Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
* Author for correspondence (e-mail: mitorres{at}vet.ucm.es)
Accepted 15 April 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cerebellum, Granule cells, NO-sensitive guanylyl cyclase, N-methyl-D-aspartate receptors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the rat, most granule cells undergo post-mitotic migration and establish
synaptic connections over the first three postnatal weeks
(Altman, 1972). Many of the
morphological and physiological characteristics of native cerebellar
development are closely replicated in vitro
(Alaimo-Beuret and Matus, 1985
;
Cumming et al., 1984
).
Cerebellar granule cells express both Ca2+ permeable
N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors
(Garthwaite et al., 1986
;
Pearce et al., 1987
;
Meier and Jorgensen, 1986
),
and NMDA receptor stimulation is essential for their developmental survival
and differentiation, playing a primary role in neuroplasticity
(Kleinschmidt et al., 1987
;
Balazs et al., 1988
;
Contestabile, 2000
). In the
central nervous system, NO formation is typically coupled to the activation of
NMDA receptors (Garthwaite and Boulton,
1995
), to which the neuronal nitric oxide synthase (nNOS) is
anchored through interaction with the postsynaptic density-95 protein
(Brenman et al., 1996
;
Craven and Bredt, 1998
;
Sheng and Pak, 2000
). Thus, NO
has been proposed as a messenger for development and synaptic plasticity, as
well as for cell death downstream to NMDA receptor activation
(Bredt and Snyder, 1994
;
Holscher, 1997
;
Dawson et al., 1993
;
Contestabile, 2000
). The
activity of these receptors dynamically regulates nNOS expression in the
cerebellum and cerebellar granule cells
(Virgili et al., 1998
;
Baader and Schilling, 1996
).
Many recent lines of evidence lend support to interaction of the
2ß1 isoform of NOGCR with PSD-95
and related proteins through PDZ domains, suggesting a synaptic localization
of this enzyme (Russwurm et al.,
2001
; Burette et al.,
2002
). In fact, this enzyme was found to be asymmetrically
localized to the developing apical dendrite of pyramidal neurons
(Polleux et al., 2000
) and
within growth cones of B5 Helisoma neurons
(Wagenen and Rehder, 2001
) and
pharmacological blockade of cGMP pathway resulted in a disruption of
hippocampal mossy fiber development
(Mizuhashi et al., 2001
).
We hypothesized the possibility of a development-dependent differential
expression of NOGCR subunits in cerebellar granule cells, with NMDA
receptor stimulation or blockade leading to long-lasting functional changes in
guanylyl cyclase activity. Here we report that during granule cell
differentiation there is a change in NOGCR subunit
expression, and that NMDA stimulation is positively correlated with guanylyl
cyclase expression and activity.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular cyclic GMP measurements
Before the assay, cells were washed twice in Locke's solution, pH 7.4
(composition in mM: NaCl 140, KCl 4.4, CaCl2 2.5, MgSO4
1.2, KH2PO4 1.2, NaHCO3 4, glucose 5.6, EDTA
0.01, glycine 0.003 and HEPES 10) and kept in this medium for 60 minutes at
37°C. The cells were then preincubated for 30 minutes at 37°C in
Locke's solution containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma,
St Louis, MO) and subjected to the appropriate stimulus: DEA/NO (Molecular
Probes Europe, Leiden, The Netherlands) or NMDA. When the effect of
L-NG nitroarginine methyl ester (L-NAME; Cayman Chemical, Ann
Arbor, MI), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson,
Ballwin, MO), D-()-2-amino-5-phosphonovaleric acid (D-AP5; Tocris
Cookson) or dizocilpine maleate (MK-801; Sigma, RBI) was tested, these
compounds were added to the incubated medium 60, 30 or 10 minutes before the
stimulation as indicated in the legends to figures. Incubation was stopped by
aspirating the medium and by adding 300 µl of 6% trichloroacetic acid. The
cells were then scraped out of the well and centrifuged. The supernatants were
neutralised with 3 M KOH plus 1.5 M triethanolamine (TEA), and the cyclic GMP
content of the crude extracts was determined using a commercial
[3H]cyclic GMP radioimmunoassay kit (Amersham Bioscience Europe
GmbH, Cerdanyola, Barcelona, Spain), as described previously
(Rodríguez-Pascual et al.,
1996).
mRNA quantification
NOGCR subunit (1, ß1 and
2) mRNA levels were determined by the real time PCR (RT-PCR)
technique.
Total RNA was extracted from cerebella of rats of different postnatal days
(P7, P14 and P21) or from cells. After subjecting cells cultured for 7 or 14
days to the appropriate treatment, total RNA was extracted using the RNeasy
kit (Quiagen, GmbH, Hilden, Germany) as previously described
(Ferrero and Torres, 2002).
RNA was quantified using the RiboGreenTM RNA Quantification Kit
(Molecular Probes Europe, Leiden, The Netherlands) as previously described
(Ferrero and Torres,
2002
).
