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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurochem. Author manuscript; available in PMC 2012 September 1.
Published in final edited form as:
PMCID: PMC3166383
NIHMSID: NIHMS313439

Expression of the 2′,3′-cAMP-Adenosine Pathway in Astrocytes and Microglia

Abstract

Many organs express the extracellular 3′,5′-cAMP-adenosine pathway (conversion of extracellular 3′,5′-cAMP to 5′-AMP and 5′-AMP to adenosine). Some organs release 2′,3′-cAMP (isomer of 3′,5′-cAMP) and convert extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP and convert these AMPs to adenosine (extracellular 2′,3′-cAMP-adenosine pathway). Because astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2′,3′-cAMP, 3′,5′-cAMP, 5′-AMP, 3′-AMP and 2′-AMP were incubated with mouse primary astrocytes or primary microglia for 1 hour and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3′,5′-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2′,3′-cAMP to 2′-AMP and 3′-AMP (with 2′-AMP being the predominant product). Although both cell types converted 2′-AMP and 3′-AMP to adenosine, microglia were 5-fold and 7-fold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2′,3′-cAMP to 2′-AMP was mediated by a different ecto-enzyme than that involved in metabolism of 2′,3′-cAMP to 3′-AMP and that although CD73 mediates the conversion of 5′-AMP to adenosine, an alternative ecto-enzyme metabolizes 2′-AMP or 3′-AMP to adenosine.

Keywords: Astrocytes, Microglia, 2′,3′-cAMP, 3′,5′-cAMP, 5′-AMP, 3′-AMP, 2′-AMP, CD73, Adenosine

INTRODUCTION

Adenosine is both a neuromodulator and an immunomodulator. As a “retaliatory metabolite”, adenosine is formed in response to injurious stimuli and affects immune function so as to protect against excessive inflammation (Sitkovsky 2009, Sitkovsky et al. 2008, Chouker et al. 2008, Thiel et al. 2005, Sitkovsky et al. 2004). In the central nervous system, adenosine, via specific cell-surface receptors, has immunomodulatory effects on microglial cell (the immune cells of the central nervous system) proliferation and function (Dare et al. 2007). Adenosine acting via its A1 receptor subtype is neuroprotective (reviewed in (Stone et al. 2009)), and adenosine production by microglia protects hippocampal neurons from glutamate-induced neurotoxicity (Lauro et al. 2008).

Given the significance of adenosine production by microglia, it is important to understand the endogenous pathways by which microglia elevate local adenosine levels. One mechanism of adenosine biosynthesis is the extracellular 3′,5′-cAMP-adenosine pathway. This pathway entails receptor-mediated production of intracellular 3′,5′-cAMP, active transport of 3′,5′-cAMP to the cell surface, extracellular metabolism of 3′,5′-cAMP to 5′-AMP and finally extracellular conversion of 5′-AMP to adenosine. The completion of this pathway results in an increased level of adenosine in the local microenvironment. Studies confirm the existence of this pathway in a wide variety of cells, tissues and organs. (Chiavegatti et al. 2008, Dubey et al. 1996, Dubey et al. 1997, Dubey et al. 1998, Dubey et al. 2000a, Dubey et al. 2000b, Dubey et al. 2001, Giron et al. 2008, Hong et al. 1999, Jackson et al. 1997, Jackson & Mi 2000, Jackson et al. 2003, Jackson et al. 2006, Jackson et al. 2007b, Jackson et al. 2007a, Jackson et al. 2007c, Jackson & Mi 2008, Mi et al. 1994, Mi & Jackson 1995, Mi & Jackson 1998, Müller et al. 2008, Do et al. 2007).

Recent studies identify a positional isomer of 3′,5′-cAMP, namely 2′,3′-cAMP (Ren et al. 2009) and show that endogenous 2′,3′-cAMP derives from the degradation of mRNA and is released from organs in response to injury (Ren et al. 2009). Moreover, extracellular 2′,3′-cAMP can be metabolized to extracellular 2′-AMP and 3′-AMP, which in turn are metabolized to adenosine (Jackson et al. 2009). Thus, previous work reveals the existence of an extracellular 2′,3′-cAMP-adenosine pathway in addition to the extracellular 3′,5′-cAMP-adenosine pathway.

