Interactions of adenosine A1 and A2a receptors on renal microvascular reactivity

Akira Nishiyama, Edward W. Inscho, and L. Gabriel Navar

Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112-2699


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine vasoconstricts preglomerular arterioles via adenosine A1 receptors. Because adenosine also activates adenosine A2 receptors, its overall renal vascular actions are complex and not fully understood. The present study was performed to determine the relative contributions of adenosine A1 and A2a receptors to the responsiveness of the renal microvasculature to adenosine. Afferent and efferent arteriolar diameters were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. Basal afferent and efferent arteriolar diameters averaged 17.1 ± 0.5 (n = 35) and 17.8 ± 0.5 (n = 20) µm, respectively. Superfusion with 0.1 and 1 µmol/l adenosine did not significantly alter afferent and efferent arteriolar diameters; however, 10 µmol/l adenosine significantly reduced afferent and efferent arteriolar diameters (-8.2 ± 0.8 and -5.7 ± 0.6%, respectively). The afferent and efferent arteriolar vasoconstrictor responses to adenosine waned at a dose of 100 µmol/l, such that diameters returned to values not significantly different from control within 2 min. During adenosine A1 receptor blockade with 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902: 10 µmol/l), 10 and 100 µmol/l adenosine significantly increased afferent diameter by, respectively, 8.1 ± 1.2 and 13.7 ± 1.3% (n = 14) and efferent arteriolar diameter by 6.4 ± 1.3 and 9.3 ± 1.2% (n = 8). The afferent and efferent arteriolar vasodilatory responses to adenosine in the presence of KW-3902 were significantly attenuated by addition of the adenosine A2a receptor antagonist 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837: 15 µmol/l, n = 7 and 6, respectively). The addition of KF-17837 alone significantly enhanced afferent (n = 15) and efferent (n = 6) arteriolar vasoconstrictor responses to 1, 10, and 100 µmol/l adenosine. These results indicate the presence of adenosine A1 and A2a receptors on afferent and efferent arterioles of juxtamedullary nephrons, such that adenosine A2a receptor-mediated vasodilation partially buffers adenosine-induced vasoconstriction in both pre- and postglomerular segments of the renal microvasculature.

adenosine; afferent arterioles; efferent arterioles; KW-3902; KF-17837


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADENOSINE SERVES AN important paracrine function in regulating renal hemodynamics (1, 15, 20, 31, 32, 36). Measurements of total renal blood flow (RBF) in the whole kidney have demonstrated an effect of exogeneous adenosine on RBF characterized by an initial transient renal vasoconstriction that wanes and becomes supplanted by a gradual vasodilation (1, 20, 31, 32). The mechanism for this biphasic response remains poorly understood, although it is presumed to involve the differential binding of adenosine to A1 and A2 receptors to evoke renal vasoconstriction and vasodilation, respectively (20, 31, 32). Adenosine A2 receptors are subdivided into distinct A2a and A2b subtypes (33), which are shown to be expressed in the kidney (14, 38). Furthermore, recent studies have demonstrated adenosine A2a receptor-mediated vasodilation in isolated rabbit renal artery (29) and arcuate artery (28).

Renal microvascular responses to the adenosine A1 or A2 receptor agonists have been evaluated using several different systems. Holtz and Steinhausen (6) performed studies using the rat hydronephrotic kidney model and observed that topical application of the selective adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) reduced afferent arteriolar diameter without affecting efferent arteriolar diameter. In contrast, Dietrich and Steinhausen (5) used the same method and demonstrated CHA-induced efferent arteriolar vasoconstriction. The authors also reported that application of the selective adenosine A2 receptor agonist N-ethyl-carboxamide adenosine (NECA) dilated both afferent and efferent arterioles. Studies performed using isolated perfused rabbit afferent arterioles also demonstrated a vasoconstrictor response to CHA; however, the selective adenosine A2 receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) had no effect on afferent arteriolar diameter (35). Collectively, these observations support the presence of adenosine A1 receptors on afferent arterioles. However, the presence of adenosine A1 receptors on the efferent arteriole and the localization of adenosine A2 receptors on the renal microvasculature remain unclear.

