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Vol. 292, Issue 3, 870-876, March 2000
Instituto de Investigaciones Citologicas, Fundación Valenciana de Investigaciones Biomédicas, Valencia, Spain
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The role of phospholipase C in the molecular mechanism of glutamate neurotoxicity was assessed in primary cultures of cerebellar neurons. It is shown that 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18-OCH3), two agents that inhibit phospholipase C, prevent glutamate and N-methyl-D-aspartic acid (NMDA) neurotoxicity. It is shown that both compounds prevent glutamate neurotoxicity at concentrations lower than those required to inhibit carbachol-induced hydrolysis of inositol phospholipids. In contrast, it was a good correlation between the concentrations of U-73122 and Et-18-OCH3 required to inhibit NMDA-induced hydrolysis of phospholipids and those required to prevent glutamate and NMDA neurotoxicity. NMDA-induced hydrolysis of phospholipids is inhibited by nitroarginine, an inhibitor of nitric-oxide synthase, and is mimicked by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine. The results reported indicate that glutamate neurotoxicity would be mediated by activation of NMDA receptors, leading to activation of nitric-oxide synthase and increased formation of nitric oxide, which results in increased activity of phospholipase C. Inhibition of phospholipase C by U-73122 or Et-18-OCH3 prevents glutamate-induced neuronal death.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Glutamate
is the main excitatory neurotransmitter in mammals. However, excessive
activation of glutamate receptors is neurotoxic, leading to neuronal
degeneration and death. In many systems, including primary cultures of
cerebellar neurons, glutamate neurotoxicity is mediated by excessive
activation of NMDA receptors, leading to increased intracellular
Ca2+, which induces the neurotoxic process (Choi,
1987
, 1992; Frandsen and Schousboe, 1993). However, the molecular
mechanisms of glutamate neurotoxicity remain unclear. Glutamate
neurotoxicity is involved in the origin of several neurodegenerative
diseases (amyotrophic lateral sclerosis, Huntington's disease) and in
the neuronal damage induced by cerebral ischemia. Therefore, the
identification of the molecular mechanism of glutamate neurotoxicity
and of possible mechanisms to prevent it would have important clinical implications.
It has been shown in different systems, including primary cultures of
cerebellar neurons, that activation of metabotropic glutamate receptors
(mGluRs) prevents glutamate and NMDA neurotoxicity (Koh et al., 1991;
Siliprandi et al., 1992; Felipo et al., 1994). This protective effect
has been attributed to activation of mGluR5, one of the subtypes of
mGluRs, although a possible contribution of mGluR1 to the protective
effect has not been ruled out (Montoliu et al., 1997). Activation of
mGluR5 or mGluR1 leads to activation of G proteins that in turn
activate phosphoinositide-specific phospholipase C, resulting in
increased hydrolysis of inositol phospholipids.
The aim of this work was to assess whether the protective effect of agonists of mGluRs is mediated by activation of phospholipase C. We tested whether inhibitors of phospholipase C are able to prevent the protective effect of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), an agonist of mGluRs. Unexpectedly, we found that inhibitors of phospholipase C prevent glutamate neurotoxicity. The protective effect of these compounds has been characterized.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials. Basal Eagle's medium, fetal bovine serum, and gentamycin were from Life Technologies (Barcelona, Spain). DNase I (deoxyribonuclease I, E.C 3.1.21.1) and Dispase II were from Boehringer Mannheim (Tarrasa, Spain). 1-[6-[[(17b)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) and tACPD were from Research Biochemicals International (Natick, MA); NMDA and carbachol were from Sigma Chemical Company (St. Louis, MO); 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (ET-18-OCH3) was from Calbiochem (AMS Biotecnología, Madrid, Spain).
Primary Cultures of Cerebellar Neurons.