The RT-PCR reactions were performed in two steps. First, strand cDNA was synthesized by MultiScribeTM reverse transcriptase (Applied Biosystems, Madrid, Spain) in RT buffer containing 5.5 mM MgCl2, 500 µM per dNTP, 2.5 µM random hexamers, 0.4 U/ml RNAse inhibitor and 3.125 U/ml MultiScribeTM reverse transcriptase. Reactions were performed in a final volume of 50 µl containing 1 µg RNA with an incubation step of 10 minutes at 25°C to maximise primer-RNA template binding. The reverse transcription reaction was performed at 48°C for 30 minutes and reverse transcriptase was inactivated before the PCR reactions by heating the samples at 95°C for 5 minutes.
To perform the PCR reactions, specific primers and probes for the
NOGCR subunits (1,
2 and
ß1) were designed using published sequences
(Nakane et al., 1988
;
Nakane et al., 1990
;
Koglin and Behrends, 2000
)
with the help of the primer express software package (Applied Biosystems,
Madrid, Spain). These primers and probes were:
1 subunit
(forward, base position 2281 5'-TGC AGT GTC CCT CGG AAA-3';
reverse, base position 2463 5'-CCA TGG TTT AGA ATT AGG TCC TTG
A-3'; TaqMan probe, base position 2390 5'-(FAM)-AAG TTT GGT GGA
AGC TCC TCC CTC GA-(TAMRA)-3').
2 subunit (forward,
base position 828 5'-TCT GCA GAC CAT TCC AAC AAA G-3'; reverse,
base position 941 5'-TCC TCA CCA AAC CTC TCT TGA ATT-3'; TaqMan
probe, base position 907 5'-(FAM)-TCA GTC CGA GTA CAT TTG CAG TAG ACC
GAA-(TAMRA)-3'). ß1 subunit (forward, base position 1734
5'-TCC GAA TAT ACA TAC AGG TGT CTC AT-3'; reverse, base position
1857 5'-GGA TAG AAA CCA GAC TTG CAT TGG-3'; TaqMan probe, base
position 1827 5'-(FAM)-TCT TGC CCT TCA TGG ACA CAG GAC
CT-(TAMRA)-3'). 18S rRNA was used as an endogenous control. To amplify a
200 bp fragment, we used a commercial mixture of primers and TaqMan probe
labelled with VIC and TAMRA at the 5' and 3' ends, respectively
(Applied Biosystems, Madrid, Spain). PCR reactions were followed in an ABI
Prism 7700 Sequence Detection System (Applied Biosystems) with TaqMan Gold PCR
reagents. The reaction mixture contained TaqMan PCR buffer, 5.5 mM
MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM
dUTP, 0.025 U/µl AmpliTaq Gold DNA polymerase, 0.01 U/µl AmpErase UNG,
300 nM of each primer and 300 nM of TaqMan probe. This technique exploits the
5'-nuclease activity of AmpliTaq Gold DNA polymerase, to cleave the
TaqMan probe during the PCR reaction when the probe has hybridised to the
target. The TaqMan probe contains a reporter dye at the 5' end and a
quencher dye at the 3' end. When the two dyes are bound to the
oligonucleotide there is no emission of fluorescence. During the reaction,
cleavage of the probe separates the reporter dye and the quencher dye from the
oligonucleotide, leading to increased fluorescence of the reporter dye. The
accumulation of PCR products is directly detected by monitoring this rise in
fluorescence. The enhanced fluorescence signal is detected only when the
target sequence is complementary to the probe and is amplified during PCR,
making this technique very specific. Reactions were performed with an initial
incubation at 50°C for two minutes followed by 10 minutes at 95°C for
AmpliTaq Gold activation and 40 cycles (melting 95°C for 15 seconds,
annealing and extension 60°C for 1 minute). Fluorescence was determined at
each step of every cycle. The threshold cycle or cT value occurs
when an exponential growth PCR product is detected. Quantifications were
always normalised using endogenous control 18S rRNA to check for variability
in the initial concentration, the quality of total RNA and the conversion
efficiency of the reverse transcription reaction.
Western blotting
Cells undergoing appropriate treatment were processed as previously
described (Ferrero et al.,
2000). Cytosolic soluble fractions (
20 µg of protein) were
subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis
and electrophoretically transferred to polyvinylidene difluoride (PVDF)
membranes. After blocking non-specific binding sites with 3% bovine serum
albumin (BSA) (Boehringer Mannheim, Mannheim, Germany) in Tris-saline buffered
(TBS) containing 0.1% Tween-20 at room temperature for 1 hour, the membranes
were incubated with NOGCR, polyclonal antiserum (Cayman Chemical,
Ann Arbor, MI) (1:1000) or NOGCR ß1 subunit
polyclonal antiserum (Cayman Chemical, Ann Arbor, MI) (1:750) in blocking
buffer overnight at 4°C with constant agitation (control), or in the
presence of
1 peptide (0.1 µg/ml), ß1
peptide (0.2 µg/ml) or
1 plus ß1 peptide.
