Extracellular 2′,3′-cAMP Is a Source of Adenosine*

We discovered that renal injury releases 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) into the interstitium. This finding motivated a novel hypothesis: renal injury leads to activation of an extracellular 2′,3′-cAMP-adenosine pathway (i.e. metabolism of extracellular 2′,3′-cAMP to 3′-AMP and 2′-AMP, which are metabolized to adenosine, a retaliatory metabolite). In isolated rat kidneys, arterial infusions of 2′,3′-cAMP (30 μmol/liter) increased the mean venous secretion of 3′-AMP (3,400-fold), 2′-AMP (26,000-fold), adenosine (53-fold), and inosine (adenosine metabolite, 30-fold). Renal injury with metabolic inhibitors increased the mean secretion of 2′,3′-cAMP (29-fold), 3′-AMP (16-fold), 2′-AMP (10-fold), adenosine (4.2-fold), and inosine (6.1-fold) while slightly increasing 5′-AMP (2.4-fold). Arterial infusions of 2′-AMP and 3′-AMP increased secretion of adenosine and inosine similar to that achieved by 5′-AMP. Renal artery infusions of 2′,3′-cAMP in vivo increased urinary excretion of 2′-AMP, 3′-AMP and adenosine, and infusions of 2′-AMP and 3′-AMP increased urinary excretion of adenosine as efficiently as 5′-AMP. The implications are that 1) in intact organs, 2′-AMP and 3′-AMP are converted to adenosine as efficiently as 5′-AMP (previously considered the most important adenosine precursor) and 2) because 2′,3′-cAMP opens mitochondrial permeability transition pores, a pro-apoptotic/pro-necrotic process, conversion of 2′,3′-cAMP to adenosine by the extracellular 2′,3′-cAMP-adenosine pathway would protect tissues by reducing a pro-death factor (2′,3′-cAMP) while increasing a retaliatory metabolite (adenosine).

to 2Ј-AMP (32), and some RNases, such as ptRNase 1 are secreted by cells into the extracellular compartment and can hydrolyze 2Ј,3Ј-cAMP to 3Ј-AMP (33,34). Finally, there is a large family of ecto-nucleotidases, for example ectonucleoside-triphosphate-diphosphohydrolases, ecto-nucleotide pyrophosphatase/phosphodiesterases and alkaline phosphatases, that could possibly function not only as ecto-2Ј,3Ј-cAMPphosphodiesterases to process 2Ј,3Ј-cAMP to 2Ј-AMP and 3Ј-AMP, but also could conceivably process 2Ј-AMP and 3Ј-AMP to adenosine (35)(36)(37). This putative extracellular 2Ј,3Ј-cAMP-adenosine pathway could be extremely important in producing extracellular adenosine whenever cells are exposed to stressful stimuli that enhance mRNA turnover, thus providing the protective, "retaliatory" metabolite, adenosine, to mitigate cellular damage. Here we establish purine metabolomics using liquid chromatography-tandem mass spectrometry and using this approach demonstrate that the intact kidney, both in isolation and in vivo, expresses an extracellular 2Ј,3Ј-cAMPadenosine pathway.

EXPERIMENTAL PROCEDURES
Animals-The studies utilized adult (ϳ16 weeks-of-age), male Wistar-Koto rats that were obtained from Taconic Farms (Germantown, NY).
Isolated, Perfused Rat Kidney-The rats were anesthetized with Inactin (90 mg/kg intraperitoneally; Sigma-Aldrich), and the left kidney was isolated and perfused with Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl 2 , 1.1 mM MgCl 2 , 12 mM NaHCO 3 , 0.42 mM NaH 2 PO 4 , 5.6 mM D(ϩ)glucose) using a Hugo Sachs Elektronik-Harvard Apparatus GmbH (March-Hugstetten, Germany) kidney perfusion system as previously described (38). Briefly, all branches of the left renal artery and vein were ligated. A polyethylene (PE)-50 cannula was placed into the left renal artery, and a PE-90 cannula was placed into the left renal vein. The left kidney was removed and attached to the perfusion system, the kidneys were perfused (single pass mode) at a constant flow (5 ml/min), and perfusion pressure was monitored with a pressure transducer.
