Involvement of Bidirectional Adenosine Transporters in the Release of l‐[³H]Adenosine from Rat Brain Synaptosomal Preparations

Abstract

Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l‐[3H]adenosine, our objectives here were to determine (1) if l‐[3H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d‐adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l‐[3H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l‐[3H]Adenosine release was inhibited dose‐dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l‐[3H]adenosine alone or l‐[3H]adenosine plus unlabeled l‐adenosine, d‐adenosine, or uridine, l‐[3H]‐adenosine release was inhibited 42% by l‐adenosine, 69% by uridine, and 81% by d‐adenosine. The inhibition of l‐[3H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.

Activation of Adenosine A2AReceptor Facilitates Brain-Derived Neurotrophic Factor Modulation of Synaptic Transmission in Hippocampal Slices

“…There are two sources of extracellular adenosine: release of adenosine by facilitated diffusion, through membrane adenosine transporters that are equilibrative and bidirectional (Gu et al, 1995), and extracellular conversion of released adenine nucleotides into adenosine through a series of ectoenzymes, the last one in the cascade and the rate-limiting step for adenosine formation being ecto-5Ј-nucleotidase (Zimmermann and Braun, 1999). ATP is co-stored with most neurotransmitters, and therefore neuronal activity causes the release of ATP and extracellular formation of adenosine in most brain areas, including the hippocampus (Wieraszko et al, 1989;Cunha et al, 1996).…”

Adenosine hypothesis of schizophrenia – Opportunities for pharmacotherapy

“…Unlike a classical neurotransmitter, adenosine is neither stored in synaptic vesicles nor released by exocytosis; and it does not act exclusively on synapses (Boison et al, 2010; Fredholm et al, 2005). Its release and up-take is mediated by bi-directional nucleoside transporters whereby the direction of transport solely depends on the concentration gradient between the cytoplasm and the extracellular space (Boison et al, 2010); Gu et al, 1995). Adenosine is therefore considered as a neuromodulator affecting neural activity through multiple mechanisms– presynaptically by controlling neurotransmitter release, postsynaptically by hyper- or de-polarizing neurons, and non-synaptically mainly via regulatory effects on glial cells (Boison et al, 2010).…”

Role of The Purinergic Neuromodulation System in Epilepsy

“…However, with respect to the predominant site of action of adenosine in the brain (i.e. in syn-apses), there is simply no information about which nucleoside transporters (see [103]) or which ecto-nucleotidases might be present in different types of nerve terminals (see [104]). Also, only the few studies that used isolated nerve terminals [98,[105][106][107], or fine electrophysiological techniques able to study individual synapses [78,108] or higher frequencies of stimulation [78,[108][109][110], were able to document the importance of ATP-derived adenosine as a main source of endogenous extracellular adenosine.…”