INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 19, no.

2, 293-308 (2006)

P2Y 2 RECEPTOR UP-REGULATION INDUCED BY GUANOSlNE;OR UTP IN RAT

BRAIN CULTURED ASTROCYTES

P. BALLERINII,2, P. DI IORIOl,2, F. CACIAGLII,2, M.P. RATHBONE3, S.llANG4, E. NARGP,2,

S. BUCCELLN, P. GIULIANI!, I. D'ALIMONTEI, G. FISCHIONE!,A. MASCIULLP, S. ROMANOI
and R. CICCARELLP,2

'Dept. ofBiomedical Sciences, University G. D'Annunzio ofChieti-Pescara, Chieti; 2Ageing

Research Center, Ce.S.I., University G. D'Annunzio ofChieti-Pescara, Chieti, Italy; 3Dept. of
Medicine, McMaster University, Health Sciences Centre, Hamilton; 'Dept. ofSurgery, McMaster
University, Hamilton, Ontario, Canada

Received September 7, 2005 - Accepted March 14, 2006

Among P2 metabotropic ATP receptors, P2Y2 subtype seems to be peculiar as its up regulation triggers
important biological events in different cells types. In non-stimulated cells including astrocytes, P2Yz
receptors are usually expressed at levels lower than P2Y1 sites, however the promoter region of the P2Yz
receptors has not yet been studied and little is known about the mechanisms underlying the regulation
of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and
naturally occurring agonist for P2Y 2 sites, but also guanosine induced an up-regulation of astrocyte P2Y2
receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside
since in our previous studies it was reported to be released by cultured astrocytes and to exert different
neuroprotective effects. UTP and guanosine-evoked P2Y2 receptor up-regulation in rat brain cultured
astrocytes was linked to an increased P2Y2-mediated intracellular calcium response, thus suggesting an
increased P2Y2 activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-
mediated P2Yz up-regulation, thus indicating that de novo transcription was required. The effect of UTP
and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction
pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface
receptor mRNAs. The results show that ERKl-2/MAPK pathway playa key role in the P2Y2 receptor up-
regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in
guanosine-induced transcriptional activation ofP2Y2 mRNA and that increased intracellular calcium levels
and PKC activation may also mediate P2Y2 receptor up-regulation triggered by UTP. The extracellular
release of ATP under physiological and pathological conditions has been widely studied. On the contrary,
little is known about the release of pyrimidines and in particular of UTP. Here we show that astrocytes are
able to release UTP, either at rest or during and following hypoxialhypoglicemia obtained by submitting
the cells to glucose-oxygen deprivation (OGD). Interestingly, also P2Y2 receptor mRNAincreased by about
two-fold the control values when the cultures were submitted to OGD. It has been recently reported that
P2Y2 receptors can playa protective role in astrocytes, thus either guanosine administration or increased
extracellular concentrations of guanosine and UTP reached locally following CNS injury may increase
P2Y2-mediated biological events aimed at promoting a protective astrocyte response.
Key words: P2Y2 receptor, upregulation, guanosine, U'I'P; astrocytes
Mailing address: Patrizia Ballerini, MD, PhD,
Department of Biomedical Sciences,
Section of Pharmacology and Toxicology,
0394-6320 (2006)
University of Chieti, Copyright © by BIOLIFE, s.a.s.
Medical School, Via dei Vestini 29, Pal B, 66013 Chieti, Italy This publication and/or article is for individual use only and may not be further
Tel: +39 0871 3554014, Fax: +39 0871 3554012 reproduced without written permission from the copyright holder.
e-mail: p.ballerini@dsb.unich.it 293 Unauthorized reproduction may result in financial and other penalties
294 P. BALLERINI ET AL.
In the central nervous system (CNS), adenosine 5'-
triphosphate (ATP),beyond its well-known intracellular
roles, acts as a neurotransmitter by affecting both
neurones and glia through a great variety of cell-
surface P2 purinoceptors that have been classified as
metabotropic P2Y and ionotropic P2X receptors (1-2).
Each of these major classes comprises a number of
pharmacologically and genetically distinct subtypes,
among which, to date, eight P2Y (P2Y t 246 t t 12 13 14) and
seven P2X receptors (P2X I_7) have been' cloIied from
mammalian tissues (3-4).
Astrocytes represent the major sourceofextracellular
adenine-based purines in the CNS under physiological
conditions, and under pathological conditions such
as seizures, trauma or stroke these cells release large
amounts of adenine-based purines. Astrocytes are also
one of the main cellular targets for these compounds.
Indeed, extracellular purine-nucleotides, particularly
ATP, have been reported to be important intercellular
messengers mediating cell communication between
astrocytes (5), as well as the interaction of astrocytes
with several kinds ofadjacent cells, including microglia,
oligodendrocytes and neurons (6).
Beyond this compelling evidence for the
extracellular signalling role of ATP, increasing data
indicate that also uridine nucleotides can be released
by glial cells e.g. 1321N 1 human astrocytoma cells,
C6 glioma cells and primary rat astrocytes (7),
and may act as important extracellular autocrine/
paracrine messenger molecules via activation of
P2Y 2 , P2Y 4 and P2Y 6 receptors (7).
Furthermore, it is becoming increasingly clear that
the stimulation of specific astrocyte P2Y receptors
by extracellularly released ATP, and possibly UTP,
represents an important component ofthe physiological
and pathological glial responses including release
of neurotransmitters, regulation of extracellular
microenvironment, modulation of immune and repair
processes and reactive astrogliosis (8).
The P2Y 2 subtype seems to be unique amongst
the P2Y metabotropic ATP receptors, since data
from several cell types indicate that in physiological
conditions this receptor is weakly expressed, and
only when it is up-regulated by various stimuli
does it trigger important biological events. For
example: in rat vascular smooth muscle cells
(VSMC) P2Y 2 up-regulation induced by ATP,
growth factors and IL-l P may represent the first
step in atherosclerosis and neointima formation
(9-10); in mouse thymocytes the P2Y 2 induction
may be considered a regulatory feedback signal in
T-cell differentiation (11); in human myocardium an
increased P2Y 2 receptor mRNA in congestive heart
failure (CHF) may he a compensatory response that
increases heart inotropism; in osteoblastic cells (12)
and chondrocytes (13), an up-regulation of P2Y 2
receptor mRNA is linked to an ATP-mediated Ca 2+
wave propagation.
Although the roles of P2Y receptors in general,
and P2Y2 receptors in particular, are generally well
understood in the cardiovascular, immune and
muscular systems, little is known about the role of
P2Y2 receptors in astrocytes. In rat brain astrocytes
P2Y2 receptors are activated equally by ATP and UTP,
and have been reported to be coupled to phospholipase
C (pLC) activation, resulting in IP3 generation and
