Louisiana State University Medical Center, Department of Cell Biology and Anatomy, New Orleans, Louisiana 70112
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ABSTRACT |
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Linn, Cindy L. and
Adele C. Gafka.
Activation of metabotropic glutamate receptors modulates the
voltage-gated sustained calcium current in a teleost horizontal cell.
In the teleost retina, cone horizontal cells contain a
voltage-activated sustained calcium current, which has been proposed to
be involved in visual processing. Recently, several studies have
demonstrated that modulation of voltage-gated channels can occur
through activation of metabotropic glutamate receptors (mGluRs).
Because glutamate is the excitatory neurotransmitter in the vertebrate
retina, we have used whole cell electrophysiological techniques to
examine the effect of mGluR activation on the sustained voltage-gated calcium current found in isolated cone horizontal cells in the catfish
retina. In pharmacological conditions that blocked voltage-gated sodium
and potassium channels, as well as
N-methyl-D-aspartate (NMDA) and non-NMDA
channels, application of L-glutamate or
1-aminocyclopentane-1,3-dicarboxylic acid
(1S,3R-ACPD) to voltage-clamped cone
horizontal cells acted to increase the amplitude of the calcium
current, expand the activation range of the calcium current by 10 mV
into the cell's physiological operating range, and shift the peak
calcium current by 5 mV. To identify and characterize the mGluR
subtypes found on catfish cone horizontal cells, agonists of group I,
group II, or group III mGluRs were applied via perfusion. Group I and
group III mGluR agonists mimicked the effect of L-glutamate
or 1S,3R-ACPD, whereas group II mGluR agonists
had no effect on L-type calcium current activity. Inhibition studies
demonstrated that group I mGluR antagonists significantly blocked the
modulatory effect of the group I mGluR agonist,
(S)-3,5-dihydroxyphenylglycine. Similar results were obtained when the group III mGluR agonist,
L-2-amino-4-phosphonobutyric acid, was applied in the
presence of a group III mGluR antagonist. These results provide
evidence for two groups of mGluR subtypes on catfish cone horizontal
cells. Activation of these mGluRs is linked to modulation of the
voltage-gated sustained calcium current.
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INTRODUCTION |
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In the vertebrate retina, there is strong evidence that
L-glutamate is the excitatory amino acid released during
neurotransmission from photoreceptors onto cells in the outer plexiform
layer, including bipolar and horizontal cells. Pharmacological and
electrophysiological studies have provided new insights into the
glutamate receptor subtypes associated with these second-order neurons.
These receptor subtypes have been classified into two distinct groups
based on their signal transduction pathways: ionotropic glutamate
receptors, which are coupled directly to the opening of cationic
channels (Murphy et al. 1987), and metabotropic
glutamate receptors (mGluRs), which are G-protein-coupled receptors
(Conn et al. 1994
).
Ionotropic glutamate receptors have been further defined
pharmacologically and electrophysiologically by their sensitivity to
N-methyl-D-aspartate (NMDA),
-amino-3-hydroxyl-5-methyl-1-isoxazole-4-propionic acid, and kainic
acid. Metabotropic glutamate receptors have been divided into three
groups based on their amino acid sequence homologies, their coupling to
second messenger systems, and their agonist selectivities (Conn
and Pin 1997
). In a number of studies, group I mGluRs have been
found to stimulate phosphoinositide hydrolysis and include mGluR1a, -b,
and -c and mGluR5a and -b subtypes. Group II metabotropic receptors, as
well as group III receptors, have been linked to a decrease in
adenylate cyclase activity. Group II receptors include mGluR2 and
mGluR3 subtypes, whereas group III receptors include mGluR4a and -b,
mGluR6, mGluR7, and mGluR8 subtypes.
In the vertebrate retina, both ionotropic and metabotropic receptors
have been identified and have been found to play a role in synaptic
transmission. In bipolar cells, two physiologically characterized cell
types have been described (Miller and Slaughter 1986).
The ON bipolar cell type hyperpolarizes in response to glutamate. This response is mediated through a group III mGluR that has
high-affinity binding to L-2-amino-4-phosphonobutyric acid,
(L-AP4). Binding of glutamate or L-AP4 to this
receptor has been shown to stimulate a G protein, which activates a
phosphodiesterase to decrease the intracellular concentration of cyclic
nucleotides that directly gate cation selective channels (Nawy
and Jahr 1990
). The OFF bipolar cells depolarize in
response to light-off or glutamate application (Murakami et al.
1975
). This evoked response is mediated via an ionotropic
glutamate receptor that opens cationic channels.