Preparation of
primary cultures of cerebellar neurons was carried out as described in
detail by Miñana et al. (1998) using cerebella from 7- to
8-day-old Wistar rats. The cells were resuspended in basal Eagle's
medium containing 10% heat-inactivated fetal bovine serum (Life
Technologies), 2 mM glutamine, 100 µg/ml gentamycin, and 25 mM KCl.
Cells were counted, and cell viability was measured by using trypan
blue staining. Then cells were plated onto poly(lysine)-coated plates
(312,000 cells/cm2; 2 ml for plates of 35 mm
diameter), and after 15 min at 37°C, medium containing unattached
cells was removed and fresh medium was added. The cells were grown at
37°C in 5% CO2 atmosphere. To prevent
proliferation of non-neuronal cells, 10 µM cytosine arabinoside was
added 24 h after plating. Glucose (5.6 µmol/ml of culture
medium) was added twice per week.
Assay for Glutamate or NMDA Neurotoxicity and for Its
Prevention.
Glutamate or NMDA toxicity in cerebellar
neurons was assayed after 11 to 19 days of culture. Briefly, culture
medium was removed and kept at 37°C (conditioned medium). Cells were
washed and incubated at 37°C for 15 min with Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM
CaCl2, 5.6 mM glucose, 5 mM HEPES, pH 7.4)
containing 10 µM glycine. Then, this solution was removed and cells
were incubated in Locke's solution without glycine for 3.5 h at
37°C in the presence of 1 mM glutamate or NMDA. Preincubation with
glycine is used to obtain more reproducible results when comparing different culture preparations. Incubation with glutamate can
be done in the presence or absence of glycine with similar results. To
test the effect of the different agents, these compounds were added 15 min before addition of glutamate or NMDA. Cells were washed with
Locke's solution without glycine, and the conditioned medium
previously removed was added again. Cell viability was measured 24 h later by staining with fluorescein diacetate and propidium iodide as
described previously (Felipo et al., 1993). The percentage of surviving
neurons was calculated by assessing the ratio of fluorescein
diacetate/propidium iodide (green/red) staining directly under the
microscope. At least 800 cells were counted for each point.
Determination of Hydrolysis of Phospholipids in Cultured
Neurons.
Neurons were seeded on 35-mm 
Petri dishes.
Experiments were carried out 8 to 14 days after seeding.
Myo-[3H]inositol (2 µCi/plate, 0.1 µM
final, from Amersham, Bucks, UK) was added to the culture medium
and incubated for 48 h at 37°C in 5% CO2
atmosphere. The culture medium was removed, neurons were washed twice
with Locke's solution (see above), and 1 ml of the same solution
containing 10 mM LiCl was added. After incubation for 15 min at 37°C,
the compounds to be tested were added. Preincubations with inhibitors
before addition of carbachol or NMDA were for 15 min. After addition of
these compounds, the incubation was continued for 30 or 60 min. The
neurons were scraped off and transferred to a tube containing 1.5 ml of
chloroform/methanol (1:2, v/v), and labeled inositol phosphates were
measured as described by Fisher et al. (1984).
Statistical Analysis. Results were analyzed by one-way ANOVA and post hoc Newman-Keul test using the Prism program (Graph- Pad Software, San Diego, CA).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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We first tested whether U-73122, an inhibitor of phospholipase C
(Bleasdale et al., 1989; Smith et al., 1990), prevents the protective
effect of tACPD, an agonist of mGluR5, against glutamate neurotoxicity.
As shown in Fig. 1, tACPD prevented
completely glutamate neurotoxicity. U-73122 did not prevent the
protective effect of tACPD, but afforded per se, a significant
protection against glutamate neurotoxicity.
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The protective effects of different concentrations of U-73122 or
Et-18-OCH3, another agent that also inhibits
phospholipase C (Powis et al., 1992; Hu, 1998) against glutamate or
NMDA neurotoxicity, are shown in Fig. 2.
U-73122 alone did not affect neuronal viability at concentrations lower
than 2 µM and induced some neuronal death at higher concentrations.
