1Istituto di Ricovero e Cura a Carattere Scientifico Fondazione S. Lucia, 00179 Rome; and 2Clinica Neurologica, Università di Tor Vergata, 00133 Rome, Italy
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ABSTRACT |
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Tozzi, Alessandro,
Ezia Guatteo,
Luigi Caputi,
Giorgio Bernardi, and
Nicola B. Mercuri.
Group I mGluRs Coupled to G Proteins Are Regulated by Tyrosine
Kinase in Dopamine Neurons of the Rat Midbrain.
J. Neurophysiol. 85: 2490-2497, 2001.
Metabotropic glutamate
receptors (mGluRs) modulate neuronal function via different
transduction mechanisms that are either dependent or independent on
G-protein function. Here we investigated, using whole cell patch-clamp
recordings in combination with fluorimetric measurements of
intracellular calcium concentration
([Ca2+]i), the metabolic
pathways involved in the responses induced by group I mGluRs in
dopamine neurons of the rat midbrain. The inward current and the
[Ca2+]i increase caused
by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 µM) were permanently activated and subsequently abolished in cells loaded with the nonhydrolizable GTP-analogue GTP--S (600 µM). In addition, when GDP-
-S (600 µM) was dialyzed into the
cells to produce the blockade of the G proteins, the DHPG-dependent responses were reduced. When the tissue was bathed with the
phospholipase C inhibitor
1-[6[[(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]exyl]-1H-pyrrole-2,5-dione (10 µM), the DHPG-induced calcium transients slightly diminished but
the associated inward currents were not affected. Interestingly, a
substantial depression of the DHPG-induced inward current and transient
increase of [Ca2+]i was
caused by the protein tyrosine kinase inhibitors tyrphostin B52 (40 µM) and 4',5,7-trihydroxyisoflavone (genistein; 40 µM), whereas
genistein's inactive analogue 4',5,7-trihydroxyisoflavone-7-glucoside (40 µM) was ineffective. The blockade of the Src family of tyrosine kinase by
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (20 µM), mitogen-activated protein kinase by 2'-amino-3'
methoxyflavone (50 µM), and protein kinase C by staurosporine (1 µM) had no effect on the cellular responses caused by DHPG. The
mGluR5-selective antagonist 2-methyl-6-(phenylethynyl)-pyridine
(10-100 µM) did not affect the actions of DHPG. Thus our results
indicate that the responses, mainly mediated by mGluRs1 in dopamine
neurons, are activated by intracellular mechanisms coupled to G
proteins and regulated by tyrosine kinases.
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INTRODUCTION |
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The activation of
metabotropic glutamate receptors (mGluRs) in the CNS modulates
second-messenger pathways and, in turn, influences cellular
excitability (Anwyl 1999; Conn and Pin
1997
; Pin and Douvoisin 1995
; Schoepp and
Conn 1993
). Eight mGluRs have been identified so far. These
have been subdivided into three groups. The group I (mGluR1 and mGluR5)
activates phospholipase C (PLC), while groups II (mGluR2 and mGluR3)
and III (mGluR4, mGluR6, mGluR7, and mGluR8) are negatively coupled to
cyclic AMP (Gomeza et al. 1996
; Pin and Douvoisin
1995
; Watkins and Collingridge 1994
). With
regard to the group I mGluRs, they are believed to couple to
GTP-binding proteins (G proteins) and, by stimulating phosphoinositide hydrolysis, to mobilize calcium from intracellular stores and depolarize neurons (Bockaert et al. 1993
; Congar
et al. 1997
; Lee and Boden 1997
; Pin et
al. 1994
). Moreover, it has been suggested that the group I
mGluRs could use other intracellular systems, which do not require G
proteins, to excite different types of central neurons
(Guerineau et al. 1995
; Heuss et al.
1999
; Tempia et al. 1998
; Zheng et al.
1995
).
