We propose experiments to study glomerular mesangial cells, in culture, in order to evaluate membrane receptors and signal transduction pathways activated by the vasoconstrictors, endothelin (ET) and thromboxane A2 (TxA2). We will quantitate receptor number, affinity and specificity and correlate receptor characteristics with cellular responses to these ligands. We will also evaluate the role of GTP binding proteins to link receptors to plasma membrane enzymes such as adenylate cyclase and phospholipases A, C and D. The role of G proteins will be measured in both intact cells and with cell membranes using stable GTP analogues. The capacity of phospholipase D to mediate cellular responses to ET and TxA2 will be studied as will the possibility that phospholipase C will use phospholipid substrates in addition to polyphosphoinositides. In subsequent studies, we will evaluate the importance of these signal transduction pathways to mediate the cellular responses of contraction and proliferation. Specifically, we will attempt to mimic proliferative and contractile actions of ET and TxA2 through changes of cellular inositol phosphates, (Ca2+)i pHi and protein kinase C activity. We will also attempt to dissociate phospholipase C activation from the membrane receptor through EJ-ras transfection of the mesangial cells. These manipulations will allow dissection of the relative importance of these signal transduction events either singly or in combination to mediate mesangial contraction and proliferation. Finally, we will search for a genetic defect of mesangial and vascular smooth muscle cells in genetically hypertensive strains. We propose that hyperresponsiveness of the phospholipase C signalling pathways may mediate enhanced vasoconstriction and reduced glomerular filtration. This theory will be tested using cultured vascular smooth muscle and mesangial cells with an assessment not only of their contractile and proliferative responses to contractile agonists but also a comparison of phospholipase C activation with measurements of (Ca2+)i, pHi, release of inositol phosphates and stimulation of protein kinase C.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
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Experimental Cardiovascular Sciences Study Section (ECS)
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Case Western Reserve University
Schools of Medicine
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Rufanova, Victoriya A; Alexanian, Anna; Ostendorf, Tammo et al. (2010) Endothelin signaling via guanine exchange factor C3G in renal glomerular mesangial cells. Can J Physiol Pharmacol 88:808-16
Chahdi, Ahmed; Sorokin, Andrey (2010) The role of beta(1)Pix/caveolin-1 interaction in endothelin signaling through Galpha subunits. Biochem Biophys Res Commun 391:1330-5
Pavlov, Tengis S; Chahdi, Ahmed; Ilatovskaya, Daria V et al. (2010) Endothelin-1 inhibits the epithelial Na+ channel through betaPix/14-3-3/Nedd4-2. J Am Soc Nephrol 21:833-43
Chahdi, Ahmed; Sorokin, Andrey (2010) Endothelin-1 induces p66Shc activation through EGF receptor transactivation: Role of beta(1)Pix/Galpha(i3) interaction. Cell Signal 22:325-9
Rufanova, Victoriya A; Alexanian, Anna; Wakatsuki, Tetsuro et al. (2009) Pyk2 mediates endothelin-1 signaling via p130Cas/BCAR3 cascade and regulates human glomerular mesangial cell adhesion and spreading. J Cell Physiol 219:45-56
Rufanova, Victoriya A; Lianos, Elias; Alexanian, Anna et al. (2009) C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis. Kidney Int 75:31-40
Chahdi, Ahmed; Sorokin, Andrey (2008) Endothelin-1 couples betaPix to p66Shc: role of betaPix in cell proliferation through FOXO3a phosphorylation and p27kip1 down-regulation independently of Akt. Mol Biol Cell 19:2609-19
Chahdi, Ahmed; Sorokin, Andrey (2008) Protein kinase A-dependent phosphorylation modulates beta1Pix guanine nucleotide exchange factor activity through 14-3-3beta binding. Mol Cell Biol 28:1679-87
Sarkozi, Rita; Miller, Bradley; Pollack, Verena et al. (2007) ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells. J Cell Physiol 211:88-100
Rufanova, Victoriya A; Sorokin, Andrey (2006) CrkII associates with BCAR3 in response to endothelin-1 in human glomerular mesangial cells. Exp Biol Med (Maywood) 231:752-6

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