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• W2018018964 abstract "Adenosine and arachidonate (AA) fulfil opposite modulatory roles, arachidonate facilitating and adenosine inhibiting cellular responses. To understand if there is an inter-play between these two neuromodulatory systems, we investigated the effect of AA on extracellular adenosine metabolism in hippocampal nerve terminals. AA (30 μm) facilitated by 67% adenosine evoked release and by 45% ATP evoked release. These effects were not significantly modified upon blockade of lipooxygenase or cyclooxygenase and were attenuated (52–61%) by the protein kinase C inhibitor, chelerythrine (6 μm). The ecto-5′-nucleotidase inhibitor, α,β-methylene ADP (100 μm), caused a larger inhibition (54%) of adenosine release in the presence of AA (30 μm) compared with control (37% inhibition) indicating that the AA-induced extracellular adenosine accumulation is mostly originated from an increased release and extracellular catabolism of ATP. This AA-induced extracellular adenosine accumulation is further potentiated by an AA-induced decrease (48%) of adenosine transporters capacity. AA (30 μm) increased by 36–42% the tonic inhibition by endogenous extracellular adenosine of adenosine A1 receptors in the modulation of acetylcholine release and of CA1 hippocampal synaptic transmission in hippocampal slices. These results indicate that AA increases tonic adenosine modulation as a possible feedback loop to limit AA facilitation of neuronal excitability. Adenosine and arachidonate (AA) fulfil opposite modulatory roles, arachidonate facilitating and adenosine inhibiting cellular responses. To understand if there is an inter-play between these two neuromodulatory systems, we investigated the effect of AA on extracellular adenosine metabolism in hippocampal nerve terminals. AA (30 μm) facilitated by 67% adenosine evoked release and by 45% ATP evoked release. These effects were not significantly modified upon blockade of lipooxygenase or cyclooxygenase and were attenuated (52–61%) by the protein kinase C inhibitor, chelerythrine (6 μm). The ecto-5′-nucleotidase inhibitor, α,β-methylene ADP (100 μm), caused a larger inhibition (54%) of adenosine release in the presence of AA (30 μm) compared with control (37% inhibition) indicating that the AA-induced extracellular adenosine accumulation is mostly originated from an increased release and extracellular catabolism of ATP. This AA-induced extracellular adenosine accumulation is further potentiated by an AA-induced decrease (48%) of adenosine transporters capacity. AA (30 μm) increased by 36–42% the tonic inhibition by endogenous extracellular adenosine of adenosine A1 receptors in the modulation of acetylcholine release and of CA1 hippocampal synaptic transmission in hippocampal slices. These results indicate that AA increases tonic adenosine modulation as a possible feedback loop to limit AA facilitation of neuronal excitability. arachidonic acid arachidonyl trifluromethylketone α,β-methylene ADP bovine serum albumin 1,3-dipropyl-8-cyclopentylxanthine field excitatory postsynaptic potential N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydroxyeicosatetraenoic acid 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine S-(p-nitrobenzyl)-6-thioinosine nordihydroguaiaretic acid prostaglandin 8-phenyltheophilline phospholipase A2 high performance liquid chromatography Whenever neurotransmitter release occurs, there is also a release of adenosine as such and of ATP (1Cunha R.A. Vizi E.S. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1996; 67: 2180-2187Crossref PubMed Scopus (215) Google Scholar), which is extracellularly catabolized into adenosine (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar, 3Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurosci. 1998; 18: 1987-1995Crossref PubMed Google Scholar). Increased neuronal activity also causes an increase in extracellular arachidonic acid (AA)1 (4Lynch M.A. Clements M.P. Voss K.L. Bramham C.R. Bliss T.V.P. Biochem. Soc. Trans. 