In Vivo Rat Kidney-The rats were anesthetized with Inactin (90 mg/kg intraperitoneally) and placed on an isothermal pad, and body temperature was monitored with a rectal probe thermometer and kept at 37°C with a heat lamp. The trachea was cannulated to facilitate respiration, and a PE-50 cannula was inserted into the left carotid artery and connected to a digital blood pressure analyzer (Micro-Med, Inc., Louisville, KY) for continuous measurement of mean arterial blood pressure and heart rate. A PE-50 cannula was placed in the jugular vein, and an infusion of 0.9% saline was begun at 50 l/min for fluid replacement. A transit time flow probe (model 1 RB; Transonic Systems, Inc., Ithaca, NY) was positioned around the left renal artery and connected to a transit-time flowmeter (Transonic Systems, Inc.) to monitor renal blood flow. A 30-gauge needle connected to an infusion pump was inserted into the left renal artery, and an infusion of 0.9% saline was begun at 20 l/min. Also, a PE-10 cannula was inserted into the left ureter for the collection of urine. After a 2-h rest period, the experiments were begun. cAMPs and AMPs were dissolved in 0.9% saline and infused into the kidney at 20 l/min.

2,3-cAMP-Adenosine Pathway
sine, internal standard, and inosine. The following parameters were the same in the TSQ tune files 1 and 2: ion spray voltage, 3.8 kilovolts; ion transfer tube temperature, 270°C; source vaporization temperature, 220°C; Q2 CID gas, argon at 1.5 mTorr; sheath gas, nitrogen at 50 p.s.i.; auxillary gas, nitrogen at 40 p.s.i.; Q1/Q3 width, 0.7/0.7 units at full-width half-maximum; source CID, off; scan width, 0.5 units; and scan time, 0.05 s. The tube lens offset was 131 V for tune file 1 and 123 V for tune file 2. Five mass transitions were monitored: 330 3 136 for 2Ј,3Ј-cAMP and 3Ј,5Ј-cAMP with a collision energy of 28 volt; 348 3 136 for 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP with a collision energy of 21 volts; 268 3 136 for adenosine with a collision energy of 19 volts; 278 3 141 for 13 C 10 -adenosine as internal standard with a collision energy of 19 volts; and 269 3 137 for inosine with a collision energy of 20 volts. Calibration standard curves standards were constructed at concentrations of 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 pg/l in ultrapure water. Fig. 2 shows typical standard curves for the seven purines of interest along with a chromatogram illustrating the separation and detection of the seven purines.
Statistical Analyses-Statistical comparisons were performed on a priori contrasts with the nonparametric Wilcoxon Signed-Rank test. The criterion of significance was p Ͻ 0.05.

RESULTS
Metabolism of Exogenous 3Ј,5Ј-cAMP, 2Ј,3Ј-cAMP, 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP in the Isolated, Perfused Rat Kidney-The isolated rat kidney is a stable model for studying the vascular metabolism of purines because sampling from the renal vein is easily accomplished without perturbing the experimental system. In contrast, blood sampling from the rat renal vein in vivo would lead to exsanguination and hemodynamic instability caused by the large sample size required for analysis. Moreover, our previous studies show that purines are metabolized extensively by the vasculature of the isolated rat kidney. For example more than 65% of etheno-5Ј-AMP is converted to etheno-adenosine during a single pass through the vascular system of the isolated, perfused rat kidney (17). It is important to note that measurement of a given renal venous purine metabolite during an infusion of the parent purine into the renal artery does not capture the extent of renal metabolism because of downstream metabolism and uptake of the purine metabolite itself. Therefore, to compare the conversion of 3Ј,5Ј-cAMP to 5Ј-AMP versus 2Ј,3Ј-cAMP to 2Ј-AMP and 3Ј-AMP, we compared the effect of each cAMP on renal venous secretion of its corresponding AMP to the renal venous secretion induced by the same concentration of its corresponding AMP. Similarly we assessed the metabolism of 3Ј-AMP and 2Ј-AMP to adenosine and inosine by comparing the effects of 5Ј-AMP versus 3Ј-AMP versus 2Ј-AMP on renal venous secretion of adenosine and inosine because 5Ј-AMP is considered the most important adenosine precursor and is extensively metabolized to adenosine in the isolated, perfused rat kidney (17). Said differently, we benchmarked the metabolism of 2Ј-AMP and 3Ј-AMP to that of 5Ј-AMP.
