Nitric Oxide-Stimulated Increase in Extracellular Adenosine Accumulation in Rat Forebrain Neurons in Culture Is Associated with ATP Hydrolysis and Inhibition of Adenosine Kinase Activity

Abstract

Adenosine is a putative endogenous sleep-inducing substance, and nitric oxide has been implicated in arousal and sleep mechanisms. We found that various nitric oxide donors, including diethylamine NONOate (DEA/NO), stimulated large increases in extracellular adenosine in nearly pure cultures of forebrain neurons. The effect of DEA/NO could be blocked by 2-phenyl-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-oxide and could not be mimicked by degraded solutions of DEA/NO or by DEA itself; therefore, it was caused by nitric oxide release on hydrolysis of the parent compound. The accumulation of adenosine was not blocked by probenecid or GMP, suggesting that neither extracellular cAMP nor extracellular AMP was the source, and that adenosine was therefore the most likely species transported across the plasma membrane. To pursue this further, we tested the effect of DEA/NO on cellular ATP and found a significant fall in ATP associated with exposure to nitric oxide. In addition, exposure to DEA/NO nearly completely inhibited adenosine kinase activity. It has been found previously that adenosine kinase is inhibited by its substrate, adenosine. We found that exposure to nitric oxide increased intracellular adenosine to 125 +/- 18% of control values (p < 0.01), consistent with the possibility that in our system the inhibition of adenosine kinase is related to an increase in intracellular adenosine, and that the effect of nitric oxide on extracellular adenosine is significantly potentiated by substrate inhibition of adenosine kinase. Furthermore, nitric oxide-stimulated adenosine accumulation may be important in the regulation of behavioral state.

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

“…A rise in cytoplasmic adenosine concentration soon results in inhibition of adenosine kinase (Figure 6; 7), which leads to further accumulation of intracellular adenosine and ultimately extracellular adenosine through the operation of equilibrative transporters in the plasma membrane (Figure 6; 8). Nitric oxide has been found to produce adenosine accumulation in neuronal cultures by a similar pathway, that is, dependent upon ATP depletion and adenosine kinase inhibition [54]. In the present work we found that phosphatase 1/2A inhibitors had no effect on NMDA-induced adenosine kinase inhibition, yet significantly reduced NMDA receptor-mediated adenosine accumulation.…”

“…Previously, we showed that NO stimulated extracellular adenosine accumulation and that this effect was mediated at least in part by adenosine kinase inhibition in neuronal cultures [54]. To test whether NO-induced adenosine accumulation also requires phosphatase 1/2A activation, we tested the ability of okadaic acid to block the effect of NO.…”

“…This procedure monitors in vivo phosphorylation of [U- 14 C] adenosine (489 mCi/mmol, Amersham, Arlington Heights, IL, USA), to AMP by adenosine kinase [38,54]. Cultures were exposed to NMDA for 5 minutes, and then radioactivity was added (0.04 μM adenosine, final concentration 5 μCi/10 −6 μmol).…”

Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

“…The Bioluminescent Somatic Cell Assay kit from Sigma (catalog #FL-ASC) was used for assay of ATP, as described previously (Rosenberg et al, 2000;Zhang and Rosenberg, 2002). At selected time points after addition of drug or vehicle, medium was removed and then replaced with 200 l of cell lysis agent.…”

Retrograde adaptive resonance theory based on the role of nitric oxide in long-term potentiation

“…These properties allow NO to act without the need for presynaptic specializations, and its action is not necessarily confined to the immediate postsynaptic neuron. In conclusion, NO has opened new dimensions in our thinking about how information is transmitted by neurons (Bredt and Snyder 1992;Malenka and Nicoll 1999;Philippides et al 2000;Rosenberg et al 2000;Smith and Philippides 2000;Burette et al 2002;Yang 2003;Ledo et al 2005). In neuroscience, Long-Term Potentiation is the potentiation of the connection between two nerve cells, which lasts for an extended period of time (minutes to hours in vitro and hours to days or even months in vivo).…”