Properties of taurine release in glucose-free media in hippocampal slices from developing and adult mice

Resea ch A icle P ope ies o Tau ine Release in Glucose-F ee Media in Hippocampal Slices om De eloping and Adul Mice Simo S. Oja and Pi jo Sa ansaa i Medical School, 33014 Uni e si y o Tampe e, Finland Co espondence should be add essed o Simo S. Oja; simo.o[email p o ec ed] Recei ed 14 May 2015; Accep ed 21 July 2015 Academic Edi o : Ha i Shanke Sha ma Copy igh © 2015 S. S. Oja and P. Sa ansaa i. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. The elease o p eloaded [3H] au ine om hippocampal slices om de eloping 7-day-old and young adul 3-mon h-old mice was s udied in a supe usion sys em in he absence o glucose. These hypoglycemic condi ions enhanced he elease a bo h ages, he e ec being ma kedly g ea e in de eloping mice. A depola izing K+concen a ion accen ua ed he elease, which indica es ha i was pa ially media ed by exocy osis. The anion channel blocke s we e inhibi o y, wi nessing he con ibu ion o ion channels. NO- gene a ing agen s omen ed he elease as a sign o he pa icipa ion o exci a o y amino acid ecep o s. The o he second messenge sys ems we e appa en ly less e icien . The much g ea e au ine elease could be a eason o he well-known g ea e ole ance o de elopingne ous issue olacko glucose. 1. In oduc ion Hypoglycemia is a me abolic condi ion common du ing de elopmen and may lead o se e e neu ological de ec s in human in an s. Howe e , he e ec s o hypoglycemia on he de eloping b ain a e s ill incomple ely unde s ood [1]. The b ain de i es mos o i s ene gy om he oxida ion o glucose, bu du ing de elopmen i also has an abili y o u ilize al e na i e ene gy subs a es which may o e p o ec ion du ing hypoglycemia [2, 3]. On he o he hand, poo ese es o high-ene gy phospha es and a high me abolic a e may p edispose he de eloping b ain o hypoglycemic inju y [4, 5]. In he b ain he hippocampus is he egion mos sensi i e o lack o oxygen and glucose. Ene gy dep i a ion leads o neu onal cell dea h, caused p ima ily by exci o oxici y due o excessi e glu ama e elease [6, 7]. Tau ine (2-aminoe hanesul onic acid) is p esen a high concen a ions in he b ain. Du ing on ogenic de elopmen i s concen a ion e en exceeds ha o he main exci a o y ansmi e glu ama e [8]. I inc eases memb ane chlo ide conduc ance, causing hype pola iza ion and inhibi ing neu- onal i ing [9, 10]. Tau ine also a enua es he excessi e neu onal accumula ion o Ca2+,whichp edisposescells o damage [11] and p e en s o educes he glu ama e- induced ele a ion o in acellula Ca2+ [12] by inhibi ing he glu ama e-induced elease o Ca2+ om he in e nal pools [13] and he glu ama e-induced Ca2+ in lux h ough L-, P/Q-, and N- ypes o ol age-ga ed Ca2+ channels [14]. Tau ine hus egula es cy oplasmic and mi ochond ial calcium homeos a- sis [15] and in his manne p o ec s neu al cells agains he oxici y o exci a o y amino acids in he hippocampus [16]. Cell-damaging condi ions, including hypoglycemia, inc ease he elease o au ine [17, 18] oge he wi h ha o exci a o y amino acid neu o ansmi e s. Ou p ima y assump ion is ha au ine elease in he absence o an adequa e supply o glucose could p o ec neu al cells om inju y. In he p esen s udy we he e o e examined he gene al p ope ies o au ine eleasein hehippocampusinhypoglycemiaandhow he eleaseisa ec edbyionchannels,secondmessenge sys ems, and adenosine ecep o s. 2. Ma e ials and Me hods 2.1. Ma e ials. De eloping (7-day-old) and young adul (3- mon h-old) NMRI mice o bo h sexes we e used in he Hindawi Publishing Co po a ion Jou nal o Amino Acids Volume 2015, A icle ID 254583, 7 pages h p://dx.doi.o g/10.1155/2015/254583 2Jou nal o Amino Acids expe imen s. All e o s we e made o minimize bo h he su e ing and he numbe o he animals used. The expe - imen s con o med o he Eu opean Communi y Di ec i e (86/609/EEC) o e hical use o expe imen al animals and we e app o ed by he Commi ee o Tampe e Uni e si y o animal expe imen s. [3H]Tau ine (speci ic adioac i i y 1.15 PBq/mol) was ob ained om Ame sham In e na ional, B is ol, UK. The a ious e ec o s we e pu chased om he Toc is Bioscience (B is ol, UK) o Sigma Ald ich (S . Louis, MO). 