Localized TRPA1 channel Ca2+ signals stimulated by reactive oxygen species promote cerebral artery dilation

Abstract

Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.

Fig. 1. TRPA1 colocalizes with NOX2 in the endothelium of cerebral arteries but not in other vascular beds

(A) RT-PCR for TRPA1 (A1) and eNOS (+) mRNA in rat coronary (C), renal (R), mesenteric (M), and cerebral (CA) arteries. NTC, no complementary DNA (cDNA) template control (n = 2 rats). (B) Top: Immunolabeling for TRPA1 in coronary, renal, mesenteric, and cerebral arteries; scale bar, 10 μm. Autofluorescence of the IEL is green. TRPA1 protein (red) was only detected in the endothelium of cerebral arteries and was present within myoendothelial projections (arrows, inset; scale bar, 5 μm). Bottom: Immunolabeling was not detected when the primary antibody was omitted. n = 3 rats. (C) RT-PCR for TRPA1 mRNA in whole human cerebral arteries (CA) and smooth muscle cells isolated from human cerebral arteries (SMC) (n = 3 independent biological replicates). (D) Western blot for TRPA1 in human cerebral arteries (CA) (n = 2 independent biological replicates). (E) RT-PCR detection of human TRPA1 (A1) and eNOS (+) mRNA in primary coronary (C), renal (R), dermal (D), and cerebral artery (CA) endothelial cells. TRPA1 mRNA was detected only in CA. Representative of three independent experiments. (F) Top: Cerebral arteries immunolabeled for NOX1 (left), NOX2 (middle), or NOX4 (right) (red); scale bars, 10 μm. NOX2 and NOX4 are more abundant within myoendothelial projections compared with NOX1 (arrows, insets; bars, 5 μm). Endothelial cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue), and IEL autofluorescence is green. Bottom: No primary antibody control. n = 3 rats per group. (G) PLA experiments for TRPA1:NOX4 (top) and TRPA1:NOX2 (bottom) in cerebral artery endothelial cells. Puncta corresponding to positive PLA results are red, cellular autofluorescence is green, and DAPI-stained nuclei are blue. Puncta density is summarized at the right (n = 4 cells per group, 3 rats); *P ≤ 0.05 versus TRPA1:NOX4. (H) PLA for TRPA1:NOX4 (top) and TRPA1:NOX2 (bottom). TRPA1:NOX2 puncta within IEL fenestrations are indicated by arrows. *, an example magnified and shown in cross section in the insets. Puncta density is summarized at the right (n = 8 to 14 vessels per group, 3 rats); *P ≤ 0.05 compared with TRPA1:NOX4.

Fig. 2. ROS stimulate TRPA1 sparklets in cerebral artery endothelial cells

(A) Time-lapse image of an AITC-induced TRPA1 sparklet recorded from a cerebral artery endothelial cell; scale bar, 8 μm. (B) AITC induces a concentration-dependent increase in TRPA1 sparklet frequency in cerebral artery endothelial cells (n = 5 to 52 cells per concentration, 4 independent cell isolations). (C) Summary data showing that HC-030031 inhibits AITC-induced increases in TRPA1 sparklet frequency in cerebral artery endothelial cells (n = 10 to 24 cells, 3 independent cell isolations); *P ≤ 0.05 compared to control at baseline. (D) Active TRPA1 sparklet sites per cell before and after administration of AITC (n = 10 cells, 5 rats). (E) Amplitude (left), duration (middle), and spatial spread (right) histograms for TRPA1 sparklets (n = 762 total events, 43 independent experiments). (F) Representative recordings of change in fluorescence (F/F₀) within an ROI on primary cerebral artery endothelial cells stimulated by AITC. Dotted lines indicate the opening of one, two, or three TRPA1 channels. (G) Time-lapse image of a TRPA1 sparklet stimulated by NADPH; scale bar, 8 μm. (H) The NOX substrate NADPH induced a concentration-dependent increase in TRPA1 sparklet frequency (n = 9 to 22 cells per concentration, 4 independent cell isolations). (I) Summary data indicating that HC-030031 inhibits NADPH-induced increases in TRPA1 sparklet frequency (n = 12 to 28 cells per group, 3 independent cell isolations); *P ≤ 0.05 compared with baseline, control.

Fig. 3. ROS generated by NOX dilate cerebral arteries by activating TRPA1

(A) Representative recordings of the intraluminal diameter of an intact, pressurized cerebral artery over time. Introduction of NADPH to the bathing solution induced vasodilation, which was nearly abolished by the TRPA1 blocker HC-030031. (B) NADPH-induced vasodilation is concentration-dependent (n = 3 vessels per concentration, 3 rats). (C) Summary data indicating that NADPH-induced dilation is attenuated by HC-030031 (n = 5 vessels, 3 rats); *P ≤ 0.05 compared to control. (D and E) Representative recordings (D) and summary data (E) indicating that the NO synthase inhibitor L-NNA and the cyclooxygenase inhibitor indomethacin do not affect NADPH-induced vasodilation (n = 3 vessels, 3 rats). (F and G) Representative recordings (F) and summary data (G) showingthat NADPH-induced vasodilation (left) is inhibited when the IK channel blocker TRAM34 is present in the lumen (right) (n = 5 vessels, 3 rats); *P ≤ 0.05 compared with control. (H) Representative recordings of smooth muscle cell membrane potential (E_m) in a pressurized cerebral artery. Smooth musclecellswerehyperpolarizedbyNADPH.NADPH-inducedhyperpolarization was blocked by HC-030031. (I) Summary data (n = 4 vessels, 4 rats); *P ≤ 0.05 compared with vehicle, control; #P ≤ 0.05 compared with vehicle, NADPH.

