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J Appl Physiol 91: 552-560, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 2, 552-560, August 2001

Adenosine A1 receptors mediate plasma exudation from the oral mucosa

Israel Rubinstein1,2,3, Rinku Chandilawa1,3, Sumeet Dagar2, Dennis Hong1,3, and Xiao-Pei Gao1

1 Departments of Medicine and 2 Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, and 3 Veterans Affairs Chicago Health Care System West Side Division, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to pharmacologically characterize the adenosine receptor subtype(s) that mediates adenosine-induced increases in macromolecular efflux from the intact hamster cheek pouch. Using intravital microscopy, we found that 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), a selective adenosine receptor-1 antagonist, but not 3,7-dimethyl-1-propargylxanthine (DMPX), a selective adenosine receptor-2 antagonist, significantly attenuated adenosine-induced leaky site formation and increased clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the intact hamster cheek pouch (P < 0.05). Both compounds had no significant effects on bradykinin-induced responses. Nanomolar concentrations of R(-)-N6-(2-phenylisopropyl)-adenosine [R(-)-PIA], a selective adenosine A1 agonist, evoked significant, concentration-dependent increases in macromolecular efflux. This response was significantly attenuated by PACPX but not by DMPX. In contrast, CGS-21680, a selective adenosine A2 agonist, increased macromolecular efflux but only at micromolar concentrations. This response was significantly attenuated by DMPX but not by PACPX. Suffusion of nitroglycerin had no significant effects on R(-)-PIA- and CGS-21680-induced responses. In addition, suffusion of NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no significant effects on adenosine-induced responses. Indomethacin had no significant effects on adenosine-, R(-)-PIA-, and CGS-21680-induced increases in macromolecular efflux. Collectively, these data indicate that adenosine increases macromolecular efflux from the intact hamster cheek pouch by stimulating high-affinity adenosine A1 receptors in a specific, nitric oxide- and prostaglandin-independent fashion.

microcirculation; venules; inflammation; intravital microscopy; adenosine analogs; adenosine receptor antagonists


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CURRENT CONCEPTS SUGGEST THAT ADENOSINE, which is formed by dephosphorylation of adenosine monophosphate both intracellularly and extracellularly, plays an important role in regulating microvascular responses in the peripheral circulation, including plasma exudation, during the host inflammatory response to injury (2, 11, 18, 19, 26, 30, 33). These effects are mediated by at least two populations of cell surface adenosine A1 and A2 receptors (4, 5-7, 18, 25, 26, 30). However, studies in the literature on the relationship between adenosine receptor subtype(s) occupancy and plasma exudation are conflicting (11, 12, 17, 22, 26, 30, 32).

For instance, Watanabe et al. (30) showed that stimulation of A2 receptors increases macromolecular permeability of coronary endothelial cell monolayers in vitro. Rosengren et al. (22) showed that 8-phenyltheophylline, a nonselective adenosine receptor antagonist, potentiated leukotriene-B4-induced increases in macromolecular efflux from postcapillary venules in the intact hamster cheek pouch. However, the effects of 8-phenyltheophylline on adenosine-induced increases in macromolecular efflux were not investigated. To this end, Gawlowski and Durán (11) and Murray et al. (17) showed that micromolar concentrations of adenosine increased macromolecular efflux from the intact hamster cheek pouch. However, they did not characterize the adenosine receptor subtype(s) that mediated this response. Taken together, these disparate data suggest that the edemagenic effect of adenosine in the peripheral circulation is microvascular bed-specific.

Hence, the purpose of this study was to begin to address this issue by using a pharmacological approach to determine the adenosine receptor subtype(s) that mediates adenosine-induced increases in macromolecular efflux from the intact hamster cheek pouch.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General Methods

Preparation of animals. Adult, male golden Syrian hamsters (n = 86) weighing 131 ± 1 g were anesthetized with pentobarbital sodium (6 mg/100 g body wt; ip). A tracheotomy was performed to facilitate spontaneous breathing. The left femoral vein was cannulated to inject the intravascular tracer, fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa), and supplemental anesthesia (2-4 mg · 100 g body wt-1 · h-1). The left femoral artery was cannulated to obtain arterial blood samples and to monitor arterial blood pressure, which did not change significantly during the experiments. Body temperature was kept constant (37-38°C) throughout the experiment.

