Dual Effects of Adenosine on the Tone of Porcine Retinal Arterioles In Vitro

Abstract

Purpose.: Previous studies have shown that adenosine induces relaxation of isolated retinal arterioles mediated by the A_2A receptor, but the contributions to tone regulation of adenosine receptors located both in and around the vascular wall have not been studied in detail.

Methods.: Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a wire myograph, and the tone was recorded after addition of antagonists to the adenosine A₁, A_2A, A_2B, and A₃ receptors, followed by removal of the perivascular retinal tissue and repetition of the experiments. Additionally, these responses were studied in concentration–response experiments using specific agonists.

Results.: Adenosine induced a significant concentration-dependent relaxation at high concentrations that was independent of the perivascular retinal tissue and could be antagonized by the nonspecific adenosine receptor antagonist 8-PSPT. The selective A_2A receptor antagonist SCH 58261 and the A_2B receptor antagonist MRS 1754 significantly antagonized the relaxing effect of adenosine. Conversely, the selective A₁ receptor antagonist KW-3902 and the A₃ receptor antagonist MRS 1523 significantly increased the relaxing effect of adenosine, and the corresponding agonists contracted retinal arterioles at intermediate concentrations. The contracting effect of the A₁ receptor agonist but not the A₃ receptor antagonist depended on the presence of perivascular retinal tissue.

Conclusions.: Adenosine has complex effects on retinal vascular tone elicited both from the vascular wall and from the perivascular retina and with receptors mediating contraction at intermediate concentrations and relaxation at high concentration.

Introduction

Diseases involving the retinal vascular supply are among the major causes of blindness in the Western world. ¹ A precondition for developing new treatment for these diseases is to have knowledge of how retinal blood flow is regulated under normal conditions. Several previous studies have shown that adenosine is involved in regulating retinal vascular tone. ^{2 –5} The compound has been shown to induce vasorelaxation through a mechanism that is independent of the perivascular retinal tissue, ⁶ and the effect has been shown to be mediated by the A_2A receptor. ⁷ In organs outside the eye, adenosine can induce vasocontraction mediated by A₁ receptors, ⁸ but the contribution to tone regulation of different types of adenosine receptors located in and around retinal vessels has not been studied in detail. 

Therefore, the purpose of the present study was to investigate the contribution of adenosine receptors previously shown to be involved in the regulation of vascular tone to contraction and relaxation of retinal arterioles, respectively, and to study whether the effects depend on the perivascular retinal tissue. Porcine retinal arterioles with preserved perivascular retinal tissue were mounted in a wire myograph, and the tone was recorded after addition of increasing concentrations of adenosine in the presence of antagonists to the adenosine A₁, A_2A, A_2B, and A₃ receptors, followed by removal of the perivascular retinal tissue and repetition of the experiments. These responses were further studied using specific receptor agonists. 

Methods

Solutions

Physiological saline solution (PSS 1.6) containing (mM) NaCl 119, KCl 4.7, MgSO₄ · 7H₂O 1.17, NaHCO₃ 25, KH₂PO₄ 1.18, EDTA 0.026, glucose 5.5, and HEPES 5.5. CaCl₂ was added to result in a final concentration of 1.6 mM, pH = 7.5. Physiological saline solution 0.0 had the same composition as PSS 1.6 except that CaCl₂ had been omitted. The solutions were bubbled with 5% CO₂ in atmospheric air. Potassium PSS (KPSS) was PSS 1.6 in which equimolar NaCl was replaced by KCl, resulting in [K⁺] of 125 mM. 

Drugs

All chemicals were purchased from Sigma-Aldrich (Glostrup, Denmark) if not otherwise noted. 

Tone Modulators.

The thromboxane analogue U46619 (AH Diagnostics, Aarhus, Denmark) and KPSS were used for vasoconstriction and papaverine for vasorelaxation. 

Adenosine Agonists.

