Introduction

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Because adenosine participates in the modulation of renal hemodynamics, tubuloglomerular feedback, renin release, erythropoietin biosynthesis and tubular epithelial transport (Jackson, 1997), knowledge of the mechanisms regulating renal adenosine production would facilitate progress in the fields of renal physiology and pharmacology. In 1991 we proposed that cellular egress of cAMPa phenomenon accompanying hormonal activation of adenylyl cyclase (Barber and Butcher, 1983)functions to enhance extracellular adenosine levels in the kidney (Jackson, 1991). More specifically, our hypothesis was that once cAMP is transported out of some renal cells in response to the appropriate agonist, ecto-phosphodiesterase metabolizes cAMP to AMP, and because AMP is readily transformed to adenosine by ecto-5'-nucleotidase, extracellular levels of adenosine in the kidney increase (Jackson, 1991). Recently, we extended this concept to aortic vascular smooth muscle cells and cardiac fibroblasts and coined the phrase "cAMP-adenosine pathway" to refer in general to the metabolism of cAMP to adenosine (Dubey et al., 1996a, 1996b, 1997, 1998). However, this nomenclature requires refinement because cAMP may be converted to adenosine in the extracellular compartment (as originally hypothesized), in the intracellular compartment, or, alternatively, cAMP may be converted to AMP intracellularly and then AMP may reach the extracellular compartment and then be converted to adenosine. Therefore, we have adopted the nomenclature of "extracellular cAMP-adenosine pathway," "intracellular cAMP-adenosine pathway" and "transcellular cAMP-adenosine pathway" to refer specifically to these three respective possibilities, and use the term "cAMP-adenosine pathway" without qualification to refer to cAMP metabolism to adenosine regardless of cellular localization of the involved enzymes.

Currently, three lines of evidence support the existence in the kidney of a cAMP-adenosine pathway in general and specifically an extracellular cAMP-adenosine pathway: 1) delivery of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) into the cortical interstitium of the kidney with a microdialysis probe reduces renal interstitial levels of both adenosine and inosine (Mi et al., 1994), suggesting that a pathway involving phosphodiesterase contributes to renal adenosine formation. 2) Infusion of exogenous cAMP into the isolated perfused rat kidney enhances the renal venous secretion rates of both AMP and adenosine (Mi and Jackson, 1995), and the increase in renal adenosine secretion induced by exogenous cAMP is attenuated by inhibition of total phosphodiesterase, ecto-phosphodiesterase and ecto-5'-nucleotidase (Mi and Jackson, 1995). 3) Exogenous cAMP is converted to adenosine by cultured preglomerular vascular smooth muscle cells (Jackson et al., 1997) and mesangial cells (Dubey et al., 1997) by a pathway involving phosphodiesterase.

However, to corroborate the existence of a cAMP-adenosine pathway in the kidney a fourth line of evidence is required, i.e., hormonal stimulation of adenylyl cyclase to increase endogenous cAMP should increase renal production of adenosine and/or its metabolites (inosine and hypoxanthine) via a mechanism involving phosphodiesterase and 5'-nucleotidase. Therefore, our objective was to determine whether activation of beta adrenoceptors in the isolated, perfused rat kidney simulates renal adenosine, inosine and hypoxanthine production by a pathway that can be attenuated by inhibition of phosphodiesterase and 5'-nucleotidase.

	Methods

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Animals. Adult male Sprague-Dawley rats obtained from Charles River (Wilmington, MA) were housed at the University of Pittsburgh Animal Facility and fed Prolab RMH 3000 (PMI Feed, Inc., St. Louis, MO) containing 0.26% sodium and 0.82% potassium. All studies received prior approval by the University of Pittsburgh Animal Care and Use Committee.

Perfused rat kidney. Rats were anesthetized (sodium pentobarbital 45 mg/kg, i.p. injection), a midline incision was made, and the left kidney, left renal artery, abdominal aorta and left ureter were dissected free from surrounding tissue. The left ureter was cannulated with polyethylene-10 tubing, the abdominal aorta below the left kidney was cannulated (polyethylene-50 tubing), the suprarenal aorta was ligated, and the left kidney was flushed with 2.5 ml/min of oxygenated Tyrode's solution containing 100 U/ml of heparin. Although maintaining perfusion, the left kidney was isolated and mounted in a water-jacketed organ chamber. The organ chamber was maintained at 37°C with a thermostatically controlled water circulator (Thermocirculator, Harvard Apparatus, South Natick, MA). Kidneys were perfused (5 ml/min) using a Harvard model 1210 peristaltic pump with Tyrode's solution [composition in mM: NaCl, 137; KCl, 2.7; CaCl₂, 1.8; MgCl₂, 1.1; NaHCO₃, 12; NaH₂PO₄, 0.42; D(+)-glucose, 5.6] that was heated to 37°C with a warming coil, gassed with 95% O₂ and 5% CO₂ and passed through a bubble trap. A Statham pressure transducer (model P23ID, Statham Division, Gould Inc., Oxnard, CO) was connected to an access port located in the perfusion line immediately before the kidney so that perfusion pressure could be continuously monitored (Grass model 79D polygraph, Grass Instruments, Qunicy, MA). In pilot studies, we found that the renal venous purine secretion rate was high just after initiating perfusion, but declined steeply with time. Therefore, we allowed a 3-hr stabilization period before conducting the experiments to obtain a very low and stable baseline renal venous purine secretion rate. This was important because the release of purines by hormonal stimulation was small and could not be differentiated from basal release if baseline release was high and rapidly declining.

