Introduction

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The endogenous nucleoside adenosine exerts multiple biochemical effects in cardiovascular cells and tissues to regulate numerous physiological systems including cardiac fibroblast growth (Dubey et al., 1997), vascular smooth muscle cell growth (Dubey et al., 1998), cardiac contractility (Belardinelli et al., 1989), cardiac electrophysiology (Belardinelli et al., 1989), resistance of the heart to ischemia/reperfusion injury (Ely and Berne, 1992), vascular tone (Berne, 1980), platelet function (Becker et al., 1998), neutrophil-endothelial cell interactions (Fredholm, 1997), sympathetic neurotransmission (Westfall et al., 1990), renin release (Jackson, 1991), tubuloglomerular feedback (Osswald et al., 1991), renal medullary blood flow (Zou et al., 1999), and renal tubular transport (Kuan et al., 1993).

In contrast to the extensive knowledge about how cardiovascular systems are modulated by adenosine, knowledge of how extracellular adenosine levels are regulated is incomplete. However, given the importance of adenosine in cardiovascular cells and tissues, one would anticipate the existence of a mechanism for sensing extracellular levels of adenosine and appropriately adjusting these levels to provide the optimal concentration of adenosine in the receptor biophase.

Because cell surface adenosine receptors are positioned to "sample" the extracellular compartment, we hypothesize that adenosine receptors may function to sense and regulate extracellular adenosine levels. In this regard, the receptor-mediated effects of adenosine are transduced by four different receptor subtypes: A₁, A_2A, A_2B, and A₃ adenosine receptors (Ralevic and Burnstock, 1998). Of these receptor subtypes, the A₁ receptor has the highest affinity (K_i = 10 nM) for adenosine (Jacobson and van Rhee, 1997) and, in fact, the affinity of adenosine for the A₁ receptor is sufficiently high that normal levels of extracellular adenosine (50 to 200 nM; Zou et al., 1999) should activate this receptor. Therefore, if adenosine receptors do indeed modulate extracellular adenosine levels, the A₁ receptor would be the most logical candidate to serve such a role. Accordingly, the purpose of the present study was to test the hypothesis that A₁ adenosine receptors modulate extracellular adenosine levels in cardiovascular cells.

	Materials and Methods

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Dulbecco's modified Eagle's/F12 medium, 0.25% trypsin-EDTA solution, penicillin-streptomycin solution, Dulbecco's PBS, HEPES buffer, and sodium bicarbonate solution were purchased from GIBCO Laboratories (Grand Island, NY). Fetal calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT). Adenosine, adenine 9--D-arabinofuranoside (internal standard), 50% aqueous chloroacetaldehyde, 2-propanol, ,-methyleneadenosine-5'-diphosphate (AMPCP), and pertussis toxin were purchased from Sigma Chemical Co. (St. Louis, MO). 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), xanthine amine congener (XAC), and N-0840 were purchased from Research Biochemicals Inc. (Natick, MA).

Cardiac fibroblasts were obtained from male Sprague-Dawley rats as described previously (Dubey et al., 1997), and human aortic vascular smooth muscle cells were purchased from Clonetics Corp. (San Diego, CA). Cells, both rat and human, were plated in 75 cm² culture flasks in 10 ml of Dulbecco's modified Eagle's/F12 medium with 0.01 M HEPES, 0.12% (w/v) sodium bicarbonate, 10% fetal calf serum, and 20 U of penicillin-streptomycin (growth media). Cells were passaged no more than four times before they were plated onto 24-well culture plates in growth media. Cells were allowed to grow until confluence before being used in an experiment.

Each culture well was washed three times with 500 µl PBS buffered with 0.01 M HEPES and 0.12% (w/v) sodium bicarbonate (modified PBS); then 500 µl of the drug solutions, dissolved in modified PBS, were added to the appropriate culture wells. Cells were incubated at 37°C for 16 h, unless stated otherwise, with the drug solutions. After the incubation period, the extracellular fluid was collected for adenosine analysis.

Adenosine, and in some samples cAMP, in the extracellular fluid were measured by HPLC, using fluorometric detection. Each 200-µl sample received 10 µl of 0.5 M acetate-buffer, 10 µl of 1 µM internal standard, and 10 µl of 50% aqueous chloroacetaldehyde. The samples were then quickly centrifuged and the tubes were incubated at 80°C for 1 h. After incubation, the samples were centrifuged at 14,000 rpm for 4 min. The samples were then placed in polypropylene microvials and capped. A total of 80 µl of this solution was injected into an ISCO (Lincoln, NE) HPLC system (pump model 2350, gradient programmer model 2360, 4.6 × 250 mm C₁₈ reverse-phase column with 5-µm particle size; ChemResearch Data Management System, Lincoln, NE). The mobile phase was 10 mM citrate-buffer with 4.5% acetonitrile and was run isocratically at 1 ml/min. Fluorescence detection was achieved at an excitation wavelength of 275 nm and an emission wavelength of 420 nm using a Waters M-470 fluorescence detector. The ratio of the area under the adenosine or cAMP peaks to the area under the internal standard peak was compared with a standard curve.