Horizontal cells are second-order neurons in the outer plexiform layer
that provide a lateral neuronal pathway responsible for the
antagonistic surround response observed in bipolar cell recordings
(Werblin and Dowling 1969). In the dark, horizontal cells are depolarized by a continuous release of neurotransmitter (Trifonov 1968
) onto ionotropic glutamate receptors. In
light, neurotransmitter release from the photoreceptors is reduced, and the cells respond with a graded hyperpolarization (Kaneko and Yamada 1972
; Werblin and Dowling 1969
).
Metabotropic glutamate receptors also may be present and play a role in
horizontal cell neurotransmission. Takahashi and Copenhagen
(1992)
reported that although L-AP4 produced no
effect when applied directly to solitary teleost horizontal cells, it
was found to reduce glutamate-evoked depolarization through a
postsynaptic mechanism under specific pH conditions (Takahashi
and Copenhagen 1992
). Similar results also have been obtained
in locust muscle (Cull-Candy et al. 1976
).
Besides being linked to regulation of neurotransmitter release, mGluRs
have been shown in a number of different neuronal preparations to
modulate voltage-gated calcium currents (Chavis et al.
1995; Hay and Kunze 1994
; Sahara and
Westbrook 1993
). In teleost horizontal cells, voltage-dependent
calcium currents have been examined for their involvement in formation
of the light response as well as a potential role in maintaining the
cell's membrane potential in the dark (Sullivan and Lasater
1992
; Winslow 1989
). In catfish cone horizontal
cells, the voltage-gated calcium current consists of a sustained L-type
calcium current. Unlike other teleost species, there is no evidence for
a T-type transient calcium current in catfish cone horizontal cells
(Shingai and Christensen 1986
). Although the L-type
sustained calcium current has been characterized pharmacologically and
electrophysiologically in catfish cone horizontal cells (Shingai
and Christensen 1986
), little is known about how this current
is regulated or modulated. Because of the importance of calcium
currents in retinal signal processing, we have used a combination of
pharmacological and electrophysiological techniques to characterize the
mGluRs found in isolated catfish cone horizontal cells and to examine
the effects of mGluR activation on voltage-dependent calcium currents.
Catfish cone horizontal cells receive an exclusive input from
red-sensitive cone photoreceptors (Naka and Sakai 1985
). Our results provide evidence that at least two types of mGluRs exist in
catfish cone horizontal cells. Activation of these receptors significantly enhanced the sustained voltage-dependent calcium current
in these cells and changed the calcium current's activation range in
the hyperpolarized direction. These results have considerable implications for retinal processing in the outer retina.
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METHODS |
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Cell isolation procedures
The procedure for catfish cone horizontal cell isolation has
been previously described by O'Dell and Christensen
(1989). Briefly, dark-adapted channel catfish (Ictalurus
punctatus) were anesthetized using tricaine methanesulfonate (100 mg/ml) until the animal no longer reacted to tactile stimulation. Both
eyes were removed under dim red light. The cornea, lens, and overlying
tissue subsequently were excised from each eye and the remaining
eyecups were placed in a low-calcium catfish saline containing (in mM)
126 NaCl, 4 KCl, 0.3 CaCl2, 1 MgCl2, 15 dextrose, 2 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; adjusted to pH 7.4 with NaOH), and hyaluronidase (0.1 mg/ml; 5,270 U/mg) that acted to digest the vitreous fluid covering the
retina. After 4 min, the eyecups were placed in low-calcium catfish
saline containing cysteine-activated papain for another 4 min (0.7 mg/ml papain; 27 U/mg; prepared 30 min before use). Enzymatically
treated retinas were separated from the underlying pigment epithelium
and treated with fresh papain/cysteine saline for another 4 min. Each
retina then was cut into four pieces and stored in normal catfish
saline containing (in mM) 126 NaCl, 4 KCl, 3 CaCl2, 1 MgCl2, 15 dextrose, and 2 HEPES (adjusted to pH 7.4 with
NaOH) and 1 mg/ml bovine serum albumin at 4°C until used. Consistent
recordings were obtained from these cells for a period of 48 h
(O'Dell and Christensen 1989
).
Before recording, a piece of retina was further dissociated to yield isolated cone horizontal cells. This process involved vigorous trituration of a piece of retina in 2 ml of normal catfish saline through a series of fire-polished Pasteur pipettes of progressively smaller tip diameters. Once the retinal tissue was broken down to single cells, a sample of isolated retinal cells was applied to the recording chamber mounted on the stage of an inverted Nikon Diaphot 300 microscope. Cells were allowed to settle in the recording chamber for 5 min before recording began.