Once washed (3x10 minutes), the blots were incubated with
anti-rabbit-IgG:HRP (Amersham Biosciences Europe GmbH, Cerdanyola, Barcelona,
Spain) (1:5000) for 1 hour at 37°C. Blots were then washed (3x10
minutes) and developed with the super signal substrate (Pierce, Rockford, IL).
Chemiluminescence was directly detected using a Bio-Rad Fluor S instrument and
analysed using Bio-Rad quantity one software (Bio-Rad, Hercules, CA).
Cell viability
Cells grown in multiwell plates were subjected to the relevant or control
treatment for 24 or 48 hours. They were then washed twice in Locke's solution
and incubated for 30 minutes at 37°C in the medium containing the
viability/cytotoxicity probes (calcein-AM 1 µM and ethidium homodimer
(EthD-1) 8 µM) (Molecular Probes Europe, Leiden, The Netherlands) as
previously described (Ferrero and Torres,
2001). Live cells are identified by the presence of the ubiquitous
intracellular esterase, detected by the enzymatic conversion of the virtually
non-fluorescent, cell-permeable calcein-AM to the intensely fluorescent
calcein (Ferrero and Torres,
2001
). The polyanionic dye calcein is well retained within live
cells and produces an intense, uniform green fluorescence (ex/em 495 nm/515
nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold
fluorescence enhancement upon binding to nucleic acids, generating bright red
fluorescence in dead cells (ex/em 495 nm/635 nm). EthD-1 is excluded by the
intact plasma membrane of live cells. Background fluorescence levels are
inherently low with this assay technique, because the dyes are virtually
non-fluorescent before interacting with cells. Cell images were taken using a
Hammamatsu 4880-80 Slow Cool Scan CCD camera coupled to a Nikon Eclipse TE 200
microscope, plus the B-2A FITC filter for calcein or G-1B TRITC filter for
EthD-1.
Statistical analysis
The results are expressed as means±s.e.m. of three or more
experiments. The data were analyzed by one-way ANOVA followed by Bonferroni's
test (confidential interval 95%). The differences between mean were considered
statistically significant when P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cultures of primary dissociated cerebellar cells from postnatal day 7 (P7) rat pups, maintained as described in Material and Methods, essentially contain granule neurons. These cells are characterized by a small soma (<10 µm in diameter), scant cytoplasm and two to six, rather short, unbranched processes. After several days of culture, the cells become integrated within a dense network and form numerous synapses, as shown in Fig. 1.
|
The expression of the different NOGCR subunits was monitored in
these cell cultures by RT-PCR. As shown in
Fig. 2A, these cells express
mRNAs coding for the 1,
2 and
ß1 NOGCR subunits. The protein products
(
1 and ß1) were also detected using specific
antibodies against NOGCR. As shown in
Fig. 2B, these antibodies were
able to identify 2 bands: one that migrates farther than the 75 kDa molecular
weight marker, which corresponds to the ß1 subunit, and
another band of molecular weight equal to or slightly higher than 80 kDa.
Immunodetection of these two bands was suppressed when the ß1
or
1 peptides used as antigens were present during the
incubation with the primary antibody.
|
NOGCR functionality in these cells was checked by stimulating with NO. Cells cultured for 7 days (7 DIV) were washed and pre-incubated with 0.5 mM IBMX for 30 minutes and then stimulated with the nitric oxide donor DEA/NO. Several tests were performed to establish the DEA/NO concentration and incubation time giving rise to maximum cGMP increases in the cell cultures. Fig. 3A shows the increase in cGMP produced by increasing the DEA/NO concentration. Fig. 3B shows the time course of cGMP production induced by treatment with 1 µM DEA/NO. According to these results, 1 µM DEA/NO and 10 minutes of stimulation were selected as the best experimental conditions for the remaining tests.
|
Besides synthesising cGMP in response to exogenous NO, the granule cells were also able to produce cGMP when NMDA receptors were stimulated, as previously reported. These cGMP increases were abolished by pre-treatment with the nNOS inhibitor L-NAME (1 mM), pre-treatment with the NOGCR inhibitor ODQ (10 µM), or by removing extracellular calcium, indicating that NMDA stimulates Ca2+ influx, activating neuronal NOS, which produces NO and in turn activates NOGCR. The effect of NMDA was counteracted by the presence of two different NMDA receptor antagonists, AP5 and MK801 (Fig. 4).
