Distribution of nucleoside transporter subtypes in rat brain

Thumbnail Image
Anderson, Christopher M.
Journal Title
Journal ISSN
Volume Title
Adenosine is recognized as an important inhibitory neuromodulator in the central nervous system. Through four distinct plasma membrane receptors, endogenous adenosine is able to inhibit the release of many neurotransmitters, including glutamate following cerebral ischemia, and also affect other non-neuronal cell types in brain such as astrocytes, microglia, platelets and endothelial cells. Nucleoside transport processes are instrumental in regulating endogenous adenosine levels and the associated effects of adenosine. There are two broad categories of nucleoside transporters: equilibrative and concentrative. Two equilibrative subtypes have been cloned and termed ENT1 and ENT2. The potent nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) can be used as a marker for ENT1 but not for ENT2. Two concentrative subtypes have also been cloned. CNT1 is pyrimidine nucleoside-selective while CNT2 is purine nucleoside-selective. There are no known selective ligands for these transporters. In the absence of selective ligands for transporters other than ENT1, the cloning of rat ENT2 (rENT2), CNT1 (rCNT1) and CNT2 (rCNT2) has provided the first opportunity to investigate the distributions of specific nucleoside transporter subtypes in rat brain. The distribution of mRNA for the four cloned transporters was investigated using 'in situ' hybridization with 35S-labeled cRNA probes in rat brain and using Northern blot analysis in human brain. Transporter protein distributions were also studied. [3H]-Nitrobenzylthioinosine ([3H]NBMPR) autoradiography was employed to estimate the distribution of rENT1 in rat brain while rCNT1 and rCNT2 distributions were studied by mapping immunoreactivity of polyclonal antibodies generated in rabbits against both transporters using immunocytochemistry in rat brain. Northern analysis of human brain total RNA indicated that both hENT1 and hENT2 have a wide distribution in human brain. Both probes detected RNA species in every region tested. In general, message for all four nucleoside transporters was seen in rat hippocampus, cerebellum, cerebral cortex, striatum and other regions with varying relative abundances among regions for each transporter. Binding of [3 H]NBMPR to rat brain sections to identify rENT1 transporters supported results from ' in situ' hybridization in thalamus, cortex and striatum, however, in contrast to 'in situ' hybridization, low signal intensity was seen in hippocampus and cerebellum indicating low protein expression. Immunocytochemistry using antibodies for rCNT1 and rCNT2 generally confirmed results seen with 'in situ' hybridization. The broad and overlapping distributions of different nucleoside transporter subtypes demonstrated here indicates that the regulation of adenosine and other nucleosides important for salvage of nucleotides with signaling capabilities is achieved through multiple transport mechanisms.