Electrophysiology
At the beginning of each experiment, normal catfish saline was
exchanged for catfish saline that contained agents to enhance the
voltage-gated calcium current and blocked voltage-gated sodium and
potassium currents, as well as ionotropic glutamate receptors. This
saline consisted of (in mM) 110 NaCl, 4 KCl, 1 MgCl2, 10 BaCl2, 10 dextrose, 2 HEPES, 10 4-aminopyridine (4-AP),
0.01 tetrodotoxin, 0.1 DL-2-amino-7-phosphonoheptanoic acid
(AP-7) and 0.005 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione (DNQX) (pH
adjusted to 7.4). Using 10 mM barium instead of calcium in these
experiments acted to enhance the sustained calcium current twofold and
shifted the voltage dependence of activation by ~10 mV in the
hyperpolarized direction when compared with results obtained using 10 mM calcium (Pfeiffer-Linn and Lasater 1996). This
enhancement of the sustained calcium current and shift in voltage
dependence is characteristic of the sustained calcium current described
in other systems (Karschin and Lipton 1989
).
Catfish cone horizontal cells were identified easily based on their
characteristic morphology (Shingai and Christensen
1989). Cells were voltage clamped according to the method
described by Hamill et al. (1981)
using patch pipettes
fashioned from borosilicate glass with the use of a Narishige (Tokyo)
vertical microelectrode puller. When measured in catfish saline,
electrode resistances ranged between 3 and 8 M
. Patch electrodes
were not beveled or fire polished and contained the following solutions
(in mM): 120 CsCl and 20 TEA to block potassium channels, along with 11 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1 CaCl2, 2 MgCl2, 1 ATP, and 10 HEPES (pH adjusted to 7.4 using KOH).
Once a cone horizontal cell was voltage clamped and the membrane
currents had stabilized (~2 min), calcium channel activity was
measured by stepping the membrane potential of the cell from a holding potential of
70 mV to a membrane potential of +50 mV or by changing the membrane potential of the cell in a ramp-wise manner between the
two membrane potentials during a 500-ms period of time (Sullivan and Lasater 1992
). Using these stimulus paradigms, four
properties of the elicited calcium current were measured: the calcium
current peak amplitude, the membrane potential where calcium current
was activated, the membrane potential where 50% of peak current was observed, and the membrane potential corresponding to peak calcium current amplitude.
Metabotropic glutamate receptor test agents {L-glutamate;
L-AP4 (Sigma);
1-aminocyclopentane-1,3-dicarboxylic acid
[(1S,3R)-ACPD];(S)-3,5-dihydroxyphenylglycine [(S)3,5-DHPG];
(S)-3-carboxy-4-hydroxyphenylglycine
[(S)-3C4H-PG]; (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA);
[(2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1);
(2S,2',3'R)-2-(2',3'-dicarboxycyclopropyl)glycine
(DCG-IV); L-serine-O-phosphate
(L-SOP);
(RS)--methylserine-O-phosphate (MSOP);
(S)-4-carboxy-3-hydroxyphenylglycine
(S-4C3H-PG);
2-amino-2-methyl-4-phosphonobutanoic acid/
-methyl-AP4 (MAP4;
Tocris Cookson); and
2R,4R-pyrrolidinecarbodithioic acid (APDC;
Calbiochem)} were applied to voltage-clamped cells via a gravity fed
perfusion system and subsequently washed away using control Ringer
solution. The complete exchange of solution in the recording chamber
occurred within 500 ms. Agonist doses used in this study were chosen
after preliminary experiments to find the effective saturating
concentration. Antagonist concentrations were chosen by their relative
effectiveness against the mGluR agonist concentration used.
Recordings were obtained using an Axon Instruments Axopatch 200A
(Foster City, CA). Series resistance and capacitive artifacts were
compensated for using the amplifier controls. Series resistance could
be adjusted to 90% and thus little voltage error occurred even for
large command voltages. Leakage currents were not corrected for in
these studies because data were only obtained from cells with
relatively small leakage currents and care was taken to reject data
from cells in which the leakage current changed by more than a few
millivolts over the course of an experiment. A small junction potential
was measured (<3 mV) and was not compensated for. Data collection was
controlled by a personal computer in conjunction with the Digidata 1200 data acquisition board. Digitization and analysis of the data were
carried out using the Axon Instruments' pClamp suite of programs. Data
were filtered at 1 kHz (
3 dB) and sampled at 5 kHz.
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RESULTS |
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Activation of mGluRs
To determine if catfish cone horizontal cells contain mGluRs that
are linked to modulation of the voltage-gated calcium current, mGluR
agonists were perfused over selected voltage-clamped horizontal cells.
