Vol. 182, No. 3, 1992 February 14, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1362-1368
DIFFERENT RECEPTORS MEDIATE STIMULATION OF NITRIC OXIDEDEPENDENT CYCLIC GMP FORMATION IN NEURONS AND ASTROCYTES CULTURE
Luis Agull
IN
and Agustina Garcia’
lnstitutu de Biologia Fundamental “V. War Palas?’ and Depatfamento de Bioquimica y Biologia Molecular, Universidad Autbnoma de Barcelona, 08 193 Bella terra, Spain Received
January
7,
1992
The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive 0 1992 Academic Press, Inc. receptors in astrocytes. In 1988, glutamate
could
Garthwaite release
endothelium-derived associated
et al. (1) found that in cerebellar a diffusible
messenger
relaxing factor identified
increases in cGMP.
with properties
similar
to the
as NO, that was responsible
for the
Since then, a large number of works have appeared
leading to the recognition of NO as a neuronal messenger NO synthase (EC 1.14.23) was demonstrated NADPH- and calcium-dependent L-arginine
cells in suspension
(for reviews see 2 and 3 ).
in brain homogenates
and competitively
and shown to be
inhibited by analogs of the substrate
(4). The enzyme has been purified from rat (5) and porcine (6) cerebellum
and demonstrated
to require calmodulin.
Histochemical
studies with antibodies
raised
against the rat enzyme showed its widespread occurrence in the CNS and indicated an association with discrete neuronal populations glial cells (7). On the other hand, astrocytes nitroprusside-sensitive
guanylate
‘To whom correspondence
and vascular endothelium contain
but not with
high levels of the soluble
cyclase (8) and have been suggested as one of the
should be addressed.
Abbreviations: AMPA, cr-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; AP5, 2-amino-5phosphonovalerate; cGMP, cyclic GMP; GABA, y-aminobutyrate; IBMX, lisobutyl-3-methylxanthine; GAMS, r-D-glutamylaminomethylsulphonate; L-NMMA, No-monomethyl-L-arginine; NMDA, N-methyl-D-aspattate; NO, nitric oxide; VIP, vasoactive intestinal peptide. 0006-291x/92 $1.50 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
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targets for the NO synthesized in neurons (2). However, astrocytes in primary culture also release vasorelaxing
nitrogen oxides in response to agonists such as bradykinin,
norepinephrine
and quisqualate
norepinephrine
stimulates
(9,lO)
NO-dependent
and we have recently cGMP formation
demonstrated
in astrocyte cultures (11).
In order to shed more light into the cellular origin of the NO produced tissue and its activation of soluble guanylate cyclase, we have compared various agonists, to stimulate
that
in brain
the ability of
reported to increase cGMP in different nervous tissue preparations,
cGMP formation
through the NO pathway in primary cultures of neurons
and astrocytes from rat cerebrum. MATERIALS
AND METHODS
Materials: Dulbecco’s modified Eagle’s medium, fetal bovine serum and horse serum were obtained from Flow Laboratories. [3H]cGMP (33.3 Ci/mmol) was from New England Nuclear. Histamine, carbachol, serotonin, L-norepinephrine, dopamine, GABA, VIP, bradykinin, adenosine, L-glutamate, kainate, quisqualate, ibotenate, AMPA, AP5, GAMS, L-NMMA, IBMX, HEPES and calcium ionophores A23187 and ionomycin were from Sigma. Ro 20-1724 was a gift from Hoffmann-La Roche & Co. and cGMP antiserum was kindly provided by Dr. B. Hamprecht (Physiologisch-chemisches lnstitut der Universitat, Tubingen, F.R.G.) Methods: Astrocyte-enriched and neuronal primary cultures were prepared from Sprague-Dawley rat brain hemispheres of newborns or 18-day-old embryos, respectively, as previously described (12). Cultures of meningeal fibroblasts were prepared from meninges of newborn rats after