December 2010
Volume 51, Issue 12
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Physiology and Pharmacology  |   December 2010
Novel Cellular Bouton Structure Activated by ATP in the Vascular Wall of Porcine Retinal Arterioles
Author Affiliations & Notes
  • Mikkel Wölck Misfeldt
    From the Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark; and
  • Christian Aalkjær
    the Institute of Physiology and Biophysics and
  • Ulf Simonsen
    the Department of Pharmacology, University of Aarhus, Aarhus, Denmark.
  • Toke Bek
    From the Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark; and
  • Corresponding author: Mikkel Wölck Misfeldt, Department of Ophthalmology, Aarhus University Hospital, Norrebrogade 44, 8000 Aarhus C, Denmark; mikkel.misfeldt@ki.au.dk
Investigative Ophthalmology & Visual Science December 2010, Vol.51, 6681-6687. doi:10.1167/iovs.10-5753
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      Mikkel Wölck Misfeldt, Christian Aalkjær, Ulf Simonsen, Toke Bek; Novel Cellular Bouton Structure Activated by ATP in the Vascular Wall of Porcine Retinal Arterioles. Invest. Ophthalmol. Vis. Sci. 2010;51(12):6681-6687. doi: 10.1167/iovs.10-5753.

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

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Abstract

Purpose.: The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5′-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles.

Methods.: Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall.

Results.: Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5′-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone.