If microglia express the extracellular 3′,5′-cAMP-adenosine pathway or the extracellular 2′,3′-cAMP-adenosine pathway then a source of adenosine production by microglia may be metabolism of 3′,5′-cAMP or 2′,3′-cAMP to corresponding AMPs and metabolism of corresponding AMPs to adenosine. Moreover, it is conceivable that other glial cell types in the brain, for example astrocytes, serve as feeder cells that convert cAMPs to AMPs for further processing to adenosine by microglia. Therefore, the goal of the present study was to examine the metabolism of 3′,5′-cAMP, 2′,3′-cAMP, 5′-AMP, 3′-AMP and 2′-AMP by astrocytes and microglia. We show here that although neither cell type metabolizes extracellular 3′,5′-cAMP efficiently, both cell types express an extracellular 2′,3′-cAMP-adenosine pathway. Moreover, although astrocytes are slightly more efficient with regard to metabolizing 2′,3′-cAMP to 2′-AMP and 3′-AMP, microglia are much more efficient than astrocytes with regard to metabolizing 2′-AMP and 3′-AMP to adenosine.

METHODS

Isolation of Primary Mouse Microglial Cells and Astrocytes

Primary microglial and astrocyte cultures were isolated from postnatal day 2 mouse pups of either sex bred from C57BL/6J mice (Jackson Laboratory; Bar Harbor, ME). In brief, the animals were sacrificed and the brains removed. After the removal of the meninges, three brains per isolation were washed in phosphate-buffered saline before the cortices were isolated, trypsinized and triturated. Cells were then plated on a T-75 flask in growth media (DMEM/F12 with L-glutamate and 20% fetal calf serum). After 14 days, the mixed microglia/astrocyte cultures were trypsinized for approximately 20 minutes at 37° C. The astrocyte layer was removed leaving adherent microglia in the flask, and the trypsin was quenched with serum containing media. After a brief recovery period, the microglia were then exposed to trypsin again for approximately 20 minutes and detached to be seeded in additional plates for experiments and analysis. The detached sheet of astrocytes was placed in trypsin to disaggregate the cells, and after 10 minutes the astrocytes were triturated and placed in 75 cm2 flasks with DMEM/F12 and 10% fetal calf serum. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under the approval of the University of Pittsburgh Animal Care and Use Committee

Immunofluorescence

Although the method described above readily provides high yields of pure astrocytes, primary microglia cultures can be contaminated with astrocytes. To determine the purity of the primary microglial cultures, microglia isolated as described above were plated on 8-chamber poly-D-lysine coated glass slides. The cells were allowed to adhere overnight and then were washed twice with PBS prior to fixation with 4% paraformaldhyde for 10 minutes. The cells were then washed and subsequently permeabilized with 0.1% Trition X-100 for 10 minutes. After blocking with 10% NGS/PBS for 1 hour the cells were incubated with the indicated primary antibodies at 4° C overnight. The following day, cells were washed 3 times prior to the incubation with the fluorochrome conjugated secondary antibodies for 2 hours. The cells were washed 3 times with PBS and then mounted using an aqueous mounting medium containing DAPI (Santa Cruz Biotechnology; Santa Cruz, CA) to visualize nuclei. To determine the percentage of cells that were microglia or astrocytes, cells were probed with a rabbit anti-Iba-1 antibody (detects microglia; 1:1000 dilution; Wako; Richmond, VA) or a mouse anti-GFAP antibody (detects astrocytes; 1:250 dilution; Cell Signaling; Danvers, MA). The images were captured using a Nikon fluorescent microscope and images were processed using both SPOT and Adobe Photoshop software. For cell purity quantification, 8 fields of view from each multiple wells were analyzed for Iba-1 and GFAP positive cells.