dissociating the cells by trypsinization (12). Cells seeded in 35 mm diameter plastic Petri dishes (1.2 x 10’ cells/ dish) were used after 14-21 days (astrocytes and meningeal fibroblasts) or 8-10 days (neurons) in culture. Indirect immunofluorescence techniques revealed that >90% of the cells present in glial cultures were glial fibrillary acidic protein positive and ~5% neuron-specific enolase positive, while the majority of cells in neuronal cultures stained for neuronspecific enolase and very few for glial fibrillary acidic protein. To determine cGMP formation, culture medium was removed and the monolayers washed twice, at 5 min intervals, with 900 ~1 of incubation medium containing 118 mM NaCI, 4.7 mM KCI, 2.5 mM CaCI,, 1.2 mM MgSO,, 1.2 mM KH,PO,, 10 mM glucose and 20 mM HEPES, adjusted to pH 7.4 with NaOH. Cells were preincubated at 37°C for 5 min in 900 ~1 of the same medium in the presence of inhibitors (when used) and reactions started by adding 100 ~.rl containing 1 mM IBMX and agonists. After 1 min, reactions were terminated by aspirating the medium and adding 0.5 ml ice-cold ethanol. The ethanol extract plus an additional 0.5 ml ethanol wash were evaporated, samples resuspended in 5 mM acetate pH 4.8 and cGMP quantified by radioimmunoassay (13) using acetylated rH]cGMP. Protein was determined by the method of Lowry et al. (14). Experiments were always performed in triplicate plates and were replicated the indicated number of times in different culture preparations. Significance of differences was assesed by the paired Students t test. RESULTS
cGMP was measured several compounds
in neuron and astrocyte cultures after stimulation
in the presence of the phosphodiesterase 1363
with
inhibitor IBMX (1 mM)
Vol. 182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that we previously showed to enhance agonist responses in astrocytes (11). Basal cGMP levels were higher in neurons than in astrocytes (0.84 & 0.44 and 0.14 f 0.02 pmol/mg protein, respectively) and this difference was greatly amplified by IBMX that
increased
cGMP
accumulation
about 6-fold in neurons
but only about 2-fold in
astrocytes (5.8 f 1.2 and 0.22 f 0.02 pmol/mg of protein, respectively).
Of the different
agonists used (Table I) only glutamate (100 pM) stimulated cGMP accumulation in
neurons and this stimulation
was of the same magnitude
ionophores
A231 87 and ionomycin
compounds
elicited
significant
(100 pM) and glutamate
as that elicited by the calcium
at 10 pM. However, in astrocytes
several
responses although only those due to norepinephrine
(100 pM) were of considerable
magnitude
and comparable
to
the ones given by A23187 and ionomycin. To discard a possible contribution of contaminating fibroblasts to the norepinephrine and glutamate responses observed in glial cultures we determined the effect of these compounds in cultures of meningeal
fibroblasts where they produced stimulations
of only 132 f 4 % (n=2) and 122 & 3 %
(n=2) of basal levels, respectively. We
have
previously
shown that
the cGMP
accumulation
induced
by
norepinephrine in astrocytes is strongly inhibited by the competitive inhibitor of the NO
synthase L-NMMA
(100 pM)(l 1). As shown in Fig. 1 the same is true for the glutamate
Table I. Effect of different compounds on cGMP accumulation in neuron and astrocyte-enriched cultures cGMP (% of basal) Neurons Astrooytes
Compounds Histamine100 pM Carbachol 1 mfvl Serotonin 100 pfvl Dopamine100 pM GABA 1 mM Adenosine 100 pM VIP 1 pM Bredykinin 0.05 pM L-Norepinephrine100 pM L-Glutamate 100 pM A23187 10 pM lonomycin 10 pM
96 f 5 (2) 119f 7(2) 104f 3 (2) 102f 1 (2) 91 f 14 (2) 110f 10 (2) 121 f 10 (2) 104f 10 (2) 101 f 14 (3) 750 f 165 (7). 582 f 16 (2)’ 936 f 172 (2)’