Conclusions.: The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

Disturbances in retinal blood flow are involved in the pathophysiology of major sight-threatening diseases, such as age-related macular degeneration, primary open angle glaucoma, and diabetic retinopathy. 1 4 Retinal blood flow is regulated primarily by the tone of resistance arterioles, which is influenced by a number of signaling molecules, including purinergic compounds released from the retinal tissue. 5 10 Purines have also been shown to be involved in the retinal response to ischemia and consequently to play a role in the pathogenesis of primary open-angle glaucoma, optic neuropathy, and retinal diseases with pathologic angiogenesis. 11 14 The vasodilating effect of adenosine has been shown to be mediated by purinergic P1-receptors by a direct effect on vascular smooth muscle cells (VSMCs) but does not depend on ryanodine calcium channels, 15 whereas the vasodilating effect of adenosine 5′-triphosphate (ATP) depends on P2-receptors in the perivascular retinal tissue, suggesting hydrolysis of ATP in or near the vascular walls. 16 However, the anatomic basis for purinergic signaling during the relaxation of retinal arterioles is unknown. 
Therefore, purinergic responses in the perivascular retinal tissue during relaxation of VSMCs were studied. Porcine retinal arterioles were mounted in a myograph for isometric tone recordings, loaded with the calcium-sensitive fluorophore Oregon green, and placed in a confocal microscope to allow simultaneous recordings of vacular tone and intracellular calcium. The effects of adenosine and ATP were studied after agonists and antagonists were added to these compounds in retinal arterioles with preserved perivascular retinas and in isolated retinal arterioles. The vascular anatomy was characterized by immunohistochemistry using antibodies against smooth muscle cells (α-actin), glial cells (glial fibrillary acidic protein [GFAP], vimentin, and S-100 protein), synapses (synaptophysin), neurons (neuron-specific enolase), and basement membrane (type IV collagen). 
Materials and Methods
The study used a wire myograph mounted in a confocal microscope to investigate calcium changes in VSMCs and perivascular cell structures in porcine retinal arterioles during relaxation induced by purinergic compounds. 
Solutions and Chemicals
Solutions.
Physiological saline solution (PSS 1.6) containing 119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4.7H2O, 25 mM NaHCO3, 1.18 mM KH2PO4, 0.026 mM EDTA, 5.5 mM glucose, and 0.0 mM PSS had the same composition as PSS 1.6 except that CaCl2 had been omitted. The solutions were bubbled with a gas mixture composed of 95% atmospheric air and 5% CO2. In the experiments in which the retina was preserved, ibuprofen was added to a final concentration of 10 μM. 
Chemicals.
For experiments in the confocal myograph, adenosine, adenosine 5′-triphosphate (ATP), 8-(p-Sulfophenyl)theophylline hydrate (8-PSPT), adenosine-5′-O-(α, β-methylene diphosphate) (AOPCP), and ibuprofen were purchased from Sigma-Aldrich (Buchs, Switzerland). The thromboxane analogue U46619 (9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F) was purchased from AH Diagnostics (Aarhus, Denmark). Loading buffer was made of a composition of 7.24 μM Oregon green bapta 1-AM, 0.32% (wt/vol) dimethylsulfoxide (DMSO), 0.066% (wt/vol) chremophore EL, and 10.66 μM pluronic F-127. DMSO, chremophore EL, and pluronic F-127 were purchased from Sigma-Aldrich (Copenhagen, Denmark). Oregon green bapta 1-AM was obtained from Invitrogen (Taastrup, Denmark). For histology, the following antibodies were purchased from DAKO (Glostrup, Denmark) and Sigma-Aldrich: rabbit anti–cow S-100 (Z0311), mouse anti–human neuron-specific enolase (NSE) (M0873), rabbit anti–human von Willebrand factor (A0082), mouse anti–human smooth muscle α-actin (M0857), rabbit anti-GFAP) (Z0334), mouse anti–human collagen IV (M0785), biotinylated polyclonal goat anti-rabbit and rabbit anti–mouse immunoglobulin conjugated with TRITC (T2402; Sigma-Aldrich) secondary antibodies, and conjugated fluorescent probe streptavidin TRITC (Invitrogen). 
Tissue