The effects of adenosine or nonselective adenosine analogs on renal microvascular reactivity have also been evaluated (4, 6, 8, 9, 34, 35). The nonselective adenosine agonist, 2-chloroadenosine (2-CA), significantly reduced afferent arteriolar diameter but increased efferent arteriolar diameter in the hydronephrotic kidney (6). Using the juxtamedullary nephron preparation, Inscho et al. (8) demonstrated that superfusion of 2-CA over juxtamedullary afferent arterioles elicits vasoconstriction but that, at the higher concentrations, the vasoconstriction changes to a vasodilation. Further studies showed that adenosine reduces both afferent and efferent arteriolar diameters (4, 9). In contrast to these observations, adenosine and 2-CA caused monophasic diameter reductions of afferent arterioles isolated from rabbits (35). Thus the fact that adenosine can act as either a vasoconstrictor or a vasodilator of the renal microvasculature has led to uncertainty in the characterization of adenosine-mediated renal microvascular reactivity.

The present study was performed to delineate the interaction between adenosine A1 and A2a receptors in the control of the renal microvasculature. We hypothesized that adenosine A2a receptor-mediated vasodilation buffers adenosine-induced vasoconstriction in both afferent and efferent arterioles. To test this hypothesis, we used the in vitro blood-perfused juxtamedullary nephron technique (4, 8, 9) and determined the effects of selective adenosine A1 and A2a receptor blockade on the responsiveness of afferent and efferent arteriolar responses to adenosine. In this study, we used 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902) and 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837), which are new-generation adenosine A1 and A2a receptor antagonists with much higher degrees of selectivity for adenosine A1 and A2a receptors, respectively, compared with other available adenosine A1 and A2a receptor antagonists (10, 19, 25, 26).


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
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Assessment of afferent and efferent arteriolar reactivity. The experiments were performed in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee.

Afferent and efferent arteriolar diameters were assessed in vitro using the blood-perfused juxtamedullary nephron technique combined with videomicroscopy, as previously described (4, 8, 9). Each experiment used two male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 325-450 g, with one rat serving as the blood donor and the second rat as the kidney donor. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a cannula was inserted in the left carotid artery of the blood donor. Donor blood was collected in a heparinized (500 units) syringe via the carotid arterial cannula and centrifuged to separate the plasma and cellular fractions. The buffy coat was removed and discarded. After sequential passage of the plasma through 5- and 0.22-µm filters (Gelman Sciences, Ann Arbor, MI), erythrocytes were added to achieve a hematocrit of 33%. This reconstituted blood was passed through a 5-µm nylon mesh and thereafter stirred continuously in a closed reservoir that was pressurized with a 95% O2-5% CO2 gas mixture.

The right kidney of the kidney donor was perfused through a cannula inserted in the superior mesenteric artery and advanced into the right renal artery. The perfusate was a Tyrode solution (pH 7.4) containing 5.1% BSA and a mixture of L-amino acids as previously described (4, 8, 9). The kidney was excised and sectioned longitudinally, retaining the papilla intact with the perfused dorsal two-thirds of the organ. The papilla was reflected to expose the pelvic mucosa and tissue covering the inner cortical surface. Overlying tissue was removed to expose the tubules, glomeruli, and related vasculature of the juxtamedullary nephrons. The arterial supply of the exposed microvasculature was isolated by ligating the larger branches of the renal artery.

After the dissection was completed, the Tyrode perfusate was replaced with the reconstituted blood. Perfusion pressure was monitored by a pressure cannula centered in the tip of the perfusion cannula. Renal perfusion pressure was regulated by adjusting the rate of gas inflow into the blood reservoir and setting at 100 mmHg. The inner cortical surface of the kidney was continuously superfused with a warmed (37°C) Tyrode solution containing 1% BSA. The tissue was transilluminated on the fixed stage of a Leitz Laborlux-12 microscope equipped with a water-immersion objective (×40). Video images of the microvessels were transferred by a Newvicon camera (model NC-67M; Dage-MTI, Michigan City, IN) through an image enhancer (model MFJ-1452; MFJ Enterprises, Starkville, MS) to a video monitor (Conrac Display Systems, Covina, CA). The video signal was recorded on videotape for later analysis (Super VHS videocassette recorder; Panasonic, Secaucus, NJ). Afferent and efferent arteriolar inside diameters were measured at 15-s intervals using a calibrated digital image-shearing monitor (Instrumentation for Physiology and Medicine, San Diego, CA).