Metabolism Studies

To examine purine metabolism, either 50,000 primary astrocytes or primary microglia cells per well were washed twice with HEPES-buffered Hanks balanced salt solution and treated with 0.5 ml of Dulbecco’s phosphate-buffered saline with HEPES (25 mmol/L) and NaHCO3 (13 mmol/L) in the presence and absence of various substrates (3′,5′-cAMP, 2′,3′-cAMP, 5′-AMP, 3′-AMP, or 2′-AMP; all from Sigma-Aldrich (St. Louis, MO)). In some experiments enzyme inhibitors were included in the incubation [3-isobutyl-1-methylxanthine {IBMX, broad spectrum phosphodiesterase inhibitor (Beavo & Reifsnyder 1990)}; 1,3-dipropyl-8-p-sulfophenylxanthine {DPSPX, ecto-phophosphodiesterase inhibitor (Tofovic et al. 1991, Mi & Jackson 1995, Zacher & Carey 1999)};α,β-methylene-adenosine-5′-diphosphate {AMPCP, ecto-5′-nucleotidase (CD73) inhibitor (Zimmermann 1992)}]. After one-hour incubation at 37° C, the medium was collected, heated for 3 minutes at 100° C to denature enzymes and then frozen at −80° C until assayed by mass spectrometry. Cell protein was measured using the Thermo Scientific Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA).

Analytical Methods

The internal standard (13C10-adenosine) was from Medical Isotopes Inc. (Pelham, NH). Purines were resolved by reversed-phase liquid chromatography (Agilent Zorbax eclipse XDB-C-18 column, 3.5 μm beads; 2.1×100 mm) and quantified using a triple quadrupole mass spectrometer (LC-MS/MS; TSQ Quantum-Ultra, ThermoFisher Scientific) operating in the selected reaction monitoring mode with a heated electrospray ionization source as previously described in detail (Jackson et al. 2009). The limit of detection for purines using this assay system is approximately 0.2 nmol/L (Ren et al. 2008).

Statistical Analysis

Data were analyzed by 1-factor analysis of variance, with post hoc comparisons using a Fisher’s Least Significant Difference test. The criterion of significance was p<0.05. All values in text and figures are means ± SEM. Data were converted to nmol/L/μg protein to normalize between cell types.

RESULTS

Purity of Primary Microglia Cell Isolations

Cultures of primary microglia were established from post-natal day 2 mice, as described in the methods above. To determine the purity of the cultures, at the time of plating for purine metabolism experiments, a subset of cells was seeded onto multi-chamber glass slides for characterization via immunofluorescence (Figure 1). Using commonly employed cell-type specific antibodies, the percentage of microglia (Iba-1+) and astrocytes (GFAP+) present in the microglial cultures was determined. Quantification of 8 views from each of 3 wells showed that primary microglial cell cultures were over 98% Iba-1+ microglia with very few astrocytes remaining.

Figure 1
Cell-type characterization of primary microglial cell cultures. Microglial cells were identified using anti-Iba-1 primary and FITC-conjugated secondary antibodies (Green, DAPI, blue; Scale bar = 50 microns) (A), and astrocytes were identified using anti-GFAP ...

Metabolism of 3′,5′-cAMP and 2′,3′-cAMP to 2′-AMP, 3′-AMP, 5′-AMP

We first examined the conversion of 3′,5′-cAMP versus 2′,3′-cAMP to AMPs in both primary microglia (Figure 2, panels A, B and C) and primary astrocytes (Figure 2, panels D, E and F). Approximately 50,000 cells were seeded per well of a 24-well plate and allowed to recover for 48 hours. The cells were then incubated with 0, 3, 10 or 30 μmol/L of 3′,5′-cAMP or 2′,3′-cAMP. After 1 hour, the medium was collected and assayed for 2′-AMP, 3′-AMP, and 5′-AMP by LC-MS/MS. Incubation of either microglia or astrocytes with increasing concentrations of 2′,3′-cAMP significantly increased medium levels of 2′-AMP and 3′-AMP, but did not affect medium levels of 5′-AMP. In this regard, the 2′,3′-cAMP-induced increase in 2′-AMP was 4.4-fold and 3.5-fold more than the increase in 3′-AMP in microglia and astrocytes, respectively. In contrast to 2′,3′-cAMP, in both microglia and astrocytes 3′,5′-cAMP did not significantly increase medium levels of either 2′-AMP or 3′-AMP. Also, 3′,5′-cAMP did not increase medium levels of 5′-AMP in either microglia or astrocytes.

Figure 2
Line graphs show the concentration-dependent effects in microglia (A, B, and C) and astrocytes (D, E and F) of 3′,5′-cAMP and 2′,3′-cAMP on extracellular levels of 2′-AMP (A and D), 3′-AMP (B and E) and ...