The experiments were carried out on the tissue of domestic Danish pigs. One eye from each pig was removed at a local slaughterhouse (Danish Crown, Horsens, Denmark) immediately after the animal had been anesthetized with CO2 and killed by exsanguination. The eyes were kept in 4°C PSS 1.6, and postmortem time until the commencement of the experiments never exceeded 3 hours. The dissection procedure was carried out in PSS 0.0 as described previously. 16,17 The small arteriole was identified on the basis of the capillary free zone, and, from a location 1 to 2 mm from the optic disc, a segment approximately 2 mm long with surrounding retinal tissue attached on each side of it was dissected from the retina. 
Mounting of Tissue
The vascular segment was transferred to a myograph (120 CW; Danish Myo Technology, Aarhus, Germany) connected to an analog/digital converter, allowing the recording of isometric tone. The mounting procedure was performed in PSS 0.0 as described previously. 16 In short, a stainless steel wire with a diameter of 20 μm was carefully introduced into the lumen of the vessel and was attached to the jaws of the force transducer. In experiments using isolated vessels, the retina was gently removed with a pair of forceps (no.5; Fontax, Prilly, Switzerland). The second wire was advanced through the lumen using the first wire as a guide and was attached to the jaws of the micrometer. The chamber solution was changed to PSS 1.6, and the jaws of the myograph were lowered to the bottom of the chamber. The vessel was allowed to equilibrate for 30 to 40 minutes until a stable tone was recorded. Continuous recordings in the confocal myograph chamber during the experiments showed that the temperature could be kept at 37°C ± 0.2°C. 
Normalization and Loading
Each vessel was normalized using a standard procedure previously described by Hessellund et al. 18 In short, the vessel was normalized in 37°C PSS 0.0 to a tone corresponding to a transmural pressure of 70 mm Hg (L70). The vessel circumference was increased in four steps in PSS 0.0, and the passive tone was recorded at each step. This diameter-tone relationship was exponential, and by plotting the intercept between this curve and a linear regression based on the Laplace equation (wall tone = transmural pressure × radius), the tone at a transmural pressure of 70 mm Hg was calculated. The diameter was adjusted to 93.5% of the intercept length, at which the vessel developed maximum tone. After normalization the chamber solution was changed to PSS 1.6; the vessel was allowed to equilibrate until a stable tone had been reached, and the loading buffer was added to the chamber. The loading period was adjusted on the basis of preliminary experiments showing that in isolated arterioles the VSMCs should be loaded in 45 minutes. In a preparation with preserved perivascular retinal tissue, the perivascular cells could be loaded in 10 minutes, whereas it was not possible to load the VSMCs even after 2 hours of loading. After loading, the chamber solution was changed three times with PSS 1.6, and the vessel was allowed to equilibrate for 10 minutes before the experiments were begun. 
Confocal Microscope
The myograph was placed in an inverted confocal microscope (LSM 5 PASCAL exciter; Zeiss, Oberkochen, Germany). A 488-nm argon laser was used to excite the calcium fluorophore and emissions were collected using a 530-nm long-pass filter. The confocal images had a depth of 12 bits, yielding a dynamic range of 4096 intensity levels. The photomultiplier gain and the confocal pinhole were adjusted using the Zeiss image browser program to keep the intensity value in all pixels within this dynamic range. The initial image size was set to 512 × 512 pixels, and subsequently a region of interest (ROI) was selected so that images were recorded 2×/s to 4×/s, depending on the image size. Image acquisition of the smooth muscle cells was performed using a 63× NA = 0.75 long working distance objective, and acquisition of images of the perivascular retinal cells was performed with a 40× NA = 0.6 long working distance objective (both objectives; Plan-Neofluar; Zeiss). 
Experimental Protocols
Experiments were performed either on arterioles with preserved retinal tissue or on isolated arterioles, and at least six experiments were conducted for each experimental condition. Experimental protocols consisted of the steps outlined here and shown in Figure 1
Figure 1.
 
