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Physiology and Pharmacology  |   June 2012
GABA-Induced Relaxation of Porcine Retinal Arterioles in Vitro Depends on Inhibition from the Perivascular Retina and Is Mediated by GABAC Receptors
Author Notes
  • From the Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark. 
  • Corresponding author: Toke Bek, Department of Ophthalmology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark; toke.bek@mail.tele.dk
Investigative Ophthalmology & Visual Science June 2012, Vol.53, 3309-3315. doi:10.1167/iovs.12-9838
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      Toke Bek, Kim Holmgaard; GABA-Induced Relaxation of Porcine Retinal Arterioles in Vitro Depends on Inhibition from the Perivascular Retina and Is Mediated by GABAC Receptors. Invest. Ophthalmol. Vis. Sci. 2012;53(7):3309-3315. doi: 10.1167/iovs.12-9838.

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

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Abstract

Purpose.: To study the dependence of γ-aminobutyric acid (GABA)-induced relaxation of retinal arterioles on the glutamate agonist N-methyl-D-aspartate (NMDA), adenosine triphosphate (ATP), prostaglandin E2 (PGE2), and adenosine, and to characterize the type and location of GABA-receptor(s) mediating GABA-induced relaxation of retinal arterioles.

Methods.: Porcine retinal arterioles were mounted in a wire myograph, and the effects of agonists and antagonists to NMDA, ATP, PGE2, and adenosine on GABA-induced relaxation were studied. Additionally, experiments were conducted to study relaxation induced by agonists to specific GABA receptors, and GABA-induced relaxation in the presence of specific GABA antagonists. Finally, immunohistochemistry was performed to identify the location of GABAC receptors in the porcine retina.

Results.: GABA induced vasorelaxation during blocking of the glutamate NMDA receptor, PGE2 receptor, and ATP synthesis degradation, but not during blocking of the adenosine receptor or by agonists to any of these compounds. The vasorelaxing effect of GABA could be elicited by a specific GABAC agonist, but not by specific GABAA or GABAB agonists, and could be blocked by a specific GABAC antagonist, but not by specific GABAA or GABAB antagonists. GABAC receptor subunits could be identified in the ganglion cell layer, and at the border between the outer plexiform and inner nuclear layers.

Conclusions.: GABA-induced relaxation of porcine retinal vessels is mediated by the GABAC receptor in the perivascular retinal tissue, and depends on blocking of the glutamate NMDA receptor, prostaglandin E2 receptor, or ATP degradation.

