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Reactive Oxygen Species-mediated TRPC6 Protein Activation in Vascular Myocytes, a Mechanism for Vasoconstrictor-regulated Vascular Tone*

  • Yanfeng Ding
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Ali Winters
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Min Ding
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Sarabeth Graham
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Irina Akopova
    Affiliations
    Departments of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Shmuel Muallem
    Affiliations
    Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
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  • Yanxia Wang
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Jeong Hee Hong
    Affiliations
    Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
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  • Zygmunt Gryczynski
    Affiliations
    Departments of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Shao-Hua Yang
    Affiliations
    Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Lutz Birnbaumer
    Affiliations
    Transmembrane Signaling Group, National Institutes of Health, Research Triangle Park, North Carolina 27709
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  • Rong Ma
    Correspondence
    To whom correspondence should be addressed: 3500 Camp Bowie Blvd., Dept. of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, TX 76107. Tel.: 817-735-2516; Fax: 817-735-5084;
    Affiliations
    Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants 5 RO1 DK079968-01A2 from NIDDK (to R. M.) and 5R21CA149897-02 (to Z. G.). This work was also supported by Grant-in-aid 09GRNT2260926 from American Heart Association South Central Affiliate (to R. M.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1, movie, and additional references.
Open AccessPublished:July 15, 2011DOI:https://doi.org/10.1074/jbc.M111.248344
      Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca2+ entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca2+ entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca2+ entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H2O2 itself evoked Ca2+ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H2O2 also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H2O2 stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H2O2 triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.

      Introduction

      Canonical transient receptor potential 6 (TRPC6) is a nonselective cation channel and participates in a diverse array of cellular functions by regulating intracellular Ca2+ signaling (
      • Venkatachalam K.
      • Montell C.
      ). In particular, TRPC6 channels are highly expressed in vascular smooth muscle cells (VSMCs)
      The abbreviations used are: VSMC
      vascular smooth muscle cell
      ROS
      reactive oxygen species
      DPI
      diphenyleneiodonium
      AVP
      arginine vasopressin
      EFF
      evanescent field fluorescence
      EGFP
      enhanced GFP
      PE
      phenylephrine
      DCF
      2′,7′-dichlorodihydrofluorescein
      TIRFM
      total internal fluorescence reflection microscopy
      Ang
      angiotensin.
      and play a key role in regulating myogenic tone in vascular tissues (
      • Inoue R.
      • Okada T.
      • Onoue H.
      • Hara Y.
      • Shimizu S.
      • Naitoh S.
      • Ito Y.
      • Mori Y.
      ,
      • Dietrich A.
      • Kalwa H.
      • Fuchs B.
      • Grimminger F.
      • Weissmann N.
      • Gudermann T.
      ,
      • Saleh S.N.
      • Albert A.P.
      • Peppiatt C.M.
      • Large W.A.
      ). Multiple mechanisms are involved in TRPC6 channel activation and regulation. These include membrane receptor activation (
      • Estacion M.
      • Li S.
      • Sinkins W.G.
      • Gosling M.
      • Bahra P.
      • Poll C.
      • Westwick J.
      • Schilling W.P.
      ), Ca2+ store depletion (
      • Mizuno N.
      • Kitayama S.
      • Saishin Y.
      • Shimada S.
      • Morita K.
      • Mitsuhata C.
      • Kurihara H.
      • Dohi T.
      ), stretch (
      • Reiser J.
      • Polu K.R.
      • Möller C.C.
      • Kenlan P.
      • Altintas M.M.
      • Wei C.
      • Faul C.
      • Herbert S.
      • Villegas I.
      • Avila-Casado C.
      • McGee M.
      • Sugimoto H.
      • Brown D.
      • Kalluri R.
      • Mundel P.
      • Smith P.L.
      • Clapham D.E.
      • Pollak M.R.
      ,
      • Inoue R.
      • Jensen L.J.
      • Jian Z.
      • Shi J.
      • Hai L.
      • Lurie A.I.
      • Henriksen F.H.
      • Salomonsson M.
