January 2005
Volume 46, Issue 1
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Retina  |   January 2005
Interaction between mGluR8 and Calcium Channels in Photoreceptors Is Sensitive to Pertussis Toxin and Occurs Via G Protein βγ Subunit Signaling
Author Affiliations
  • Peter Koulen
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas; and the
    North Texas Eye Research Institute, Fort Worth, Texas.
  • Jiyuan Liu
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas; and the
  • Everett Nixon
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas; and the
  • Christian Madry
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas; and the
Investigative Ophthalmology & Visual Science January 2005, Vol.46, 287-291. doi:10.1167/iovs.04-0963
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      Peter Koulen, Jiyuan Liu, Everett Nixon, Christian Madry; Interaction between mGluR8 and Calcium Channels in Photoreceptors Is Sensitive to Pertussis Toxin and Occurs Via G Protein βγ Subunit Signaling. Invest. Ophthalmol. Vis. Sci. 2005;46(1):287-291. doi: 10.1167/iovs.04-0963.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The most recently identified metabotropic glutamate receptor (mGluR), type 8 mGluR (mGluR8), has been identified functionally as a presynaptic autoreceptor in rod photoreceptors. This study analyzed the mechanism of action underlying mGluR8 activity and modulation of the cytosolic Ca2+ concentration in mouse photoreceptors.

methods. The cytosolic Ca2+ concentration of acutely isolated rod photoreceptors was monitored optically with microspectrofluorimetry and in the presence of modulators of G protein activity.

results. mGluR8 activation by the group III mGluR agonists l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate or the physiological ligand l-glutamate produced a decrease in influx of extracellular Ca2+ into the cytosol. Pretreatment of isolated rod photoreceptors with the G protein uncoupler suramin or pertussis toxin, which inactivates Gi/o/z proteins and Gt protein/transducin, or a G protein βγ subunit–inhibiting peptide abolished this activity. Preincubation of cells with cholera toxin (CTX), an activator of Gs protein, had no effect.

conclusions. These results suggest that the function of mGluR8 of modulating the cytosolic Ca2+ concentration and thereby potentially the release of neurotransmitter from rod spherules, the axon terminal systems of rod photoreceptors, is mediated by a pertussis toxin–sensitive G protein potentially via the βγ subunit. The absence of Go and Gz proteins, as reported previously, implies a novel potential interaction between Gi2 and/or Gt protein/transducin and mGluR8 in photoreceptors. These results have potential implications for the regulatory function and pharmacologic targeting of mGluR8 in photoreceptors.