|
Differential expression of NOGCR subunits during in vitro
cell development or cerebellum development
The expression of 1,
2 and
ß1 NOGCR subunits was analysed in freshly isolated
cells and during cell culture using the quantitative RT-PCR technique. This
procedure allows relative quantification of different PCR products. As shown
in Fig. 5A, the amount of
1 mRNA was very high in freshly isolated cells, compared to
cultured cells. The
1 mRNA level diminished drastically in 7
DIV cells and then moderately increased in cells cultured for 14 DIV (all the
values were normalised to 7 DIV). Conversely,
2 and
ß1 mRNA levels in freshly isolated cells were lower than those
detected in 7 DIV cells, and increased in 14 DIV cells. While the increase in
ß1 mRNA level was moderate, the amount of
2
mRNA in 7 DIV cells was 2.3-fold higher than in freshly isolated cells and
this value underwent a further 2.94-fold increase in 14 DIV cells. These
spectacular changes in
subunit expression are summarised in
Fig. 5B, which shows the
relative contributions of each
subunit (
1 or
2) to the total mRNA coding for the
subunits. In
freshly isolated cells,
1 mRNA represented 81.6% of the
total mRNA, whereas in 7 DIV cells and 14 DIV cells this proportion dropped to
16%. In contrast,
2 mRNA varied from 18.4% in freshly
isolated cells to 84% in cultured cells.
|
Since granule cells constitute the major cellular type in cerebellum, the
expression of 1,
2 and ß1
NOGCR subunits was analysed in the cerebellum at different ages
(P7, P14 and P21). As it is shown in Fig.
6 at P7 the most abundant mRNA was that encoding for
1, and the level of this mRNA decreased with cerebellum age.
However, both
2 and ß1 mRNA increased in
parallel with cerebellum development. The ratio
2
mRNA/
1 mRNA varied from 0.58 at P7 to 13 at P21.
|
NOGCR subunit expression is regulated via NMDA
receptors
Since it has been previously reported that NMDA receptor activation or
blockade could affect rat cerebellar granule cell differentiation and regulate
nNOS expression, we decided to try to establish whether the changes elicited
by NMDA in these cells might alter NOGCR expression. Cells kept in
culture for 7 or 14 days were treated for the indicated times with NMDA, and
NOGCR subunit expression analysed. As shown in
Fig. 7A and
Fig. 8A, treatment of 7 DIV
cells with NMDA for 24 or 48 hours caused upregulation of both mRNA and
1 protein (3- and 2-fold, respectively). However, the same
treatment failed to affect
1 mRNA or protein levels in 14
DIV cells. Incubating cells with NMDA, led also to an increase in
2 and ß1 subunit mRNAs after both 7 and 14
days of culture, the increase being much higher in 7 DIV cells
(Fig. 7B,C). This upregulation
caused by NMDA was prevented by the presence of two different NMDA receptor
antagonists, AP5 and MK801. The raised ß1 NOGCR
subunit mRNA level was accompanied by an increase in the corresponding protein
(Fig. 8B).
|
|
NMDA receptor activation upregulates cGMP production in response to
exogenous NO
NMDA receptor stimulation or blockade is closely linked to cell
developmental survival and death
(Contestabile, 2000). After
the different treatments, we evaluated cell viability using a two-colour
fluorescence cell viability assay based on the simultaneous distinction
between live and dead cells. Cells (7 DIV or 14 DIV) subjected to NMDA
treatment for less than 48 hours showed no signs of toxicity (results not
shown). When treatment was prolonged to 48 hours or longer cells showed
evident signs of toxicity, reflected by an enhanced proportion of cells
labelled with EthD-1 in the presence of NMDA.
Fig. 9 shows images of 7 DIV
and 14 DIV cells incubated with calcein-AM, which labels live cells, and
EthD-1, which enters cells with damaged membranes and produces bright red
fluorescence in damaged cells. As shown in these images, in 7 DIV cells the
number of EthD-1 labelled cells is very low and is twice the quantity in cells
treated with NMDA. However, the rate of EthD-1 labelled cells increased in 14
DIV cells (control) and NMDA treatment lead to a significant increase, as
detailed in Fig. 9B.
|
The response to NO stimulation (1 µM DEA/NO, 10 minutes) was evaluated in cells subjected to different treatments, to establish whether the changes observed in the expression levels of the different NOGCR subunits were accompanied by modified enzyme activity. As shown in Fig. 10, NMDA pretreatment led to enhanced cGMP responses to exogenous NO, while in cells pretreated with AP5, the amount of DEA/NO-elicited cGMP was lower than that achieved in untreated cells. Protein levels determined to correct for possible changes in cell numbers, only differed after 72 hours of treatment, cGMP levels being corrected to take into account cell loss. The cGMP content of non-NO stimulated cells treated with NMDA or AP5 for different periods of time was comparable to that observed in untreated cells (control), indicating that although acute NMDA stimulation leads to cGMP synthesis at these times the cyclic nucleotide levels have dropped to basal by phosphodiesterase activity.