calcium discharge from intracellular stores (14), and
to modulate the release of bioactive substances such
as eicosanoids (15). Moreover, UTP increases cell
proliferation in C6 glioma cells (16) and Muller cells
(17). Very recent findings indicate that, as in other
cells, P2Y2 receptor upregulation in astrocytes may be
important and could increase its effects. For example, a
marked induction ofmRNA for P2Y2 receptor has been
reported during hippocampal astrocyte development,
which could indicate a role for this receptor in synaptic
stabilisation by perisynaptic astrocytes. Furthermore,
the up-regulation of P2Y2 mRNA in astrocytes treated
with IL-lP, or in Cx43 knock-out (KO) astrocytes, is
associated with an ATP-mediated intercellular calcium
propagation, which is considered to be a mechanism of
cell-to-cell communication, alternative to that mediated
by gap junctions (18).
Despite these findings, the organisation and
promoter sequences of P2Y2 gene have not been
characterised, and little is known regarding factors
and mechanisms that regulate the expression of this
receptor. We recently provided evidence for the
presence in both rat brain and astrocyte membranes of
a new cell-surface metabotropic purine receptor which
recognises guanosine as its naturally occurring agonist
(19-21) and whose activation in astrocytes increases
the release of adenine-based purines (22), stimulates
astrocyte proliferation (23), increases synthesis and
release of trophic factors (24-25) and protects against
staurosporine-induced apoptosis (26). In the present
 Int. J. ImmunopathoI. Pharmacol.
study we show that this guanine-nucleoside together
with UTP and ATP, the naturally occurring agonists for
P2Y2sites, regulates the expression ofP2Y2 receptors in
rat brain cultured astrocytes. We also report the results
of a series of experiments designed to elucidate the
signalling pathway underlying the P2Y2 up-regulation
evoked by guanosine and UTP.
It is known that in the brain the levels of
extracellular adenine-based purines can increase up to
IOO-fold during pathological events such as cerebral
ischemia (27). We have recently shown that in rat brain
cultured astrocytes, hypoxialhypoglicemia results in
a significant overproduction of not only adenine but
also guanine-based purines (28). Thus, based on these
findings we also evaluated whether in this in vitro
model of stroke, the levels of astrocyte P2Y 2 receptor
mRNA were increased.
MATERIALS AND METHODS
Materials
Ethylene glycol-bistjl-aminoethyl ether)-N,N,N',N' ,-
tetracetic acid (EGTA) and poly-D-lysine, were from Sigma
(Sigma-Aldrich, Milan, Italy), {1-[2-(5-Carboxyoxazol-2yl)-
6aminobenzofuran-5-oxyl]-2(2'-amino-5'-methylphenoxy)-
ethane-N,N,N'N'-tetracetic Acid Penta(acetoxymethyl)
Ester} (fura-2/AM), [1,2-bis(o-Aminophenoxy)ethane-
N,N,N' ,N' -tetraacetic Acid Tetra(acetoxymethyl)Ester]
(BAPTNAM) and Bisindolylmaleimide I (GF109203X)
were purchased from Calbiochem (Calbiochem-
Novabiochem Corp., CA, USA). DNase-I, RNase-free was
purchased from Roche (Monza, Italy), RETROscript™ was
fromAmbionInc. (Texas, USA), PCR reaction buffer, dNTPs
and Ampli'Iaqfiold" DNA polymerase were supplied by
Perkin-Elmer (Branchburg, NJ, USA), oligonucleotide
primers were from MGW-Biotech (Ebersberg, Germany),
Hybond-N nylon membranes, megaprime DNA labelling
system, [5.3H]uridine and [32P]-dCTP were from Amersham
Biosciences (Milan, Italy), 2-(2-amino-3-methoxy phenyl)-
4H-l-benzopyran-4-one (pD98059) was from Research
Biochemical Incorporated (Sigma-Aldrich, Milan, Italy).
Dulbecco's modified Eagle medium (DMEM), horse serum
(HS) and TRIzol Reagent were from GibcoBRL (Life
Technologies, Milan, Italy). Disposable materials for tissue
cultures were purchased from NUNC (Mascia Brunelli,
Milan, Italy).
Astrocyte cultures
Primary cultures of astrocytes were prepared from 18
to 19-day old rat fetuses as previously reported (28) (work
approved by the Animal Research Ethics Board of the
295
University of Chieti and carried out in accordance with
the European Animal Care Guidelines). After decapitation
the cortices were quickly removed and pooled into 2 ml
Dulbecco Modified Eagle's Medium (DMEM) added with
glucose (6 gil), NaHC03 (3.7 gil) and horse serum (20%).
The tissue was mechanically dissociated and the obtained
cell suspension was centrifuged at 200 g for 3 min. The
resultant pellet was resuspended in the growth medium
indicated above, containing also 5 roM L-leucine methyl
ester in order to restrain microglia contamination (29).
Cells were seeded onto poly-D-lysine (10 ug/ml) coated
flasks and maintained at 37 DC in a humidified incubator
under 95% air and 5% CO 2. Growth medium was renewed
after 24 h, subsequently every 3 days, serum concentration
being diminished to 5%. On the T" and the 13th day in vitro
(DIY), the cells were shaken for 3 h at 80 rpm on a plate
shaker to minimise microglia contamination. For Northern
blot analysis and calcium measurements, confluent primary
cultures of astrocytes on the 14th DIV were trypsinised
(0.025% trypsin/O.04% EDTA dissolved in PBS, 10-20 min,
37DC) and replated at a concentration of 1.5 x 106 cells/dish
(100 mm Petri dishes).
The assays on astrocytes were performed 4 days after
replating. More than 98% of the cultured cells were identified
as astrocytes. using immunofluorescent staining technique
for GFAP (30). Microglia contamination was determined by
labellingmicroglialcells with a macrophage cell-specific F4/80
biotinylated monoclonal antibody, followed by staining with
avidin-Texas red(30).
Efflux ofpyrimidines
Astrocytes grown on coverslips were incubated for 30
min at 37 DC in 20 ml ofKrebs solution (in roM: 118.5 NaCl,
4.7 KCl, 2.5 CaC~, 1.2 MgS04, 1.2 ~PO, 10 glucose,
25 NaHC0 3) oxygenated with 02 plus CO 2 (95%-5%)
containing P'3H]uridine (3 ~M for a specific activity of25.5
Ci/mmol). At the end of incubation, the cells were quickly
washed with ice-cold unlabelled medium to avoid a non-
specific contamination. Two coverslips were then transferred
into each of six superfusion chambers (0.5 ml vol) provided
with two platinum electrodes able to deliver a uniform field
electrical stimulation. The flow rate was 0.5 ml/min at 37 DC
and, after an initial 30-min washout, the collection of 10 min
fractions began, which continued throughout the remainder
ofthe experiment. After a further 30-min superfusion period,
field electrical stimulation (biphasic square wave pulses,
duration 5 min, amplitude 20 V at a current of 30 mNcm2,
frequency 10 Hz) was delivered for 5 min. At the end of the
experiments, cell proteins were dissolved in 0.1 M NaOH;
aliquots were used to assay the protein amount (31).
HPLC analysis
Pyrimidine nucleotides were identified by HPLC using
296 P. BALLERINI ET AL.
a reverse-phase, ion pair technique. HPLC was carried out
using a reverse-phase analytical column (LiChrospher
100 RP-18 5 urn, Merck) protected by a guard column
(LiChrospher RP-18, 5 urn, Merck), using a Kontron
Instruments HPLC apparatus. Elution was carried out
