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Mise en évidence d’une interaction fonctionnelle entre le récepteur GABAA et le récepteur du peptide vasoactif urotensine II : implication potentielle dans la fonction astrocytaire.

Abstract : Considered as passive cells in the central nervous system, astrocytes play a pivotal role in the neurovascular unit controlling metabolic support to neurons, synaptic function and/or neurogenesis and synaptogenesis, via the expression of a plethora of receptors/ion channels/transporters and the release of autocrine/paracrine factors. Reactive astrogliosis observed during cerebral lesions such as ischemia, could be controlled by vasoactive factors locally over-expressed. Urotensin II (UII) and its paralog, the urotensin II-related peptide (URP), are two neuropeptides currently considered as the most potent vasoactive factors characterized. Previous studies conducted in our team have showed that Rat cortical astrocytes expressed functional UT receptors, and that UII activates Gq/PLC/Ca2+ and Gi/o transduction pathways, T-type calcium channels, and stimulates cell proliferation. The distinct UT affinities for UII and URP, and the UT-associated different couplings to G-proteins would be explained by receptor plasticity regulating by astrocyte proliferation, involving cross-talk of UT with other receptors. Receptor plasticity has ever been suggested for the GABAA receptor, whose functional expression at the plasma membrane is inversely proportional to the degree of astrocyte proliferation, after a stroke or in malignant astrocytes. These data suggest that the GABAA receptor works as a repressor of cell proliferation. A study demonstrating that the UII modulates the GABAergic function in Aplysia neurons suggested a possible interaction between the mitogenic effects of UII and the repressor activity of the GABAA receptor on astrocyte proliferation in physiopathological conditions. Thus, this project aimed at i) demonstrating that UT and the GABAA receptor are coexpressed in astrocytes and investigating the existence of a bidirectional functional interaction between the two receptors, ii) characterizing the mechanism of the UII-induced inhibition of the GABAergic function in astrocytes from cerebellum co-cultured with neurons, and in a cell line coexpressing the human UT and GABAA receptor subunits, and iii) identifying theIn cultured cortical astrocytes, we showed for the first time that UII binding induces a dose-dependant stimulation of cAMP production. This effect is suppressed by the specific AC antagonist SQ22536, and sustained in the presence of the PKA inhibitor H89, suggesting the existence of a negative feed-back of the AC/cAMP/PKA system on UT functioning. Moreover, UII induces a concentration-dependant stimulation of the PIPs turnover, reduced in the presence of H89. These data suggest that UII stimulates cAMP formation and activates PKA, that would provoke a switch of UT coupling from Gs to Gi on the one hand, and Gq, on the other hand. We demonstrated that activation of the GABAA receptor counteracts the effects of UII on both PIPs turnover and cAMP production, thus providing the evidence that UT function can be regulated by the GABAA receptor in astrocytes. Double labeling studies performed on slices from Rat cerebellum revealed that UT and the γ1 and γ2 subunits of the GABAA receptor mainly co-localize in glial processes in the molecular layer and in Purkinje cells. On astrocyte/neuron co-cultures from cerebellum, UT and the γ1 subunit are predominantly expressed in Purkinje cells and astrocytes. In this coculture model, we showed that UII reduces the membrane depolarization and induces a dose dependant inhibition of the chloride current evoked by the GABAA receptor agonist, isoguvacine. On a CHO cell line that stably express the human UT (CHO-UT) and transiently transfected with different combinations of cDNA encoding GABAA receptor subunits, UII i) significantly inhibits the chloride current from the concentration of 10-13 M (β3γ2 ≈ α2β3γ2 ≈α2β1γ2 > α2β3γ1 > α2β1γ1), ii) is more potent than the γ subunit is associated to the β3 subunit in the GABAergic complex (α2β3γ2 ≈ α2β1γ2 >> α2β3 ≈ α2β1), and iii) fails to inhibit the chloride current in the absence of gamma subunits. In CHO-UT expressing α2β3γ2 subunits, a 1-sec UII perfusion inhibited the current, and this effect is correlated to the effect of DMCM, a direct allosteric modulator of GABAergic complexes containing a γ subunit. Intracellular calcium mobilization assays on CHO-UT showed that UII provokes an irreversible intracellulaire calcium concentration ([Ca2+]c) increase whereas URP transiently stimulates the [Ca2+]c. UII and URP irreversibly and reversibly inhibits the isoguvacine-evoked current, respectively. We demonstrated that the peptide antagonist [Orn5]-URP is a partial agonist both on the [Ca2+]c and the GABAergic function, and blocks the prolonged effects of UII on each assays. The non-peptidic antagonist palosuran developed by Actelion, fails to modulate the [Ca2+]c, totally abolishes the UII-induced [Ca2+]c increase but mimics the effect of UII on the GABAergic function, i.e. a significant inhibition of the chloride current. The effect of UII, characterized by a fast inhibition (<1s after UII perfusion), followed by a run-down of the Iso-evoked current, is not relayed via G-proteins coupled to UT. However, the long-term UII-induced inhibition partially involves calcium, intracellular kinases/phosphatases and dynamine, a protein known to mediate the GABAA receptor internalization. On CHO-UT transfected with α2β3HAγ2 subunits, we confirmed that UII induces the internalization of the GABAA receptor. We also generated four C-terminal truncated mutants of the UT receptor lacking the 19 (UT370), 38 (UT351), 57 (UT332) and 70 (UT319) amino acids, respectively, and showed that the co-expression of UT370 with the GABAA receptor strongly reduces the fast UII-induced inhibition, and delays the long-term depression of the chloride current. This suggests that the rapid and direct effect of UII involved the distal fragment 370-389 of the UT C-terminus. When the GABAA receptor is coexpressed with UT351, UT332 or UT319, UII totally failed to inhibit the chloride current, indicating that the 351-370 UT sequence plays a major role in the internalization of the GABAA receptor. Taken together, these data demonstrates for the first time the existence of a bidirectional functional interaction between the GABAA receptor and the G protein-coupled receptor for UII, associated with astrocyte plasticity. The UII mechanisms, which do not entirely require classical GPCR-associated signaling pathways, may involve a more “direct” cross-talk between the C-terminal fragment of UT and the γ subunit associated to the GABAA complex. Thus, the extinction of the GABAA receptor expressed at the plasma membrane triggered by the vasoactive peptide UII, would play a major role in astrocyte proliferation following cerebral lesions
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Thomas Lefebvre. Mise en évidence d’une interaction fonctionnelle entre le récepteur GABAA et le récepteur du peptide vasoactif urotensine II : implication potentielle dans la fonction astrocytaire.. Sciences du Vivant [q-bio]. Université de Rouen, 2010. Français. ⟨tel-02351434⟩

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