|
We then went on to explore the effect of 48 hours of treatment with the indicated concentrations of NMDA on the NO-elicited cGMP increases in cells cultured for 7 or 14 days. As shown in Fig. 11, NMDA enhanced the NO response in a dose-dependent manner in both 7 DIV and 14 DIV cells. Nevertheless, the kinetics of this effect was slightly different; in 7 DIV cells, the maximum effect was achieved at 250 µM NMDA and the effect produced by lower NMDA concentrations was smaller than that produced in 14 DIV cells, which showed a maximum response to NMDA levels close to 100 µM. AP5 treatment caused a reduction in the NO-elicited cGMP increases, but this effect did not depend on AP5 concentration; all concentrations generally used to block NMDA receptors reducing the NO response to a similar extent (40%). When higher concentrations were employed (0.5 or 1 mM), the reduction observed was higher than 50%. These results are summarized in Table 1.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Herein we show that cultured cerebellar granule cells express several
NOGCR subunits and that, when stimulated with NMDA, these cells
synthesize cGMP in response to exogenously added or endogenously synthesised
NO. Thus, as well as expressing NOGCR subunits, it seems these
subunits form an active enzyme. We were able to detect 1,
ß1 and
2 subunit mRNA, along with the
presence of
1 and ß1 subunits at the protein
level. Since there are no antibodies specific for the
2
subunit of this enzyme, we could not explore
2 subunit
expression at the protein level. Thus, assuming that functionally active
NOGCR requires
and ß subunits, and given that the only
isoforms known to occur naturally as heterodimers in mammalian tissue are
1ß1 and
2ß1, the coexistence of these two
heterodimers in these cells is plausible. Although
1 and
2 differ in their primary structure and cellular
localisation,
1ß1 and
2ß1 heterodimers show similar behaviour and
NO activation (Russwurn et al., 1998). Nevertheless, the fact that
1 and
2 show very different patterns of
expression, may suggest that each subunit has a specific role at each
developmental stage, and that their expression is regulated by development.
Although there is scarce data on the expression patterns of the different
NOGCR subunits during nervous system development, the presence of
1 mRNA in prenatal rat brain has been demonstrated
(Smigrodzki and Levitt, 1996
).
This subunit also occurs in the granule cell layer of the adult rat cerebellum
(Burgunder and Cheung, 1994
).
Weak ß1 expression has been described in the embryo, which
increases after birth to be widely expressed in the adult
(Giuili et al., 1994
). The
level of
2 mRNA expression in adult animals appears to be
less than in 8-day-old animals, labelling being concentrated in the granule
cell layer (Gibb and Garthwaite,
2001
).
Assuming that 1 and
2 subunits can form
heterodimers with the ß1 subunit with similar affinity, the
most abundant guanylyl cyclase heterodimer in non-differentiated granule cells
is likely to be
1ß1. During development
these cells show a change in
subunit expression to the
2ß1 isoform, which is the most abundant in
mature cells. It has been recently demonstrated that the
2
subunit can interact with PSD-95 protein through PDZ domains, clustering
guanylyl cyclase to nNOS and NMDA receptors and leading to the formation of a
signalling microdomain (Russwurm et al.,
2001
). If the
2 subunit of the NOGCR
heterodimer is responsible for synaptic localisation of the enzyme, an
increase in
2 mRNA and protein concurrent with cellular
development and differentiation when neural ramifications and synaptic
connections appear would be conceivable. The presence of
2ß1 NOGCR in a signalling
microdomain comprised of the NMDA receptor, nNOS and NOGCR would
favour the cell response to low concentrations of NO generated at synaptic
terminals. Indeed, it has been recently shown that very low concentrations of
NO are needed to fully activate NOGCR
(Bellamy et al., 2002
).
Glutamate is widely recognized as a good candidate for a role in regulating
the development of synaptic connections. In particular, its action on the NMDA
receptor plays a critical role in anatomical and physiological
activity-dependent pattern formation and plasticity during development
(Contestabile, 2000). We
observed that NMDA receptor stimulation led to the upregulation of
NOGCR subunit mRNA, and that the effect of NMDA was more potent in
7 DIV than in 14 DIV cells. Moreover, in 7 DIV cells, NMDA enhanced the levels
of the three mRNAs, while it only increased the levels of
2
and ß1 subunit mRNAs in 14 DIV cells. NMDA also increased
1 and ß1 subunit levels and NO-stimulated
cGMP synthesis by granule neurons. The effect of NMDA was clearly different in
7 DIV and 14 DIV cells: while the mRNA increases were more pronounced in 7 DIV
cells, the increases in NO-stimulated cGMP were higher in 14 DIV cells, and
the maximum effect was achieved at lower NMDA concentrations. Conversely, NMDA
treatment caused small cytotoxic effects in 7 DIV cells but enhanced the
relative number of cells labelled with EthD-1 in 14 DIV cells. This difference
might be explained by changes in the expression of the different subunits of
the NMDA receptor in cells cultured for different times, which could account
for differences in the affinity for NMDA
(Janssens and Lesage, 2001
).