2.2. Release Expe imen s. Co onal slices 0.4 mm hick weigh- ing 15–20 mg we e manually p epa ed om he mouse hip- pocampus wi h a issue slice o S adie-Riggs ype. The slices we e immedia ely imme sed in 5 mL o oxygena ed medium and incuba ed wi h 0.01 mM [3H] au ine (50 MBq/L) a 37∘C o 30 min unde agi a ion. The s anda d K ebs-Ringe - Hepes medium con ained (in mmol/L) NaCl 127, KCl 5, CaCl2 0.8, MgSO41.3, Na2HPO41.3, N-2-hyd oxye hylpipe azine- N󸀠-2-e hanesulphonic acid (Hepes) 15, NaOH 11, and D- glucose10(pH7.4).Thesliceswe e hen ans e edin o 0.25 mL cups and supe used wi h he abo e medium a a a e o 0.25 mL/min o 50 min in a sys em in which eely loa ing shaken slices we e kep unde a con inuous low o oxygen in o de o p ese e hei iabili y [19]. Hypo- glycemic condi ions we e induced by omi ing glucose om he supe usion media. Po assium s imula ion was applied om 30 o 50 min wi h 50 mM K+. In ou expe imen al se up his K+concen a ion has yielded he bes and mos ep oducible esponses in GABA and au ine elease [19]. This high K+concen a ion may cause elease no only om neu ons bu om glial cells as well [20]. Howe e , au ine elease is ypically slow a onse and p olonged and high K+concen a ions abo e hose p e ailing in i o should be used in in i o expe imen s [21]. The di e en e ec o s we e added o he medium a he onse o supe usions, as explained in he able legends. The supe usion medium was pooled du ing he i s 20min, whe ea e 2min ac ions (0.5 mL) we e collec ed di ec ly in o small scin illa ion ials wi h a ac ion collec o . A e supe usion he slices we e weighed, homogenized in ice-cold 5% (w/ ) ichlo oace ic acid solu ion, and cen i uged, and he clea supe na an s we e used o scin illa ion coun ing. The e luen samples we esubjec ed o hesameanalyses. 2.3. Es ima ion o E lux Ra e Cons an s. Desa u a ion cu es o labeled au ine om he slices we e plo ed as a unc ion o ime on he basis o he adioac i i ies emaining in heslicesa e supe usionand eco e edin hecollec ed supe usa e ac ions [19]. Du ing supe usion he elease o labeled au ine o igina es ini ially om he ex acellula spaces in slices. This sou ce is g adually exhaus ed du ing he i s 20minand he eleasesubsequen lyoccu s om he in acellula pools. The e lux a e cons an s o au ine o he ime in e als o 20 o 30 min (𝑘1,ini ial eleasephase)and 34–50 min (𝑘2,la e eleasephase)we ecompu edasnega i e slopes o he eg essionlineso heloga i hmo adioac i i y emaining in he slices e sus supe usion ime. The e we e no di e ences be ween he esul s om male and emale mice and he esul s om bo h sexes we e he e o e combined. 2.4. S a is ical Analysis. The signi icance o he esul s was analyzed wi h wo-way analysis o a iance (ANOVA) using SPSS s a is ics, e sion 17.0, compu e p og am. The analyses we e done by g ouping he esul s acco ding o he na u e o hee ec o ss udied,po assiums imula ion,chlo idechannel blocke s, NO-gene a ing agen s, adenosine agonis s, and second messenge s. When signi ican e ec s we e de ec ed, he pos hoc Bon e oni es was applied o b ing ou he di e ences be ween he sample means. They we e conside ed signi ican when he calcula ed 𝑝 alues we e less han 0.05 o 0.01. 3. Resul s Hypoglycemia signi ican ly enhanced au ine elease in bo h age g oups. In adul 3-mon h-old mice he ac ional elease a e cons an in he p esence o glucose in medium was o he supe usion pe iod o 34–50 min (1.54±0.04)×10−3 (𝑛=18) andin heabsenceo glucose(1.93 ± 0.18)×10−3,𝑛=11, signi ican ly di e en a a le el o p= 0.042. The hypoglycemia e ec was mo e ma ked in 7-day-old mice, he co esponding cons an s being (0.38±0.20)×10−3,𝑛=11,and(1.20±0.12) ×10−3,𝑛=11,p= 0.000, espec i ely. S imula ion by 50 mM K+enhanced he eleasein heabsenceo glucose(d =3, 𝐹 = 23.007,𝑝 = 0.000). The enhancemen s we e 60 pe cen and 63 pe cen in adul (𝑝 = 0.000) and de eloping (𝑝= 0.001) mice, espec i ely (see Table 1). The anion channel blocke s gene ally inhibi ed he elease (d =7,𝐹 = 13.562,𝑝 = 0.000). O hem, DIDS signi ican ly inhibi ed he K+-s imula ed au ine eleaseinbo hadul (𝑝 = 0.013) and de eloping (𝑝 = 0.044) mice, being in adul micealsoe ec i eon heuns imula ed elease(𝑝= 0.047) (Table 1). SITS, he blocke o chlo ide anspo , was e en mo e e ec i e in all expe imen al si ua ions (in adul mice, uns imula ed elease 𝑝 = 0.11,s imula ed elease, 𝑝 = 0.035, and in de eloping mice he co esponding da a 𝑝 = 0.016 and 𝑝 = 0.017). Ano he anspo inhibi o 9-AC was no e ec i e. All ni ic oxide gene a o s, SNAP, SNP, and hyd oxylamine, we e s ong s imula o s in au ine elease in bo h adul (d =7,𝐹 = 32.190,𝑝 = 0.000)and de eloping (d =7,𝐹 = 13.732,𝑝 = 0.000) mice and in bo h heuns imula edandK +-s imula ed elease (Table 2). On he o he hand, all adenosine ecep o agonis s es ed, CHA, R- PIA and CGS 21680, and iluzole, which inhibi s glu ama e elease and GABA up ake, we e wi hou any e ec s (Table 3). O compounds in ol ed in he second messenge sys- ems, genis ein, wi h i s main known ac i i y as a y osine kinase inhibi o , was no e ec i e du ing he pe iod o 34– 50 (Table 4), whe eas quinac ine, a nonselec i e inhibi o o bo h monoamine oxidases A and B, inhibi ed he uns im- ula ed and K+-s imula ed elease in bo h age g oups a all s ages o supe usion (d =6,𝐹 = 23.648,𝑝 = 0.000). The p o ein kinase C ac i a o PMA only inhibi ed he uns imula ed eleaseinadul mice(𝑝 = 0.036), whe eas he p o ein kinase inhibi o chele y h ine was no e ec i e. Jou nal o Amino Acids 3 Table 1: E ec s o ion channel inhibi o s on au ine elease om hippocampal slices om 3-mon h-old and 7-day-old mice in hypoglycemia. E ec o s E lux a e cons an s (×10−3min−1)±SEM 3-mon h-old 7-day-old 𝑘1𝑘2𝑘1𝑘2 Basal (con ol) 2.36 ±0.11 (20) 1.93 ±0.18 (11) 1.34 ±0.08 (31) 1.20 ±0.11 (11) +50 mM K+(con ol) 2.92 ±0.21 (10) 1.90 ±0.10 (12) DIDS 0.5 mM 1.34 ±0.15∗∗ (6) 1.31 ±0.10∗(4) 1.12 ±0.14 (8) 1.16 ±0.13 (4) +50 mM K+1.81 ±0.12∗∗ (4) 1.45 ±0.06∗(4) SITS 2.0 mM 1.04 ±0.07∗∗ (8) 0.93 ±0.06∗∗ (4) 0.80 ±0.09∗∗ (6) 0.62 ±0.06∗∗ (4) +50 mM K+1.50 ±0.21∗∗ (4) 0.90 ±0.12∗∗ (4) 9-AC 0.2 mM 2.67 ±0.17 (5) 2.74 ±0.49 (4) 1.82 ±0.23 (8) 1.07 ±0.09 (4) +50 mM K+3.53 ±0.32 (4) 1.84 ±0.10 (4) The d ugs we e added a he beginning o supe usion and 50 mM K+a 30 min. The esul s show he e lux a e cons an s ±SEM (×10−3min−1) o he ime in e als o 20–30 min (𝑘1) and 34–50 min wi hou he excess o K+o in he p esence o 50 mM K+(𝑘2) wi h he numbe o independen expe imen s in pa en hesis. Abb e ia ions: DIDS, diiso hiocyanos ilbene-2󸀠2-disulphona e; SITS, 4-ace amido-4󸀠-iso hiocyanos ilbene-2󸀠2-disulphona e; 9- AC, 9-an h aceneca boxylic acid. Signi icance o di e ences om he co esponding con ols: ∗𝑝 < 0.05,∗∗𝑝 < 0.01. Table 2: E ec s o ni ic oxide gene a o s on au ine elease om hippocampal slices om 3-mon h-old and 7-day-old mice in hypoglycemia. E ec o s E lux a e cons an s (×10−3 min−1)±SEM 3-mon h-old 7-day-old 𝑘1𝑘2𝑘1𝑘2 Basal (con ol) 2.36 ±0.11 (20) 1.93 ±0.18 (11) 1.34 ±0.08 (31) 1.20 ±0.11 (11) +50 mM K+(con ol) 2.92 ±0.21 (10) 1.90 ±0.10 (12) SNAP 1.0 mM 4.31 ±0.22∗∗ (7) 5.44 ±0.14∗∗ (4) 2.84 ±0.11∗∗ (8) 2.17 ±0.07∗∗ (4) +50 mM K+5.35 ±0.15∗∗ (4) 2.34 ±0.08∗∗ (4) SNP 1.0 mM 3.17 ±0.13∗∗ (15) 4.23 ±0.38∗∗ (8) 1.80 ±0.21∗(7) 1.65 ±0.13∗(4) +50 mM K+4.41 ±0.77∗∗ (4) 1.98 ±0.10∗∗ (4) Hyd oxylamine 5.0 mM 5.16 ±0.17∗∗ (7) 6.04 ±0.76∗∗ (7) 3.82 ±0.33∗∗ (8) 3.44 ±0.16∗∗ (4) +50 mM K+7. 03 ±0.28∗∗ (4) 3.84 ±0.10∗∗ (4) The e ec o s we e added a he beginning o supe usion and 50 mM K+a 30 min. The esul s show he e lux a e cons an s ±SEM (×10−3 min−1) o he ime in e als o 20–30 min (𝑘1) and 34–50 min wi hou he excess o K+o in he p esence o 50 mM K+(𝑘2) wi h he numbe o independen expe imen s in pa en hesis. SNAP: S-ni oso-N-ace ylpenicillamine; SNP: sodium ni op usside. Signi icance o di e ences om he co esponding con ols: ∗𝑝 < 0.05, ∗∗𝑝 < 0.01. Table 3: E ec s o adenosine agonis s and iluzole on au ine elease om hippocampal slices om 3-mon h-old and 7-day-old mice in hypoglycemia. E ec o s E lux a e cons an s (×10−3 min−1)±SEM 3-mon h-old 7-day-old 𝑘1𝑘2𝑘1𝑘2 Basal (con ol) 2.36 ±0.11 (20) 1.93 ±0.18 (11) 1.34 ±0.08 (31) 1.20 ±0.11 (11) +50 mM K+(con ol) 2.92 ±0.21 (10) 1.90 ±0.10 (12) CHA 0.5 mM 2.98 ±0.12 (6) 1.85 ±0.30 (6) 1.48 ±0.07 (10) 1.11 ±0.06 (7) +50 mM K+2.42 ±0.27 (4) 2.00 ±0.06 (4) R-PIA 0.1 mM 2.46 ±0.17 (6) 1.92 ±0.21 (6) 1.97 ±0.19 (6) 1.41 ±0.12 (4) +50 mM K+3.63 ±0.51 (4) 2.12 ±0.11 (4) CGS 21680 10.0 mM 2.59 ±0.22 (4) 2.20 ±0.20 (4) 1.75 ±0.16 (6) 1.38 ±0.20 (4) +50 mM K+3.44 ±0.32 (4) 2.28 ±0.11 (4) Riluzole 0.1 mM 2.47 ±0.17 (7) 1.86 ±0.27 (4) 1.86 ±0.15∗(6) 1.38 ±0.10 (4) +50 mM K+2.50 ±0.35 (4) 2.23 ±0.15 (4) The agonis s we e added a he beginning o supe usion and 50 mM K+a 30 min. The esul s show he e lux a e cons an s ±SEM (×10−3 min−1) o he ime in e als o 20–30 min (𝑘1) and 34–50 min wi hou he excess o K+o in he p esence o 50 mM K+(𝑘2) wi h he numbe o independen expe imen s in pa en hesis. CHA: N6-cyclohexyladenosine; R-PIA: (R(−)N6-(2-phenylisop opyl)adenosine; CGS 21680: 4-[2-[[6-amino-9-(N-e hyl-𝛽-D- ibo u anu onamidosyl)-9H-pu in-2-yl]amino]e hyl]benzenep opanoic acid hyd ochlo ide. Signi ican di e ence om he con ol: ∗𝑝 < 0.05. 4Jou nal o Amino Acids Table 4: E ec s o compounds in ol ed in he second messenge sys ems on au ine elease om mouse hippocampal slices in hypoxia. Concen a ion (mM) E lux a e cons an s (×10−3 min−1)±SEM 3-mon h-old 7-day-old 𝑘1𝑘2𝑘1𝑘2 Basal (con ol) 2.36 ±0.11 (20) 1.93 ±0.18 (11) 1.34 ±0.08 (31) 1.20 ±0.11 (11) +50 mM K+(con ol) 2.92 ±0.21 (10) 1.90 ±0.10 (12) Genis ein 0.001 2.59 ±0.13 (7) 2.06 ±0.18 (7) 1.92 ±0.16∗(7) 1.44 ±0.16 (4) +50 mM K+3.07 ±0.25 (4) 2.27 ±0.06 (4) Quinac ine 0.01 1.20 ±0.08∗∗ (7) 0.83 ±0.05∗∗ (4) 0.75 ±0.07∗∗ (8) 0.61 ±0.06∗∗ (4) +50 mM K+1.12 ±0.13∗∗ (4) 0.79 ±0.05∗∗ (4) PMA 0.00001 2.31 ±0.20 (8) 1.77 ±0.11 (4) 1.80 ±0.15∗(8) 1.33 ±0.17 (4) +50 mM K+1.95 ±0.17∗(4) 1.85 ±0.03 (4) Chele y h ine 0.001 2.49 ±0.17 (7) 2.07 ±0.14 (4) 1.73 ±0.18 (8) 1.53 ±0.04 (4) +50 mM K+3.40 ±0.51 (4) 2.01 ±0.09 (4) IBMX 1.0 1.85 ±0.16 (8) 2.51 ±0.29 (4) 1.99 ±0.24 (6) 1.33 ±0.11 (4) +50 mM K+2.70 ±0.13 (4) 1.85 ±0.03 (4) RO 20-1724 0.2 3.46 ±0.12∗∗ (8) 3.43 ±0.15∗∗ (4) 1.96 ±0.26 (7) 1.27 ±0.17 (4) +50 mM K+4.56 ±0.30∗∗ (4) 2.65 ±0.33∗(4) Zap inas 0.1 2.31 ±0.15 (8) 2.00 ±0.20 (8) 1.00 ±0.13 (6) 0.66 ±0.12∗(4) +50 mM K+2.53 ±0.06 (4) 2.04 ±0.23 (4) ODQ 0.01 2.51 ±0.18 (7) 2.01 ±0.13 (4) 1.50 ±0.17 (8) 1.19 ±0.08 (8) +50 mM K+2.50 ±0.25 (4) 2.04 ±0.06 (4) Alloxan 5.0 2.81 ±0.31 (7) 2.19 ±0.06 (4) 1.70 ±0.17 (6) 1.58 ±0.25 (4) +50 mM K+3.13 ±0.10 (4) 1.86 ±0.07 (4) The agonis s we e added a he beginning o he supe usion and 50 mM K+a 30 min. The esul s show he e lux a e cons an s ±SEM (×10−3 min−1) o he ime in e als o 20–30 min (𝑘1)and o 34–50minwi hou heexcesso K +o in he p esence o 50 mM K+(𝑘2) wi h he numbe o independen expe imen s in pa en hesis. PMA: 4𝛽-pho bol 12-my is a e 13-ace a e; IBMX: 3-isobu yl-1-me hylxan hine; ODQ: 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one; RO 20-1724: 4-(3-bu oxy-4-me hoxyphenyl)-2-imidazolidone. Signi icance o di e ences om he co esponding con ols: ∗𝑝 < 0.05,∗∗𝑝 < 0.01. The nonspeci ic inhibi o o cAMP and cGMP phosphodi- es e ases and he nonselec i e adenosine ecep o an agonis IBMX was no e ec i e du ing he pe iod o 34–50 min. The selec i e inhibi o o cGMP-insensi i e phosphodies e ase, ype IV, RO 20-1724 enhanced bo h he uns imula ed (𝑝= 0.008)ands imula ed(𝑝 = 0.005) elease in adul mice and alsosligh ly hes imula ed elease(𝑝 = 0.038)inde eloping mice (Table 4). The selec i e inhibi o o cGMP-speci ic phosphodies e ases V and VI (PDE5/6) and he agonis a he G p o ein-coupled ecep o 35 zap inas signi ican ly (𝑝= 0.035) educed he uns imula ed elease in 7-day-old mice, whe eas ODQ, he po en and selec i e inhibi o o NO- sensi i e guanylyl cyclase, was wi hou e ec . Alloxan, he unspeci ic inhibi o o adenylyl cyclases, was likewise no e ec i e du ing he pe iod o 34–50 min. 4. Discussion The p esen esul s show au ine elease o be enhanced in hypoxia a bo h ages s udied, he e ec being mo e p onounced in he de eloping hippocampus. The elease is la gelymedia edbyanionchannels,since heanionchannel inhibi o s ma kedly educed i . The ni ic acid gene a o s s ongly s imula ed he elease. The e ec s o he second messenge sys em we e mo e a iable. These ac ions we e also in many cases di e en in adul and de eloping mice. The enhancemen could be due o se e al mechanisms, including Ca2+-dependen exocy osis, Ca2+-independen elease ia e e sal o ca ie -media ed up ake, indisc im- ina e opening o ion channels which allow he passage o au ine molecules, o leakage h ough damaged plasma memb anes. Depola iza ion by K+-s imula ion was now able o u he po en ia