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'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As 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'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h 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'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, 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 the 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'- or 3'-AMP to adenosine.

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 primary and RED-conjugated secondary antibodies (Red, DAPI, blue) (B). Also presented is a merged image of the same field of view showing both cell types (C). Quantification of random fields of view reveals that the primary cultures are over 98% Iba-1+ microglial cells with only few residual astrocytes present (***, p<0.001) (D).

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 5′-AMP (C and F). P-values in panels are from analysis of variance. ^ap<0.05, compared with basal (0). Values represent means ± SEM for 6 experiments.

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 (C and D) on the metabolism of 2′,3′-cAMP to 2′-AMP (A and C) and 3′-AMP (B and D). P-values in panels are from analysis of variance. ^ap<0.05, compared with basal; ^bp<0.05, compared with no inhibitor. Values represent means ± SEM for 6 experiments.

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 from analysis of variance. ^ap<0.05, compared with basal (0). Values represent means ± SEM for 6 experiments.

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), 3′-AMP (B and E) and 5′-AMP (C and F) to adenosine. P-values in panels are from analysis of variance. ^ap<0.05, compared with basal (0); ^bp<0.05, compared with 5′-AMP alone. Values represent means ± SEM for 6 experiments.

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, compared with basal (0). Values represent means ± SEM for 6 experiments.