1991; 19: 391-396Crossref PubMed Scopus (45) Google Scholar), a free fatty acid that is found sterified in the sn-2 position of membrane phospholipids and can potentially be released by a number of phospholipases, in particular by phospholipases A2 (5Dennis E.A. Rhee S.G. Billah M.M. Hannun Y.A. FASEB J. 1991; 5: 2068-2077Crossref PubMed Scopus (475) Google Scholar). Adenosine and AA fulfil opposite roles in regulating cell to cell communication in the nervous system. For instance, in the hippocampus, AA facilitates while adenosine A1 receptor activation inhibits intracellular free calcium accumulation and the release of excitatory neurotransmitters (6Damron D.S. Dorman R.V. Neurochem. Res. 1993; 18: 1231-1237Crossref PubMed Scopus (12) Google Scholar, 7Freeman E.J. Terrian D.M. Dorman R.V. Neurochem. Res. 1990; 15: 743-750Crossref PubMed Scopus (69) Google Scholar, 8Ambrósio A.F. Malva J.O. Carvalho A.P. Carvalho C.M. Eur. J. Pharmacol. 1997; 340: 301-310Crossref PubMed Scopus (62) Google Scholar); AA facilitates while adenosine inhibits synaptic transmission and plasticity (4Lynch M.A. Clements M.P. Voss K.L. Bramham C.R. Bliss T.V.P. Biochem. Soc. Trans. 1991; 19: 391-396Crossref PubMed Scopus (45) Google Scholar, 9de Mendonça A. Ribeiro J.A. Life Sci. 1997; 60: 245-251Crossref PubMed Scopus (77) Google Scholar); AA has been proposed to be a mediator of epileptogenic phenomena (10Bazan N.G. Ann. N. Y. Acad. Sci. U. S. A. 1989; 559: 1-16Crossref PubMed Scopus (92) Google Scholar) whereas adenosine is considered an endogenous anticonvulsant (11Dragunow M. Prog. Neurobiol. 1988; 31: 85-108Crossref PubMed Scopus (169) Google Scholar); AA potentiates hypoxic damage (12Chan P.H. Fishman R.A. Chen S. Chew S. J. Neurochem. 1983; 41: 1550-1557Crossref PubMed Scopus (12) Google Scholar), while adenosine is an endogenous neuroprotector (13Fredholm B.B. Int. Rev. Neurobiol. 1997; 40: 259-280Crossref PubMed Google Scholar). However, despite this parallel between the genesis and opposite actions of adenosine and AA, it is not known whether there is an inter-play between these two neuromodulatory systems. We now report that AA causes an increase in the extracellular accumulation of adenosine upon stimulation of nerve terminals from the rat hippocampus. This AA-induced increase of extracellular adenosine seems to be partly protein kinase C-mediated and to be due to a combination of an AA-induced facilitation of ATP release and an increased extracellular formation of adenosine from released ATP together with an AA-induced inhibition of extracellular adenosine removal. This AA-induced increase in endogenous extracellular adenosine is likely to be the basis of the greater tonic facilitatory effects of adenosine A1 receptor antagonists on acetylcholine release and synaptic transmission caused by AA. AA, bovine serum albumin (BSA, fatty acid free), phospholipase A2 (EC 3.1.1.4, PLA2) from bee venom, melittin, indomethacin, nordihydroguaiaretic acid (NDGA), arachidic acid, linolenic acid, prostaglandin E2(PGE2), prostaglandin F2α(PGF2α), (±)-5-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE), 12(R)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), α,β-methylene ADP (AOPCP),S-(p-nitrobenzyl)-6-thioinosine (NBTI), veratridine, adenosine deaminase (type VI, 2000U/ml), 2-chloroadenosine, and the luciferin-luciferase solution (FL-AAM) were from Sigma. Arachidonyl trifluromethylketone (AACOCF3), chelerythrine, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62), and [N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide (HA1004) were from Calbiochem (La Jolla, CA), dipyridamole was from Boehringer Ingelheim, KH2PO4 was from BDH (Aristar, Poole, United Kingdom) and methanol (Chromosolv) was from Riedel-de-Haën (Seelze, Germany). Hemicholinium-3, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 8-phenyltheophilline (8-PT), and N 6-cyclopentyladenosine were from RBI (Natick, MA). [2,3,8′-3H]Adenosine (specific activity 56.8–57.6 Ci/mmol), [2-3H]adenosine (specific activity 62.4–64.5 Ci/mmol), and [5,6,8,9,11,14,15-3H]arachidonic acid (specific activity 218 Ci/mmol) were obtained from Amersham Pharmacia Biotech. All other reagents were of the highest purity available. Free fatty acids were made up into a 30 mm stock solution and AACOCF3was made up into a 10 mm stock solution in ethanol, aliquoted, and stored under nitrogen atmosphere at −20 °C. Indomethacin was made up into a 20 mm stock in methanol and nordihydroguaiaretic acid was made up into a 20 mm stock