To visualize the microcirculation of the cheek pouch, we used a method previously described in our laboratory and by other investigators (1, 6, 8-11, 13-15, 17, 20-24, 27, 28, 32). Briefly, the left cheek pouch was spread gently over a small plastic baseplate, and an incision was made in the outer skin to expose the cheek pouch membrane. The avascular connective tissue layer was removed, and a plastic chamber was positioned over the baseplate and secured in place by suturing the skin around the upper chamber. This chamber contained the suffusion fluid. This arrangement forms a triple-layered complex: the baseplate, the upper chamber, and the cheek pouch membrane exposed between the two plates. After these initial procedures, the hamster was transferred to a heated microscope stage. The chamber was connected to a reservoir containing warmed bicarbonate buffer (37-38°C), which allowed continuous suffusion of the cheek pouch. The buffer was bubbled continuously with 95% N2-5% CO2 (pH 7.4). The chamber was also connected via a three-way valve to an infusion pump (Sage Instruments, Cambridge, MA) that allowed for the constant administration of drugs into the suffusate.

Determination of clearance of macromolecules. Cheek pouch microcirculation was visualized with an Olympus microscope (Jacobs Instruments, Shawnee Mission, KS) coupled to a 100-W mercury light source at a magnification of ×40. Fluorescence microscopy was accomplished with the aid of filters that matched the spectral characteristics of FITC-dextran as previously described (15). Macromolecular leakage was determined by extravasation of FITC-dextran, which appeared as fluorescent "spots" or leaky sites around post-capillary venules. The number of leaky sites was determined by counting three random microscopic fields every minute for the first 7 min and then at 5-min intervals for 30-60 min after each intervention (see below). The total number of leaky sites was averaged and expressed as the number of leaky sites per 0.11 cm2 of cheek pouch corresponding to the area of one microscopic field (15).

In experiments in which clearance of FITC-dextran was calculated, the suffusion fluid was collected at 5-min intervals throughout the experiment by a fraction collector (Microfractionator, Gilson Medical Electronics, Middleton, WI). Samples were collected in glass test tubes, and the concentration of FITC-dextran was determined. Arterial blood samples were collected in heparinized capillary tubes (70-µl volume; Scientific Products, McGaw Park, IL) 5 min before and 5, 30, 60, 120, 180, and 240 min after injection of FITC-dextran. The concentration of FITC-dextran was determined in all plasma samples. To quantitate the concentration of FITC-dextran in the plasma and suffusate, a standard curve for FITC-dextran concentrations vs. percent emission was performed on a spectrophotofluorometer (Perkin-Elmer, Norwalk, CT). The standard was FITC-dextran that was prepared on a weight per volume basis. With the bicarbonate buffer used as background, a standard curve was generated for each experiment, and each curve was subjected to linear regression analysis. The percent emission for unknown samples (plasma and suffusate) was measured on the spectrophotofluorometer, and the concentration of FITC-dextran was calculated from the standard curve. In preliminary experiments, minimal fluorescence signal (<2% above background) was detected when drugs were added to the buffer and when plasma and suffusate samples were examined before the addition of FITC-dextran. Clearance of FITC-dextran was determined by calculating the ratio of suffusate (ng/ml) to plasma (mg/ml) concentration of FITC-dextran and multiplying this ratio by the suffusate flow rate (2 ml/min).

Experimental Protocols

Effects of adenosine agonists on macromolecular efflux. The purpose of these studies was to characterize the adenosine receptor subtype that mediates the adenosine-induced increase in macromolecular efflux. To accomplish this goal, we used selective A1 and A2 agonists and antagonists. In the first series of experiments, buffer was suffused for 30 min (equilibration period), FITC-dextran was injected intravenously, and the number of leaky sites and clearance of FITC-dextran were determined for 30 min. Then, increasing concentrations of R(-)-N6-(2-phenylisopropyl)-adenosine [R(-)-PIA; 1.0 and 10.0 nM], a selective A1 agonist (4-7, 25, 26), were suffused in a nonsystematic fashion. Each concentration was suffused for 10 min. The number of leaky sites was determined every minute for 10 min and at 5-min intervals for 45 min thereafter. Clearance of FITC-dextran was determined before and every 5 min during and after suffusion of R(-)-PIA for 45 min. The time interval between subsequent suffusions of R(-)-PIA was at least 45 min. After suffusion of R(-)-PIA was stopped and the number of leaky sites returned to baseline, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; 1.0 and 10 µM), a selective A1 antagonist (4-7, 25, 26), or 3,7-dimethyl-1-propargylxanthine (DMPX; 10 µM), a selective A2 antagonist (4-7, 18, 25, 26), was suffused for 30 min, and suffusion of R(-)-PIA (1.0 and 10.0 nM) was repeated as outlined above. The number of leaky sites and clearance of FITC-dextran were determined during each intervention.

