RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m302401200
ISSN1083-351X
AutoresDolly Mehta, Gias U. Ahmmed, Biman C. Paria, Michael Holinstat, T. Voyno-Yasenetskaya, Chinnaswamy Tiruppathì, Richard D. Minshall, Asrar B. Malik,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+] i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability. We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+] i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability. The increase in cytosolic calcium concentration ([Ca2+] i) 1The abbreviations used are: [Ca2+] i , intracellular calcium concentration; IP3R, inositol trisphosphate receptor; IP3, inositol trisphosphate; TRPC, transient receptor potential channel; ER, endoplasmic reticulum; SOC, store-operated Ca2+ channel; PM, plasma membrane; HPAEC, human pulmonary arterial endothelial cells; HMEC, human microvessel endothelial cells; HBSS, Hanks' balance salt solution; C3, C3 transferase; Abs, antibodies; RBD, Rhotekin-Rho binding domain; GFP, green fluorescent protein; FBS, fetal bovine serum; GST, glutathione S-transferase; DAPI, 4,6-diamidino-2-phenylindole; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. activated by depletion of Ca2+ stores is required for signaling multiple processes in non-excitable cells (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar). The increase in [Ca2+] i regulates responses ranging from tension development through activation of actin and myosin motors to modulation of cell-cell and cell-extracellular matrix adhesive forces (2Dudek S.M. Garcia J.G. J. Appl. Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus (843) Google Scholar, 3Lum H. Malik A.B. Am. J. Physiol. 1994; 267: L223-L241Crossref PubMed Google Scholar, 4Moy A.B. Van Engelenhoven J. Bodmer J. Kamath J. Keese C. Giaever I. Shasby S. Shasby D.M. J. Clin. Invest. 1996; 97: 1020-1027Crossref PubMed Scopus (172) Google Scholar). For example in endothelial cells, increase in [Ca2+] i triggered by depletion of Ca2+ stores is essential for the increase in endothelial permeability induced by thrombin (5Sandoval R. Malik A.B. Naqvi T. Mehta D. Tiruppathi C. Am. J. Physiol. 2001; 280: L239-L247Crossref PubMed Google Scholar, 6Tiruppathi C. Freichel M. Vogel S.M. Paria B.C. Mehta D. Flockerzi V. Malik A.B. Circ. Res. 2002; 91: 70-76Crossref PubMed Scopus (332) Google Scholar). [Ca2+] i increases rapidly after the release of Ca2+ stores in the endoplasmic reticulum (ER). This is followed by a more sustained response secondary to Ca2+ entry through plasmalemmal channels (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 7Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 8Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 9Putney Jr., J.W. Bird G.S. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (486) Google Scholar, 10Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 11Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar). The initial release of cytosolic Ca2+ occurs following the heterotrimeric G protein-coupled receptor-mediated generation of inositol 1,4,5-trisphosphate (IP3), which in turn binds to inositol 1,4,5-trisphosphate receptor (IP3R) on ER, and signals Ca2+ release from ER stores. Depletion of ER Ca2+ induces activation of store-operated channels (SOC), resulting in Ca2+ entry and replenishment of ER stores (8Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 11Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (767) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar). Molecular cloning and functional expression studies showed that the Drosophila transient receptor potential channel (TRPC) family of proteins are prominent plasmalemmal Ca2+ channels in non-excitable cells (10Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (767) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 14Putney Jr., J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (358) Google Scholar). These channels are activated in response to stimulation of G protein-coupled receptors (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 10Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (767) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 15Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14669-14671Crossref PubMed Scopus (139) Google Scholar, 16Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). TRPC-1, -2, -4, and -5 are likely candidates for endogenous SOCs because they are activated by Ca2+ store depletion (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (767) Google Scholar, 14Putney Jr., J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (358) Google Scholar, 17Berridge M.J. Lipp P. Bootman M.D. Science. 