Desensitization of adenosine A(1) receptors in rat immature cortical neurons.

Adenosine plays an important neuroprotective role in brain, usually mediated by the activation of adenosine A1 receptors. Prolonged activation of a G-protein-coupled receptor generally leads to the partial loss of the responsiveness of receptor-mediated transduction pathways (desensitization). Rat immature cortical neurons were treated with 100 nM⁻N6-phenylisopropyladenosine (R-PIA), a selective A1 receptor agonist, and the effect on adenosine A1 receptor/adenylyl cyclase pathway was studied. Incubation with R-PIA for 6, 12, 24 and 48 h elicited a time-dependent decrease in adenosine A1 receptors in plasma membranes (92, 58, 43 and 26% of control, respectively), which was associated with variations in microsomal fraction (21, 56, 124 and 233% of control, respectively), suggesting the internalization and down-regulation of adenosine A1 receptors. Moreover, real-time PCR assays showed a significant increase in mRNA levels coding adenosine A1 receptor after the longest treatment period (48 h). In addition, αGi1₋2 protein levels detected in microsomes and mRNA levels coding αGi1 protein were increased after 48 h of treatment with R-PIA, suggesting the synthesis of new αGi1 proteins. Finally, adenylyl cyclase inhibition elicited by 2-Chloro-N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, was significantly reduced after 12, 24 and 48h of treatment (37, 24 and 23%, respectively) as compared to controls (54%), suggesting the desensitization of adenosine A1 receptor/adenylyl cyclase pathway. These results suggest that adenosine A1 receptors desensitize slowly after prolonged receptor activation in immature cortical neurons, showing mechanisms of desensitization similar to those described not only in fetal but also in adult rat brain.

Glutamate differently modulates excitatory and inhibitory adenosine receptors in neuronal and glial cells.

Adenosine is a neuromodulator which acts through adenosine receptors regulating functions such as inhibition of glutamate release. Adenosine A(1) and A(2A) receptor activations most often regulate opposing actions. Primary rat cortical neurons and rat C6 cells, an astrocytic derived cell line, were exposed to 100muM l-glutamate, and cell viability and transduction pathways mediated by both A(1) and A(2A) receptors were analyzed. Glutamate-induced excitotoxic damage was found only in cortical neurons, with C6 cells preserved. In C6 cells, adenosine A(1) and A(2A) receptors were increased and decreased, respectively. Consequently, A(1)-mediated adenylyl cyclase inhibition and A(2A)-mediated adenylyl cyclase stimulation were, respectively, increased and decreased after glutamate exposure. In cortical neurons, glutamate treatment increased both A(1) and A(2A) receptors. Moreover, adenylyl cyclase responsiveness to A(1) or A(2A) receptor agonists was heightened in these cells, in which pharmacological activation of AC induced cell death. Finally, activation of A(1) receptor or blockade of A(2A) receptor during glutamate treatment partially prevented the glutamate-induced cell death detected in cultured cortical neurons. Results show that adenosine receptors are regulated by glutamate, and that this regulation is dependent on the cell type, suggesting that adenosine receptors might be promising targets in the therapy against excitotoxic cell death.

Glutamate differently modulates metabotropic glutamate receptors in neuronal and glial cells.

Glutamate is an excitatory neurotransmitter implicated in learning and memory processes, but at high concentrations it acts as an excitotoxin causing degeneration and neuronal death. The aim of this work was to determine the excitotoxic effect of glutamate and the regulation of metabotropic glutamate receptors (mGluR) during excitotoxicity in neurons and C6 glioma cells. Results show that glutamate causes excitotoxic damage only in cortical neurons. Loss of cell viability in neurons was glutamate concentration- and time-dependent. Total mGluR levels were significantly reduced in these cells when exposed to glutamate. However, in C6 cells, which have been used as a model of glial cells, these receptors were regulated in a biphasic manner, decreased after 6 h, and increased after 24/48 h of treatment. Results show a cell dependent mGluR regulation by glutamate exposure which could mediate the vulnerability or not to glutamate mediated excitotoxicity.