e au ine elease a bo h ages unde hypoglycemia. This may signi y elease ia exocy osis, which is also p ese ed in hippocampal slices when hey a e exposed o e en mo e d as ic cell-damaging condi ions such as ischemia ( he absence o glucose and oxygen a mosphe e) [17, 18]. In de eloping mice au ine elease in he absence o glucose is app oxima ely he same as in he p esence o ni ogen a mosphe e wi hou glucose, whe eas in adul s hese ischemic condi ions ha e had e en mo e impac [18]. Bo h neu ons and glial cells ha e been shown o con ain au ine [22], and hus only a pa o he eleased au ine may o igina e om neu ons. K+s imula ion o au ine elease has also been shown o be associa ed wi h cell swelling [23]. Indeed, he olume- egula ed anion channels may con ibu e, oge he wi h he e e sed unc ion o as ocy ic glu ama e anspo e s,up o80pe cen o he o al eleaseo au ine and exci a o y amino acids in global ce eb al ischemia in a s [24]. In acellula swelling ac i a es s e ch-sensi i e ( olume- egula ed) ion channels and is accompanied by he elease Jou nal o Amino Acids 5 o bo h ino ganic and o ganic osmoly es, including au ine [25]. Fo ins ance, he olume- egula ed anion channels ha e been shown o be he p edominan con ibu o s o he elease o exci a o y amino acids in he ischemic co ical penum- b a [26]. The swelling-induced inc ease in au ine elease is a di usional p ocess wi hou any ca ie in ol emen [27]. In bo h he de eloping and adul hippocampus he p esy- nap ically ac ing iono opic glu ama e agonis s signi ican ly inc ease he elease o au ine in a ecep o -media ed manne [28, 29]. Fo ins ance, o he iono opic ecep o s, NMDA ecep o ac i a ion in pa icula has been assumed o play a cen al ole in hypoglycemia-induced glu ama e elease [30]. Ac i a ion o NMDA ecep o s allows Ca2+ o en e he cells. NO syn hase is a Ca2+-dependen enzyme [31], being ac i a ed in he p esence o Ca2+ and henp oducing NO.NOs imula essolubleguanyla ecyclaseandin his manne omen s he p oduc ion o 3󸀠,5󸀠-cyclic guanosine monophospha e [32], which enhances au ine elease [33]. The p esen ac i a o y e ec o he NO gene a o s s udied is in conce wi h his sequence o e en s. NO also egula es neu onal ac i i y by ac i a ing calcium-dependen po assium channels [34, 35]. NO can be he endogenous opene o leak K+channels in he hippocampus as shown in basal o eb ain choline gic neu ons [36]. The inc ease in ex acellula K+ enhances au ine elease. The NO dono s did no ma kedly omen he K+-e oked elease now. The e ec was appa en ly al eady maximal a he K+concen a ion used in his s udy. NO p oduced by an exogenous NO dono has been shown o block bo h e odo oxin-sensi i e and e odo oxin- esis an Na+cu en s in ba o ecep o neu ons [37] and NO o block di e en Na+channels in ba o ecep o neu- ons [38]. In con as , NO dono s SNP and SNAP ha e signi ican ly inc eased he mean pe sis en cu en in excised inside-ou pa ches om cul u ed hippocampal neu ons [39]. Such esul s sugges ha , depending on cell ype, NO may modula e Na+cu en s di e en ly. Tau ine e lux has been shown o be dependen on he p esence o bo h Na+and Cl−ions [40]. The educ ion o elease by he Cl−channel an agonis s SITS and DIDS indica es ha he elease may also occu h ough anion channels, simila o he olume- sensi i e au ine elease in as ocy es and neu ons [41, 42]. The p esen a enua ing e ec o quinac ine on he elease is u he mo e in line wi h his assump ion since quinac ine has been shown o inhibi he hypo onically induced whole cell Cl−cu en s in human submandibula gland (HSG) cells [43]. The a ious second messenge s exe ed less p onounced e ec s on au ine elease. In keeping wi h his he e ec s o di e en me abo opic glu ama e ecep o agonis s and an agonis s ha e also no been pa icula ly e ec i e in au ine elease in he hippocampus [44]. The p esynap ic adenosine ecep o s, pa icula ly o he A1class, a e known o egula e neu o ansmi e elease [45]. The A1 ecep o s ha e also enhanced au ine elease in he adul hippocampus, bu only when i was subjec ed o K+s imula ion in ischemia [46]. In he p esen s udy he nonspeci ic an agonis o adenosine ecep o s likewise exhibi ed only mino e ec s. 5. Conclusions The elease o au ine in he hypoglycemic hippocampus o bo h young and