In Fig. 1A, the membrane
potential of one such horizontal cell was changed in a ramp-wise
fashion from a holding potential of 70 to +50 mV during a 500-ms
period of time in the presence and absence of 100 µM
L-glutamate. When this procedure was conducted in bathing
saline that favored calcium current expression and blocked other
voltage-gated channels and ionotropic glutamate receptors (see
METHODS), a current/voltage relationship of the horizontal
cell was produced that clearly illustrated the inward calcium current.
In Fig. 1A, the current trace obtained after L-glutamate application was superimposed on top of the
control current trace. Under control conditions, the voltage-gated
calcium current activated at a membrane potential of
32 mV, and the
peak current was reached at
11 mV. Two minutes after
L-glutamate application, the calcium current's amplitude
significantly increased at all membrane potentials between
40 and 0 mV. The peak calcium current amplitude increased from
220 to
403
pA, representing a 83% increase. Furthermore, in the presence of
L-glutamate, the calcium current activated at
38 mV
instead of
32 mV, 50% of the peak current occurred at
25 mV
instead of
15 mV, and the peak calcium current shifted from
11 to
20 mV. These calcium current changes were typical of data obtained
from 21 other voltage-clamped cells where the calcium current's
activation changed from a mean membrane potential of
29 ± 4 (SD) mV to
39 ± 4 mV, the membrane potential representing 50%
of the peak current measured changed from a mean of
14 ± 2 mV
to
26 ± 3 mV, the mean peak current shifted from
10 ± 4 mV to
25 ± 5 mV, and the peak calcium current amplitude increased by a mean of 80 ± 15% (Table
1) from control conditions. Recovery of
these effects was almost complete within 5 min after application (Fig.
1A, *). Although there was a significant change in
the calcium current's activation range and amplitude after L-glutamate application, there was no change in the
current/voltage relationship measured at voltages positive to 0 mV.
Inactivation properties of the calcium current were not modulated by
L-glutamate.
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To characterize the calcium current activation more carefully,
normalized chord conductances were generated from the data shown in
Fig. 1A using the equation
g/gmax = I/(Vm Erev), where g = conductance,
gmax = maximal conductance, I = current, V = voltage potential, and
Erev = calcium reversal potential (Fig. 1B). Control and L-glutamate chord conductances
were fit by the Boltzmann equation, g = gmax/1 + exp[(V
V1/2)/k], where
V1/2 is the half-activation value and
k is the slope factor. Before L-glutamate
application, gmax = 7 pS, k = 3.1/mV, and V1/2 =
16 mV. These results
obtained from the L-type calcium current in catfish cone horizontal
cells are similar to Boltzmann values obtained from L-type calcium
currents found in other preparations (Griffith et al.
1994
). After L-glutamate application,
V1/2 increased to
28 mV, while
gmax and the slope factor were unchanged. These values indicate that L-glutamate modifies the voltage
dependency of the activation range in catfish cone horizontal cells and
does not affect new channel populations.
The current trace shown in Fig. 1C demonstrates that
ionotropic glutamate receptors are not responsible for the modulatory effect seen by L-glutamate application. Catfish cone
horizontal cells contain NMDA as well as non-NMDA ionotropic glutamate
receptors (O'Dell and Christensen 1989). Activation of
these receptors allows an influx of cations into the cell and produces
a corresponding inward depolarizing current. To block these receptor
channels, a cone horizontal cell was voltage clamped in catfish saline
containing AP-7 and DNQX (Fig. 1C). Application of
L-glutamate under these conditions produced no inward
current when the membrane potential was held at
70 mV
(n = 21) but did modulate the amplitude and activation
range of the voltage-gated calcium current (Fig. 1C). This
suggests that the ionotropic glutamate receptors on the voltage-clamped horizontal cells were blocked completely and that
L-glutamate's modulatory effect on the voltage-gated
calcium current must be due to activation of a mGluR.
In an effort to determine the concentration range over which glutamate
was active, a dose-response curve was generated. Various concentrations
of L-glutamate were applied to isolated horizontal cells
the membrane potentials of which were changed in a ramp-wise fashion to
elicit maximal calcium current activity. As shown in Fig.
2, L-glutamate's effect was
plotted as mean expansion of the calcium current's activation range
from control values against agonist concentration. The data points were
curve fit using the Hill equation
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Pharmacological characterization of the mGluR subtypes
To characterize pharmacologically the specific type of mGluRs
found on catfish cone horizontal cells, we analyzed the effects of
various metabotropic agonists on calcium current activity in selected
voltage-clamped cells. In clonal mammalian cell lines expressing each
of the three mGluR groups, 1S,3R-ACPD is a potent agonist for specific subtypes of group I and II metabotropic receptors but is a weak agonist for group III subtypes (Okamoto et al.