Rat kidneys were isolated and perfused with Tyrode's solution. After a 1-h stabilization period, renal venous perfusate was collected for 1 min. Then either 3Ј,5Ј-cAMP, 2Ј,3Ј-cAMP, 5Ј-AMP, 3Ј-AMP, or 2Ј-AMP (10 mol/liter) was administered into the renal artery, and 4 min into the treatment, another 1-min venous perfusate sample was collected. After a rest period of 20 min with no treatments, this protocol was performed with another purine. This procedure was repeated every 20 min until all five purines had been administered. The purines were administered in random order. The base-line renal perfusion pressure was 47 Ϯ 3 mm Hg, which is normal for the isolated, perfused rat kidney at physiological flow rates because the isolated kidney is maximally vasodilated in the absence of endogenous vasoconstrictors and renal sympathetic tone (39). None of the treatments affected renal perfusion pressure. Renal artery administration of 3Ј,5Ј-cAMP increased renal venous 5Ј-AMP secretion (p ϭ 0.0051; Fig. 3A), but not 3Ј-AMP secretion (Fig. 3B). The renal artery administration of 5Ј-AMP and 3Ј-AMP also increased renal venous secretion of 5Ј-AMP (p ϭ 0.0195; Fig. 3A) and 3Ј-AMP (0.0118; Fig.  3B), respectively. The administration of 3Ј,5Ј-cAMP increased 5Ј-AMP secretion about half as effectively as an infusion of 5Ј-AMP (p ϭ 0.0322), suggesting that approximately half of the 3Ј,5Ј-cAMP was converted to 5Ј-AMP during a single pass through the intact renal vasculature. Renal artery administration of 2Ј,3Ј-cAMP increased renal venous 3Ј-AMP secretion (p ϭ 0.0117; Fig.  3C) and 2Ј-AMP secretion (p ϭ 0.0222; Fig. 3D). The renal artery administration of 3Ј-AMP and 2Ј-AMP also increased renal venous secretion of 3Ј-AMP (p ϭ 0.0118; Fig. 3C) and 2Ј-AMP (p ϭ 0.0222; Fig. 3D), respectively. The administration of 10 mol/liter of 2Ј,3Ј-cAMP increased 3Ј-AMP secretion numerically less, but not statistically less, than an infusion of 3Ј-AMP and increased 2Ј-AMP secretion approximately one-third as effectively as an infusion of 2Ј-AMP (p ϭ 0.0269), suggesting that 2Ј,3Ј-cAMP was converted quantitatively to 3Ј-AMP plus 2Ј-AMP during a single pass through the intact renal vasculature, with 3Ј-AMP being the dominant pathway at 10 mol/liter.
Because both 2Ј,3Ј-cAMP and 3Ј,5Ј-cAMP were converted efficiently into their respective AMPs, we examined whether 3Ј-AMP and 2Ј-AMP were as efficiently converted to adenosine and inosine as was 5Ј-AMP (the most important precursor of adenosine and previously considered the main source of adenosine). Importantly, renal artery administration of all three AMPs increased renal venous secretion of adenosine (p ϭ 0.0015, p ϭ 0.002, and p ϭ 0.0022 for 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP, respectively; Fig. 4A) and inosine (p ϭ 0.0022, p ϭ 0.0022, and p ϭ 0.0068 for 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP, respectively; Fig. 4B), and the ability of 3Ј-AMP and 2Ј-AMP to increase renal venous secretion of adenosine was not significantly different compared with 5Ј-AMP or each other (Fig. 4A). Also, both 3Ј-AMP and 2Ј-AMP increased renal venous secretion of inosine, and the ability of 3Ј-AMP to increase inosine secretion was similar to that achieved by 5Ј-AMP, although

2,3-cAMP-Adenosine Pathway
2Ј-AMP was less efficient in this regard (p ϭ 0.0068 and p ϭ 0.0005 versus 5Ј-AMP and 3Ј-AMP, respectively; Fig. 4B). None of the AMPs altered renal perfusion pressure. These results suggested that 2Ј-AMP and 3Ј-AMP were converted to adenosine and inosine in the intact renal vasculature as efficiently as was 5Ј-AMP.