An alteration of the functional interbalance between glutamate and
dopamine in the basal ganglia has been implicated in motor and
psychiatric disorders (Albin et al. 1989;
Carlsson and Carlsson 1990
; Davis et al.
1991
). Therefore the activity of mGluRs in the ventral midbrain
are critically involved in the expression of motor behaviors and
sensitization to amphetamine by influencing the dopaminergic output in
the terminal fields (Kim and Vezina 1998
; Swanson
and Kalivas 2000
; Vezina and Kim 1999
).
Metabotropic glutamate receptors are found on dopamine neurons
(Shigemoto et al. 1992
; Testa et al.
1994
) where they provide vital important support for growth
(Plenz and Kitaj 1998
), mediate presynaptic and
postsynaptic effects (Bonci et al. 1997
), and control
neuronal discharge (Meltzer et al. 1997
; Mercuri
et al. 1993
). In addition, the stimulation of excitatory fibers
within the ventral midbrain induces a slow metabotropic depolarizing
and/or hyperpolarizing potential in dopamine cells that is mediated by
group I mGluRs (Fiorillo and Williams 1998
; Shen
and Johnson 1997
). In line with these results, we have
previously demonstrated that mGluR I agonists produce a
sodium-dependent inward current and promote release of calcium from
intracellular stores in these neurons (Guatteo et al.
1999
; Mercuri et al. 1993
). Although we have
hypothesized that these two effects are not interdependent
(Guatteo et al. 1999
), their molecular mechanisms are
not completely known yet. Therefore the purpose of the present study is
to investigate the transduction processes mediating the cellular
responses caused by the activation of group I mGluRs in rat dopamine
neurons maintained in vitro in a slice preparation, using
the whole cell patch-clamp recordings in combination with fluorimetric
measurements of [Ca2+]i.
Thus our principal goals are to assess if G proteins are involved in
the effects of the activation of mGluRs I, if PLC activation is
required for the membrane current production and calcium release from
the internal stores, and if protein tyrosine kinase (PTK) activation is
an important factor in the intracellular pathways leading to the
expression of the (S)-3,5-dihydroxyphenylglycine (DHPG)-induced responses.
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METHODS |
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Tissue preparation
The preparation of the midbrain tissue was performed as
described previously (Mercuri et al. 1995). Albino
Wistar rats of either sex (150-200 g) were anesthetized with ketamine
and killed. All experiments were carried out according to guidelines of
the Comitato Etico of the Tor Vergata University on the use of animals in research. The brain was rapidly removed, and horizontal slices (thickness, 250 µm) were cut in 10-14°C physiological saline using a vibratome and starting from the ventral surface of the midbrain. The
artificial cerebrospinal fluid solution (ACSF) contained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 18 NaHCO3; gassed with 95%
O2-5% CO2. After
incubation in a reservoir (1-2 h, 34°C), a single slice was
transferred into a recording chamber and completely submerged in ACSF
with continuously flowing (2.5 ml/min) solution at 33-34°C (pH 7.4).
The chamber was mounted on the stage of an upright microscope Axioskop
(Carl Zeiss, Oberkochen, Germany) equipped for infrared video
microscopy (Hamamatsu, Hamamatsu City, Japan) and video
microfluorimetry (ImproVision, Coventry, UK).
Electrophysiology
Whole cell patch-clamp recordings were obtained with an
amplifier (Axopatch 1D, Axon Instruments, Foster City, CA) from
visually identified dopamine neurons (Guatteo et al.