1994). To determine if 1S,3R-ACPD
recognized the glutamate receptors on isolated catfish cone horizontal
cells, calcium current activity was recorded before and after
1S,3R-ACPD (50 µM) was perfused over selected
voltage-clamped cells. Figure
3A demonstrates that 1S,3R-ACPD mimicked L-glutamate's
effects on calcium current activity in catfish cone horizontal cells.
Two minutes after 1S,3R-ACPD application, the
calcium current peak amplitude increased by 33% and the calcium
current activated at a membrane potential of
45 mV instead of
35
mV. As seen with L-glutamate application,
1S,3R-ACPD also caused a corresponding shift in
the peak of the calcium current. Partial recovery of these effects
occurred 5 min after 1S,3R-ACPD application (Fig.
3A, *). Similar results were obtained from six voltage-clamped cells (Table 1, Fig. 3B), suggesting that
catfish cone horizontal cells contain at least a group I or a group II type of mGluR.
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As 1S,3R-ACPD had been shown to activate multiple
mGluR subtypes, it was unclear whether catfish cone horizontal cells
contain more than one mGluR subtype. To determine if multiple mGluR
subtypes exist on catfish cone horizontal cells, several metabotropic
agonists for group I, group II, and group III metabotropic receptors
were applied to selected voltage-clamped cells. The current traces in
Fig. 4A illustrate the effect
of applying a group I agonist, (S)3,5-DHPG (100 µM), to a
cone horizontal cell. Two minutes after (S)3,5-DHPG
application, the peak calcium current amplitude significantly increased
by 55%, the peak current shifted from 16 to
24 mV and the calcium
current's activation range expanded by
5 mV. Five minutes after
(S)3,5-DHPG application (Fig. 4A, *), recovery is
almost complete. Similar results were obtained for six other voltage-clamped cells (Table 1), suggesting that catfish cone horizontal cells contain a group I mGluR. On the other hand, group II
agonists had no effect when applied to voltage-clamped horizontal cells. These results are illustrated in Fig. 4B. The two
superimposed current traces were obtained before and after the group II
agonist, (S)-3C4H-PG, was applied. Two minutes after
application, 100 µM (S)3C4H-PG did not affect the peak
current nor change the calcium current activation range in the
hyperpolarized direction (n = 6; Table 1). Similar
results also were obtained using another group II agonist, L-CCG-1. As
reported in Table 1, application of 50 µM L-CCG-1 had no significant
effect on calcium current amplitude and no effect on the activation
range or peak calcium current. However, at higher concentrations (>100
µM) L-CCG-1 shifted the calcium current's activation range and
increased calcium current amplitude. Although L-CCG-1 has been found to
be selective for group II mGluR subtypes in some preparations
(Wright and Schoepp 1996
), other studies have found that
L-CCG-1 activated group I and group III mGluR subtypes as well
(Gomeza et al. 1996
; Hayashi et al.
1992
). Because of the possibility of nonspecific effects, further experiments using more specific group II mGluR agonists (DCG-IV
and APDC) were performed. As DCG-IV also activated NMDA receptors
(Hayashi et al. 1992
), preliminary studies verified that
NMDA currents were pharmacologically blocked (see METHODS). Under these conditions, application of DCG-IV (10 µM) had no
significant effect on calcium current activity (n = 4).
Among the group II mGluR agonists, APDC is considered to be a specific
group II agonist (Conn and Pin 1997
; Schoepp et
al. 1995
). Application of APDC (25 µM) had no effect on
calcium current activity (n = 4). Taken together, these
results support the hypothesis that activation of group I mGluRs on
catfish cone horizontal cells are linked to modulation of the
voltage-gated calcium current by 1S,3R-ACPD. There is no evidence to suggest that group II mGluR subtypes are involved in modulation of the voltage-gated calcium current in catfish
cone horizontal cells.
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As previously mentioned, there is some evidence that suggests group III
mGluRs exist on vertebrate horizontal cells and affect glutamate-evoked
depolarization (Takahashi and Copenhagen 1992). To
determine if activation of a group III mGluR modulates the voltage-gated calcium current in catfish cone horizontal cells, the
potent group III agonist, L-AP4, was perfused over
voltage-clamped horizontal cells. In Fig. 4C, the two
current traces shown were obtained before and after 100 µM
L-AP4 was applied to a selected cone horizontal cell. Like
1S,3R-ACPD and (S)3,5-DHPG,
L-AP4 mimicked L-glutamate's effects. In the
example shown, the peak calcium current amplitude increased by 50%,
shifted by
7 mV and the calcium current activation range changed by
5 mV. Similar results were obtained from eight other cells using
L-AP4 and from five voltage-clamped cells using another
group III agonist, L-SOP (100 µM; Table 1); this suggests
that more than one subtype of mGluR may be involved in the modulation
of the calcium current in catfish cone horizontal cells.
