Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca2+: Comparison of Endothelial Cells from Large-Sized and Small-Sized Arteries

We compared the Ca2+ responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H2O2) caused an increase in cytosolic Ca2+ levels ([Ca2+]i) in both cell types. The [Ca2+]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP3) receptors. Removal of Ca2+ from the bath also decreased the [Ca2+]i rises in response to H2O2. In addition, treatment of endothelial cells with H2O2 reduced the [Ca2+]i responses to subsequent challenge of ATP. The decreased [Ca2+]i responses to ATP were resulted from a pre-depletion of intracellular Ca2+ stores by H2O2. Interestingly, we also found that Ca2+ store depletion was more sensitive to H2O2 treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca2+]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H2O2 but not by hydroxyl radical. H2O2 contribution in HX-XO-induced [Ca2+]i rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H2O2 could induce store Ca2+ release via phospholipase C-IP3 pathway in endothelial cells. Resultant emptying of intracellular Ca2+ stores contributed to the reduced [Ca2+]i responses to subsequent ATP challenge. The [Ca2+]i responses were more sensitive to H2O2 in endothelial cells of small-sized arteries than those of large-sized arteries

TRPC5 channels participate in pressure-sensing in aortic baroreceptors

published_or_final_versio

Plasma Membrane Mechanical Stress Activates TRPC5 Channels

<div><p>Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca²⁺ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between −20 and −40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.</p></div

Effect of extracellular Ca²⁺ on H₂O₂-induced [Ca²⁺]_i rises in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces of [Ca²⁺]_i rises in response to 5 mM H₂O₂ in the primary cultured aortic ECs (A) and MAECs (B) that were bathed in N-PSS (1 mM Ca²⁺), 0.5Ca²⁺-PSS (0.5 mM Ca²⁺) or 0Ca²⁺-PSS (nominal Ca²⁺-free). Fluorescence intensity before H₂O₂ application was normalized to 1 as F0. <b>C and D</b>. Summary of the maximal [Ca²⁺]_i changes to H₂O₂ as expressed in F1/F0. Mean±SEM of 4 to 8 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to N-PSS.</p

Depleting effect of H₂O₂ on store Ca²⁺ content in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces showing the [Ca²⁺]_i rises in response to 30 µM ATP. The cells were pre-treated with or without 1 mM H₂O₂ for 30 min in N-PSS. Control had no H₂O₂ treatment. Cells were transferred to 0Ca²⁺-PSS and then challenged by ATP. Fluorescence intensity before ATP application was normalized to 1 as F0. <b>C and D</b>. Summary of data showing the effect of H₂O₂ (500 µM or 1 mM as indicated) on ATP-induced maximal [Ca²⁺]_i rises in aortic ECs and MAECs as expressed in F1/F0. <b>E and F</b>. Summary of data showing the effect of H₂O₂ treatment on store Ca²⁺ content as determined by Mag-fluo4 fluorescence in aortic ECs and MAECs. The cells were treated with or without 500 µM H₂O₂ for 30 min. Mean±SEM of 3 to 17 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to control.</p

Effect of H₂O₂ pre-treatment on ATP-induced [Ca²⁺]_i rises in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces showing the [Ca²⁺]_i rises in response to 30 µM ATP. The cells were pre-treated with or without H₂O₂ (500 µM or 1 mM as indicated) in N-PSS for 30 min, followed by ATP challenge. Control had no H₂O₂ treatment. Fluorescence intensity before ATP application was normalized to 1 as F0. <b>C and D</b>. Summary of data showing the ATP-induced maximal [Ca²⁺]_i rises as in A and B, expressed in F1/F0. <b>E and F.</b> Summary of data showing the ATP-induced maximal [Ca²⁺]_i rises after the cells were treated with 500 µM H₂O₂ for different period of time in N-PSS. Mean±SEM of 3 to 7 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to control.</p

H₂O₂-induced IP₃ production in a H₂O₂ concentration-dependent manner in aortic ECs and MAECs.

<p>The intracellular IP₃ production was measured in aortic ECs and MAECs after different concentration of H₂O₂ challenge (500 µM, 2 mM and 5 mM), according to the protocols described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025432#s4" target="_blank">Methods</a>. Mean±SEM of 3 independent experiments.</p

Effect of U73122 on H₂O₂-induced [Ca²⁺]_i rises in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces showing the [Ca²⁺]_i rises in response to 5 mM H₂O₂ with or without U73122 or U73343. The cells were pre-treated with or without 10 µM U73122 or 10 µM U73343 for 30 min in N-PSS. Control had no U73122 and U73343. Fluorescence intensity before H₂O₂ application was normalized to 1 as F0. <b>C and D</b>. Summary of data showing the effect of 10 µM U73122 or 10 µM U73343 on H₂O₂-induced maximal [Ca²⁺]_i rises in aortic ECs (C) and MAECs (D) as expressed in F1/F0. Mean ± SEM of 3 to 4 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to U73343.</p

Effect of catalase and DMSO on H₂O₂-induced [Ca²⁺]_i rises in aortic ECs and MAECs.

<p><b>A and B</b>. Representative traces of H₂O₂-induced [Ca²⁺]_i rises in the presence or absence of catalase or DMSO in N-PSS. 2000 U/ml catalase or 2% DMSO was added 30 min prior to the addition of H₂O₂ (5 mM). Fluorescence intensity before application of H₂O₂ was normalized to 1 as F0. <b>C and D</b>. Summary of data showing the effect of 2000 U/ml catalase and 2% DMSO treatment on H₂O₂-induced maximal [Ca²⁺]_i rises in aortic ECs (C) and MAECs (D) as expressed in F1/F0. Mean±SEM of 3 to 4 independent experiments (10 to 15 cells per experiment). *, <i>P</i><0.05 as compared to control.</p