1999) using pipettes made from 1.5-mm borosilicate glass (WPI,
Sarasota, FL) and pulled with a PP 83 Narishige puller (Tokyo). The
resistance of the patch pipette was around 4 M
when filled with a
standard solution containing (in mM) 145 K+-gluconate, 0.1 CaCl2, 2 MgCl2, 10 HEPES, 0.75 EGTA, 2 Mg-ATP, and 0.3 Na3GTP (pH 7.35). In a subset of experiments,
Na3GTP (GTP) was substituted with a
nonhydrolizable analogue GTP-
-S trilithium salt (GTP-
-S) or with
GDP-
-S trilithium salt (GDP-
-S, both 0.6 mM). For
microfluorimetry (see following text), 125 µM calcium green-1
exapotassium salt (Molecular Probes, Leiden, The Netherlands) was added
to the pipette solution. Series resistance was partially compensated
using the amplifier. The membrane voltage and current were digitized at
5 kHz through a Digidata 1200B A/D converter acquired and analyzed
using pClamp and Axoscope software (Axon Instruments).
Microfluorimetry
The fluorescent ion indicator calcium green-1 was loaded into
the cell via the patch pipette and excited via a ×40 water-immersion objective (Olympus, Hamburg, Germany) by epi-illumination with light
provided by a 75-W Xenon lamp. Excitation light was band-pass filtered
at 480 nm, and emission light passed a barrier filter of 500 nm and was
detected by a CCD camera (Photonic Science, Millham, UK). A set of
images was acquired at 6- or 12-s interval. Time courses of
fluorescence values were calculated over regions of the neuron that
included the cell body ("regions of interest," defined as those
pixels that exhibit at least 20-30% of maximal specific fluorescence)
and were corrected for background fluorescence measured from image
regions free of dye fluorescence (IonVision software, ImproVision,
Coventry, UK). The fluorescent signal was relatively homogenous over
the cell body and initial portion of the proximal dendrites; thus the
analysis was restricted to this part of the neuron. Recordings of
changes of [Ca2+]i are
expressed as percentage of (Ft F0)/F0
values (
F/F%), where
Ft is the emitted fluorescence at time
t, and F0 is the emitted
fluorescence at time 0 of the cell excited at 480-nm
wavelength. Data analysis was done using Origin 4.1 software (Microcal,
Northampton, MA).
Statistical analysis
Values in the text are expressed as means ± SE. Student's t-test or one-way/two-way ANOVA test for repeated measures, followed by the Tukey test for post hoc analysis when differences were significant were used. Significance levels of 0.01-0.05 (*) or 0.001 (**) were considered. Traces show the most representative example within a set of experiments. Bars in the plots represent averaged responses (calcium transients and currents) obtained from each single experiment and under each condition.
Drug application
DHPG, 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate
(CPCCOEt), 2-methyl-6-(phenylethynyl)-pyridine (MPEP),
4',5,7-trihydroxyisoflavone (Genistein), and
N-(2-phenylethyl)-3,4-dihydroxybenzyldenecyanoacetamide (Tyrphostin B52) were obtained from Tocris Cookson (Bristol, UK); 2'-amino-3' methoxyflavone (PD 98059) was obtained from Calbiochem (Milan, Italy).
1-[6[[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]exyl]-1H-pyrrole-2,5-dione (U73122) was from RBI (Milan, Italy).
4',5,7-Trihydroxyisoflavone-7-glucoside (Genistin), staurosporine and
dopamine were from Sigma (Milan, Italy).
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine
(PP1) was from Alexis (Postfach, Switzerland).
CPCCOEt, PD98052, genistein, genistin, tyrphostin B52, U73122, PP1, and staurosporine were dissolved in DMSO just before a set of experiments, and the final concentration of each compound included DMSO 1:1000 diluted. DHPG was applied via a glass pipette, which was positioned close to the cell body of the patched neuron and connected to a pneumatic pico-pump set at 10-20 psi, 2 s (Picospritzer, WPI, Sarasota, FL). The other drugs were bath-applied by switching the solution to one containing known concentrations of drugs. An exchange of the solution in the recording chamber occurred in about 1 min.
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RESULTS |
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Properties of the dopamine cells
Patch-clamp whole cell recordings were performed on 76 visually
and electrophysiologically identified "principal" dopamine neurons
in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These cells were identified by their fusiform cell body and long unbranched proximal dendrites extending in the plane
of the slice (Grace and Onn 1989). In current-clamp mode
they fired spontaneously at 1.6 ± 0.2 Hz (n = 5).