Inhibition of mGluRs
To test further the specificity of the mGluR subtypes, group I and
group III mGluR agonists were applied to voltage-clamped cone
horizontal cells in the presence of various mGluR antagonists (Fig.
5). Calcium currents were elicited before
and after agonists application by changing the membrane potential of
each voltage-clamped cell in a ramp-wise manner between 70 and +50
mV. The mean change in calcium current amplitude after agonist
application was normalized and displayed as histograms. Figure 5,
left, illustrates the inhibitory effect that group I mGluR
antagonists have on (S)3,5-DHPG's action. When 200 µM
AIDA was present in the bathing solution, 100 µM
(S)3,5-DHPG had little effect on calcium current amplitude
compared with control conditions, resulting in a mean increase of only
3% (n = 8). Likewise, in the presence of another group
I mGluR antagonist, S-4C3H-PG (200 µM),
(S)3,5-DHPG increased calcium current amplitude by a mean of
only 9% (n = 4) compared with control values.
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Effective antagonists also were found that blocked the calcium current modulation caused by application of group III agonists. As shown by Fig. 5, right, with MAP4 (500 µM) present in the bathing solution, application of L-AP4 had virtually no effect on the sustained calcium current (n = 6). Similarly, another group III antagonist, MSOP (200 µM), significantly reduced the effect L-AP4 had on modulating the calcium current amplitude (n = 4). Taken together, these results support the hypothesis that catfish cone horizontal cells contain group I and group III mGluRs.
Multiple mGluR subtypes
The pharmacological results presented in this study suggest that multiple mGluR subtypes can be found on catfish cone horizontal cells. However, this pharmacological data cannot rule out the possibility that there is a single mGluR in catfish retina that is distinct from previously cloned mammalian mGluR subtypes and can be activated by both (S)3,5-DHPG and L-AP4. To address this issue, we have shown that the effects of (S)3,5-DHPG and L-AP4 are differentially sensitive to subtype-selective antagonists. In Fig. 6, current traces from voltage-clamped horizontal cells were obtained before (*) and after application of group I (Fig. 6A) or group III (Fig. 6B) mGluR agonists. In Fig. 6A (left), 2 min after perfusion of L-AP4, the voltage-gated calcium current's activation range expanded in the hyperpolarized direction and the calcium current amplitude significantly increased from control conditions. Figure 6A, middle and right, demonstrates that MAP4 is specific for group III mGluR subtypes but not group I mGluR subtypes. In Fig. 6A, middle, two current traces were obtained from another voltage-clamped cell in saline containing the group III antagonist, MAP4. In the presence of the metabotropic group III antagonist, L-AP4 had no effect on calcium current activity. MAP4 blocked the group III metabotropic receptors. However, MAP4 did not block (S)3,5-DHPG's modulatory effect (right). Even with 500 µM MAP4 in the extracellular saline, (S)3,5-DHPG significantly modulated calcium current activity. Thus MAP4 appears to be specific for group III mGluR subtypes but not group I mGluR subtypes. Similar results were obtained from four other voltage-clamped cells.
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Figure 6B demonstrates that AIDA is specific for group I mGluR subtypes. The two current traces shown (left) illustrate currents obtained before and after application of the group I agonist, (S)3,5-DHPG. (S)3,5-DHPG modulated the calcium current activity in the characteristic manner. The center current traces were obtained from another voltage-clamped cone horizontal cell in saline containing 200 µM AIDA. AIDA blocked the typical modulatory change associated with (S)3,5-DHPG application. Although AIDA eliminated (S)3,5-DHPG's effect on calcium current activity, it did not block group III mGluR subtypes. As seen in Fig. 6B, right, even with 200 µM AIDA in the bathing saline, L-AP4 modulated calcium current activity. These results were typical of data collected from four other voltage-clamped cells. Taken together, these results demonstrate that there are at least two mGluR subtypes on catfish cone horizontal cells.
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DISCUSSION |
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In the present study, we have used pharmacological and
electrophysiological techniques to determine if mGluRs exist on catfish cone horizontal cells and to examine whether these receptors were linked to modulation of the voltage-gated sustained calcium current. In
conditions where glutamate ionotropic currents were blocked pharmacologically, we found that L-glutamate, as well as
group I and group III mGluR agonists, modulated the voltage-gated
sustained calcium current in catfish cone horizontal cells. The
modulation consisted of a significant increase in the calcium current
amplitude at all membrane potentials and a change in the calcium
current's activation range toward a more hyperpolarized membrane
potential. This expansion of the calcium current's activation range
also acted to change the peak calcium current's membrane potential by
at least 5 mV.