Introduction
The functional integration of visual input in the retina depends on inhibitory signaling, which is mediated by a number of neurotransmitters, including γ-aminobutyric acid (GABA). 1 The inhibitory action of GABA is mediated by metabotropic GABAB receptors identified on neuronal elements in all layers of the retina, 2 and by ionotropic GABAA and GABAC receptors on bipolar cell axon terminals. Additionally, GABAA receptors have been identified on dendrites of retinal amacrine and ganglion cells, and GABAC receptors on postsynaptic membranes in the outer plexiform layer. 3 Evidence suggests that the two ionotropic GABA receptors differ by GABAA mediating fast transient inhibitory responses, whereas GABAC receptors are more sensitive, and mediate slowly onset and sustained inhibitory responses. 4,5  
It has been shown previously that GABA may induce relaxation of retinal arterioles during inhibition of the glutamate N-methyl-D-aspartate (NMDA) receptor in the perivascular retinal tissue, 6 and that stimulation of this receptor can induce relaxation, which is mediated by adenosine, and depends on adenosine triphosphate (ATP) and prostaglandin E (PGE2). 7,8 Additionally, a recent study has shown that lack of the GABAC receptor rho-1 subunit may increase retinal vascular permeability similarly to the pathological changes observed in retinal hypoxic conditions, 9 suggesting a role of GABAC for retinal vascular function. However, to our knowledge the detailed mechanisms underlying GABAergic control of retinal vascular tone have not been elucidated in detail. 
Therefore, the purpose of the study was to investigate how changes in retinal vascular tone induced by GABA depends on agonists and antagonists to NMDA, ATP, PGE2, and adenosine, and to identify the type and location of GABA receptor(s) involved in GABA-induced changes in tone on porcine retinal arterioles in vitro. 
Materials and Methods
Solutions and Compounds
Solutions
Physiological saline solution (PSS) containing (in mM) NaCl 119, KCl 4.7, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, CaCl2 1.6, EDTA 0.026, glucose 5.5, and HEPES 5.0 (pH 7.4), was used for transportation, storage, and the pharmacologic experiments. During dissection and normalization of the vessel diameter, a similar solution was used in which CaCl2 had been omitted (Ca2+-free PSS). 
Compounds
Tone Modulators
All compounds were purchased from Sigma-Aldrich (Vallensbaek, Denmark), apart from prostaglandin agonists and antagonists, which were purchased from Cayman Chemicals (Tallinn, Estonia). 
Pre-contraction was performed with the thromboxane analogue U46619 (9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F), and the additional modulation of tone was performed with the following: 
Agonists: The glutamate agonist NMDA, ATP, adenosine, and PGE2, an agonist to the EP1-4 receptors. 
Antagonists: The glutamate NMDA-receptor antagonist DL-2-amino-5-phosphonopentanoic acid (DL-APV), the ectonucleotidase inhibitor adenosine-5′-O-methylenediphosphate (AOPCP), the adenosine receptor blocker 8-P-sulfophenyl-theophyllin (8-PSPT), the unspecific prostaglandin synthesis inhibitor ibuprofen, and the selective EP1 receptor antagonist SC-19220 (14060). 
U46619 and NMDA were dissolved in distilled water as stock solutions 103 times the highest concentration used in the experiments, and stored frozen for later use. All other compounds were prepared on the day of the experiment. DL-APV was dissolved in 5 × 10−5 NaOH, and the other compounds were dissolved in PSS. 
Gaba-Mediators
All compounds were purchased from Tocris Bioscience (Bristol, UK), apart from 1,2,5,6-tetrahydropyridin-4-yl)methylphosphonic acid (TPMPA), isoguvacine, and GABA, which were purchased from Sigma-Aldrich, and 2-methyl-imidazole acetic acid 10 (structure 8a) generously provided by Bente Frølund, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. 
GABA-agonists included GABA, the GABAA-agonist isoguvacine, the GABAB-agonist baclofen, and the GABAC agonist 2-methyl-imidazole acetic acid. 
GABA-antagonists included the GABAA-antagonists bicuculline and picrotoxin, the GABAB-antagonist GCGP 55845, and the GABAC-antagonist TPMPA. All compounds were prepared on the day of the experiment and dissolved in PSS. 
Antibodies
Numbers in parentheses refer to the product list of from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Antibodies included goat anti-human GABAA Rρ1 C-20, and GABAC Rρ3 Y-16 primary antibodies, and rabbit anti-goat IgG-AP (sc-2949) secondary antibody. 
Tissue
The technique has been described in detail previously, 6,8 and all experiments adhered to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Porcine eyes were collected from a local slaughterhouse and were transported to the laboratory in 4°C PSS within one hour. The eyes were bisected by a frontal section through the equator, the vitreous was removed, and the retina was detached from the underlying pigment epithelium by injection of PSS between these two structures. The first order arteriole was located by its smaller diameter, as compared to the adjoining venule and the capillary-free zone surrounding the arteriole. Subsequently, an arteriolar segment with a length of 2 mm with approximately 2 mm retinal tissue attached on each side of the vessel was dissected from the retina. 
Mounting Procedure
The vascular segment was placed in the chamber of a small vessel myograph (610M Multi-Myograph, Danish Myo Technology, Aarhus, Denmark) and mounted on 25 μm diameter tungsten wires. After mounting, the preparation was suspended freely in PSS between the myograph jaws, and bubbling of the bath was commenced with a mixture of 95% atmospheric air and 5% CO2 to result in a pH of 7.4. 
Normalization
Normalization was performed according to procedures described previously. 11 In short, the arteriolar diameter was increased in four steps in Ca2+-free PSS solution, and the passive tensions (corresponding to transmural pressures between 0 and approximately 70 mm Hg) were measured. This diameter-tension relationship was exponential, and the intercept between this curve and a straight line based on the Laplace equation (wall tension = transmural pressure × radius) with the transmural pressure set to 70 mm Hg was calculated. Using the built-in micrometer screw, the jaws of the myograph were adjusted to 93.5% of the intercept length, at which the arteriole can develop the maximum tone (i.e., the optimal length for contraction of the vascular smooth muscle cells). 
Experimental Protocols
One arteriole was used from each animal, and for each experimental condition at least six observations were obtained. Three different experimental series were conducted as shown in Figure 1. Each experiment consisted of the following steps: 
Figure 1. 
 
The experimental protocol.
Figure 1. 
 