They showed a hyperpolarizing/outward response to dopamine (30 µM)
and a pronounced hyperpolarization-activated inward current,
Ih (Grace and Onn 1989
;
Johnson and North 1992
; Lacey et al.
1989
; Mercuri et al. 1995
; Yung et al.
1991
). When held at
60 mV, the average holding current was
58 ± 12 pA, n = 8. In general, no
electrophysiological differences were found in neurons of the SNc
(n = 46) and VTA (n = 30), and those
cells responded to the mGluR agonist in the same way.
DHPG-induced responses on dopaminergic neurons
The specific mGluR group-I agonist DHPG (Conn and Pin
1997; Schoepp et al. 1994
) was locally applied
via a glass pipette placed close to the soma of neurons voltage-clamped
at
60 mV (Guatteo et al. 1999
). Figure
1B represents a dopamine
neuron in the whole-cell configuration. Repeated puffs of DHPG (100 µM, 20 psi, 2 s) induced a transient inward current associated
with a rapid increase in [Ca2+]i (Fig.
1C). Mean amplitudes were 249 ± 17 pA
(n = 37) for inward currents (Fig.
2B, bottom) and
70 ± 3%
F/F (n = 37)
for calcium transients (Fig. 2B, top).
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The current reached a peak in 1.9 ± 0.2 s and decay time
constant () was 2.4 ± 0.5 s (n = 3). The
[Ca2+]i reached a peak in
5.7 ± 0.3 s and recovered with a decay time constant of
12.4 ± 0.8 s (n = 3). Both decay time
constants (
) were obtained by fitting the traces with a
monoexponential function. To avoid desensitization (Guatteo et
al. 1999
), the cellular responses to the agonist were usually
evoked every 2 min and remained constant up to 30 min from the onset of
the recording.
Cellular responses to DHPG are G-protein dependent
To examine whether signaling pathways linked to activation of
group I mGluR involve G proteins (Conn and Pin 1997), we
exchanged the intracellular solution containing GTP with a solution
containing GTP-
-S (600 µM), a nonhydrolysable analogue of GTP that
keeps G proteins in a long-term activated state (Lacey et al.
1988
). At the first DHPG puff, 2-6 min after the establishment
of whole cell configuration, a transient increase in
[Ca2+]i coupled to an
inward current occurred. However, at the following puffs, the
corresponding current and calcium rise did not fully return to the
baseline, leading to a progressive increase in the holding current and
[Ca2+]i. Figure
2A shows that 6 min after the onset of recordings (3rd DHPG
puff) was sufficient to completely abolish both DHPG-induced signals.
Indeed, mean values for calcium transients at 4 and 6 min (2nd and 3rd
puff) were reduced to 3 ± 1%
F/F and to
1 ± 0.1%
F/F, n = 11, **P < 0.001. Mean values for inward currents at 4 and
6 min were reduced to 48 ± 8 pA and to 18 ± 7 pA,
n = 12, **P < 0.001.
Under these conditions, a depolarizing pulse (30 mV, 20 s) applied
to the membrane of the recorded cell was still able to cause a
[Ca2+]i increase,
suggesting that the calcium influx from the extracellular space was not
affected. When GTP--S was dialyzed, the first DHPG puff application
(at 2 min) evoked a smaller calcium transient (%
F/F = 30 ± 10, n = 11)
respect to the puff at 2 min in control cells (%
F/F = 70 ± 3, n = 37;
ANOVA, Tukey test, **P < 0.001), whereas no
significant changes of the current amplitude were measured at this time
point (Fig. 2B, bottom, 249 ± 17 pA,
n = 37 for controls and 262 ± 33 pA,
n = 12 in GTP-
-S, ANOVA, Tukey test,
P = 0.99).