adul mice appea s o be a complex p ocess media ed by se e al di e en mechanisms, such as exocy osis, ca ie e e sal, di usion ia ion channels, and p obably leakage h ough pa ially damaged cell memb anes. Hypoglycemia also e okes he elease o exci a o y amino acids [6, 18]. Glu ama e elease o e ac i a es i s ecep o s and unc ions as an exci o oxic agen . The e is a a ionale he e in ha he ac i a ion o exci a o y amino acid ecep o s causes a concomi an inc ease in au ine elease. Tau ine is inhibi o y in na u e and coun e ac s his excessi e exci a ion. In he de eloping b ain au ine elease is much la ge in magni ude han in he adul b ain, which could be a eason o he well- knowng ea e ole anceo de elopingne ous issue olack o glucose. Abb e ia ions 9-AC: 9-An aceneca boxyla e AIDA: (RS)-1-Aminoindan-1,5- dica boxyla e AMPA: 2-Amino-3-hyd oxy-5-me hyl-4- isoxazolep opiona e 2R,4R-APDC: (2R,4R)-4-Aminopy olidine-2,4- dica boxyla e cAMP: Cyclic adenosine monophospha e ATPase: Adenosine iphospha ase cGMP: Cyclic guanosine monophospha e CGS 21680: 4-[2-[[6-Amino-9-(N-e hyl-𝛽-D- ibo u anu onamidosyl)-9H- pu in-2- yl]amino]e hyl]benzenep opanoic acid hyd ochlo ide CHA: N6-Cyclohexyladenosine DHPG: (S)-3,5-Dihyd oxyphenylglycine DIDS: Diiso hiocyanos ilbene-2,2󸀠- disulphona e DMPX: 3,7-Dime hyl-1-p opa gylxan hine EDTA: E hylenediamine e aace a e EGLU: (2S)-2-Cyclop opyl-4- phosphonophenylglycine GABA: 𝛾-Aminobu y a e GAT: GABA anspo e HA: Hyd oxylamine Hepes: N-2-Hyd oxye hylpipe azine-N󸀠- 2-e hanesulphonic acid IBMX: 3-Isobu yl-1-me hylxan hine L-AP4: L(+)-2-Amino-4- phosphonobu y a e L-NAME: N6-Ni o-L-a ginineme hyles e L-SOP: O-Phospho-L-se ine MK-801 (dizocilpine): (5S,10R)-(+)-Me hyl-10,11- dihyd o-5H- dibenzo(a,d)cyclohep en-5,10- amine 6Jou nal o Amino Acids NBQX: 2,3-Dioxo-6-ni o-1,2,3,4- e ahyd obenzo[ ]quinoxaline-7- sul onamide NMDA: N-Me hyl-D-aspa a e ODQ: 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxalin-1- one PDE: Phosphodies e ase PMA: 4𝛽-Pho bol 12-my is a e 13-ace a e PKC: P o einkinaseC RO 20-1724: 4-(3-Bu oxy-4-me hoxyphenyl)-2- imidazolidone R-PIA: R(−)N6-(2-Phenylisop opyl)adenosine SITS: 4-Ace amido-4󸀠-iso hiocyanos ilbene- 2,2󸀠-disulphona e SNAP: S-Ni oso-N-ace ylpenicillamine SNP: Sodium ni op usside. Con lic o In e es s The au ho s decla e ha he e is no con lic o in e es s ega ding he publica ion o his pape . Acknowledgmen s The skill ul echnical assis ance o M s. I ma Ran amaa and he in aluable help o D . Saka i Oja in s a is ical analyses a e g a e ully acknowledged. Re e ences [1] M. Co nbla h, J. M. Hawdon, A. F. Williams e al., “Con o e - sies ega ding de ini ion o neona al hypoglycemia: sugges ed ope a ional h esholds,” Pedia ics, ol.105,no.5,pp.1141–1145, 2000. [2] A. Nehlig and A. P. de Vasconcelos, “Glucose and ke one body u iliza ion by he b ain o neona al a s,” P og ess in Neu obiology, ol.40,no.2,pp.163–221,1993. [3] R. C. Vannucci and S. J. Vannucci, “Hypoglycemic b ain inju y,” Semina s in Neu obiology, ol.6,pp.73–79,2001. [4] J. E. McGowan and J. M. Pe lman, “Glucose managemen du ing and a e in ensi e deli e y oom esusci a ion,” Clinics in Pe ina ology, ol.33,no.1,pp.183–196,2006. [5]C.M.Bu ns,M.A.Ru he o d,J.P.Boa dman,andF.M. Cowan, “Pa e ns o ce eb al inju y and neu ode elopmen al ou comes a e symp oma ic neona al hypoglycemia,” Pedi- a ics, ol.122,no.1,pp.65–74,2008. [6] M. Sza kowski and D. A well, “T igge ing and execu ion o neu onal dea h in b ain ischaemia: wo phases o glu ama e elease by di e en mechanisms,” T ends in Neu osciences, ol. 17,no.9,pp.359–365,1994. [7] J.-M. Lee, G. J. Zip el, and D. W. Choi, “The changing landscape o ischaemic b ain inju y mechanisms,” Na u e, ol.399,pp.A7– A14, 1999. [8] P. Sa ansaa i and S. S. Oja, “Tau ine and neu al cell damage,” Amino Acids, ol.19,no.3-4,pp.509–526,2000. [9]S.S.Oja,P.Kon o,andP.L ¨ ahdesm¨ aki, “Amino acids as inhibi o y neu o ansmi e s,” P og ess in Pha macology, ol.1, no. 3, pp. 1–119, 1977. [10] S. Oja, E. R. Ko pi, and P. Sa ansaa i, “Iodi ica ion o chlo ide lux ac oss b ain memb anes by inhibi o y amino acids in de eloping and adul mice,” Neu ochemical Resea ch, ol.15,no. 8, pp. 797–804, 1990. [11] P. Kon o and S. S. Oja, “E ec s o au ine on he in lux and e lux o calcium in b ain slices o adul and de eloping mice,” In e na ional Jou nal o Neu oscience, ol.38,no.1-2,pp.103– 109, 1988. [12] W. Q. Chen, H. Jin, M. Nguyen e al., “Role o au ine in egula ion o in acellula calcium le el and neu op o ec i e unc ion in cul u ed neu ons,” Jou nal o Neu oscience Resea ch, ol.66,no.4,pp.612–619,2001. [13] J.