CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation

Abstract

Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t_1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

Keywords: Adenosine; CD39KO; CD73KO; PAP; TNAP; hippocampus; in vivo; mechanosensitive; sex differences; spontaneous adenosine.

Figure 1.. Spontaneous adenosine in the hippocampus.

Example adenosine events in females of (A) wild-type (B) CD73KO, and (C) CD39KO mice. Adenosine can be identified in cyclic voltammograms (top) by its primary oxidation peak at 1.3 V on the cathodic scan and secondary oxidation peak at 1.2 V on the anodic scan. Concentration vs. time traces (middle) were derived from corresponding 3-D color plots (bottom). Adenosine oxidations are the green/purple area in the middle of the color plot. (D) Number of adenosine events per hour (One-way ANOVA, n = 8 animals (4M/4F)/genotype, overall effect p = 0.0002, Tukey’s test, ** p<0.01, ***p < 0.001). (E) Inter-event time distributions. The underlying inter-event time distributions of adenosine were significantly different (Kruskal-Wallis test, overall effect p < 0.0001, Dunn’s test, **** p < 0.0001). (F) Mean concentrations of each adenosine event did not vary by geneotype (One-way ANOVA, n = 8 animals/genotype, p = 0.77). (G) Average t_1/2 did not significantly differ by genotype (One-way ANOVA, n = 8 animals/genotype, p = 0.13).

Figure 2.. Mechanically-stimulated adenosine in the hippocampus.

Example of mechanically-stimulated adenosine in female (A) wild-type, (B) CD73KO, and (C) CD39KO mice. Electrode was lowered 0.1 mm at 30 s (arrow). Concentration vs. time traces show the peak concentration of adenosine. Four consecutive stimulations were performed every 15 min for totally 4 stimulations. (D) Concentration of mechanically-stimulated adenosine, shown by stimulation number. Red dashed lines are the average for all 4 stimulations for each genotype. There was no significant effect of genotypes or stimulation number on concentration of mechanically-stimulated adenosine (Two-way repeated measure ANOVA, n = 8 animals (4M and 4F)/genotype, p = 0.65 and p = 0.072, respectively). (E) t_1/2 of mechanically-stimulated adenosine, by stimulation number. Red line is average for all stimulations. There was no significant effect of genotypes or stimulation number on t_1/2 of mechanically-stimulated adenosine (Two-way repeated measure ANOVA, n=8 (4M/4F) p = 0.43 and p = 0.71, respectively).

Figure 3.. Sex differences in spontaneous (A-B) and mechanically-stimulated (C) adenosine.

(A) Number of spontaneous adenosine events per hour, broken down by sex in each genotype. There is a significant interaction of genotype and sex on number of adenosine events (Two-way ANOVA, n = 4 animals/group, F _{(2, 18)} = 7.9, p = 0.0034, Tukey post- test data is marked for important differences and full post-test data is in Table S1). (B) Concentration of individual adenosine transients in each genotype. There is a significant interaction of genotype and sex on concentration of adenosine events (Two-way ANOVA, n = 200 transients/group, F _{(2, 1194)} = 32, p < 0.0001. Tukey’s post-test data shown, with full post-test data given in Table S2). (C) Concentration of mechanically-stimulated adenosine. There is a significant interaction of genotype and sex on concentration of mechanically-stimulated adenosine (Two-way ANOVA, n = 4 animals/group, F _{(2, 18)} =4.7, p = 0.023). Tukey’s post-test data is shown and full post-test results are in Table S3. * p<0.05 ** p<0.01, ***p < 0.001, ****p < 0.0001

Figure 4.. mRNA expression of other ATP breakdown enzymes in the hippocampus of wild-type (WT) and CD73KO mice.

(A) The mRNA expression of TNAP in CD73KO males is significantly higher than in males of WT (2-way ANOVA, Tukey’s test, n = 5 animals/group, p=0.015), but TNAP is not upregulated in females (p=0.92). (B) For s-PAP, there are no significant differences by genotype or sex. (C) For TM-PAP, there are no significant differences by genotype or sex.