Subcellular localization of adenosine A 1 receptors in nerve terminals and synapses of the rat hippocampus

Abstract

Adenosine is a neuromodulator in the CNS that mainly acts through pre- and postsynaptic A 1 receptors to inhibit the release of excitatory neurotransmitters and NMDA receptor function. This might result from a highly localized distribution of A 1 receptors in the active zone and postsynaptic density of CNS synapses that we now investigated in the rat hippocampus. The binding density of the selective A 1 receptor antagonist, [ 3H]1,3-dipropyl-8-cyclopentylxanthine ([ 3H]DPCPX), was enriched in membranes from Percoll-purified nerve terminals ( B max=1839±52 fmol/mg protein) compared to total membranes from the hippocampus ( B max=984±31 fmol/mg protein), the same occurring with A 1 receptor immunoreactivity. [ 3H]DPCPX binding occurred mainly to the plasma membrane rather than to intracellular sites, since the binding of the membrane permeable A 1 receptor ligand [ 3H]DPCPX to intact hippocampal nerve terminals ( B max=1901±192 fmol/mg protein) was markedly reduced ( B max=321±30 fmol/mg protein) by the membrane impermeable adenosine receptor antagonist, 8-sulfophenyltheophilline (25 μM). Further subcellular fractionation of hippocampal nerve terminals revealed that A 1 receptor immunoreactivity was strategically located in the active zone of presynaptic nerve terminals, as expected to understand the efficiency of A 1 receptors to depress neurotransmitter release. A 1 Receptors were also present in nerve terminals outside the active zone in accordance with the existence of a presynaptic A 1 receptor reserve. Finally, A 1 receptor immunoreactivity was evident in the postsynaptic density together with NMDA receptor subunits 1, 2A and 2B and with N-and P/Q-type calcium channel immunoreactivity, emphasizing the importance of A 1 receptors in the control of dendritic integration.