We also examined the effects of IBMX (1 mmol/L; broad-spectrum phosphodiesterase inhibitor) and DPSPX (1 mmol/L; ecto-phosphodiesterase inhibitor) on conversion of 2′,3′-cAMP to 2′-AMP and 3′-AMP in both primary microglia (Figure 3, panels A and B) and primary astrocytes (Figure 3, panels C and D). IBMX did not affect the conversion of 2′,3′-cAMP to either 3′-AMP or 2′-AMP in either microglia or astrocytes. DPSPX attenuated the metabolism of 2′,3′-cAMP to 2′-AMP in both microglia and astrocytes, but did not reduced the metabolism of 2′,3′-cAMP to 3′-AMP in either microglia or astrocytes.

Figure 3
Bar graphs illustrate the effects of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L; broad spectrum phosphodiesterase inhibitor) and 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 1 mmol/L; ecto-phosphodiesterase inhibitor) in microglia (A and B) and astrocytes ...

Metabolism of 2′-AMP, 3′-AMP and 5′-AMP to Adenosine

We next examined the conversion of 2′-AMP, 3′-AMP and 5′-AMP to adenosine in primary microglia (Figure 4, panels A, B and C) and primary astrocytes (Figure 4, panel D, E and F). In these experiments, microglia and astrocytes in 24-well plates were incubated with 0, 3 or 10 μmol/L of the AMPs. After 1 hour, the medium was collected and assayed for adenosine by LC-MS/MS. 2′-AMP, 3′-AMP and 5′-AMP produced a concentration-dependent increase in extracellular levels of adenosine in both microglia and astrocytes; however, the conversion of 2′-AMP, 3′-AMP and 5′-AMP to adenosine was 7.3-fold, 5.1-fold and 3.1-fold, respectively, more efficient in microglia compared with astrocytes.

Figure 4
Line graphs show the concentration-dependent effects in microglia (A, B and C) and astrocytes (D, E and F) of 2′-AMP (A and D), 3′-AMP (B and E) and 5′-AMP (C and F) on extracellular levels of adenosine. P-values in panels are ...

We also examined the effects of AMPCP (0.1 mmol/L; broad-spectrum ecto-5′-nucleotidase inhibitor) on conversion of 2′-AMP, 3′-AMP and 5′-AMP to adenosine in both primary microglia (Figure 5, panels A, B and C) and primary astrocytes (Figure 5, panels D, E and F). AMPCP did not affect the conversion of either 2′-AMP or 3′-AMP to adenosine in either microglia or astrocytes. However, AMPCP markedly suppressed metabolism of 5′-AMP to adenosine in both microglia and astrocytes.

Figure 5
Bar graphs depict the effects in microglia (A, B and C) and astrocytes (D, E and F) of α,β-methylene-adenosine-5′-diphosphate (AMPCP, 0.1 mmol/L; ecto-5′-nucleotidase inhibitor) on metabolism of 2′-AMP (A and D), ...

Metabolism of 2′,3′-cAMP and 3′,5′-cAMP to Adenosine

Finally, we examined the conversion of 2′,3′-cAMP and 3′,5′-cAMP to adenosine in primary microglia (Figure 6, panel A) and primary astrocytes (Figure 6, panel B). Microglia and astrocytes in 24-well plates were incubated with 0, 3, 10 or 30 μmol/L of the cAMPs. After 1 hour, the medium was collected and assayed for adenosine by LC-MS/MS. 3′,5,-cAMP did not significantly increase extracellular levels of adenosine. In contrast, 2′,3′-cAMP caused a concentration-dependent increase in extracellular levels of adenosine in both microglia and astrocytes. However, microglia were 3-fold more efficient with regard to converting 2′,3′-cAMP to adenosine.

Figure 6
Line graphs show the concentration-dependent effects of 3′,5′-cAMP and 2′,3′-cAMP on extracellular levels of adenosine in microglia (A) and astrocytes (B). P-values in panels are from analysis of variance. ap<0.05, ...

DISCUSSION

Our findings show that incubation of microglia or astrocytes with exogenous 2′,3′-cAMP induces an increase in the levels of 3′-AMP, 2′-AMP and adenosine in the medium, whereas incubation of microglia or astrocytes with 3′,5′-cAMP has little affect on extracellular levels of 5′-AMP or adenosine. We conclude that both microglia and astrocytes express the extracellular 2′,3′-cAMP-adenosine pathway, but not the extracellular 3′,5′-cAMP-adenosine pathway.