These changes may also be linked to a switch in signalling pathways triggered
by NMDA receptor activation, as has been previously proposed for the dual
effects of NMDA receptor activation on polysialylated neural cell adhesion
molecule expression during postnatal brainstem development
(Bouzioukh et al., 2001
). This
switch related to development might also explain why neonatal blockade of NMDA
receptors decreases nNOS expression in the cerebellum
(Virgili et al., 1998
), and
similar treatment of 14 DIV granule cells resulted in the drastic upregulation
of nNOS expression (Baader and Schilling,
1996
).
The present study does not address the molecular mechanism involved in the
NMDA-induced upregulation of NOGCR subunit expression, although
this is one of our future objectives. An upregulation of 1
protein levels by NMDA via the NO signalling pathway was recently demonstrated
in spinal cord, implying cGMP production
(Tao and Johns, 2002
).
Further, regulation of gene expression by NO/cGMP through activation of PKG
has been demonstrated in neuronal and glial cells
(Gudi et al., 1999
). Some
evidence has been recently shown that NO signalling may be functionally
coupled to CREB activation in nerve cells
(Ciani et al., 2002
). In mouse
cerebellar granule cells NMDA caused a downregulation of nNOS expression by a
mechanism that involves Ca2+ entry
(Baader and Schilling, 1996
).
However, a mechanism triggered by Ca2+ entry and a mechanism
involving NO and cGMP synthesis is not incompatible since in neural cells NO
synthesis is coupled to Ca2+ entrance through NMDA receptors
(Garthwaite and Boulton,
1995
). Conversely, we previously demonstrated that the prolonged
exposure to exogenous NO of bovine chromaffin cells causes a downregulation of
1 and ß1 mRNAs involving cGMP and PKG
activation (Ferrero and Torres,
2002
). We also found that PKA positively modulated the levels of
these mRNAs in these cells, and NMDA-stimulated cAMP synthesis has been
demonstrated in primary neuronal cultures of rat cerebral cortex and
hippocampus (Suvarna and O'Donnell,
2002
). Furthermore, cAMP response element-binding protein (CREB)
phosphorylation has been observed in granule neurons stimulated with glutamate
(Baader and Schilling, 1996
).
Thus, the possible involvement of a signalling pathway involving cGMP/PKG or
cAMP/PKA and CREB phosphorylation will be evaluated, although other pathways
cannot be precluded since NMDA receptor stimulation upregulates homer 1a mRNA
via the mitogen-activated protein kinase cascade in cultured mouse cerebellar
granule cells (Sato et al.,
2001
).
In conclusion, our findings provide evidence for development-dependent
NOGCR subunit expression in cerebellar granule cells and its
regulation via NMDA receptor stimulation. The dynamics of NOGCR
subunit expression and the fact that the NOGCR 2
subunit directs the enzyme to the synaptic membranes, all point to a finely
tuned role as mediator of NO effects in synaptic transmission in the mature
granule cell.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alaimo-Beuret, D. and Matus, A. (1985). Changes in the cytoplasmic distribution of microtubule-associated protein 2 during the differentiation of culture cerebellar granule cells. Neuroscience 14,1103 -1115.[CrossRef][Medline]
Altman, J. (1972). Postnatal development of the cerebellar cortex in the rat I. The external germinal layer and the transitional molecular layer. J. Com. Neurol. 145,353 -398.[Medline]
Baader, S. L. and Schilling, K. (1996). Glutamate receptors mediate dynamic regulation of nitric oxide synthase expression in cerebellar granule cells. J. Neurosci. 16,1440 -1449.[Abstract]
Balazs, R., Jorgensen, O. S. and Hack, N. (1988). N-Methyl-D-aspartate promotes the survival of cerebellar granule cells in culture. Neuroscience 27,437 -451.[CrossRef][Medline]
Baptista, C. A., Hatten, M. E., Blazeski, R. and Mason, C. A. (1994). Cell-cell interactions influence survival and differentiation of purified Purkinje cells in vitro. Neuron 12,243 -260.[Medline]
Bellamy, T. C. and Garthwaite, J. (2001).
"AMP-Specific" phosphodiesterase contributes to cGMP degradation
in cerebellar cells exposed to nitric oxide. Mol.
Pharmacol. 59,54
-61.
Bellamy, T. C., Griffiths, C. and Garthwaite, J.
(2002). Differential sensitivity of guanylyl cyclase and
mitochondrial respiration to nitric oxide measured using clamped
concentrations. J. Biol. Chem.
277,31801
-31807.
Bouzioukh, F., Tell, F., Rougon, G. and Jean, A. (2001). Dual effects of NMDA receptor activation on polysialylated neural cell adhesion molecule expression during brainstem postnatal development. Eur. J. Neurosci. 14,1194 -1202.[CrossRef][Medline]
Bredt, D. S. and Snyder, S. H. (1994). Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium. Neuron 13,301 -313.[Medline]
Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., Froehner, S. C. and Bredt, D. S. (1996). Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 84,757 -767.[Medline]
Brewer, G. J. (1995). Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42,674 -683.[Medline]
Burette, A., Zabel, U., Weinberg, R. J., Schmidt, H. H. H. W.
and Valtschanoff, J. G. (2002). Synaptic localization of
nitric oxide synthase and soluble guanylyl cyclase in the hippocampus.