2000; 287: 1604-1605Crossref PubMed Scopus (164) Google Scholar). TRPC1 activates Ca2+ entry upon store depletion in a variety of cell types including endothelial cells (12Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (767) Google Scholar, 18Wu S. Sangerman J. Li M. Brough G.H. Goodman S.R. Stevens T. J. Cell Biol. 2001; 154: 1225-1233Crossref PubMed Scopus (66) Google Scholar, 19Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 20Moore T.M. Brough G.H. Babal P. Kelly J.J. Li M. Stevens T. Am. J. Physiol. 1998; 275: L574-L582Crossref PubMed Google Scholar, 21Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Crossref PubMed Scopus (154) Google Scholar, 22Rosado J.A. Brownlow S.L. Sage S.O. J. Biol. Chem. 2002; 277: 42157-42163Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Despite their involvement in the mechanism of Ca2+ entry, the signals by which store depletion activate TRPC1-induced Ca2+ entry are unclear. It is believed that interaction of IP3R with TRPC is required for activation of store depletion-induced Ca2+ entry (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 7Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 8Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 16Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 23Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 24Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 25Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (563) Google Scholar). Interaction may be in the form of chemical or conformation coupling. Both models help to explain activation of Ca2+ entry through TRP channels (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 7Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 8Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 16Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In the chemical-coupling model, Ca2+ store depletion induces release of diffusible messenger(s) from ER that activate SOC (26Parekh A.B. Terlau H. Stuhmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (320) Google Scholar, 27Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (789) Google Scholar, 28Su Z. Csutora P. Hunton D. Shoemaker R.L. Marchase R.B. Blalock J.E. Am. J. Physiol. 2001; 280: C1284-C1292Crossref PubMed Google Scholar, 29Xie Q. Zhang Y. Zhai C. Bonanno J.A. J. Biol. Chem. 2002; 277: 16559-16566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); however, identity of these mediator(s) is not known. In the conformational coupling model, store depletion causes a IP3R conformational change that enables it to interact with TRPC, thereby resulting in channel opening (7Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 8Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 13Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 16Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). If a change in IP3R conformation is the sole requirement for its coupling to TRPC and its activation, Ca2+ entry through these channels should be essentially complete upon store depletion. However, patch clamp and whole cell fluorescence studies showed that complete activation of Ca2+ entry through SOC was achieved several tens to hundreds seconds after Ca2+ store depletion (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 23Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 30Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1294) Google Scholar, 31Hoth M. Penner R. J. Physiol. 1993; 465: 359-386Crossref PubMed Scopus (662) Google Scholar, 32Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar, 33McDonald T.V. Premack B.A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar, 34Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar). Furthermore, this model fails to explain Ca2+ entry by TRPC1 as these channels have been shown to be localized within intracellular membranes (35Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar). These inconsistencies suggest that coupling involves other events such as translocation and docking of IP3R and TRPC1 at the plasma membrane (1Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (339) Google Scholar, 23Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 24Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 34Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar). Thus, it is possible that proteins capable of trafficking IP3R and TRPC1 to the membrane induce association of components of