Afferent and efferent arteriolar diameters were measured at a location as close to the glomerulus as possible (within 100 µm of the glomerulus). Efferent arterioles were studied between the glomerulus and the first bifurcation. Treatments were administered by superfusing the tissue with a solution containing the agent to be tested or vehicle (0.2% DMSO). Superfusion directly over the adventitial side of afferent and efferent arterioles permits investigation of the actions of adenosine from the interstitial side as would occur if adenosine is serving as a paracrine agent.

Experimental protocols. After a 15-min equilibration period, an experimental protocol was initiated consisting of consecutive 5-min treatment periods. Steady-state diameter determinations were calculated from the average of measurements obtained during the final 2 min of each 5-min treatment period and were used for statistical analysis. We also evaluated the rapid responses to adenosine occurring during the first 45 s.

Afferent and efferent arteriolar responsiveness to increasing concentrations (0.1, 1, 10, and 100 µmol/l) of adenosine was assessed. After the control dose-response relationship to adenosine was obtained, the kidneys were superfused with the control buffer solution for 20 min to allow restoration of control diameters. Next, the effects of adenosine A1 or A2a blockade on afferent and efferent arteriolar responses to adenosine were determined. The selective adenosine A1 antagonist KW-3902 (10 µmol/l; Kyowa-Hakko Kogyo, Tokyo, Japan; see Refs. 19, 25) or the selective adenosine A2a antagonist KF-17837 (15 µmol/l; Kyowa-Hakko Kogyo; see Refs. 10, 19, and 26) was added to the superfusate for 5 min, and the afferent and efferent responses to adenosine (0.1-100 µmol/l) were reassessed in the presence of these adenosine antagonists. In a separate experimental series, the afferent and efferent arteriolar responses to adenosine (0.1-100 µmol/l) were also assessed before and during superfusion with KW-3902 (10 µmol/l) plus KF-17837 (15 µmol/l).

The assessment of afferent arteriolar responsiveness to adenosine (0.1-100 µmol/l) was repeated to determine the afferent arteriolar responses to repeated administration of adenosine. After the first dose-response relationship to adenosine was obtained, the kidney was superfused for 20 min with the control buffer solution. Next, the second afferent arteriolar dose-response relationship to adenosine was determined. To avoid the possibility that prolonged exposure to extracellular adenosine results in desensitization of adenosine receptors, the response of afferent arterioles to a single application of 10 µmol/l adenosine was repeated in a separate group of vessels. This adenosine concentration was chosen because it evoked vasoconstriction from each of the afferent arterioles previously studied (4, 9). After the first response to 10 µmol/l adenosine was obtained, the kidney was superfused for 20 min with the control buffer solution. Subsequently, the second afferent arteriolar response to 10 µmol/l adenosine was evaluated.

In a separate experimental series, the responses of afferent and efferent arterioles to 10 µmol/l adenosine were assessed before and during treatment with KW-3902 (10 µmol/l) and KW-3902 (10 µmol/l) plus KF-17837 (15 µmol/l). After afferent and efferent arteriolar responses to a single application of 10 µmol/l adenosine were assessed, the kidney was superfused for 20 min with the control buffer solution. Next, KW-3902 was added to the superfusate for 5 min, and the afferent and efferent arteriolar responses to 10 µmol/l adenosine were reassessed in the presence of KW-3902. Subsequently, KF-17837 was added to the superfusate containing KW-3902 and 10 µmol/l adenosine.

Drugs. KW-3902 and KF-17837 were supplied by Kyowa Hakko Kogyo. Adenosine was obtained from Sigma Chemical (St. Louis, MO).

Statistical analysis. Analysis of changes in afferent and efferent arteriolar responses to adenosine were performed using the one-way ANOVA for repeated measures combined with the Newman-Keuls post hoc test. Within each series, differences in afferent and efferent arteriolar responses to adenosine between group means were determined using the two-way ANOVA for repeated measures combined with the Newman-Keuls post hoc test. Differences between groups from different experimental series were determined using Student's t-test for unpaired data. P < 0.05 was considered statistically significant. Data are presented as means ± SE.