applying a linear gradient from 100% solvent A (60 mM
KHl04 and 5 mM tetrabutylammonium phosphate, pH
6.0) to 100% solvent B (30% methanol plus 70% solvent
A) over a 15 min period (flow rate 1.5 ml/min). The
amount of different pyrimidine compounds was measured
on the basis of the absorption at 254 nm by comparison
with appropriate standards.
An on-line radiochemical detector (Flo-one 500
TR, Packard Instrument) was used for determining
radioactivity. The liquid scintillation cocktail (Ultima-
Flo M, Canberra Packard) was pumped at 3 ml/min
and mixed continuously with the HPLC eluate. The
mixture was passed through a 500 ul detector flow cell.
Background radioactivity was subtracted from all radio-
chromatograms. Radio-chromatograms were integrated
and the cpm under each peak was obtained.
Combined oxygen/glucose deprivation (OGD) protocol
Cells were maintained in standard culture condition
for a first phase of stabilisation, as described in the
previous section. A glucose-free bicarbonate-buffered
Krebs solution, widely used to achieve an OGD condition
(27), was then added to the cultures after a gentle
cell-washing with the same buffer. This medium was
previously bubbled with 95% N/5% CO2 at 3 I/min for
5 min and pre-warmed at 37°C. Hypoxia was induced by
placing cells in a humidified, sealed chamber (Billups-
Rothenberg, Del Mar, CA, USA) at 37°C, which was
flushed with 95% N/5% CO2 for 5 min. In this condition,
all but 0.3% oxygen tension could be removed, as
indicated by a P02 meter (Beckman oxygen analyser OM-
11, Beckman Instruments). At the end of the OGD period
(30 min), cultures were returned to standard conditions
for the indicated period. In each experiment, cultures
exposed to OGD were always compared with normoxic
controls supplied with Krebs solution containing glucose.
The release of purines was assayed in the media collected
every 30 min, starting from 30 min before and up to 90
min after OGD. Cell protein content was evaluated by
Bradford method (1976).
Cell viability determination
Cell viability of cultures, normoxic and submitted to
OGD or challenged with A23187, was determined by the
measurement of lactate dehydrogenase (LDH) activity in
the culture media collected at the end of the experimental
procedures, using a kit from Boehringer Mannheim
(Mannheim, Germany). Total LDH release, corresponding
to complete astrocyte death, was determined at the end of
each experiment following cell-freezing at -70°C and
rapid thawing, and was considered to be 100%. Data from
LDH activity were compared with the count of living
cells in sister cultures by the determination of trypan
blue exclusion. One hour after stimulation the cel1s were
trypsinised, suspended in the culture medium and mixed
with trypan blue (0.4%, Sigma) (1: 1, v/v). Viability
of the cells was then determined in a hemocytometer.
Approximately 100 cel1swere counted per culture and the
proportion of negative staining cells was expressed as a
percentage of total cell counts.
RNA isolation
For RNA determination, astrocytes in 100-mm dishes
were incubated for 24 h in a medium containing 1% FBS
to equilibrate the cel1s and then treated with the various
agents for the times and concentrations indicated in the text
and figure legends. Total RNA was isolated from cel1s using
TRIzol reagent (Life Technologies, Milan Italy) fol1owing
the supplier's instructions. The resulting RNA pel1et was
final1y washed with 70% ice-cold ethanol, air dried and
re-dissolved in 30 III diethyl-pyrocarbonate (DEPC)-
treated water. The RNA concentration was determined
spectrophotometrically.
RT-PCR analysis
10 ug RNA was treated with 10 U DNase 1- RNase
free (Roche, Monza, Italy) to remove any genomic DNA
contaminants. cDNA was synthesized, using random
hexamers as primers, by using RETROscript ™ (Arnbion
Inc, TX, USA) according to the manufacturer's instructions.
The resultant cDNA (2 Ill) was amplified in a 50 ul reaction
volume containing 2 U Taq polymerase (ArnpliTaq Gold™,
Applied Biosystems, CA, USA), dNTPs (200 IlM each)
(Applied Biosystems), 1.5 mM MgCI2, 5 III lOXpolymerase
chainreactionbufferand primersusedatfinalconcentrationof
I mM (MWG Biotech, Ebersberg,Germany). The fol1owing
primers were used: P2Y2 forward 5'-TITGCTCCCTITf-
3' and reverse 5'-GTTGCCTTAGATACGATTCC-3' ;
P2Y4 forward 5'-ATGGACTCATGGCCCGGCGA-3' and
reverse 5'-CTTGGTGGGTGTCCGCCCAC-3'; P2Y6
forward 5' -TATAACTACGCCAGAGGGGACCAC-3'
and reverse 5'-GGCGGGCCATGCGACAGTAG-3'. The
mixture was overlaid with 25 ul of mineral oil to prevent
evaporation. Temperature cycling proceeded as fol1ows: I
cycle at 94°C for 5 min and 30 cycles at 94°C for 1min,
55°C for 1 min and noc for I min, fol1owed by noc for 10
min. PCR products were detectedby electrophoresison a 1%
agarose gel containing ethidium bromide.
Northern blot analysis
RNA (15 ug/lane) was fractionated on formaldehyde
 Int. J. Immunopathol. Pharmacol.
denaturing 1% agarose gel and blotted overnight onto
Hybond-N nylon membrane (Amersham Pharmacia
Biotech, Buckinghamshire, UK). The filter was then UV
cross-linked in UV Stratalinker 1800 (Stratagene). The
blots were prehybridized for at least 8 hours at 42°C in
a solution containing 50% formammide, 5x Denhardt's
Solution, 0.1% SDS and 100 ug/ml salmon sperm DNA.
The hybridization of cDNA probes was performed in a
buffer of the same composition containing 106 cpm/ml of
radiolabelled probes. The cDNAs obtained by extracting
PCR products from agarose gel slices (Millipore, Bedfore,
MA USA) were radiolabelled by the method of random
priming (Megaprime DNA labelling system, Amersham
Pharmacia Biotech) using alfa 32P-dCTP (Amersham
Pharmacia Biotech). After hybridization, the membranes
were washed in O.1X SSC containing 0.1% SDS at 50°C
and exposed to Kodak Biomax MS autoradiography film
at -80°C with intensifying screen for 2 days. Subsequent
to hybridization with P2Y2 cDNA probe, Northern blots
were hybridized with a probe for GAPDH to allow
correction for the recovery of RNA in each sample.
Experiments were performed at least in triplicate. For the
purpose of quantification, autoradiograms were scanned
using laser densitometry. P2Y 2 mRNA signals were
normalized against GAPDH content by determining the
ratio of the respective optical densities.
Calcium measurements
Intracellular calcium concentration ([Ca2+]) was measured
with the fluorescent indicator Fura-2, as described previously
(32). In brief, cultures were washed twice with phosphate-
buffered saline, and cells were detached from the flasks with
a 0.025 % trypsin-EDTA solution (37°C, 5 min). The cells
were then washed twice by centrifugation in a standard buffer
containing 125 mM NaC!, 5 mM KCl, 1 mM MgS04 , 1 mM
KHl04, 1 mM CaCI2, 5.5 mM glucose and 20 mM HEPES,
pH 7.4. 3 IlM of the cell-permeable acetoxy methyl ester of
Fura-2 (Fura-2/AM, Sigma, Milan, Italy) and 250 IlM of
sulfinpyrazone were then added to the cell suspension for
30 min at 37°C. After centrifugation at room temperature,
the cells were resuspended in the standard buffer containing
Sulfinpyrazone (250 IlM). [Ca2+1measurements were carried