      • Morita H.
      • Kawarabayashi Y.
      • Mori M.
      • Mori Y.
      • Ito Y.
      ), membrane lipids (
      • Hofmann T.
      • Obukhov A.G.
      • Schaefer M.
      • Harteneck C.
      • Gudermann T.
      • Schultz G.
      ), and trafficking (
      • Cayouette S.
      • Lussier M.P.
      • Mathieu E.L.
      • Bousquet S.M.
      • Boulay G.
      ,
      • Graham S.
      • Ding M.
      • Ding Y.
      • Sours-Brothers S.
      • Luchowski R.
      • Gryczynski Z.
      • Yorio T.
      • Ma H.
      • Ma R.
      ). The distinct activation/regulation mechanisms may be tissue/cell type-specific and thus render TRPC6 a specific function in a particular site. For instance, mechanosensitive TRPC6 residing in glomerular podocytes (
      • Reiser J.
      • Polu K.R.
      • Möller C.C.
      • Kenlan P.
      • Altintas M.M.
      • Wei C.
      • Faul C.
      • Herbert S.
      • Villegas I.
      • Avila-Casado C.
      • McGee M.
      • Sugimoto H.
      • Brown D.
      • Kalluri R.
      • Mundel P.
      • Smith P.L.
      • Clapham D.E.
      • Pollak M.R.
      ,
      • Winn M.P.
      • Conlon P.J.
      • Lynn K.L.
      • Farrington M.K.
      • Creazzo T.
      • Hawkins A.F.
      • Daskalakis N.
      • Kwan S.Y.
      • Ebersviller S.
      • Burchette J.L.
      • Pericak-Vance M.A.
      • Howell D.N.
      • Vance J.M.
      • Rosenberg P.B.
      ) and mesangial cells (
      • Sours S.
      • Du J.
      • Chu S.
      • Ding M.
      • Zhou X.J.
      • Ma R.
      ,
      • Graham S.
      • Ding M.
      • Sours-Brothers S.
      • Yorio T.
      • Ma J.X.
      • Ma R.
      ) may regulate hydrostatic pressure-driven ultrafiltration in response to changes in glomerular capillary pressure. Most likely, different mechanisms may exist in the same cell and work together in a synergistic way to regulate the cell function more precisely and efficiently (
      • Inoue R.
      • Jensen L.J.
      • Jian Z.
      • Shi J.
      • Hai L.
      • Lurie A.I.
      • Henriksen F.H.
      • Salomonsson M.
      • Morita H.
      • Kawarabayashi Y.
      • Mori M.
      • Mori Y.
      • Ito Y.
      ). We recently demonstrated that TRPC6 also was a redox-sensitive channel that was activated by H2O2 in a TRPC6-expressing cell line (
      • Graham S.
      • Ding M.
      • Ding Y.
      • Sours-Brothers S.
      • Luchowski R.
      • Gryczynski Z.
      • Yorio T.
      • Ma H.
      • Ma R.
      ). However, the physiological relevance of activation of the channel by reactive oxygen species (ROS) is completely unknown.
      ROS are produced in G protein-coupled receptor-signaling pathway (
      • Lyle A.N.
      • Griendling K.K.
      ,
      • Seshiah P.N.
      • Weber D.S.
      • Rocic P.
      • Valppu L.
      • Taniyama Y.
      • Griendling K.K.
      ), a pathway also linked to TRPC6 channel activation. ROS not only function as an intracellular signaling molecule in a variety of cells but are also associated with many diseases, such as hypertension (
      • Lyle A.N.
      • Griendling K.K.
      ,
      • Lassègue B.
      • Griendling K.K.
      ). In blood vessels, all types of vascular cells can produce ROS that modulate vasoactive agent-induced endothelial cell and myocyte responses (
      • Griendling K.K.
      • Sorescu D.
      • Lassègue B.
      • Ushio-Fukai M.
      ). In VSMCs, ROS play an important role in mediating vasoactive hormone-induced proliferation and hypertrophy (
      • Lyle A.N.