In the mammalian retina, l-glutamate is the major excitatory neurotransmitter and is used in the vertical pathway for processing of visual information by photoreceptor, bipolar, and ganglion cells. The same cells also use glutamatergic neurotransmission to activate modulatory horizontal pathways involving GABAergic and glycinergic interneurons in the inner and outer plexiform layers. 1 2 3 4 5 Effects of l-glutamate are mediated by ionotropic (iGluRs) and metabotropic glutamate receptors (mGluRs) in the central nervous system, including the retina. Both classes of glutamate receptors can be further subdivided into groups based on sequence homology, signaling mechanism, and pharmacologic profile. 6 7 8 9 Among the mGluRs, which have been divided into groups I, II, and III both inhibitory and excitatory functions have been determined that are critically linked to the receptor’s synaptic localization (pre- versus postsynaptic) and the selective differential activation of second messenger pathways. 9 10 11 12 mGluRs play critical roles in determining and fine tuning retinal physiology. 13 14 15 The most recently identified mGluR, mGluR8, was cloned from a retinal cDNA library 16 and was identified by chromosomal mapping as potentially relevant in neurodegenerative diseases of the retina. 17 This finding and the unique pharmacologic profile of mGluR8 with mixed properties of both group II and III mGluRs 18 19 20 together generated interest in the investigation of mGluR8’s role in retina physiology. 21 We have identified mGluR8a as the first, and so far the only, glutamate receptor in mammalian photoreceptors localized to the axon terminals. 22 23 mGluR8’s role as a presynaptic autoreceptor regulating synaptic strength in terminals of photoreceptors was postulated based on the observed decrease of the cytosolic Ca2+ concentration in photoreceptors after mGluR8 activation. 22 However, except for the exclusion of cAMP- or protein-phosphorylation–mediated signaling mechanisms and an indirect regulation of L-type calcium channels, little is known about downstream signaling events after mGluR8 activation that ultimately lead to a modulation of the cytosolic Ca2+ concentration. 22 In the present study, we therefore investigated whether mGluR8 activity in rod photoreceptor is G protein dependent and if so which class or type of G protein couples mGluR8 to the decrease in Ca2+ influx into the cytosol. 
Materials and Methods
Experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and guidelines for the welfare of experimental animals issued by the NIH and the University of North Texas Health Science Center at Fort Worth. 
Dissociation of Photoreceptor Cells
Rod photoreceptor cells were mechanically and enzymatically isolated and identified by using morphologic and/or immunochemical criteria, as described previously. 22 Briefly, retinas from adult outbred albino Swiss Webster mice (Harlan, Indianapolis, IN) were dissected and incubated in extracellular solution (ECS; in mM: NaCl, 137; KCl, 5; CaCl2, 2; Na2HPO4, 1; MgSO4, 1; HEPES, 10; glucose, 22; pH 7.4) containing 1g/L pronase (P5147; Sigma-Aldrich, St. Louis, MO) and 2 mM EGTA, for 15 minutes at 37°C. After washing the tissue with ECS containing 0.5% bovine serum albumin (BSA), 2 mM EGTA, and 10 mg/L DNase (Sigma-Aldrich) cells were obtained by trituration of the tissue. Isolated cells were plated on poly-d-lysine–coated coverslips and incubated in ECS at 37°C for 30 minutes before the experiments began. 
Calcium Imaging
Measurement of intracellular Ca2+ concentrations by optical imaging was performed as described previously. 22 24 25 In brief, rod photoreceptor cells were incubated in 2 μM cell-permeant fluorescent dye (Fluo-3-acetoxymethylester; Molecular Probes, Eugene, OR) in ECS at 37°C for 30 minutes and washed with ECS. Intracellular Ca2+ concentrations were measured for up to 6 hours after dissociation. Using time-lapse videomicroscopy (model IX70; Olympus, Tokyo Japan; ORCA-ER, a high-resolution 12-bit digital B/W cooled CCD camera, Hamamatsu, Hamamatsu, Japan; Lambda DG-4 Ultra High Speed Wavelength Switcher with appropriate filter sets; Sutter Instrument Co., Novato, CA; SimplePCI Imaging Software, ver. 5.2; Compix Inc., Imaging Systems/Hamamatsu Photonics Management Corp., Bridgewater, NJ) the fluorescence intensity of the Ca2+ indicator dye in loaded cells was recorded over time while cells were constantly perfused with ECS in a gravity-driven perfusion system at a flow rate of 1 mL/min. Ca2+ transients were identified as the ratio of the fluorescence intensity during drug application (F) over the average baseline fluorescence intensity 10 seconds before drug application (F/F0) with an image acquisition rate of 500 ms. Using standard one-way or multiple ANOVA at least three independent experiments with at least five cells each were performed for each experimental condition. 
Chemicals Used and Their Application before and during Calcium-Imaging Experiments