At concentrations between 0.5 and 2 µM, U-73122 afforded a
significant protection against glutamate and NMDA neurotoxicity. The
protection was nearly complete for 1 µM U-73122.
Et-18-OCH3 alone did not affect neuronal
viability at concentrations lower than 20 µM.
Et-18-OCH3 at 5 to 10 µM afforded complete
protection against glutamate and NMDA neurotoxicity.
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To assess whether there was a correlation between the protection against glutamate neurotoxicity and the inhibition of phospholipase C, we determined simultaneously, in another set of experiments using sister plates from the same neuronal cultures, the effects of different concentrations of U-73122 or Et-18-OCH3 on glutamate neurotoxicity and on the hydrolysis of phospholipids. To increase the sensitivity of the assay, the hydrolysis of phospholipids in neurons was stimulated by addition of carbachol, which activates muscarinic receptors associated with activation of phospholipase C.
As shown in Fig. 3 under the conditions
used, 1 µM U-73122 inhibits carbachol-induced activation of
phospholipase C only very slightly; higher concentrations inhibit it in
a dose-dependent manner (IC50 = 3.6 µM).
The inhibition was complete at 7 µM, a dose that is toxic for the
neurons. It can be seen that at 0.5 to 1 µM U-73122 did not inhibit
significantly carbachol-induced hydrolysis of phospholipids but almost
completely prevented glutamate- and NMDA-induced neuronal death.
Similar results were obtained for Et-18-OCH3; the
concentrations that prevent glutamate or NMDA neurotoxicity did not
inhibit carbachol-induced hydrolysis of phospholipids. These results
show that two different inhibitors of phospholipase C prevent
glutamate- and NMDA-induced neuronal death at concentrations lower than
that required to inhibit carbachol-induced hydrolysis of
phospholipids.
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In cultured cerebellar neurons,
glutamate can activate different types of receptors associated
with activation of phospholipase C. Glutamate neurotoxicity in
these cells is mediated mainly by excessive activation of NMDA
receptors. Activation of NMDA receptors by glutamate or NMDA leads to
hydrolysis of phospholipids (Nicoletti et al., 1986; Hokin et al.,
1996; Fragoso and Lopez-Colome, 1999). We then tested whether
U-73122 inhibits activation of phospholipase C induced by NMDA and
whether there is a correlation between the doses required to inhibit
phospholipase C and to prevent glutamate neurotoxicity. As shown in
Fig. 3, U-73122 and Et-18-OCH3 inhibited NMDA-induced hydrolysis of phospholipids at concentrations lower than
those required to inhibit carbachol-induced phospholipase C activation.
Moreover, it was a good correlation between the doses required to
inhibit NMDA-induced hydrolysis of phospholipids and to prevent
glutamate- and NMDA-induced neuronal death. NMDA-induced hydrolysis of
phospholipids was inhibited by 60% at 1 µM U-73122, which prevented
nearly completely glutamate- and NMDA-induced neuronal death.
NMDA-induced hydrolysis of phospholipids was also inhibited by more
than 50% by 10 µM Et-18-OCH3, which completely prevented glutamate
and NMDA neurotoxicity.
These results confirm that activation of NMDA receptors activates
signal transduction pathways leading to activation of some form of
phospholipase C. Another possible mechanism to explain the NMDA-induced
hydrolysis of phospholipids could be that activation of NMDA receptors
induces the release of glutamate that subsequently activates
metabotropic glutamate receptors coupled to activation of phospholipase
C. We found that NMDA-induced hydrolysis of phospholipids is completely
prevented by
(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine hydrogen maleate
(MK-801), an antagonist of NMDA receptors, but was not reduced at all
by (±)-
-methyl-4-carboxyphenylglycine (MCPG), an antagonist of
metabotropic glutamate receptors (Fig. 4). The hydrolysis of phospholipids was
increased by NMDA to 187 ± 14% of control. Preincubation with
MK-801 completely prevented it (106 ± 5% of control), whereas
MCPG did not affect NMDA-induced hydrolysis of phospholipids at all
(181 ± 20% of control). These results indicate that NMDA-induced
hydrolysis of phospholipids is not mediated by NMDA-induced glutamate
release and subsequent activation of metabotropic glutamate receptors
coupled to phospholipase C, but to direct activation by NMDA of
intracellular signal transduction pathways leading to activation of
phospholipase C.
