Adenosine Transport by Rat and Guinea Pig Synaptosomes: Basis for Differential Sensitivity to Transport Inhibitors

Abstract

Adenosine transport by rat and guinea pig synaptosomes was studied to establish the basis for the marked differences in the potency of some transport inhibitors in these species. An analysis of transport kinetics in the presence and absence of nitrobenzylthioinosine (NBTI) using synaptosomes derived from several areas of rat and guinea pig brain indicated that at least three systems contributed to adenosine uptake, the Km values of which were approximately 0.4, 3, and 15 microM in both species. In both species, the system with the Km of 3 microM was potently (IC50 of approximately 0.3 nM) and selectively inhibited by NBTI. This NBTI-sensitive system accounted for a greater proportion of the total uptake in the guinea pig than in the rat and was inhibited by dipyridamole, mioflazine, and related compounds more potently in the guinea pig. Preliminary experiments with other species indicate that adenosine transport in the mouse is similar to that in the rat, whereas in the dog and rabbit, it is more like that in the guinea pig. In the rat, none of the systems appeared to require Na+, but the two systems possessing the higher affinities for adenosine were inhibited by veratridine- and K(+)-induced depolarization. The transport systems were active over a broad pH range, with maximal activity between pH 6.5 and 7.0. Our results are consistent with the possibility that adenosine transport systems may be differentiated into uptake and release systems.

Dipyridamole increases oxygen-glucose deprivation-induced injury in cortical cell culture.

“…Rather, our data support the alternative idea that dipyridamole-sensitive adenosine transporters serve to export adenosine during reduced energy conditions. The observed effective concentration of dipyridamole, 10 /xmol/L, is higher than expected from prior studies with synaptosomes 31 but is consistent with studies with intact neuronal systems.…”

Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

“…The es transporter is also more sensitive than the ei transporter to other inhibitors, such as dilazep, dipyridamole and draflazine [16,19,20]. However, unlike that seen using NBMPR, the actual potency of these latter compounds for inhibiting the es transporter is very species-dependent [21][22][23][24][25][26]. Dipyridamole, for example, has a K i of approx.…”

“…Relative to hENT1, mENT1 was also missing two amino acids (Lys-Gly) from the large central intracellular loop ; however, another mENT1 isoform (mENT1b ; Figure 1 and see below) was also identified in the present study that did not have this particular two-amino-acid deletion. In general, the major differences in ENT1 sequence between species (rat, mouse and human) occurred in the C-terminal half of the large extracellular loop between TM1 and TM2 and in the fifth extracellular loop between TM9 and TM10 ; these differences may contribute to the distinctive affinities of the transporters from the three species for the cardioprotective agents dipyridamole, dilazep and draflazine [21][22][23][24][25][26]34]. In this regard, Sundaram et al [37] have used hENT1\rENT1 chimaeric constructs to identify a region bordered by residues 100 and 231 in hENT1 as being critical for the interaction of dipyridamole and dilazep with the transport protein.…”

Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity

“…Significant species differences have been noted in the sensitivity of the es/ENT1 transporter to inhibitors such as dipyridamole and draflazine (Hammond and Clanachan 1985;Plagemann and Woffendin 1988;Baer et al 1990; Ogbunude and Baer 1990;Shank and Baldy 1990). The human transporter is one of the most sensitive with K i values for inhibition by dipyridamole in the low nanomolar…”