involve the dilazep-sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol-induced motor disturbances in the rat.     

Histidine Transport into Rat Brain Synaptosomes

Abstract: Histidine transport and metabolism in rat brain synptosomes were investigated to study the possible role of histidine uptake in the synthesis of the putative neurotransmitter histamine (HA). Histidine uptake was found to be regionally distributed and temperature sensitive and was not totally independent of sodium or possium ions. Transport was inhibited by metabolic inhibitors, as well as by promethazine and quinacrine. A number of other HA-related agents and several histidine metabolites had no effect. Kinetic analyses of histidine transport revealed the presence of both high- and lowaffinity systems in cerebral cortex. Histidine uptake increased following preexposure of synaptosomes to depolarizing concentrations of potassium. This effect was dependent on the presence of calcium ions during the preincubation. No newly formed [³H]HA was detectable in rat brain synaptosomes following [³H]histidine transport. Lesions of the medial forebrain bundle did not alter histidine uptake in the hippocampus or cerebral cortex. Ontogenic studies indicated that the histidine uptake system developed rapidly and reached a peak during postnatal days 12–17. Overall, the present findings do not support a role for histidine transport in the regulation or maintenance of neurotransmitter pools of HA in rat brain.     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Transport of Histidine into Synaptosomes of the Rat Central Nervous System

Abstract: Histidine transport into synaptosomes was studied in order to characterize this aspect of histamine synthesis in neurons. Histidine transport was found to be independent of sodium, calcium, and magnesium ions and dependent upon potassium and chloride ions. Histidine transport was also found to be energy dependent, and subcellular fractionation studies suggested it was highly localized to nerve terminals. Kinetic analysis of histidine transport in several brain regions indicated the presence of two uptake sites, a high-affinity site with a K _m of approximately 35 μ M and a low-affinity site with a K _m in the millimolar range. Density of the high-affinity site, as reflected by V_max, correlates well with density of proposed histaminergic innervation. Rate of histidine transport was not altered by prior depolarization of the synaptosomes, indicating that histidine transport probably does not play a regulatory role in histamine synthesis.     

Uptake of Agmatine into Rat Brain Synaptosomes: Possible Role of Cation Channels

Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy- and temperature-dependent and saturable with a K _m of 18.83 ± 3.31 m M and a V _max of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na⁺,K⁺-ATPase inhibitor) or removal of extracellular Na⁺ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion-channel agents on agmatine uptake, only Ca²⁺-channel blockers inhibited the uptake, whereas a reduction in extracellular Ca²⁺ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na⁺-independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.     

Adenosine Transport by Primary Cultures of Neurons from Chick Embryo Brain

Abstract: The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [³H]adenosine. During the 5-25-s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis-Menten relationship and yielded a K_m of 13 μM and V_max of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10-100-fold higher than [³H]adenosine. Adenosine derivatives ( N ⁶-cyclohexyl-, N⁶-benzyl-, N⁶-methyl-, and 2-chloroadenosine) were more effective inhibitors; p -nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high-affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.     

Characteristics of Tryptophan Accumulation by Isolated Rat Forebrain Synaptosomes

Uptake of 10 microM L-tryptophan into isolated rat brain synaptosomes was studied to assess its effect on the rate of serotonin synthesis from tryptophan. The initial rate of uptake was rapid, being two orders of magnitude above the rate of tryptophan hydroxylation. Uptake was highly concentrative, the concentration ratio across the plasma membrane at equilibrium being approximately 9. This concentration ratio was decreased to about 1 in the presence of high concentrations of amino acids transported by the L-type neutral amino acid uptake system. A mixture of the large neutral amino acids at physiological concentrations decreased the internal tryptophan concentration to 58% of that in their absence. Large tryptophan concentration ratios were observed in experiments in which Na+ in the medium was replaced with choline+. The concentrative uptake of tryptophan was energy-dependent, being decreased by inclusion of cyanide and omission of glucose. The concentration gradient was abolished by veratridine or rotenone. Time courses of the changes in ATP content and tryptophan concentration ratio on addition of these and other agents established that tryptophan uptake is probably not driven by ATP hydrolysis or efflux of other amino acids, but by the plasma membrane potential.     