-Y.Wu,W.Chen,X.W.Tange al.,“Modeo ac iono au ine and egula ion dynamics o i s syn hesis in he CNS,” Ad ances in Expe imen al Medicine and Biology, ol.483,pp.35–44,2000. [14] H. Wu, Y. Jin, J. Wei, H. Jin, D. Sha, and J.-Y. Wu, “Mode o ac ion o au ine as a neu op o ec o ,” B ain Resea ch, ol.1038,no.2, pp. 123–131, 2005. [15] A. El Id issi and E. T enkne , “Tau ine egula es mi ochond ial calcium homeos asis,” Ad ances in Expe imen al Medicine and Biology, ol.526,pp.527–536,2003. [16] E. D. F ench, A. Vezzani, W. O. Whe sell J ., and R. Schwa cz, “An i-exci o oxic ac ions o au ine in he a hippocampus s udied in i o and in i o,” Ad ances in Expe imen al Medicine and Biology, ol.203,pp.349–362,1986. [17] P. Sa ansaa i and S. S. Oja, “Enhanced au ine elease in cell- damaging condi ions in he de eloping and ageing mouse hippocampus,” Neu oscience, ol.79,no.3,pp.847–854,1997. [18] P.Sa ansaa iandS.S.Oja,“Releaseo endogenousglu ama e, aspa a e, GABA, and au ine om hippocampal slices om adul and de eloping mice unde cell-damaging condi ions,” Neu ochemical Resea ch, ol. 23, no. 4, pp. 563–570, 1998. [19] P.Kon oandS.S.Oja,“Tau ineandGABA elease ommouse ce eb al co ex slices: po assium s imula ion eleases mo e au ine han GABA om de eloping b ain,” De elopmen al B ain Resea ch, ol.37,no.1-2,pp.277–291,1987. [20] G. Le i, “Release o pu a i e ansmi e amino acids,” in Handbook o Neu ochemis y, A. Laj ha, Ed., ol. 6, pp. 463–509, Plenum P ess, New Yo k, NY, USA, 2nd edi ion, 1984. [21] P. Sa ansaa i and S. S. Oja, “Release o GABA and au ine om b ain slices,” P og ess in Neu obiology, ol.38,no.5,pp.455– 482, 1992. [22] S. S. Oja and P. Sa ansaa i, “Tau ine,” in Handbook o Neu o- chemis y and Molecula Neu obiology,S.S.Oja,P.Sa ansaa i, and A. Schousboe, Eds., ol. 6, pp. 155–206, Sp inge , New Yo k, NY, USA, 3 d edi ion, 2007. [23] A.Schousboe,J.Mo ´ an, and H. Pasan es-Mo ales, “Po assium- s imula ed elease o au ine om cul u ed ce ebella g anule neu ons is associa ed wi h cell swelling,” Jou nal o Neu oscience Resea ch, ol.27,no.1,pp.71–77,1990. [24] H. K. Kimelbe g, N. B. Nes o , and P. J. Feus el, “Inhibi ion o elease o au ine and exci a o y amino acids in ischemia and neu op o ec ion,” Neu ochemical Resea ch, ol.29,no.1,pp. 267–274, 2004. [25] H. Pasan es-Mo ales, S. Ala ez, R. S. Olea, and J. Mo ´ an, “Con ibu ion o o ganic and ino ganic osmoly es o olume egula ion in a b ain cells in cul u e,” Neu ochemical Resea ch, ol.18,no.4,pp.445–452,1993. [26] P. J. Feus el, Y. Jin, and H. K. Kimelbe g, “Volume- egula ed anion channels a e he p edominan con ibu o s o elease o exci a o y amino acids in he ischemic co ical penumb a,” S oke, ol.35,no.5,pp.1164–1168,2004. Jou nal o Amino Acids 7 [27] R. S´ anchez-Olea, C. Pe˜ na, J. Mo ´ an, and H. Pasan es-Mo ales, “Inhibi ion o olume egula ion and e lux o osmo egula o y amino acids by blocke s o Cl− anspo in cul u ed as ocy es,” Neu oscience Le e s, ol.156,no.1-2,pp.141–144,1993. [28] T. Smi no a, J. S innak e, and J. Malle , “Cha ac e iza ion o a p esynap ic glu ama e ecep o ,” Science, ol.262,no.5132,pp. 430–433, 1993. [29] S. S. Oja and P. Sa ansaa i, “Modula ion o au ine elease in glucose- ee media by glu ama e ecep o s in hippocampal slices om de eloping and adul mice,” Amino Acids, ol.44, no. 2, pp. 533–542, 2013. [30]R.N.Icho d,M.V.Johns on,andR.J.T ays man,“MK801 dec eases glu ama e elease and oxida i e me abolism du ing hypoglycemic coma in pigle s,” De elopmen al B ain Resea ch, ol. 128, no. 2, pp. 139–148, 2001. [31] D. S. B ed , C. D. Fe is, and S. H. Snyde , “Ni ic oxide syn hase egula o y si es. Phospho yla ion by cyclic AMP-dependen p o ein kinase, p o ein kinase C, and calcium/calmodulin p o ein kinase; iden i ica ion o la in and calmodulin binding si es,” TheJou nalo BiologicalChemis y, ol.267,no.16,pp. 10976–10981, 