137 f 10 (7)’ 105f 7 (3) 89 f 16 (3) 135f 2(3)*
97 f 3 (3) 139f22 (3) 163 f 15 (4)’ 121 f 10 (4)*
373 f 207 f 242 f 304 f
29 (8). 25 (8)* 37 (2). 15 (3)’
Basal and stimulatedcGMP accumulationswere measuredin the presence of 1 mM IBMX (or 100 pM Ro 20-1724 when adenosinewas used) as described in Methods. Results,expressed as percent of basal cGMP in each case, are meansf SEM of the experiments indicated in brackets. Basal values were 5.8 f 1.2 (n=7) and 0.22 f 0.02 (n=12) pmols/mgprotein in the presenceof IBMX and 1.2 f 0.3 (n=2) and 0.10 f 0.01 (n-3) pmols/mg protein in the presence.of Ro 20-1724 in neuronal and aettocyte cultures, respectively. Significantly different from basal (~0.05). l
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250
cx 1
700 600
100 0
0 GLU
NMDA
KA
QA
IBOT
GLU
AMPA
NMDA
KA
QA
IBOT AMPA
Fia. 1. Effect of glutamate receptor agonists on cGMP accumulation in neurons (A) and astrocytes (6). Inhibition by L-NMMA. Cell cultures preincubated in the absence (empty bars) or in the presence of 100 uM L-NMMA (filled bars) were assayed for 1 min in the presence of IBMX without (basal) or with NMDA, kainate (KA), quisqualate (QA), ibotenate (IBOT) and AMPA at 100 uM. Results, expressed as percent over basal, are means f SEM of 4-6 experiments (without L-NMMA) or 2-3 experiments (with L-NMMA). Basal values in the absence and presence of L-NMMA were 4.2 f 0.1 and 1.6 f 0.9 pmols/mg protein in neurons and 0.22 f 0.04 and 0.19 f 0.01 pmols/mg protein in astrocytes. Significantly different from basal (~~0.05). l
responses
and in astrocytes. On the other hand, basal cGMP content
in both neurons
in the presence of IBMX was reduced in the presence of L-NMMA
by 60 % in neurons
but was not affected in astrocytes. In order to investigate
if different receptors were involved in the response to
glutamate
in the two cell types we assayed different agonists and antagonists
excitatory
aminoacid
ibotenate
and AMPA, all at 100 uM, significantly
receptors.
As shown in Fig 1. NMDA, stimulated
kainate,
of
quisqualate,
cGMP accumulation
in
neurons, but NMDA and kainate were the most effective. In contrast, only quisqualate and ibotenate
elicited significant
effects in astrocytes.
induce a response even in the absence of magnesium glycine,
conditions
(unpublished
that
potentiate
observations).
Agonist
In these cells, NMDA did not and the presence response
of 1 pM
5-8 times
the NMDA
in neurons
responses
in both cell types were L-NMMA-
sensitive (Fig. 1). As shown in Fig. 2, the glutamate inhibited ionotropic
by the NMDA
response in neuronal cultures was completely
receptor antagonist
receptor antagonist
AP5 (1 mM) whereas the non-NMDA
GAMS (1 mM) had a small inhibitory effect. In these cells,
basal cGMP was decreased by 20 % in the presence of AP5 and was not affected by GAMS.
Neither
accumulation
antagonist
affected
the basal or the quisqualate
in astrocytes. 1365
stimulated
cGMP
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120
I
100
E zp m
80
5
60
::
40
E L0
20
.-E
R
COMMUNICATIONS
ASTROCYTES
NEURONS 5 .2
RESEARCH
0 GLU
GLU+ AI’5
GLU+ GAMS
QUIS
WE+ AP5
QUIS+ GAMS
Fia. 2. Effect of glutamate receptor antagonists. Cell cultureswere preincubatedwith 1 mM AP5 or 1 mM GAMS for 5 min before incubationfor 1 minwith 100 pM glutamate (neurons) or 100 pM quisqualate (astrocytes). Results, expressed as percent of the agonist stimulation in each case, are means f SE of triplicate determinations in a representative experiment that was replicated with similar results. Basal and agonist stimulatedcGMP levels were 4.2 f 0.2 and 20.9 f 1.6 pmols/mgprotein in neuronsand 0.23 f 0.01 and 0.53 f 0.07 pmols/mgprotein in astrocytes. Basal levels were only affected by AP5 in neurons (3.4 f 0.3 pmols/mgprotein).