The agonists included adenosine, the A₁ receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA), the A_2A receptor agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl]phenyl] propanoic acid (CGS-21680), the A₃ receptor agonist 1-deoxy-1-[6-[((3-iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide,N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), and the A₃ receptor agonist 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA). 

Adenosine Antagonists.

The nonselective adenosine receptor antagonist 8-(p-sulphophenyl) theophyllin hydrate (8-PSPT), the selective A₁ receptor antagonist 1,3-dipropyl-8-(3-noradamantyl)xanthine, 8-(Hexahydro-2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione MK-7418 Rolofylline (KW-3902), the A_2A receptor antagonist 7-(2-phenylethyl)-5-amino-2-(2-fyryl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (SCH 58261), the A_2B receptor antagonist 8-[4-[((4-cyanophenyl) carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine hydrate (MRS 1754), and the A₃ receptor antagonist 3-propyl-6-thyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS 1523) were used. 

Stock solutions were prepared by dissolving in demineralized water (adenosine and 8-PSPT) or dimethyl sulfoxide (DMSO) (all other compounds) according to the recommendations from the manufacturers. 

Tissue

The procedures have been described in detail elsewhere. ⁹ Porcine eyes were collected daily from a local slaughterhouse (Danish Crown, Horsens, Denmark). The eyes were removed immediately after the pigs had been anaesthetized with CO₂ and killed by exsanguination, and were placed in PSS (1.6) at 4°C. The subsequent transport time to the laboratory was approximately 45 minutes. The following dissection was performed in PSS (0.0) and started within 1 hour post mortem. The eye was bisected by a frontal section immediately behind the equator using a razor blade, followed by removal of the vitreous body without touching the retina. A 2-mm segment of an arteriole within 5 mm from the optic disc was identified under a microscope (Stemi 2000; Zeiss, Hamburg, Germany), and using a microblade, the segment was cut to include 1 mm of perivascular retinal tissue on each side of the segment. Altogether 98 vascular segments from 98 pigs were studied; the segments had a mean diameter of 156 μm and a range of 130 to 170 μm. 

Mounting Procedure

The preparation was placed in PSS (0.0) in the chamber of a wire myograph (620M; DMT, Aarhus, Denmark) and (620M; DMT) for isometric tone measurements and was mounted between two tungsten wires with a diameter of 25 μm. Subsequently, PSS (0.0) was replaced with PSS (1.6) and the tissue chamber was heated to 37°C. Bubbling of the chamber with a mixture of 95% atmospheric air and 5% CO₂ was commenced, and the arterioles were allowed to equilibrate for 30 to 35 minutes in order to achieve a stable tone. 

Normalization

The chamber solution was changed to PSS (0.0), and the arteriole was normalized using a standardized procedure previously described. ¹⁰ In short, the procedure consisted of two parts. First, the arteriole was stretched in three to four steps corresponding to intraluminal pressures between 0 and 70 mm Hg, which is within the physiologic pressure range. ¹¹ At each step the passive tone was recorded after 2 minutes in order for a stable tone to develop; the internal circumference (IC) was calculated using the equation IC = π × D × 2 × (D + d), D being the diameter of the wire and d being the distance between the two wires. The plotting of wall tension against IC revealed an exponential curve, and the intercept between this curve and a straight line using the Laplace equation (wall tension = transmural pressure × radius) set to a transmural pressure of 70 mm Hg could be used to calculate L₇₀. ^12,13 Subsequently, the micrometer screw was used to adjust the diameter to 93.5% of L₇₀, where porcine retinal arterioles have been shown to elicit maximal contractility of smooth muscle cells and sensitivity to agonists. ¹⁰ After normalization, the chamber solution was replaced with PSS (1.6) and the arteriole was allowed to equilibrate for at least 10 minutes or until stable tone had been reached. 

Only one arteriole from each animal was used. For each experimental condition, at least six observations were made. The total time for each experiment never exceeded 5 hours. Three different experimental protocols were followed. 

Protocol A

Protocol A, shown in Figure 1, consisted of the following steps: 

Figure 1

 

The experimental protocol followed.

