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,Shao-Hua Yang,Lutz Birnbaumer,Rong Ma

doi:10.1074/jbc.m111.248344

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.

Highlights

The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by reactive oxygen species (ROS) was a physiological mechanism for regulating vascular tone by vasoconstrictors
Ca2ϩ influx from the extracellular compartment may participate in the initial response, these results suggest that the TRPC6 channel may not be involved in arginine vasopressin (AVP)-induced Ca2ϩ release and the initial stage of Ca2ϩ entry
Live movies showing a real time We have shown that H2O2 stimulated Ca2ϩ entry, which change in evanescent field fluorescence (EFF) of TRPC6 and EGFP were presented in sup- was mediated by TRPC6 in A7r5 cells (Fig. 6, A and B)

Summary

EXPERIMENTAL PROCEDURES

The KO and WT mice were euthanized with ketamine (95 mg/kg)/xylazine (5 mg/kg), and the thoracic aortas were removed for vessel contraction assay or isolation of VSMCs. Cell Culture and Transient Transfection—Both human embryonic kidney 293 (HEK293T) and A7r5 cells were purchased from ATCC (Manassas, VA) and were cultured as described previously (11). A7r5 cells, grown on a coverslip (22 ϫ 22 mm), were loaded with fura-2 by incubation for ϳ50 min at room temperature in the dark in physiological saline solution containing 2 ␮M acetoxymethyl ester of fura-2 (fura-2/AM) and 0.018 g/dl pluronic F-127 (Molecular Probes, Eugene, OR). A7r5 cells grown in 60-mm plates with various treatments as indicated in Fig. 3C were washed three times with cold Hanks’ balanced salt solution and loaded with 15 ␮M DCF diacetate (Molecular Probes, Eugene, OR) for 10 min at 37 °C in the dark. Statistical analysis was performed using SigmaStat (Jandel Scientific, San Rafael, CA)

RESULTS

DISCUSSION

Birnbaumer and Rong Ma