    RESULTS
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Effects of extracellular adenosine on afferent and efferent arteriolar diameters. Figure 1 shows the afferent and efferent arteriolar responses to increasing concentrations of adenosine (0.1, 1, 10, and 100 µmol/l). Basal afferent and efferent arteriolar diameters were similar in both arterioles and averaged 17.1 ± 0.5 and 17.8 ± 0.5 µm (n = 35 and 20, respectively). Superfusion with 0.1 µmol/l adenosine did not alter afferent and efferent arteriolar diameters (17.1 ± 0.5 and 17.7 ± 0.5 µm, respectively). Increases in superfusate adenosine concentration to 1 µmol/l caused a transient reduction in arteriolar diameter by 4.9 ± 1.1% within the initial 45 s (P < 0.05). With continued application of 1 µmol/l adenosine, this effect waned, and afferent arteriolar diameter returned to values that were only 3.2 ± 0.5% below control values (not significant). Afferent arteriolar diameter was significantly reduced with 10 µmol/l adenosine by 11.3 ± 1.2% to 15.2 ± 0.5 µm within the initial 45 s and remained significantly smaller than control. Afferent diameter averaged 15.8 ± 0.5 µm (91.7 ± 0.7% of the control diameter) over the last 2 min of adenosine treatment. In contrast, 1 µmol/l adenosine did not alter efferent arteriolar diameter significantly during the treatment period. Furthermore, 10 µmol/l adenosine elicited a slowly developing vasoconstrictior response in efferent arterioles that decreased efferent arteriolar diameter by 5.7 ± 0.6% to 16.7 ± 0.5 µm. The afferent and efferent arteriolar vasoconstrictor responses to 10 µmol/l adenosine waned at a dose of 100 µmol/l, such that diameters returned to values not significantly different from control within 2 min (17.5 ± 0.5 and 17.9 ± 0.5 µm).


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Fig. 1.   Effects of adenosine on afferent (A and B, n = 35) and efferent (C and D, n = 20) arteriolar diameters. Time course of afferent (A) and efferent (C) arteriolar responses to adenosine. Data are average vessel diameters measured at 15-s intervals. Steady-state changes in afferent (B) and efferent (D) arteriolar diameters in response to adenosine. Steady-state diameter determinations were calculated from the average of measurements obtained during the final 2 min of each 5-min treatment period. Data are expressed as %changes of the control diameters. C, control; R, recovery. *P < 0.05 vs. control period.

The time control experiments (n = 4) demonstrated no differences in afferent arteriolar responses to repeat administration of adenosine (0.1-100 µmol/l; data not shown). The afferent arteriolar response to a single application of 10 µmol/l adenosine was also determined (n = 7). Administration of 10 µmol/l adenosine elicited a rapid vasoconstrictor response, with afferent diameter declining by -11.8 ± 2.5% from 18.0 ± 1.0 to 15.9 ± 1.0 µm within the initial 45 s. With continued application of 10 µmol/l adenosine, afferent diameter increased slightly but remained significantly smaller than control. Afferent diameter averaged 16.5 ± 0.9 µm (91.5 ± 0.8% of the control diameter) over the last 2 min of adenosine treatment. Removal of adenosine from the superfusate bathing solution allowed recovery of afferent diameter to 17.9 ± 0.9 µm. No differences in the repeat afferent arteriolar vasoconstrictor response to 10 µmol/l adenosine were observed (data not shown).

Effects of adenosine A1 receptor blockade on afferent and efferent arteriolar responses to adenosine. Responsiveness of afferent (n = 14) and efferent (n = 8) arterioles to increasing concentrations of adenosine (0.1-100 µmol/l) were examined before and during adenosine A1 receptor blockade with KW-3902. As depicted in Fig. 2, 10 µmol/l adenosine significantly decreased afferent arteriolar diameter by 9.2 ± 1.4% and efferent arteriolar diameter by 5.5 ± 0.9%. KW-3902 treatment (10 µmol/l) did not alter basal afferent or efferent arteriolar diameters (17.3 ± 0.7 to 17.5 ± 0.7 µm and 18.4 ± 0.5 to 18.6 ± 0.7 µm). In the presence of KW-3902, 0.1 and 1 µmol/l adenosine also did not alter afferent or efferent arteriolar diameters; however, 10 and 100 µmol/l adenosine significantly increased afferent arteriolar diameter by 8.1 ± 1.2% to 19.0 ± 0.9 µm and by 13.7 ± 1.3% to 19.9 ± 0.8 µm, respectively, and efferent arteriolar diameter by 6.4 ± 1.3% to 19.8 ± 0.7 µm and by 9.3 ± 1.2% to 20.4 ± 0.8 µm, respectively (steady state). During treatment with KW-3902, 0.1-100 µmol/l adenosine did not elicit any rapid vasoconstrictor responses within the initial 45 s.