DISCUSSION The results presented here show that cGMP content can be regulated in both neurons and astrocytes by neuromodulator substances through a mechanism that requires an active NO synthase since responses are strongly inhibited by the arginine analog L-NMMA. Basal as well as stimulated cGMP levels are higher in neurons than in astrocytes and even more so in the presence of the phosphodiesterase inhibitor IBMX that was used throughout this study to enhance agonist responses. Since soluble nitroprusside-sensitive guanylate cyclase appears to be abundant in astrocytes (8), these cells must contain less NO synthase than neurons and this could explain why it was not detected with the antibodies so far used in immunocytochemical studies (7). That the responses observed in astrocyte cultures are not due to the few contaminating neurons is clear since different receptors are involved in the two cell types. In neurons, the glutamate effect seems to be mediated predominantly by NMDA receptors in agreement with results reported in rat cerebellar neurons in suspension (1). Although NMDA and kainate are equally effective and quisqualate, ibotenate and AMPA have a small but significant effect, the glutamate response is inhibited completely by the NMDA-specific antagonist AP5 but very little by the non-NMDA ionotropic receptor antagonist GAMS. Even the basal cGMP content is decreased by APS and also by LNMMA what seems to indicate that it is in part a result of stimulation by glutamate released during spontaneous neuronal activity. In contrast, in astrocytes NMDA, kainate 1366
Vol. 182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
and AMPA have no effect and the glutamate quisqualate.
response is mimicked
This latter response is not inhibited
AMPA-insensitive
RESEARCH COMMUNICATIONS
by ibotenate
by AP5 or GAMS, thus quisqualate
receptors appear be involved. In addition,
neurons do not respond
to norepinephrine
while astrocytes show a stronger response than with glutamate
is predominantly
mediated
preparation).
by a,-adrenoceptors
(11 and Agull
that
and Garcia,
in
Our results agree with those of Murphy et al. (9,lO) who reported that a,-
adrenoceptors cultures.
and
and quisqualate
receptors mediate NO release in rat cortical astrocyte
These two types of receptors have been shown to increase intracellular
calcium concentrations
free
in forebrain astrocytes by inducing both calcium mobilization
and
extracellular
calcium influx (15-17) and in both cases calcium influx is required in order
to observe
stimulation
preparation)
as occurs in other CNS preparations
of NO-dependent
cGMP
Although our results clearly demonstrate
formation
(Agullo
that the cGMP NO-sensitive
responses observed in astrocytes, which are considerably be assessed at present. More detailed cellular localization observations
in
(2).
can operate both in neurons and in astrocytes, the physiological
preliminary
and Garcia,
relevance
pathway of the
lower than in neurons, cannot studies are underway and
indicate that large regional differences may exist in astrocyte
responsivness.
Acknowledaments: This work was supported in part by grants from DGICYT 0079) and Fundacion M.F. Roviralta (Barcelona, Spain).
(PM 89-
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Garthwaite J., Charles S.L. and Chess-Williams Ft. (1988) Nature 336, 385-388. Garthwaite J. (1991) Trends Neufosci. 14, 60-67. Snyder S.H. and Bredt D.S. (1991) Trends Pharmacol. Sci. 12,125-128. Knowles R.G., Palacios M., Palmer R.M.J. and Moncada S. (1989) Proc. Nat/. Acad. Sci. USA 86, 5159-5162. Bredt D.S. and Snyder S.H. (1990) Proc. Nat/. Acad. Sci. USA 87, 682-685. Mayer B., John M. and Bdhme E. (1990) FfBS Left 277,215-219. Bredt. D.S., Hwang P.M. and Snyder S.H. (1990) Nature 347, 768-770. De Vente J., Bol J.G.J.M., Berkelmans H.S., Schipper J. and Steinbusch H.M.W. (1990) Eur. J. Neurosci. 2, 845-862. Murphy S., Minor R.L.Jr., Welk G. and Harrison D.G. (1990) J. Neurochem. 55, 349-351. Murphy S., Minor R.L.Jr., Welk G. and Harrison D.G. (1991) J. Cardiovasc. Pharmacol. 17(53), S265S268. AguIlt L. and Garcia A. (1991) Eur. J. Pharmacol. 206, 343-346. Agulld L., Picatoste F. and Garcia A. (1990) J. Neurochem. 55, 1592-1598. 1367
Vol.