      • Griendling K.K.
      ). With respect to vascular contractile function, many studies have demonstrated that H2O2 evoked constriction in a variety of vascular beds (
      • Ardanaz N.
      • Pagano P.J.
      ). However, the molecular mechanism for ROS-induced vasoconstriction is poorly understood.
      Because both TRPC6 and ROS play an important role in regulation of vascular function, we used VSMCs and freshly isolated blood vessel segments as a model in this study to investigate the physiological significance of activation of the TRPC6 channel by ROS and the underlying mechanism. The findings from this study for the first time suggest that ROS is a physiological intermediator to mediate vasoconstrictor-induced vessel contraction by activating TRPC6 in VSMCs.

      DISCUSSION

      Multiple mechanisms are involved in ROS-induced vessel contraction. In this study we proposed TRPC6 being a novel target of ROS in a physiological regulation of vascular tone. This conclusion is based on several major findings from our in vitro and ex vivo assays as shown. 1) AVP-induced Ca2+ entry was comparably suppressed by knockdown of TRPC6 or NADPH oxidase inhibitors in A7r5 cells (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, A and B). 2) H2O2 could mimic AVP responses in A7r5 cells, i.e. stimulating Ca2+ entry and inward membrane currents, and importantly, the ROS-dependent responses were significantly inhibited by either knockdown of TRPC6 or blocking TRPC6 channel with a specific antibody (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7, A–D). 3) In primary aortic VSMCs, AVP and Ang II evoked robust inward currents and Ca2+ influx, which were significantly inhibited by catalase and DPI, respectively (Fig. 4). Application of H2O2 also caused a similar current response in TRPC6 WT VSMCs but not in TRPC6 KO cells (Fig. 6, E and F). 4) Ex vivo vessel contraction assays showed that catalase significantly suppressed PE-induced aortic contraction (Fig. 5), and consistent with this, H2O2 itself or selective and direct activation of TRPC6 channels by hyperforin reproduced PE responses in aortas from WT but not TRPC6 KO mice (FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7).
      It is generally accepted that diverse pathways activated by ROS are convergent to increase [Ca2+]i, which triggers smooth muscle contraction (
      • Ardanaz N.
      • Pagano P.J.
      ). For instance, ROS can activate voltage-operated Ca2+ channels (
      • Horowitz A.
      • Menice C.B.
      • Laporte R.
      • Morgan K.G.
      ), stimulate Ca2+ release from the internal stores (
      • Favero T.G.
      • Zable A.C.
      • Abramson J.J.
      ), and inhibit Ca2+-ATPase on the sarcoplasmic reticulum (
      • Grover A.K.
      • Samson S.E.
      • Fomin V.P.
      ). TRPC6 is a Ca2+-permeable cation channel (
      • Venkatachalam K.
      • Montell C.
      ). Our findings are fully compatible with the Ca2+-dependent mechanism. Suppression of TRPC6 attenuated and genetic removal of TRPC6 abolished ROS-induced intracellular Ca2+ response, membrane currents, and vessel constriction, suggesting a necessity of TRPC6 for ROS effects in VSMCs. The TRPC6-mediated increase in [Ca2+]i involves multiple mechanisms. These include the following: 1) allowing Na+ to enter the cells, which either depolarizes the membrane and opens voltage-operated Ca2+ channels (
      • Soboloff J.
      • Spassova M.
      • Xu W.
      • He L.P.
      • Cuesta N.
      • Gill D.L.
      ,
      • Welsh D.G.
      • Morielli A.D.
      • Nelson M.T.
      • Brayden J.E.
      ) or activates a reverse mode of Na+-Ca2+ exchangers (
      • Poburko D.
      • Liao C.H.
      • Lemos V.S.
      • Lin E.
      • Maruyama Y.
      • Cole W.C.
      • van Breemen C.
      ); 2) directly conducting Ca2+ influx. Which TRPC6 mechanism underlies the ROS-associated Ca2+ increase observed in the current study is unknown. However, the previous findings that voltage-operated Ca2+ channels contributed to H2O2-induced Ca2+ response (
      • Horowitz A.