In another series of experiments, after the equilibration period, increasing concentrations of CGS-21680 (1.0 and 10.0 nM, and 2.5 and 5.0 µM), a selective A2 agonist (4-7, 18, 25, 26), were suffused on the cheek pouch for 10 min each in a nonsystematic fashion. The number of leaky sites and clearance of FITC-dextran were determined as outlined above. The time interval between subsequent suffusions of CGS-21680 was at least 45 min. After suffusion of CGS-21680 was stopped and the number of leaky sites returned to baseline, PACPX or DMPX (10 µM each) was suffused for 30 min, and suffusion of CGS-21680 (2.5 and 5.0 µM; see below) was repeated. The number of leaky sites and clearance of FITC-dextran were determined during each intervention.

In preliminary studies, we determined that repeated suffusions of R(-)-PIA (1.0 and 10.0 nM) and CGS-21680 (1.0 and 10.0 nM, and 2.5 and 5.0 µM) were associated with reproducible results (data not shown). In addition, suffusion of PACPX (1.0 and 10.0 µM) and DMPX (10 µM) for 30 min and saline (vehicle) for the entire duration of the experiment was not associated with visible leaky site formation and significant increases in clearance of FITC-dextran. The concentrations of R(-)-PIA, CGS-21680, PACPX, and DMPX used in these studies are based on preliminary studies and reports in the literature (4-7, 18, 25, 26, 30).

Effects of adenosine receptor antagonists on adenosine-induced responses. The purpose of these studies was to determine the adenosine receptor subtype mediating the adenosine-induced increase in macromolecular efflux. The experimental design was similar to that outlined above except that adenosine (1.0 µM) was suffused for 10 min before and after suffusion of PACPX or DMPX (10.0 µM each) for 30 min. The number of leaky sites and clearance of FITC-dextran were determined during each intervention. In preliminary studies, we determined that repeated suffusions of adenosine (1.0 µM) were associated with reproducible results. The concentration of adenosine used in these experiments is based on previous studies in our laboratory and reports in the literature (1, 8-11, 17, 22, 32).

Specificity of adenosine receptor antagonist- and agonist-induced responses. The purpose of these studies was to determine whether PACPX and DMPX attenuation of the agonist-induced increase in macromolecular efflux is specific. To accomplish this goal, we devised two strategies. In the first series of experiments, we determined whether PACPX or DMPX attenuates leaky site formation and increase in clearance of FITC-dextran evoked by bradykinin, a potent phlogistic mediator (3, 8, 26, 32). The experimental design was similar to that outlined above except that bradykinin (0.5 µM) was suffused for 10 min before and 30 min after suffusion of PACPX or DMPX (10 µM each). The number of leaky sites and clearance of FITC-dextran were determined during each intervention. In preliminary studies, we determined that repeated suffusions of bradykinin (0.5 µM) were associated with reproducible results. The concentration of bradykinin used in these experiments is based on previous studies in our laboratory and a report in the literature (8, 17, 32).

In another series of experiments, we sought to determine whether R(-)-PIA- and CGS-21680-induced increases in macromolecular efflux are mediated, in part, by local changes in vasomotor tone (16, 17, 21, 28, 29). The experimental design was similar to that outlined above except that R(-)-PIA (1.0 and 10.0 nM) or CGS-21680 (2.5 and 5.0 µM) was suffused for 10 min each followed by 45-min suffusion of buffer (control) and repeated suffusion of R(-)-PIA (1.0 and 10.0 nM) or CGS-21680 (2.5 and 5.0 µM) together with nitroglycerin (1.0 µM), a potent vasodilator in the cheek pouch (23, 27). The number of leaky sites and clearance of FITC-dextran were determined during each intervention. In preliminary studies, we determined that suffusion of nitroglycerin (1.0 µM) for 10 min increases arteriolar diameter of second-order (resistance) arterioles in the cheek pouch by ~30% and that this response is not affected by cosuffusion of R(-)-PIA (1.0 and 10.0 nM) or CGS-21680 (2.5 and 5.0 µM).