Experimental protocols used.
Figure 1.
 
Experimental protocols used.
Protocol A: Arterioles with Preserved Perivascular Retina.
First, the vessel was precontracted with U46619 (1 μM) and was allowed to equilibrate for at least 10 minutes or until a stable tone had been achieved. Second, the ectonucleotidase inhibitor AOPCP (2.5 × 10−4 M) or the calcium channel blocker ryanodine (10 μM) was added, and the vessel was allowed to equilibrate for at least 10 minutes. Third, concentration-response experiments were performed with ATP or adenosine added successively in the following concentrations (in M): 1 × 10−8, 1 × 10−7,1 × 10−6, 1 × 10−5, and 1 × 10−4
Control experiments were repeated with both ATP and adenosine in the absence of the blockers in step 2. Those in which the added drugs were replaced with PSS did not show any changes in spontaneous tone over time. 
Protocol B: Isolated Vessels.
First, precontraction was performed with U46619 (1 μM), as in protocol A. Second, the vessels were incubated with the adenosine A1/A2 receptor blocker 8-PSPT (500 μM) or the P2 receptor antagonist suramin (100 μM) for 10 minutes. Results after the addition of suramin were disregarded from further analysis because addition of this compound disintegrated the tissue in the preparation. Third, adenosine or ATP was added successively in the same concentrations used for ATP in protocol A. Control experiments were repeated without incubation step 2. 
Data Sampling and Analysis
Sampling of tone and fluorescence was begun immediately after the addition of U46619 and again after a stable tone had been achieved (∼5–10 minutes after precontraction, after the addition of a blocker, and immediately after the addition of each new concentration of ATP or adenosine. 
Active Wall Tone
The active wall tone was defined as the tone produced by U46619 or the tone after the addition of a purinergic receptor blocker, subtracted by the tone immediately before the precontraction. The change in tone was evaluated as the relative change produced at each concentration step of either ATP or adenosine. When the concentration-response experiments resulted in a sigmoid curve, the data were additionally fitted to the Michaelis-Menten equation to calculate the EC50 value, and the time from the application of ATP or adenosine until the tone had reached this EC50 value was calculated. In each concentration response experiment, the concentration that produced half-maximum dilatation was identified, and the time from the application until the response had fully developed at this concentration was recorded. 
Calcium-Dependent Fluorescence
The confocal plane was placed near the bending of the vessel around the wire so that the different layers of the vascular wall were extended to fill out the image field. Subsequently, a region of interest (ROI) containing VSMCs was defined for the image recording, and the average size of this ROI was 0.06 mm2 (range, 0.05–0.1). Sampling was performed at 4 to 6 Hz for 2 to 4 minutes, depending on the image size. From the recordings, the fraction of active cells in the ROI was calculated. A cell was defined to be active when fast Fourier analysis of the oscillations of the fluorescence identified the frequency to be between 0.06 and 0.66 Hz, and the amplitude of the oscillations was at least 10% of the average fluorescence during the sampling period. 
In vessels with preserved retinal tissue, recordings were made with the focal plane displaced laterally to include perivascular structures in a zone extending at least 50 μm external to the VSMC layer. In each preparation, an ROI was selected to include an area of approximately 0.6 mm2 (mean, 0.66 mm2; range, 0.49–1.01; n = 24), which was expected to contain a sufficient number of cellular elements, and fluorescence inside elements in this area was recorded. Distances in the ROI were defined using the built-in scale in the acquisition software of the confocal microscope. 
Immunohistochemistry
Additional experiments were conducted using protocol A for loading with calcium-sensitive fluorophore, excluding step 2. The vessel segments were washed with PBS and fixated in 4% paraformaldehyde for 10 minutes. The chamber solution was changed to PBS containing 0.3% Triton-X, and two segments were incubated for 24 hours with each of the following antibodies: rabbit anti–human S100 protein 1:100, rabbit anti–cow GFAP polyclonal antibody 1:100, mouse anti–human α-actin 1:100, mouse anti–human synaptophysin 1:100, or mouse anti–human type IV collagen primary antibody for 24 hours at 5°C. Subsequently, the preparations were incubated for another 24 hours with either the biotinylated polyclonal goat anti–rabbit secondary antibody (1:100) or the conjugated TRITC anti–mouse secondary antibody (1:100), respectively. Finally, the S-100 and GFAP preparations were incubated for 2 hours using the streptavidin-TRITC fluorescent probe (1:100). 
As positive controls for the immunohistochemical reactions, the following were used: retinal Müller cells (GFAP and vimentin), retinal vascular endothelial cells (vWF), VSMCs (actin), ganglion cells (NSE), basement membrane (collagen IV), perivascular glial cells (S-100 protein), and synapses in choroidal nerves (synaptophysin). The TRITC-labeled preparations were examined by fluorescence microscopy using a He-Ne 543-nm excitation laser, and emission was recorded at 570 nm. From each vessel the images with green fluorescence, indicating intracellular calcium, and the images with red fluorescence, due to immunoreactivity, were recorded with a depth resolution extending from an upper level at the bouton layer tangential to the surface of the vessel to a depth at which a clear cross-section of the sides of the vessel was observed and were finally composed to demonstrate the relationship between green fluorescing cellular structures and structural markers in the vascular wall. Only the immunohistochemical reactions to α-actin, vimentin, and GFAP will be reported because only these markers contributed to the localization of green fluorescent cellular components in the vascular wall; consequently, only these markers will be reported. 
Statistical Analysis
For all experiments, repeated-measures ANOVA was used to test whether the addition of adenosine or ATP resulted in a concentration-dependent change in the tone and the fraction of active cells. Dunnett's multiple-comparison test was used to test for differences between the concentration response curves. 
For each concentration step in the experiments from protocol A, one-way ANOVA was used to test for significant differences between the tone and the fraction of active boutons obtained during the four different experimental conditions (ATP, ATP+AOPCP, ATP + ryanodine, adenosine). If findings were significant, Bonferroni's test was used to identify which of the experimental conditions produced a response that differed significantly from the others. 
For each concentration step in the experiments from protocol B, two-way ANOVA was used to test for significant differences between the tone and the fraction of active VSMCs, with the purines adenosine and ATP as one variable and the presence or not of the blocker 8PSPT as the other variable. Bonferroni's test was used to identify which of the experimental conditions produced a response that differed significantly from the others. Differences in EC50 values were tested using Student's unpaired t-test. 
Results
Vessels with Preserved Perivascular Retina
Hyperfluorescence was recorded from a population of abundant round boutons that did not show immunoreactivity to any of the antibodies tested. The combined loading with fluorophore and immunohistochemical staining showed that the boutons were located in a layer of vimentin-positive thin processes (Fig. 2A) immediately external to the smooth muscle cell layer (Fig. 2B) and internal to the retinal glial cell layer (Fig. 2C). The boutons did not show any nuclear staining, and it was not possible to trace the boutons or the vimentin-positive processes to cell nuclei inside or outside the vascular wall. In the recordings from the pharmacologic experiments, the boutons had a density of 36 to 105/0.1 mm2 and a mean diameter of 7.5 μm (range, 5–10 μm), and the fluorescence intensity in the boutons showed an increase after the addition of ATP (Figs. 3A, 3B); no response was observed after the addition of adenosine (Figs. 3C, 3D). 
Figure 2.
 
