Ontogenetic profile of ecto-5'-nucleotidase in rat brain synaptic plasma membranes.
ABSTRACT Ecto-5'-nucleotidase (CD73; EC 188.8.131.52, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K(m) and V(max), suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (V(max)/K(m)). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.
Article: The Janus face of caffeine.[Show abstract] [Hide abstract]
ABSTRACT: Caffeine is certainly the psychostimulant substance most consumed worldwide. Over the past years, chronic consumption of caffeine has been associated with prevention of cognitive decline associated to aging and mnemonic deficits of brain disorders. While its preventive effects have been reported extensively, the cognitive enhancer properties of caffeine are relatively under debate. Surprisingly, there are scarce detailed ontogenetic studies focusing on neurochemical parameters related to the effects of caffeine during prenatal and earlier postnatal periods. Furthermore, despite the large number of epidemiological studies, it remains unclear how safe is caffeine consumption during pregnancy and brain development. Thus, the purpose of this article is to review what is currently known about the actions of caffeine intake on neurobehavioral and adenosinergic system during brain development. We also reviewed other neurochemical systems affected by caffeine, but not only during brain development. Besides, some recent epidemiological studies were also outlined with the control of "pregnancy signal" as confounding variable. The idea is to tease out how studies on the impact of caffeine consumption during brain development deserve more attention and further investigation.Neurochemistry International 09/2013; · 2.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Elements of the nucleoside system (nucleoside levels, 5'-nucleotidases (5'NTs) and other nucleoside metabolic enzymes, nucleoside transporters and nucleoside receptors) are unevenly distributed in the brain, suggesting that nucleosides have region-specific functions in the human brain. Indeed, adenosine (Ado) and non-Ado nucleosides, such as guanosine (Guo), inosine (Ino) and uridine (Urd), modulate both physiological and pathophysiological processes in the brain, such as sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington's disease, Alzheimer's disease and Parkinson's disease. Interactions have been demonstrated in the nucleoside system between nucleoside levels and the activities of nucleoside metabolic enzymes, nucleoside transporters and Ado receptors in the human brain. Alterations in the nucleoside system may induce pathological changes, resulting in central nervous system (CNS) diseases. Moreover, several CNS diseases such as epilepsy may be treated by modulation of the nucleoside system, which is best achieved by modulating 5'NTs, as 5'NTs exhibit numerous functions in the CNS, including intracellular and extracellular formation of nucleosides, termination of nucleoside triphosphate signaling, cell adhesion, synaptogenesis and cell proliferation. Thus, modulation of 5'NT activity may be a promising new therapeutic tool for treating several CNS diseases. The present article describes the regionally different activities of the nucleoside system, demonstrates the associations between these activities and 5'NT activity and discusses the therapeutic implications of these associations.Current Medicinal Chemistry 08/2013; · 3.72 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ecto-5'-nucleotidase (e-5NT), a glycosylphosphatidylinositol-linked membrane protein, catalyzes a conversion of AMP to adenosine, which influences nearly every aspect of brain physiology, including embryonic and postnatal brain development. The present study aimed to investigate a pattern of expression, activity and kinetic properties of e-5NT in the hippocampal formation and synaptic plasma membrane (SPM) preparations in rats at postnatal days (PDs) 7, 15, 20, 30 and 90. By combining gene expression analysis and enzyme histochemistry, we observed that e-5NT mRNA reached the adult level at PD20, while the enzyme activity continued to increase beyond this age. Further analysis revealed that hippocampal layers rich in synapses expressed the highest levels of e-5NT activity, while in layers populated with neuronal cell bodies, the enzyme activity was weak or absent. Therefore, activity and expression of e-5NT were analyzed in SPM preparations isolated from rats at different ages. The presence of two protein bands of about 65 and 68 kDa was determined by immunoblot analysis. The 65-kDa band was present at all ages, and its abundance increased from PD7 to PD20. The 68-kDa band appeared at PD15 and increased until PD30, coinciding with the increase of e-5NT activity, substrate affinity and enzymatic efficiency. Since distinct e-5NT isoforms may derive from different patterns of the enzyme protein N-glycosylation, we speculate that long-term regulation of e-5NT activity in adulthood may be effectuated at posttranslational level and without overall change in the gene and protein expression.Journal of Molecular Neuroscience 02/2014; 54(1). · 2.76 Impact Factor
Int. J. Devl Neuroscience 29 (2011) 397–403
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience
journal homepage: www.elsevier.com/locate/ijdevneu
Ontogenetic profile of ecto-5?-nucleotidase in rat brain synaptic plasma
Ivana Stanojevi´ ca,∗, Ivana Bjelobabab, Nadeˇ zda Nedeljkovi´ cc, Dunja Drakuli´ ca, Snjeˇ zana Petrovi´ ca,
Mirjana Stojiljkovi´ cb,c, Anica Horvata
aLaboratory for Molecular Biology and Endocrinology, Institute of Nuclear Sciences “Vinca”, University of Belgrade, Mike Petrovica 12-14, 11000 Belgrade, Serbia
bDepartment of Neurobiology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stevana 142, 11000 Belgrade, Serbia
cInstitute for Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
a r t i c l e i n f o
Received 13 December 2010
Received in revised form 28 February 2011
Accepted 8 March 2011
Synaptic plasma membranes
a b s t r a c t
Ecto-5?-nucleotidase (CD73; EC 184.108.40.206, e-5NT) is regarded as the key enzyme in the extracellular for-
mation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor
in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning
AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM)
preparations isolated from whole female rat brain at different ages. We observed pronounced increase
in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-
day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed
the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum rel-
ative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme
ing postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic
due to the increase in the enzyme catalytic efficiency (Vmax/Km). Finally, double immunofluorescence
staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for
the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that
e-5NT may be a part of general scheme of brain development and synapse maturation and provide ratio-
studies concerning localization of e-5NT in the brain.