All samples were in quadruplicate or greater repeats, and the data are expressed as mean ± S.E. Statistical analysis was performed with either a two-way or one-way ANOVA using a Fisher's least-significant difference test between groups for multiple comparisons, depending on the experimental design. Additionally, Student's t test or an Aspin-Welch unequal variance t test were used when appropriate. Values with p < .05 were considered significant.

	Results

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To determine whether structurally distinct A₁ receptor antagonists increase extracellular levels of adenosine, rat cardiac fibroblasts (Fig. 1A) or human aortic vascular smooth muscle cells (Fig. 1B) were incubated with either DPCPX, XAC, or N-0840 (10 nM concentration for each A₁ receptor antagonist). In rat cardiac fibroblasts, the three adenosine receptor antagonists similarly increased extracellular adenosine levels, whereas in human aortic vascular smooth muscle cells the effects of XAC were modestly greater than the effects of DPCPX and N-0840.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effects of three structurally distinct A1 receptor antagonists, each at 10 nM, on extracellular levels of adenosine in rat cardiac fibroblasts (A) and in human aortic vascular smooth muscle cells (B). Values are mean ± S.E. for four observations. *p < .05, compared with control; p < .05, compared with either DPCPX or N-0840.

To assess whether the effects of A₁ receptor antagonists on extracellular adenosine levels are concentration-dependent, extracellular adenosine levels were measured in rat cardiac fibroblasts incubated with either 0, 1, 10, or 100 µM DPCPX. As demonstrated in Fig. 2, DPCPX induced a concentration-dependent increase in extracellular adenosine levels.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of three different concentrations of 8-DPCPX on extracellular levels of adenosine in rat cardiac fibroblasts. Values are mean ± S.E. for six observations. *p < .05, compared with basal.

To investigate the participation of inhibitory G proteins and ecto-5-nucleotidase in the extracellular adenosine response to A₁ receptor blockade, the effects of DPCPX (10 nM) on extracellular adenosine levels were examined in rat cardiac fibroblasts coincubated with either pertussis toxin (200 ng/ml; inhibitor of G_i) or AMPCP (1 µM; inhibitor of ecto-5'-nucleotidase). As illustrated in Fig. 3, both pertussis toxin and AMPCP abolished the DPCPX-induced increase in extracellular adenosine levels.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of 10 nM DPCPX on extracellular levels of adenosine in normal rat cardiac fibroblasts and in rat cardiac fibroblasts coincubated with 200 ng/ml pertussis toxin or 1 µM AMPCP. Values are mean ± S.E. for eight observations. *p < .05, compared with control.

To determine the effects of A₁ receptor antagonism on extracellular adenosine levels in cells exposed to elevated endogenous adenosine levels, the effects of DPCPX on extracellular adenosine levels were investigated in rat cardiac fibroblasts treated with isoproterenol to activate the cAMP-adenosine pathway (Jackson, 1991). As shown in Fig. 4, incubation of rat cardiac fibroblasts with isoproterenol (10 µM; a -adrenoceptor agonist) markedly increased extracellular levels of both cAMP and adenosine, a finding consistent with activation of the cAMP-adenosine pathway. In rat cardiac fibroblasts in which endogenous adenosine levels were increased by isoproterenol, DPCPX (10 nM) did not significantly increase extracellular adenosine levels (Fig. 5). Moreover, a statistically significant interaction between DPCPX and isoproterenol was detected by two-factor ANOVA, indicating that the effects of DPCPX on extracellular adenosine were significantly reduced by isoproterenol.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of isoproterenol (10 µM) on extracellular levels of cAMP (A) and adenosine (B) in rat cardiac fibroblasts. Values are mean ± S.E. for six observations. <DL, less than detection limit; *p < .05, compared with respective control.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of 10 nM DPCPX on extracellular levels of adenosine in control rat cardiac fibroblasts and in cardiac fibroblasts treated with 10 µM isoproterenol. Values are mean ± S.E. for six observations.

To assess the time course of the effects of A₁ receptor antagonism on extracellular adenosine levels, rat cardiac fibroblasts were incubated with DPCPX (10 nM) for either 2, 8, or 16 h. As shown in Fig. 6, there was a time lag in the extracellular adenosine response to DPCPX of greater than 8 h but less than 16 h.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of incubation time with DPCPX (10 nM) on extracellular levels of adenosine in rat cardiac fibroblasts. Values are mean ± S.E. for six observations. *p < .05, compared with respective control.