13. 14. 15. 16. 17.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Brooker G., Harper J.F., Terasaki W.L. and Moylan RD. (1979) In Advances in Cyc/jc Nuckotide Research (Greengard P. and Robison G.A., Ed.) Vol. X, pp. l33. Raven Press, New York. Lowry O.M., Roebrough N.J., Farr A.L. and Randall R.J. (1951) J. Biol. Chem. 193, 265-275. Glaum S.R., Holzwarth J.A. and Miller R.J. (1990) Proc. Nat/. Acad. Sci. USA87, 3454-3458. Jensen A.M. and Chiu S.Y. (1990 J. Neurosci 10, 1165-l 175. Enkvist M.O.K., Holopanien I. and h kerman K.E.O. (1989) Brain Res. 500,46-54.
1368
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1362-1368
DIFFERENT RECEPTORS MEDIATE STIMULATION OF NITRIC OXIDEDEPENDENT CYCLIC GMP FORMATION IN NEURONS AND ASTROCYTES CULTURE
Luis Agull
IN
and Agustina Garcia’
lnstitutu de Biologia Fundamental “V. War Palas?’ and Depatfamento de Bioquimica y Biologia Molecular, Universidad Autbnoma de Barcelona, 08 193 Bella terra, Spain Received
January
7,
1992
The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive 0 1992 Academic Press, Inc. receptors in astrocytes. In 1988, glutamate
could
Garthwaite release
endothelium-derived associated
et al. (1) found that in cerebellar a diffusible
messenger
relaxing factor identified
increases in cGMP.
with properties
similar
to the
as NO, that was responsible
for the
Since then, a large number of works have appeared
leading to the recognition of NO as a neuronal messenger NO synthase (EC 1.14.23) was demonstrated NADPH- and calcium-dependent L-arginine
cells in suspension
(for reviews see 2 and 3 ).
in brain homogenates
and competitively
and shown to be
inhibited by analogs of the substrate
(4). The enzyme has been purified from rat (5) and porcine (6) cerebellum
and demonstrated
to require calmodulin.
Histochemical
studies with antibodies
raised
against the rat enzyme showed its widespread occurrence in the CNS and indicated an association with discrete neuronal populations glial cells (7). On the other hand, astrocytes nitroprusside-sensitive
guanylate
‘To whom correspondence
and vascular endothelium contain
but not with
high levels of the soluble
cyclase (8) and have been suggested as one of the
should be addressed.
Abbreviations: AMPA, cr-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; AP5, 2-amino-5phosphonovalerate; cGMP, cyclic GMP; GABA, y-aminobutyrate; IBMX, lisobutyl-3-methylxanthine; GAMS, r-D-glutamylaminomethylsulphonate; L-NMMA, No-monomethyl-L-arginine; NMDA, N-methyl-D-aspattate; NO, nitric oxide; VIP, vasoactive intestinal peptide. 0006-291x/92 $1.50 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
1362
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
targets for the NO synthesized in neurons (2). However, astrocytes in primary culture also release vasorelaxing
nitrogen oxides in response to agonists such as bradykinin,
norepinephrine
and quisqualate
norepinephrine
stimulates
(9,lO)
NO-dependent
and we have recently cGMP formation
demonstrated
in astrocyte cultures (11).