© 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
Adenosine is an important neuromodulator and homeostatic
regulator in the nervous system (Ribeiro and Sebastiao, 2010),
exerting effects in development, cell proliferation, migration, dif-
ferentiation and synaptic network formation (for review see,
Zimmermann, 2006). By acting on its own P1 receptor family
coupled to either inhibition (A1and A3) or activation (A2Aand
A2B) of adenylate cyclase (Cunha et al., 1996a; Cunha, 2005),
adenosine modulates neuronal activity by inhibiting or facilitat-
ing synaptic transmission (Stone, 1981; Ribeiro and Sebastiao,
Abbreviations: e-5NT, ecto-5?-nucleotidase; AMP, adenosine monophosphate;
SPM, synaptic plasma membrane.
∗Corresponding author at: Laboratory of Molecular Biology and Endocrinology,
Institute of Nuclear Sciences “Vinˇ ca”, P.O. Box 522, 11001 Belgrade, Serbia.
Tel.: +381 113443619, fax: +381 112455561.
E-mail address: email@example.com (I. Stanojevi´ c).
2010). Extracellular adenosine originates from two sources; it
can be released directly through bidirectional nonconcentra-
tive adenosine transporters (Brundege and Dunwiddie, 1996)
or it can arise from released ATP and adenine nucleotides,
by extracellular catabolism via ecto-nucleotidase enzyme path-
way (Cunha et al., 1996b). Several ecto-nucleotidase enzyme
families contribute to the extracellular catabolism of adenine
nucleotides. Currently known ecto-nucleotidases include ecto-
nucleotide pyrophosphatase/phosphodiesterase family (E-NPP)
and ecto-nucleosidetriphosphate diphosphohydrolase family (E-
NTPDase), that hydrolyze extracellular ATP and ADP to AMP and
ecto-5?-nucleotidase (Zimmermann, 2000), that hydrolyzes AMP
to adenosine. This catabolic pathway constitutes the predomi-
nant way of extracellular adenosine formation at nerve terminals
(Zimmermann, 1996; Cunha et al., 1996b). Ecto-5?-nucleotidase is
the rate limiting enzyme in this pathway, since its feed-forward
inhibition by ATP and ADP controls the timing and extent of adeno-
sine formation (James and Richardson, 1993) and consequently
time course of neuromodulatory effects of adenosine.
0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
Ecto-5?-nucleotidase (CD73; EC 220.127.116.11, e-5NT) is an ecto-
enzyme that is anchored to the extracellular surface of cell
membrane through a glycosyl phosphatidylinositol (GPI) linkage.
has distinct role during brain development and plasticity. E-5NT is
expressed on the surface of migrating nerve cells during postna-
tal development (Fenoglio et al., 1995; Schoen et al., 1988) and it
Schoen and Kreutzberg, 1995). Several studies clearly documented
that e-5NT activity increase in brain during ontogeny. Thus, up to
Paula Cognato et al., 2005; Mackiewicz et al., 2006), hippocampus
(Cunha, 2001; de Paula Cognato et al., 2005), spinal cord (Torres
et al., 2003) and in most brain regions of aged compared to young
rats (Fuchs, 1991). Alterations of e-5NT activity were also observed
tex (Naidoo and Pratt, 1954). However, although all cited studies
measured specific activity of e-5NT in postnatal stages, none has
investigated changes in its kinetic properties with ontogeny.
Contrary to the unequivocal findings on age-related increase,
cellular distribution of e-5NT in the brain is still matter of con-
troversy. Namely, previous studies on the enzyme distribution
in brain faced inconsistencies between enzyme histochemical
and immunocytochemical staining. Enzyme histochemical stain-
ing and biochemical studies revealed broad distribution of e-5NT
and its association with myelin, astrocytes, activated microglia
and neurons (for review, see Zimmermann, 1992; Zimmermann,
1996; Langer et al., 2008). However, immunohistochemical meth-
ods demonstrated more restricted enzyme localization at glial
structures, such as perivascular endfeets (Schoen et al., 1987,
whereas neuronal localization was rarely observed (Nacimiento
and Kreutzberg, 1990; Bjelobaba et al., 2007). The same paradox
exists in respect to the synaptic localization of e-5NT. Although
biochemical studies repeatedly demonstrated presence of AMP
hydrolyzing activity in the presynaptic elements (Cunha et al.,
1992; Zimmermann, 1992; James and Richardson, 1993; Cunha,
2001; de Paula Cognato et al., 2005; Schmatz et al., 2009; Sigueira
et al., 2010), immunocytochemical studies have shown only spo-
1987; Schoen and Kreutzberg, 1997; Zimmermann et al., 1993).