L-[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.     

Tryptophan Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na⁺ gradient [Na⁺] outside > [Na⁺] inside and is maximal when both Na⁺ and Cl⁻ are present. The uptake represents transport into an os-motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo-larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na⁺, K⁺-activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4-dihydroxyphenylalanine.     

Zinc Uptake into Synaptosomes

Zinc uptake was studied in synaptosomes, isolated by the Ficoll flotation technique, using the radiotracer 65Zn. True uptake of zinc could be discriminated from binding to the outside of the synaptosomes by the absence of accumulation at 0 degree C and the dependency of the rate of uptake on the medium osmolarity. The zinc uptake, studied in the presence of various zinc-complexing agents, showed saturation kinetics when analyzed in terms of [Zn]free, yielding Km = 0.25 microM. The zinc uptake was independent of both ATP and the Na+ gradient. No efflux of zinc could be demonstrated from preloaded synaptosomes due to the formation of insoluble zinc complexes inside the synaptosomes. The results are discussed in terms of the modulation of diverse neurochemical processes by zinc.     

Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Synaptosomes Is Mediated by Glutamate Uptake and Not by Receptors

L-Glutamate (10 microM-1 mM) released endogenous adenosine from rat cortical synaptosomes. Studies with excitatory amino acid antagonists, (+)-5-methyl-16,11,dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), 6,7-dinitroquinoxaline-2,3-dione (DNQX), Mg2+, and agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate, indicated that this release was not receptor mediated. D,L-2-Amino-4-phosphonobutanoic acid (APB) also did not affect glutamate-evoked adenosine release. Inhibition of glutamate uptake by dihydrokainate or replacement of extracellular Na+ blocked glutamate-evoked adenosine release. D-aspartate, which is a substrate for the glutamate transporter but is not metabolized, also released adenosine, suggesting that release was due to amino acid transport and not to its subsequent metabolism. D-Glutamate, a relatively poor substrate for the transporter, was correspondingly less potent than L-glutamate at releasing adenosine. Glutamate-evoked adenosine release was not Ca2+ dependent or tetrodotoxin sensitive and did not appear to occur on the bidirectional nucleoside transporter. Inhibition of ecto-5'-nucleotidase virtually abolished glutamate-evoked adenosine release, indicating that adenosine was derived from extracellular metabolism of released nucleotide(s). However, L-glutamate did not release ATP and did not appear to release cyclic AMP. Therefore, transport of glutamate into presynaptic terminals releases some other nucleotide which is converted extracellularly to adenosine. This adenosine could act at P1-purinoceptors to modulate glutamatergic neurotransmission.     

Morphine Activates ω-Conotoxin-Sensitive Ca2+ Channels to Release Adenosine from Spinal Cord Synaptosomes

Abstract: Morphine-induced release of adenosine from the spinal cord is believed to contribute to spinal antinociception. Although this release is Ca²⁺ dependent, little is known of the nature of this dependence. In this study, the effects of the dihydropyridine L-type Ca²⁺ channel agonist Bay K 8644 and the antagonist nifedipine, the N-type Ca²⁺ channel antagonist ω-conotoxin, and ruthenium red, a blocker of Ca²⁺ influx induced by capsaicin, on release of adenosine evoked by morphine were determined. The effect of partial depolarization with a minimally effective concentration of K⁺ on morphine-evoked release of adenosine also was examined. Morphine 10^?5-10^?4M produced a dose-dependent enhancement of adenosine release from dorsal spinal cord synaptosomes. Following the addition of 6 mM K⁺ (total K⁺ concentration of 10.7 mM), 10^?6M morphine also enhanced release, and an additional component of action at 10^?8M was revealed. Release was Ca²⁺-dependent as it was not observed in the absence of Ca²⁺ and presence of EGTA. Bay K 8644 (10 nM) and nifedipine (100 nM) had no effect on the release of adenosine evoked by morphine, but ω-conotoxin (100 nM) markedly reduced such release in both the absence and the presence of the additional 6 mM K⁺. Morphine-evoked adenosine release was not altered in the presence of a partially effective dose of capsaicin, nor by ruthenium red. These results indicate that morphine can stimulate two distinct phases of adenosine release from the spinal cord (nanomolar and micromolar), and that both phases of release are due to Ca²⁺ entry via ω-conotoxin-sensitive N-type Ca²⁺ channels.     