in ethanol. Dipyridamole, NBTI, chelerythrine, HA1004, and KN-62 were made up into 5 mm stock solutions in dimethyl sulfoxide, DPCPX was made up into a 5 mm stock solution in 99% dimethyl sulfoxide and 1% NaOH (1 m) and veratridine was made up into a 5 mm stock in 2.5 m in maleic acid. Aqueous dilutions of these stock solutions were made daily. The maximal concentrations of ethanol, methanol, or dimethyl sulfoxide used were devoid of effects on [3H]adenosine and [3H]acetylcholine release as well as on synaptic transmission. Hippocampal synaptosomes from 6-week-old male Wistar rats (140 g) were prepared by differential sucrose-Percoll density gradient centrifugations, as described previously (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar). The synaptosomes were resuspended in oxygenated Krebs solution of the following composition (mm): NaCl 124, KCl 3, NaH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 26, glucose 10, pH 7.4, and gassed with a 95% O2 and 5% CO2 mixture. This synaptosomal suspension was equilibrated at 37 °C for 10 min, loaded with [2,3,8′-3H]adenosine (3 μCi/ml, 0.05 μm) for 20 min at 37 °C and then layered over Whatman GF/C filters into four parallel 90-μl superfusion chambers (adapted from Swinny filter holders, Millipore, Bedford, MA) through the aid of a roller pump (flow rate of 0.6 ml/min, kept constant throughout the experiment). The chamber volume plus dead volume was approximately 0.6 ml. A series of four parallel superfusion chambers was used to enable both control and test conditions to be performed in duplicate from the same batch of synaptosomes. After setting up the synaptosomes, a 40-min washout period was performed before starting sample collection. The effluent was then collected (release period) in 3-min fractions. To quantify tritium release, 500 μl of the each of the effluent samples were analyzed by scintillation counting, after addition of 5 ml of scintillation liquid (Scintran T, Wallac, Turku, Finland). In some experiments, [3H]adenosine in the effluent samples was separated. This procedure consisted in the lyophilization of 1.2 ml of the effluent, resuspension in a 70% (v/v) methanol solution to extract adenosine, HPLC separation of adenosine, and collection of the HPLC eluent corresponding to the adenosine peak that was scintillation counted to determine the amount of [3H]adenosine (see Ref. 1Cunha R.A. Vizi E.S. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1996; 67: 2180-2187Crossref PubMed Scopus (215) Google Scholar). The synaptosomes were stimulated for 2 min with 20 mmK+ (isomolar substitution of Na+ by K+ in the superfusion Krebs solution) at 21 min after starting sample collection. In some experiments, veratridine stimulation (10 μm for 2 min) was used instead of K+ stimulation. Tested drugs, applied through the superfusion Krebs solution, were added 9 min before K+stimulation, and removed 13 min after the offset of stimulation. When testing the modifications of the effect of a drug by a modifier, this modifier was applied 15 min before starting sample collection and was present throughout the experiment. At the end of the experiments, the filters were removed from the superfusion chambers and analyzed by scintillation counting for determination of tritium retained by the synaptosomes. Radioactivity was expressed in terms of disintegrations per second per mg of protein (Bq/mg) in each chamber. The fractional release was expressed in terms of the percentage of total radioactivity present in the preparation at the beginning of the collection of each sample. The amount of radioactivity (expressed as fractional release) released by K+ stimulation was calculated by integration of the area of the peak upon subtraction of the estimated basal tritium release from the total release of tritium obtained upon K+stimulation. The basal release was assumed to decline linearly from 2 min before onset of stimulation to the 8th min after onset of stimulation. Hippocampal synaptosomes were resuspended and equilibrated at 37 °C in 2 ml of Krebs/HEPES solution of the following composition (mm): NaCl 124, KCl 3, NaH2PO4 1.25, MgSO4 1, CaCl2 2, HEPES 26, glucose 10, pH 7.4. Aliquots of 230 μl of synaptosomes (0.52–0.61 mg of protein) were placed in a reaction tube to which 