Therefore, in the present study, we have readdressed the question
of the developmental changes in the activity, kinetic properties
and the expression of e-5NT in the synaptic plasma membranes
isolated from whole brain of female rats. As syntaxin is presynap-
tic membrane-bound protein involved in synaptic anchoring and
exocytosis, also considered a reliable marker of synaptogenesis
(Bennett et al., 1992, 1993; Calakos et al., 1994; Sudhof, 1995), we
provided evidence of pre-synaptic localization of e-5NT by means
of double immunofluorescence staining for e-5NT and syntaxin in
purified synaptosomes isolated from female rat brain at different
2. Materials and methods
Female rats of the Wistar strain were used in the study: juvenile (15-day-old)
(n=9), pre-pubertal (30-day-old) (n=9), young adult (60-day-old) (n=9) and adult
on the floor, under standard conditions: 12h light/dark cycle, constant temperature
(22±2◦C) and free access to food and water. All animals were treated in accordance
with the principles from Guide for Care and Use of Laboratory Animals (NIH Pub-
lication No. 80-23) and the Belgrade University Animal Care and Use Committee
approved the protocols. Efforts were made to minimize the number of used animals
and their suffering.
2.2. Synaptosomes and synaptic plasma membranes preparations
After decapitation using a small animal guillotine (Harvard Apparatus, Hollis-
ton, MA, USA), whole brains from the same age group (3 brains/group/isolation)
were rapidly removed for immediate synaptosome and synaptic plasma mem-
brane (SPM) isolation, starting with ice-cold medium (0.32mol/l sucrose, 5mmol/l
Tris–HCl, pH 7.4). Synaptosomes from pooled brain homogenates of same group
were purified according to modified method of Cotman and Matthews (1971), as
described previously (Horvat et al., 2010). Parts of purified synaptosomes from all
ages were resuspended in medium (in mmol/l): 140 NaCl, 5 KCl, 1.2 NaH2PO4, 5
NaHCO3, 1 MgCl2, 10 glucose, 10 Tris–HCl, pH 7.4. The contamination of SPM frac-
tions, based on morphological and biochemical markers, were less than 7% (Horvat
et al., 1995). Another part of synaptosomal fraction was proceeded for SPM prepa-
ration, as described previously (Horvat et al., 2010). Synaptosomes and SPM protein
levels at different ages were determined according to Markwell et al. (1978) using
bovine serum albumin as a standard. Samples of SPM were kept at −70◦C until use.
Three independent isolations were made for each age group.
2.3. Enzyme assays
Ecto-5?-nucleotidase assay was described previously (Horvat et al., 2010).
Briefly, reaction mixture contained 50mmol/l Tris–HCl buffer, pH 7.4, 5mmol/l
MgCl2, 1.0mmol/l AMP and 80?g SPM. The reaction mixture was pre-incubated
for 10min at 37◦C. The reaction was started by the addition of AMP and stopped
after 30min by the addition of 22?l 3mol/l perchloric acid. The samples were
chilled on ice 10min and taken for the assay of released inorganic phosphate (Pi)
(Pennial, 1966), using KH2PO4as a reference standard. Incubation time and protein
concentration were chosen in order to ensure the linearity of the reaction. Activa-
tion of e-5NT in the presence of increasing AMP concentrations (0.05–2.5mmol/l)
was determined in the same reaction mixture without varying other conditions.
All age samples were run in triplicate in 6 independent determinations from three
independent SPM isolations.
2.4. Western blot analysis
Samples were adjusted to a final SPM protein concentration of 4mg/ml, mixed
with Laemmli’s sample buffer and boiled for 5min. Samples (50?g of proteins)
were subjected to 10% polyacrylamide gel electrophoresis (PAGE) in the presence
of sodium dodecyl sulphate (SDS), electrophoretically transferred to polyvinyli-
dene difluoride (PVDF) membranes (0.45?m, from Millipore) for 1h at 100V. After
blocking in 5% bovine serum albumin in Tris-buffered saline (50mmol/l Tris–HCl
pH 7.4, 150mmol/l NaCl, 0.05% Tween-20, TBS-T) for 1h at room temperature, the
membranes were incubated overnight at 4◦C with primary antibody against e-5NT
(1:1000 dilution, anti-CD73 goat polyclonal, Santa Cruz Biotechnology, Inc., SCB).
After four washing periods for 5–10min with TBS-T, the membranes were incu-
bated with secondary anti-goat horseradish peroxidase (HRP)-conjugated antibody
antibody (SCB) was used (1:2000 dilution) as an equal loading control, on the same
membrane as e-5NT. Negative control with omitted primary antibody was done.
Finally, immunopositive bands were visualized with 3,3?-diaminobenzidine (DAB;
Sigma). Image J (image analysis software) was used for semi-quantitative analysis.
The finale value of e-5NT relative protein abundance was normalized based on the
respective ?-actin. Values were expressed as mean relative intensity±SEM.
