Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism

Abstract

Adenosine is a neuromodulator that regulates neurotransmission in the brain and central nervous system. Recently, spontaneous adenosine release that is cleared in 3-4 sec was discovered in mouse spinal cord slices and anesthetized rat brains. Here, we examined the clearance of spontaneous adenosine in the rat caudate-putamen and exogenously applied adenosine in caudate brain slices. The V max for clearance of exogenously applied adenosine in brain slices was 1.4 ± 0.1 μmol/L/sec. In vivo, the equilibrative nucleoside transport 1 (ENT1) inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI) (1 mg/kg, i.p.) significantly increased the duration of adenosine, while the ENT1/2 inhibitor, dipyridamole (10 mg/kg, i.p.), did not affect duration. 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyrido[2,3-d]byrimidin-4-amine dihydrochloride (ABT-702), an adenosine kinase inhibitor (5 mg/kg, i.p.), increased the duration of spontaneous adenosine release. The adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (10 mg/kg, i.p.), also increased the duration in vivo. Similarly, NBTI (10 μmol/L), ABT-702 (100 nmol/L), or EHNA (20 μmol/L) also decreased the clearance rate of exogenously applied adenosine in brain slices. The increases in duration for blocking ENT1, adenosine kinase, or adenosine deaminase individually were similar, about 0.4 sec in vivo; thus, the removal of adenosine on a rapid time scale occurs through three mechanisms that have comparable effects. A cocktail of ABT-702, NBTI, and EHNA significantly increased the duration by 0.7 sec, so the mechanisms are not additive and there may be additional mechanisms clearing adenosine on a rapid time scale. The presence of multiple mechanisms for adenosine clearance on a time scale of seconds demonstrates that adenosine is tightly regulated in the extracellular space.

Keywords: Adenosine deaminase; adenosine kinase; equilibrative nucleoside transporter; voltammetry.

Figure 1

Stability of spontaneous, transient adenosine release in vivo using fast‐scan cyclic voltammetry. (A) Concentration versus time trace of spontaneous adenosine release in vivo. The gray shading shows when adenosine levels are above 10% baseline and the clearance of adenosine is fit with a single exponential decay (red). (B) A characteristic CV of adenosine detected with fast‐scan cyclic voltammetry. The primary oxidation is observed at 1.40 V and the secondary oxidation is observed at 1.0 V. (C) The duration of transient adenosine release was measured in the caudate‐putamen of anesthetized rats over a 5 h period. The durations are placed into 1 h time bins. The fourth and fifth hour are significantly higher than the first 3 h (one‐way ANOVA post‐Bonferroni test, n = 4, P < 0.05). (D) Concentration of spontaneous adenosine release were placed into hour bins. There is a significant difference in concentration over time (one‐way ANOVA, n = 4, P < 0.05), but not between the second and third hours (one‐way ANOVA post‐Bonferroni test, n = 4, P > 0.05). CV, cyclic voltammogram; ANOVA, analysis of variance. ****P < 0.001

Figure 2

Inhibition of clearance of spontaneous, transient adenosine release by NBTI. (A) Concentration versus time trace of spontaneous adenosine release pre‐ (top) and post‐NBTI (bottom, 1 mg/kg i.p.) which inhibits ENT1. Gray shading indicates when adenosine is above 90% of the baseline with exponential decay fit (red trace). (B) CVs of adenosine for predrug (top) and postdrug (bottom) transients. (C) The duration of adenosine significantly increased (unpaired t‐test, n = 6, P = 0.0327). (D) The decay rate (k, sec⁻¹) following NBTI administration (blue) is significantly smaller than predrug values (white) (unpaired t‐test, n = 8, P = 0.0024). (E) A histogram of relative frequency versus interevent time (time between consecutive transients) with 30 sec bins. Exponential fits are displayed for predrug (black) and post‐NBTI (blue) with no significant difference in the underlying distributions (Kolmogorov–Smirnov test, n = 8 rats, P = 0.2882). (F) Spontaneous transient adenosine concentration predrug (white bars) and post‐NBTI (blue bars) did not significantly change (unpaired t‐test, n = 8, P = 0.3585). NBTI, S‐(4‐nitrobenzyl)‐6‐thioinosine; ENT1, equilibrative nucleoside transport 1; CV, cyclic voltammogram. *P < 0.05, ****P < 0.001

Figure 3

Inhibition of clearance of spontaneous, transient adenosine release through ENT1/2. (A) A predrug transient (top) and post‐dipyridamole (ENT1/2 inhibitor, 10 mg/kg i.p., bottom) concentration versus time graph with duration shaded gray and exponential decay fit in red. (B) CVs of adenosine before and after dipyridamole. (C) Duration before (white) and after dipyridamole (green) did not significantly change (unpaired t‐test, n = 6 animals, P = 0.6921). (D) The decay rate following dipyridamole significantly decreased (unpaired t‐test, n = 6 animals, P = 0.0141). (E) Interevent time histogram before (black) and after (green) dipyridamole. The underlying distributions are not significantly different (KS test, n = 6 animals, P = 0.3141). (F) Concentration of spontaneous adenosine did not significantly change postdrug (unpaired t‐test, n = 6 animals, P = 0.4893). ENT, equilibrative nucleoside transport; CV, cyclic voltammogram; KS, Kolmogorov–Smirnov. *P < 0.05