Inasmuch as the cAMPs were converted efficiently into AMPs and the AMPs were converted efficiently to adenosine plus inosine, we predicted that the cAMPs would increase adenosine and inosine levels comparably. In support of this prediction, renal artery infusions of both 3Ј,5Ј-cAMP and 2Ј,3Ј-cAMP increased both adenosine (p ϭ 0.0022 and p ϭ 0.0015 for 3Ј,5Ј-cAMP and 2Ј,3-cAMP, respectively; Fig. 4C) and inosine (p ϭ 0.0022 and p ϭ 0.0024 for 3Ј,5Ј-cAMP and 2Ј,3-cAMP, respectively; Fig. 4D) secretion, without affecting renal perfusion pressure. The increases in adenosine and inosine induced by 3Ј,5Ј-cAMP and 2Ј,3Ј-cAMP were not significantly different (p ϭ 0.4697 and p ϭ 0.6772 for adenosine and inosine, respectively).
The aforementioned experiments were conducted with cAMPs at a concentration of 10 mol/liter. To determine whether the profile of metabolism of 3Ј,5Ј-cAMP versus 2Ј,3Ј-cAMP to AMPs, adenosine and inosine would differ at higher concentrations, in a separate group of isolated, perfused kidneys we examined the effects of 30 mol/liter of 3Ј,5Ј-cAMP and 2Ј,3Ј-cAMP on downstream metabolites. The baseline renal perfusion pressure was 48 Ϯ 1 mm Hg, and the cAMPs at 30 mol/liter did not alter renal perfusion pressure. 3Ј,5Ј-cAMP profoundly increased renal venous secretion of 5Ј-AMP (p ϭ 0.0033; Fig. 5A), but not 3Ј-AMP (Fig. 5B) nor 2Ј-AMP (Fig. 5C), indicating that even at very high concentrations, 3Ј,5Ј-cAMP was not metabolized to 3Ј-AMP but only to 5Ј-AMP. In contrast, 30 mol/liter of 2Ј,3Ј-cAMP had no effect on 5Ј-AMP secretion ( Fig. 5A) but greatly increased renal venous secretion of 3Ј-AMP (p ϭ 0.0033; Fig. 5B) and 2Ј-AMP (p ϭ 0.0033; Fig. 5C). With regard to 2Ј,3Ј-cAMP conversion to AMPs, with 10 mol/liter of 2Ј,3Ј-cAMP the dominant recovered product was 3Ј-AMP (p ϭ 0.0010 versus 2Ј-AMP; Fig. 4, C and D), whereas at 30 mol/liter the dominant recovered product was 2Ј-AMP (p Ͻ 0.0001 versus 3Ј-AMP; Fig. 5, B and C). This indicated that whether 3Ј-AMP or 2Ј-AMP was the dominant recovered product depended in part on the concentration of 2Ј,3Ј-cAMP. At this 3-fold higher concentration, both cAMPs markedly increased the renal venous secretion of adenosine (p ϭ 0.0033 for both cAMPs; Fig. 5D) and inosine (p ϭ 0.0033 for both cAMPs; Fig. 5E) with 2Ј,3Ј-cAMP being more effective with regard to increasing inosine secretion (p ϭ 0.0003; Fig. 5E). Importantly, the increases in AMPs, adenosine, and inosine induced by the cAMPs were not linearly related to input concentrations (compare Figs. 3 and 4 with Fig. 5), suggesting that at higher levels of input the downstream metabolites were less likely to be captured by alternative metabolic pathways.