The experimental protocol.
  • A.  
    Addition of tone modulator(s): a tone modulator was added to the chamber to be present throughout the experiment, and the arteriole was allowed to equilibrate for 10 minutes in PSS for the tone to stabilize.
  • B.  
    Pre-contraction: the vessel was contracted by adding 10−6 M U46619.
  • C.  
    Concentrations-response experiments: a GABA agonist was added to the tissue chamber in increasing concentrations, with one log unit interval between 10−9 and 10−3 M. Each new concentration was added after at least 3 minutes or when the tone had stabilized.
  • D.  
    Removal of the perivascular tissue: the myograph was moved to a stereo microscope, and the perivascular retinal tissue was removed gently within two minutes using two pairs of fine forceps (Inox 5; DJ-Instruments, Copenhagen, Denmark) without touching the arterioles.
  • E.  
    Repetition of the concentration-response experiment: the myograph was replaced in the recording unit and the procedures described in steps A to C were repeated on the isolated arteriole.
  • F.  
    Maximal relaxation: finally, papaverine 10−4 M was added to determine maximal relaxation.
Series 1
  •  
    Step A. The tone modulator was the glutamate NMDA receptor antagonist DL-APV in the following concentrations: 5 × 10−5 M, 10−4 M, or none (control).
  •  
    Step C. The agonist was GABA or none (time control).
Series 2
  •  
    Step A. The tone modulators were the ectonucleotidase inhibitor AOPCP 10−4 M, adenosine receptor blocker 8-PSPT 5 × 10−4 M, prostaglandin synthesis inhibitor ibuprofen 10−5 M, selective EP1 receptor antagonist SC-19220 5 × 10−5 M, glutamate agonist NMDA 10−3 M, ATP 10−3 M, adenosine 10−3 M, and PGE2 10−3 M.
  •  
    Step B. The experiments with the agonists NMDA, ATP, and adenosine were repeated without contraction with U46619.
  •  
    Step C. The agonist was GABA.
Series 3
  •  
    Step A. The tone modulators were DL-APV 5 × 10−5 M combined with each of the following: the GABAA-antagonists bicuculline 10−5 M and picrotoxin 1.5 × 10−5 M; the GABAB-antagonist GCGP55845 5 × 10−6 M, bicuculline, and GCGP55845 together; and the GABAC-antagonist TPMPA 10−5 M, or none (control). The antagonist concentrations were defined on the basis of the EC50 provided by the manufacturer to ensure full occupancy of the receptor.
  •  
    Step C. The agonists were GABA in experiments with DL-APV and the antagonists 1-5, and the following specific agonists in experiments with DL-APV only: the GABAA agonist isoguvacine, GABAB agonist baclofen, and GABAC agonist 5-methyl-imidazole-4-acetate.
Immunohistochemistry
Two porcine eyes from different animals were divided by a sagittal section near the optic disk and were fixated in 4% paraformaldehyde for 4 hours. One hemiglobe of each eye was dehydrated in ethanol, passed to petroleum, and embedded in paraffin. Subsequently, histological sections with a thickness of four microns were sampled from the cut edge of the eye. After storage for at least 3 months, the sections were deparaffined and rehydrated, followed by demasking of antibodies in citrate buffer at pH 6.0 at 60°C for one minute. The sections were incubated with each of the goat-anti-human/mouse primary antibodies against GABAC and GABAA primary antibodies in concentrations of 1:100, 1:200, 1:400, and 1:800 overnight at 4°C. Subsequently, endogenous peroxidase was blocked with 3% H2O2 for 10 minutes, and the sections were incubated with rabbit-anti-goat secondary antibody for 30 minutes, with horseradish peroxidase labeled anti-rabbit antibody for 30 minutes, and was developed with diaminobenzidine. The labeling was amplified with CuSO4 for 5 minutes followed by counterstaining with hematoxylin. 
Data Analysis
The tone produced by the mounted arterioles was sampled at 1 Hz and displayed on a computer monitor as a function of time during the experiments. The data were stored in a Microsoft Excel (Hellerup, Denmark) file for subsequent analysis. The tone obtained after addition of each concentration of an agonist was normalized to the tone produced after addition of 10−6 M U46619 (100%) and the tone after relaxation with papaverine (0%). The normalized tone was plotted as a function of the agonist concentration. Using the software GraphPad Prism version 4.02 (La Jolla, CA), the data from experiments showing saturation kinetics at high agonist concentrations could be fitted to the Michaëlis-Menten equation to obtain the EC50
Statistical Analysis
In all experimental series repeated measurements ANOVA was used to test whether addition of agonist changed the vascular tone significantly. Subsequently, in series 1, one-way ANOVA was used to test whether the tone obtained at the different experimental conditions differed at each GABA concentration, and when a significant difference was found the analysis was followed by pair-wise comparisons of all combinations of two of the studied variables using Student's unpaired t-test. In series 2 and 3, Student's paired t-test was used to test whether the response at each agonist concentration differed before and after removal of the perivascular retina. Dunnett's correction for multiple comparisons was performed for repeated t-tests at different agonist concentrations within the same experiment. 
Results
Figure 2 shows the results from series 1. It appears that in the presence of perivascular retinal tissue GABA alone (solid line) did not change vascular tone significantly (P = 0.22, repeated measurements ANOVA), which did not differ from the time controls (not shown). In the presence of 5 × 10−5 M DL-APV, GABA-induced significant relaxation at the highest concentration where the normalized tone was reduced to approximately half of the maximum level (broken line). DL-APV in a concentration of 10−4 M further increased the relaxing effect of GABA to become significant at the four highest concentrations (dotted line). 
Figure 2. 
 