1.  
   Addition of antagonist. One of the following was added to be present throughout the experiment: the unselective adenosine receptor antagonist 8-PSPT (5 × 10⁻⁵ M), the selective A₁ receptor antagonist KW-3902 (3 × 10⁻⁵ M), the A_2A receptor antagonist SCH 58261 (10⁻⁶ M), the A_2B receptor antagonist MRS 1754 (10⁻⁶ M), the A₃ receptor antagonist MRS 1523 (10⁻⁵ M), or none (control).
2.  
   Preconstriction: The thromboxane analogue U46619 (10⁻⁶ M) was added. If the resulting tone was below 0.2 mN/mm, the arteriole was considered to be nonviable and was discarded. Experiments were repeated in absence of preconstriction.
3.  
   Concentration–response experiment: Adenosine was added successively in the concentrations (M) 10⁻⁸, 3 × 10⁻⁸, 10⁻⁷, 3 × 10⁻⁷, 10⁻⁶, 3 × 10⁻⁶, 10⁻⁵, 3 × 10⁻⁵, 10⁻⁴. After each addition of a higher concentration, the arteriole was allowed to equilibrate for 3 minutes or until stable tone was obtained.
4.  
   Removal of perivascular retinal tissue: The chamber solution (PSS 1.6) was changed twice. Under a microscope the perivascular retina was carefully detached from the vessel using two forceps. The procedure for removing the retinal tissue never exceeded 15 minutes. Subsequently, the chamber solution was changed three times and the isolated arterioles were allowed to equilibrate for 35 to 40 minutes.
5.  
   Repetition: Steps 2 and 3 were repeated on the isolated arteriole.
6.  
   Testing for viability: PSS (1.6) was replaced with KPSS followed by equilibration for 5 to 8 minutes. If the resulting increase in tone was less than 0.2 mN/mm, the arteriole was regarded as nonviable and was discarded.
7.  
   Maximal relaxation: Finally, papaverine (10⁻⁴ M) was added in order to obtain total relaxation.

Protocol B

The procedures in protocol A were repeated with the following modifications: 

In step 1, one of the following was added to be present throughout the experiment: the selective A₁ receptor antagonist KW-3902 (3 × 10⁻⁵ M), the A_2A receptor antagonist SCH 58261 (10⁻⁵ M), the A_2B receptor antagonist MRS 1754 (10⁻⁵ M), the A₃ receptor antagonist MRS 1523 (10⁻⁵ M), or none (control). For this protocol the concentrations of the A_2A and A_2B antagonists were 10 times higher than in protocol A in order to ensure blockage of these compounds if present. 

In step 3, concentration–response experiments were performed using selective agonists in the concentrations (M) 10⁻⁸, 3 × 10⁻⁸, 10⁻⁷, 3 × 10⁻⁷, 10⁻⁶, 3 × 10⁻⁶, 10⁻⁵, and 3 × 10⁻⁵, and 10⁻⁴. The A₁ receptor agonist CCPA was tested in the presence of the A₁, A_2A, and A_2B receptor antagonists, the A_2A receptor agonist CGS-21680 in the presence of the A_2A and the A_2B receptor antagonists, and the A₃ receptor agonists IB-MECA and Cl-IB-MECA in the presence of the A₃ receptor antagonist. IB-MECA was unable to antagonize MRS 1523 and was therefore not used further. 

Control Experiments

Reproducibility.

Concentration–response experiments with adenosine in the presence and absence of the antagonist 8-PSPT were conducted both in the beginning and by the end of the study, which were separated by 4 months. Since there was no significant change in the results over time, the data from these different series were pooled. 

Time Controls.

The procedures in protocol A with and without the addition of 8-PSPT were repeated, except that adenosine was omitted from the concentration–response experiment. In the presence of perivascular retinal tissue there was a significant (P < 0.05) decrease in arteriolar tone of approximately 20% between 20 and 40 minutes after the beginning of the experiment that could be blocked by the adenosine receptor blocker 8-PSPT. No such time-dependent decrease in vascular tone was observed in the isolated arterioles. 

