Activation of Metabotropic Glutamate Receptors Modulates the Voltage-Gated Sustained Calcium Current in a Teleost Horizontal Cell

Cindy L. Linn and Adele C. Gafka

Louisiana State University Medical Center, Department of Cell Biology and Anatomy, New Orleans, Louisiana 70112


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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.


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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), alpha -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.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega . 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(beta -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)-alpha -methylserine-O-phosphate (MSOP); (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3H-PG); 2-amino-2-methyl-4-phosphonobutanoic acid/alpha -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.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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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Fig. 1. Effect of L-glutamate on voltage-gated calcium currents in catfish horizontal cells. A: membrane potential of a cone horizontal cell was shifted in a ramp-wise fashion between -70 to +50 mV during a 500-ms period of time and clearly showed the sustained calcium current. Sodium and potassium currents all have been suppressed pharmacologically as have ionotropic glutamate currents. Three current traces have been superimposed. Note that 2 min after 100 µM L-glutamate application, the amplitude of the voltage-gated calcium current significantly increased, the calcium current activation range expanded in the hyperpolarized direction, and the peak calcium current shifted. The voltage ramp is shown below the current. *, recovery trace obtained 5 min after L-glutamate application. B: chord conductances were generated from the data presented in A using the equation: g/gmax = I/(Vm -Erev), where g = conductance, gmax = maximal conductance, I = current, V = voltage potential, and Erev = calcium reversal potential. , data points obtained under control conditions; , data points obtained 2 min after application of 100 µM L-glutamate in the presence of ionotropic glutamate blockers. Data points were fit using 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. C: 100 µM L-glutamate (up-arrow ) was applied to a voltage-clamped cone horizontal cell in bathing solution containing pharmacological blockers of sodium and potassium currents, as well as 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione (DNQX; 10 µM) and DL-2-amino-7-phosphonoheptanoic acid (AP-7; 100 µM) to block ionotropic glutamate channels. Note that application of L-glutamate alone produced no membrane current. However, L-glutamate application did result in an enhancement of the calcium current amplitude when the membrane potential was changed from a holding potential of -70 to -10 mV to activate calcium current activity 2 min later. The step paradigm is shown below the current trace.


                              
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Table 1. Effect of various agonists on calcium current activity

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
[Expansion of activation range] = {[Expansion of activation range]<SUB>max</SUB> ×([<IT>A</IT>]<SUP><IT>n</IT></SUP><IT>&cjs0823;  EC</IT><SUB><IT>50</IT></SUB><IT> + </IT><IT>A</IT><SUP><IT>n</IT></SUP>)}
where A is the agonist concentration applied, EC50 is the concentration of agonist producing a half-maximal response, and n is the Hill coefficient. At concentrations as low as 25 µM, L-glutamate significantly potentiated the sustained calcium current. The EC50 for glutamate's action was calculated to be 42 µM, n equaled 1.38, and the peak expansion of the calcium current amplitude changed by a mean of -9.5 mV from control values.



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Fig. 2. Dose-response curve obtained from isolated cone horizontal cells. Various concentrations of L-glutamate were perfused onto selected voltage-clamped horizontal cells. After a stabilization period, the membrane potential of each cell was changed in a ramp-wise manner between -70 and +50 mV. Data points represent the average expansion of the calcium current's activation range measured 2 min after L-glutamate application. To obtain these points, the threshold of the calcium current's activation range was defined as the membrane potential where 5% of the peak calcium current was attained. The mean difference in calcium current's activation threshold, before and after L-glutamate application, then was plotted as a data point. Points were curve fit using the Hill equation. Data points represent the means from 8 to 22 cells and vertical bars represent standard deviations from the mean.