As calcium channel gating represents an important mechanism for
regulation of cytoplasmic calcium concentration and many
calcium-dependent intracellular processes, other studies have looked at
the role of mGluRs on calcium channel activity. In the majority of
these studies, L-type sustained calcium currents were reduced with
stimulation of mGluRs (Akopian and Witkovsky 1996;
Sayer et al. 1992
), although it has been noted in
cerebellar granule cells that application of
1S,3R-ACPD increased calcium-channel activity
(Zegarra-Moran and Moran 1993
). The enhancement of
calcium channel activity also is seen in this study in catfish cone
horizontal cells when mGluRs are stimulated with specific agonists. It
is interesting to note that other studies have found that
L-glutamate application to isolated catfish cone horizontal
cells reduced calcium current activity (Dixon et al.
1993
) by raising intracellular proton concentration. In these
studies, glutamate was not applied in the presence of ionotropic
blockers. Therefore glutamate activated both NMDA and non-NMDA
receptors as well as metabotropic receptors. This suggests that
activation of the glutamate receptors on catfish cone horizontal cells
produces opposing modulatory effects on the L-type calcium current. When ionotropic currents are activated, the net effect on the
calcium current is to reduce the calcium current amplitude. On the
other hand, activation of mGluRs alone potentiates the calcium current
amplitude. This would indicate that there exists an interactive
regulatory mechanism in catfish cone horizontal cells that acts to
modulate calcium channel activity. One possible mechanism of
interaction may involve the timing of modulatory events. For instance,
in catfish cone horizontal cells, if ionotropic effects were faster
than metabotropic-receptor-triggered events, initially the calcium
current would be modified by ionotropic events, only later to be
modified by metabotropic responses. Further studies are needed to
support this idea.
Evidence of multiple mGluRs on catfish cone horizontal cells
Pharmacological studies presented here suggest that at least two
groups of mGluRs are found on catfish cone horizontal cells and
activation of either group modulates the voltage-gated calcium channel
in a similar manner. This is based on studies demonstrating that
application of specific agonists for group I and group III metabotropic
receptors modulates the voltage-gated calcium current in the same
fashion (Table 1). Furthermore when these agonists were applied in the
presence of specific antagonists for the two groups of metabotropic
receptors, the modulatory action virtually was eliminated (Fig. 5).
However, the existence of two mGluR channels in catfish cone horizontal
cells must be regarded with caution because none of the agents used in
this study were truly specific for a particular metabotropic receptor
subtype. In fact, numerous reports using these same metabotropic
agonists and antagonists have demonstrated varied efficacies and
concentration dependencies (Kemp et al. 1994;
Sekiyama et al. 1996
). To address this issue, we have
shown that the effects of (S)3,5-DHPG and L-AP4
are differentially sensitive to subtype-selective antagonists (Fig. 6).
These results support the hypothesis that there are at least two types
of mGluR subtypes on catfish cone horizontal cells.
mGluR modulation of voltage-gated calcium current
The results presented in this study support the hypothesis that at
least two mGluR subtypes are found to modulate the voltage-gated calcium current in a similar manner. How could activation of two different receptors result in similar modulation? One possibility is
that the two different mGluR subtypes are linked to different second-messenger systems. In fact, studies from expression systems suggest that activation of group I mGluR subtypes mediate
phosphoinositide hydrolysis responses in brain slices and cultured
cells (Miller et al. 1995). Inositol trisphosphate
(IP3) production in turn releases calcium from
intracellular stores (Sugiyama et al. 1987
) that can act
directly to modulate calcium current activity. However, other studies
have observed that activation of group I mGluRs have induced large
increases in L-type calcium current amplitudes via activation of
ryanodine receptors, which generate a retrograde signal that modifies
L-type calcium channel activity (Chavis et al. 1996
).
This mechanism is independent of IP3 and classical protein
kinases. On the other hand, activation of group III mGluR subtypes have
been found to inhibit forskolin-induced increases in adenosine
3',5'-cyclic monophosphate (cAMP) accumulation in brain slices and
neuronal cultures (Schoepp et al. 1994
). This mechanism
of modulation could occur in catfish cone horizontal cells if the
voltage-gated calcium channels were substrates for cAMP-dependent
protein kinase A (PKA). Biochemical studies have supported this
by demonstrating that multiple types of voltage-gated calcium channels
are substrates for protein kinase C (PKC) as well as
cAMP-dependent PKA (Catterall 1991
). Further
pharmacological and electrophysiological experiments need to be
conducted in the catfish retina to determine which of these mechanisms
link activation of mGluRs to modulation of the voltage-gated calcium
currents.