Relaxation induced by GABA in porcine retinal arterioles with preserved perivascular retinal tissue without DL-APV (solid line), and in the presence of DL-APV in a concentration of 5 × 10−5 M (broken line) and 10−4 M (dotted line). The asterisk indicates the GABA concentration where 5 × 10−5 M DL-APV induced significantly more relaxation in the absence of DL-APV (control), whereas double crosses indicate concentrations where 10−4 M DL-APV induced significantly more relaxation than control.
Figure 2. 
 
Relaxation induced by GABA in porcine retinal arterioles with preserved perivascular retinal tissue without DL-APV (solid line), and in the presence of DL-APV in a concentration of 5 × 10−5 M (broken line) and 10−4 M (dotted line). The asterisk indicates the GABA concentration where 5 × 10−5 M DL-APV induced significantly more relaxation in the absence of DL-APV (control), whereas double crosses indicate concentrations where 10−4 M DL-APV induced significantly more relaxation than control.
Figure 3 shows the results from series 2. It appears that GABA-induced relaxation in the presence of 5 × 10−5 M DL-APV disappeared after removal of the perivascular retinal tissue (Fig. 3, top left). GABA also induced relaxation depending on the perivascular retinal tissue in the presence of the ectonucleotidase inhibitor AOPCP (Fig. 3, top right) and the EP2 receptor blocker SC19220 (Fig. 3, middle left), but was unable to induce relaxation after addition of the COX-inhibitor ibuprofen (Fig. 3, middle right) and the adenosine receptor antagonist 8-PSPT (Fig. 3, bottom). The agonists NMDA, PGE2, ATP, and adenosine had no effect on GABA-induced vascular tone, neither on contracted nor non-contracted arterioles (not shown). 
Figure 3. 
 
GABA-induced relaxation in the presence of perivascular retinal tissue and DL-APV (top left), the ectonucleotidase inhibitor AOPCP (top right), the EP2 receptor blocker SC19220 (middle left), but not in the presence of the COX-inhibitor ibuprofen (middle right), and the adenosine receptor antagonist 8-PSPT (bottom).
Figure 3. 
 
GABA-induced relaxation in the presence of perivascular retinal tissue and DL-APV (top left), the ectonucleotidase inhibitor AOPCP (top right), the EP2 receptor blocker SC19220 (middle left), but not in the presence of the COX-inhibitor ibuprofen (middle right), and the adenosine receptor antagonist 8-PSPT (bottom).
Figure 4 shows the effect of specific GABA agonists (left column) and antagonists (right column) on retinal vascular tone in the presence of 5 × 10−5 M DL-APV. 
Figure 4. 
 
Left: the effect of specific agonists to, respectively, GABAA (upper), GABAB (middle), and GABAC (lower) receptors. Right: The effect of GABA after specific inhibition of each of the three GABA receptors A (upper), B (middle), and C (lower).
Figure 4. 
 