In order to shed more light into the cellular origin of the NO produced tissue and its activation of soluble guanylate cyclase, we have compared various agonists, to stimulate
that
in brain
the ability of
reported to increase cGMP in different nervous tissue preparations,
cGMP formation
through the NO pathway in primary cultures of neurons
and astrocytes from rat cerebrum. MATERIALS
AND METHODS
Materials: Dulbecco’s modified Eagle’s medium, fetal bovine serum and horse serum were obtained from Flow Laboratories. [3H]cGMP (33.3 Ci/mmol) was from New England Nuclear. Histamine, carbachol, serotonin, L-norepinephrine, dopamine, GABA, VIP, bradykinin, adenosine, L-glutamate, kainate, quisqualate, ibotenate, AMPA, AP5, GAMS, L-NMMA, IBMX, HEPES and calcium ionophores A23187 and ionomycin were from Sigma. Ro 20-1724 was a gift from Hoffmann-La Roche & Co. and cGMP antiserum was kindly provided by Dr. B. Hamprecht (Physiologisch-chemisches lnstitut der Universitat, Tubingen, F.R.G.) Methods: Astrocyte-enriched and neuronal primary cultures were prepared from Sprague-Dawley rat brain hemispheres of newborns or 18-day-old embryos, respectively, as previously described (12). Cultures of meningeal fibroblasts were prepared from meninges of newborn rats after dissociating the cells by trypsinization (12). Cells seeded in 35 mm diameter plastic Petri dishes (1.2 x 10’ cells/ dish) were used after 14-21 days (astrocytes and meningeal fibroblasts) or 8-10 days (neurons) in culture. Indirect immunofluorescence techniques revealed that >90% of the cells present in glial cultures were glial fibrillary acidic protein positive and ~5% neuron-specific enolase positive, while the majority of cells in neuronal cultures stained for neuronspecific enolase and very few for glial fibrillary acidic protein. To determine cGMP formation, culture medium was removed and the monolayers washed twice, at 5 min intervals, with 900 ~1 of incubation medium containing 118 mM NaCI, 4.7 mM KCI, 2.5 mM CaCI,, 1.2 mM MgSO,, 1.2 mM KH,PO,, 10 mM glucose and 20 mM HEPES, adjusted to pH 7.4 with NaOH. Cells were preincubated at 37°C for 5 min in 900 ~1 of the same medium in the presence of inhibitors (when used) and reactions started by adding 100 ~.rl containing 1 mM IBMX and agonists. After 1 min, reactions were terminated by aspirating the medium and adding 0.5 ml ice-cold ethanol. The ethanol extract plus an additional 0.5 ml ethanol wash were evaporated, samples resuspended in 5 mM acetate pH 4.8 and cGMP quantified by radioimmunoassay (13) using acetylated rH]cGMP. Protein was determined by the method of Lowry et al. (14). Experiments were always performed in triplicate plates and were replicated the indicated number of times in different culture preparations. Significance of differences was assesed by the paired Students t test. RESULTS
cGMP was measured several compounds
in neuron and astrocyte cultures after stimulation
in the presence of the phosphodiesterase 1363
with
inhibitor IBMX (1 mM)
Vol. 182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that we previously showed to enhance agonist responses in astrocytes (11). Basal cGMP levels were higher in neurons than in astrocytes (0.84 & 0.44 and 0.14 f 0.02 pmol/mg protein, respectively) and this difference was greatly amplified by IBMX that
increased
cGMP
accumulation
about 6-fold in neurons
but only about 2-fold in
astrocytes (5.8 f 1.2 and 0.22 f 0.02 pmol/mg of protein, respectively).