J. Neurosci. 22,8961
-8970.
Burgunder, J. M. and Cheung, P. T. (1994). Expression of soluble guanylyl gene in adult rat brain. Eur. J. Neurosci. 6,211 -217.[Medline]
Ciani, E., Guidi, S., Bartesaghi, R. and Contestabile, A. (2002). Nitric oxide regulates cGMP-dependent CREB phosphorylation and Bcl-2 expression in cerebellar neurons: implication for a survival role of nitric oxide. J. Neurochem. 82,1282 -1289.[CrossRef][Medline]
Contestabile, A. (2000). Roles of NMDA receptor activity and nitric oxide production in brain development. Brain. Res. Rev. 32,476 -509.[CrossRef][Medline]
Craven, S. E. and Bredt, D. S. (1998). PDZ proteins organize synaptic signaling pathways. Cell 93,495 -498.[Medline]
Cumming, R., Burgoyne, R. D. and Lytton, N. A.
(1984). Immunofluorescence distribution of -tubulin,
ß-tubulin and microtubule associated protein 2 during in vitro maturation
of cerebellar granule cell neurons. Neuroscience
12,775
-782.[CrossRef][Medline]
Cyr, M., Ghribi, O., Thibault, C., Morissette, M., Landry, M. and di Paolo, T. (2001). Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors. Brain Res. Rev. 37,153 -161.[Medline]
Dawson, V. L., Dawson, T. M., Bartley, D. A., Uhl, G. R. and Snyder, S. H. (1993). Mechanism of nitric oxide-mediated neurotoxicity in primary brain cultures. J. Neurosci. 13,2651 -2661.[Abstract]
Ferrero, R. and Torres, M. (2001). Prolonged exposure to YC-1 induces apoptosis in adrenomedullary endothelial and chromaffin cells through a cGMP-independent mechanism. Neuropharmacology 41,895 -906.[CrossRef][Medline]
Ferrero, R. and Torres, M. (2002). Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels. B. M. C. Biochemistry 3,26 .
Ferrero, R., Rodríguez-Pascual, F., Miras-Portugal, M. T. and Torres, M. (2000). Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation. J. Neurochem. 75,2029 -2039.[CrossRef][Medline]
Garthwaite, J. and Boulton, C. L. (1995). Nitric oxide signalling in the central nervous system. Annu. Rev. Physiol. 57,683 -706.[CrossRef][Medline]
Garthwaite, J., Garthwaite, G. and Hajos, F.
(1986). -Aminobutyric acid affects the developmental
expression of neuron-associated proteins in cerebellar granule cell culture.
J. Neurochem. 46,1256
-1262.[Medline]
Gibb, B. J. and Garthwaite, J. (2001). Subunits of the nitric oxide receptor, soluble guanylyl cyclase, expressed in rat brain. Eur. J. Neurosci. 13,539 -544.[CrossRef][Medline]
Giuili, G., Luzi, A., Poyard, M. and Guellaën, G. (1994). Expression of mouse brain soluble guanylyl cyclase and NO synthase during ontogeny. Dev. Brain. Res. 81,269 -283.[Medline]
Gudi, T., Hong, G. K-P., Vaandrager, A. B., Lhomann, S. M. and
Pilz, R. B. (1999). Nitric oxide and cGMP regulate gene
expression in neuronal and glial cells by activating type II cGMP-dependent
protein kinase. FASEB J.
13,2143
-2152.
Holscher, C. (1997). Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity. Trends.Neurosci. 20,298 -303.
Janssens, N. and Lesage, A. S. J. (2001). Glutamate receptor subunit expression in primary neuronal and secondary glial cultures. J. Neurochem. 77,1457 -1474.[CrossRef][Medline]
Kleinschmidt, A., Bear, M. F. and Singer, W. (1987). Blockade of NMDA receptors disrupts experience-dependent plasticity of kitten striate cortex. Science 238,355 -358.[Medline]
Koglin, M. and Behrends, S. (2000). Cloning and
functional expression of the rat 2 subunit of soluble
guanylyl cyclase. Biochim. Biophys. Acta.
1494,286
-289.[Medline]
Lev-Ram, V., Jiang, T., Wood, J., Lawrence, D. S. and Tsien, R. Y. (1997). Synergies and coincidence requirements between NO, cGMP and Ca2+ in the induction of cerebellar long-term depression. Neuron 18,1025 -1038.[Medline]
Meier, E. and Jorgensen, O. S. (1986). Gamma-aminobutyric acid affects the developmental expression of neuron-associated proteins in cerebellar granule cell culture. J. Neurochem. 45,1256 -1262.
Mizuhashi, S., Nishiyama, N., Matsuki, N. and Ikegaya, Y.