mGluR8 agonists (l-2-amino-4 phosphonobutyrate, l-AP4; l-serine-O-phosphate, l-SOP; l-glutamate, l-Glu) were obtained from Axxora, LLC (San Diego, CA), and were bath applied in a bolus application directly into the recording chamber. To analyze the involvement of G proteins in the recorded calcium signals, cells were incubated 2 hours before recording and maintained during recordings in ECS containing G-protein modulators (100 μM suramin, 300 μg/L pertussis toxin [PTX], or 400 μg/L cholera toxin [CTX]; EMD Biosciences, La Jolla, CA). 
Peptide Delivery to Dissociated Photoreceptor Cells
To analyze the involvement of the G protein βγ subunit in the observed calcium-signaling pathway, a 28-mer peptide corresponding to a C-terminal portion of the β-adrenergic receptor kinase (WKKELRDAYREAQQLVQRVPKMKNKPRS) was synthesized as its N-terminally acylated and C-terminally amidated form. 26 Dissociated photoreceptor cells were incubated with 100 μM of the 28-mer peptide and a protein-delivery reagent (Chariot; Active Motif, Carlsbad, CA) for two hours at 37°C after the manufacturer’s recommendation. After peptide delivery or control incubation with protein delivery reagent alone cells were washed in ECS and used for calcium imaging experiments. 
Results
Pharmacology of the mGluR8-Mediated Decrease in Intracellular Ca2+ Concentration
As previous studies have shown, mGluR8 is so far the only glutamate receptor found to be expressed by mammalian photoreceptors. 22 23 These studies postulated a role for mGluR8 as a presynaptic autoreceptor that controls Ca2+-dependent neurotransmitter release through modulation of l-type calcium channel activity. However, besides excluding cAMP as a second messenger and an involvement of protein phosphorylation, the identification of the underlying mechanism of action is still lacking. In the present study, we investigated the G-protein pharmacology associated with mGluR8 activity. As in previous studies, 22 we isolated mouse photoreceptors and monitored their intracellular Ca2+ concentration with microfluorimetry, in the presence or absence of G protein modulators (Fig. 1) . We confirmed previous results 22 showing that mGluR8 activation by either the physiological ligand l-glutamate (Fig. 2a)or the group III mGluR agonists l-AP4 (Fig. 1a 1b 1c 1d Fig. 2d)or l-SOP (Fig. 2g)resulted in a significant decrease in the intracellular Ca2+ concentration of rod photoreceptors. Figure 1shows montages of recorded rod photoreceptor cells with their key morphologic features, soma, and inner segment. The outer segment was typically lost, and the axon terminal was typically retracted into the soma (Figs. 1a 1e) . After preincubation with buffer in control experiments, cells responded to an l-AP4 stimulus with a decrease in emission fluorescence intensity correlating to a decrease in intracellular free Ca2+ (Figs. 1b 1c 1d) . However, when cells had been pretreated with PTX, l-AP4-induced activation of mGluR8 had no effect on the intracellular Ca2+ concentration of rod photoreceptors (Figs. 1f 1g 1h) . The effect of PTX was independent of the mGluR8 agonist used and was consistently observed for l-glutamate (Fig. 2b)and l-SOP (Fig. 2h)in addition to l-AP4 (Figs. 1f 1g 1h 2e) . Alternatively to PTX, which inactivates Gi/o proteins and Gt protein/transducin, we pretreated cells with the G protein uncoupler suramin before stimulating them with mGluR8 agonists. Regardless of the type of agonist used, suramin blocked the effect of mGluR8-mediated reduction of intracellular free Ca2+ levels (Figs. 2c 2f 2i) . Preincubation of rod photoreceptor cells with CTX, an activator of Gs protein had no effect on mGluR8 activity (Table 1)
Mediation of the Effects of mGluR8 by the βγ Subunit of Heterotrimeric G Proteins
To identify which subunit(s) of the PTX-sensitive heterotrimeric G protein potentially mediate(s) mGluR8 activity, we used G protein βγ subunit inhibition in isolated rod photoreceptor cells. To this end, we delivered a 28-mer peptide corresponding to a C-terminal portion of the β-adrenergic receptor kinase (β-ARK peptide) into photoreceptors using the protein-delivery reagent (Chariot; Active Motif). β-ARK peptide has been shown to interact specifically with the βγ subunit of signal-transducing heterotrimeric G proteins and to block βγ activity effectively. 26 Peptide delivery of β-ARK peptide into rod photoreceptor cells abolished mGluR8 activity (Table 1) . Controls treated with the protein delivery reagent alone showed no effect on mGluR8 activity. 
In all groups—control, PTX, CTX, suramin, and β-ARK peptide–treated cells—subsequent exposure to the L-type calcium channel blocker nifedipine still decreases intracellular Ca2+ levels, as reported previously, 22 indicating that the use of pharmacologic modulators of G protein function had no direct effect on L-type calcium channel activity. 
Discussion