the complex and trigger Ca2+ entry. The monomeric GTP-binding proteins regulating SOC activation (24Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 36Bird G.S. Putney Jr., J.W. J. Biol. Chem. 1993; 268: 21486-21488Abstract Full Text PDF PubMed Google Scholar, 37Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 38Rosado J.A. Sage S.O. Biochem. J. 2000; 347: 183-192Crossref PubMed Scopus (92) Google Scholar, 39Fernando K.C. Gregory R.B. Katsis F. Kemp B.E. Barritt G.J. Biochem. J. 1997; 328: 463-471Crossref PubMed Scopus (23) Google Scholar) may be important in signaling the interaction of IP3R and TRPC1, and thus in activation of Ca2+ entry. Activation of monomeric Rho family GTP-binding proteins, Rho, Rac, and Cdc42, depends on the GTP/GDP exchange cycle (40Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar, 41Ridley A.J. Traffic. 2001; 2: 303-310Crossref PubMed Scopus (234) Google Scholar, 42Hall A. Science. 1998; 280: 2074-2075Crossref PubMed Scopus (166) Google Scholar). These proteins can traffic from cytosol to plasma membrane on activation and they also regulate vesicle trafficking (40Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar, 41Ridley A.J. Traffic. 2001; 2: 303-310Crossref PubMed Scopus (234) Google Scholar, 42Hall A. Science. 1998; 280: 2074-2075Crossref PubMed Scopus (166) Google Scholar). We and others have shown that thrombin rapidly induces activation of RhoA (but not Rac or Cdc42) in endothelial cells (43van Nieuw Amerongen G.P. Draijer R. Vermeer M.A. van Hinsbergh V.W. Circ. Res. 1998; 83: 1115-1123Crossref PubMed Google Scholar, 44van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar, 45Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). 2P. Kouklis, unpublished observations. In the present study, we addressed the possibility that RhoA induces the interaction of IP3R and TRPC1 required for activation of store depletion-induced Ca2+ entry. We observed that RhoA associated with IP3R and TRPC1 at the plasma membrane after thrombin stimulation of endothelial cells. Plasma membrane translocation of IP3R and TRPC1 and store depletion-induced Ca2+ entry were dependent on Rho because inhibition of Rho activation prevented these responses. We also showed that Rho-activated Ca2+ entry has an important functional consequence in mediating the thrombin-induced increase in transendothelial permeability. Materials—Human α-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Human pulmonary arterial endothelial cells (HPAEC) and endothelial growth medium were obtained from Clonetics (San Diego, CA). Human microvessel endothelial cells (HMEC), a human dermal microvascular endothelial cell line, were obtained from Dr. Edwin W. Ades (National Center for Infectious Diseases, Center for Disease Control, Atlanta, GA). LipofectAMINE, Opti-MEM I, trypsin, Hanks' balanced salt solution (HBSS), and pD-SRed1-N1 (dsREd) plasmid cDNAs were obtained from Invitrogen. Superfect transfection reagent was obtained from Qiagen Inc. (Valencia, CA). Anti-RhoA polyclonal and monoclonal antibodies and IgG were from Santa Cruz Biotechnology (San Diego, CA), anti-IP3 receptor polyclonal antibody and 3-deoxy-3-fluoro-d-myoinositol 1,4,5-trisphosphate were from Calbiochem (La Jolla, CA), and TRPC1 polyclonal antibody and blocking peptide was purchased from Alamonos Lab (Jerusalem, Israel) or Sigma. Green fluorescent protein (pGREEN LANTERN-1, GFP) was purchased from Clontech (Palo Alto, CA). Alexa-phalloidin, Alexa-bound secondary Abs, Fluo3-AM, Fura2-AM, and DAPI were purchased from Molecular Probes (Eugene, OR). C3 transferase was obtained from Cytoskeleton Inc. (Denver, CO). Endothelial Cell Culture—HPAEC were cultured in endothelial growth medium supplemented with 10% FBS. HMEC were grown in endothelial basal medium MCDB-131 supplemented with 10% FBS. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air until they formed confluent monolayer. Cells from each primary flask were detached with 0.05% trypsin containing 0.02% EDTA, resuspended in fresh culture medium, and passaged as described below. In all experiments, HPAEC between passages 5 and 8 were used. Cell Transfection—C3 transferase was introduced into endothelial monolayer by transfecting C3 using LipofectAMINE (46Borbiev T. Nurmukhambetova S. Liu F. Verin A.D. Garcia J.G. Anal. Biochem. 