Purines modulate growth of plasma membrane extensions in isolated astrocytes

It has been shown that astrocytes exert their function in the tripartite synapse and make contact with brain vessels by plasma membrane (PM) extensions (perisynaptic processes and perivascular endfeet, respectively). Such PM extensions express aquaporin-4 (AQP-4) and K+ inward rectifying (Kir) channels, regulating both water and K+ homeostasis. Astrocyte activity is largely modulated by purines, such as adenosine. Therefore in the present study we focused on effects of purines in PM extension growth in astrocytes. In western blot analysis, we observed an increased expression of P2Y1 receptor over time in cultured hippocampal astrocytes. In isolated cells, we validated functional P2Y1 receptor (endogenous Gq protein coupled receptor) by monitoring calcium flux in living cells loaded with Fluo-4 calcium indicator, after stimulation with the selective agonist 2-methylthioadenosine diphosphate (2MeSADP, 100 μM). In order to evaluate PM extensions in astrocytes, living cells were stained with the fluorescent PM dye CellMask Orange, stimulated with 2MeSADP and imaged for 20 min. Number and length of PM extensions increased over time and such effect was prevented by pre-treatment with selective P2Y1 antagonist, MRS 2179 (1 μM). Since calcium mobilization by Gq-mediated pathway might contribute to PM extension growth, we performed experiments in presence of calcium chelator EGTA in the buffer. In cells loaded with Fluo-4 indicator and incubated with 10 μM EGTA for 10 min, astrocytes stimulated with 2MeSADP did not show either intracellular calcium increase or PM extension growth. In a different set of imaging experiments, cells treated with phospholipase C inhibitor U73122 (3 μM) showed a dramatic decrease of PM extensions in length and number. Based on this observation we hypothesized that reduction of PM extensions will affect both AQP-4 expression and Kir function. Western blot analysis of AQP-4, in conditions tested in imaging experiments, did not show any changes in protein level. Patch-clamp recordings were performed in isolated astrocytes applying voltage-steps (-180 mV to 60 mV) to elicit Kir current in basal condition and after U73122 application. A dramatic increase in current through K+ channels at negative potentials (-180 mV to -100 mV) was observed within 3 min in U73122. At positive potentials (20 mV to 60 mV), K+ current was reduced over time up to 10 min. These data show a dynamic modulation of astrocytes PM extensions by adenosine through P2Y1 receptors and the Gq-mediated pathway, suggesting their role in astrocyte buffering function, crucial for brain homeostasis.