To better establish the linkage between the mGluR I activation and G
proteins, six neurons were also patched with a solution in which GTP
was substituted with GDP--S, an inactive analogue of GDP
(Eckstein et al. 1979
). Figure
3A shows the DHPG-induced calcium increase (top) and inward current
(bottom) after 8 and 30 min from the onset of the recording
of a cell dialyzed with GDP-
-S. Figure 3B reports mean
values of the inward current and [Ca2+]i at 8 min
(I = 86 ± 14 pA, bottom, %
F/F = 60 ± 9, n = 6, top) and after 30 min from the onset of the recording
(I = 24 ± 3 pA, bottom, %
F/F = 21 ± 6, n = 6, top) compared with control values reported in Fig.
2B. Note that calcium transients at 8 min were comparable in
amplitudes to controls (ANOVA, P = 0.1), whereas inward
currents at the same time point were already significantly reduced
(ANOVA, Tukey test, **P < 0.001). Both signals were
almost abolished within 30 min of GDP-
-S dialysis with respect to
the signals measured at 8 min (ANOVA, Tukey test, *P < 0.05). This clearly suggests that G proteins are involved in the
effects induced by DHPG.
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Metabotropic glutamate responses are partially dependent on phospholipase C but not on protein kinase C activation
mGluRs I are known to be coupled to the hydrolysis of membrane
phosphoinositides by PLC and to the subsequent production of intracellular inositol-(1,4,5)-triphosphate (IP3)
that enhances the release of calcium from intracellular stores.
However, bath application of the membrane permeable PLC inhibitor
U73122 (10 µM) (Chen et al. 1994; Lee and Boden
1997
; Netzeband et al. 1997
) for 15-20 min had
a small reducing effect on the DHPG-induced calcium rise but did not
change the DHPG-induced current (Fig. 4).
In fact, in the presence of U73122 the amplitude of calcium transients
was reduced to 82 ± 7% of control, n = 9 (t-test, **P < 0.001) (Fig. 4B,
top), but the amplitude of the inward currents was not
affected by the drug (102 ± 3% of control, n = 9, t-test, P = 0.8, bottom). The
hydrolysis of phosphatidil inositol leads to diacylglycerol (DAG)
production that in turn stimulates PKC. To investigate whether PKC
activation is involved in the DHPG-induced calcium release and/or
membrane currents, we bath applied staurosporine (1 µM) (Bonci
and Williams 1997
), a potent inhibitor of PKC, and evaluated
DHPG-induced calcium and current amplitudes at 6-8 and 14-16 min
after the onset of staurosporine application. Staurosporine had no
effect on both DHPG-induced inward current (93 ± 11% of control,
at 6-8 min and 96 ± 10% of control at 14-16 min, ANOVA, P = 0.99) and calcium increase (%
F/F = 108 ± 9, at 6-8 min and 96 ± 10, at 14-16 min, n = 3, ANOVA, P = 0.86, data not shown).
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Tyrosine kinase mediates the DHPG-induced responses
It has been recently reported that group I mGluRs can modulate
either a nonselective cationic current in CA3 hippocampal cells or
Ca2+ mobilization in Chinese hamster ovary
(CHO) cells by the phosphorylation of tyrosine substrates
(Heuss et al. 1999; Umemori et al. 1997
). To test for this possibility in dopaminergic neurons, we investigated the effects of the nonspecific tyrosine kinase inhibitor genistein (Wang and Salter 1994
) on the DHPG-induced responses.
Bath applications of genistein for 4-16 min (40 µM) reversibly
reduced the inward current and elevation in
[Ca2+]i (Fig.
