Question: In the last decades astrocytes have emerged as important synergic cells in brain function, having roles in trophic support, synaptic activity and vascular modulation. Most of these functions are mediated by astrocyte plasma membrane (PM) extensions, expressing both aquaporin-4 and K inward rectifying (Kir) channels. Since astrocytes might be exposed to different stimuli and several pathways are activated by G protein coupled receptors (GPCR), we asked whether PM extensions and their function might be altered over time upon metabotropic receptor activation/deactivation. Methods: Isolated rat hippocampal astrocytes were used for live cell imaging experiments and whole-cell patch-clamp recordings. For imaging, cells were stained with the PM dye CellMask Orange and monitored for 20 min after drug treatments. In patch-clamp experiments, astrocyte Kir current was recorded applying voltage-steps (-180 mV to 60 mV) following 0 mV holding potential. Results: We found that PM extensions are modulated in number and length by GPCR, mainly by Gq-mediated pathway. To elicit Gq activation we selected endogenous purinergic receptor P2Y1. In western blot analysis, we observed an increased expression of P2Y1 receptor in cultured cells up to three weeks. In live cell imaging experiments, we validated functional P2Y1 receptor by monitoring calcium flux with Fluo-4 indicator. Activation of P2Y1 by the selective agonist 2-methylthioadenosine diphosphate (2MeSADP, 100 mM) showed increased number of PM extensions that was prevented by pre-treatment with selective P2Y1 antagonist, MRS 2179 (1 mM). To test the effective contribution of Gq-mediated pathway, we performed live cell imaging experiments in cells treated with phospholipase C inhibitor U73122 (3 mM). We observed a dramatic decrease of PM extensions in length and number. Based on this observation we hypothesized that reduction of extensions will alter K channel activity. Kir current was recorded 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. Cells were monitored for 10 min after U73122 application and such effect was achieved after 3 min treatment. At positive potentials (20 mV to 60 mV), K current was reduced over time up to 10 min. Conclusions: These data suggest that astrocytes extensions are dynamically modulated by GPCR and that astrocytes K buffering function might be altered by impaired Gq-mediated pathways.

Short-term modulation of astrocyte plasma membrane extensions by GPCRs

CHISARI, Mariangela;SORTINO, Maria Angela
2015

Abstract

Question: In the last decades astrocytes have emerged as important synergic cells in brain function, having roles in trophic support, synaptic activity and vascular modulation. Most of these functions are mediated by astrocyte plasma membrane (PM) extensions, expressing both aquaporin-4 and K inward rectifying (Kir) channels. Since astrocytes might be exposed to different stimuli and several pathways are activated by G protein coupled receptors (GPCR), we asked whether PM extensions and their function might be altered over time upon metabotropic receptor activation/deactivation. Methods: Isolated rat hippocampal astrocytes were used for live cell imaging experiments and whole-cell patch-clamp recordings. For imaging, cells were stained with the PM dye CellMask Orange and monitored for 20 min after drug treatments. In patch-clamp experiments, astrocyte Kir current was recorded applying voltage-steps (-180 mV to 60 mV) following 0 mV holding potential. Results: We found that PM extensions are modulated in number and length by GPCR, mainly by Gq-mediated pathway. To elicit Gq activation we selected endogenous purinergic receptor P2Y1. In western blot analysis, we observed an increased expression of P2Y1 receptor in cultured cells up to three weeks. In live cell imaging experiments, we validated functional P2Y1 receptor by monitoring calcium flux with Fluo-4 indicator. Activation of P2Y1 by the selective agonist 2-methylthioadenosine diphosphate (2MeSADP, 100 mM) showed increased number of PM extensions that was prevented by pre-treatment with selective P2Y1 antagonist, MRS 2179 (1 mM). To test the effective contribution of Gq-mediated pathway, we performed live cell imaging experiments in cells treated with phospholipase C inhibitor U73122 (3 mM). We observed a dramatic decrease of PM extensions in length and number. Based on this observation we hypothesized that reduction of extensions will alter K channel activity. Kir current was recorded 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. Cells were monitored for 10 min after U73122 application and such effect was achieved after 3 min treatment. At positive potentials (20 mV to 60 mV), K current was reduced over time up to 10 min. Conclusions: These data suggest that astrocytes extensions are dynamically modulated by GPCR and that astrocytes K buffering function might be altered by impaired Gq-mediated pathways.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11769/112583
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