2.5. Synaptosome immunofluorescence
Double labeling of e-5NT and syntaxin was performed as follows. Synapto-
somal pellet, concentrations of 1mg/ml, was resuspended in 1ml of phosphate
buffered saline (PBS) (in mmol/l): 137 NaCl, 2.6 KCl, 1.5 KH2PO4, 8.1 Na2HPO4, pH
7.4 and was allowed to attach to poly-l-lysine-coated cover slips for 30min. Then,
the synaptosomes were fixed for 15min with 4% paraformaldehyde in PBS. After
several washes in PBS, synaptosomes were incubated for 1h in PBS containing 5%
normal donkey serum, 3% BSA, and 0.1% Triton X-100. Incubation with the appro-
priate primary antibody was carried overnight at 4◦C. Anti-ecto-5?-nucleotidase
antibody was applied at 1:50 dilution, and anti-syntaxin antibody (anti-syntaxin
After washing in PBS, synaptosomes were incubated for 2h at room temperature
with the appropriate fluorescent secondary antibodies (donkey anti-rabbit Alexa
Fluor 488, donkey anti-goat Alexa Fluor 555; dilution 1:200; Invitrogen, Carlsbad,
CA,). Finally, synaptosomes were washed in PBS and mounted on glass slides with
the omission of the primary antibodies. Synaptosomes were viewed under a Zeiss
Axiovert microscope equipped with camera and EC Plan-Neofluor 100× objective
and using an Apotome system for obtaining optical sections.
2.6. Data analysis
Data obtained for the enzyme activities are presented as mean activ-
ity (nmol Pi/mg/min)±SEM from three independent SPM preparations (n=9
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
Fig. 1. Developmental profile of e-5NT activity in SPM preparations isolated from
female rat brain at different ages. Enzyme activity was assayed as described in
Section 2 in a presence of 1.0mmol/l AMP. Bars represent mean activity (nnmol
Pi/mg/min)±SEM. from six different determinations performed in triplicate. (#)
Indicates significant difference from juvenile (15-day-old) rats (ANOVA followed by
Tukey’s test, F=116.4, p<0.001).
ing activity in each membrane preparation was performed by computer-assisted
least square fitting of the data to the Michaelis–Menten equation, while kinetic
parameters Vmax (apparent maximum activity) and Km (apparent Michaelis con-
stant) values were calculated from Eadie–Hofstee semi-reciprocal plot of V vs. V/[S],
by Tukey’s post hoc test (considering p<0.05 as significant) was used to determine
the significant changes in enzyme activities, kinetic parameters and relative protein
abundance between animal groups.
3.1. Developmental profile of e-5NT activity in synaptic plasma
AMP hydrolyzing activity showed gradual and significant
increase in rats at different ages (Fig. 1). Low level of the enzyme
activity was found in the SPM preparation isolated from juvenile
rats (7.57±0.40nmol Pi/mg/min), but increased more than 2-fold
in pre-pubertal rats (17.54±3.12nmol Pi/mg/min) and was 3-fold
higher in young adults (21.70±5.51nmol Pi/mg/min) compared to
juvenile animals (F[3,74]=116.4, p<0.001). Adult rats had nearly
the same level of e-5NT activity (27.62±0.60nmol Pi/mg/min) as
3.2. Kinetic analysis of e-5NT in SPM preparation at different ages
We further assessed kinetic properties of the e-5NT during mat-
activation with raising AMP concentrations (0.05–2.5mmol/l). As
expected, maximum enzyme activity at the plateau increased con-
sistently with postnatal development.
The kinetic parameters Vmax and Km were calculated from
the Eadie–Hofstee transformation (insets in Fig. 2A–D) of the
data from Michaelis–Menten plots at different ages (Table 1).
Analysis of variance showed significant effect of maturation for
Vmax (F[3.8]=124.3, p<0.001), whereas Km values did not differ
Fig. 2. Michaelis–Menten plots of initial velocities vs. raising AMP concentrations at day 15 (A), day 30 (B), day 60 (C) and day 90 (D). The enzyme assay was performed as
described in Section 2 in presence of 0.05–2.5mmol/l AMP. Bars represent mean activity (nmol Pi/mg/min)±SEM. from six independent experiments performed in triplicate.
Solid lines represent best fit obtained by using Origin 7.5 software package. Inset (A–D): linear semi-reciprocal Eadie–Hofstee plots of V vs. V/[S] from data presented in the
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
Kinetic parameters Km, Vmaxand Km/Vmaxfor ecto-5?-nucleotidase activity at differ-
Young adults (60)
0.32 ± 0.02
0.29 ± 0.02
0.30 ± 0.09
0.49 ± 0.07*
8.70 ± 0.24
19.15 ± 0.43#
26.98 ± 2.70#
37.51 ± 2.61#
27.3 ± 0.1
66.0 ± 0.4#
89.9 ± 2.0#
76.5 ± 6.0#
Kinetic parameters Kmand Vmaxwere obtained by Eadie–Hofstee transformation of
the data presented in Fig. 2A–D, whereas Vmax/Kmvalues were obtained from the
*p<0.05 – indicates significant difference from juvenile rats.
#p<0.001 – indicates significant difference from juvenile rats.
Fig. 3. Western blot analysis of SPM preparations isolated from whole adult brains.
Proteins (50?g per lane) were resolved on 10% gel, transferred to PVDF membranes
and probed with anti-e-5NT antibody. E-5NT antibody specifically stained one band
at about 68kDa. Lane 1 shows molecular weight markers, and both lanes 2 and 3
are SPM from 90 days old rat brains.
between juvenile, pre-pubertal and young adults, but significantly
increased in adult animals (F[3,8]=7.71, p<0.05), compared to
all age groups. Vmax/Km ratios showed that enzyme physiologi-
cal efficiency increased 2–3-fold in adult compared to juvenile rats
3.3. E-5NT immunodetection
The specificity of the antibody used for immunodetection of e-
5NT was tested using SPM fraction isolated from adult rat brain.
