Measurement and regulation of in vivo levels of endogenous adenosine in rat brain

Abstract

Elevated brain levels of endogenous adenosine occur during pathological e ents such as hypoxia, ischemia and seizure activity and are thought to be neuroprotective. Thus increasing levels of adenosine under excitotoxic conditions may lead to the development of a therapeutic strategy that enhances levels of adenosine in a site- and event-specific manner. We validated the use of high-energy (10 kW) focused microwave irradiation as a means of obtaining precise and accurate measurements of in vivo levels of adenosine. Unilateral intrastriatal injections of drugs were administered to male Sprague-Dawley rats and, following microwave irradiation, striata ipsilateral and contralateral to injections were excised and adenosine levels measured using fluorescence-HPLC. The ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA) and kainate, but not amino-3-hydroxy-5-methyl-4-isoxazol propionic acid dose-dependently increased levels of adenosine in injected striata. The effects of kainate were mediated through NMDA receptors probably because of kainate-induced glutamate release. Use of the 5$\sp\prime$-nucleotidase inhibitor, $\alpha,\beta$-methyleneADP, and the adenosine transport inhibitor, dilazep, showed that adenosine came from both extracellular and intracellular sources during basal and NMDA-stimulated conditions. Both dilazep and the adenosine deaminase inhibitor, 2$\sp\prime$-deoxycoformycin, increased basal and NMDA-induced levels of adenosine. The adenosine kinase inhibitor 5$\sp\prime$-amino-5$\sp\prime$-deoxyadenosine increased basal, but not NMDA-evoked levels of adenosine. Potentiation of adenosine levels in a site- and event-specific manner might best be achieved using a combination of adenosine deaminase and adenosine kinase inhibition or of adenosine deaminase and adenosine transport inhibition. Nitric oxide was found to play a role in regulating basal, but not NMDA-evoked levels of adenosine. Free radicals had a role in both basal and NMDA-induced levels of adenosine. To summarize, striatal levels of adenosine can be increased by ionotropic glutamate receptor regulation, manipulated using inhibitors of adenosine transport and metabolism, and regulated by nitric oxide and free radicals.