Solvents.

The time control experiments described above were repeated in the presence of the solvents DMSO or ethanol, in dilutions similar to those used for the experiments in protocol A and B, and showed no significant differences from the results observed in the time control experiments (not shown). 

Data Analysis

The active tone was defined as the tone produced after preconstriction with U46619 (and in nonpreconstricted vessels the tone produced by KPSS) minus the tone measured in PSS (0.0) (vessels with preserved perivascular retinal tissue) or the tone after addition of papaverine (isolated vessels). Subsequently, in order to compare the different experiments, the normalized tone for all experiments was calculated as the active tone divided by the tone produced by U46619 (preconstricted vessels) or KPSS (nonconstricted vessels). Finally, for each agonist concentration, the normalized tone obtained in the time control experiments was subtracted from the normalized tone obtained during the intervention. 

Statistical Analysis

Repeated measures two-way ANOVA was used to test for significant difference between the tone responses induced by adenosine in the presence or absence of perivascular tissue or in the presence of an antagonist as one variable and the agonist concentration as the other variable. The analysis was repeated with all concentrations apart from the highest as the starting value in the repeated measures analysis. When a significant difference was found, the most deviant data set was removed until the statistical significance disappeared. Finally, tests for differences between each concentration were performed using Student's unpaired t-test. Data are presented as mean ± SEM values if not otherwise stated. 

Results

None of the interventions affected the tone of nonconstricted arterioles. 

Figure 2 shows that adenosine induced a significant (P < 0.05) concentration-dependent relaxation that was independent of the presence of perivascular retinal tissue. The relaxing effect of adenosine could be antagonized by the nonspecific adenosine receptor antagonist 8-PSPT, and the effect was significant at the two highest adenosine concentrations. 

Figure 2

 

Adenosine induced relaxation alone (solid lines, n = 22) and in the presence of the unspecific antagonist 8-PSPT (broken lines, n = 6) of isolated retinal arterioles (squares) and arterioles with perivascular retinal tissue (triangles). Bars indicate mean ± SEM. The asterisk indicates the range of individual concentrations with significant change in tone induced by the antagonist on isolated vessels, and the double cross indicates the effect on arterioles with preserved perivascular retinal tissue.

Figure 3 shows the results after addition of specific adenosine receptor antagonists. The selective A_2A receptor antagonist SCH 58261 had no effect on adenosine-induced relaxation in the presence of perivascular retinal tissue, but antagonized the relaxing effect of adenosine at the concentrations 10⁻⁶ and 3 × 10⁻⁶ M in isolated arterioles (Fig. 3A). The selective A_2B receptor antagonist MRS 1754 significantly antagonized the relaxing effect of adenosine on isolated arterioles at all concentrations and on arterioles with preserved perivascular retinal tissue at the three highest concentrations (Fig. 3B). The selective A₁ receptor antagonist 3902 significantly increased the relaxing effect of adenosine at concentrations up to 10⁻⁶ M in the presence of the perivascular retina and reduced the relaxing effect of adenosine at concentrations up to 3 × 10⁻⁶ M in isolated arterioles (Fig. 3C). The A₃ receptor antagonist MRS 1523 had no effect on adenosine-induced relaxation in the presence of perivascular retinal tissue, but significantly increased the relaxing effect of adenosine in isolated arterioles at the three highest concentrations (Fig. 3D). 

Figure 3

 

Adenosine induced relaxation in the presence of (broken lines) (A) the A_2A antagonist SCH 58261 (n = 6), (B) the A_2B receptor antagonist MRS 1754 (n = 9), (C) the A₁ receptor antagonist KW-3902 (n = 6), and (D) the A₃ receptor antagonist MRS 1523 (n = 10), as compared to the response of adenosine alone (solid gray lines). Bars indicate mean ± SEM. Asterisks indicate the ranges of individual concentrations with significant changes in tone induced by the antagonist on isolated vessels; double crosses indicate the effect on arterioles with preserved perivascular retinal tissue.