50 μl of luciferin-luciferase solution (FL-AAM from Sigma dissolved in 5 ml of water, kept at 4 °C) and 10 μl of Krebs/HEPES containing the test drug were added. The mixture was placed in a luminometer (Wallac 1250 from LKB Turku, Finland) and the electrical signal generated by the photomultiplier recorded. After obtaining a stable baseline, the photomultiplier entry was closed, 10 μl of a Krebs/HEPES solution with concentrated KCl (to attain a final concentration of 20 mm) were added, the mixture resuspended, the photomultiplier entry was opened again, and the variation in signal recorded between 15 and 30 s was used to estimate the evoked release of ATP. The calibration curve for ATP was linear between 5 and 1000 pm (see Ref. 1Cunha R.A. Vizi E.S. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1996; 67: 2180-2187Crossref PubMed Scopus (215) Google Scholar). Time course kinetic studies of the extracellular catabolism of adenine nucleotides were performed as described previously (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar, 3Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurosci. 1998; 18: 1987-1995Crossref PubMed Google Scholar). Briefly, the hippocampal synaptosomes were resuspended in 1 ml of Krebs/HEPES solution and aliquots of 100 μl were added to incubation vials with 400 μl of Krebs/HEPES solution, to which AA (30 μm) and/or the protein kinase inhibitor chelerythrine (6 μm) were added, and kept at 37 °C. After 10 min incubation, the initial substrate, i.e. ATP (30 μm) or AMP (10 μm), was added. The kinetic protocols consisted of a 5- or 10-min incubation period with sample collection (50 μl) at 0, 1, 2.5, 5, 7.5, and 10 min for AMP and at 0, 0.5, 1, 2, 3, and 5 min for ATP. The zero time was defined as the sample collected immediately after (approximately 2–5 s) addition of the initial substrate. Each collected sample was centrifuged (14,000 × g for 10 s) and the supernatant (40 μl) was ice-stored for HPLC analysis (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar, 3Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurosci. 1998; 18: 1987-1995Crossref PubMed Google Scholar). After the 5- or 10-min incubation, the synaptosomes were pelleted by centrifugation (14,000 × g for 10 s). The pelleted synaptosomes were homogenized in 200 μl of 2% (v/v) Triton X-100 to determine total lactate dehydrogenase activity and protein content (14Spector T. Anal. Biochem. 1978; 86: 142-146Crossref PubMed Scopus (1356) Google Scholar). The remaining bathing solution was used to quantify lactate dehydrogenase activity, an index of cellular disruption (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar), which was always lower than 3% of total lactate dehydrogenase activity in the synaptosomes. The synaptosomes were resuspended in 1 ml of Krebs/HEPES solution and equilibrated at 37 °C. All adenosine transport assays were conducted at 37 °C in a total volume of 300 μl, containing ∼0.15 mg of protein, basically as described by Gu et al. (15Gu J.G. Kala G. Geiger J.D. J. Neurochem. 1993; 60: 2232-2237Crossref PubMed Scopus (12) Google Scholar), with minor modifications. Transport was initiated by addition of 0.25–2 μm [2-3H]adenosine, which was added at least 10 min after exposure of synaptosomes to tested drugs, and incubations were for 15 s. Transport was terminated by addition of 5 ml of ice-cold inhibitor/stop mixture (100 μmdipyridamole and 1 mm adenosine in Krebs/HEPES solution), followed by filtration through Whatman GF/C filters and washing of the reaction tube and filter with 5 ml of ice-cold inhibitor/stop mixture. The filters were then analyzed by scintillation counting for determination of tritium retained by the synaptosomes after addition of 5 ml of scintillation mixture. Adenosine transport was calculated as the difference between total adenosine incorporation into synaptosomes and the nonspecific component of adenosine fixation by synaptosomes for each concentration of [2-3H]adenosine, determined in the presence of 100 μm dipyridamole and 1 mmadenosine. To determine the kinetic values for adenosine transport (K m and V max values), [2-3H]adenosine transport was performed in the presence of 6 concentrations of [2-3H]adenosine ranging from 0.25 to 2 μm. The apparent K m andV max values were calculated by nonlinear regression analysis with the Raphson-Newton method using the GraphPad Prism software. One 400-μm hippocampal slice, obtained as described previously (3Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurosci. 