2,3-cAMP-Adenosine Pathway
Metabolism of Exogenous 3Ј,5Ј-cAMP, 2Ј,3Ј-cAMP, 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP in the In Vivo Rat Kidney-In the experiments using isolated, perfused kidneys, we did not collect urine samples because of the well known instability and unreliability of tubular function in this model system. It was important, therefore, to compare the metabolism of 2Ј,3Ј-cAMP versus 3Ј,5Ј-cAMP in the tubular compartment by examining the effects of the cAMPs and AMPs on urinary excretion of purines in the intact animal. Accordingly, we anesthetized rats, and while maintaining body temperature and monitoring arterial blood pressure, heart rate, and renal blood flow to assure hemodynamic stability, we infused into the renal artery 3Ј,5Ј-cAMP or 2Ј,3Ј-cAMP. We used a cross-over experimental design with 30-min basal, treatment, and recovery periods for each cAMP (90 nmol/kg/min) while collecting urine via a polyethylene tubing inserted into the left ureter. The order of treatment was random. The basal arterial blood pressure, heart rate, renal blood flow, and urine flow rate were physiologically normal (126 Ϯ 8 mm Hg, 369 Ϯ 5 beats/min, 6.4 Ϯ 1.0 ml/min, and 0.18 Ϯ 0.05 ml/30 min, respectively) and were not altered by the cAMPs. Administration of 2Ј,3Ј-cAMP into the renal artery did not increase the urinary excretion rate of 3Ј,5Ј-cAMP but did increase the urinary excretion rate of 2Ј,3Ј-cAMP (p ϭ 0.0010; Fig. 7). In contrast, renal artery infusions of 3Ј,5Ј-cAMP increased urinary excretion of 3Ј,5Ј-cAMP (p ϭ 0.0022) but not 2Ј,3Ј-cAMP (Fig. 7). Importantly, 3Ј,5Ј-cAMP increased the urinary excretion of 3Ј,5Ј-cAMP more so than 2Ј,3Ј-cAMP increased the urinary excretion of 2Ј,3Ј-cAMP (p ϭ 0.0049). These data suggested that in vivo the tubular compartment metabolized 2Ј,3Ј-cAMP more efficiently than 3Ј,5Ј-cAMP.
To examine the metabolism of the cAMPs to AMPs by the in vivo kidney, we measured the urinary excretion rate of 5Ј-AMP, 3Ј-AMP, and 2Ј-AMP in response to the renal artery administration of the cAMPs. 2Ј,3Ј-cAMP did not increase the urinary excretion of 5Ј-AMP but profoundly increased the urinary excretion of 3Ј-AMP (p ϭ 0.0022) and 2Ј-AMP (p ϭ 0.0010;  Fig. 8), with 2Ј-AMP greater than 3Ј-AMP (p ϭ 0.0010). 3Ј,5Ј-cAMP did not increase the urinary excretion of either 3Ј-AMP or 2Ј-AMP but slightly increased the urinary excretion of 5Ј-AMP (p ϭ 0.0269; Fig. 8). The 2Ј,3Ј-cAMP-induced increases in 3Ј-AMP and 2Ј-AMP were greater than the 3Ј,5Ј-cAMP-induced increases in 5Ј-AMP (p ϭ 0.0015 and p ϭ 0.0010, respectively). 2Ј,3Ј-cAMP markedly and significantly increased the urinary excretion rate of adenosine (p ϭ 0.0022), whereas 3Ј,5Ј-cAMP did not (Fig. 9). The 2Ј,3Ј-cAMP-induced increase in adenosine was significantly greater that the 3Ј,5Ј-cAMP-induced increase in adenosine (p ϭ 0.0005). These data suggested that in vivo in the tubular compartment, 2Ј,3Ј-cAMP was metabolized to AMPs and hence to adenosine more efficiently than was 3Ј,5Ј-cAMP.

