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
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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.
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