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International audience; The orphan Glutamate receptor Delta2 (GluD2) intrinsic ion channel activity is indirectly triggered by glutamate through stimulation of the metabotropic glutamate receptor 1 (mGlu1), in cerebellar Purkinje cells. However, the mechanisms of GluD2 ion channel opening are entirely unknown. In this work, we investigated the signaling pathways underlying the mGlu1-induced GluD2 current, performing whole-cell voltage-clamp recordings from mGlu1 and GluD2 transfected HEK293 cells. We show that the activation of GluD2 channels via DHPG-induced mGlu1 stimulation is Gαq-dependent. Moreover, inhibition of the downstream components of the mGlu1 canonical signaling pathway PLC and PKC with U73122 and GF109203X, respectively, strongly reduced the DHPG-induced GluD2 current. These results were further confirmed on endogenous receptors at the Parallel Fiber - Purkinje Cell cerebellar synapse, indicating that the opening of the GluD2 channel by mGlu1 receptor mobilizes the canonical Gq-PLC-PKC pathway. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

International audience; We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.

International audience; We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.

Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca2+concentration ([Ca2+]i; EC503 and 6 μM, respectively). The order of potency for nucleotide analogs was ATP = UTP > adenosine 5′- O-[thiotriphosphate] ≫ ADP > UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca2+]iresponses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca2+]iresponse to ATP. Thus ATP- and UTP-stimulated [Ca2+]imobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca2+]iresponse to ATP.