mGluR8 has a unique distribution pattern in the retina, 16 22 23 potential relevance for neurodegenerative diseases of the retina, 17 and a unique pharmacological profile, 18 19 20 all of which make mGluR8 interesting for the investigation of retinal physiology. mGluR8 activity has been associated with the activation of Gi and Go proteins and the modulation of second-messenger systems. 16 18 27 28 In addition to the coupling to adenylyl cyclase, a regulation of potassium and calcium channel activity by mGluR8 resulting in numerous effects of the receptor in the central nervous system has been reported in several studies. 18 22 28 29 30 31 The present study focused on two related aspects of mGluR8 function, the modulation of the intracellular free Ca2+ levels and negative feedback signaling to regulate neurotransmitter release presynaptically. 22 28 32 33 34 The latter process being Ca2+-dependent ties in directly with the modulation of the intracellular Ca2+ concentration by mGluR8 through inhibition of plasma membrane calcium channel activity. The results of the present study support a mechanism of action for mGluR8 potentially using the βγ subunit of signal-transducing heterotrimeric G proteins. 
We explored pharmacologic characteristics of mGluR8 in rod photoreceptor cells using pharmacologic tools for G protein signaling. With our findings that the mGluR8-mediated reduction of intracellular free Ca2+ levels is PTX sensitive and CTX insensitive, the question arises as to which signal-transducing heterotrimeric G proteins mGluR8 potentially couples to exert its effects on the intracellular Ca2+ concentration in rod photoreceptors. Several studies indirectly indicate that of the PTX-sensitive Gi family of G proteins, Go, 35 36 37 38 39 and Gz 40 are not expressed by photoreceptors. There is the possibility that photoreceptors express the PTX-sensitive Gi2 but not Gi1 or Gi3. 41 Even though the immunolocalization data presented in Oguni et al. 41 do not conclusively prove expression of Gi2 by photoreceptor cells, such an expression combined with data presented in the present study suggests a potential role for Gi2 in mediating mGluR8’s effects. Of the other G proteins that have been described in photoreceptors, Gt/transducin 42 43 and G11, 44 only Gt is PTX sensitive 45 46 and represents a potential signaling partner of mGluR8 using a PTX-sensitive and CTX-insensitive signaling pathway, as shown in the present study. This opens up the possibility that Gt/transducin, besides its major function in photoreceptor outer segments, 42 43 may also have a secondary function in photoreceptor somata and the outer plexiform layer. Diurnal changes in Gt/transducin expression levels that have been reported for Gt/transducin’s function in phototransduction 47 48 could therefore also affect mGluR8 signaling. 
Indirect physiological evidence indicates the involvement of PTX-sensitive G proteins in the signaling mediated by neurotransmitters other than l-glutamate systems in vertebrate photoreceptors. 49 50 51 In a related study, activation of glutamate transporters in vertebrate rod photoreceptors was able to inhibit presynaptic Ca2+ currents. 52 In the same study, 52 no effect on mGluRs was detected, which is potentially due to species differences in receptor pharmacology and/or distribution. 
The second finding of the present study, the involvement of the βγ subunit of signal-transducing heterotrimeric G proteins in the mGluR8-mediated reduction of intracellular Ca2+ levels, is supported by evidence from several studies identifying Gα-independent signaling pathways. G protein–gated inwardly rectifying K+ (GIRK) channels, 53 several voltage-dependent calcium channels, 54 55 56 57 and the fusion machinery of vesicular release 58 have been shown to be direct targets of Gβγ activity. With the effects of Gβγ on the fusion machinery of vesicular release being a calcium-independent process 58 and the clear dependence of mGluR8 activity in rod photoreceptors on extracellular calcium, 22 Gβγ’s effects potentially target voltage-dependent calcium channels directly or GIRK channels, thereby indirectly influencing voltage-dependent calcium channels. Expression of voltage-dependent calcium channels in photoreceptors has been described, and predominantly L-type channels had been identified. 59 60 61 In a recently published study, the interdependence of intracellular Ca2+ concentration, plasma membrane depolarization, and neurotransmitter vesicle release kinetics were studied in rod photoreceptors. 62 In this study, the direct coupling of intracellular Ca2+ concentration to plasma membrane depolarization 62 expands the physiological relevance of L-type calcium channels in rod photoreceptors. 59 60 61 63 64 However, despite the strong evidence for L-type calcium channels as the source of the standing dark current of Ca2+ into the cytosol from the extracellular medium, the involvement of other sources cannot be excluded. 22  
Even though the expression of GIRK channels has not been shown directly, the presence of inwardly rectifying K+ currents in rod photoreceptors 63 64 allows for a potential involvement of Gβγ-regulated GIRK channel activity in the mGluR8-mediated regulation of intracellular free-Ca2+ levels in rod photoreceptors. The exact identity of the Gβγ subunits involved has yet to be determined, but, based on previous studies, potentially involves Gβ1 and Gγ1 in rod photoreceptors 65 and may involve different subunits in cone photoreceptors. 65 66 67 Even though the results from the experiments using the G protein βγ subunit inhibiting (β-ARK) peptide are conclusive, an involvement of Gi family α subunits cannot be excluded because of a lack of inhibitors that selectively inhibit Gi family α but not βγ subunits. 
 