Effects of a nitric oxide synthase inhibitor on adenosine-induced responses. The purpose of these studies was to determine whether products of the L-arginine-nitric oxide (NO) biosynthetic pathway mediate, in part, adenosine-induced increase in macromolecular efflux from the cheek pouch (5, 14, 15, 20, 24, 31). The experimental design was similar to that outlined above except that adenosine (1.0 µM) was suffused for 10 min before and after suffusion of NG-nitro-L-arginine methyl ester (L-NAME; 100 µM), a nonselective NO synthase inhibitor (20, 24), for 30 min onto the cheek pouch. The number of leaky sites and clearance of FITC-dextran were determined during each intervention. In preliminary studies, we determined that suffusion of L-NAME (10.0 µM) for 30 min was associated with no visible leaky site formation and significant increase in clearance of FITC-dextran. The concentration of L-NAME used in these studies is based on a previous study in our laboratory and a report in the literature (20, 24).

Effects of indomethacin on adenosine- and adenosine agonist-induced responses. The purpose of these studies was to determine whether products released through the cyclooxygenase pathway of arachidonic acid metabolism mediate, in part, the increase in macromolecular efflux evoked by adenosine, R(-)-PIA, and CGS-21680. The experimental design was similar to that outlined above except that adenosine (1.0 µM), R(-)-PIA (1.0 and 10.0 nM), or CGS 21680 (2.5 and 5.0 µM) was suffused for 10 min before and 30 min after indomethacin (10 mg/kg) was infused intravenously over a 30-min period using an infusion pump (total volume = 1.0 ml). The number of leaky sites and clearance of FITC-dextran were determined during each intervention. In preliminary studies, we determined that infusion of indomethacin (10 mg/kg) over a 30-min period was associated with no visible leaky site formation and significant increase in clearance of FITC-dextran. The concentration of indomethacin used in these studies is based on previous studies in our laboratory and a report in the literature and has been shown to inhibit cyclooxygenase in the cheek pouch (1, 8, 9, 21, 23).

Drugs. FITC-dextran, adenosine, bradykinin, and indomethacin were obtained from Sigma Chemical (St. Louis, MO). R(-)-PIA, CGS-21680, PACPX, and DMPX were obtained from RBI/Sigma (Natick, MA). Nitroglycerin was obtained from American Regent Laboratories (Shirley, NY). Indomethacin was dissolved in 5% Na2CO3. All other drugs were dissolved in saline. Drugs were prepared fresh before each experiment and were diluted in saline to the desired concentrations.

Data and statistical analyses. When a test compound was suffused over the cheek pouch, we determined the maximal change in the number of leaky sites and used it as the response to that compound. Data are expressed as means ± SE except for body weight, which is expressed as means ± SD. Because the number of leaky sites returned to baseline (nil) between successive applications of test compounds, all vehicle (saline) control data are expressed as a single value for each experimental condition. Statistical analysis was performed using two-way ANOVA and the Newman-Keuls test for multiple comparisons. A P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Adenosine Agonists on Macromolecular Efflux

Suffusion of R(-)-PIA (1.0 and 10.0 nM) elicited a significant, concentration-dependent leaky site formation and increase in clearance of FITC-dextran (Fig. 1; each group, n = 6, P < 0.05). These responses were significantly attenuated by PACPX (1.0 and 10.0 µM) (Fig. 1; each group, n = 6, P < 0.05). The number of leaky sites decreased significantly from 15 ± 1 per 0.11 cm2 during suffusion of R(-)-PIA alone to 3 ± 1 per 0.11 cm2 during suffusion of R(-)-PIA and PACPX (Fig. 1A; P < 0.05). Likewise, clearance of FITC-dextran decreased significantly from 44.9 ± 6.2 ml/min × 10-6 during suffusion of R(-)-PIA alone to 25.6 ± 2.8 ml/min × 10-6 during suffusion of R(-)-PIA and PACPX (Fig. 1B; P < 0.05). By contrast, DMPX (10.0 µM), a selective A2 antagonist, had no significant effects on R(-)-PIA (1.0 and 10.0 nM)-induced responses (Fig. 1; each group, n = 4; P > 0.5).