1998; 18: 1987-1995Crossref PubMed Google Scholar), was transferred to a 1-ml recording chamber for submerged slices and continuously superfused with gassed Krebs solution, kept at 30 °C, at a flow rate of 3 ml/min. Drugs were added to this superfusion solution. Electrophysiological recordings of field excitatory post-synaptic potentials (fEPSP) were obtained as described previously (3Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurosci. 1998; 18: 1987-1995Crossref PubMed Google Scholar). Stimulation (rectangular pulses of 0.1 ms applied once every 15 s) was delivered through a bipolar concentric electrode placed on the Schaffer fibers, in thestratum radiatum near the CA3/CA1 border. Orthodromically evoked fEPSPs were recorded through an extracellular microelectrode (4m NaCl, 2–5 MΩ resistance) placed in the stratum radiatum of the CA1 area. The intensity of the stimulus was adjusted to evoke a fEPSP of 0.7–1 mV without appreciable population spike contamination. Recordings were obtained with an Axoclamp 2B amplifier coupled to a DigiData 1200 interface (Axon Instruments, Foster City, CA) and averages of 8 consecutive responses were continuously monitored on a personal computer with the LTP 1.01 software (16Anderson W.W. Collingridge G.L. Neurosci. Abstr. 1997; 23: 665Google Scholar). Responses were quantified as the initial slope of the averaged fEPSPs. Superfusion of a slice with drugs was started after responses were stable for at least 20 min. The release of [3H]acetylcholine was performed as described previously (17Cunha R.A. Milusheva E. Vizi E.S. Ribeiro J.A. Sebastião A.M. J. Neurochem. 1994; 63: 207-214Crossref PubMed Scopus (142) Google Scholar, 18Almeida T. Cunha R.A. Ribeiro J.A. Brain Res. 1999; 826: 104-111Crossref PubMed Scopus (48) Google Scholar). Briefly, rat hippocampal slices were loaded with [3H]choline (30 μCi/ml, 0.3 μm) for 30 min, washed, placed in 100-μl Perspex chambers, and superfused with oxygenated Krebs solution containing 10 μmhemicholinium-3 at 30 °C with a flow rate of 0.6 ml/min. The preparations were stimulated twice (S1 and S2) at 6 and 36 min with supramaximal monopolar square-wave pulses with a duration of 3 ms and an amplitude of 40 V, delivered with a frequency of 5 Hz for a period of 2 min (600 pulses), through platinum electrodes. Drugs were added to the superfusion medium 15 min before S2 and remained in the bath up to the end of the experiment. When we evaluated the modification of the effect of a drug by a modifier, this modifier was applied 15 min before sample collection was started and hence was present during S1 and S2. At the end of the release experiments, 5 ml of scintillation mixture (Scintran T) was added to a 500-μl aliquot of each eluent fraction and to 100 μl of the homogenized slices (sonicated in 500 μl of 3m perchloric acid and 2% Triton X-100). Radioactivity was expressed in terms of disintegrations per minute per mg of protein in each chamber (dpm/mg). Protein was quantified according to Spector (14Spector T. Anal. Biochem. 1978; 86: 142-146Crossref PubMed Scopus (1356) Google Scholar). The fractional release was expressed in terms of the percentage of total radioactivity present in the tissue at the time of sample collection. The release of tritium evoked by each period of electrical stimulation, i.e. the evoked release (expressed as fractional release), was calculated by integration of the area of the peak upon subtraction of the estimated basal tritium outflow from the total outflow due to electrical stimulation. The evoked release of tritium under these conditions is Ca2+- and tetrodotoxin-sensitive and is mostly constituted by [3H]acetylcholine (17Cunha R.A. Milusheva E. Vizi E.S. Ribeiro J.A. Sebastião A.M. J. Neurochem. 