1992. [32] E. M. Schuman and D. V. Madison, “Ni ic oxide and synap ic unc ion,” Annual Re iew o Neu oscience, ol.17,pp.153–183, 1994. [33] P. Sa ansaa i and S. S. Oja, “Tau ine elease in he de eloping and adul mouse hippocampus: in ol emen o cyclic guanosine monophospha e,” Neu ochemical Resea ch, ol.27,no.1-2,pp. 15–20, 2002. [34] V. M. Bolo ina, S. Najibi, J. J. Palacino, P. J. Pagano, and R. A. Cohen, “Ni ic oxide di ec ly ac i a es calcium-dependen po assiumchannelsin ascula smoo hmuscle,”Na u e, ol. 368, no. 6474, pp. 850–853, 1994. [35] L. R. Zhong, S. Es es, L. A inian, and V. Rehde , “Ni ic oxide egula es neu onal ac i i y ia calcium-ac i a ed po assium channels,” PLoS ONE, ol. 8, no. 11, A icle ID e78727, 2013. [36]Y.Kang,Y.Dempo,A.Ohashie al.,“Ni icoxideac i a es leak K+cu en s in he p esumed choline gic neu on o basal o eb ain,” Jou nal o Neu ophysiology, ol. 98, no. 6, pp. 3397– 3410, 2007. [37] Z.Li,M.W.Chapleau,J.N.Ba es,K.Biele eld ,H.-C.Lee,andF. M. Abboud, “Ni ic oxide as an au oc ine egula o o sodium cu en s in ba o ecep o neu ons,” Neu on, ol.20,no.5,pp. 1039–1049, 1998. [38] M. Rengana han, T. R. Cummins, and S. G. Waxman, “Ni ic oxide blocks as , slow, and pe sis en Na+channels in C- ype DRG neu ons by S-ni osyla ion,” Jou nalo Neu ophysiology, ol.87,no.2,pp.761–775,2002. [39] A. K. M. Hamma s om and P. W. Gage, “Ni ic oxide inc eases pe sis en sodium cu en in a hippocampal neu ons,” Jou nal o Physiology, ol.520,pa 2,pp.451–461,1999. [40] X. Han, A. B. Pa e s, D. P. Jones, I. Zeliko ic, and R. W. Chesney, “The au ine anspo e : mechanisms o egula ion,” Ac a Physiologica, ol.187,no.1-2,pp.61–73,2006. [41] H. K. Kimelbe g, S. K. Gode ie, S. Higman, S. Pang, and R. A. Waniewski, “Swelling-induced elease o glu ama e, aspa a e, and au ine om as ocy e cul u es,” The Jou nal o Neu o- science, ol.10,no.5,pp.1583–1591,1990. [42] A. Schousboe, R. Sanchez-Olea, J. Mo ´ an, and H. Pasan es- Mo ales, “Hyposmola i y-induced au ine elease in ce ebella g anule cells is associa ed wi h di usion and no wi h high- a ini y anspo ,” Jou nal o Neu oscience Resea ch, ol.30,no. 4, pp. 661–665, 1991. [43] S. Fa he azi, K. T. Izu su, R. B. Wellne , and C. M. Bel on, “Hypo onically ac i a ed chlo ide cu en in HSG cells,” Jou nal o Memb ane Biology, ol.142,no.2,pp.181–193,1994. [44] P. Sa ansaa i and S. S. Oja, “In ol emen o me abo opic glu- ama e ecep o s in au ine elease in he adul and de eloping mouse hippocampus,” Amino Acids, ol.16,no.2,pp.165–179, 1999. [45] R. A. Cunha, “Adenosine as a neu omodula o and as a homeo- s a ic egula o in he ne ous sys em: di e en oles, di e en sou ces and di e en ecep o s,” Neu ochemis y In e na ional, ol.38,no.2,pp.107–125,2001. [46] P.Sa ansaa iandS.S.Oja,“In e ac ionso au ineandadeno- sine in he mouse hippocampus in no moxia and ischemia,” Ad ances in Expe imen al Medicine and Biology, ol.526,pp. 445–451, 2003. Submi you manusc ip s a h p://www.hindawi.com Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Ana omy Resea ch In e na ional Pep ides In e na ional Jou nal o Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com In e na ional Jou nal o Volume 2014 Zoology Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Molecula Biology In e na ional Genomics In e na ional Jou nal o Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 The Scien i ic Wo ld Jou nal Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Bioin o ma ics Ad ances in Ma ine Biology Jou nal o Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Signal T ansduc ion Jou nal o Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 BioMed Resea ch In e na ional E olu iona y Biology In e na ional Jou nal o Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Biochemis y Resea ch In e na ional A chaea Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Gene ics Resea ch In e na ional Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Ad ances in Vi ology Hindawi Publishing Co po a ion h p://www.hindawi.com Nucleic Acids Jou nal o Volume 2014 S em Cells In e na ional Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 Enzyme Resea ch Hindawi Publishing Co po a ion h p://www.hindawi.com Volume 2014 In e na ional Jou nal o Mic obiology