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  		Fig. 1.   Effects of 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine [PACPX; 1.0 µM (vertically hatched bars) and 10.0µM (horizontally hatched bars) each group, n = 6], a selective adenosine receptor A1 antagonist, and 3,7-dimethyl-1-propargylxanthine [DMPX; 10.0 µM; (diagonally hatched bars) each group, n = 4], a selective adenosine receptor A2 antagonist, on leaky site formation (A) and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; B) from the in situ hamster cheek pouch elicited by R(-)-N6-(2-phenylisopropyl)-adenosine [R(-)-PIA], a selective adenosine receptor A1 agonist. Values are means ± SE. *P < 0.05 compared with saline (vehicle); dagger P < 0.05 compared with R(-)-PIA alone.

Suffusion of CGS-21680, a selective A2 agonist, at concentrations similar to R(-)-PIA (1.0 and 10.0 nM) was not associated with visible leaky site formation and a significant increase in clearance of FITC-dextran (data not shown). However, suffusion of higher concentrations of CGS-21680 (2.5 and 5.0 µM) evoked significant, concentration-dependent leaky site formation and increase in clearance of FITC-dextran (Fig. 2; each group, n = 7, P < 0.05). These responses were significantly attenuated by DMPX (10.0 µM). The number of leaky sites decreased significantly from 11 ± 2 per 0.11 cm2 during suffusion of CGS-21680 (5.0 µM) alone to 5 ± 1 per 0.11 cm2 during suffusion of CGS-21680 (5.0 µM) and DMPX (10.0 µM, Fig. 2A; each group, n = 7, P < 0.05). Likewise, clearance of FITC-dextran decreased significantly from 30.0 ± 4.9 ml per min × 10-6 during suffusion of CGS-21680 (5.0 µM) alone to 18.3 ± 4.0 ml per min × 10-6 during suffusion of CGS-21680 (5.0 µM) and DMPX (10.0 µM; Fig. 2B; each group, n = 7, P < 0.05). By contrast, PACPX (10.0 µM) had no significant effects on CGS-21680-induced responses (Fig. 2; each group, n = 4, P > 0.5).


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  		Fig. 2.   Effects of DMPX [(10.0 µM horizontally hatched bars); each group, n = 7] and PACPX [(10.0 µM gray bars); each group, n = 4] on leaky site formation (A) and increases in clearance of FITC-dextran (B) from the in situ hamster cheek pouch evoked by CGS-21680 [(2.5 and 5.0 µM black bars)], a selective adenosine receptor A2 agonist. Values are means ± SE. *P < 0.05 compared with saline (vehicle); dagger P < 0.05 compared with CGS-21680 alone.

Effects of adenosine receptor antagonists on adenosine-induced responses. Suffusion of adenosine (1.0 µM) evoked leaky site formation and significantly increased clearance of FITC-dextran (Fig. 3; n = 6, P < 0.05). Pretreatment with PACPX (10.0 µM) significantly attenuated adenosine-induced responses. The number of leaky sites decreased significantly from 10 ± 1 per 0.11 cm2 during suffusion of adenosine alone to 3 ± 1 per 0.11 cm2 during suffusion of adenosine and PACPX (Fig. 3A; each group, n = 6, P < 0.05). Likewise, clearance of FITC-dextran decreased significantly from 33.9 ± 3.6 ml per min × 10-6 during suffusion of adenosine alone to 20.0 ± 2.5 ml per min × 10-6 during suffusion of adenosine and PACPX (Fig. 3B; each group, n = 6, P < 0.05). By contrast, suffusion of DMPX (10.0 µM) had no significant effects on adenosine-induced responses (Fig. 3; each group, n = 4, P > 0.5).


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  		Fig. 3.   Effects of PACPX [10.0 µM (horizontally hatched bars); each group, n = 6] and DMPX [(gray bars) 10.0 µM; each group, n = 4] on adenosine [1.0 µM (black bars)]-induced leaky site formation (A) and increase in clearance of FITC-dextran (B) from the in situ hamster cheek pouch. Values are means ± SE. *P < 0.05 compared with saline (vehicle); dagger P < 0.05 compared with adenosine alone.

Specificity of Adenosine Receptor Antagonist- and Agonist-induced Responses

Suffusion of bradykinin (0.5 µM) elicited leaky site formation and significantly increased clearance of FITC-dextran (Fig. 4; each group, n = 4, P < 0.05). However, pretreatment with PACPX (10.0µM) or DMPX (10.0 µM) had no significant effects on bradykinin-induced responses (Fig. 4; each group, n = 4, P > 0.5). Suffusion of nitroglycerin (1.0 µM) had no significant effects on R(-)-PIA (1.0 and 10.0 nM)- and CGS-21680 (2.5 and 5.0 µM)-induced leaky site formation and increase in clearance of FITC-dextran (Fig. 5; each group, n = 5, P > 0.5).