Compositions of images of arterioles after loading with Oregon green (green) and successive immunohistochemistry to components of the vascular wall (red). Each image has been recorded with a depth resolution extending from the upper surface of the bouton layer (left) to a depth at which a clear cross-section of the vascular wall can be observed (right). Scale bars, 10 μm. (A) The green boutons are interspersed in a network of vimentin-positive red processes in the tangential view (left) and disappear in the heavy red signal corresponding to the cross-section (right). (B) The green boutons are located superior to the α-actin–positive red VSMCs in the tangential view (left) and external to these cells in the cross-section (right). (C) The green boutons are located inferiorly to the GFAP-positive red perivascular glial cells not visible in the tangential view (left) and external to these cells in the cross-section (right).
Figure 2.
 
Compositions of images of arterioles after loading with Oregon green (green) and successive immunohistochemistry to components of the vascular wall (red). Each image has been recorded with a depth resolution extending from the upper surface of the bouton layer (left) to a depth at which a clear cross-section of the vascular wall can be observed (right). Scale bars, 10 μm. (A) The green boutons are interspersed in a network of vimentin-positive red processes in the tangential view (left) and disappear in the heavy red signal corresponding to the cross-section (right). (B) The green boutons are located superior to the α-actin–positive red VSMCs in the tangential view (left) and external to these cells in the cross-section (right). (C) The green boutons are located inferiorly to the GFAP-positive red perivascular glial cells not visible in the tangential view (left) and external to these cells in the cross-section (right).
Figure 3.
 
(A) Confocal scanning image of the bouton layer after loading with Oregon green. Arrows: two boutons. (B) Trace of the calcium response in the two boutons after application of ATP. (C) Confocal scanning image of the bouton layer. (D) Fluorescence trace in the two boutons shown in (C) after application of adenosine. Scale bars, 30 μm.
Figure 3.
 
(A) Confocal scanning image of the bouton layer after loading with Oregon green. Arrows: two boutons. (B) Trace of the calcium response in the two boutons after application of ATP. (C) Confocal scanning image of the bouton layer. (D) Fluorescence trace in the two boutons shown in (C) after application of adenosine. Scale bars, 30 μm.
The changes in vascular tone and the fraction of active perivascular boutons during purinergic stimulation are shown in Figure 4. Figure 4A shows that ATP induced a significant reduction in the vascular tone (P < 0.01) that was not significantly different from the relaxation induced by adenosine. The relaxing effect of ATP was reduced significantly by the ectonucleotidase inhibitor AOPCP (P < 0.05) at the two highest concentrations and nonsignificantly by the calcium channel blocker ryanodine (P = 0.93). Figure 4B shows that ATP induced a concentration-dependent reduction in the fraction of active boutons (P < 0.01). In the presence of AOPCP and ryanodine, the effect of ATP was significantly reduced (P < 0.001). Adenosine had no effect on the fraction of active boutons. 
Figure 4.
 
