Schwann Cells Metabolize Extracellular 2',3'-cAMP to 2'-AMP

Abstract

The 3',5'-cAMP-adenosine pathway (3',5'-cAMP→5'-AMP→adenosine) and the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2',3'-cAMP-adenosine pathway via their robust expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; converts 2',3'-cAMP to 2'-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2',3'-cAMP-adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2',3'-cAMP-adenosine pathway to the 3',5'-cAMP-adenosine pathway in Schwann cells, we examined the metabolism of 2',3'-cAMP, 2'-AMP, 3'-AMP, 3',5'-cAMP, and 5'-AMP in primary rat Schwann cells in culture. Addition of 2',3'-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2'-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2',3'-cAMP to 3'-AMP or 3',5'-cAMP to either 3'-AMP or 5'-AMP. Although Schwann cells slightly converted 2',3'-cAMP and 2'-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2',3'-cAMP and extracellular 2'-AMP. These findings indicate that Schwann cells do not have a robust 3',5'-cAMP-adenosine pathway but do have a 2',3'-cAMP-adenosine pathway; however, because the pathway mostly involves 2'-AMP formation rather than 3'-AMP, and because the conversion of 2'-AMP to adenosine is slow, metabolism of 2',3'-cAMP mostly results in the accumulation of 2'-AMP. Accumulation of 2'-AMP in peripheral nerves postinjury could have pathophysiological consequences.

Fig. 1.

Schwann cell culture purity and CNPase expression. Cultures were probed with the Schwann cell markers S100 red (A) and CNPase green (B) to confirm the purity of the primary rat Schwann cell cultures (4′,6-diamidino-2-phenylindole, blue; scale bar = 50 μm). (C) Western blots for TNAP, CNPase, and α-tubulin on Schwann cell lysates from cultures grown in either growth or defined medium. Defined medium did not alter expression of either TNAP or α-tubulin, but increased expression of CNPase. (D) Quantification of CNPase expression between the cultures grown in growth medium versus defined medium (normalized to α-tubulin as a loading control) revealed a *significant increase in CNPase cultures grown in defined medium. Ab, antibody; CNP1, CNPase isoform type 1; CNP2, CNPase isoform type 2.

Fig. 2.

Schwann cell 2′,3′-cAMP and 3′,5′-cAMP metabolism. Line graphs show the concentration-dependent effects in Schwann cells of 2′,3′-cAMP and 3′,5′-cAMP on the extracellular levels of 2′-AMP (A), 3′-AMP (B), and 5′-AMP (C). For visual comparisons of 2′-AMP versus 3′-AMP and 5′-AMP, the main graphs show results using the same y-axis scale. However, since levels of 3′-AMP and 5′-AMP were very low, also shown are the levels of 3′-AMP and 5′-AMP using an expanded (magnified) y-axis scale. Values represent the mean ± S.E.M., n = 6 for each group. ^aP < 0.05 versus basal. ANOVA, analysis of variance.

Fig. 3.

Effects of phosphodiesterase inhibitors on metabolism of 2′,3′-cAMP and 3′,5′-cAMP in Schwann cells. Bar graphs show the effects of IBMX (1 mM) and DPSPX (1 mM) on Schwann cell metabolism of 2′,3′-cAMP to 2′-AMP (A), 2′,3′-cAMP to 3′-AMP (B), and 3′,5′-cAMP to 5′-AMP (C). Values represent the mean ± S.E.M., n = 6 for each group. ^aP < 0.05 versus PBS; ^bP < 0.05 versus no inhibitor; ANOVA, analysis of variance; No Inhib, no inhibitor present in the medium.

Fig. 4.

Schwann cell AMP and cAMP metabolism. Line graphs show the concentration-dependent effects in Schwann cells of AMPs (A) and cAMPs (B) on the extracellular levels of adenosine. Values represent the mean ± S.E.M., n = 6 for each group. ^aP < 0.05 versus basal; ANOVA, analysis of variance.

Fig. 5.

Effects of CD73 inhibitor and alkaline phosphatase inhibitor on Schwann cell metabolism of AMPs to adenosine. Bar graphs show the effect of AMPCP (CD73 inhibitor; 0.1 mM) and levamisole (TNAP inhibitor; 1 mM) on the conversion of 2′-AMP (A), 3′-AMP (B), and 5′-AMP (C) to adenosine by the primary Schwann cell cultures. Values represent the mean ± S.E.M., n = 6 for each group. ^aP < 0.05 versus PBS; ^bP < 0.05 versus no inhibitor. ANOVA, analysis of variance; No Inhib, no inhibitor present in the medium.

Fig. 6.

Production of endogenous 2′,3′-cAMP, 2′-AMP, 3′,5′-cAMP, and 5′-AMP by sciatic nerves. Ex vivo transected sciatic nerve pieces were incubated for 0, 1, or 3 hours. At the indicated times, the medium was collected and the nerve sections were extracted. Both medium and nerve extracts were analyzed by liquid chromatography–tandem mass spectrometry for 2′,3′-cAMP (A), 2′-AMP (B), 3′,5′-cAMP (C), and 5′-AMP (D). Values represent the mean ± S.E.M., n = 7 for each group. ^aP < 0.05 versus PBS; ANOVA, analysis of variance.

Fig. 7.

Production of endogenous adenosine by sciatic nerves. Ex vivo transected sciatic nerve pieces were incubated for 0, 1, or 3 hours. At the indicated times, the medium was collected and the nerve sections were extracted. Both medium and nerve extracts were analyzed by liquid chromatography–tandem mass spectrometry for adenosine. Adenosine did not change in nerve extracts (A) but decreased in the medium (B). Values represent the mean ± S.E.M., n = 7 for each group. ^aP < 0.05 versus PBS; ANOVA, analysis of variance.

Fig. 8.

Inhibition of CNPase activity by DPSPX. Bar graph shows the effect of 1 and 10 mM DPSPX on recombinant human CNPase metabolism of 2′,3′-cAMP to 2′-AMP. Values represent the mean ± S.E.M., n = 6 for each group. ^aP < 0.05 versus no inhibitor; No enzyme, no CNPase present; No Inhibitor, no DPSPX present in the medium.