Of the different
agonists used (Table I) only glutamate (100 pM) stimulated cGMP accumulation in
neurons and this stimulation
was of the same magnitude
ionophores
A231 87 and ionomycin
compounds
elicited
significant
(100 pM) and glutamate
as that elicited by the calcium
at 10 pM. However, in astrocytes
several
responses although only those due to norepinephrine
(100 pM) were of considerable
magnitude
and comparable
to
the ones given by A23187 and ionomycin. To discard a possible contribution of contaminating fibroblasts to the norepinephrine and glutamate responses observed in glial cultures we determined the effect of these compounds in cultures of meningeal
fibroblasts where they produced stimulations
of only 132 f 4 % (n=2) and 122 & 3 %
(n=2) of basal levels, respectively. We
have
previously
shown that
the cGMP
accumulation
induced
by
norepinephrine in astrocytes is strongly inhibited by the competitive inhibitor of the NO
synthase L-NMMA
(100 pM)(l 1). As shown in Fig. 1 the same is true for the glutamate
Table I. Effect of different compounds on cGMP accumulation in neuron and astrocyte-enriched cultures cGMP (% of basal) Neurons Astrooytes
Compounds Histamine100 pM Carbachol 1 mfvl Serotonin 100 pfvl Dopamine100 pM GABA 1 mM Adenosine 100 pM VIP 1 pM Bredykinin 0.05 pM L-Norepinephrine100 pM L-Glutamate 100 pM A23187 10 pM lonomycin 10 pM
96 f 5 (2) 119f 7(2) 104f 3 (2) 102f 1 (2) 91 f 14 (2) 110f 10 (2) 121 f 10 (2) 104f 10 (2) 101 f 14 (3) 750 f 165 (7). 582 f 16 (2)’ 936 f 172 (2)’
137 f 10 (7)’ 105f 7 (3) 89 f 16 (3) 135f 2(3)*
97 f 3 (3) 139f22 (3) 163 f 15 (4)’ 121 f 10 (4)*
373 f 207 f 242 f 304 f
29 (8). 25 (8)* 37 (2). 15 (3)’
Basal and stimulatedcGMP accumulationswere measuredin the presence of 1 mM IBMX (or 100 pM Ro 20-1724 when adenosinewas used) as described in Methods. Results,expressed as percent of basal cGMP in each case, are meansf SEM of the experiments indicated in brackets. Basal values were 5.8 f 1.2 (n=7) and 0.22 f 0.02 (n=12) pmols/mgprotein in the presenceof IBMX and 1.2 f 0.3 (n=2) and 0.10 f 0.01 (n-3) pmols/mg protein in the presence.of Ro 20-1724 in neuronal and aettocyte cultures, respectively. Significantly different from basal (~0.05). l
1364
Vol.
182,
No.
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AND
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RESEARCH
COMMUNICATIONS
250
cx 1
700 600
100 0
0 GLU
NMDA
KA
QA
IBOT
GLU
AMPA
NMDA
KA
QA
IBOT AMPA
Fia. 1. Effect of glutamate receptor agonists on cGMP accumulation in neurons (A) and astrocytes (6). Inhibition by L-NMMA. Cell cultures preincubated in the absence (empty bars) or in the presence of 100 uM L-NMMA (filled bars) were assayed for 1 min in the presence of IBMX without (basal) or with NMDA, kainate (KA), quisqualate (QA), ibotenate (IBOT) and AMPA at 100 uM. Results, expressed as percent over basal, are means f SEM of 4-6 experiments (without L-NMMA) or 2-3 experiments (with L-NMMA). Basal values in the absence and presence of L-NMMA were 4.2 f 0.1 and 1.6 f 0.9 pmols/mg protein in neurons and 0.22 f 0.04 and 0.19 f 0.01 pmols/mg protein in astrocytes. Significantly different from basal (~~0.05). l
responses
and in astrocytes. On the other hand, basal cGMP content
in both neurons
in the presence of IBMX was reduced in the presence of L-NMMA
by 60 % in neurons
but was not affected in astrocytes. In order to investigate
if different receptors were involved in the response to
glutamate
in the two cell types we assayed different agonists and antagonists
excitatory
aminoacid
ibotenate
and AMPA, all at 100 uM, significantly
receptors.
As shown in Fig 1. NMDA, stimulated
kainate,
of
quisqualate,
cGMP accumulation
in
neurons, but NMDA and kainate were the most effective. In contrast, only quisqualate and ibotenate
elicited significant
effects in astrocytes.
induce a response even in the absence of magnesium glycine,
conditions
(unpublished
that
potentiate
observations).
Agonist
In these cells, NMDA did not and the presence response
of 1 pM
5-8 times
the NMDA
in neurons
responses
in both cell types were L-NMMA-
sensitive (Fig. 1). As shown in Fig. 2, the glutamate inhibited ionotropic
by the NMDA
response in neuronal cultures was completely
receptor antagonist
receptor antagonist
AP5 (1 mM) whereas the non-NMDA
GAMS (1 mM) had a small inhibitory effect. In these cells,
basal cGMP was decreased by 20 % in the presence of AP5 and was not affected by GAMS.