As shown in Fig. 3, the antibody recognizes one prominent band
at 68kDa corresponding closely to the molecular weight pre-
viously shown for e-5NT from mammalian brain (Vogel et al.,
1992). Fig. 4 presents representative Western blot membrane
(Fig. 4A) and relative protein abundance of e-5NT in SPM prepa-
rations isolated at different postnatal stages (Fig. 4B). Analysis
of the data revealed significant negative effect of maturation, in
young adult and adult (F[3,40]=5.728, p<0.01), for the abun-
dance of e-5NT in the whole brain SPM compared to juvenile
rats. Namely, the enzyme protein was more abundant in the
SPM preparation isolated from juvenile rats and its abundance
consistently decreased with age, being the lowest in adult ani-
3.4. Inmunocytochemical localization of e-5NT in purified
In order to verify whether e-5NT was indeed associated with
SPM, we analyzed the distribution of e-5NT and presynaptic mem-
Fig. 4. Western blot of e-5NT (A) and relative protein abundance (B) in the SPM
preparations isolated from whole brains of rats at different ages. The finale value of
e-5NT relative protein abundance was normalized based on the respective ?-actin.
Bars represent mean±SEM. from six independent experiments. (**) Indicates dif-
ference from juvenile animals (ANOVA followed by Tukey’s test, F=5.728, p<0.01).
brane marker syntaxin at different ages. Our Western blot results
brains was lower compared to SPM from pre-pubertal, young adult
and adult rats (data not shown). As shown in Fig. 5, presence of
double-labeled structures suggests that e-5NT was indeed present
in the presynaptic membrane compartment. However, we have
also observed single labeled, e-5NT – or syntaxin-positive struc-
tures, indicating that at part of the e-5NT was not associated with
presynaptic membranes. The relative number of double labeled, e-
5NT-syntaxin-positive synaptosomes was the highest in juvenile
rats and declines with the maturation.
The present study confirmed what was previously shown
(Fuchs, 1991; Torres et al., 2003; Mackiewicz et al., 2006), that
AMP hydrolyzing activity consistently and significantly increases
in the synaptic plasma membranes (SPM) isolated from whole
female rat brain during ontogeny. As extracellular AMP hydrol-
ysis in synaptic cleft occurs through the action of extracellular
surface-located e-5NT, increase in this enzyme activity indicates
efficient production of adenosine. However, only when extra-
cellular ATP and ADP levels decrease below the threshold of
inhibition of e-5NT, adenosine will be formed in a considerable
amount (James and Richardson, 1993). Since the production of
AMP is possibly altered with maturation, it is difficult to con-
clude whether the increased e-5NT activity, which was measured
in vitro, will or will not result in increased extracellular adenosine
Since the most pronounced increase in e-5NT activity coincides
with the onset of gonadal function in female rats (Baker, 1979), it
is possible that e-5NT is under the regulation of gonadal steroids.
Since the study of Torres et al. (2003) reported similar prominent
increase in the enzyme activity in the spinal cord synaptosomes of
both male and female rats, and unpublished data from our labora-
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
(green) antibodies, and the merged (yellow) of two images. C – control, the background fluorescence in the absence of the primary antibodies; T – phase contrast image of
synaptosomes. Scale bar=3?m in all images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
with the phase of oestrus cycle, we concluded that female gonadal
steroids were not related to the observed alterations. On the other
same developmental period was observed in almost all brain areas
the other body tissues or cell types (Fuchs, 1991), it was reasonable
to conclude that such phenomena did not reflect a general mech-
anism of cellular aging, but rather some developmental processes,
which are unique to the brain.
The novel finding of our study was a direct negative correlation
between e-5NT activity and its protein abundance in the SPM at
different ages. In other words, e-5NT protein was the most abun-
dant in SPM at juvenile stage when its specific activity was the
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
lowest, and the least abundant in the adult stage when the enzyme
specific activity was the highest. What could be the physiological
explanation of the observed discrepancy? Like many GPI-anchored
such as cell adhesion. In the developing cerebral cortex, between 3
and 9 weeks of age, e-5NT carries HNK-1 epitope, which is impli-
1986; Vogel et al., 1991; Olmo et al., 1992; Sadej et al., 2006).
However, the epitope disappears from the enzyme at 12 weeks
of age and afterwards (Vogel et al., 1993). The transient appear-
ance of the HNK-1 epitope in e-5NT protein coincides with the
juvenile to pre-pubertal periods in our study, when the enzyme
displays the highest abundance and the lowest activity. Thus, the
high abundance of e-5NT during juvenile period may be connected
with its roles other than catalytic activity, such as synaptogenesis
postnatal development (Vogel et al., 1993) and these interactions
may modulate the enzyme catalytic activity (Dieckhoff et al., 1986;
Olmo et al., 1992; Sadej et al., 2006). Consecutively, decrease in
the enzyme relative abundance may be the results of developmen-
tal down-regulation during later stages of ontogeny, when e-5NT
Our finding raises yet another question: if e-5NT expression
in SPM progressively decline during the ontogeny, what could be
the mechanism of the increase in its catalytic activity with aging?