Fig. 4. ROS-derived lipid peroxidation metabolites stimulate TRPA1 sparklets and dilate cerebral arteries

(A) Proposed pathway and pharmacological interventions for activation of TRPA1 by NOX-generated ROS metabolites. (B) NADPH-induced increases in TRPA1 sparklet frequency in endothelial cells at baseline (n = 27 to 60 cells per group, 5 independent cell isolations) were attenuated by inhibition of NOX with apocynin (n = 8 to 10 cells per group, 3 independent cell isolations) and gp91ds-tat (n = 7 to 9 cells per group, 3 independent cell isolations) compared to vehicle and a scrambled peptide (scr. gp91ds-tat), respectively; H₂O₂ degradation with extracellular catalase (n = 9 to 11 cells per group, 3 independent cell isolations); and iron chelation with deferoxamine (n = 8 to 12 cells per group, 3 independent cell isolations); *P ≤ 0.05 compared with baseline, control. (C to F) Representative traces and summary data indicating inhibition of NADPH-induced vasodilation by apocynin (n = 5 vessels, 3 rats) (C), gp91ds-tat (n = 5 vessels, 3 rats) (D), catalase (n = 5 vessels, 3 rats) (E), and deferoxamine (n = 5 vessels, 3 rats) (F); *P ≤ 0.05 compared with vehicle control or scr. gp91ds-tat.

Fig. 5. Lipid peroxidation products activate TRPA1 sparklets in endothelial cells and dilate cerebral arteries

(A) Cerebral artery mounted en face and immunolabeled for 4-HNE–modified proteins (red, top). 4-HNE labeling was present in perinuclear regions and within IEL fenestrations (arrows, inset; scale bar, 5 μm). Immunolabeling was not detected in the absence of primary antibody (bottom); scale bar, 10 μm. Endothelial cell nuclei are stained with DAPI (blue), and autofluorescence of the IEL is green (n = 3 rats). (B) Time-lapse image of a 4-HNE–induced TRPA1 sparklet; scale bar, 8 μm. (C) 4-HNE–induced increases in TRPA1 sparklet frequency are concentration-dependent (n = 9 to 11 cells per concentration, 3 rats). (D) 4-HNE–induced increases in TRPA1 sparklet frequency are abolished by the TRPA1 blocker HC-030031 (n = 19 to 31 cells, 4 rats); *P ≤ 0.05 compared with baseline, control. (E) Representative recording of 4-HNE–induced vasodilation of a pressurized cerebral artery. 4-HNE–induced dilation (left) was inhibited by HC-030031 (right). (F) Concentration-response curve for 4-HNE–induced dilation in cerebral arteries (n = 3 to 5 vessels per group, 5 rats). (G) Summary data indicating that 4-HNE–induced dilation is abolished by HC-030031 (n = 5 vessels, 4 rats); *P ≤ 0.05 compared with control.

Fig. 6. ROS-derived lipid peroxidation products fail to dilate cerebral arteries from endothelial cell–specific TRPA1-knockout mice

(A) Western blot for TRPA1 protein (138 kD) in cerebral arteries from control and eTRPA1^−/− mice (n = 3 mice per group). (B) TRPA1 immunolabeling in the cerebral endothelium (top) and DRG neurons (bottom) from control and eTRPA1^−/− mice (n = 3 mice per group). (C) Summary data indicating the effect of AITC on the frequency of sparklets recorded from endothelial cells isolated from control compared with eTRPA1^−/− mice (n = 20 cells per group, 3 independent cell isolations per group). (D and E) 4-HNE (D) and NADPH (E) dilate cerebral arteries from control but not eTRPA1^−/− mice (n = 5 vessels per group, 3 mice per group); *P ≤ 0.05 compared with control. (F) Proposed signaling pathway: In endothelial cells (EC), NOX generates O₂⁻, which is rapidly dismutated to H₂O₂. In the presence of iron, H₂O₂ undergoes the Fenton reaction to yield OH•. Oxidation of membrane lipids by OH• generates lipid peroxidation products (LPP), which activate TRPA1 sparklets through binary-coupled TRPA1 channels. Ca²⁺ domains created by TRPA1 sparklets stimulate outward K⁺ currents through IK channels to hyperpolarize the EC plasma membrane (E_m). Electrotonic spread of EC hyperpolarization through myoendothelial gap junctions (MEGJ) causes smooth muscle cell (SMC) hyperpolarization and vasodilation.