      • Menice C.B.
      • Laporte R.
      • Morgan K.G.
      ) suggest that membrane depolarization could be involved. It could explain why there was no difference in vessel contraction by high KCl between WT and TRPC6-deficient vessels (Fig. 7C).
      We further provided evidence that ROS activate TRPC6 in VSMCs by stimulating the translocation of channel vesicles to the plasma membrane. This is supported by correlation of the time course of the vesicle trafficking with the development of membrane current and [Ca2+]i in response to H2O2. Membrane trafficking of TRPC6 occurs after activation of Gq protein-coupled receptors (
      • Cayouette S.
      • Lussier M.P.
      • Mathieu E.L.
      • Bousquet S.M.
      • Boulay G.
      ). Because ROS are also generated in this signaling pathway (
      • Seshiah P.N.
      • Weber D.S.
      • Rocic P.
      • Valppu L.
      • Taniyama Y.
      • Griendling K.K.
      ), we reason that inserting the TRPC6 channel into the plasma membrane by ROS may be a general mechanism in the Gq-coupled receptor-signaling pathway in TRPC6-enriched cells, such as podocytes (
      • Winn M.P.
      • Conlon P.J.
      • Lynn K.L.
      • Farrington M.K.
      • Creazzo T.
      • Hawkins A.F.
      • Daskalakis N.
      • Kwan S.Y.
      • Ebersviller S.
      • Burchette J.L.
      • Pericak-Vance M.A.
      • Howell D.N.
      • Vance J.M.
      • Rosenberg P.B.
      ). How ROS stimulate migration of TRPC6 to the cell surface remains unknown. Several possibilities exist, the first of which is that ROS could directly oxidize TRPC6 protein or membrane proteins or lipids that results in movement and binding of TRPC6 to the plasma membrane. Second, ROS could indirectly alter the phosphorylation-dephosphorylation state of the TRPC6 protein or membrane components that promote physical interactions between TRPC6 and the plasma membrane. Oxidative activations of protein-tyrosine kinase Src (
      • Suzaki Y.
      • Yoshizumi M.
      • Kagami S.
      • Koyama A.H.
      • Taketani Y.
      • Houchi H.
      • Tsuchiya K.
      • Takeda E.
      • Tamaki T.
      ) and oxidative inactivation of protein-tyrosine phosphatases (
      • Rhee S.G.
      • Chang T.S.
      • Bae Y.S.
      • Lee S.R.
      • Kang S.W.
      ) have been described as a downstream mechanism in ROS-dependent cellular responses, particularly in VSMCs (
      • Seshiah P.N.
      • Weber D.S.
      • Rocic P.
      • Valppu L.
      • Taniyama Y.
      • Griendling K.K.
      ). A recent study demonstrated that TRPC6 was activated by tyrosine phosphorylation (
      • Hisatsune C.
      • Kuroda Y.
      • Nakamura K.
      • Inoue T.
      • Nakamura T.
      • Michikawa T.
      • Mizutani A.
      • Mikoshiba K.
      ). However, whether stimulation of trafficking is the sole mechanism in ROS-TRPC6 pathway is unknown. In fact, that blocking vesicle translocation did not completely abolish the H2O2-evoked and TRPC6-mediated Ca2+ entry in A7r5 cells (Figs. 6, A and B, and 8D) indicates an additional nontrafficking mechanism involved.
      We noted that there is a difference in the recovery rate between vasoconstrictor- and H2O2-induced inward currents in native VSMCs. AVP and Ang II currents had a prolonged recovery process (Fig. 4, A and D), and H2O2 currents were more transient (Fig. 6E). This discrepancy may be due to different profiles of changes in intracellular ROS in response to the distinct treatments. With direct application of H2O2, intracellular ROS rose rapidly and was scavenged immediately, resulting in a transient current response. However, when the cells were treated with agonists, ROS were continuously generated as long as these agents were present. Thus, a high level of ROS was sustained, and membrane currents were maintained. Another possibility is that vasoconstrictors activate TRPC6 through a ROS-independent mechanism. Indeed, diacylglycerol is a well known physiological activator of TRPC6 (
      • Hofmann T.