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The above results also suggest that carbachol and NMDA induce
hydrolysis of phospholipids by activating different forms of phosphoinositide-specific phospholipase C. It has been shown that activation of muscarinic receptors by carbachol leads to activation of
G proteins that activate phospholipase C-
(Berstein et al., 1992).
In cortical cultured neurons, glutamate induces antigenic changes
to phospholipase C-
, which are mediated by activation of NMDA
receptors and are prevented by nitroarginine, an inhibitor of
nitric-oxide synthase (NOS) (Shimohama et al., 1995). The authors suggest that nitric oxide (NO) formation, which is secondary to NMDA receptor activation, leads to alteration of phospholipase C-
(Shimohama et al., 1995).
We then assessed whether NMDA-induced hydrolysis of phospholipids in
primary cultures of cerebellar neurons is mediated by increased
formation of NO. We tested whether it is prevented by nitroarginine and
whether the NO-generating agent
S-nitroso-N-acetyl-penicillamine (SNAP) is also
able to induce phospholipid hydrolysis. As shown in Fig.
5, nitroarginine reduced significantly
NMDA-induced hydrolysis of phospholipids, indicating that it is
mediated by activation of NOS and formation of NO. NMDA increased the
hydrolysis of phospholipids by 72 ± 32%. However, in the
presence of nitroarginine, NMDA did not significantly increase the
hydrolysis of phospholipids (22 ± 15%). Moreover, as shown in
Fig. 5, SNAP also induced the hydrolysis of phospholipids, which
increased by 55 ± 20%.
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NO modulates the activity of different enzymes. One of the enzymes activated by NO in neurons is guanylate cyclase. To assess whether NMDA-induced hydrolysis of phospholipids is mediated by increased cyclic GMP, we tested whether 1H-[1,2,4]oxadiaxolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, prevents NMDA-induced hydrolysis of phospholipids. As shown in Fig. 5, ODQ did not affect basal nor NMDA-induced hydrolysis of phospholipids.
It is also possible that SNAP-induced hydrolysis of phospholipids
could be mediated by release of glutamate and subsequent activation of
metabotropic glutamate receptors coupled to activation of phospholipase
C. To assess this possibility, we tested whether SNAP-induced
hydrolysis of phospholipids is prevented by antagonists of different
types of glutamate receptors. As shown in Fig.
6, antagonists of NMDA,
(S)--amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) or metabotropic glutamate receptors [MK-801;
6,7-dinitroquinoxaline-2,3-dione (DNQX); and MCPG,
respectively] did not prevent SNAP-induced hydrolysis of
phospholipids.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The results reported show that inhibitors of phospholipase C do not prevent the protective effect of agonists of metabotropic glutamate receptors against glutamate neurotoxicity, indicating that the protective effect is not mediated by activation of phospholipase C but to activation of other pathways associated with these receptors (e.g., modulation of adenylate cyclase, phospholipase D, or calcium channels).
Unexpectedly, the experiments carried out showed that inhibitors of phospholipase C prevent glutamate- and NMDA-induced neuronal death in primary cultures of cerebellar neurons. This indicates that activation of NMDA receptors leads to activation of some form of phospholipase C. This effect may be due to direct activation of signal transduction pathways associated with NMDA receptors, leading to activation of phospholipase C. Another possible mechanism to explain the NMDA-induced hydrolysis of phospholipids may be that activation of NMDA receptors induces the release of glutamate that subsequently activates metabotropic glutamate receptors coupled to activation of phospholipase C. The results shown in Fig. 4 indicate that NMDA-induced activation of phospholipase C is not mediated by release of glutamate but is directly coupled to activation of NMDA receptors.