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Fig. 2.   Steady-state changes in afferent (A and B, n = 14) and efferent (C and D, n = 8) arteriolar diameters in response to adenosine before and during administration of adenosine A1 receptor antagonist 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902). open circle , Adenosine alone; , adenosine + KW-3902. Data are expressed in µm (A and C) and as %changes of the control diameters (B and D). *P < 0.05 vs. control period. dagger P < 0.05 vs. adenosine alone.

Responsiveness of afferent (n = 14) and efferent (n = 6) arterioles to increasing concentrations of adenosine (0.1-100 µmol/l) was examined before and during adenosine A2a receptor blockade with KF-17837. KF-17837 treatment (15 µmol/l) did not alter basal afferent and efferent arteriolar diameters. During treatment with KF-17837, 0.1 µmol/l adenosine did not alter afferent arteriolar diameter; however, 1, 10, and 100 µmol/l adenosine significantly reduced afferent arteriolar diameters (Fig. 3A). As summarized in Fig. 3B, reductions in afferent arteriolar diameters (averaged over the last 2 min of treatment) to 1, 10, and 100 µmol/l adenosine were 6.9 ± 0.6, 13.4 ± 1.1, and 8.4 ± 1.5%, respectively. Compared with the responses observed with adenosine alone, the magnitude of the afferent arteriolar vasoconstriction to 10 µmol/l adenosine was significantly augmented during KF-17837 administration (Fig. 3B). However, KF-17837 did not significantly alter afferent arteriolar rapid responses (within the initial 45 s) to adenosine. During treatment with KF-17837, 0.1 µmol/l adenosine did not alter efferent arteriolar diameter; however, 1, 10, and 100 µmol/l adenosine significantly reduced efferent arteriolar diameter by -5.4 ± 0.8, -12.3 ± 1.2, and -7.5 ± 1.1%, respectively (Fig. 3C). The magnitude of the efferent arteriolar vasoconstriction to 10 µmol/l adenosine was significantly augmented during KF-17837 administration (Fig. 3C).


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Fig. 3.   Effects of adenosine A2a receptor blockade with 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837) on afferent and efferent arteriolar responses to adenosine. A: time course of afferent arteriolar responses to adenosine before and during administration of KF-17837 (n = 14). Data are average vessel diameters measured at 15-s intervals. SE bars have been omitted for clarity. open circle , Adenosine alone; , adenosine + KF-17837. B: steady-state changes in afferent arteriolar diameter in response to adenosine before and during administration of KF-17837 (n = 14). Data are expressed as %changes of the control diameters. C: steady-state changes in efferent arteriolar diameter in response to adenosine before and during administration of KF-17837 (n = 6). Data are expressed as %changes of the control diameters. *P < 0.05 vs. control period. dagger P < 0.05 vs. adenosine alone.

Afferent (n = 7) and efferent (n = 6) arteriolar responses to 0.1, 1, 10, and 100 µmol/l adenosine were also assessed during both adenosine A1 and A2a receptor blockade, as shown in Fig. 4. Basal afferent and efferent arteriolar diameters were similar in both arterioles and averaged 16.6 ± 1.1 and 16.9 ± 1.2 µm, respectively. Treatment with KW-3902 (10 µmol/l) plus KF-17837 (15 µmol/l) did not alter basal afferent and efferent arteriolar diameters (16.8 ± 1.2 and 17.1 ± 1.4 µm, respectively). During treatment with KW-3902 plus KF-17837, 0.1, 1, and 10 µmol/l adenosine did not alter afferent and efferent arteriolar diameters. However, 100 µmol/l adenosine significantly increased afferent and efferent arteriolar diameters by 5.9 ± 1.0% to 17.8 ± 1.1 µm and by 5.8 ± 0.9% to 18.1 ± 1.5 µm, respectively (steady state). On the basis of group comparisons, the afferent and efferent arteriolar responses to 100 µmol/l adenosine in kidneys treated with KW-3902 plus KF-17837 were significantly smaller than those observed in kidneys treated with KW-3902 alone, as shown in Figs. 2 and 4 (P < 0.05).