Neither
accumulation
antagonist
affected
the basal or the quisqualate
in astrocytes. 1365
stimulated
cGMP
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120
I
100
E zp m
80
5
60
::
40
E L0
20
.-E
R
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ASTROCYTES
NEURONS 5 .2
RESEARCH
0 GLU
GLU+ AI’5
GLU+ GAMS
QUIS
WE+ AP5
QUIS+ GAMS
Fia. 2. Effect of glutamate receptor antagonists. Cell cultureswere preincubatedwith 1 mM AP5 or 1 mM GAMS for 5 min before incubationfor 1 minwith 100 pM glutamate (neurons) or 100 pM quisqualate (astrocytes). Results, expressed as percent of the agonist stimulation in each case, are means f SE of triplicate determinations in a representative experiment that was replicated with similar results. Basal and agonist stimulatedcGMP levels were 4.2 f 0.2 and 20.9 f 1.6 pmols/mgprotein in neuronsand 0.23 f 0.01 and 0.53 f 0.07 pmols/mgprotein in astrocytes. Basal levels were only affected by AP5 in neurons (3.4 f 0.3 pmols/mgprotein).
DISCUSSION The results presented here show that cGMP content can be regulated in both neurons and astrocytes by neuromodulator substances through a mechanism that requires an active NO synthase since responses are strongly inhibited by the arginine analog L-NMMA. Basal as well as stimulated cGMP levels are higher in neurons than in astrocytes and even more so in the presence of the phosphodiesterase inhibitor IBMX that was used throughout this study to enhance agonist responses. Since soluble nitroprusside-sensitive guanylate cyclase appears to be abundant in astrocytes (8), these cells must contain less NO synthase than neurons and this could explain why it was not detected with the antibodies so far used in immunocytochemical studies (7). That the responses observed in astrocyte cultures are not due to the few contaminating neurons is clear since different receptors are involved in the two cell types. In neurons, the glutamate effect seems to be mediated predominantly by NMDA receptors in agreement with results reported in rat cerebellar neurons in suspension (1). Although NMDA and kainate are equally effective and quisqualate, ibotenate and AMPA have a small but significant effect, the glutamate response is inhibited completely by the NMDA-specific antagonist AP5 but very little by the non-NMDA ionotropic receptor antagonist GAMS. Even the basal cGMP content is decreased by APS and also by LNMMA what seems to indicate that it is in part a result of stimulation by glutamate released during spontaneous neuronal activity. In contrast, in astrocytes NMDA, kainate 1366
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AND BIOPHYSICAL
and AMPA have no effect and the glutamate quisqualate.
response is mimicked
This latter response is not inhibited
AMPA-insensitive
RESEARCH COMMUNICATIONS
by ibotenate
by AP5 or GAMS, thus quisqualate
receptors appear be involved. In addition,
neurons do not respond
to norepinephrine
while astrocytes show a stronger response than with glutamate
is predominantly
mediated
preparation).
by a,-adrenoceptors
(11 and Agull
that
and Garcia,
in
Our results agree with those of Murphy et al. (9,lO) who reported that a,-
adrenoceptors cultures.
and
and quisqualate
receptors mediate NO release in rat cortical astrocyte
These two types of receptors have been shown to increase intracellular
calcium concentrations
free
in forebrain astrocytes by inducing both calcium mobilization
and
extracellular
calcium influx (15-17) and in both cases calcium influx is required in order
to observe
stimulation
preparation)
as occurs in other CNS preparations
of NO-dependent
cGMP
Although our results clearly demonstrate
formation
(Agullo
that the cGMP NO-sensitive
responses observed in astrocytes, which are considerably be assessed at present. More detailed cellular localization observations
in
(2).
can operate both in neurons and in astrocytes, the physiological
preliminary
and Garcia,
relevance
pathway of the
lower than in neurons, cannot studies are underway and
indicate that large regional differences may exist in astrocyte
responsivness.
Acknowledaments: This work was supported in part by grants from DGICYT 0079) and Fundacion M.F. Roviralta (Barcelona, Spain).
(PM 89-
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