Figure 1.
 
The decrease in the intracellular Ca2+ concentrations of rod photoreceptor cells induced by application of l-AP4, a group-III mGluR agonist, was PTX sensitive. Ca2+ transients in isolated mouse rod photoreceptor cells ester loaded with the Ca2+-indicator dye fluo-3 were measured optically with time-lapse video microscopy by monitoring changes in the intensity of the fluorescent tracer’s emission wavelength maximum. Montages of recordings from two representative experiments are displayed with (ad) showing control preincubated cells and (eh) showing cells that had been preincubated with PTX. Selected images visualize the intracellular Ca2+ concentration at (time point: 0 seconds; b, f), during (time point: 5 seconds; c, g), and after (time point: 20 seconds; d, h) application of l-AP4. Whereas in control cells (bd) the high intracellular Ca2+ concentration drops significantly after stimulation with the mGluR8 agonist, the same application of l-AP4 has no effect in cells pretreated with PTX (fh). All cells of the control and experimental groups showed a constant low-level decrease in fluorescence intensity due to bleaching of the indicator dye that is not stimulus related. (a, e) Differential interference contrast (DIC) images of the cells indicating their morphology. Arrows: selected typical rod photoreceptor cells. Scale bars, 10 μm.
Figure 1.
 
The decrease in the intracellular Ca2+ concentrations of rod photoreceptor cells induced by application of l-AP4, a group-III mGluR agonist, was PTX sensitive. Ca2+ transients in isolated mouse rod photoreceptor cells ester loaded with the Ca2+-indicator dye fluo-3 were measured optically with time-lapse video microscopy by monitoring changes in the intensity of the fluorescent tracer’s emission wavelength maximum. Montages of recordings from two representative experiments are displayed with (ad) showing control preincubated cells and (eh) showing cells that had been preincubated with PTX. Selected images visualize the intracellular Ca2+ concentration at (time point: 0 seconds; b, f), during (time point: 5 seconds; c, g), and after (time point: 20 seconds; d, h) application of l-AP4. Whereas in control cells (bd) the high intracellular Ca2+ concentration drops significantly after stimulation with the mGluR8 agonist, the same application of l-AP4 has no effect in cells pretreated with PTX (fh). All cells of the control and experimental groups showed a constant low-level decrease in fluorescence intensity due to bleaching of the indicator dye that is not stimulus related. (a, e) Differential interference contrast (DIC) images of the cells indicating their morphology. Arrows: selected typical rod photoreceptor cells. Scale bars, 10 μm.
Figure 2.
 