To address this question we performed kinetic analysis of e-5NT
activity and calculated kinetic constants, Km and Vmax at differ-
ent ages. Kinetic analysis showed significant progressive increase
in Vmaxbetween juvenile, pre-pubertal and young adults without
changes in apparent Kmand further increase of Vmaxin adult age
with only slight increase in Kmvalue. Therefore, since immunode-
tection showed significant decline in abundance of the enzyme
protein with maturation, we concluded that increase in e-5NT
activity in SPM with development was entirely due to the increase
in the enzyme catalytic efficiency (Vmax/Km). The changes we
have observed in the enzyme kinetic properties during maturation
might be caused by changes in the enzyme protein conformation
and topography that has been found during the brain develop-
ment (Schoen et al., 1988; Zimmermann, 1992; Vogel et al., 1993).
Namely, functional e-5NTs are a homodimers with highly diverse
pattern of glycosylation (Fini et al., 2003; Strater, 2006), that could
influence the enzyme kinetic properties. In addition, e-5NT dimers
associate with each other and with other ectonucleotidases, P1 and
complexes (Schicker et al., 2009) that contribute to e-5NT fine-
sible for changes in the kinetics we observed during the ontogeny
(Kenworthy and Edinin, 1998).E-5NT enzyme assay and the West-
ern blot analysis in SPM fraction suggest that the enzyme is
associated with the presynaptic membrane compartment. Yet,
while the SPM fraction was of satisfactory purity, we could not
exclude that the fraction contains some glial or post-synaptic
“contamination” that could contribute to the enzyme assay and
protein with individual syntaxin-positive synaptosomes at all ages
and thus verify unambiguously that the enzyme was associated
with the presynaptic plasma membrane compartment. Using an
localized e-5NT activity on the glial cells and at the main types of
asymmetric synapses; the percentage of labeled synapse increased
until adulthood (Bailly et al., 1995). In addition, neuronal syntaxin
syntaxin positive synaptosomes without e-5NT signal and in much
lesser extent, e-5NT immunosignal associated with unidentifiable
syntaxin-negative structures. Therefore, the immunofluorescence
in the part of excitatory synapses at all ages.
The e-5NT might regulate local levels of its product adeno-
sine, that acts as a potent trophic factor regulating development,
1996), neurite extension (Abbracchio et al., 1989), neurogenesis
1996), as well as a neuromodulator (Zimmermann, 1996) affecting
the level of neuronal activity. Furthermore, there is considerable
evidence that the e-5NT is associated with the neural surface dur-
ing synaptic plasticity and remodeling (review in Zimmermann,
2006) and plays a significant role in cellular contacts during synap-
tic formation. Low enzyme activity and high expression in early
postnatal development, indicated e-5NT adhesion role in synapto-
genesis and possibly participation in the substrate selection of the
developing synapses. Its function may thus go beyond its activity
as an adenosine-producing enzyme. When the intensive formation
of synapses is completed, then the e-5NT has mainly hydrolyzing
activity that is seen in adulthood. In this respect, changes in the
e-5NT activity and abundance during ontogeny suggest that the
enzyme may be part of general scheme of brain development and
In summary, the results presented in this study provide
direct evidence for the existence of e-5NT in the presynaptic
compartment. The e-5NT protein is enriched in the presynap-
tic compartment in early postnatal development and decreases
with maturation, whereas its activity follows the reverse pat-
tern of expression. Such changes could have broad influence
on the process of brain development and maturation and addi-
tionally provide rationale for the inconsistency between enzyme
immunohistochemical and biochemical studies concerning the
cellular and subcellular localization of e-5NT in the nervous sys-
Conflict of interest
The authors declare no conflict of interest.
This study was supported by Serbian Ministry of Science and
Technology projects nos. 173044 and 41014.
Abbracchio, M.P., Cattabeni, F., Clementi, F., Sher, E., 1989. Adenosine receptors
during cell differentiation. Neuroscience 30, 819–825.
Abbracchio, M.P., Ceruti, S., Barbieri, D., Franceschi, C., Malorni, W., Biondo, L., Burn-
stock, G., 1995. A novel action for adenosine: apoptosis of astroglial cells in rat
brain primary cultures. Biochem. Biophys. Res. Commun. 213, 908–915.
Bailly, Y., Schoen, S.W., Delhaye-Bouchaud, N., Kreutzberg, G.W., Mariani, J., 1995.
5?-Nucleotidase activity as a synaptic marker of parasagittal compartmentation
in the mouse cerebellum. J. Neurocytol. 24, 879–890.
Baker, D.J., 1979. Reproduction and breeding. In: Baker, D.J., Lindsey, J.R., Wisbroth,
S.H. (Eds.), The Laboratory Rat Biology and Diseases, vol. 1. Academic Press, New
York, pp. 154–167.
Bennett, M.K., Calakos, N., Scheller, R.H., 1992. Syntaxin: a synaptic protein impli-
cated in docking of synaptic vesicles at presynaptic active zones. Science 275,
Bennett, M.K., Garcia-Araras, J.E., Elferink, L.A., Peterson, K., Fleming, A.M., Hazuka,
C.D., Scheller, R.H., 1993. The syntaxin family of vesicular transport receptors.
Cell 74, 863–873.
Bjelobaba, I., Stojiljkovic, M., Pekovic, S., Dacic, S., Lavrnja, I., Stojkov, D., Rakic,
Lj., Nedeljkovic, N., 2007. Immunohistological determination of ecto-nucleoside
triphosphate diphosphohydrolase1 (NTPDase1) and 5?-nucleotidase in rat hip-
pocampus reveals overlapping distribution. Cell. Mol. Neurobiol. 27, 731–743.