Figure 4A shows that in the presence of perivascular retinal tissue, the selective A₁ receptor agonist CCPA induced a significant contraction at the intermediate concentrations 10⁻⁶ to 10⁻⁵ M and had a relaxing effect similar to that of adenosine at high concentrations, whereas the contracting effect was absent in isolated arterioles. The contracting effect of CCPA at intermediate concentrations could be blocked by the A₁ receptor antagonist KW-3902. In the presence of perivascular retinal tissue, the A_2A receptor antagonist SCH 58261 and the A_2B receptor antagonist MRS 1754 significantly reduced the tone at both intermediate and high CCPA concentrations (not shown). As shown in Figure 4B, the selective A_2A receptor agonist CGS-21680 induced concentration-dependent relaxation similar to that for adenosine in the presence of perivascular retinal tissue, which was significantly reduced but still present at the highest concentration (P < 0.04) in isolated vessels. The effect could be antagonized by the A_2A receptor antagonist SCH 58261 and was significant at 3 × 10⁻⁶ M, whereas the A_2B receptor antagonist MRS 1754 had no effect on the response. As seen in Figure 4C, the selective A₃ receptor agonist Cl-IB-MECA induced relaxation of retinal arterioles, which could be blocked by the A₃ receptor antagonist MRS 1523 at 3 × 10⁻⁵ M on isolated arterioles and at 10⁻⁵ M on arterioles with preserved perivascular retina. 

Figure 4

 

The tone response induced by (A) the A₁ receptor agonist CCPA alone (broken line) and in the presence of (solid line) the A₁ receptor antagonist KW-3902 (n = 6 for both series), (B) the A_2A receptor agonist CGS-21680 alone (broken line) and in the presence of (solid line) the A_2A receptor antagonist SCH 58261 (n = 18 and n = 8, respectively), and (C) the A₃ receptor agonist Cl-IB-MECA alone (broken line) and in the presence of (solid line) the A₃ receptor antagonist MRS 1523 (n = 8 and n = 13, respectively). Squares indicate isolated retinal arterioles, and triangles indicate arterioles with preserved perivascular retinal tissue. Bars indicate mean ± SEM. Asterisks indicate significant changes in tone of isolated arterioles and double crosses the effect on vessels with preserved perivascular retinal tissue.

Discussion

The experiments of the present study extend previous studies using the wire myograph to investigate the tone regulation of retinal arterioles. ^6,14,15 Adenosine was shown to induce vasorelaxation that was independent of the presence of perivascular tissue, indicating a direct effect on the arteriolar wall. In previous studies it has been shown that 8-PSPT can reduce this relaxing effect ⁶ and that the vasodilating effect of adenosine on isolated porcine retinal arterioles is mediated by A_2A receptors but not by A₁ receptors. ⁷ These findings were confirmed and extended by the present study. Thus, the A_2A receptor agonist CGS-21680 had a relaxing effect on retinal arterioles, similar to that of adenosine in the presence of perivascular retinal tissue, which was significantly reduced on isolated vessels. The fact that this response was unaffected by the A_2B receptor antagonist supports that the observed A_2A receptor-mediated relaxation was specific, whereas the minimal blocking of the response by the A_2A receptor antagonist suggests that the efficacy of this antagonist was limited. This conclusion is also supported by the limited effect of the A_2A receptor antagonist SCH 58261 on adenosine-induced relaxation and the finding that the selective A_2B receptor antagonist MRS 1754 could reduce adenosine-induced relaxation in isolated arterioles at all concentrations and in arterioles with perivascular retinal tissue at the highest concentrations. The results can therefore be interpreted as indicating that relaxation of retinal arterioles mediated by A₂ receptors may involve an effect of A_2A receptors in the perivascular retina and A_2B receptors in the vascular wall. These findings also confirm in vivo studies showing that adenosine-induced relaxation of retinal arterioles can be mediated by A₂ receptors, ^16–19 but indicate that the mechanism of action is complex and involves several receptor populations, as well as anatomical structures both in the vascular wall and in the perivascular tissue. 