The results shown in Figs. 2 and 3 show that both U-73122 and Et-18-OCH3 prevented glutamate neurotoxicity at concentrations lower than those required to inhibit carbachol-induced hydrolysis of phospholipids but high enough to inhibit NMDA-induced hydrolysis of phospholipids.
These results indicate that carbachol and NMDA induce hydrolysis of
phospholipids by activating different forms of phospholipase C that
have different sensitivity to the inhibitors tested. It has
been shown that carbachol induces hydrolysis of inositol phospholipids by activating muscarinic receptors associated with activation of G proteins that activate phospholipase C- (Berstein et al., 1992).
NMDA also induces hydrolysis of phospholipids in cerebellum, but the
mechanism is different and is mediated by increased formation of NO.
This was previously reported by Smith and Li (1993) and is confirmed by
the results shown in Figs. 5 and 6. Shimohama et al. (1995) have shown
that glutamate induces antigenic changes of phospholipase C- that
are prevented by blocking NMDA receptors with MK-801 or by preventing
NOS activation with nitroarginine. These results indicate that
activation of NMDA receptors leads to a NO-mediated alteration in
phospholipase C- and support the possibility that NMDA-induced
hydrolysis of phospholipids could be mediated by activation of
phospholipase C-. As shown in Fig. 5, NMDA-induced hydrolysis of
phospholipids is also prevented by inhibiting NOS with nitroarginine.
Moreover, in agreement with previous reports (Smith and Li,
1993), the NO-generating agent SNAP also induces a hydrolysis
of phospholipids (Fig. 5). The results reported in this study, together
with those of Shimohama et al. (1995), indicate that changes in
NMDA-induced hydrolysis of phospholipids and in antigenicity of
phospholipase C- are parallel, suggesting that activation of NMDA
receptors would lead to NO-mediated activation of phospholipase C-.
The results shown in this article indicate that the protective effect
of phospholipase C inhibitors is not due to inhibition of phospholipase
C-, indicating that this form of phospholipase C does not mediate
glutamate neurotoxicity. The participation of other forms of
phospholipase cannot be ruled out on the basis of the results reported;
however, these results, together with other findings reported in the
literature, support the idea that the protective effect of inhibitors
of phospholipase C would be due to prevention of NMDA-induced
activation of phospholipase C-. Shimohama et al. (1995) have shown
that it was a good correlation between the antigenic changes in
phospholipase C- and the neurotoxic effects of glutamate, supporting
the possibility that activation of phospholipase C- would be
involved in the process of glutamate neurotoxicity.
The mechanism by which activation of NMDA receptors leads to activation
of phospholipase C- involves activation of NOS and increased
formation of NO, because it is prevented by nitroarginine and mimicked
by a NO-generating agent (Fig. 5). It was shown previously that
nitroarginine prevents glutamate neurotoxicity in primary cultures of
cerebellar neurons (Marcaida et al., 1995), indicating that interfering
with the pathway by which NMDA leads to activation of
phospholipase C- at the level of NOS also prevents glutamate neurotoxicity.