out in a thermostatically-regulated and magnetically-stirred
fluorometer cuvette at a density of lx10 6 cells/m!. Fura-2
fluorescence was monitored with excitation wavelengths at
340 and 380 nm and the emission wavelength at 510 nm
by a FluoroMax ™ spectrofluorometer. The 340/380 nm
fluoroscence signalling ratios (R) were converted off-line
to an estimate of [Ca2+ 1 using the formula [Ca2+1 = Kd ~
(R-Rmin)/(Rmax-R). Calibrations were obtained by adding
0.1% Triton X-IOO to solutions containing 1.5 mM CaCl2
(Rmax) or no added Ca2+ (Rmin). Kd (a dissociation constant
ofFura-2 for Ca2+) was assumed to equal 224 nm, according
297
to Grynkiewicz et al. (1985) (33) and ~ (equivalent to 380
nm at Rmin/380 nm at Rmax) was 4.2.
Statistical Analysis
The statistical significance was established by
analysing data by Student's two-tailed t-test, using Prism
(version 3.03) program (GraphPad Software, San Diego,
CA, USA) with P<0.05 considered to be significant.
RESULTS
Release of uridine-nucleotides from rat cortical
astrocytes
Under basal conditions, cultures of rat brain
astrocytes not only released adenine and guanine-based
purines as we previously reported (30), but they also
released pyrimidines. The release of [3H]pyrimidines
was assayed in cultures of astrocytes following 30 min
of [3H]uridine (specific activity 25.5 Ci/mmol) loading.
On-line radiochemical detection ofpyrimidines present
in the superfusion medium of rat cultured astrocytes
showed that, as for their adenine and guanine-based
counterparts, after a 5 min application of the field
electrical stimulation (10 Hz) the release ofUTP, UDP
and UMP significantly increased; the value for UTP
being about 3-fold more than basal (Fig.I). As already
reported (34), electrical stimulation did not significantly
increase LDH release into the culture medium 15 min
after the beginning ofthe stimulus (data not shown).
Expression of P2Y] and P2Y2 receptor mRNA in
rat cortical astrocytes in resting conditions and after
cell exposure to AT?, UTP and Guanosine
Total RNA was extracted from rat cortical
astrocytes and the expression of P2Y!' the main
functionally active astrocyte P2 metabotropic ATP
receptor (35) was examined along with that of
P2Y 2 by Northern blotting. In untreated cultures,
hybridization with specific probes gave rise to two
signals (Fig. 2A), one of an estimated size of 4.2 kb,
corresponding to P2Y 1 receptor, and a weaker one of
3.6 kb corresponding to the P2Y 2 site, in agreement
with that found in other rat tissues (36-37).
To evaluate whether mRNA expression of
these two metabotropic receptors is regulated in
an activity-dependent manner by stimulation with
their naturally occurring agonists, whose release was
reported above, quiescent, serum-starved rat brain
cortical astrocytes were treated for 1 h with: i) 100
IlM ATP, an agonist for both P2Y 1 and P2Y 2 sites;
298 P. BALLERINI ET AL.
ii) 100 11m UTP, the principal phyisiological agonist
for P2Y 2 and P2Y4 receptors (38); iii) 300 11M
Guanosine (Guo), a concentration that we reported
to induce maximal increase of cell proliferation (30),
maximal antiapoptotic effect (26) and maximal Akt/
PKB phosphorylation (26) in cultured astrocytes.
The expression of the two receptor mRNAs was then
tested at the indicated times. All three compounds
used were able to induce P2Y 2 mRNA expression
(Figs. 2 B, C, D). Both ATP and Guo significantly
up-regulated the P2Y l receptor mRNA (Figs. 2 Band
D) whereas, as expected, changes in P2Y] mRNA
levels after cell stimulation with the P2Y 2 agonist
UTP were minimal (Fig. 2e). In controls, levels
of the GAPDH mRNA, chosen as housekeeping
gene, were found not to be modified by the above
mentioned treatments (data not shown).
We focused our attention on the P2Y 2 since in other
cell types it is only weakly expressed at rest (11, 18)
but undergoes significant up-regulation in relation to
some characteristic functional conditions or certain
pathological events (9, 11, 18).
UTP-induced expression of the P2Y 2 receptor
900
c=JUTP **
750 ITIID UDP
CI)
~UMP
~ 600
...(,) 450
CI)
s:::: * free medium for 24 hrs, the response to 100 11M
';je. 300
* UTP increased intracellular calcium concentrations
150
o [Ca 2+1measurement after 5 or 6 h respectively, UTP
Fig. 1. Effect ofthe electrical stimulation on the efflux of
FHjuridine-nucleotides from cultured astrocytes. After a
stabilization period of60 min astrocytes were challenged
with electrical stimulation (biphasic square wave pulses..
duration 5 min, amplitude 20 Vat a current of30 mA/cm 2,
frequency 10 Hz). Supernatants were collected at timed
intervals (10 min). The basal release of [JH]uridine-
nucleotides was: UTP= 4.6±0.69 FH]pmol/lOmin/mg
proteins (n=6), UDP= 3.1±0.5 [JH]pmol/lOmin/mg
proteins and UMP= 15.5±2.6 FH]pmol/1Omin/mg
proteins. Data are expressed as percentage increase from
basal values and shown as ±SEM, *p<0.05, **p< 0.005,
n=6. (Student s t test).
mRNA peaked at 5 hrs (2.6±0.38 fold the control
values, n=3, p< 0.05) after cells were exposed for
1 h to the nucleotide, and decreased nearly to the
basal level at 24 hrs (about 1.15±0.2 fold the control
values, n=3) (Fig. 2e). On the other hand, Guo
up-regulated P2Y 2 receptor mRNA with a different
temporal pattern. The Guo-induced expression
peaked at 6 hrs after astrocyte exposure for 1 h to
the nucleoside (2.97±0.31 fold the control values,
n=3, p<0.05) and decreased to nearly the basal level
by 24 hrs (about 1.10±0.13 fold the control values,
n=3) (Fig. 2D). Therefore, subsequent experiments
were carried out using 6 hr and 7 hr time-periods
from the beginning of incubation time for UTP and
Guo respectively.
UTP-mediated effects were abolished (98±16%
of control, n=3, p < 0.05, Fig. 3) by pre-treatment of
astrocytes for 30 min with suramin (50 11M) which has
been reported to act as an antagonist at P2Y l and P2Y 2
receptors, and to be much less effective, or ineffective,
at P2Y4 receptor (39-40). We could not carry out
parallel experiments for the induction of the P2Y 2
receptor by Guo because, to date, no antagonists are
available for the putative Guo receptor.
Rat brain astrocytes have been reported to
respond to exogenous UTP with an increase in
[Ca"], Fig. 4 shows that UTP and Guo-evoked P2Y 2
up-regulation in rat brain cultured astrocytes was
also linked to an increased P2Y 2-mediated calcium
response. After astrocyte incubation with a serum-
([Ca 2+]) by 35±7.7 nM over the basal value. When
UTP (100 11M) or Guo (300 11M) were added to the
cell medium for 1 h and the cells were harvested for
was able to increase [Ca 2+ 1to a significantly higher
extent (Fig. 4).
Transcriptional regulation of P2Y] receptor
mRNA induction
To assay whether the up-regulation of P2Y 2
mRNA, induced by UTP and Guo was due to
transcriptional or postranscriptional mechanisms,
actinomycin D (Act D) (10 ug/ml), an inhibitor of
RNA polymerase, was added to the cell medium 1
h before treatments. This drug reduced the basal
levels ofP2Y 2 mRNA and, as shown in Fig. 5, in the
 Int. J. Immunopathol. Pharmacol. 299