Protocol. The protocol consisted of two 10-min experimental periods. Just before the first experimental period and during the last minute of each 10-min experimental period, perfusate exiting the renal vein was collected on ice and frozen at 40°C for later analysis of purines. During the first and second 10-min experimental periods, (±)-isoproterenol HCl (Sigma Chemical Company, St. Louis, MO) was added to the perfusate at 1 and 10 µM, respectively, which provided concentrations of ()-isoproterenol of approximately the K_d and 10 times the K_d of ()-isoproteronol for beta adrenoceptors. In some experiments no inhibitors were added to the perfusate. In other experiments, either DL-propranolol HCl (0.1 µM; a beta adrenoceptor agonist, IBMX (1 mM; a phosphodiesterase inhibitor) or AMPCP (0.1 mM; an ecto-5'-nucleotidase inhibitor) was added to the perfusate 20 min before the first 10-min experimental period. All three inhibitors were obtained from Sigma. The concentrations of IBMX and AMPCP were selected on the basis of our previous studies (Mi and Jackson, 1995) in which we demonstrated that IBMX and AMPCP blocked the conversion of exogenous cAMP to adenosine in the perfused rat kidney. The concentration of DL-propranolol provided a concentration of L-propranolol that was approximately 10-fold greater than the K_B of L-propranolol for beta adrenoceptors. In some kidney perfusion experiments, no isoproterenol was added to the perfusate (time controls) during the experimental periods.

Sample analysis. The concentrations of adenosine, inosine, hypoxanthine, xanthine and uric acid in the perfusate were measured with an Isco (Lincoln, NE) high-pressure liquid chromatographic system (pump model 2350, gradient programmer model 2360, 4.6 × 250 mm C₁₈ column with 5-µm particle size, ChemResearch Data Management System) using UV detection as previously described (Mi and Jackson, 1995). The renal venous secretion rate of adenosine, inosine, hypoxanthine, xanthine and uric acid was calculated by multiplying the concentration of each substance in the venous perfusate by the perfusion rate.

Data analysis. Multiple comparisons were performed by 1- or 2-factor analysis of variance with repeated measures as appropriate. A Wilcoxon Signed-Rank test was used to determine whether isoproterenol-induced release of purines was different than zero. All statistical analyses were performed using the Number Cruncher Statistical System (Kaysville, UT), and all values in the text, figures and tables refer to means ± S.E.M.

	Results

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In time-control experiments, i.e., no isoproterenol was added to the perfusate, the renal venous secretion rates of adenosine, inosine, hypoxanthine, xanthine and uric acid and perfusion pressure were stable during the two 10-min experimental periods (table 1). The renal venous secretion rates of purines (defined as the secretion rates of adenosine + inosine + hypoxanthine) decreased slightly during the first experimental period but returned to baseline values during the second experimental period.

Figures 1 and 2 illustrate the effects of isoproterenol on the renal venous secretion rates of adenosine (fig. 1, top), inosine (fig. 1, bottom), hypoxanthine (fig. 2, top) and purines (fig. 2, bottom). A concentration of 10 µM isoproterenol significantly increased the renal venous secretion rates of all four measures of purine biosynthesis, whereas 1 µM isoproterenol significantly increased the renal venous secretion rates of hypoxanthine and purines. The renal venous secretion rate of purines was the most discriminating measure of the effects of isoproterenol. Using this index, not only was a significant difference observed with regard to the effects of low concentrations of isoproterenol, but the effects of low and high concentrations of isoproterenol were significantly different. As shown in table 2, isoproterenol did not significantly increase renal venous secretion rates of xanthine or uric acid, and perfusion pressure was very stable increasing only 3 mm Hg over the experimental protocol.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Bar graph illustrating the effects of isoproterenol on renal venous secretion rate of adenosine (top) and inosine (bottom). Values represent means and S.E.M. for 31 perfused kidneys. The P values in upper left corner of each panel are for the overall 1-factor analysis of variance with repeated measures, and the asterisk indicates significant differences between the indicated groups by multiple comparisons using the Fisher LSD test.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Bar graph illustrating the effects of isoproterenol on renal venous secretion rate of hypoxanthine (top) and the sum of adenosine + inosine + hypoxanthine (bottom). Values represent means and S.E.M. for 31 perfused kidneys. The P values in upper left corner of each panel are for the overall 1-factor analysis of variance with repeated measures, and the asterisk indicates significant differences between the indicated groups by multiple comparisons using the Fisher LSD test.