Previous studies demonstrate that the extracellular 3′,5′-cAMP-adenosine pathway is mediated by an ecto-phosphodiesterase that is sensitive to inhibition by both IBMX and DPSPX (Jackson & Raghvendra 2004). The current study shows that IBMX has little effect on the conversion of 2′,3′-cAMP to 3′-AMP or 2′-AMP by either microglia or astrocytes. Thus the 2′,3′-cAMP-phosphodiesterase that mediates the 2′,3′-cAMP-adenosine pathway is distinct from the 3′,5′-cAMP-phosphodiesterase that mediates the 3′,5′-cAMP-adenosine pathway. 2′,3′-cAMP is hydrophilic and would not be expected to penetrate cell membranes, so enzymes that metabolize 2′,3′-cAMP must be ecto-enyzmes, i.e., ecto-2′,3′-cAMP-phosphodiesterases.

In both microglia and astrocytes, DPSPX reduces the conversion of 2′,3′-cAMP to 2′-AMP, yet has no effect on the metabolism of 2′,3′-cAMP to 3′-AMP. This suggests that there are two separate ecto-2′,3′-cAMP-phosphodiesterases in microglia and astrocytes. One metabolizes 2′,3′-cAMP to 3′-AMP (i.e., ecto-2′,3′-cAMP-2′-phosphodiesterase) and is resistant to inhibition by both IBMX and DPSPX. A second metabolizes 2′,3′-cAMP to 2′-AMP (i.e., ecto-2′,3′-cAMP-3′-phosphodiesterase) and is resistant to inhibition by IBMX, but is modestly inhibited by DPSPX. These findings are consistent with observations in preglomerular vascular smooth muscle and glomerular mesangial cells where degradation of 2′,3′-cAMP to 2′-AMP is partially inhibited by DPSPX, but that to 3′-AMP is not (Jackson et al. 2010).

That different enzymes mediate the metabolism of 2′,3′-cAMP to 2′-AMP versus 3′-AMP has implications. If 3′-AMP and 2′-AMP have different biological effects apart from their conversion to adenosine, the relative expression of ecto-2′,3′-phosphodiesterases and their relative kinetic properties would determine whether extracellular 2′,3′-cAMP is converted predominantly to 3′-AMP or to 2′-AMP. Indeed, the current study demonstrates that both microglia and astrocytes convert 2′,3′-cAMP preferentially to 2′-AMP, rather than 3′-AMP.

Incubating primary microglia or astrocytes with exogenous 3′-AMP or 2′-AMP (hydrophilic compounds) induces an increase in extracellular adenosine. This suggests that these AMPs can be metabolized to adenosine in the extracellular biophase and supports the existence of the extracellular 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine) in these cell types. This conclusion is confirmed by the increase in extracellular adenosine levels following incubation of microglia or astrocytes with 2′,3′-cAMP.

Astrocytes convert 2′,3′-cAMP to 2′-AMP and 3′-AMP more efficiently than microglia, yet microglia convert 2′-AMP and 3′-AMP to adenosine more efficiently than astrocytes. This suggests that astrocytes collaborate with microglia to provide a 2′,3′-cAMP-adenosine pathway that affects microglia function via adenosine formation. In this regard, astrocytes may function to metabolize 2′,3′-cAMP to 2′-AMP and 3′-AMP which can then diffuse to nearby microglia and be converted to adenosine in the biophase of adenosine receptors on microglia.

The present study shows that in both microglia and astrocytes, AMPCP inhibits the conversion of extracellular 5′-AMP to extracellular adenosine, but has little effect on the metabolism of extracellular 3′-AMP or 2′-AMP to extracellular adenosine. This suggests that the ecto-2′/3′-nucleotidase that mediates the conversion of 3′-AMP or 2′-AMP to adenosine is distinct from the ecto-5′-nucleotidase (CD73) that mediates the conversion of extracellular 5′-AMP to adenosine. Conceivably there exists distinct ecto-2′-nucleotidases and ecto-3′-nucleotidases and ecto-nucleotidases that recognize both 2′-AMP and 3′-AMP.