A 4.2 Kb
3.6Kb O.D.
21

GAPDH 13

B ATP (100 J.1M) C UTP (100 J.1M) D Guo (300 J.1M)

1/1 300 1/1 300 400

'iH-'~Gi 275 0 P2Y, 'iH-'~Gi 275 0 P2Y 1 .!!!
'i ~Gi"'50 0 P2Y,
:; ~ ~ 250 • P2Y2
:;~~250 • P2Y2
~ ~~300 • P2Y2
e la
u_ > 225 e la
u_ > 225 ~Ia>
g~ 5
:z:: E.c
200
175
g:sCIlia:g 200
:z:: E.c 175
O'!.! iij250
1 1
:z::C .c(1200
00'"
II.:!:: 0 150
00'"
II.:!:: 0 150 0 0 0 150
~:!::
~
c( 1/1 c( 1/1
~~loo
0 0
Cl5i~125 Cl 5i ~ 125
'0 100
~ '0 100
~ ClCll-
'0 50
75 ~ 75 ~ 0 ~
0 3 6 9 1218 21 24 0 3 6 9 1218 21 24 0 3 6 9 1218 21 24
Hours Hours Hours

Fig. 2. A representative Northern blot probed for P2Y, and P2Y] nucleotide receptors in rat cultured astrocytes is
shown in panel A. Total RNA (15 Ilg/Iane) was extractedfrom cultured rat astrocytes, electrophoresed on a 1% agarose/
formaldehyde gel, blotted on Hybond membrane and hybridized with either P2Y,. P2Y] or GAPDH eDNA probes. In the
inset densitometric analysis ofNorthern blots for P2Y, and P2Y] receptors are shown. Panel B) C) and D) Time course of
the induction ofP2Y, and P2Y] receptor mRNA in serum-starved rat cultured astrocytes. Starved astrocytes (as described
in Materials and Methods) were exposed to ATP (100 11M), UTP (100 1lA1), and Guanosine (300 11M) for I hour and
RNA extracted at indicated times. Valuesfrom densitometric analysis ofthe expression levels ofeach P2Y receptor were
normalized to those ofGAPDH mRNA. Each value represents mean ± S.E.M of 3 independent experiments.

presence of Act D, neither UTP (100 11M) nor Guo
(300 11M) were able to exert any stimulatory effect on
P2Y 2 mRNA expression. This indicates that de novo
transcription is required for the P2Y 2 induction.
Role of calcium, protein kinase C (PKC) and
of Extracellular Signal-Regulated Kinase (ERK)
signaling in UTP-mediated P2Y] mRNA induction
In astrocytes the activation of P2Y Z receptors
is linked to a mobilization of intracellular Ca2+
via the phospholipase CP-IP3 pathway (40-41).
Moreover, several studies have reported that an
increase in [Caz+]j can play an important role in
activity-regulated gene expression in neuronal cells
(42). To address whether P2Y z mRNA up-regulation
is controlled by a rise in [Ca"], the cultures were
stimulated with 100 /lM UTP after a pre-incubation
with BAPTA/AM (5 /lM) for 30 min. Chelation
of intracellular calcium by BAPTA/AM markedly
reduced P2Y z mRNA induction mediated by UTP
(Fig. 6).
The effect of UTP was also evaluated in cells
pre-incubated with GF 102903X (10 /lM for 20
min) an inhibitor of both Ca't-dependent and Ca2+-
independent PKC. This drug completely inhibited
the UTP-mediated P2Y z mRNA induction (Fig. 6).
Accordingly, astrocyte stimulation with 100 nM
of the potent PKC activator phorbol 12-myristate
acetate (PMA) for 3 h significantly increased the
levels of P2Y 2 mRNA (3.6 ±0.33 fold the basal
value). Astrocyte pre-treatment with GF 102903X
blocked this effect, indicating that PKC is implicated
in the mechanisms underlying the up-regulation of
P2Y 2 (inset of Fig. 6).
Activation of P2Yz receptor sites in glial cells
has been reported to be also linked to the MEK/
ERK pathway (43). Inhibition of this cascade by
pretreating the cultured astrocytes with the specific
300 P. BALLERINI ET AL.