Left: the effect of specific agonists to, respectively, GABAA (upper), GABAB (middle), and GABAC (lower) receptors. Right: The effect of GABA after specific inhibition of each of the three GABA receptors A (upper), B (middle), and C (lower).
In the presence of the perivascular retinal tissue, the GABAA agonist isoguvacine (upper left) and the GABAB agonist baclofen (middle left) had no significant effect on vascular tone, whereas the GABAC agonist 5-methyl-imidazol-4-acetate (lower left) induced significant relaxation of the retinal arterioles at the highest concentration. Conversely, blocking of the GABAA receptor with picrotoxin (upper right) and bicuculline (not shown), and blocking the GABAB receptor with GCGP55845 (middle right), as well as bicuculline and GCGP558er together (not shown) were unable to eliminate GABA-induced vasorelaxation, while blocking of the GABAC receptor with TPMPA (lower right) eliminated this relaxation. 
Figure 5 shows immunoreactivity to the ionotropic GABA receptor subunits. Figure 5A shows immunoreactivity to the GABAC receptor subunit in cells located in the ganglion cell layer, and at the border zone between the outer plexiform and the inner nuclear layers. Figure 5B shows immunoreactivity to the GABAA receptor corresponding to the plexiform layers and the nerve fiber layer. None of the antibodies showed immunoreactivity to components of the retinal vascular walls. 
Figure 5. 
 
Immunoreactivity to subunits of (A) the GABAC receptor in a sub-population of cells in the ganglion cell layer, and (B) the GABAA receptor located in the plexiform layers. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
Figure 5. 
 
Immunoreactivity to subunits of (A) the GABAC receptor in a sub-population of cells in the ganglion cell layer, and (B) the GABAA receptor located in the plexiform layers. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
Discussion
The results of the study suggest that GABA may have a role for the regulation of retinal vascular tone, and that this effect may be mediated by the GABAC receptor through a mechanism that involves inhibition of glutamate, ATP, and PGE2. The finding that the relaxing effect of GABA on porcine retinal arterioles depended on the NMDA receptor blocker DL-APV confirms previous findings. 6 The study showed that this effect of DL-APV was concentration-dependent, and the subsequent experiments aimed at elucidating the mechanism of action of GABA-induced vasorelaxation were conducted with a DL-APV concentration similar to that used previously, which was confirmed to generate a baseline tone from which contraction and relaxation could be studied. 
Pre-contraction was performed with a concentration of U46619 that ensured full contraction so that possible minimal relaxation of the arterioles could be studied. 12 It has been shown previously that NMDA may stimulate the release of adenosine to relax retinal vascular smooth muscle cells through a pathway that involves ATP and PGE2 in the perivascular retinal tissue. 7,8 However, our study showed that blocking of the effect of NMDA, ATP, and PGE2, but not of adenosine, was a precondition for GABA to induce vasorelaxation. This finding of relaxing compounds that block the effect of other relaxing compounds suggests that the regulation of relaxation in retinal arterioles is complex and involves several mutually interacting pathways. The physiologic background for this finding is unknown, but may reflect a more generalized pattern of vascular tone regulation where changes in neuronal activity via the neurotransmitters glutamate and GABA modulate relaxation, whereas contraction is mediated by other mechanisms. Thus, recent studies have shown that the regulation of the diameter of arterioles and capillaries in the brain involve interactions between the neurotransmitters glutamate and GABA, prostaglandin E, and purinergic compounds, 13,14 with astrocytes acting as a bridge to relay information about neural activity to the blood vessels. Our findings indicate that similar mechanisms may be involved in regulating retinal blood flow, and cellular elements involved in the regulation of retinal vascular tone that may constitute a retinal correlate to these brain astrocytes have been identified recently external to vascular smooth muscle cells in the retinal vascular wall. 15  
Experiments were conducted without pre-contraction to study whether GABA could induce contraction in the presence of the agonists NMDA, ATP, and PGE2. However, the fact that these agonists could not counteract the observed vasorelaxation facilitated by antagonists to these specific signaling molecules indicates that the studied compounds are involved only in relaxation of retinal arterioles, and that vasocontraction is stimulated by other pathways. It also is noteworthy that GABA-induced relaxation was mediated by the specific PGE2 receptor blocker SC19220, but was blocked by the unspecific COX inhibitor ibuprofen. This confirms the results from other studies and suggests that several COX products with opposite effects are involved in the tone regulation of retinal arterioles. 8  
The vasorelaxing effect of GABA was investigated using specific agonists and antagonists to the three GABA receptors, and picrotoxin and bicuculline were found to be useful as specific GABAA receptor antagonists, although it has been shown in some experimental setups that this receptor may be less sensitive to bicuculline in mammals. 4 On the basis of these experiments, it was shown that GABA-induced vasorelaxation was mediated by the GABAC receptor. The specific role of this receptor in retinal signal transmission is unknown, but several studies have shown that the GABAC receptor can mediate inhibitory signaling in the retina as a response to light stimulation in fish 16,17 and in mice. 18 Our findings, and that of a recent study showing that lack of GABAC receptor function increases the permeability of retinal arterioles microvessels, 9 suggest that the specific function of the GABAC receptor involves the regulation of retinal vascular tone. This is opposed to the role of the GABAA and GABAB receptors, which have been found to be involved only in retinal neurotransduction. 19  
The findings might be interpreted that the biological effect of GABA is modulated by the relative distribution of different GABA receptor subtypes. In previous studies GABAC receptors have been identified on bipolar cell axon terminals, and on postsynaptic membranes in the outer plexiform layer 3 and amacrine cells. 20,21 This is in accordance with the present immunohistochemical finding of GABAC receptor subunits located in the inner retinal layers, and the reference staining of GABAA receptors located in all synapse layers. Although it cannot be excluded that the use of goat anti-human antibodies on porcine retinal tissue may have influenced the specificity of the immunohistochemical staining pattern, the fact that no immunoreactivity was found on components of the retinal vascular wall is in agreement with the fact that GABA-induced vasorelaxation disappeared after removal of the perivascular retinal tissue. 
Altogether, the findings suggest that GABAC receptors in the porcine perivascular retina mediate vasorelaxation in a complex interaction with another vasorelaxing pathway that involves glutamate, ATP, PGE2, and several COX products. Future studies should aim at exploring the anatomical background and the individual reaction steps in this chain of events. A full elucidation of the elements involved in the regulation of retinal vascular tone may be important for developing new drugs for improving pathological changes in retinal blood flow. 
Acknowledgments
Technicians Svetlana Teplaia and Asnakech Tadesse assisted with the study. 
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Footnotes
 Supported by a grant from the VELUX foundation (TB).
Footnotes
 Disclosure: T. Bek, P; K. Holmgaard, P
Figure 1. 
 
