Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone

Yanfeng Ding, Ali Winters, Min Ding, Sarabeth Graham, Irina Akopova, Shmuel Muallem, Yanxia Wang, Jeong Hee Hong, Zygmunt Gryczynski, Shaohua Yang, Lutz Birnbaumer, Rong Ma

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Abstract

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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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, H 2 O 2 itself evoked Ca 2+ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H 2 O 2 also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H 2 O 2 stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H 2 O 2 triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.

Original languageEnglish
Pages (from-to)31799-31809
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number36
DOIs
StatePublished - 9 Sep 2011

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Vasoconstrictor Agents
Vascular Smooth Muscle
Knockout Mice
Muscle Cells
Smooth Muscle Myocytes
Blood Vessels
Muscle
Arginine Vasopressin
Reactive Oxygen Species
Chemical activation
Constriction
Catalase
Proteins
Membranes
NADPH Oxidase
Induced currents
Phenylephrine
Thoracic Aorta
Angiotensin II
Small Interfering RNA

Cite this

Ding, Yanfeng ; Winters, Ali ; Ding, Min ; Graham, Sarabeth ; Akopova, Irina ; Muallem, Shmuel ; Wang, Yanxia ; Hong, Jeong Hee ; Gryczynski, Zygmunt ; Yang, Shaohua ; Birnbaumer, Lutz ; Ma, Rong. / Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 36. pp. 31799-31809.
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abstract = "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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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, H 2 O 2 itself evoked Ca 2+ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H 2 O 2 also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H 2 O 2 stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H 2 O 2 triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.",
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Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone. / Ding, Yanfeng; Winters, Ali; Ding, Min; Graham, Sarabeth; Akopova, Irina; Muallem, Shmuel; Wang, Yanxia; Hong, Jeong Hee; Gryczynski, Zygmunt; Yang, Shaohua; Birnbaumer, Lutz; Ma, Rong.

In: Journal of Biological Chemistry, Vol. 286, No. 36, 09.09.2011, p. 31799-31809.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone

AU - Ding, Yanfeng

AU - Winters, Ali

AU - Ding, Min

AU - Graham, Sarabeth

AU - Akopova, Irina

AU - Muallem, Shmuel

AU - Wang, Yanxia

AU - Hong, Jeong Hee

AU - Gryczynski, Zygmunt

AU - Yang, Shaohua

AU - Birnbaumer, Lutz

AU - Ma, Rong

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