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Fig. 4.   Steady-state changes in afferent (A and B, n = 7) and efferent (C and D, n = 6) arteriolar diameters in response to adenosine before and during administration of KW-3902 + KF-17837. open circle , Adenosine alone; , adenosine + KW-3902 + KF-17837. Data are expressed in µm (A and C) and as %changes of the control diameters (B and D). *P < 0.05 vs. control period. dagger P < 0.05 vs. adenosine alone.

Afferent (n = 7) and efferent (n = 6) arteriolar responses to acute application of 10 µmol/l adenosine were assessed before (Fig. 5A) and during KW-3902 treatment alone and in combination with KF-17837 treatment (Fig. 5B). Basal afferent and efferent arteriolar diameters were similar in both arterioles and averaged 17.1 ± 0.9 and 17.6 ± 1.5 µm, respectively. Adenosine alone (10 µmol/l) significantly reduced afferent arteriolar diameter by 8.1 ± 1.3% to 15.7 ± 0.8 µm and efferent arteriolar diameter by 6.7 ± 1.1% to 16.5 ± 1.5 µm (Fig. 5A). Removal of adenosine from the superfusate solution allowed recovery of afferent diameter to 17.0 ± 1.1 µm and efferent diameter to 17.0 ± 1.9 µm. Similar to the studies described above, KW-3902 (10 µmol/l) treatment did not alter afferent and efferent arteriolar diameters (17.1 ± 1.1 and 17.0 ± 1.7 µm, respectively). During treatment with KW-3902, 10 µmol/l adenosine significantly increased afferent arteriolar diameter by 10.5 ± 1.1% to 18.9 ± 1.2 µm and efferent arteriolar diameter by 5.8 ± 1.0% to 18.1 ± 1.9 µm. The adenosine-induced afferent and efferent arteriolar vasodilation was recovered by the addition of 15 µmol/l KF-17837 (afferent arterioles: to 17.6 ± 1.2 µm, efferent arterioles: to 19.5 ± 1.8 µm), as shown in Fig. 5B.


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Fig. 5.   Steady-state changes in afferent (n = 7) and efferent (n = 6) arteriolar diameters in response to acute application of 10 µmol/l adenosine before (A) and during (B) treatment with KW-3902 or KW-3902 + KF-17837. Open bars, afferent arterioles; filled bars, efferent arterioles. *P < 0.05 vs. control period. dagger P < 0.05 vs. KW-3902 alone. Data presented as %changes of the control diameters (A) and from the diameter of KW-3902 alone (B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, the mRNAs for A1 and A2a receptors were detected in renal arterial tissue (29); however, the localization and functional role of these receptors in the renal microvasculature remain unclear. The present experiments using the blood-perfused juxtamedullary nephron preparation establish that superfusion with 10 µmol/l adenosine evokes sustained afferent and efferent arteriolar vasoconstriction, whereas with higher concentrations of adenosine the vasoconstrictor effect is attenuated. To evaluate the possible interaction between adenosine A1 receptor-mediated vasoconstriction and A2a receptor-mediated vasodilation, we examined the afferent and efferent arteriolar responses to adenosine during selective adenosine A1 and A2a receptor blockade.

In agreement with the previous studies (4, 9), we observed that adenosine constricts both afferent and efferent arterioles of juxtamedullary nephrons. Although adenosine A1 receptor-mediated afferent arteriolar vasoconstriction has been shown in several experimental models (4, 5, 6, 8, 9, 11, 34, 35), the efferent arteriolar response to adenosine A1 receptor activation has been less consistent (4-6, 11). Some studies reported efferent arteriolar vasoconstriction in response to adenosine or the adenosine A1 agonist CHA (4, 5), whereas others observed no change with these compounds (6, 11). Dietrich and Steinhausen (5) found that CHA constricts proximal efferent arterioles (within 50 µm of the glomerulus), whereas no effect was observed on distal efferent arterioles (near the welling point). In the present study, the measurement sites along the efferent arterioles were selected at a location as close to the glomerulus as possible. Therefore, one possible explanation for the discrepancy between the current report and others (6, 11) may be due to regional heterogeneity of the adenosine A1 receptor population or to the sensitivity of the efferent arterioles studied. It is also possible that there is a heterogeneity in the distribution of vascular adenosine A1 receptors between superficial and juxtamedullary nephrons, as suggested by other investigators (27, 35).