2,3-cAMP-Adenosine Pathway
In another protocol, we infused into the renal artery, in a cross-over experimental design, either vehicle, 5Ј-AMP, 3Ј-AMP, or 2Ј-AMP. In this experiment, we had to decrease the    NOVEMBER 27, 2009 • VOLUME 284 • NUMBER 48 infusion rate of AMPs to 9 nmol/kg/min (one-tenth the dose of cAMPs) to avoid hemodynamic effects (hypotension and bradycardia), which occurred with all three AMPs when infused at 90 nmol/kg/min. The basal arterial blood pressure, heart rate, renal blood flow, and urine flow rate were physiologically normal (128 Ϯ 9 mm Hg, 392 Ϯ 9 beats/min, 7.5 Ϯ 0.9 ml/min, and 0.17 Ϯ 0.03 ml/30 min, respectively) and were not altered by the AMPs at 9 nmol/kg/min. Importantly, all three AMPs similarly increased the urinary excretion rate of adenosine (p ϭ 0.0391, p ϭ 0.0273, and p ϭ 0.0078 for 2Ј-AMP, 3Ј-AMP, and 5Ј-AMP, respectively; Fig. 10). These findings indicated that in vivo 2Ј-AMP and 3Ј-AMP were as efficacious as 5Ј-AMP with regard to being converted to adenosine.

DISCUSSION
Tissue Injury Activates mRNA Breakdown by RNases-Tissue injury increases mRNA breakdown. For example, Almeida et al. (42) report that incubation of liver tissue at 37°C results in extensive RNA degradation, with mRNA levels falling to onetenth those before incubation. Akahane et al. (40,41) report that normothermic ischemia/reperfusion injury increases mRNA breakdown within 1-3 h in bone and skeletal muscle. Studies by Chevyreva et al. (43) show that in human brain tissue, low pH markedly increases mRNA breakdown, and Catts et al. (44) report similar findings in mouse brain. mRNA breakdown is most likely a universal response to cellular, tissue, and organ injury.
2Ј,3Ј-cAMP May Be Formed by RNA Breakdown-mRNA is degraded by the action of RNases that catalyze the hydrolysis of the P-O 5Ј bond of mRNA. This hydrolysis reaction may involve transphosphorylation of mRNA to form 2Ј,3Ј-cyclic nucleotides (such as 2Ј,3Ј-cAMP). Thompson et al. (28) used 31 P NMR spectroscopy to monitor the accumulation of the 2Ј,3Ј-cyclic nucleotides during the transphosphorylation and hydrolysis reactions catalyzed by various RNases. 2Ј,3Ј-Cyclic nucleotides were found to accumulate during catalysis by RNAses of widely disparate phylogenetic origin. Thus at least with isolated RNases acting on isolated mRNA, it appears that 2Ј,3Ј-cyclic nucleotides are not enzyme-bound intermediates but are true reaction products that are released by RNases.
mRNA Breakdown Would Likely Produce Large Quantities of 2Ј,3Ј-cAMP-Most mRNAs contain a poly-A tail of ϳ150 adenine repeats (45), and mRNA turnover is initiated by hydrolysis of the poly-A tail by RNases (27). A typical mammalian cell contains 363,000 molecules of mRNA (45). Therefore, a typical mammalian cell would contain 5.445 ϫ 10 7 potential molecules of 2Ј,3Ј-cAMP stored in mRNA poly-A tails, which when divided by Avogadro's number (6.022 ϫ 10 23 ) is 0.9042 ϫ 10 Ϫ16 mol/cell. The average mammalian cell has a water volume of 2.800 ϫ 10 Ϫ12 liter (46). Therefore, the potential concentration of 2Ј,3Ј-cAMP achieved in a mammalian cell by releasing the 2Ј,3Ј-cAMP stored in the poly-A tails of mRNA would be 32.29 mol/liter. In addition, the average mRNA is 1500 bases in length (45), 25% of which is adenine. Therefore, there are 1500 ϫ 0.25 ϫ 363,000 ϭ 1.361 ϫ 10 8 potential molecules of 2Ј,3Ј-cAMP in the non-poly-A tail part of mRNAs. If these were mobilized, then the concentration of 2Ј,3Ј-cAMP would increase by another 80.71 mol/liter. Therefore, each cell has the capacity to generate a total concentration of about 113.0 mol/ liter of 2Ј,3Ј-cAMP by degrading mRNA. Importantly, the poly-A tail would provide a rapidly releasable pool of 2Ј,3Ј-cAMP, whereas the body of the mRNA would provide a slowly releasable form.