The subsequent events by which NO leads to activation of phospholipase
C- remain unclear. The results shown in Fig. 6 indicate that the
effect is not mediated by NO-induced release of glutamate and
subsequent activation of metabotropic glutamate receptors. It has been
shown that phospholipase C- is modulated by different mechanisms
(Pawelczyk, 1999). For example, it is modulated by the GTP-binding
protein rho-A (Hodson et al., 1998), by changes in
Ca2+ ion concentrations (Allen et al., 1997), and
by sphingosine (Matecki and Pawelczyk, 1997). It has also been
reported that phospholipase C- contains a calmodulin-like structure
that could play a role in the regulation of the enzyme (Richard et al.,
1997). NO modulates the activity of different enzymes; e.g., it
activates guanylate cyclase (Katsuki et al., 1977), inhibits aconitase
(e.g., Drapier, 1997), and also modulates the activity of GTP-binding
proteins such as Ras (Yun et al., 1998). As shown in Fig. 5,
guanylate cyclase does not mediate the effect of NO on phospholipase
C-, because it is not prevented by ODQ, an inhibitor of guanylate cyclase. One possibility is that NO could modulate the activity of some
of the different small GTP-binding proteins (e.g., Rho-A) in a way
similar to its effect on Ras. The Rho-A protein (or other small G
proteins) could then modulate the activity of phospholipase C-
(Homma and Emori, 1995; Schmidt et al., 1997; Illenberger et
al., 1998; Shibatohge et al., 1998). Another possibility is that
reactive nitrogen species derived from NO mediate NO-dependent activation of phospholipase C (Wright et al., 1996).
The results reported support the idea that glutamate and NMDA
neurotoxicity may be mediated by NO-mediated activation of
phospholipase C- and show clearly that inhibitors of phospholipase C
prevent glutamate neurotoxicity.
It is also shown in Fig. 2 that doses of U-73122 and
Et-18-OCH3 slightly higher than the doses that
afford significant neuroprotection are neurotoxic. This indicates that
the neurons can only tolerate some degree of inhibition of some form of
phospholipase C and that a strong inhibition of this phospholipase C is
not compatible with neuronal survival. It is possible that the
phospholipase C that is necessary for neuronal survival is not
phospholipase C- but phospholipase C- or another phospholipase.
If this is the case, and glutamate neurotoxicity is mediated by
activation of phospholipase C-, the development of new inhibitors
that are more specific for phospholipase C- and do not inhibit other
phospholipases would be of great clinical interest.
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Footnotes |
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Accepted for publication December 6, 1999.
Received for publication October 20, 1999.
1 This work was supported in part by grants from the Plan Nacional de Investigación y Desarrollo (SAF97-0001 and PM98-0065) of the Ministerio de Educación y Cultura of Spain and of Fundació La Marató de TV3. M.L. and P.M. are fellows of Conselleria de Educación de la Generalitat Valenciana.
Send reprint requests to: Vicente Felipo, Laboratory of Neurobiology, Instituto de Investigaciones Citologicas, FVIB, Amadeo de Saboya, 4, 46010 Valencia, Spain. E-mail: vfelipo{at}ochoa.fib.es
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Abbreviations |
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mGluR, metabotropic glutamate receptor;
AMPA, (S)--amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic
acid;
ET-18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine;
MCPG, (±)--methyl-4-carboxyphenylglycine;
MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo
[a,d]cyclohepten-5,10-imine hydrogen
maleate;
NMDA, N-methyl-D-aspartic acid;
NO, nitric oxide;
NOS, nitric-oxide synthase;
ODQ, 1H-[1,2,4]oxadiaxolo[4,3-a]quinoxalin-1-one;
SNAP, S-nitroso-N-acetyl-penicillamine;
tACPD, trans-(±)-1-amino-1,3-cyclopentanedicarboxylic
acid;
U-73122, 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione;
DNQX, 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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1.
J Biol Chem
267:
8081-8088 stimulating activities.
EMBO J
14:
286-291[Medline].This article has been cited by other articles:
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  T. Munsch, M. Freichel, V. Flockerzi, and H.-C. Pape Contribution of transient receptor potential channels to the control of GABA release from dendrites PNAS, December 23, 2003; 100(26): 16065 - 16070. [Abstract] [Full Text] [PDF]
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, treatments that afforded significant protection
(P < .001) against glutamate
neurotoxicity; a, significantly different
(P < .001) from control untreated neurons.