(2001). Cyclic nucleotide-mediated regulation of hippocampal
mossy fiber development: a target-specific guidance. J.
Neurosci. 21,6181
-6194.
Nakane, M., Saheki, S., Kuno, T., Ishii, K. and Murad, F. (1988). Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung. Biochem. Biophys. Res. Commun. 157,1139 -1147.[Medline]
Nakane, M., Arai, K., Saheki, S., Kuno, T., Buechler, W. and
Murad, F. (1990). Molecular cloning and expression of cDNAs
coding for soluble guanylate cyclase from rat lung. J. Biol.
Chem. 265,16841
-16845.
Novelli, A., Nicoletti, F., Wroblewski, J. T., Alho, H., Costa, E. and Guidotti, A. (1987). Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells. J. Neurosci. 7,40 -47.[Abstract]
Pearce, I. A., Cambray-Deakin, M. A. and Burgoyne, R. D. (1987). Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells. FEBS Lett. 233,143 -147.[CrossRef]
Polleux, F., Morrow, T. and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404,567 -573.[CrossRef][Medline]
Pons, S., Trejo, J. L., Martínez-Morales, J. R. and
Martí, E. (2001). Vitronectin regulates sonic hedgehog
activity during cerebellum development through CREB phosphorylation.
Development 128,1481
-1492.
Rodríguez-Pascual, F., Miras-Portugal, M. T. and Torres, M. (1996). Effect of cGMP-increasing agents, nitric oxide and C-type natriuretic peptide on bovine chromaffin cell function: inhibitory role mediated by cyclic GMP-dependent protein kinase. Mol. Pharmacol. 49,1058 -1070.[Abstract]
Russwurm, M., Behrends, S., Harteneck, C. and Koesling, D. (1998). Functional properties of a naturally occurring isoform of soluble guanylyl cyclase. Biochem. J. 335,125 -130.[Medline]
Russwurm, M., Wittaus, N. and Koesling, D.
(2001). Guanylyl cyclase/PSD-95 interaction targeting of the
nitric oxide-sensitive 2ß1 guanylyl cyclase
to synaptic membranes. J. Biol. Chem.
276,44647
-44652.
Sato, M., Suzuki, K. and Nakanishi, S. (2001).
NMDA receptor stimulation and brain-derived neurotrophic factor upregulate
homer 1a mRNA via the mitogen-activated protein kinase cascade in cultured
cerebellar granule cells. J. Neurosci.
21,3797
-3805.
Sheng, M. and Pak, D. T. (2000). Ligand-gated ion channel interactions with cytoskeletal and signalling proteins. Annu. Rev. Physiol. 62,755 -778.[CrossRef][Medline]
Smigrodzki, R. and Levitt, P. (1996). The alpha 1 subunit of soluble guanylyl cyclase is expressed prenatally in the rat brain. Brain. Res. Dev. Brain. Res. 97,226 -234.[Medline]
Southam, E., Morrios, R. and Garthwaite, J. (1992). Sources and targets of nitric oxide in rat cerebellum. Neurosci. Lett. 137,241 -244.[CrossRef][Medline]
Suvarna, N. U. and O'Donnell, J. M. (2002).
Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4
and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral
cortex and hippocampus. J. Pharmacol. Exp. Ther.
302,249
-256.
Tao, Y. X. and Jhons, R. A. (2002). Activation and up-regulation of spinal cord nitric oxide receptor, soluble guanylate cycles, after formalin injection into the rat hind paw. Neurocience 112,439 -446.[CrossRef][Medline]
Vincent, S. R. (1996). Nitric oxide and the synaptic plasticity: NO news from the cerebellum. Behav. Brain. Sci. 19,362 -367.
Virgili, M., Facchinetti, F., Sparapani, M., Tregnago, M., Lucchi, R., Dall'Olio, R., Gandolfi, O. and Contestabile, A. (1998). Neuronal nitric oxide synthase is permanently decreased in the cerebellum of rats subjected to chronic neonatal blockade of N-methyl-D-aspartate receptors. Neuroscience Lett. 258, 1-4.[CrossRef][Medline]
Wagenen, S. V. and Rehder, V. (2001). Regulation of neuronal growth cone filopodia by nitric oxide depends on soluble guanylyl cyclase. J. Neurobiol. 46,206 -219.[CrossRef][Medline]
Wong, M. and Moss, R. L. (1994). Patch-clamp analysis of direct steroidal modulation of glutamate receptor-channels. J. Neuroendocrinol. 6,347 -355.[Medline]
Wong, J. K., Kennedy, P. R. and Belcher, S. M. (2001). Simplified serum- and steroidal-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons. J. Neurosci. Methods 110, 45-55.[CrossRef][Medline]
Wood, P. L. (1991). Pharmacology of the second messenger, cyclic guanosine 3',5'-monophosphate, in the cerebellum. Pharmacol. Rev. 43, 1-25.[Medline]