      • Obukhov A.G.
      • Schaefer M.
      • Harteneck C.
      • Gudermann T.
      • Schultz G.
      ).
      It was reported that TRPC3, which has higher constitutive activity, was up-regulated in the aorta of TRPC6 KO mice (
      • Dietrich A.
      • Mederos Y.
      • Schnitzler M.
      • Gollasch M.
      • Gross V.
      • Storch U.
      • Dubrovska G.
      • Obst M.
      • Yildirim E.
      • Salanova B.
      • Kalwa H.
      • Essin K.
      • Pinkenburg O.
      • Luft F.C.
      • Gudermann T.
      • Birnbaumer L.
      ). Thus, it could be argued that the lesser H2O2 effects in the TRPC6-deficient aortic VSMCs and vessels were due to an inhibition to TRPC3. However, studies have shown that TRPC3 itself is not redox-sensitive (
      • Yoshida T.
      • Inoue R.
      • Morii T.
      • Takahashi N.
      • Yamamoto S.
      • Hara Y.
      • Tominaga M.
      • Shimizu S.
      • Sato Y.
      • Mori Y.
      ). We also found that H2O2 did not change membrane currents in TRPC3 overexpressing HEK293 cells (data not shown). Moreover, our preliminary study in mesenteric arteries from TRPC6 KO mice showed no difference in H2O2-stimulated contraction in the presence and absence of Pyr3, a selective TRPC3 channel blocker (
      • Kiyonaka S.
      • Kato K.
      • Nishida M.
      • Mio K.
      • Numaga T.
      • Sawaguchi Y.
      • Yoshida T.
      • Wakamori M.
      • Mori E.
      • Numata T.
      • Ishii M.
      • Takemoto H.
      • Ojida A.
      • Watanabe K.
      • Uemura A.
      • Kurose H.
      • Morii T.
      • Kobayashi T.
      • Sato Y.
      • Sato C.
      • Hamachi I.
      • Mori Y.
      ).
      Both TRPC6 and ROS play an important role in regulating vascular function. In this study we provided evidence to show an interplay between the two molecules, which has not been described before. To our knowledge, this is the first time to report that TRPC6 is a target of ROS in the physiological regulation of vascular tone. TRPC6 channel activation/regulation involves multiple mechanisms such as membrane receptor activation (
      • Estacion M.
      • Li S.
      • Sinkins W.G.
      • Gosling M.
      • Bahra P.
      • Poll C.
      • Westwick J.
      • Schilling W.P.
      ), Ca2+ store depletion (
      • Mizuno N.
      • Kitayama S.
      • Saishin Y.
      • Shimada S.
      • Morita K.
      • Mitsuhata C.
      • Kurihara H.
      • Dohi T.
      ), stretch (
      • Inoue R.
      • Jensen L.J.
      • Jian Z.
      • Shi J.
      • Hai L.
      • Lurie A.I.
      • Henriksen F.H.
      • Salomonsson M.
      • Morita H.
      • Kawarabayashi Y.
      • Mori M.
      • Mori Y.
      • Ito Y.
      ), membrane lipids (
      • Hofmann T.
      • Obukhov A.G.
      • Schaefer M.
      • Harteneck C.
      • Gudermann T.
      • Schultz G.
      ) and trafficking (
      • Cayouette S.
      • Lussier M.P.
      • Mathieu E.L.
      • Bousquet S.M.
      • Boulay G.
      ,
      • Graham S.
      • Ding M.
      • Ding Y.
      • Sours-Brothers S.
      • Luchowski R.
      • Gryczynski Z.
      • Yorio T.
      • Ma H.
      • Ma R.
      ). We now elongated the list by adding ROS. Because both TRPC6 and ROS are also engaged in physiology and pathology of many other tissues and organs in addition to the vascular system, the information from this study may be generalized to all G protein-coupled receptor-signaling pathways and ROS-related diseases.

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