including microglia and astrocytes, Guo causes a

...
-
Q

C
rapid and time-dependent increase in ERK I/ERK2
Q
<:J phosphorylation (19) . Recently, this effect was also
reported in PC 12 cells (44). Thus to investigate
3.6 Kb
the possible involvement of this pathway in Guo-
mediated up-regulation of P2Y 2 mRNA expression,
GAPDH the effect of the MEK 1/2 inhibitor P098059 was
tested . Pretreatment of astrocytes with 30 JlM
UTP (100 JlM)
P098059, reported to reduce the antiapoptotic effect
of Guo in cultured astrocytes (26) markedly reduced
the Guo-induced P2Y 2 up-regulation (by about 75%)
(Fig. 7).
350 We recently found that Guo increases cyclicAMP
.....
e300 * (cAMP) formation and protein kinase A (PKA)
'8250
= activation in astrocytes (45). To further examine
~
... the mechanism underlying the direct Guo-mediated
r; 200 P2Y 2 induction, astrocytes were pretreated with
~
KT5720, a membrane-permeable PKA inhibitor.
~ 150
~
(J
At a concentration of 1 11M this drug reduced the
.5100 Guo-mediated effect by about 40% (Fig . 7). When
~
e..... 50 the PKA inhibitor was added in combination with
the MEK inhibitor P098059, the Guo-mediated
o P2Y 2 up-regulation was completely abolished
UTP (100 JIM) + + (Fig. 7) suggesting that, even though the MAPK
Suramin (50 JIM) + + pathway predominates, PKA is also involved in the
up-regulation of P2Y 2 mRNA by Guo . On the other
Fig. 3. Effict of suramin on the P2Y, receptor mRNA hand, as expected, BAPTA/AM failed to modify
up-regulation evoked by UTP (100 JlM) in cultured rat the Guo effect (0.98± 1.2 % of the Guo-induced
astrocytes, evaluated by Northern blot analysis. Serum- increase in P2Y 2 mRNA levels , n=2) . Indeed , in
starved cells were pretreated with 50 JlM suramin for 30 our experimental conditions, Guo at concentrations
min and then treated with 100 JlM UTP for I h as described
of up to 500 11M did not increase [Ca"] , (data not
in Materials and Methods . A representative experiment is
reported in the upper panel. In the bottom panel changes in
shown). This is in contrast to the effect of guanosine
mRNA levels/or P2Y! receptor were normalized to those of on astrocytes differentiated by culturing the cells in
GAPDH mRNA and expressed as percentage o/mRNA levels the presence of dibutyryl cyclic AMP (dBcAMP) for
in untreated cultures. Each value represents mean ± S.E.M at least 3-4 weeks (46).
of 3 independent experiments (p<0.05 vs untreated cells; Effect of combined oxygen/glucose deprivation
·p <0.05 vs UTP treated cells; Student s t test). (aGO) P2Y I and P2Y 2 receptor mRNA expression._
There have been no previous reports of the
MEK inhibitor P098059 (30 JlM for 30 min) influence of hypoxialhypoglicemia on P2Y I and
abrogated P2Y 2 mRNA up-regulation caused by UTP P2Y 2 expression in neuronal and non-neuronal cells.
(Fig. 6). These results suggest that ERK activation We evaluated whether oxygen-glucose deprivation
is involved in UTP-mediated P2Y 2 mRNA induction (aGO) could affect the mRNA levels of P2Y 1
via upstream activation of PKC (Fig. 6). and P2Y 2 receptors in rat cortical astrocytes. P2Y I
receptor expression which, as reported above, was
Extracellular signal-regulated kinase (ERK) strongly induced by both ATP and Guo as early as
signaling and cyclicAMP-dependent protein kinase after 60 min of agonist stimulation, was not modified
(PKA) in guanosine -mediated P2Y! mRNA induction 60 min after the onset ofhypoxialhypoglicemia (Fig.
We previously reported that in cultured glial cells, 8). On the other hand, aGO strongly induced P2Y 2
 Int. J. Immunopsthol. Phsrmseol. 301

200 e... Act ino my ci n D

UT P (100 JlM) ce o
::
Q"
f-
0
::
175 ... o =.J o

~ 150 * * 3.6 Kb

+
M 125 GAPD"
eo:
U 100
eo:
-.....
Q
~
75
50 400

25
o ~ 200
Fig. 4. The effects of UTP and Guanosine on [Ca"], in-
crease evoked hy 100 ~M UTP were evaluated in Fura-Z
pre-loaded serum-starved astrocytes as descrihed in
Materials and Methods. Della calcium represent the net
increases in [Ca"], calculated by subtracting the basal
level of [Ca"], from peak levels. Data are the means ±
s.E.M. from three determinations-. Significantly different
from controls 'p < 0.05 (Student s I lest).
mRNA by 6 h after the insult (I.9±0.14 fold the
control values, n=3, p<0.05) (Fig. 8).
In our previous study, we found that aGO for 30
min enhanced the release ofboth adenine and guanine-
based purines during the hypoxialhypoglicemia and
in the following 30 min from cultured astrocytes
(Ciccarelli et al., 1999). Interestingly, UTP efflux
was also significantly increased in these experimental
conditions, being 3.2±5 .7 fold the basal values (n=3)
under aGO (inset of Fig. 8). The concentration of
UTP also remained elevated in the 30 min following
the hypoxialhypoglicemia. As previously reported,
LOH release into the culture medium was increased
only by 15.6±4% as compared to that measured in
the medium coming from normoxic control (=22±5
mOD/min).
DISCUSSION
The role of P2Y2 receptors is not well elucidated.
Phylogenetically and structurally it has been
included in the group I of P2Y receptors which also
encompasses the subtypes P2Y P2Y 4 , P2Y 6 and
"
P2Y 11• These receptors have a relatively high level
of structural divergence with the group II subtypes
-=... 350
g 300
.-
b 250
'.-~" 150
c
.- 100
't.
50
o
UT P ( 100 llM) - + +
G uo (300 1ll\1) - + +
Act ino my ci n ( 10 m g/ml ) - + +
Fig. 5. Eflect ofActinomycin D on UTP. and Guanosin e-
mediated induction ofP2 Y! receptor mRNA in rat cultured
astrocytes evaluated by Northern blot analysis. Serum-
starved cells were pretreated with the inhibitor of RNA
polymerase (10 ~g/ml) for I h and then treated with 100
~M UTP or 300 ~M Guanosine for I h as described in
Materials and Methods. A representative experiment is re-
ported in the upper panel. In the bottom pan el changes in
mRNA levels for P2Y! receptors were normalized to those
of GAPDH mRNA and expressed as percentage of mRNA
levels in untreated cultures. Each value represents mean
± s.E.M. of 3 independent exp eriments (P<0.05 and
"p <O.OI vs untreated cells ; "p< 0.05 and sp-ul. O! vs UTP
and guanosine-treated cells respectively ; Student :~ t test).
represented by the P2Y 12 and P2Y 13 sites and by the
recently cloned and characterized P2Y 14 subtype (3).
Human and rodent P2Y 2 receptors, like rodent P2Y 4
receptors, have a mixed selectivity for adenine-
and pyrimidine-nucleotides (3). This is different
from both the adenine-nucleotide-preferring P2Y
receptors, which include human and rodent P2Y, .
P2Y I 2 and P2Y 13 and human P2Y" that respond to
ATP/AOP, and the pyrimidine-preferring receptors,
human P2Y 4 and P2Y 6 that respond to UTP/UOP.
The P2Y 2 subgroup of receptors is widely
expressed in a number of mammalian non-neural
302 P. BALI-ERINI ET AI..