2000; 285: 260-264Crossref PubMed Scopus (22) Google Scholar). After rinsing the monolayer with Opti-MEM I, cells were incubated with Opti-MEM I medium containing 3.5 μg/ml LipofectAMINE for 45 min. C3 transferase (2–4 μg/ml) was then added to this medium and cells were allowed to incubate for 6–8 h after which media was replaced with 10% FBS, endothelial growth medium and cells were used for experiments the following day. The EGFP vector containing wild type RhoA was a generous gift from Dr. M. Philips (New York University School of Medicine, NY), pCMV5 vector and vector containing dominant-negative (N19dn) Rho mutant were provided by Dr. T. Kozasa (University of Illinois at Chicago, IL). Transfection was performed on 50–70% confluent HPAEC grown on 4-well Lab-Tek chambers or 24-well plates using Superfect reagent following the supplier's protocol. The efficiency of transfection in HPAEC ranged from 10 to 20%. We also determined whether Rho regulates Ca2+ entry by modulating the activity of TRPC1. We overexpressed TRPC1 by 3-fold in HMEC as in these cells a high efficiency of transfection can be obtained. Briefly, HMEC grown to 50% confluence were incubated with LipofectAMINE-TRPC1 cDNA or LipofectAMINE-pcDNA3.1 vector complexes for 4 h. LipofectAMINE-DNA complexes were made by incubating 4 μg of LipofectAMINE with 0.5 μg of plasmid DNA in 0.2 ml of Opti-MEM I for 45 min at 22 °C. LipofectAMINE-DNA complexes were diluted with 0.8 ml of Opti-MEM before being added to HMEC, pre-washed 2 times with Opti-MEM I, for 4 h. To end the transfection procedure, 2 ml of MCDB 131-medium supplemented with 10% FBS was added to each well for up to 16 h after which they were fed again with fresh 10% FBS-MCDB medium and allowed to grow further till confluence (47Ellis C.A. Malik A.B. Gilchrist A. Hamm H. Sandoval R. Voyno-Yasenetskaya T. Tiruppathi C. J. Biol. Chem. 1999; 274: 13718-13727Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Cells overexpressing TRPC1 were then treated without or with C3 to determine the role of Rho in modulating TRPC1-induced Ca2+ entry. Transendothelial Resistance Measurement—The time course of endothelial cell retraction in real time, a measure of increased endothelial permeability, was determined as described (48Tiruppathi C. Malik A.B. Del Vecchio P.J. Keese C.R. Giaever I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7919-7923Crossref PubMed Scopus (379) Google Scholar). HPAE cells (200,000 cells) grown to confluence on gelatin-coated small gold electrode (4.9 × 10–4 cm2) were left untreated or treated with C3, as reported previously (45Mehta D. Rahman A. Malik A.B. J. Biol. Chem. 2001; 276: 22614-22620Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Cells were then stimulated with thrombin to measure changes in electrical resistance of endothelial monolayer in real time. The small electrode and larger counter electrode were connected to a phase-sensitive lock-in amplifier. A constant current of 1 μA was supplied by a 1-V, 4000-Hz alternating current connected serially to 1 mΩ resistor between the small electrode and the larger counter electrode. The voltage between the small electrode and large electrode was monitored by lock-in amplifier, stored, and processed on a computer. Data are presented as change in resistive (in-phase) portion of impedance normalized to its initial value at time. [Ca2 + ]i Measurement—Increase in [Ca2+] i was measured using the Ca2+-sensitive fluorescent dyes fura-2AM or fluo3-AM. For loading cells with fura-2AM, cells grown on 25-mm coverslips were incubated with 3 μm fura for 15 min at 37 °C. Cells were then washed 2 times with HBSS and imaged using an Attoflor Ratio Vision digital fluorescence microscopy system (Atto Instruments, Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and a F-Fluar ×40, 1.3 NA oil immersion objective. Regions of interest in individual cells were marked and excited at 334 and 380 nm with emission at 520 nm. The 334/380 excitation ratio that increased as a function of [Ca2+] i was captured at 5-s intervals. For loading cells with fluo-3AM, cells were grown on 4-well Lab-Tek chambers and incubated with 3 μm fluo-3AM for 15 min at 37 °C. Cells were then washed 2× with HBSS and viewed using a LSM510 Zeiss confocal microscope with 63X 1.2 NA water immersion objective. A series of time lapse confocal images were acquired at 12.5-s intervals following thrombin stimulation using 488 and 568 nm excitation laser lines for fluo3 and dsRed fluorescence, respectively. We used the Zeiss Multitrack imaging configuration to acquire these images independent of each other to avoid cross-talk between the two fluorescent indicators. Patch Clamping of Endothelial Cells—Patch clamp in a whole cell configuration was performed