Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(betagamma-subunits)-phosphatidylinositol 3-kinase branch, and 2) the G(q)-PLC-(InsP(3) receptor)/Ca2+ -PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation.
Azarashvili T, Krestinina O, Galvita A, Grachev D, Baburina Y, Stricker R, Evtodienko Y, Reiser G. Ca 2ϩ -dependent permeability transition regulation in rat brain mitochondria by 2Ј,3Ј-cyclic nucleotides and 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase. Am J Physiol Cell Physiol 296: C1428 -C1439, 2009. First published April 8, 2009 doi:10.1152/ajpcell.00006.2009.-Recent evidence indicates that 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes, is also present in neural and nonneural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria and studied the effects of CNP substrates, 2Ј,3Ј-cyclic nucleotides, on functional parameters of rat brain mitochondria. 2Ј,3Ј-cAMP and 2Ј,3Ј-cNADP stimulated Ca 2ϩ overload-induced Ca 2ϩ release from mitochondrial matrix. This Ca 2ϩ release under threshold Ca 2ϩ load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2Ј,3Ј-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondriainduced cell death. 2Ј,3Ј-cAMP effects were observed on the functions of rat brain mitochondria only when PT was developed. This demonstrates involvement of 2Ј,3Ј-cAMP in PT regulation in rat brain mitochondria. We also discovered that, under PT development, the specific enzymatic activity of CNP was reduced. Thus we hypothesize that suppression of CNP activity under threshold Ca 2ϩ load leads to elevation of 2Ј,3Ј-cAMP levels that, in turn, promote PT development in rat brain mitochondria. Similar effects of 2Ј,3Ј-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knockdown oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for Ca 2ϩ overload-induced Ca 2ϩ release. Thus our results reveal a new function for CNP and 2Ј,3Ј-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT. oligodendrocyte mitochondria; 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase; permeability transition; calcium transport THE ENZYME 2Ј,3Ј-CYCLIC NUCLEOTIDE 3Ј-phosphodiesterase (CNP, EC 3.1.4.37) accounts for ϳ2-5% of the total protein in the central nervous system myelin and 0.5-1% of peripheral nervous system myelin (36). CNP catalyzes the hydrolysis of 2Ј,3Ј-cyclic nucleotides to form the corresponding 2Ј-monophosphates (3). CNP was shown to be an integral protein of myelin of oligodendrocytes in the central nervous system and of peripheral myelin in Schwann cells (36,39). The majority of studies investigating the role of CNP were focused exclusively on the expression of CNP in oligodendrocytes and Schwann cells and the involvement of CNP in myelinogenesis. However, there is increasing evidence showing that this enzyme is present in a variety of other cell types. CNP-like enzyme activity was found ...
We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C‐terminus than centaurin‐α from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide‐specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane‐associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki=1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment. © 1997 Federation of European Biochemical Societies
Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.
Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growthregulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1b and tumour necrosis factor-a secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C 2 -ceramideinduced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.
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