I. Stanojevi´ c et al. / Int. J. Devl Neuroscience 29 (2011) 397–403
Braun, N., Brendel, P., Zimmermann, H., 1995. Distribution of 5?-nucleotidase in the
developing mouse retina. Dev. Brain Res. 88, 79–86.
Bronte, V., Macino, C., Zambon, A., Rosato, A., Mandruzzato, S., Zanovello, P., Collavo,
D., 1996. Protein tyrosine kinases and phosphatases control apoptosis induced
by extracellular adenosine 5?-triphosphate. Biochem. Biophys. Res. Commun.
Brundege, J.M., Dunwiddie, T.V., 1996. Modulation of excitatory synaptic trans-
mission by adenosine released from single hippocampal pyramidal neurons. J.
Neurosci. 16, 5603–5612.
Cammer, W., Sacchi, R., Kahn, S., 1985. Immunocytochemical localization of 5?-
nucleotidase in oligodendroglia and myelinated fibers in the central nervous
system of adult and young rats. Dev. Brain Res. 20, 89–96.
Calakos, N., Bennett, M.K., Peterson, K.E., Scheller, R.H., 1994. Protein–protein
interactions contributing to the specificity of intracellular vesicular trafficking.
Science 263, 1146–1149.
Cotman, C.W., Matthews, D.A., 1971. Synaptic plasma membranes from rat brain
synaptosomes: isolation and partial characterization. Biochim. Biophys. Acta
Cunha, R.A., Sebastiao, A.M., Ribeiro, J.A., 1992. Ecto-5?-nucleotidase is associated
with cholinergic nerve terminals in the hippocampus but not in the cerebral
cortex of the rat. J. Neurochem. 59, 657–666.
Cunha, R.A., Correia-de-Sa, P., Sebastiao, A.M., Ribeiro, J.A., 1996a. Preferential acti-
vation of excitatory adenosine receptors at rat hippocampal and neuromuscular
synapses by adenosine formed from released adenine nucleotides. Brit. J. Phar-
macol. 119, 253–260.
Cunha, R.A., Vizi, E.S., Ribeiro, J.A., Sebastiao, J.A., 1996b. Preferential release of ATP
frequency stimulation of rat hippocampal slices. J. Neurochem. 67, 2180–2187.
Cunha, R.A., 2001. Regulation of the ecto-nucleotidase pathway in rat hippocampal
nerve terminals. Neurochem. Res. 26, 979–991.
Cunha, R.A., 2005. Neuroprotection by adenosine in the brain: from A1 receptor
activation to A2A receptor blockade. Purinergic Signal. 1, 111–134.
de Paula Cognato, G., Bruno, A.V., Vuaden, F.C., Sarkis, J.J., Bonan, C.D., 2005.
Ontogenic profile of ectonucleotidase activities from brain synaptosomes of
pilocarpine-treated rats. Int. J. Dev. Neurosci. 23, 703–709.
Dieckhoff, J., Mollenhauer, J., Kühl, U., Niggemeyer, B., von der Mark, K., Mannherz,
AMPase activity of 5?-nucleotidase from chicken gizzard smooth muscle. FEBS
Lett. 195, 82–86.
Fenoglio, C., Scherini, E., Vaccarone, R., Bernocchi, G., 1995. A reevaluation of the
bellum, with a cerium-based method. J. Neurosci. Methods 59, 253–263.
ical and mass spectrometric characterization of soluble ecto-5?-nucleotidase
from bull seminal plasma. Biochem. J. 372, 443–451.
Fuchs, J.L., 1991. 5?-Nucleotidase activity increases in aging rat brain. Neurobiol.
Aging 12, 523–530.
membranes of rat brains. Experientia 51, 11–15.
Horvat, A., Stanojevi´ c, I., Drakuli´ c, D., Velickovi´ c, N., Petrovi´ c, S., Milosevi´ c, M., 2010.
Effect of acute stress on NTPDase and 5?-nucleotidase activities in brain synap-
tosomes in different stages of development. Int. J. Dev. Neurosci. 28, 175–182.
James, S., Richardson, P.J., 1993. Production of adenosine from extracellular ATP at
the striatal cholinergic synapse. J. Neurochem. 60, 219–227.
Kenworthy, A.K., Edinin, M., 1998. Distribution of a glycosylphosphatidylinositol-
anchored protein at the apical surface of MDCK cells examined at a resolution of
<100A using imaging fluorescence resonance energy transfer. J. Cell Biol. 142,
Langer, D., Hammer, K., Koszalka, P., Schrader, J., Robson, S., Zimmermann, H., 2008.
Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res.
Mackiewicz, M., Nikonova, E.V., Zimmermann, J.E., Romer, M.A., Cater, J., Galante,
R.J., Pack, A.I., 2006. Age-related changes in adenosine metabolic enzymes in
sleep/wake regulatory areas of the brain. Neurobiol. Aging 27, 351–360.
Markwell, M.A., Haas, S.A., Lieber, L., Tolbert, N.A., 1978. A modification of the Lowry
ples. Anal. Biochem. 87, 206–210.
Nacimiento, W., Kreutzberg, G.W., 1990. Cytochemistry of 5?-nucleotidase in the
Exp. Neurol. 109, 362–373.
Naidoo, D., Pratt, O.E., 1954. The development of adenosine-5?-phosphatase activity
with the maturation of rat cerebral cortex. Enzymologia 16, 298–304.