1994; 63: 207-214Crossref PubMed Scopus (142) Google Scholar, 18Almeida T. Cunha R.A. Ribeiro J.A. Brain Res. 1999; 826: 104-111Crossref PubMed Scopus (48) Google Scholar). The effect of drugs on the release of tritium was expressed by alterations of the ratio between the evoked release due to the second stimulation period and the evoked release due to the first stimulation period (S2/S1 ratio). For the determination of the energy charge, the synaptosomes were incubated without or with AA, PLA2, or melittin for 30 min, homogenized by sonication, and adenine nucleotides were extracted and analyzed by HPLC (17Cunha R.A. Milusheva E. Vizi E.S. Ribeiro J.A. Sebastião A.M. J. Neurochem. 1994; 63: 207-214Crossref PubMed Scopus (142) Google Scholar, 18Almeida T. Cunha R.A. Ribeiro J.A. Brain Res. 1999; 826: 104-111Crossref PubMed Scopus (48) Google Scholar). To evaluate cellular integrity, the synaptosomes were placed in the superfusion chambers and superfused for 30 min with gassed Krebs without or with AA, PLA2, or melittin in a close-circuit manner. Cellular disruption was determined by comparing the lactate dehydrogenase activity in the superfusion medium with that found in the synaptosomes upon their solubilization, by sonication, with 2% (v/v) Triton X-100 (2Cunha R.A. Sebastião A.M. Ribeiro J.A. J. Neurochem. 1992; 59: 657-666Crossref PubMed Scopus (68) Google Scholar). To study the metabolism of AA in hippocampal synaptosomes, [3H]AA (5 μm, 21.8 μCi/ml) was incubated for 20 min at 37 °C with hippocampal synaptosomes (0.36–0.42 mg protein) in a final volume of 200 μl, in the absence or presence of indomethacin (20 μm) and/or NDGA (50 μm). The samples were then diluted to a concentration of 80% (v/v) ethanol, vortexed, and kept on ice for 20 min. The samples were then centrifuged at 1,000 × g for 10 min to pellet the precipitated protein material and the supernatant was evaporated to dryness on a rotary evaporator at 37 °C. Extraction of lipid material was performed by resuspension in 300 μl of 30% (v/v) methanol, a procedure that yields good recoveries (86 ± 3%,n = 4, using synaptosomes previously boiled for 30 min) of [3H]AA, as previously reported (19Henke D.C. Kouzan S. Eling T.E. Anal. Biochem. 1984; 140: 87-94Crossref PubMed Scopus (80) Google Scholar). Separation of eicosanoids (100 μl injected) in standards and samples was achieved by an adapted reverse-phase HPLC method (see Ref. 19Henke D.C. Kouzan S. Eling T.E. Anal. Biochem. 1984; 140: 87-94Crossref PubMed Scopus (80) Google Scholar) using a Beckman Gold system equipped with a Lichrospher C-18 (5 μm) column, imposing a sequence of isocratic elutions (flow rate:1.25 ml/min) starting with 55% (v/v) methanol in buffered water (prepared by adding 1 ml of glacial acetic acid to 1 liter of water and adjusting the pH to 5.8 with 1 m NH4OH) for 22 min followed by 100% methanol for 14 min. The retention times of the tested eicosanoids, identified by UV detection at 220 nm, were: PGE2 7.77 ± 0.06 min, PGF2α 10.51 ± 0.09 min, 5-HETE and 12-HETE co-eluting at 24.76 ± 0.09 min, and AA 26.08 ± 0.07 min. The HPLC eluent of the chromatographed lipid extracts of synaptosomes incubated with [3H]AA was collected every minute and analyzed by scintillation counting to monitor the disappearance of tritium from [3H]AA and the appearance of tritium in other eicosanoid peaks. The values are presented as mean ± S.E. To test the significance of the effect of an agonist versuscontrol, a paired Student's t test was used. When making comparisons from a different set of experiments with control, one-way analysis of variance (ANOVA) was used, followed by Dunnett's test.p < 0.05 was considered to represent a significant difference. The stimulation of hippocampal synaptosomes with K+ (20 mm for 2 min) resulted in an increase of tritium (Fig. 1 A, open symbols) and of [3H]adenosine (Fig. 1 B, open symbols) in the superfusion effluent, which represents 1.12 ± 0.03% (n = 12) and 0.164 ± 0.007% (n = 12) of total tritium retained by the synaptosomes, respectively. The addition of AA (30 μm) to the superfusate 9 min before K+ stimulation caused a 28 ± 3% (n = 4) decrease in the basal outflow of tritium (Fig. 1 A) and a 16 ± 5% (n = 4) decrease in the basal outflow of [3H]adenosine (Fig.1 B); in contrast, in the presence of AA (30 μm), the evoked tritium outflow and the evoked [3H]adenosine outflow were enhanced by 69 ± 3% (n = 6) and 57 ± 5% (n = 4), respectively (Fig. 1). AA (30 μm) also enhanced the veratridine-evoked tritium outflow by 62 ± 4% (n= 3, data not shown), which excludes direct effects of AA on K+ channels (20Ordway R.W. Singer J.J. Walsh Jr., J.V. Trends Neurosci. 