Fig. 6. Role of calcium. ERKI-2IMAPK and PKC acti-

e vation in UTP mediated up-regulation of P2 Y! receptor
! mRNA in cultured astrocytes evaluated by Northern blot
0'
U "b analysis. Serum-starved astrocytes were pretreatedfor 30
e or.
o
ee min with: i) the intracellular Ca" chelator BAPTAIAM
c
o
U ..
0'
Q P." " 0. 1 ~ \I (50 11M), ii) the MEK inhibitor PD98059 (30 11M) or iii)
the PKC inhibitor GFI02903X (10 11M) and then treated
"2V, 3.6 kb
with 100 11M UTP for I h as described in Materials and
Methods. A representative experiment is reported in the
GA I' D11
upper panel. In the bottom panel changes in mRNA levels
for P2Y! receptor were normalized to those of GAPDH
TI'(100 I'M ) mRNA and expressed as percentage ofmRNA levels in un-
treated cultures. Data represent mean ± SE.M of 4 inde-
000
pendent experiments (p<O.OI vs untreated cells; ·p<0.05
E 350 and ItIIp <0.005 vs UTP- treated cells). A representative
~ 300 Northern blot showing the effect ofPMA (0.1 11M) on P2Y!
~ 250
receptor mRNA expression in cultured astrocytes is shown
~ 200
in the inset. The serum-starved cells were treated for 3 h
~ 1 50

.c
"-: 100
50
with PMA and then total RNA was extractedfor Northern
blot analysis as described in Materials and Methods. Va-
o lues from densitometric analysis normalized to those of
TP (100 I'~I ) + + + +
GAPDH mRNA- showed an increase of3.6±O.31 fold the
8AI'TA · A~1 (S I'~I ) +
Pll980S9 (30 I'~ I ) +
basal values, n=3.
G F I 02903X (10 I'M ) +

and neural cell types. In different cell types the been studied, and little is known on the cellular
stimulation of P2Y 2 and of P2Y 4 receptors has pathways or of the factors involved in the regulation
been reported to enhance [Ca2+l; (5), to trigger cell of P2Y 2 receptor expression. Therefore, the study
proliferation (47-48) and to activate outwardly of the molecular mechanisms underlying changes
rectifying chloride currents (49-50). The activation in P2Y 2 mRNA levels is important to expand the
of P2Y 2 also increases the bio-synthetic release of understanding of their functional role and their
prostanoids and the efflux of adenine- and non- regulation. Accordingly, we evaluated the signal
adenine-based nucleotides (51-54). Most of these transduction pathways which mediated the effects
effects are shared with those mediated by other P2Y of UTP, a naturally occurring agonist for P2Y 2
receptors such as the P2Y I subtype, thus making it receptors . We also focused our attention on the
difficult to determine the functional specificity of possible effects mediated by Guo. This nucleoside
P2Y 2 sites and to appreciate their role in the different has been reported to exert several biological effects
pathophysiological events. However, a peculiarity increasing astrocyte proliferation (30), increasing
of P2Y 2 receptors is that in non-stimulated cells production of trophic factors by these cells (19,
of several types, including thymocytes, VSMCs, 57), protecting against apoptosis triggered by
cardiomyocytes and astrocytes, they are usually staurosporine (26) and stimulating the uptake
expressed at lower levels than P2Y I sites (9, 11, of glutamate by astrocytes (58). Together, these
18), but their expression increases substantially actions of Guo imply that it may have an important
both in relation to some characteristic physiological neuroprotective role. Recently we reported a novel
conditions such as cell development, cell high affinity binding site for this purine nucleoside
differentiation or activation (11, 55), cell stimulation in membranes of both whole rat brain and astrocytes
with growth factors or by IL-l P (9- I0), and to (20-21). Thus, at least some of the effects mediated
pathological events such as vascular injury and by extracellular Guo may be exerted by its binding to
congestive heart failure (9, 56). these specific cell membrane sites. Our laboratories
The promoter region of the P2Y 2 has not yet are currently investigating the structure and the
 Int. J. Immunopalhol. Pharmacol. 303

ATP receptor in astrocytes, with a different temporal

pattern. As far as ATP is concerned, it is possible
that following extracellular nucleotide breakdown,
adenosine might activate its A I, A2 and A3 astrocyte
"0
l- receptors, thus participating in the regulation of P2Y 2
ee mRNA expression in these cells. We cannot exclude
U
this possibility. On the other hand, UTP, which does
3.6 kb not activate P2Y) receptors, dramatically increased
GA POH the levels ofP2Y 2 mRNA and only slightly increased
those of P2Y) receptors, therefore we chose to use
UTP in the following experiments. Among P2Y
G uo (300 JlM)
receptors, it is known that this pyrimidine-nucleotide
400
is also active on P2Y 4 receptors whereas P2Y 6 sites
E 350 are more sensitive to UDP than UTP (38). The results
of RT-PCR analysis indicate that in our experimental
§ 300
<J
conditions primary astrocytes cultures express
.b
~ 200
250
mRNA for these three receptor subtypes (data not
shown) in agreement with Fumagalli et al (59) and
~ 150
Dixon et al (60) . Our results showed that suramin
.~ 100
(Fig . 3), which has been reported to discriminate
~ 50
between P2Y 2 and P2Y 4 sites by blocking only
o ...L..J'--.L..L ---l--Ll:
the former and not the latter (38-39) was able to
G ua nosine (300 Ill ) + + + + abrogate UTP-mediated P2Y 2 mRNA up-regulation.
1'098059 (30 Jll\1 ) + +
KT5720 (10 Jll\1 )
These findings make it unlikely that either P2Y 6 or
+ +
P2Y 4 receptors could have contributed to the P2Y 2
Fig. 7. Role of ERKI-2/MAPK and PICA activation in mRNA up-regulation and to the subsequent increase
Guanosine mediated up-regulation of P2Y, receptor in [Ca 2+J; triggered by UTP. In the presence of the
mRNA in cultured astrocytes evaluated by No~thern blot mRNA polymerase inhibitor ActO, neither UTP
analysis . Serum-starved astrocytes were pretreatedfor 30
nor Guo were able to induce any increase in P2Y 2
min with: i) the MEK inhibitor PD9805 (30 11M). ii) the
mRNA levels, thus indicating that their effects in
PKA inhibitor KT5720 (l 11M) or iii) a combination of
KT5720 (lIlM) plus PD98059 (30 11M) and then treated astrocytes were mediated through de novo synthesis
with 300 11M Guanosine for I h as described in Materials rather than through inhibition of the degradation rate
and Methods. A representative experiment is reported in for P2Y 2 mRNA. The transcriptional induction of
the upper panel. In the lower panel changes in mRNA lev- the expression of P2Y 2 receptors on the astrocyte
els for P2Y! receptor were normalized to those ofGAPDH plasma membranes was linked to an enhanced UTP-
mRNA and expressed as percentage of mRNA levels in mediated Ca 2+ response, suggesting that there exists
untreated cultures. Data represent mean ± S.E.M of 4 in- a tight relationship between the level of expression
dependent experiments (p <0.05. ••p< 0.005 vs untreated and the rate of activity, in agreement with results
cells: #p< O. 05 and # # p<O.OOI vs Guanosine-treated cells:
obtained by Hou et al in VSMCs where P2Y 2 up-
Student :~ t test).
regulation was triggered by cell treatment with IL-
signal transduction pathways of this new putative IP for 26 h (10).
purinergic receptor. The inhibitor analysis of signaling cascades
Our data show that Guo, like UTP and ATP, which including ERK, PKC and PKA reported to be
are equally active on rat P2Y 2 sites (3), induced an involved in the regulation of other cell surface
increase in P2Y 2 mRNA which peaked 6 hand 5 h receptor mRNAs, including AT2 angiotensin, alpha
after astrocyte exposure for I h to these compounds. adrenergic, beta adrenergic and skeletal muscle
ATP and Guo were also able to up-regulate the acetylcholine receptor subunits (61-64), was
mRNA of P2Y) site, the most studied metabotropic performed on both Guo and UTP induced P2Y 2
304 P. BALLERINI ET AL.