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  		Fig. 4.   Effects of PACPX (diagonally hatched bars; 10.0 µM) and DMPX (horizontally hatched bars; 10.0 µM) on bradykinin (black bars; 0.5 µM)-induced leaky site formation (A) and increase in clearance of FITC-dextran (B) from the in situ hamster cheek pouch. The differences in the number of leaky sites (A) and clearance of FITC-dextran (B) between bradykinin, PACPX, and DMPX are not statistically significant (P > 0.5). Values are means ± SE. *P < 0.05 compared with saline (vehicle).



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  		Fig. 5.   Effects of nitroglycerin (1.0 µM) on leaky site formation (a) and increase in clearance of FITC-dextran (b) from the in situ hamster cheek pouch elicited by R(-)-PIA (diagonally hatched bars; 10.0 nM; A) and CGS-21680 (black bars; 5.0 µM; B). Values are means ± SE for n = 5 for each group. *P < 0.05 compared with saline (vehicle; open bars).

Effects of an NO Synthase Inhibitor on Adenosine-induced Responses

Suffusion of L-NAME (100 µM) had no significant effects on adenosine (1.0 µM)-induced leaky site formation (9 ± 1 per 0.11 cm2 before and 9 ± 1 per 0.11 cm2 after suffusion of L-NAME; each group, n = 4, P > 0.5) and increase in clearance of FITC-dextran (35.4 ± 3 ml per min × 10-6 before and 31.8 ± 4.8 ml per min × 10-6 after suffusion of L-NAME; each group, n = 4, P > 0.5).

Effects of Indomethacin on Adenosine Agonist-induced Responses

Pretreatment with indomethacin (10 mg/kg iv) had no significant effects on adenosine (1.0 µM)-, R(-)-PIA (1.0 and 10.0 nM)-, and CGS-21680 (2.5 and 5.0 µM)-induced leaky site formation and increase in clearance of FITC-dextran (Fig. 6; each group, n = 6, P > 0.5).


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  		Fig. 6.   Effects of indomethacin (10 mg/kg iv) on leaky site formation (a) and increase in clearance of FITC-dextran (b) evoked by adenosine (1.0 µM), R(-)-PIA (A; black bars = PIA alone; diagonally hatched bars = PIA with indomethacin), and CGS-21680 (B; black bars = CGS-21680 alone; diagonally hatched bars = CGS-21680 with indomethacin). Values are means ± SE for n = 6 for each group. *P < 0.05 compared with saline (vehicle).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The new finding of this study is that high-affinity A1 receptors mediate the increase in macromolecular efflux elicited by adenosine from postcapillary venules in the intact hamster cheek pouch. This conclusion is based on the following observations. First, we found that adenosine-induced responses were significantly attenuated by PACPX, a selective A1 receptor antagonist (4-7, 25, 26), but not by DMPX, a selective A2-receptor antagonist (4-7, 18, 25, 26). Moreover, PACPX and DMPX had no significant effects on bradykinin-induced increases in macromolecular efflux in the same microvascular bed, which is mediated by bradykinin B2 receptors (3, 9, 32).

Second, nanomolar concentrations of R(-)-PIA (4-7, 25, 26) evoked a significant concentration-dependent increase in macromolecular efflux from the cheek pouch. This response was significantly attenuated by PACPX but not by DMPX. The 100-fold higher concentration of adenosine necessary to evoke a similar increases in macromolecular efflux as elicited by R(-)-PIA suggests that uptake and subsequent catabolism of adenosine in the cheek pouch microcirculation could interfere with functional expression of A1-receptor occupancy by adenosine (12, 25). Although CGS-21680 (4-7, 18, 25, 26) also elicited a significant concentration-dependent increase in macromolecular efflux, it was observed only at micromolar concentrations. This response was significantly attenuated by DMPX but not by PACPX. The effects of R(-)-PIA and CGS-21680 were not related to nonspecific effects on the microvascular endothelium because macromolecular efflux returned to baseline once suffusion of both drugs was stopped.

