Unlike AK inhibition the inhibition of ADA activities by EHN
Unlike AK inhibition, the inhibition of ADA activities by EHNA had little effect on spinal reflex potentials, although EHNA did increase extracellular adenosine levels. One explanation for this discrepancy is that the adenosine increase seen herein was not sufficient to inhibit reflex potentials. In this study, the time course of the adenosine increase by EHNA tended to be slower than that by AK. Alternatively, we measured adenosine released from the whole spinal cord preparation, and thus the elevation of adenosine levels by EHNA could occur not only at synaptic regions expressing A1 receptors, but also in various parts of the spinal cord. Therefore, it is suspected that the elevation of adenosine levels by EHNA occurs at different regions in the spinal cord from that by AK. ADA on the cell surface (as ecto-ADA) was also reported in many types of cells (Franco et al., 1997, Franco et al., 1998). Although ecto-ADA is argued to play a minor role in adenosine metabolism compared with cytosolic ADA (Arch and Newsholme, 1978), EHNA may augment extracellular adenosine levels by blockade of ecto-ADA more rapidly than by blockade of intracellular ADA, resulting in the adenosine increase at different regions from that by ABT-702. In addition, AK was appeared to similarly express in both dorsal and ventral horn neurons, while the highest expression of ADA was found in motoneurons at the ventral horn, at where MSRs were mediated. MSRs were less sensitive to the activation of A1 receptors than sVRPs, which may also contribute to the different effect between EHNA and ABT-702. On the other hand, there was no apparent difference in astrocytic AK expression between dorsal and ventral horn. AK was scarcely expressed in astrocytic processes, which should surround synaptic regions, suggesting that the neuronal AK is more important for synaptic regulation than the astrocytic AK. In the mouse brain, it has been shown that AK expression shifts from neurons to astrocytes during postnatal development (Studer et al., 2006). The astrocytic AK may become more important in the spinal cord of mature animals. Further investigation is needed to determine its underlying mechanisms. On the other hand, EHNA substantially enhanced the ABT-702-mediated adenosine release and inhibition of reflex potentials. Within cells, ADA probably acts more efficiently under conditions in which intracellular adenosine levels are excessively increased, because the Km values for ADA are higher than those for AK in the rat qx 314 (Phillips and Newsholme, 1979). ENTs transport adenosine in a transmembrane adenosine gradient-dependent fashion (Baldwin et al., 2004, King et al., 2006). We showed that NBTI/DIP increased extracellular adenosine levels and inhibited sVRPs, suggesting that adenosine levels, at least in the vicinity of the cell membrane, are higher in the extracellular vs. the intracellular space. Thus, ENTs apparently function as uptake transporters under normal conditions. To the contrary, NBTI/DIP decreased the adenosine increase in response to ABT-702. In the presence of ABT-702 and increasing intracellular adenosine levels due to AK inhibition, ENTs are thought to transport adenosine from the cell interior to the exterior through an inversed adenosine gradient across the membrane. Therefore, ABT-702 failed to inhibit sVRPs in the presence of NBTI/DIP. These results indicate that ENTs function as adenosine efflux pathways in response to ABT-702. On the other hand, NBTI/DIP did not significantly inhibit the adenosine increase by EHNA. This result also supports our speculation that the EHNA-evoked adenosine increase is mediated at least in part by ecto-ADA inhibition. Like ABT-702, adenosine and its analogs inhibit spinal reflex potentials (Nakamura et al., 1997, Otsuguro et al., 2009). A1 receptors seem to be responsible for these actions, because the rank order of the inhibitory potencies of these agonists is consistent with the rank order of their affinities for A1 receptors. Furthermore, the inhibitory effects of adenosine were competitively antagonized by 8CPT, an A1 receptor antagonist, as were those of CHA and ABT-702. However, unlike ABT-702, the inhibition by adenosine was enhanced by the ENT inhibitors. The effect of CHA, a stable A1 receptor agonist, was not changed by them, indicating that the ENT inhibitors did not affect the downstream signaling of A1 receptor activation. Extracellular adenosine is controlled by a rapid uptake into cells (Arch and Newsholme, 1978). It is likely that exogenous adenosine is rapidly removed from local extracellular spaces near A1 receptors by ENTs, and thus the ENT inhibitors instantly enhanced the adenosine effect. In clinical applications, ENT inhibitors might exert different effects in patients treated with AK inhibitors from A1 receptor agonists. In addition, chronic activation of A1 receptors reportedly influences the activities and/or expression levels of adenosine receptors and ENTs (Hettinger et al., 1998, Sheth et al., 2014, Hughes et al., 2015). Therefore, the long-term treatment of ABT-702 may affect the activities and expression of molecules in purinergic systems, and thus lead to unexpected effects.