	Discussion

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This study demonstrates that three structurally distinct A₁ receptor antagonists increase extracellular adenosine levels in two different types of cardiovascular cells, i.e., cardiac fibroblasts and vascular smooth muscle cells, and in two species, rats and humans. The extracellular adenosine responses to A₁ receptor antagonists are concentration dependent, and are attenuated by pertussis toxin, a toxin that inactivates inhibitory G proteins, and by AMPCP, an inhibitor of ecto-5'-nucleotidase. This study also demonstrates that augmentation of extracellular adenosine levels attenuates the increase in extracellular adenosine induced by A₁ receptor antagonists. Finally, this study shows that the effects of A₁ receptor antagonists on extracellular adenosine levels develop with a time lag of several hours.

Our working hypothesis is that high-affinity A₁ receptors detect elevated levels of adenosine in the biophase of the cell surface and engage a signal transduction process that ultimately decreases extracellular adenosine levels. We postulate that this negative feedback system functions to tightly regulate extracellular adenosine concentrations. Because cells normally produce adenosine and because A₁ receptors are high-affinity receptors, in the present study we tested our working hypothesis using A₁ receptor antagonists, rather than A₁ receptor agonists. Our logic was that interrupting the feedback system with A₁ receptor antagonists would provide a clearer assessment of the extent to which the putative feedback system works, because basal activation of A₁ receptors by endogenous adenosine would confound the interpretation of studies using A₁ receptor agonists.

If A₁ receptors mediate a negative feedback on extracellular adenosine levels, then blockade of A₁ receptors should increase extracellular levels of adenosine. The results of the present study establish beyond a reasonable doubt that blockade of A₁ receptors does indeed increase extracellular adenosine levels. Several aspects of the current investigation support this conclusion. First, three structurally dissimilar A₁ receptor antagonists similarly increase extracellular adenosine levels. Because it is very unlikely that the three compounds used in the present study would have shared "nonspecific effects", these results support an A₁ receptor-mediated mechanism. Second, the concentrations of the A₁ receptor antagonists required to increase extracellular adenosine levels are low, i.e., 10 nM, which is consistent with a receptor-mediated mechanism of action. Third, the effects of DPCPX on extracellular adenosine levels are concentration dependent, a hallmark of receptor-mediated effects. Fourth, the effects of DPCPX are abolished by inactivating inhibitory G proteins with pertussis toxin. Because A₁ receptors are well known to signal via pertussis toxin-sensitive inhibitory G proteins (for review see Ralevic and Burnstock, 1998), this finding is consistent with a receptor-dependent mechanism of action for the effects of A₁ receptor antagonists on extracellular adenosine levels.

A fifth line of evidence supporting the conclusion that blockade of A₁ receptors increases extracellular adenosine levels is that when endogenous adenosine levels are elevated, the effects of DPCPX on extracellular adenosine levels are diminished. The cAMP-adenosine pathway is a mechanism for increasing endogenous adenosine in the local biophase of the receptors by stimulating adenylyl cyclase. Stimulation of adenylyl cyclase leads to cellular cAMP egress followed by local metabolism of the extracellular cAMP to AMP by ecto-phosphodiesterase and local metabolism of AMP to adenosine by ecto-5'-nucleotidase (Jackson, 1991; Mi et al., 1994; Mi and Jackson, 1995, 1998). Our previous studies support the existence of a cAMP-adenosine pathway in both cardiac fibroblasts (Dubey et al., 1996a) and aortic vascular smooth muscle cells (Dubey et al., 1996b). The present study demonstrates that stimulation of adenylyl cyclase with isoproterenol increases both extracellular cAMP levels and extracellular adenosine levels, consistent with activation of the cAMP-adenosine pathway. Moreover, the present study shows that when extracellular adenosine levels are elevated, the effects of DPCPX on extracellular adenosine levels are reduced. This is consistent with an A₁ receptor mechanism of action for DPCPX. Because DPCPX is a competitive A₁ receptor antagonist, increasing the local concentrations of adenosine should decrease the ability of a fixed concentration of DPCPX to modulate extracellular adenosine levels.