Taken together, these data suggest that, under normal physiological conditions, adenosine stimulates high-affinity A1 receptors in the cheek pouch microcirculation, which promote plasma exudation from the bloodstream into the extravascular space. However, at higher concentrations, such as detected during the host inflammatory response to injury (33), adenosine may also stimulate low-affinity A2 receptors, thereby amplifying plasma exudation and tissue dysfunction. Clearly, additional studies are warranted to support or refute this hypothesis.

The hamster cheek pouch is an established animal model to study the effects of phlogistic mediators, including adenosine and bradykinin, on macromolecular efflux from postcapillary venules in the in situ peripheral microcirculation (1, 6, 8-11, 13-15, 17, 20-24, 27, 28, 32). In this model, solute efflux is determined by two reproducible parameters, leaky site formation and clearance of FITC-dextran, thereby providing quantitative appraisal of macromolecular transport across postcapillary venules in the cheek pouch during experimental interventions. Importantly, successive suffusions of test compounds at appropriate time intervals are associated with reproducible formation of leaky sites and increases in clearance of FITC-dextran in the absence of tachyphylaxis. Consequently, changes in macromolecular efflux can be tested repeatedly in the same cheek pouch so that each animal serves as its own control. This, in turn, reduces the overall number of animals required to perform the study and facilitates data analysis.

The increase in macromolecular efflux evoked by adenosine agonists may have been related, in part, to changes in vasomotor tone and/or increases in venular driving pressure in the cheek pouch. However, this possibility seems unlikely because suffusion of nitroglycerin, a potent vasodilator in the cheek pouch (23, 27), had no significant effects on R(-)-PIA- and CGS-21680-induced increases in macromolecular efflux. Moreover, both PACPX and DMPX had no significant effects on bradykinin-induced leaky site formation and increases in clearance of FITC-dextran, indicating that their attenuating effects on adenosine- and adenosine agonist-induced responses were not related to changes in vasomotor tone and/or venular driving pressure in the cheek pouch. Collectively, these findings are consistent with previous studies in the cheek pouch and other microvascular beds and species in which the effects of various phlogistic mediators on microvascular transport are dissociated from their effects on microvascular tone (13, 16, 17, 21, 28, 29).

The cellular target(s) and intracellular signal transduction pathway(s) that mediate the increases in macromolecular efflux from postcapillary venules in the cheek pouch evoked by adenosine and adenosine analogs were not elucidated in this study. However, these processes were not mediated by local elaboration of NO or prostaglandins because L-NAME (20, 24) and indomethacin, at a concentration previously shown to inhibit cyclooxygenase in the cheek pouch (8, 21, 23), had no significant effects on adenosine-, R(-)-PIA-, and CGS-21680-induced responses. Additional studies using cellular, biochemical, and molecular biology techniques are needed to elucidate the putative target cell(s) and metabolic pathway(s) in the cheek pouch microcirculation.

The results of this and previous studies in the literature suggest that the effects of adenosine and adenosine analogs on macromolecular efflux from postcapillary venules are species- and microvascular bed-specific (2, 5, 6, 11, 12, 17, 19, 22, 26, 30). For instance, Allison et al. (2) showed that adenosine attenuates macromolecular efflux evoked by infusion of phorbol myristate acetate in the canine lung. In addition, Haselton et al. (12) showed that adenosine decreases permeability of bovine aortic endothelial monolayers by activating adenosine A2 receptors. However, these cells are derived from large conduit arteries that play no role in regulation of macromolecular efflux. Further studies using different species and distinct microvascular beds are indicated to address these issues.

In summary, we found that, under normal physiological conditions, adenosine increases macromolecular efflux from the intact hamster cheek pouch through stimulation of high-affinity adenosine A1 receptors in a specific, NO- and prostaglandin-independent fashion. We suggest that selective adenosine A1-receptor antagonists could attenuate plasma exudation elicited by adenosine in the inflamed oral mucosa.


    ACKNOWLEDGEMENTS

This study was supported, in part, by grants from the National Institutes of Health (DE-10347), American Heart Association of Metropolitan Chicago, and Laerdal Foundation for Acute Medicine.


    FOOTNOTES

Dr. Rubinstein is a recipient of a Research Career Development Award from the National Institutes of Health (DE-00386) and a University of Illinois Scholar Award.

Address for reprint requests and other correspondence: I. Rubinstein, Dept. of Medicine (M/C 787), Univ. of Illinois at Chicago, 840 South Wood St., Chicago, Illinois 60612-7323 (E-mail: IRubinst{at}uic.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 22 September 2000; accepted in final form 28 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(2):552-560
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