Cultures of nearly pure primary microglia or astrocytes do not express the 3′,5′-cAMP-adenosine pathway. However, there is ample evidence for the extracellular 3′,5′-cAMP-adenosine pathway in the brain. For example, in rat cerebral cortex in dissociated cell culture, 3′,5′-cAMP is converted to 5′-AMP and adenosine (Rosenberg & Dichter 1989). Also, in cerebral cortical cultures, isoproterenol (Rosenberg et al. 1994), norepinephrine (Rosenberg & Li 1995), epinephrine (Rosenberg & Li 1995) and forskolin (Rosenberg & Li 1996) increase extracellular 3′,5′-cAMP and adenosine. The extracellular 3′,5′-cAMP pathway also exists in the hippocampus (Brundege et al. 1997). In superfused hippocampal slices, both forskolin and exogenous 3′,5′-cAMP increase extracellular adenosine levels and induce adenosine-mediated electrophysiological effects (Brundege et al. 1997). Moreover, studies indicate that the 3′,5′-cAMP-adenosine pathway limits D1 receptor expression in CAD catecholaminergic cells (Do et al. 2007). Therefore, it is likely that the 3′,5′-cAMP-adenosine pathway exists in the brain, either in neurons or as a result of interactions among several cell types. Because the present study demonstrates robust metabolism of 5′-AMP to adenosine by microglia, if neurons metabolize 3′,5′-cAMP to 5′-AMP, microglia would provide ecto-5-nucleotidase activity to metabolize 5′-AMP to adenosine. Consequently, microglia may be involved in a functioning 3′,5′-cAMP-adenosine pathway when in collaboration with other CNS cell types.

Adenosine activates four subtypes of cell-surface adenosine receptors (A1, A2A, A2B and A3). Our recent study shows that A1 receptor stimulation reduces microglial activation after experimental traumatic brain injury (Haselkorn et al. 2009). Seven days after controlled cortical impact injury, Iba-1 staining identifies marked microglial proliferation that is significantly enhanced in nearly all brain regions in A1 receptor knockout mice. Two prior studies using A1-receptor-null mice in other unrelated models of CNS inflammation report similar magnitudes of increases in microglial proliferation in knockout mice versus wild-type (Tsutsui et al. 2004, Synowitz et al. 2006). Moreover, our previous work demonstrates that in BV-2 cells (immortalized mouse microglia cell line) in culture, cell proliferation, as assessed by 3H-thymidine incorporation, is inhibited by either A1 receptor or A2B receptor activation (Haselkorn et al. 2009). Thus injury-induced formation of adenosine resulting from activation of the extracellular 2′,3′-cAMP-adenosine pathway in microglia may be critical with regard to limiting the aggressiveness of the microglial response to injury. This would be analogous to the now well established role of adenosine as an anti-inflammatory autocoid that helps resolve inflammation in peripheral tissues (Rajakariar et al. 2009). Finally, a paracrine role for microglia-derived adenosine may protect neurons from glutamate-induced excitotoxicity (Lauro et al. 2008).

Recent studies show that 2′,3′-cAMP enhances opening of the mitochondrial permeability transition pore (Azarashvili et al. 2009), a process that is linked to apoptosis and necrosis. Thus the cellular transport of 2′,3′-cAMP by injured neurons into the brain interstitial space following an insult may be important in removing this potentially lethal intracellular compound. Rapid metabolism of extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP by astrocytes and microglia may facilitate the disposal of neuronal intracellular 2′,3′-cAMP in the area of injury by maintaining a low extracellular concentration of 2′,3′-cAMP and thereby providing a more favorable intracellular-to-extracellular gradient for efflux of intracellular 2′,3′-cAMP. In summary, the characterization of extracellular 2′,3′-cAMP metabolism presented here reveals a novel function of microglia and astrocytes which may be important in their response to brain injury.

Acknowledgments

This work was supported by NIH grants NS070003 (PMK/EKJ), DK068575 (EKJ), DK079307 (EKJ), NS38087 (PMK), NS30318 (PMK), T32 HD040686 (JE), and by the Swiss National Science Foundation #32-64040.00 (RKD), 320000-117998 (RKD), and Oncosuisse OCS-01551-08-2004 (RKD).

Footnotes

No author had a conflict of interest.

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