The mGluR8 mediated decrease in the intracellular Ca2+ concentration of isolated rod photoreceptor cells was sensitive to G protein modulation. Traces of individual representative experiments from control cells (a, d, g) and cells that had been pretreated with PTX (b, e, h) or suramin (c, f, i) are shown. The fluorescence intensity of the Ca2+ indicator dye was corrected for bleaching and normalized as fluorescence intensity over baseline fluorescence intensity (F/F0) as a measure of changes in the intracellular Ca2+ concentration over time. Single bolus applications (arrows) of mGluR8 the agonists l-glutamate (Glu; ac), l-AP4 (df), and l-SOP (gi) lead to significant reduction of the intracellular Ca2+ concentration of isolated rod photoreceptor cells (a, d, g). In contrast, application of the same stimulus has no effect on cells pretreated with PTX (b, e, h) or suramin (c, f, i) during and after drug application.
Figure 2.
 
The mGluR8 mediated decrease in the intracellular Ca2+ concentration of isolated rod photoreceptor cells was sensitive to G protein modulation. Traces of individual representative experiments from control cells (a, d, g) and cells that had been pretreated with PTX (b, e, h) or suramin (c, f, i) are shown. The fluorescence intensity of the Ca2+ indicator dye was corrected for bleaching and normalized as fluorescence intensity over baseline fluorescence intensity (F/F0) as a measure of changes in the intracellular Ca2+ concentration over time. Single bolus applications (arrows) of mGluR8 the agonists l-glutamate (Glu; ac), l-AP4 (df), and l-SOP (gi) lead to significant reduction of the intracellular Ca2+ concentration of isolated rod photoreceptor cells (a, d, g). In contrast, application of the same stimulus has no effect on cells pretreated with PTX (b, e, h) or suramin (c, f, i) during and after drug application.
Table 1.
 
Summary of the Pharmacologic Modulation of mGluR8 Responses in Rod Photoreceptor Cells
Table 1.
 
Summary of the Pharmacologic Modulation of mGluR8 Responses in Rod Photoreceptor Cells
Drug n F/F0 (%) SEM (%) SL
l-Glutamate 16 88 7 *
 Suramin/Glu 16 102 2 NS
 PTX/Glu 16 102 3 NS
 CTX/Glu 19 90 6 *
 β-ARK peptide/Glu 18 101 1 NS
l-SOP 30 91 4 *
 Suramin/l-SOP 20 101 1 NS
 PTX/l-SOP 21 100 1 NS
 CTX/l-SOP 17 92 3 *
 β-ARK peptide/l-SOP 16 100 2 NS
l-AP4 33 89 4 *
 Suramin/l-AP4 24 100 1 NS
 PTX/l-AP4 28 101 1 NS
 CTX/l-AP4 25 88 5 *
 β-ARK peptide/l-AP4 19 99 2 NS
The authors thank Margaret, Richard, and Sara Koulen for generous support and encouragement. 
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Figure 1.
 
The decrease in the intracellular Ca2+ concentrations of rod photoreceptor cells induced by application of l-AP4, a group-III mGluR agonist, was PTX sensitive. Ca2+ transients in isolated mouse rod photoreceptor cells ester loaded with the Ca2+-indicator dye fluo-3 were measured optically with time-lapse video microscopy by monitoring changes in the intensity of the fluorescent tracer’s emission wavelength maximum. Montages of recordings from two representative experiments are displayed with (ad) showing control preincubated cells and (eh) showing cells that had been preincubated with PTX. Selected images visualize the intracellular Ca2+ concentration at (time point: 0 seconds; b, f), during (time point: 5 seconds; c, g), and after (time point: 20 seconds; d, h) application of l-AP4. Whereas in control cells (bd) the high intracellular Ca2+ concentration drops significantly after stimulation with the mGluR8 agonist, the same application of l-AP4 has no effect in cells pretreated with PTX (fh). All cells of the control and experimental groups showed a constant low-level decrease in fluorescence intensity due to bleaching of the indicator dye that is not stimulus related. (a, e) Differential interference contrast (DIC) images of the cells indicating their morphology. Arrows: selected typical rod photoreceptor cells. Scale bars, 10 μm.
Figure 1.
 