Phosphate Ion Transport in Rabbit Brain Synaptosomes

Abstract: Synaptosomes (vesicles of nerve endings) isolated from rabbit brain were studied as a model system for the uptake of inorganic phosphate. The phosphate uptake showed a sodium-dependent, saturable component with a K _t of 0.29 m m , The sodium-dependent component was larger at pH 6 than at pH 7.4 or 8. Application of potassium salts, ouabain, monensin, nigericin or FCCP decreased the uptake. The results indicate that the sodium-sensitive phosphate influx is dependent on the Na⁺ gradient and on the membrane potential, which might act, preferentially, on the transport of the monovalent phosphate ion.     

[3H]Adenosine Transport in Synaptoneurosomes of Postmortem Human Brain

Abstract: Abstract: [³H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [³H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with K_t and V_max values of 89 μM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μM [³H]adenosine as substrate, intrasynaptoneurosomal concentrations of [³H]adenosine were 0.26 μM at 5 s and 1 μM at 600 s. Metabolism of accumulated [³H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (μM) of total accumulated ³H-purines ([³H]-adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of ³H-purines was higher than the initial concentration of 1 μM [³H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′-iodotubercidin, metabolism of the transported [³H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total ³H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μM, respectively. Thus, the apparently “concentrative'’accumulation of ³H-purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.     

The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5-Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes

The effect of the serotonergic receptor agonist 1-(m-trifluoromethylphenyl)piperazine (TFMPP) was studied on the K(+)-evoked [3H]acetylcholine [( 3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5-1,000 microM) inhibited the evoked ACh release in a dose-dependent manner (IC50 = 81.8 microM). The inhibitory effect of TFMPP was mimicked by CGS-12066B (10, 30, and 100 microM), a 5-hydroxytryptamine1B (5-HT1B)/5-HT1D receptor agonist; 1-(m-chlorophenyl)piperazine (100 microM), a 5-HT1C/5-HT1B receptor agonist; and 5-carboxamidotryptamine (10 microM), a nonselective 5-HT1 receptor agonist. 8-Hydroxy-2-(di-n-propylamino)tetralin (10 and 100 microM), a 5-HT1A receptor agonist, and quipazine (10 and 100 microM), a 5-HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 microM), metergoline (0.1 microM), methysergide (0.5 and 1 microM), or yohimbine (1 and 10 microM), blocked the TFMPP effect dose-dependently. In contrast, methiotepine (0.3 and 1 microM), propranolol (1 microM), ketanserin (0.1 microM), mesulergine (0.1 microM), ICS 205930 (0.1 and 1 microM), and spiroperidol (1 and 7 microM) did not affect the TFMPP-induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K(+)-evoked ACh release is modulated by a 5-HT1 receptor distinct from the 5-HT1A, 5-HT1B, and 5-HT1C subtypes.     

Introduction of Impermeant Molecules into Synaptosomes Using Freeze/Thaw Permeabilization

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.     

Some Biochemical Properties of the Rapid Adenosine Uptake System in Rat Brain Synaptosomes

Abstract: The rapid uptake of adenosine into rat brain cortical synaptosomes is mediated by a facilitated diffusion process. The carrier mediated uptake is sensitive to pH and temperature. The average Q₁₀ value for the system is approximately 1.77 and the necessary activation energy ( E _a) is estimated to be 8870 cal/mol. These values are essentially in agreement with values reported for adenosine uptake carriers of other tissues. Substrate specificity of the uptake system in the CNS demonstrates that nucleotides do not interact with the carrier until they have been hydrolyzed to nucleosides. Structural modification of the purine moiety at the "2" position did not have a profound effect on the ability of the molecule to serve as a substrate for the uptake system. Competitive inhibition by sulfhydryl reagents, p -chloromercuribenzoate, and N- ethylmaleimide on adenosine uptake suggests a direct involvement of sulfhydryl group(s) in the uptake mechanism. Other purines and pyrimidines also inhibited adenosine uptake, suggesting that a variety of nucleosides can interact with a common carrier system.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.