The finding that the selective A₁ receptor antagonist KW-3902 increased the vasorelaxing effect of adenosine at low and intermediate concentrations in the presence of the retinal tissue can be interpreted as a result of blocking of a constricting effect of adenosine mediated by A₁ receptors in the perivascular retinal tissue. Conversely, the fact that KW-3902 reduced the relaxing effect of adenosine at high concentrations may imply the existence of A₁ receptors mediating relaxation in the arteriolar wall. These findings were confirmed by experiments using the specific A₁ receptor agonist CCPA, which contracted the vessels at intermediate concentrations in the presence of perivascular retinal tissue and relaxed the vessels at high concentrations, both on isolated retinal arterioles and on arterioles with preserved perivascular tissue. Therefore, the findings suggest that A₁ receptors may have a dual effect, mediating both vasorelaxation and constriction, depending on the agonist concentration and the anatomical localization in the retina. The finding that A_2A and A_2B receptor antagonists reduced the tone induced by CCPA at both intermediate and high concentrations in the presence of perivascular retinal tissue indicates that these antagonists may have cross-reacted with the effects mediated by A₁ receptors. 

The fact that the A₃ receptor antagonist MRS 1523 increased the vasorelaxing effect of adenosine in isolated arterioles, but had no effect in the presence of the perivascular retina, might suggest that adenosine can induce vasoconstriction mediated by A₃ receptors in the arteriolar wall and is in accordance with findings from the rat aorta. ^12,20 This interpretation is supported by the finding that blocking of the A₃ receptor using the specific antagonist MRS 1523 also increased the relaxing effect of the A₃ receptor agonist Cl-IB-MECA. However, the persistence of some relaxation indicates that CL-IB-MECA had vasoactive effects other than those inhibited by MRS 1523, and a detailed evaluation of the role of the A₃ receptor in tone regulation of retinal arterioles therefore needs further investigation. 

The findings of the present study suggest that several adenosine receptor types are involved in modulating retinal arteriolar tone, depending on the concentration and site of action in the tissue elements around the retinal arterioles. The dual effects with adenosine-induced contraction mediated via some receptors and relaxation via other receptors are in accordance with findings from the kidney ⁸ and the fallopian tube, ²¹ and with studies of other vasoactive compounds in both smaller retinal ²² and ciliary ²³ vessels. It has been shown that the release of adenosine is involved in the pathogenesis of experimental retinal ischemia ²⁴ and in diseases characterized by impairment of retinal blood flow. ^19,25,26 It is possible that the differentiated vessel response in retinal vascular disease with vasoconstriction in the retinal periphery and vasodilation in the macular area ²⁷ may involve dual responses by compounds with effects depending on concentration and anatomical site of action. The identity of these sources of tone regulation might potentially be evaluated using techniques that allow the study of receptor-mediated activity in individual retinal cell types. Recently, a perivascular cell structure has been identified that displays intracellular calcium activity during adenosine triphosphate (ATP)-induced relaxation of porcine retinal arterioles, which suggests a role of this structure in the control of retinal vascular tone. ^4,5 However, this cell structure showed only limited activity during relaxation induced by adenosine, which suggests that other cell structures in the perivascular retina are involved in the adenosine-induced changes in tone. The identification of these cell structures should be the subject of future studies. In conclusion, the present study has shown that adenosine plays an important role in modulating retinal arteriolar tone and that purinergic compounds might be important mediators for pharmacological intervention in diseases involving disturbances in the retinal blood flow. 

Acknowledgments

The authors thank Jens Leipziger, Institute of Biomedicine, University of Aarhus, for assistance in the use of purinergic compounds and proofreading the manuscript. 

Supported by the Danish Medical Research Council, the VELUX Foundation, and Jochum and Marie Jensen's Foundation. 

Disclosure: M.J. Riis-Vestergaard, None; M.W. Misfeldt, None; T. Bek, None