Serendipitous Discovery That Intact Tissues/Organs Release 2Ј,3Ј-cAMP-While investigating 3Ј,5Ј-cAMP secretion by the isolated, perfused rat kidney, we noticed a chromatographic peak while measuring 3Ј,5Ј-cAMP by selective reaction monitoring using high performance liquid chromatograpy-tandem mass spectrometry that was at the incorrect retention time for 3Ј,5Ј-cAMP (26). We investigated this unknown substance and confirmed that it was 2Ј,3Ј-cAMP and that two different stimuli that are known to activate RNA breakdown, i.e. energy depletion and rapamycin, increased the release of 2Ј,3Ј-cAMP into the extracellular compartment (26).

2,3-cAMP-Adenosine Pathway
The Extracellular 2Ј,3Ј-cAMP-Adenosine Pathway-Our long term interest in the renal extracellular 3Ј,5Ј-cAMP-adenosine pathway (Fig. 1, left side) caused us to conceive by analogy the possibility of a renal 2Ј,3Ј-cAMP-adenosine pathway (Fig. 1,  right side), and the goal of the present study was to test this hypothesis. In this regard, the results of the present study show that the intact kidney metabolizes exogenous 2Ј,3Ј-cAMP to 3Ј-AMP, 2Ј-AMP, adenosine, and inosine and converts exogenous 3Ј-AMP and 2Ј-AMP to adenosine and inosine. Importantly, these findings apply to both kidneys perfused in vitro as well as kidneys in vivo. Moreover, at least in the intact kidney, 3Ј-AMP and 2Ј-AMP generate adenosine as readily as 5Ј-AMP, hitherto considered the most important adenosine precursor. However, the relative efficacy of 3Ј-AMP, 2Ј-AMP, and 5Ј-AMP as adenosine precursors may change under different conditions and in different cells/tissues/organs.
Our results suggest that whether exogenous 2Ј,3Ј-cAMP is metabolized predominantly down the 2Ј-AMP limb or 3Ј-AMP limb of the extracellular 2Ј,3Ј-cAMP-adenosine pathway is concentration-dependent. Low concentrations of 2Ј,3Ј-cAMP may favor the 3Ј-AMP limb, whereas higher concentrations may tilt the flux of 2Ј,3Ј-cAMP metabolism toward the 2Ј-AMP limb. An alternative, but not mutually exclusive, possibility is that with higher concentrations of 2Ј,3Ј-cAMP, 2Ј-AMP accumulates more than 3Ј-AMP because downstream metabolism of 2Ј-AMP becomes saturated. Thus concentration-dependent recovery of 2Ј-AMP versus 3Ј-AMP likely stems from the relative kinetic parameters and expression levels of the enzymes involved in metabolizing 2Ј,3Ј-cAMP, 2Ј-AMP, and 3Ј-AMP.
In the isolated perfused kidney, renal artery infusions of 2Ј,3Ј-cAMP and 3Ј,5Ј-cAMP similarly increase renal venous levels of adenosine. In contrast, infusions of 2Ј,3Ј-cAMP into the renal artery of kidneys in vivo increase urinary excretion of adenosine much more than do comparable infusions of 3Ј,5Ј-cAMP. Because comparable infusions of 2Ј-AMP, 3Ј-AMP, and 5Ј-AMP similarly increase urinary adenosine excretion in vivo, the relative inability of 3Ј,5Ј-cAMP to elevate urinary adenosine excretion is likely due to inefficient conversion of 3Ј,5Ј-cAMP to 5Ј-AMP rather than inefficient conversion of 5Ј-AMP to adenosine. This conclusion is confirmed by the greater urinary recovery of 3Ј,5Ј-cAMP and the lower urinary excretion of 5Ј-AMP following renal artery infusions of 3Ј,5Ј-cAMP compared with the lesser urinary recovery of 2Ј,3Ј-cAMP and the greater urinary excretion of 2Ј-AMP and 3Ј-AMP during renal artery infusions of 2Ј,3Ј-cAMP. The reason for the similar ability of 2Ј,3Ј-cAMP versus 3Ј,5Ј-cAMP to produce adenosine in isolated kidneys but the greater ability of 2Ј,3Ј-cAMP to do so in the in vivo kidney may relate to the fact that in the isolated, perfused kidney we measure metabolites in the renal vein, whereas in the kidney in vivo we examine metabolites in urine. Renal vein levels of metabolites likely reflect vascular metabolism, whereas urine levels of metabolites likely reflect renal epithelial metabolism. At any rate, the extracellular 2Ј,3Ј-cAMP pathway appears to be quantitatively important regardless of the site of sampling.