5A) to 39 ± 11%
(n = 10, t-test, *P < 0.01)
and 47 ± 12% (n = 10, t-test,
*P < 0.01) of controls, respectively (Fig. 5B). The effect of genistein partially washed out within 5 min. To further clarify the involvement of PTKs, we bath applied
tyrphostin B52 (40 µM), a compound that is selective for epidermal
growth factor receptor (EGFR)-kinase and other PTKs
(Gazit et al. 1991
). Tyrphostin B52 significantly
reduced, in a poorly reversible manner, both DHPG-induced inward
current (I = 25 ± 12%, n = 6, t-test, *P < 0.05) and
[Ca2+]i elevation (%
F/F = 32 ± 3%, n = 6, t-test, *P < 0.01).
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Figure 5B shows mean amplitudes of calcium (top)
and current (bottom) signals evoked by DHPG application,
expressed as percentage of control, in the presence of genistein (40 µM), tyrphostin B52 (40 µM), and the genistein's inactive analogue
genistin (40 µM). Note that genistin did affect neither the current
nor the calcium amplitudes (I = 84 ± 5% of
control, n = 4, t-test, P = 0.4; F/F = 98 ± 4% of control,
n = 4, t-test, P = 0.8), and
this was tested in two of the experiments in which genistein was used.
To assess the possible involvement of the Src-family of
tyrosine kinase (Boxall et al. 1996) in modulating the
DHPG-induced responses, we performed experiments in the presence of the
specific Src-kinase inhibitor PP1 (20 µM) (Hanke et al.
1996
; Heuss et al. 1999
). In the presence of
this compound, DHPG produced an inward current and calcium signal that
were not significantly different from control (not shown). Indeed,
intracellular calcium transients were 110 ± 13% of control at
6-8 min and 105 ± 16% of control at 14-16 min
(n = 3, ANOVA, P = 0.92), and the
inward currents were 95 ± 5% of control at 6-8 min and
90.5 ± 5% of control at 14-16 min (n = 3, ANOVA, P = 0.93).
Another possible target of the tyrosine kinase-dependent and mGluR
I-dependent intracellular pathway is the activation of the MAPK
cascade. For this reason, we investigated whether the inhibition of the
extracellular signal regulated kinase (ERK) (Ferraguti et al.
1999; Fiore et al. 1993
) might be involved in the induction of the effects of mGluRs. PD 98059 (50 µM), a specific inhibitor of the two best characterized members of ERK, ERK1/ERK2 (Alessi et al. 1995
) did not change the inward current
and increase of [Ca2+]i
caused by locally applied DHPG. In the presence of PD 98059, calcium
transients were 106 ± 13% of control at 6-8 min and 86 ± 6% of control at 14-16 min (n = 4, ANOVA,
P = 0.42, not shown) and the inward currents were
74 ± 13% of control at 6-8 min and 89 ± 7% of control at
14-16 min (n = 4, ANOVA, P = 0.76, not shown).
Pharmacology of metabotropic glutamate receptors
DHPG is a specific agonist for group I mGluRs, therefore the
mGluR1 or the mGluR5 subtypes, most likely, mediate the responses described in the preceding text. To differentiate between these two
receptors, we took advantage of the fact that the compound CPCCOEt
preferentially antagonizes responses mediated by mGluR1 (Casabona et al. 1997) while MPEP those mediated by
mGluR5 (Gasparini et al. 1999
). We previously reported
that CPCCOEt (100 µM) depressed the DHPG-induced inward current and
the elevation in [Ca2+]i
(Guatteo et al. 1999
, Fig. 5). Now we found that the
specific mGluR5 antagonist MPEP 10 µM (Gasparini et al.
1999
; Heuss et al. 1999
) did not antagonize the
inward current or the DHPG-mediated Ca2+ signals
(I = 102 ± 1% of control, t-test,
P = 0.13, Fig.
6B, bottom;
F/F% = 127 ± 13% of control,
n = 4, t-test, P = 0.09). MPEP up to 100 µM did not reduce the DHPG-induced inward currents and
the calcium transients (n = 2, data not shown).
|
This pharmacological profile identifies the receptor mediating the DHPG-induced responses in dopamine neurons as mGluR1-like.