Changes in tone (A) and fraction of active cells (B) after application of adenosine, ATP, or ATP in vessels incubated with either AOPCP or ryanodine. *Significant differences between the effect of ATP and ATP + AOPCP. #Significant differences between the effect of adenosine and both ATP + AOPCP and ATP + ryanodine.
Figure 4.
 
Changes in tone (A) and fraction of active cells (B) after application of adenosine, ATP, or ATP in vessels incubated with either AOPCP or ryanodine. *Significant differences between the effect of ATP and ATP + AOPCP. #Significant differences between the effect of adenosine and both ATP + AOPCP and ATP + ryanodine.
Isolated Vessels
The results from experiments on isolated retinal arterioles are shown in Figure 5. Figure 5A shows that both ATP and adenosine induced significant relaxation of vascular tone without a blocker (P < 0.001) and that 8-PSPT reduced the relaxation induced by 10−4 M ATP and of 10−5 M adenosine. Figure 5B shows that both ATP and adenosine induced a significant reduction in the fraction of active VSMCs (P < 0.001) and that 8-PSPT reduced the fraction of active VSMCs at a concentration of 10−6 M and 10−5 M of the two compounds. 
Figure 5.
 
Changes in tone (A) and fraction of active cells (B) after application of ATP or adenosine in the presence or absence of 8-PSPT. *Significant differences between the effect of ATP and ATP after incubation with 8-PSPT. #Significant differences between the effect adenosine and adenosine combined with 8-PSPT.
Figure 5.
 