We also examined the effects of propranolol, IBMX and AMPCP on isoproterenol-induced purine biosynthesis. Each inhibitor study included two groups, a group in which the inhibitor was added to the Tyrode's solution perfusing the kidney and a separate control group that was randomized in with each inhibitor group. Because the renal venous secretion rate of purines was the most discriminating measure of the effects of isoproterenol, we focused on this measure to reduce the number of animals required to achieve adequate statistical power. The beta adrenoceptor antagonists propranolol (fig. 3), the phosphodiesterase inhibitor IBMX (fig. 4) and the ecto-5'-nucleotidase inhibitor AMPCP (fig. 5) significantly reduced the isoproterenol-induced increase in the renal venous secretion rate of purines. Neither propranolol, IBMX nor AMPCP altered basal perfusion pressure (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Bar graph illustrating the effects of propranolol (0.1 µM) on isoproteronol-induced renal venous secretion rate of the sum of adenosine + inosine + hypoxanthine. Values represent means and S.E.M. for the isoproterenol-induced purine secretion (i.e., secretion in the presence of isoproterenol minus baseline secretion) in six perfused kidneys. The P value in the right panel is for the effect of propranolol in a 2-factor analysis of variance with repeated measures, and the asterisk indicates significantly different from zero by Wilcoxon Signed-Rank test.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Bar graph illustrating the effects of IBMX (1 mM) on isoproterenol-induced renal venous secretion rate of the sum of adenosine + inosine + hypoxanthine. Values represent means and S.E.M. for the isoproterenol-induced purine secretion (i.e., secretion in the presence of isoproterenol minus baseline secretion) in five to six perfused kidneys. The P value in the right panel is for the effect of IBMX in a 2-factor analysis of variance with repeated measures, and the asterisk indicates significantly different from zero by Wilcoxon Signed-Rank test.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Bar graph illustrating the effects of AMPCP (0.1 mM) on isoproterenol-induced renal venous secretion rate of the sum of adenosine + inosine. (AMPCP interfered with hypoxanthine measurement.) Values represent means and S.E.M. for the isoproterenol-induced purine secretion (i.e., secretion in the presence of isoproterenol minus baseline secretion) in five to six perfused kidneys. The P value on the right is for the effect of AMPCP in a 2-factor analysis of variance with repeated measures, and the asterisk significantly different from zero by Wilcoxon Signed-Rank test.

	Discussion

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Our previous studies (Mi and Jackson, 1995; Jackson et al., 1997) indicate that exogenous cAMP, when added to the perfusate, is converted to adenosine in the isolated perfused rat kidney by a mechanism involving the metabolism of cAMP to AMP and AMP to adenosine by the enzymes phosphodiesterase and 5'-nucleotidase, respectively. In the present study, our purpose was to determine whether endogenous cAMP is converted to adenosine in the kidney via this pathway.

To test the hypothesis that stimulation of renal adenylyl cyclase promotes the conversion of endogenous cAMP to adenosine in the kidney, we examined the ability of isoproterenol to stimulate purine biosynthesis in the isolated perfused rat kidney. Because isoproterenol is a well-known beta adrenoceptor agonist that activates adenylyl cyclase by interacting with beta-adrenoceptors, if our hypothesis is correct isoproterenol should, in a concentration-dependent manner, increase purine biosynthesis. This prediction of course is justifiable only if beta adrenoceptors exist in the same renal tissues in which exogenous cAMP is converted to adenosine. In this regard, we previously demonstrated that exogenous cAMP is converted to adenosine at least in part by the vascular compartment (Jackson et al., 1997), and work by others indicates that beta adrenoceptors do reside in the rat renal vasculature (Amenta et al., 1983; Healy et al., 1985; Lakhlani et al., 1994). Therefore, our approach of addressing the stated hypothesis seemed reasonable.

Consistent with our hypothesis, isoproterenol caused an increase in renal purine biosynthesis, as assessed by the renal venous secretion rate of adenosine, inosine, hypoxanthine and the sum of adenosine + inosine + hypoxanthine. These effects of isoproterenol were observed with concentrations that selectively activate beta adrenoceptors (Williams and Lefkowitz, 1978) and at concentrations that caused little, if any, change in renal perfusion pressure. Therefore, the effects of isoproterenol were not likely mediated by other receptors such as alpha adrenoceptors that are activated only by much higher concentrations of isoproterenol (Williams and Lefkowitz, 1978). Moreover, the complete inhibition of the isoproterenol-induced purine biosynthesis by the appropriate concentrations of the beta adrenoceptor antagonist propranolol further supports the conclusion that the effects of isoproterenol were mediated via beta adrenoceptors.