The experimental protocol.
Figure 1. 
 
The experimental protocol.
Figure 2. 
 
Relaxation induced by GABA in porcine retinal arterioles with preserved perivascular retinal tissue without DL-APV (solid line), and in the presence of DL-APV in a concentration of 5 × 10−5 M (broken line) and 10−4 M (dotted line). The asterisk indicates the GABA concentration where 5 × 10−5 M DL-APV induced significantly more relaxation in the absence of DL-APV (control), whereas double crosses indicate concentrations where 10−4 M DL-APV induced significantly more relaxation than control.
Figure 2. 
 
Relaxation induced by GABA in porcine retinal arterioles with preserved perivascular retinal tissue without DL-APV (solid line), and in the presence of DL-APV in a concentration of 5 × 10−5 M (broken line) and 10−4 M (dotted line). The asterisk indicates the GABA concentration where 5 × 10−5 M DL-APV induced significantly more relaxation in the absence of DL-APV (control), whereas double crosses indicate concentrations where 10−4 M DL-APV induced significantly more relaxation than control.
Figure 3. 
 
GABA-induced relaxation in the presence of perivascular retinal tissue and DL-APV (top left), the ectonucleotidase inhibitor AOPCP (top right), the EP2 receptor blocker SC19220 (middle left), but not in the presence of the COX-inhibitor ibuprofen (middle right), and the adenosine receptor antagonist 8-PSPT (bottom).
Figure 3. 
 
GABA-induced relaxation in the presence of perivascular retinal tissue and DL-APV (top left), the ectonucleotidase inhibitor AOPCP (top right), the EP2 receptor blocker SC19220 (middle left), but not in the presence of the COX-inhibitor ibuprofen (middle right), and the adenosine receptor antagonist 8-PSPT (bottom).
Figure 4. 
 
Left: the effect of specific agonists to, respectively, GABAA (upper), GABAB (middle), and GABAC (lower) receptors. Right: The effect of GABA after specific inhibition of each of the three GABA receptors A (upper), B (middle), and C (lower).
Figure 4. 
 
Left: the effect of specific agonists to, respectively, GABAA (upper), GABAB (middle), and GABAC (lower) receptors. Right: The effect of GABA after specific inhibition of each of the three GABA receptors A (upper), B (middle), and C (lower).
Figure 5. 
 
Immunoreactivity to subunits of (A) the GABAC receptor in a sub-population of cells in the ganglion cell layer, and (B) the GABAA receptor located in the plexiform layers. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
Figure 5. 
 
Immunoreactivity to subunits of (A) the GABAC receptor in a sub-population of cells in the ganglion cell layer, and (B) the GABAA receptor located in the plexiform layers. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer.
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