Changes in tone (A) and fraction of active cells (B) after application of ATP or adenosine in the presence or absence of 8-PSPT. *Significant differences between the effect of ATP and ATP after incubation with 8-PSPT. #Significant differences between the effect adenosine and adenosine combined with 8-PSPT.
Discussion
The regulation of the retinal microcirculation has been shown to depend on the release of several compounds from the retinal tissue, including prostaglandins (PGs), ATP/adenosine, and nitric oxide (NO). 8,16,19 21 Indeed, some in vitro studies have suggested the existence of a retinal relaxing factor released from retinal tissue around retinal arterioles to inhibit the contraction of the vessels. 5,6 However, the anatomic basis for the release of these retinal modulators of vascular tone has not been studied in detail. 
The present study used an experimental model that has been shown to be suitable for studying the relation between intracellular calcium transients in VSMCs and the tone in isolated porcine retinal arterioles in vitro. 15 It was, therefore, expected that the model could be extended to study the relation between vascular tone and calcium transients in the perivascular retinal tissue. However, as shown previously, this required blocking of the COX enzyme to prevent the effect of inhibitors of vascular tone released from the perivascular retinal tissue. 19 Therefore, the interpretation of the results should take into consideration that possible influences of COX products were not investigated. 
The study identified a layer of hyperfluorescent boutons interspersed in a network of vimentin-positive processes encircling the vessel that was located immediately external to the VSMCs but internal to the GFAP-positive glial cells. It was not possible to trace these boutons or processes encircling the retinal arterioles to a cell nucleus in or around the vascular wall, and these structures were not found to display immunoreactivity to any of the studied markers of neuronal and glial cells in or around retinal vessels. The lack of identification of the cellular origin of the boutons may be attributed to insufficient loading with fluorophore or lack of resolution of processes connecting the boutons with cell bodies in the retina. These cell bodies may potentially be related to pericytes or may be of neuronal or glial origin, which is in accordance with recent findings of purinergic signaling in these cells in the retina. 7 The lack of immunoreactivity in the newly identified boutons may explain the lack of focus on these structures in the literature 22 24 and prompts further studies to identify the cells from which they originate. 
The study showed that the calcium activity in the perivascular boutons depended on ryanodine channels, whereas an effect of this compound on vascular tone was less evident. This might have been because of the low active tone (∼0.4 N/m) generated after loading with fluorophore, which might have reduced the responsiveness of the vessels. Thus, previous findings suggest that the observed finding is not due to a direct effect of ryanodine on smooth muscle cells. 13  
The effect of ATP on the calcium activity in the perivascular boutons was paralleled by a reduction in vascular tone, both of which could be blocked by the ectonucleotidase inhibitor AOPCP. This suggests that the perivascular boutons may be involved in the physiological regulation of retinal vascular tone. The ATP-induced increase in [Ca2+]i in the boutons located immediately external to the VSMCs is similar to responses previously demonstrated in astrocytes from rat cortical slices 25 27 and in rat retinal glial cells, 28 and ATP has been shown to have vasoactive properties in retinal microvasculature. 29 Additionally, ATP-induced relaxation of porcine retinal arterioles has previously been shown to be blocked by inhibiting the degradation of ATP, an effect that depended on the presence of the perivascular retinal tissue. 16 In the present study, the calcium response in the boutons external to the VSMC was attenuated by inhibiting the hydrolysis of ATP using AOPCP and could not be elicited by adenosine, suggesting that ATP degradation products other than adenosine, possibly ADP or AMP, may be involved in eliciting the response. Therefore, full elucidation of the relaxing effect of purines on retinal arterioles requires investigation of the site and mode of synthesis of all degradation products from ATP in the retinal vascular wall. 
The study showed that relaxation of isolated arterioles induced by adenosine was accompanied by a decrease in the fraction of active VSMCs. This decrease may be attributed to adenosine receptor–mediated inhibition of IP3-dependent Ca2+ release from the sarcoplasmic reticulum. 15,30 33 In a previous study, ATP was found not to have an effect on the tone of isolated retinal arterioles, 16 and the present findings of a vasodilating effect of ATP might have been attributed to the fact that the compound was tested in a concentration 30× higher. It is conceivable that this effect was caused by the degradation of ATP to adenosine because the effect could have been blocked by an adenosine receptor blocker. 
The findings of the present study suggest that the perivascular boutons may be involved in the regulation of retinal vascular tone and, thus, may be involved in the pathogenesis of retinal vascular disease. 34 36 Previous clinical and experimental studies have shown that the metabolism of purines is disturbed in diabetic retinopathy and in other retinal diseases. 37 39 Thus, adenosine is significantly upregulated in the retina within a few minutes after the onset of retinal ischemia, 40 and evidence suggests that the inhibition of purine receptors may be involved in the formation of diabetic macular edema in streptozotocin diabetic rats. 41 This is substantiated by the finding that hyperglycemia is associated with elevated concentrations of ATP in rat retinal cells, which indicates a higher release of ATP from retinal cells or a downregulation of ATP hydrolysis, which may contribute to the disturbances in blood flow observed in diabetic retinopathy. 42 The present findings suggest that these changes may be related to perturbations in the functioning of a signaling pathway, where ATP stimulates perivascular cells with a consequent hydrolysis to adenosine, which again affects the tone of retinal VSMCs by store-operated calcium channels. 43 This pathway may be a possible target for future pharmacologic intervention in diseases with disturbed retinal blood flow. 
In conclusion, the present study has demonstrated that the vasodilating effect of purines in porcine retinal tissue involves calcium activity in a layer of boutons located external to the VSMC and internal to the perivascular retinal glial cells, which can be stimulated by ATP but not by adenosine. This knowledge contributes to a deeper understanding of the regulation of retinal blood flow as a precondition for understanding the pathogenesis of retinal vascular diseases such as diabetic retinopathy. 
Footnotes
 Supported by the Boehms Foundation, the Frikke Foundation, the Danish Eye Research Foundation, the Eye Foundation Save the Sight, the Reventlow Poulsen Foundation, and the VELUX Foundation.
Footnotes
 Disclosure: M.W. Misfeldt, None; C. Aalkjær, None; U. Simonsen, None; T. Bek, None
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Figure 1.
 