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DISCUSSION |
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Intracellular events leading to the DHPG-induced responses
The main finding of the present study is that in midbrain
dopaminergic neurons, the inward currents and associated intracellular calcium transients, induced by the activation of group I mGluRs, are
G-protein dependent and apparently require a protein tyrosine kinase.
Although G-protein-unlinked inward currents are reported to be caused
by mGluRs agonists in hippocampal CA3 and dorsolateral septal neurons
(Guerineau et al. 1995; Zheng et al.
1995
), the fact that the DHPG-induced current and intracellular
calcium signal are blocked by the dialysis with GTP-
-S and strongly
reduced by the intracellular diffusion of GDP-
-S, confirms the
involvement of G proteins when group I mGluRs are activated in dopamine
neurons. This evidence is also in agreement with the results previously published by Shen et al. (Shen and Johnson 1997
)
concerning the G-protein dependency of the mGluR-induced excitatory
postsynaptic currents in VTA dopamine neurons. Moreover, as already
reported by these authors, we observed that GDP-
-S has a longer time
course than GTP-
-S in affecting mGluR-mediated responses. We might
speculate that, as shown in other cell types (Gruol et al.
1996
; Saugstad et al. 1998
; Umemori et
al. 1997
), the mGluR-activated responses in dopamine cells are
possibly mediated by Gq, a class of G-protein insensitive
to pertussis toxin (Pin and Douvoisin 1995
).
Furthermore, the stimulation of group I mGluRs is usually believed to
activate, via G proteins, PLC, which in turn catalyzes IP3 and DAG production from membrane
phosphatides. IP3 induces the release of
Ca2+ from internal stores and DAG the activation
of PKC (Bockaert et al. 1993; Casabona et al.
1997
; Irving et al. 1992
; Kawabata et al.
1998
; Linden et al. 1994
; Nakanishi
1994
; Netzeband et al. 1997
; Pin and
Douvoisin 1995
). However, the involvement of this classical
intracellular pathway in the DHPG-induced responses is not consistent
with our experimental evidence. In fact, the insensitivity of the
DHPG-evoked inward current to U73122 and to staurosporine, together
with the slight effect of U73122 but not staurosporine in reducing
[Ca2+]i, demonstrates
that the DHPG-induced effects are mainly independent of PLC and PKC
activation. This is also supported by the fact that both drugs were
used at saturating concentration, as currently reported in the
literature (Bonci and Williams 1997
; Lee and
Boden 1997
; Tempia et al. 1998
). Accordingly,
group I mGluR-mediated responses not coupled to PLC and unlinked to PKC
have been already described in Purkinje cells and in heterologous
expression systems (Casabona et al. 1997
; Hirono
et al. 1998
; Tempia et al. 1998
).
Protein kinase-dependent responses
It is known that the brain contains high levels of PTK activity
(Hirano et al. 1988). Antibody staining for
phosphotyrosine demonstrates that developmental and regional PTK
variations within the brain are present (Cudmore and Gurd
1991
). It has been already reported that DHPG induces a
G-protein-independent cationic current that requires the
phosphorylation of tyrosine in CA3 hippocampal neurons (Heuss et
al. 1999
). A tyrosine kinase-dependent reduction of
IAHP is also induced by mGluRs in
dentate granule neurons (Abdul-Ghani et al. 1996
). In
agreement with these studies, we found that the inhibition of tyrosine
kinase activity by genistein and by tyrphostin B52 reduced both the
mGluR-mediated inward current and the
[Ca2+]i signal in
dopamine neurons. The lack of effect of the inactive analogue genistin
also confirms a specific modification of the tyrosine phosphorylation
sites by DHPG. It is not known which tyrosine kinase is involved in the
responses caused by mGluR (Boxall and Lancaster 1998
).