The Physiological Role of 2Ј,3Ј-cAMP-Adenosine Pathway-Studies in isolated mitochondria demonstrate that 2Ј,3Ј-cAMP opens mitochondrial permeability transition pores (47), and the opening of these mitochondrial pores is considered to be a significant contributor to ischemia/reperfusion injury leading to cell death via both apoptosis and necrosis (48). The extracellular 2Ј,3Ј-cAMP-adenosine pathway would protect cells from apoptosis and necrosis by two mechanisms: 1) removal of 2Ј,3Ј-cAMP from the cell (efflux to the extracellular compartment) and 2) conversion of extracellular 2Ј,3Ј-cAMP to 2Ј-AMP and 3Ј-AMP and hence to adenosine. Because adenosine is well known to act on cell surface receptors to induce cellular protection by engaging a host of signal transduction systems (49), the production of adenosine by the extracellular 2Ј,3Ј-cAMPadenosine pathway would provide this retaliatory metabolite to protect both the injured cell (autocrine action) and neighboring cells (paracrine action). This concept is supported by the findings of the present study that metabolic poisons increase the release from the intact kidney of 2Ј,3Ј-cAMP, 3Ј-AMP, 2Ј-AMP, and adenosine. However, it is conceivable that there are three different types of 2Ј,3Ј-cAMP-adenosine pathways that serve to protect cells (Fig. 11), and the present study addresses the existence of only one, i.e. the extracellular 2Ј,3Ј-cAMP-adenosine pathway. In this regard, it is likely that some 2Ј,3Ј-cAMP is metabolized intracellularly, before efflux can occur, to produce intracellular 2Ј-AMP and 3Ј-AMP. Intracellularly produced 2Ј-AMP and 3Ј-AMP could be transported to the extracellular compartment and metabolized to adenosine (transcellular 2Ј,3Ј-cAMP-adenosine pathway) or metabolized intracellularly to adenosine followed by efflux of adenosine FIGURE 11. Schematic summarizing the possible biochemical steps in the postulated 2,3-cAMP-adenosine pathways. The figure illustrates the concept that 2Ј,3Ј-cAMP may be exported from the cell and metabolized to adenosine extracellularly (extracellular 2Ј,3Ј-cAMP-adenosine pathway), may be metabolized to adenosine intracellularly (intracellular 2Ј,3Ј-cAMP-adenosine pathway), or may be metabolized to 2Ј-AMP and 3Ј-AMP inside the cell followed by export of 2Ј-AMP and 3Ј-AMP to the interstitial compartment with subsequent metabolism of these AMPs to adenosine (transcellular 2Ј,3Ј-cAMP-adenosine pathway). These processes would reduce the intracellular levels of 2Ј,3Ј-cAMP (a breakdown product of mRNA that is known to open mitochondrial permeability transition pores (mPTP), thus causing apoptosis and necrosis) while increasing the extracellular levels of adenosine (a retaliatory metabolite that protects cells from injury in an autocrine and paracrine fashion). (a), breakdown of mRNA (e.g. by RNases); (b) and (f), active transport (e.g. by multidrug resistance proteins); (c) and (e), hydrolysis of cyclic phosphodiester (e.g. by CNPase or ptRNase 1), (d) and (g) dephosphorylation (e.g. by various ecto-and endo-2Ј/3Ј-nucleotidases); and (h), transport of adenosine (e.g. by equilibrative nucleoside transporters).