Experimental protocols used.
Figure 1.
 
Experimental protocols used.
Figure 2.
 
Compositions of images of arterioles after loading with Oregon green (green) and successive immunohistochemistry to components of the vascular wall (red). Each image has been recorded with a depth resolution extending from the upper surface of the bouton layer (left) to a depth at which a clear cross-section of the vascular wall can be observed (right). Scale bars, 10 μm. (A) The green boutons are interspersed in a network of vimentin-positive red processes in the tangential view (left) and disappear in the heavy red signal corresponding to the cross-section (right). (B) The green boutons are located superior to the α-actin–positive red VSMCs in the tangential view (left) and external to these cells in the cross-section (right). (C) The green boutons are located inferiorly to the GFAP-positive red perivascular glial cells not visible in the tangential view (left) and external to these cells in the cross-section (right).
Figure 2.
 
Compositions of images of arterioles after loading with Oregon green (green) and successive immunohistochemistry to components of the vascular wall (red). Each image has been recorded with a depth resolution extending from the upper surface of the bouton layer (left) to a depth at which a clear cross-section of the vascular wall can be observed (right). Scale bars, 10 μm. (A) The green boutons are interspersed in a network of vimentin-positive red processes in the tangential view (left) and disappear in the heavy red signal corresponding to the cross-section (right). (B) The green boutons are located superior to the α-actin–positive red VSMCs in the tangential view (left) and external to these cells in the cross-section (right). (C) The green boutons are located inferiorly to the GFAP-positive red perivascular glial cells not visible in the tangential view (left) and external to these cells in the cross-section (right).
Figure 3.
 
(A) Confocal scanning image of the bouton layer after loading with Oregon green. Arrows: two boutons. (B) Trace of the calcium response in the two boutons after application of ATP. (C) Confocal scanning image of the bouton layer. (D) Fluorescence trace in the two boutons shown in (C) after application of adenosine. Scale bars, 30 μm.
Figure 3.
 
(A) Confocal scanning image of the bouton layer after loading with Oregon green. Arrows: two boutons. (B) Trace of the calcium response in the two boutons after application of ATP. (C) Confocal scanning image of the bouton layer. (D) Fluorescence trace in the two boutons shown in (C) after application of adenosine. Scale bars, 30 μm.
Figure 4.
 
Changes in tone (A) and fraction of active cells (B) after application of adenosine, ATP, or ATP in vessels incubated with either AOPCP or ryanodine. *Significant differences between the effect of ATP and ATP + AOPCP. #Significant differences between the effect of adenosine and both ATP + AOPCP and ATP + ryanodine.
Figure 4.
 
Changes in tone (A) and fraction of active cells (B) after application of adenosine, ATP, or ATP in vessels incubated with either AOPCP or ryanodine. *Significant differences between the effect of ATP and ATP + AOPCP. #Significant differences between the effect of adenosine and both ATP + AOPCP and ATP + ryanodine.
Figure 5.
 
Changes in tone (A) and fraction of active cells (B) after application of ATP or adenosine in the presence or absence of 8-PSPT. *Significant differences between the effect of ATP and ATP after incubation with 8-PSPT. #Significant differences between the effect adenosine and adenosine combined with 8-PSPT.
Figure 5.
 
Changes in tone (A) and fraction of active cells (B) after application of ATP or adenosine in the presence or absence of 8-PSPT. *Significant differences between the effect of ATP and ATP after incubation with 8-PSPT. #Significant differences between the effect adenosine and adenosine combined with 8-PSPT.
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