Figure 4

The effect of adenosine kinase on clearance of transient adenosine release. (A) Concentration versus time of spontaneous, transient adenosine release before (top) and after (bottom) ABT‐702 (5 mg/kg, i.p.). (B) CVs of adenosine pre and postdrug. (C) The duration significantly increased (unpaired t‐test, n = 5 animals, P = 0.0155) post‐ABT‐702 (orange). (D) The decay rate significantly decreased after administration of ABT‐702 (unpaired t‐test, n = 5 animals, P = 0.0127). (E) Interevent time histogram of predrug (black) and postdrug (orange) with no significant difference between the underlying distributions (KS test, n = 5 animals, P = 0.0414). (F) The concentration of spontaneous adenosine release significantly increased (unpaired t‐test, n = 5 animals, P = 0.0483) after ABT‐702 administration. ABT‐702, 5‐(3‐bromophenyl)‐7‐[6‐(4‐morpholinyl)‐3‐pyrido[2,3‐d]byrimidin‐4‐amine dihydrochloride; CV, cyclic voltammogram; KS, Kolmogorov–Smirnov. *P < 0.05

Figure 5

The effect of adenosine deaminase on clearance of transient adenosine release. (A) Effect of adenosine deaminase inhibition with EHNA (10 mg/kg i.p.) on spontaneous adenosine release. Concentration versus time plots before and after inhibition with gray indicating the duration of release and red showing the exponential decay fit. (B) CVs for adenosine pre‐ and post‐EHNA. (C) The duration of transient adenosine significantly increased pre and postdrug (unpaired t‐test, n = 6 animals, P = 0.0049). (D) Decay rates were significantly different after EHNA administration (unpaired t‐test, n = 6 animals, P < 0.0001). (E) Interevent time histogram with predrug trace (black) and post‐EHNA (red). EHNA had no significant effect on the underlying distribution (n = 6 animals, KS test, P = 0.8506). (F) The concentration of transient adenosine decreased following adenosine deaminase (red) inhibition (unpaired t‐test, n = 6 animals, P = 0.0404). EHNA, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine; CV, cyclic voltammogram; KS, Kolmogorov–Smirnov. *P < 0.05, **P < 0.01, ****P < 0.0001

Figure 6

The effect of simultaneous inhibition of adenosine kinase, adenosine deaminase, and ENT1 on clearance of transient adenosine release. (A) Effect of ABT‐702 (5 mg/kg), EHNA (10 mg/kg), and NBTI (1 mg/kg) on the duration of spontaneous, transient adenosine release. The inhibition of adenosine deaminase, adenosine kinase, and ENT1 significantly increased the duration of adenosine release (unpaired t‐test, n = 5 animals, P < 0.0001). (B) The decay rates were significantly smaller following the cocktail administration (unpaired t‐test, n = 5 animals, P = 0.0315). (C) The interevent distribution was not significantly different before and after inhibition (n = 5 animals, KS test, P = 0.3485). (D) The concentration of released adenosine did not change (unpaired t‐test, n = 5 animals, P = 0.1951). ENT1, equilibrative nucleoside transport 1; ABT‐702, 5‐(3‐bromophenyl)‐7‐[6‐(4‐morpholinyl)‐3‐pyrido[2,3‐d]byrimidin‐4‐amine dihydrochloride; EHNA, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine; NBTI, S‐(4‐nitrobenzyl)‐6‐thioinosine; KS, Kolmogorov–Smirnov. *P < 0.05, ****P < 0.0001

Figure 7

Application of exogenous adenosine in caudate‐putamen brain slices. (A) Concentration versus time trace of increasing amounts of adenosine picospritzed onto brain slices. The concentrations range from 3.4 to 310 femtomol and are fit with single exponential decays (dashed lines). (B) Velocity of clearance from multiple electrodes plotted versus applied adenosine concentration. The concentrations were placed into 100 nmol/L bins. The Michaelis–Menten equation was fit to the curve in order to determine a V _max of 1.4 μmol/L/sec for adenosine clearance.

Figure 8

Clearance rate of exogenously applied adenosine during inhibition. Adenosine was picospritzed onto the brain slices and clearance rates were fit with a single exponential curve before and after drug application. (A) The ENT1 inhibitor, NBTI (10 μmol/L, blue), significantly decreased the clearance rate (paired t‐test, n = 4, P = 0.0002). (B) The ENT1/2 inhibitor, dipyridamole (10 μmol/L, green), did not have an effect on clearance rate (n = 6, paired t‐test, P = 0.5209). (C) The adenosine kinase inhibitor, ABT‐702 (100 nmol/L, orange), significantly decreased the clearance rate (n = 5, paired t‐test, P = 0.0053). (D) The adenosine deaminase inhibitor, EHNA (20 μmol/L, red), significantly decreased the clearance rate (n = 4, paired t‐test, P = 0.0036). ENT1, equilibrative nucleoside transport 1; NBTI, S‐(4‐nitrobenzyl)‐6‐thioinosine; ABT‐702, 5‐(3‐bromophenyl)‐7‐[6‐(4‐morpholinyl)‐3‐pyrido[2,3‐d]byrimidin‐4‐amine dihydrochloride; EHNA, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine. **P < 0.01, ***P < 0.001