We found that adenosine dilates both afferent and efferent arterioles during adenosine A1 receptor blockade. These results are consistent with the recent observations that adenosine dilates isolated rabbit afferent arterioles in the presence of the adenosine A1 receptor antagonist 6-oxo-3-[2-phenylpyrazole(1,5-a)pyridine-3-yl]-l(6H)-pyridazinebutyric (37). We also observed that afferent and efferent arteriolar vasodilatory responses to adenosine during adenosine A1 receptor blockade were significantly attenuated by adenosine A2a receptor inhibition. Thus these data support the hypothesis that adenosine A1 and A2a receptors are expressed on both afferent and efferent arterioles and support the postulate that adenosine A2a receptor-mediated vasodilation may buffer adenosine A1 receptor-mediated vasoconstriction. These counteracting actions may help explain why some studies have failed to show significant effects of nonselective adenosine agonists or antagonists in various studies.

As reported previously (9, 34), we observed a biphasic afferent arteriolar response to adenosine characterized by a transient vasoconstriction that wanes within a minute. One possible explanation for the waning vasoconstrictor response is the delayed activation of a secondary vasodilatory response. During adenosine A2a receptor blockade, the rapid afferent arteriolar vasoconstrictor responses to adenosine (within the initial 45 s) were not altered; however, the constrictor responses to adenosine averaged over the last 2 min of treatment were significantly augmented. These results indicate that adenosine A2a receptors participate in the apparent waning of the initial afferent arteriolar vasoconstrictor response to adenosine. In contrast, adenosine did not elicit a rapid vasoconstrictor response in efferent arterioles. The reason for the discrepancy between afferent and efferent arteriolar responses to adenosine is unclear; however, the fact that adenosine A2a receptor blockade augmented adenosine-induced efferent arteriolar vasoconstriction suggests that a similar vasodilatory effect of adenosine A2a receptors also exists on efferent arterioles.

The presence of adenosine A2 receptors on afferent and efferent arterioles is supported by studies performed in vivo (1, 13) and in vitro (5, 6, 16, 17). Miura et al. (13) demonstrated that renal arterial administration of the selective adenosine A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine significantly increased RBF without affecting glomerular filtration rate in anesthetized dogs. Thus these results indicate that adenosine A2a receptors mediate afferent and efferent arteriolar vasodilation. Similar results were reported by Murray and Churchill (16, 17) using the nonselective adenosine A2 receptor agonist NECA in the isolated perfused rat kidney. It was also demonstrated that NECA significantly increased both afferent and efferent arteriolar diameters in the hydronephrotic rat kidney (5, 6). In contrast to these observations, Weihprecht et al. (35) showed that NECA constricts afferent arterioles and the more selective A2 analog DPMA had no effect on the isolated afferent arteriolar diameter. The reason for the discrepancy between these results is unclear; however, it is possible that such varied responses may be due to the relatively low selectivity of NECA and DPMA for adenosine A2 receptors and may reflect some interaction between the A2 agonists and adenosine A1 receptors. Recently, Yaoita et al. (37) showed that, during adenosine A1 receptor blockade, adenosine dilates isolated afferent arterioles preconstricted with norepinephrine but does not affect the diameter of afferent arterioles not treated with norepinephrine. Therefore, another possibility exists that adenosine A2 receptor-mediated afferent arteriolar reactivity could be influenced by the basal vascular tone. Indeed, Weihprecht et al. (35) performed studies using nonpreconstricted isolated afferent arterioles and assessed the response to an adenosine A2 agonist.