Neary, J.T., Burnstock, G., 1996. Purinoceptors in the regulation of cell growth and
differentiation. Drug Dev. Res. 39, 407–412.
Olmo, N., Turnay, J., Risse, G., Deutzmann, R., von der Mark, K., Lizarbe, M.A., 1992.
Modulation of 5?-nucleotidase activity in plasma membranes and intact cells
by the extracellular matrix proteins laminin and fibronectin. Biochem. J. 282,
Pennial, R., 1966. An improved method for determination of inorganic phosphate by
the isobutanol–benzene extraction procedure. Anal. Biochem. 14, 87–90.
Ribeiro, J.A., Sebastiao, A.M., 2010. Modulation and metamodulation of synapses by
adenosine. Acta Physiol. 199, 161–169.
Sadej, R., Spychala, J., Skladanowski, A.C., 2006. Expression of ecto-5?-nucleotidase
Schicker, K., Hussl, S., Chandaka, G.K., Kosenburger, K., Yang, J.W., Waldhoer, M.,
Sitte, H.H., Boehm, S., 2009. A membrane network of receptors and enzymes for
adenine nucleotides and nucleosides. Biochem. Biophys. Acta 1793, 325–334.
Schoen, S.W., Graeber, M.B., Kreutzberg, G.W., 1987. Light and electron microscopi-
cal immunocytochemistry of 5?-nucleotidase in rat cerebellum. Histochemistry
natal ontogeny of rat cerebellum: a marker for migrating nerve cells? Dev. Brain
Res. 39, 125–136.
Schoen, S.W., Kreutzberg, G.W., 1995. Evidence that 5?-nucleotidase is associated
with malleable synapses – an enzyme cytochemical investigation of the olfac-
tory bulb of adult rats. Neuroscience 65, 37–50.
Schoen, S.W., Kreutzberg, G.W., 1997. 5?-Nucleotidase enzyme cytochemistry as a
tool for revealing activated glial cells and malleable synapses in CNS develop-
ment and regeneration. Brain Res. Protoc. 1, 33–43.
Schmatz, R., Mazzanti, C.M., Spanevello, R., Stefanello, N., Gutierres, J., Maldon-
ado, P.A., Correa, M., da Rosa, C.S., Becker, L., Bagatini, M., Goncalves, J.F.,
Jaques Jdos, F., Schetinger, M.R., Morsch, V.M., 2009. Ectonucleotidase and
acetylcholinesterase activities in synaptosome from the cerebral cortex of
streptozotocin-induced diabetic rats and treated with resveratrol. Brain Res.
Bull. 80, 371–376.
Sesack, S.R., Snyder, C.L., 1995. Cellular and subcellular localization of syntaxin-like
immunoreactivity in the rat striatum and cortex. Neuroscience 67, 993–1007.
Sigueira, I.R., Elsner, V.R., Rilho, L.S., Bahlis, M.G., Bertoldi, K., Rozisky, J.R., Batasttini,
A.M., Torres, I.L., 2010. A neuroprotective exercise protocol reduces the adenine
nucleotides hydrolysis in hippocampal synaptosomes and serum of rats. Brain
Res. 1316, 173–180.
Stone, T.W., 1981. Physiological roles for adenosine and adenosine 5?-triphosphate
in the nervous system. Neuroscience 6, 523–555.
Strater, N., 2006. Ecto-5?-nucleotidase: structure function relationships. Purinergic
Signal. 2, 343–350.
Sudhof, T.C., 1995. The synaptic vesicle cycle: a cascade of protein–protein interac-
tions. Nature 375, 645–653.
Torres, I.L., Battastini, A.M., Buffon, A., Furstenau, C.R., Siqueira, I., Sarkis, J.J., Dalmaz,
as function of age. Int. J. Dev. Neurosci. 21, 425–429.
Vogel, M., Kowalewski, H., Zimmermann, H., Janetzko, A., Margolis, R.U., Wollny,
H.E., 1991. Association of HNK-1 epitope with 5?-nucleotidase from Torpedo
marmorata (electric ray) electric organ. Biochem. J. 278, 199–202.
Vogel, M., Kowalewski, H., Zimmermann, H., Hooper, N.M., Turner, A.J., 1992. Solu-
and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-
anchored ectoenzyme by phospholipase C cleavage. Biochem. Res. 284,
Vogel, M., Zimmerman, H., Singer, W., 1993. Transient association of the HNK-1
epitope with 5?-nucleotidase during development of the cat visual cortex. Eur.
J. Neurosci. 5, 1423–1425.
Weaver, D.R., 1996. A1-adenosine receptor gene expression in fetal rat brain. Dev.
Brain Res. 94, 205–223.
Biochem. J. 285, 345–365.
Zimmermann, H., Vogel, M., Laube, U., 1993. Hippocampal localization of ecto-5?-
nucleotidase as revealed by immunocytochemistry. Neuroscience 55, 105–112.
Zimmermann, H., 1996. Biochemistry, localization and functional roles of ecto-
nucleotidases in the nervous system. Prog. Neurobiol. 49, 589–618.
Zimmermann, H., 2000. Extracellular metabolism of ATP and other nucleotides.
Naunyn Schmiedebergs Arch. Pharmacol. 362, 299–309.
Zimmermann, H., 2006. Nucleotide signaling in nervous system development.
Pflugers. Arch. 452, 573–588.