Adenosine plays a major modulatory and neuroprotective role in the mammalian CNS. During cerebral metabolic stress, such as hypoxia or ischemia, the increase in extracellular adenosine inhibits excitatory synaptic transmission onto vulnerable neurons via presynaptic adenosine A 1 receptors, thereby reducing the activation of postsynaptic glutamate receptors. Using a combination of extracellular and whole-cell recordings in the CA1 region of hippocampal slices from 12-to 24-d-old rats, we have found that this protective depression of synaptic transmission weakens with repeated exposure to hypoxia, thereby allowing potentially damaging excitation to both persist for longer during oxygen deprivation and recover more rapidly on reoxygenation. This phenomenon is unlikely to involve A 1 receptor desensitization or impaired nucleoside transport. Instead, by using the selective A 1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and a novel adenosine sensor, we demonstrate that adenosine production is reduced with repeated episodes of hypoxia. Furthermore, this adenosine depletion can be reversed at least partially either by the application of exogenous adenosine, but not by a stable A 1 agonist, N 6 -cyclopentyladenosine, or by endogenous means by prolonged (2 hr) recovery between hypoxic episodes. Given the vital neuroprotective role of adenosine, these findings suggest that depletion of adenosine may underlie the increased neuronal vulnerability to repetitive or secondary hypoxia/ischemia in cerebrovascular disease and head injury.Key words: adenosine; hypoxia; ischemia; sensor; depletion; replenishment; glutamate; hippocampus; head injury; TBI; stroke; TIA; neuroprotection; adenosine deaminase; nucleoside phosphorylase; xanthine oxidase Extracellular adenosine in the CNS increases during pathological stimuli such as head injury (Nilsson et al., 1990;Headrick et al., 1994), epileptic seizures (Winn et al., 1980;Dunwiddie, 1999), and hypoxia/ischemia (Berne et al., 1974;Rudolphi et al., 1992;Sweeney, 1997;Von Lubitz, 1999). The increase in extracellular adenosine inhibits glutamate release via presynaptic adenosine A 1 receptors (Fowler, 1989;Katchman and Hershkowitz, 1993;Zhu and Krnjevic, 1993;Pearson and Frenguelli, 2000). In addition, simultaneous activation of postsynaptic A 1 receptors activates a potassium conductance leading to membrane hyperpolarization, thereby intensifying the magnesium block of the NMDA subtype of glutamate receptor (Erdemli et al., 1998;Von Lubitz, 1999). Together, these actions exert a powerful neuroprotective "retaliatory" (Newby, 1984) influence during traumatic or metabolic stress.Manipulations that increase extracellular adenosine, such as adenosine uptake inhibition (Gidday et al., 1995), inhibition of adenosine metabolizing enzymes (Phillis and O'Regan, 1989;Miller et al., 1996;Jiang et al., 1997), or activation of A 1 receptors by exogenous A 1 agonists (Rudolphi et al., 1992;Sweeney, 1997;Von Lubitz, 1999;de Mendonca et al., 2000) are all neuroprotective. Conversely, antagonism of ...

The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine ␣ 1a -adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by ␤-arrestin-dependent endocytosis. Internalization of the ligand did not occur in ␤-arrestin-deficient cells; was blocked or reversed by another ␣ 1 ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human ␣ 1a -AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with ␤-arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in ␤-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are ␤-arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that ␣ 1a -ARs recycle rapidly by an agonist-independent, constitutive, ␤-arrestin-dependent process and that this can transport "␣-blockers" into cells carrying these receptors.