DPCPX, XAC, and DPCPX (10 nM) caused similar effects on extracellular adenosine levels (Fig. 1), except that in human aortic vascular smooth muscle cells, the effects of 10 nM XAC were somewhat greater than the effects induced by 10 nM DPCPX and 10 nM N-0840. Inasmuch as the K_i values of DPCPX and XAC for A₁ receptors are 0.9 and 1.3 nM, respectively (Daly and Jacobson, 1995), it is reasonable that 10 nM DPCPX and 10 nM XAC would have similar effects on extracellular adenosine levels. The literature is unclear, however, regarding the K_i of N-0840 for A₁ receptors, with K_is reported as low as 10 nM (May et al., 1992) and as high as 380 nM (Barrett et al., 1993). If the higher-affinity estimate for N-0840 is correct, then our finding that N-0840 has similar effects on extracellular adenosine compared with DPCPX and XAC is reasonable. If the lower-affinity estimate for N-0840 is correct, other differences between N-0840 and DPCPX/XAC, such as protein binding, may account for the similar effects of N-0840 compared with DPCPX and XAC. It is interesting that, in human aortic vascular smooth muscle cells, XAC had a somewhat greater effect on extracellular adenosine compared with DPCPX and N-0840. There are at least three possible explanations for this finding: 1) a type I statistical error occurred and the apparent greater effect of XAC is an artifact; 2) the greater hydrophilicity of XAC resulted in higher concentrations of XAC in the receptor biophase; or 3) XAC has affinity for A₂ receptors (K_i = 63 nM) (Daly and Jacobson, 1995), and this contributed to the effects of XAC.

The current investigation reveals three important aspects of the signal transduction mechanism by which A₁ receptors modulate extracellular levels of adenosine. First, the fact that pertussis toxin prevents DPCPX-induced increases in extracellular adenosine suggests that inhibitory G proteins are involved in the signal transduction process. Second, the delayed increase in extracellular adenosine after the application of DPCPX suggests that signal transduction may involve changes in protein synthesis. Although the time lag between blockade of A₁ receptors and the increase in extracellular adenosine levels is long (>8 h) even for new protein synthesis, there are previous reports of de novo protein synthesis having long time courses (8 to 36 h; Pantopoulos et al., 1996; Nilsen et al., 1999). It is also possible that blockade of A₁ receptors rapidly increases protein synthesis, but that the new proteins must undergo significant post-translational modifications and/or cellular trafficking, processes that could entail a significant time lag. It is also possible that blockade of A₁ receptors increases extracelluar adenosine levels by reducing the rate of synthesis of key proteins. If such is the case, and if the half-lives of the effected proteins are long, a significant time lag would result. For example, if blockade of A₁ receptors decreases the production of adenosine deaminase and if hours are required for adenosine deaminase levels to decrease after production is reduced, this would entail a significant time lag. A third important aspect of the signal transduction mechanism by which A₁ receptors modulate extracellular adenosine levels is that somehow ecto-5'-nucleotidase is involved. It is possible that A₁ receptors govern pertussis toxin-sensitive G-protein modulation of gene products involved in regulating ecto-5'-nucleotidase activity, the availability of substrate for the ecto-5'-nucleotidase, and/or the stability of extracellular adenosine formed from ecto-5'-nucleotidase. Interestingly, A₁ receptors are complexed not only with inhibitory G proteins, but also with an ecto-adenosine deaminase (Saura et al., 1996, 1998). Thus, it is conceivable that A₁ receptors modulate the activity of ecto-adenosine deaminase and thereby regulate extracellular levels of adenosine formed from ecto-5'-nucleotidase. Additional studies are required to gain a more complete understanding of the mechanism by which A₁ receptors regulate extracellular adenosine levels.

Our finding that A₁ receptors modulate extracellular levels of adenosine has physiological and pharmacological significance. From a physiological perspective, these results suggest that the extracellular level of the endogenous agonist for A₁ receptors, i.e., adenosine, is tightly regulated by the degree of activation of the A₁ receptor. This may represent an important feedback mechanism to ensure a highly regulated level of extracellular adenosine. From a pharmacological perspective, our results raise the possibility that some effects of A₁ receptor antagonists are mediated indirectly via increased activation of other adenosine receptor subtypes by the elevated extracellular levels of adenosine. In this regard, the findings by Neely et al. (1996) that selective and nonselective A₁ receptor antagonists significantly reduce infarct size in animals subjected to myocardial ischemia/reperfusion injury may be an example of how A₁ receptor antagonists produce effects apparently consistent with adenosine receptor activation (Pitarys et al., 1991; Norton et al., 1991; Forman et al., 1993), rather than inhibition. However, if such is the case, the time course for A₁ receptor modulation of extracellular adenosine levels in vivo must be more rapid than in vitro, because A₁ receptor antagonists could reduce myocardial ischemia/reperfusion injury by this mechanism only if the time course were less than 3 h, rather than greater than 8 h as observed in the present study.

In summary, this study demonstrates that A₁ receptors regulate extracellular levels of adenosine by a mechanism involving inhibitory G proteins and ecto-5'-nucleotidase. This mechanism may represent an important feedback loop for ensuring an appropriate level of extracellular adenosine, and may contribute to the pharmacology of A₁ receptor antagonists.