Heuss et al. (1999)
have shown that the nonreceptor Src-family PTK is responsible for the synaptic activation of mGluR1 in
CA3 pyramidal cell of rat hippocampus. Here, we report that Src-kinase
seems not to contribute to the mGluR transduction pathway in dopamine
cells because the Src-kinase specific inhibitor PP1, applied at the
same concentration reported by these authors (Heuss et al.
1999
), does not affect the responses that depend on the activation of mGluR I. MAPKs have been also reported to be regulated by
mGluRs or by PKA/PKC in CHO, cortical glial, and hippocampal CA1 cells
(Ferraguti et al. 1999
; Kurino et al.
1995
; Peavy and Conn 1998
; Roberson et
al. 1999
). Conversely, our data obtained using the ERK1/2
inhibitor PD 98059, used at same concentration that was effective in
other types of neurons, exclude the possibility that MAPKs are directly
implicated in the mechanisms responsible for the generation of the
DHPG-induced inward current and release of
[Ca2+]i in the
dopaminergic cells.
One possibility might be that, in dopamine neurons, the targets of PTK
are either the mGlu receptors or the G proteins. In fact,
Umemori et al. (1997) demonstrated that PTK inhibitors
act before the activation of a Gq/11 protein and
that stimulation of Gq/11-protein-coupled
receptor induces phosphorylation of a tyrosine residue on the
G
q/11 subunit, which is essential for the
activation of the G protein. Alternatively, PTK could directly phosphorylate the metabotropic glutamate receptor protein
(Orlando et al. 1999
) as it has been described for the
N-methyl-D-aspartate receptors (Lu et al.
1999
). Another possibility is that the responses to DHPG might
be dependent on Homer proteins, known to be linked to group I mGluRs
and to tyrosine phosphatases (Tu et al. 1998
). Thus it
could be possible that the level of tyrosine phosphorilation regulate
these transduction pathways.
Subtype of receptors
The results obtained with subtype specific antagonists suggest
that the metabotropic receptor responsible for the induction of the
inward current and increase in
[Ca2+]i is mGluR1-like.
In fact, while CPCCOEt (mGluR1 antagonist) reduced the
electrophysiological and Ca2+ responses to DHPG
(Guatteo et al. 1999), MPEP (mGluR5 antagonist) was
ineffective. These findings are also consistent with a prominent expression of mGluR1 in dopamine cells (Kosinski et al.
1998
; Testa et al. 1994
).
Conclusions
Taken together, our electrophysiological and fluorimetric
observations suggest that tyrosine kinase regulates the
G-protein-dependent inward current and intracellular calcium signal
caused by the activation of mGluR I in dopamine cells. Although no
functional relationship exists between intracellular
Ca2+ increase and the inward current response
(Guatteo et al. 1999), here we demonstrate that both
phenomena share common metabolic pathways that involve G protein and
tyrosine kinase activation. The induction of the inward current has
obvious enhancing effects on the excitability of the cells, while the
release of calcium from intracellular stores can cause a secondary
membrane hyperpolarization (Fiorillo and Williams 1998
).
It might be also speculated that the phosphorilation of proteins by
mGluR-linked PTK are involved in trophic processes and in enduring
changes of synaptic signals (Berridge 1998;
Boxall et al. 1996
; Merlin et al. 1998
;
Nakanishi 1994
; Phenna et al. 1995
) on
the dopaminergic cells, and this in turn could influence motor behavior
(Conquet et al. 1994
) and sensitization to drugs of
abuse (Vezina and Kim 1999
).
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ACKNOWLEDGMENTS |
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We thank Dr. Marco Capogna for critical reading of the manuscript and M. Federici for technical assistance.
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FOOTNOTES |
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Address for reprint requests: N. B. Mercuri, Experimental Neurology Laboratory, IRCCS Fondazione S. Lucia, 00179 Rome, Italy (E-mail: mercurin{at}med.uniroma2.it).
Received 4 December 2000; accepted in final form 9 March 2001.
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REFERENCES |
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