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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Fig. 3. Effect of 1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) on calcium current activation range. A: membrane potential of a voltage-clamped cone horizontal cell was changed in a ramp-wise fashion between -70 and +50 mV to evoke the sustained calcium current. Three current traces were superimposed representing a current trace obtained under control conditions (down-arrow ), a current trace obtained 2 min after perfusion of 50 µM 1S,3R-ACPD and a recovery current trace obtained 5 min after 1S,3R-ACPD application (*). Note that 1S,3R-ACPD produced an enhancement of the calcium current amplitude, an expansion of the calcium current's activation range and a shift of the peak current. B: this figure represent the normalized mean calcium currents obtained from 4 voltage-clamped cone horizontal cells. Each cell's membrane potential was held at a holding membrane potential of -70 mV and subsequently was changed in a ramp-wise manner to +50 mV to evoke the voltage-gated calcium current. Because the actual amplitude of calcium current varied depending on cell size, the calcium current obtained at each membrane potential between -70 and +50 mV was normalized and plotted as a current/voltage relationship. , normalized mean data points obtained under control conditions; , normalized mean data points obtained 2 min after 50 µM 1S,3R-ACPD application. Vertical bars represent standard deviations.

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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Fig. 4. Effect of various metabotropic glutamate agonists on calcium current activity. A: membrane potential of a voltage-clamped cone horizontal cell was changed in a ramp-wise fashion between -70 and +50 mV to evoke the sustained calcium current. Three current traces were superimposed representing a current trace obtained under control conditions (down-arrow ), a current trace obtained 2 min after perfusion of 100 µM (S)-3,5-dihydroxyphenylglycine, [(S)3,5-DHPG] and a recovery current trace obtained 5 min after (S)3,5-DHPG application (*). Note that (S)3,5-DHPG produced an enhancement of the calcium current amplitude, a hyperpolarizing change in the calcium current activation range and a shift of the peak current. B: in the presence of the group II metabotropic glutamate agonist, (S)-3-carboxy-4-hydroxyphenylglycine, [(S)-3C4H-PG; 100 µM] failed to effect the amplitude or activation range of the voltage-gated calcium current. There was no significant difference between the control current trace shown here and the trace obtained after (S)-3C4H-PG was perfused over a voltage-clamped cell. C: perfusion of 100 µM L-2-amino-4-phosphonobutyric acid, (L-AP4) mimicked L-glutamate's effect on calcium current activity. Three current traces were superimposed representing a current trace obtained under control conditions (down-arrow ), a current trace obtained 2 min after perfusion of 100 µL-AP4 and a recovery current trace obtained 5 min after agonist application (*). Two minutes after perfusion of the group III metabotropic glutamate agonist, the calcium current's amplitude was potentiated, the activation range changed, and a shift of the calcium current peak amplitude occurred.

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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Fig. 5. Effect of metabotropic glutamate antagonists on the voltage-gated calcium current. Left: mean normalized calcium current increase from control conditions that occurred when cone horizontal cells were exposed to a mGluR agonist, (S)3,5-DHPG (100 µM). Note that in the presence of group I mGluR antagonists, (RS)-1-aminoindan-1,5-dicarboxylic acid, (AIDA; 200 µM) or S-4C3H-PG (200 µM), (S)3,5-DHPG's modulatory effect was reduced significantly. Right: mean normalized calcium current increase observed when cells were exposed to a group III mGluR agonist, L-AP4 (100 µM). Note that L-AP4's effect was reduced significantly in the presence of mGluR antagonists, 2-amino-2-methyl-4-phosphonobutanoic acid/alpha -methyl-AP4 (MAP4, 500 µM) or (RS)-alpha -methylserine-O-phosphate (MSOP, 200 µM). Means were collected from between 5 and 22 cells. Error bars represent SD.

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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Fig. 6. Evidence for multiple mGluR subtypes on catfish cone horizontal cells. Membrane potential of voltage-clamped cone horizontal cells were changed in a ramp-wise fashion to elicit the sustained calcium current before (*) and after application of group I or group III mGluR agonists. Two current traces were superimposed to note any changes in calcium current amplitudes or shifts in activation range. A, left: 100 µM L-AP4 modulated the voltage-gated calcium current in that voltage-clamped cell. Middle: traces obtained in the presence of 500 µM MAP4. In the presence of the group III antagonist, L-AP4's modulatory activity was blocked. Right: traces also were obtained in the presence of 500 µM MAP4. However, MAP4 did not block the modulatory action of (S)3,5-DHPG. B, left: group I agonist, (S)3,5-DHPG modulated the voltage-gated calcium current in a characteristic manner. However, when the bathing solution of voltage-clamped cells contain the group I antagonist, AIDA, (S)3,5-DHPG's action was blocked (middle). Right: AIDA did not affect the group III metabotropic agonist, L-AP4.

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.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society