1991; 14: 96-100Abstract Full Text PDF PubMed Scopus (345) Google Scholar) as a possible cause for the AA-induced increase in adenosine evoked release. Under control conditions (i.e. in the absence of added AA), omission of Ca2+ in the superfusate and addition of 100 nm EGTA, caused a 81 ± 8% increase of the basal outflow of tritium and a 72 ± 4% decrease of the K+-evoked outflow of tritium. The effect of the absence of Ca2+ and presence of EGTA (100 nm) on [3H]adenosine outflow was similar to that of tritium release, with a 124 ± 9% increase of the basal outflow and a 76 ± 6% decrease of the K+-evoked outflow of [3H]adenosine (n = 4). This Ca2+ dependence of the evoked outflow of tritium and of [3H]adenosine was also observed in the presence of AA (30 μm), when the absence of Ca2+ and presence of EGTA (100 nm) caused a 82 ± 4% decrease of the K+-evoked tritium outflow and a 86 ± 6% decrease of the K+-evoked [3H]adenosine outflow (n = 3). Thus, in the absence of Ca2+ and presence of 100 nm EGTA, AA (30 μm) did not cause a significant (p > 0.05) increase in K+-evoked [3H]adenosine outflow. AA (3–30 μm) caused a concentration-dependent facilitation of the K+-evoked outflow of both tritium and [3H]adenosine (Fig. 1 C). However, a higher concentration of AA (100 μm) caused a lower facilitation mainly of the evoked outflow of [3H]adenosine but also of tritium (Fig. 1 C). To investigate whether the effect of AA on tritium outflow was influenced by detergent-like effects or oxidative phosphorylation uncoupling caused by AA (e.g. Ref.21Breukel A.I.M. Besselsen E. Lopes da Silva F. Ghijsen W.E.J.M. Brain Res. 1997; 773: 90-97Crossref PubMed Scopus (21) Google Scholar), we tested the effect of AA on the energy charge and on the release of lactate dehydrogenase, an intracellular marker, from superfused rat hippocampal synaptosomes. As shown in TableI, AA (30 μm) did not significantly change either the energy charge or the release of lactate dehydrogenase. In contrast, AA (100 μm) decreased the energy charge by 8.8 ± 0.1% (n = 4,p < 0.05) and caused the release of 12 ± 2% (n = 4, p < 0.05) of total lactate dehydrogenase during the 30-min superfusion period (Table I). Thus, as was previously proposed to occur (18Almeida T. Cunha R.A. Ribeiro J.A. Brain Res. 1999; 826: 104-111Crossref PubMed Scopus (48) Google Scholar, 21Breukel A.I.M. Besselsen E. Lopes da Silva F. Ghijsen W.E.J.M. Brain Res. 1997; 773: 90-97Crossref PubMed Scopus (21) Google Scholar), the lower effects of AA (100 μm) on the outflow of tritium and [3H]adenosine might probably be due to a modification of the homeostasis and viability of the synaptosomes.Table ILack of effect of AA (30 μm), of phospholipase A2(PLA2, 2 units/ml) and of melittin (1 μm) and modification by AA (100 μm) of the energy charge and release of lactate dehydrogenase, an intracellular marker, from rat superfused hippocampal synaptosomesEnergy charge% total lactate dehydrogenase releasedCONTROL0.784 ± 0.0024 ± 1AA (30 μm)0.786 ± 0.0014 ± 1AA (100 μm)0.715 ± 0.004 1-ap < 0.05 versus control (i.e. absence of any added drug).12 ± 2 1-ap < 0.05 versus control (i.e. absence of any added drug).PLA2 (2 units/ml)0.785 ± 0.0022 ± 1Melittin (1 μm)0.782 ± 0.0035 ± 2The values are mean ± S.E. of four experiments.1-a p < 0.05 versus control (i.e. absence of any added drug). Open table in a new tab The values are mean ± S.E. of four experiments. Since AA caused virtually identical effects on tritium outflow and [3H]adenosine outflow, the tritium outflow was used as a measure of [3H]adenosine outflow in the remaining experiments. It was also observed that the effect of AA on the K+-evoked tritium outflow were consistent from experiment to experiment, whereas there was a large variation in the effects of AA on the basal tritium outflow, which precluded a systematic investigation of the effect of AA on basal tritium" @default.
• W2018018964 created "2016-06-24" @default.
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• W2018018964 title "Modification by Arachidonic Acid of Extracellular Adenosine Metabolism and Neuromodulatory Action in the Rat Hippocampus" @default.
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