Physiological implications
The data presented in this study support the hypothesis that
glutamate potentiates the calcium current amplitude in catfish cone
horizontal cells and expands the calcium current activation range by an
average of 10 mV. This has significant physiological implications for
horizontal cells as it has been proposed that the L-type calcium
current in teleost horizontal cells may play a role in shaping the
light response and could help to maintain the membrane potential of the
horizontal cell at depolarized levels in the dark (Pfeiffer-Linn
and Lasater 1996
; Sullivan and Lasater 1992
).
These proposals were based on the characteristic current/voltage relationship of the voltage-gated calcium currents found in teleost horizontal cells. In these studies, under control conditions, the
sustained calcium current activated at the edge of the physiological operating range of the cells (
30 to
20 mV) (Werblin and
Dowling 1969
; Kaneko 1970
). As previously noted
in other studies, the result of enhancing the amplitude of the calcium
current in this range would be to greatly enhance calcium entry into
the cell and affect any calcium-dependent process (Pfeiffer-Linn
and Lasater 1996
).
Besides enhancing the calcium current at the foot of the physiological
operating range of the cells, activating mGluRs in catfish cone
horizontal cells also acted to expand the calcium current's activation
range in the hyperpolarized direction. In other preparations, a change
in activation range has been associated with a displacement of charged
molecules in the vicinity of the channel (Zhou and Jones
1995), a change in seal resistance or in leakage currents
through the cell membrane (Ben-Tabou et al. 1994
), a
conformational change induced by binding of permeant ions
(Prod'hom et al. 1989
), or a direct action on the
calcium channel itself which causes a change in the voltage-dependency of the channel (Zamponi and Snutch 1996
).
Generally, the displacement of charged molecules causes a shift in a calcium current's activation range when calcium is substituted for barium. However, in the experiments presented here, there were no divalent cation substitutions and all studies were conducted in the presence of 10 mM barium. Therefore we do not believe that screened surface charges were affected. We also find no evidence that the change in activation range was due to an increase in leak conductance. As shown in Fig. 1, application of a mGluR agonist enhanced the voltage-gated calcium current without affecting the preceding leak currents. Likewise, mGluR agonists had no affect on the amplitude of the leak currents that were produced by 20-mV hyperpolarizations from the holding potential (data not shown, n = 8); this suggests that there was no significant leak current produced after agonist application.
We also do not believe that permeant calcium ions cause
conformational changes to the calcium channel itself that result in a
change of the activation range. If this was the mechanism involved in
the calcium current expansion, application of Bay K 8644 also would
produce an expansion of the activation range. However, in teleost cone
horizontal cells, Bay K 8644 significantly enhances the calcium current
amplitude at depolarized membrane potentials without producing a
substantial change in the activation range or a change in the calcium
current's peak membrane potential (Pfeiffer-Linn and Lasater
1996).
Instead, the change in activation range could be due to a change in the
voltage dependency of the cell as a result of activated mGluRs. In this
scenario, L-glutamate would bind to a mGluR and activate a
second-messenger system to affect, directly or indirectly, a site on
the calcium channel and change the voltage dependency of the channel.
This hyperpolarizing change in the activation range brings the calcium
current's activation range much more into the operating range of the
horizontal cell in the intact retina (Kaneko 1970;
Yang et al. 1988
). In the intact retina, the horizontal
cells respond to light with a hyperpolarization. At light
OFF, in response to glutamate being released from the photoreceptors, the cell is depolarized due to cation permeability through ionotropic glutamate receptors. At the same time, in the catfish, glutamate released from the photoreceptors also would bind to
the mGluRs expanding the voltage-gated calcium currents activation
range in a hyperpolarized direction. Thus during light OFF,
the voltage-gated calcium current is activated and likely would help to
shape the repolarization phase of the light response. Also, because a
sustained calcium current is involved, once activated, the calcium
current would help to maintain the resting membrane potential in the
dark.
Summary
In this study, we have provided evidence to support the hypotheses that catfish cone horizontal cells contain two groups of mGluRs, that activation of these receptors modulate the voltage-dependent L-type calcium current in these cells, and that modulation causes an expansion of the calcium current's activation range toward the hyperpolarized direction. These results suggest that glutamate can modulate activity at the same synapse at which it elicits synaptic responses.
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ACKNOWLEDGMENTS |
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The authors thank Dr. David M. Linn and Dr. Jeff McGee for helpful discussions throughout the course of this study.
This work was supported by National Eye Institute Grant EY-11133 awarded to C. L. Linn.
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FOOTNOTES |
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Address for reprint requests: C. L. Linn, Dept. of Cell Biology and Anatomy, Louisiana State Medical Center, 1901 Perdido St., New Orleans, LA 70112.
Received 24 December 1997; accepted in final form 19 October 1998.
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REFERENCES |
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