An important aspect of our experimental design was minimizing the basal activation of adenylyl cyclase and reducing the basal production of purines so that the effects of an agonist on purine biosynthesis could be detected. We have previously demonstrated that IBMX, when delivered locally into the interstitium of the rat kidney using a microdialysis probe, reduces adenosine and inosine levels in the extracellular space by approximately 40% (Mi et al., 1994). These results suggest that in vivo in the rat kidney the cAMP-adenosine pathway is already markedly activated so that detecting the effects of additional stimulation of adenylyl cyclase on renal adenosine levels in vivo is problematic. Indeed in pilot studies, we were unable to increase adenosine levels in the interstitial space in vivo in the rat kidney by adding isoproterenol to the dialysate. However, we reasoned that by isolating the kidney and perfusing it with Tyrode's solution we could reduce the influence of endogenous activators of adenylyl cyclase on adenylyl cyclase activity and increase the sensitivity of our experimental approach. Moreover, by allowing the perfused kidney to stabilize for 3 hr before exposing it to isoproterenol we were able to achieve a low and stable basal purine biosynthesis rate.

We have shown previously that the conversion of exogenous cAMP to adenosine in perfused rat kidneys is blocked by inhibition of phosphodiesterase with IBMX, by inhibition of ecto-phosphodiesterase with 1,3-dipropyl-8-p-sulfophenylxanthine and by inhibition of ecto-5'-nucleotidase with AMPCP (Mi and Jackson, 1995; Jackson et al., 1997). These studies suggest that rat kidneys support a cAMP-adenosine pathway in general and more specifically an extracellular cAMP-adenosine pathway that involves the conversion of cAMP to AMP by ecto-phosphodiesterase and the conversion of AMP to adenosine by ecto-5'-nucleotidase. If isoproterenol-induced purine biosynthesis is the result of presenting endogenous cAMP to the extracellular cAMP-adenosine pathway, then inhibition of phosphodiesterase and ecto-5'-nucleotidase should abolish isoproterenol-induced purine biosynthesis. As predicted, in our study we observed that both inhibition of phosphodiesterase with IBMX and inhibition of ecto-5'-nucleotidase with AMPCP completely prevented isoproterenol-induced purine biosynthesis. Because AMPCP is a selective inhibitor of ecto-5'-nucleotidase and does not inhibit intracellular forms of 5'-nucleotidase (Zimmermann, 1992), most likely the synthesis of adenosine in response to isoproterenol occurred in the extracellular compartment. However, because IBMX inhibits intracellular and extracellular phosphodiesterases we cannot rule out the possibility that some cAMP was metabolized to AMP within cells and then transported out of cells into the extracellular compartment. Nonetheless, whether metabolism of cAMP to adenosine occurs via a transcellular and/or extracellular cAMP-adenosine pathway, either mechanism represents a cAMP-adenosine pathway that could be important in regulating renal function.

Many different families of phosphodiesterases exist, and selective inhibitors of many of these isoforms are available (Burns et al., 1996). However, because it is not known which phosphodiesterase isoforms mediate the cAMP-adenosine pathway, in our study we used a "broad spectrum" phosphodiesterate inhibitor, IBMX, to inhibit all isoforms of this enzyme superfamily. In this regard, we recently demonstrated that IBMX markedly increases isoproterenol-induced cAMP secretion from the isolated perfused rat kidney, with a concentration-response relationship that does not plateau until concentrations of IBMX are more than 0.3 mM (Jackson et al., 1997). Accordingly, in our study we used 1 mM IBMX to fully inhibit phosphodiesterases. Papaverine is an alternative broad spectrum phosphodiesterase inhibitor (Beavo and Reifsnyder, 1990); however, because papaverine potently inhibits renal adenosine transport (Coulson and Trimble, 1986), we chose not to use this agent. It is possible that the high concentration of IBMX used in our study could have inhibited isoproterenol-induced purine secretion by a mechanism other than phosphodiesterase inhibition. Additional studies are required, therefore, to identify the phosphodiesterase isoforms that mediated the cAMP-adenosine pathway so that lower concentrations of more specific inhibitors can be used to elucidate the physiological importance of this biochemical mechanism.

In summary, our results support the existence of a renal cAMP-adenosine pathway that is initiated by activation of endogenous cAMP biosynthesis and proceeds with the conversion of cAMP to AMP and hence to adenosine. Because adenosine exerts numerous actions within the mammalian kidney, the cAMP pathway may contribute importantly to the regulation of renal function.