'0 Q KT5720 showed an additive inhibitory effect,

.. 0
=
8 u (J
suggesting that, even though the ERK-I /2-MAPK
pathway predominates, PKA is also involved in Guo-
P2Y. 4.2 kb stimulated P2Y2 mRNA up-regulation. These data are
in agreement with both our recent findings indicating
GAPDH _~.
that Guo increases the formation of cAMP (45) and
P2Y2 3.6 kb with the results by Martin K.A., 1997, which reported
that in human myeloid lymphocytes P2Y2 mRNA
GAPDH levels were transiently up-regulated by the cAMP
analogue BtcAMP.
250 The MAPK cascade also plays a key role in the up-
P2Y1
=.. P2Y2 c::::::J Control
_CGOD
regulation mediated by UTP as indicated by the effect
-=.. 200 of astrocyte pretreatment with PD98059. Chelation of
.....
Q

150
intracellular calcium with BAPTNAM significantly,
..
Q though not completely, decreased the UTP-mediated

...
I II
01 100
.S
50
~
0 linked to the activation of Ca2+-dependent and Ca2 ' -
0 indepedent PKC isoforms (43). These data fit well
Fig. 8. mRNA levels ofP2Y} and P2Y2 receptors in cultured
astrocytes submitted to combined oxygen/glucose deprivation
(OGD) and evaluated by Northern blot analysis as reported
in Materials and Methods. A representative experiment is
reported in the upper panel. In the lower panel changes in
mRNA levels for P2Y} and P2Y 2 receptors were normalized
to those of GAPDH mRNA and expressed as percentage of
mRNA levels in cultures maintained in nonnoxic conditions.
Data represent mean ± SE.M. of 4 independent experiments
(p< 0.05; Students t test). In the inset the effect of com-
bined oxygen/glucose deprivation (OGD) on the release of
[3HjUTPfrom cultured astrocytes is shown. The value meas-
ured in the 30 min before hypoxia/hypog/icemia represents
the basal one (UTP= 108±J6 [3Hjpmol/30min/mg proteins;
n=3) and is reported as 100%. Data are expressed as per-
centage increase from basal values and shown as ±SEM,
*p<O.05(Student s t test).
mRNA up-regulation.
Consistent with our previous findings showing that
ERKIIERK2 phopsphorylation is an important event
involved in some Guo-mediated effects in astrocytes
including the synthesis of trophic factors and the
protection against starusporine-induced apoptosis (26,
65), our results suggest that ERKI-2/MAPK pathway
represents a key mediator that links the activation of
Guo metabotropic purinoceptors to the transcriptional
activation of P2Y2 mRNA. The combination of the
MEK inhibitor PD98059 with the PKA inhibitor
induction of P2Y2 mRNA. It has been reported that
P2Y2 receptors in astrocytes and Hela cells can be
with our findings showing that GF I02903X, a non-
specific PKC isoform inhibitor, abrogated UTP effect.
Moreover, the addition of the PKC activator PMA to
the medium ofquiescent astrocytes markedly increased
the levels ofP2Y2 mRNA thus stressing the hypothesis
ofPKC involvement in the mechanisms underlying the
regulation ofP2Y 2 expression in these cells. These data
suggest that in rat cortical astrocytes the UTP-mediated
transcriptional upregulation of P2Y 2 seems to involve
a MAPK pathway downstream, the activation of Ca2 ' -
dependent and Ca2+-independent PKC isoforms.
Our previous study showed that rat brain cortical
astrocytes are able to release both adenine and
guanine-based purines (30). We report evidence
that these cells are also able to release pyrimidines
in resting conditions and under a field electrical
stimulation, in agreement with the observations
of Lazarowski et al who found that mechanical
stimulation triggered UTP release in 1321N I
human astrocytoma cells, C6 rat glioma cells and
rat primary astrocytes (7) . In resting astrocytes,
the cellular constitutive release of UTP could
reach local nucleotide concentrations high enough
to provide a mechanism by which it may exert a
tonic stimulation of its receptors, among which the
P2Y 2 site. Dubyak and colleagues recently reported
that ATP concentrations in the bulk medium of
platelets stimulated by the addition of thrombin
underestimated by at least I order of magnitude the
 Int. J. Immunopathol. Pharmacol.
ATP concentration near the cell surface. Moreover,
under conditions of increased extracellular UTP
accumulation, this nucleotide could provide a stronger
autocrine control on astrocyte responsiveness in
terms of cell proliferation, IP3 generation and [Ca"],
rise and release of bioactive substances, not only by
activating its receptors such as P2Y2 ones, but also
by up-regulating them.
The extracellular release of ATP under
physiologic.al and pathological conditions has been
the subject of many investigations. In contrast, little
is known about the release of pyrimidines, and in
particular of UTP, from biological samples. In this
study we confirmed (30) that both adenine- and
guanine- based purines are released from cultured·
astrocytes during, and 30 min following, hypoxial
hypoglicemia, and it was also found that UTP is
released under the same experimental conditions.
P2Y2 mRNA evaluated 6 hrs after the cells were
submitted to glucose-oxygen deprivation increased
by about two-fold the control values whereas the
P2Y, mRNA was not modified.
It is known that a number of pathological events
in the brain, including ischemic injury, can cause
astrocytes to undergo reactive changes. In the
initial stages, reactive astrocytes can contribute
to neuronal regeneration by releasing different
neuroactive substances, including neurotrophic
factors, but in the chronic stages they can also be
implicated in the progression of neurodegeneration
and in neuronal cell death. It has been reported
that P2Y receptors can be involved in both the
development and regeneration of nervous tissue in
the CNS (66), whereas P2X receptors playa role in
neurodegenerative events (8). Recently a protective
role for P2Y 2 in astrocytes has been shown, its
activation being able to increase the expression of
genes implicated in nervous system development,
including ephrin A3, in cell survival, including Bcl-2
and in neuroprotection includingseveralneurotrophins,
neuropetides and growth factors (66). Moreover, the
stimulation of P2Y2 in 1321Nl cells promoted the
release of sAPPa, a soluble, non-amyloidogenic N-
terminal fragment with neuroprotective and mitogenic
effects (67). Thus, a P2Y2 up-regulationoccurringafter
Guo administration or under pathological conditions
could represent an early response to injury aimed at
initiating a protective astrocyte response in the CNS
305
. and promoting neuronal viability.
ACKNOWLEDGEMENTS
This research was supported by grants from the
Italian Ministero dell'Istruzione, dell'Universita e
della Ricerca (MIUR): PRIN 2003053992; Center of
Excellence on Ageing (CEA), University of Chieti-
Pescara; Italy.
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