on HMEC attached to a coverslip. Patch electrodes made from 1.5-mm borosilicate glass tubing without filament (Narishige, Japan) had a resistance typically between 3 and 6 mΩ when filled. Cell membrane and pipette capacitative transients were subtracted from the records by the amplifier circuitry before sampling. Voltages were not compensated for liquid junction potentials. Membrane currents were measured with an EPC-7 amplifier in conjunction with pClamp 8.1 software and a Digidata 1322 A/D converter (Axon Instruments, Foster City, CA). The currents were filtered at 2 kHz (low-pass bessel filter) and sampled at an interval of 10 ms. Coverslips with cells were perfused at a rate of ∼2 ml/min, whereas the bath solution was continuously removed by a vacuum line. Complete solution changes were achieved within 10 s. The standard extracellular solution contained (mm) 135 sodium glutamate, 1 MgCl2, 4 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 (NaOH). The pipette was filled with (mm) 135 N-methyl-d-aspartic acid glutamate, 10 CsCl, 10 BAPTA, 1 MgCl2, 1 ATP, 10 HEPES, pH 7.2 (CsOH). After formation of the giga-ohm seal the patch was ruptured by negative suction and the whole cell patch configuration was achieved. SOC currents were then measured at a holding potential of –50 mV by depleting the store with 30 μm 3-deoxy-3-fluoro-d-myoinositol-IP3 (non-metabolizable IP3) included in the pipette solution. All experiments were performed at room temperature. Co-immunoprecipitation of Rho, IP3R, and TRPC1—HPAEC grown in 100-mm dishes were serum starved followed by quick washing in ice-cold phosphate-buffered saline. Cells were then lysed in buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 0.25 mm EDTA, pH 8.0, 1% deoxycholic acid, 0.5% Nonidet P-40, 0.1% SDS, 1 mm NaF, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml each of leupeptin, aprotinin, and pepstatin A. The lysate was scraped and centrifuged at 4 °C at 14,000 × g for 10 min. Cell lysate containing an equal amount of protein was then incubated with rabbit IgG or incubated with anti-rabbit polyclonal Rho, IP3 receptor, or TRPC1 Abs for 3–4 h followed by addition of protein A-agarose beads overnight at 4 °C. Beads were collected by centrifugation, washed 3 times with lysis buffer without detergents after which proteins were eluted from the beads by boiling the samples suspended in Laemmli sample buffer. Each sample was then electrophoresed on 10 or 4–20% linear gradient SDS-PAGE gels, transferred to nitrocellulose for Western blotting with IP3 receptor, TRPC1, or Rho Abs. Specificity of the TRPC1 Ab as described recently (22Rosado J.A. Brownlow S.L. Sage S.O. J. Biol. Chem. 2002; 277: 42157-42163Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) was confirmed by using peptide immunogen as a negative control. Measurement of Rho Activity—pGEX-2T containing Rhotekin-Rho binding domain was provided by Dr. M. A. Schwartz (Scripps Research Institute, La Jolla, CA). Bacterial expressed GST-Rhotekin Rho binding domain protein (GST-RBD) was purified from isopropyl-1-thio-β-d-galactopyranozide (1 mm)-induced DH5α cells previously transformed with the appropriate plasmid as described (49Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). Confluent HPAE cells grown in 100-mm dishes were stimulated for the indicated times with 50 nm thrombin. Cells were then quickly washed with ice-cold Tris-buffered saline and lysed in buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, 10 mm MgCl2, 10 μg/ml each of aprotinin and leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 14,000 × g at 4 °C for 2 min and equal volumes of cell lysates were incubated with GST-RBD beads (15 μg) at 4 °C for 1 h. The beads were washed three times with wash buffer (50 mm Tris, pH 7.4, 1% Triton X-100, 150 mm NaCl, 10 mm MgCl2, 10 μg/ml each of aprotinin and leupeptin, and 0.1 mm phenylmethylsulfonyl fluoride), and bound Rho was eluted by boiling each sample in Laemmli sample buffer. Eluted samples from beads and total cell lysate were then electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose, blocked with 5% nonfat milk, and analyzed by Western blotting using a polyclonal anti-RhoA, anti-IP3R, or anti-TRPC1 Abs. In addition, cell lysate from each sample was Western blotted with anti-RhoA Ab to confirm equal protein loading in each lane. Immunofluorescence Studies—Cells were fixed for 15 min with 2% paraformaldehyde in HBSS containing 10 mm HEPES buffer, pH 7.4, at room tempe
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