Because adenosine A2a receptor blockade significantly augmented the vasoconstrictor response to 10 µmol/l adenosine, we anticipated that 100 µmol/l adenosine would further reduce afferent and efferent arteriolar diameters in the presence of the adenosine A2a receptor antagonist. However, the magnitude of the afferent and efferent arteriolar response to 100 µmol/l adenosine during adenosine A2a receptor blockade was actually less than the response to 10 µmol/l adenosine (Fig. 3, A-C). We also observed that 100 µmol/l adenosine slightly but significantly dilated both afferent and efferent arterioles during combined administration of adenosine A1 and A2a receptor blockers (Fig. 4). These data suggest the possibility that the dose of the adenosine A2a receptor antagonist KF-17837 (15 µmol/l) is insufficient to prevent the vasodilatory responses to 100 µmol/l adenosine. KF-17837 has great selectivity for the adenosine A2a receptors compared with other adenosine A2a receptor antagonists (10, 19, 26); however, Scatchard analysis demonstrated that KF-17837 functions as a competitive inhibitor (26). Therefore, we could not use a higher concentration of KF-17837 in the present experimental setting because high concentrations of KF-17837 may affect other adenosine receptors. Another possibility is that the low-affinity adenosine A2b receptors may be activated at high adenosine concentrations and elicit a vasodilatory response. Tang et al. (34) reported that there are two different types of adenosine-induced afferent arteriolar vasodilatory responses in the in vitro perfused hydronephrotic rat kidney: one response corresponds to activation of low-affinity adenosine A2b receptors and the second response is mediated by high-affinity adenosine A2a receptors. The authors suggested that adenosine concentrations >10 µmol/l are required for adenosine A2b receptor-mediated afferent arteriolar vasodilation in in vitro preparations (34). Although recent studies indicate that adenosine A2a-mediated afferent (34) and arcuate arteriolar (28) vasodilation are linked to the activation of ATP-sensitive potassium channels, the higher concentrations of adenosine may also activate potassium channels via adenosine A2b receptors, leading to vasodilation, as shown for other vessels (12, 18).

The microdialysis experiments performed in anesthetized rats (3, 30, 38), dogs (21-23), and rabbits (24) indicate that resting renal interstitial adenosine concentrations normally remain in the 0.1-1 µmol/l range. Therefore, the fact that a significant vasoconstriction to adenosine occurs at 10 µmol/l suggests the possibility that endogenous adenosine concentrations are lower than appears necessary for significant adenosine-mediated renal vasoconstriction in resting conditions. In the present study, we observed that KW-3902 treatment did not alter afferent or efferent arteriolar diameters under resting conditions. These results are consistent with previous observations that intrarenal administration of KW-3902, which prevents the vasoconstrictor influence of exogeneous adenosine, did not alter basal RBF or glomerular filtration rate (2), thus indicating that the basal influence of endogeneous adenosine is relatively low. Recent studies have shown, however, that renal interstitial adenosine concentrations are increased by chronic salt loading (30, 38) and during acute renal failure (21, 23, 24). Therefore, it is possible that endogeneous adenosine contributes to renal hemodynamic changes in some pathological circumstances.

In summary, the availability of more selective adenosine antagonists has allowed us to demonstrate that extracellular adenosine can evoke vasoconstrictor and vasodilator actions on both afferent and efferent arterioles of juxtamedullary nephrons. During adenosine A1 receptor blockade, adenosine elicits afferent and efferent arteriolar vasodilation with a slightly greater effect on afferent arterioles. These vasodilator effects are markedly attenuated by adenosine A2a receptor blockade. During adenosine A2a receptor blockade, the adenosine-induced vasoconstrictor responses are enhanced. These data indicate the presence of adenosine A1 and A2a receptors on juxtamedullary afferent and efferent arterioles and are consistent with the hypothesis that adenosine A2a receptor-mediated vasodilation buffers adenosine A1 receptor-mediated vasoconstriction in the renal microvasculature.


    ACKNOWLEDGEMENTS

We thank Anthony K. Cook for technical assistance. We are also grateful to Dr. Akira Karasawa (Kyowa Hakko Kogyo, Tokyo, Japan) for supplying KW-3902 and KF-17837 and for helpful advice in the performance of these studies.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-18426 and DK-44628, by an award from the American Heart Association, Southeast Affiliate (A. Nishiyama), and by the National American Heart Association (E. W. Inscho). E. W. Inscho is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: A. Nishiyama, Dept. of Physiology, no. SL-39, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: anishiy{at}tulane.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 September 2000; accepted in final form 7 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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