The decrease in the intracellular Ca2+ concentrations of rod photoreceptor cells induced by application of l-AP4, a group-III mGluR agonist, was PTX sensitive. Ca2+ transients in isolated mouse rod photoreceptor cells ester loaded with the Ca2+-indicator dye fluo-3 were measured optically with time-lapse video microscopy by monitoring changes in the intensity of the fluorescent tracer’s emission wavelength maximum. Montages of recordings from two representative experiments are displayed with (ad) showing control preincubated cells and (eh) showing cells that had been preincubated with PTX. Selected images visualize the intracellular Ca2+ concentration at (time point: 0 seconds; b, f), during (time point: 5 seconds; c, g), and after (time point: 20 seconds; d, h) application of l-AP4. Whereas in control cells (bd) the high intracellular Ca2+ concentration drops significantly after stimulation with the mGluR8 agonist, the same application of l-AP4 has no effect in cells pretreated with PTX (fh). All cells of the control and experimental groups showed a constant low-level decrease in fluorescence intensity due to bleaching of the indicator dye that is not stimulus related. (a, e) Differential interference contrast (DIC) images of the cells indicating their morphology. Arrows: selected typical rod photoreceptor cells. Scale bars, 10 μm.
Figure 2.
 
The mGluR8 mediated decrease in the intracellular Ca2+ concentration of isolated rod photoreceptor cells was sensitive to G protein modulation. Traces of individual representative experiments from control cells (a, d, g) and cells that had been pretreated with PTX (b, e, h) or suramin (c, f, i) are shown. The fluorescence intensity of the Ca2+ indicator dye was corrected for bleaching and normalized as fluorescence intensity over baseline fluorescence intensity (F/F0) as a measure of changes in the intracellular Ca2+ concentration over time. Single bolus applications (arrows) of mGluR8 the agonists l-glutamate (Glu; ac), l-AP4 (df), and l-SOP (gi) lead to significant reduction of the intracellular Ca2+ concentration of isolated rod photoreceptor cells (a, d, g). In contrast, application of the same stimulus has no effect on cells pretreated with PTX (b, e, h) or suramin (c, f, i) during and after drug application.
Figure 2.
 
The mGluR8 mediated decrease in the intracellular Ca2+ concentration of isolated rod photoreceptor cells was sensitive to G protein modulation. Traces of individual representative experiments from control cells (a, d, g) and cells that had been pretreated with PTX (b, e, h) or suramin (c, f, i) are shown. The fluorescence intensity of the Ca2+ indicator dye was corrected for bleaching and normalized as fluorescence intensity over baseline fluorescence intensity (F/F0) as a measure of changes in the intracellular Ca2+ concentration over time. Single bolus applications (arrows) of mGluR8 the agonists l-glutamate (Glu; ac), l-AP4 (df), and l-SOP (gi) lead to significant reduction of the intracellular Ca2+ concentration of isolated rod photoreceptor cells (a, d, g). In contrast, application of the same stimulus has no effect on cells pretreated with PTX (b, e, h) or suramin (c, f, i) during and after drug application.
Table 1.
 
Summary of the Pharmacologic Modulation of mGluR8 Responses in Rod Photoreceptor Cells
Table 1.
 
Summary of the Pharmacologic Modulation of mGluR8 Responses in Rod Photoreceptor Cells
Drug n F/F0 (%) SEM (%) SL
l-Glutamate 16 88 7 *
 Suramin/Glu 16 102 2 NS
 PTX/Glu 16 102 3 NS
 CTX/Glu 19 90 6 *
 β-ARK peptide/Glu 18 101 1 NS
l-SOP 30 91 4 *
 Suramin/l-SOP 20 101 1 NS
 PTX/l-SOP 21 100 1 NS
 CTX/l-SOP 17 92 3 *
 β-ARK peptide/l-SOP 16 100 2 NS
l-AP4 33 89 4 *
 Suramin/l-AP4 24 100 1 NS
 PTX/l-AP4 28 101 1 NS
 CTX/l-AP4 25 88 5 *
 β-ARK peptide/l-AP4 19 99 2 NS
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