Hydrogen Peroxide Elicits Constriction of Skeletal Muscle Arterioles by Activating the Arachidonic Acid Pathway

<div><p>Aims</p><p>The molecular mechanisms of the vasoconstrictor responses evoked by hydrogen peroxide (H₂O₂) have not been clearly elucidated in skeletal muscle arterioles.</p><p>Methods and Results</p><p>Changes in diameter of isolated, cannulated and pressurized gracilis muscle arterioles (GAs) of Wistar-Kyoto rats were determined under various test conditions. H₂O₂ (10–100 µM) evoked concentration-dependent constrictions in the GAs, which were inhibited by endothelium removal, or by antagonists of phospholipase A (PLA; 100 µM 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid), protein kinase C (PKC; 10 µM chelerythrine), phospholipase C (PLC; 10 µM U-73122), or Src family tyrosine kinase (Src kinase; 1 µM Src Inhibitor-1). Antagonists of thromboxane A2 (TXA2; 1 µM SQ-29548) or the non-specific cyclooxygenase (COX) inhibitor indomethacin (10 µM) converted constrictions to dilations. The COX-1 inhibitor (SC-560, 1 µM) demonstrated a greater reduction in constriction and conversion to dilation than that of COX-2 (celecoxib, 3 µM). H₂O₂ did not elicit significant changes in arteriolar Ca²⁺ levels measured with Fura-2.</p><p>Conclusions</p><p>These data suggest that H₂O₂ activates the endothelial Src kinase/PLC/PKC/PLA pathway, ultimately leading to the synthesis and release of TXA2 by COX-1, thereby increasing the Ca²⁺ sensitivity of the vascular smooth muscle cells and eliciting constriction in rat skeletal muscle arterioles.</p></div

Endothelial mechanisms of H₂O₂-evoked vasoconstriction of skeletal muscle arterioles.

<p>Arteriolar diameter was recorded in response to H₂O₂ without pretreatment (control, as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103858#pone-0103858-g001" target="_blank">Fig. 1A</a>, closed symbols) or after test incubations (open symbols) for at least 15 min in the presence of PLA inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (100 µM, n = 5 arterioles from 5 different animals, id:130±11 µm; panel A), or in the presence of PKC inhibitor chelerythrine (10 µM, n = 5 arterioles from 5 different animals, id: 164±11 µm; panel B), or in the presence of PLC inhibitor U-73122 (10 µM, n = 4 arterioles from 4 different animals, id: 126±10 µm; panel C), or in the presence of Src kinase inhibitor Src inhibitor-1 (5 µM, n = 5 arterioles from 5 different animals, id: 143±12 µm; panel D). Asterisks denote significant differences from the control.</p

H₂O₂-induced vasoconstriction is mediated by TXA2.

<p>The role of TXA2 receptors was tested by comparing H₂O₂-induced vascular responses under control conditions (closed symbols) with those in the presence of TXA2 receptor antagonist SQ-29548 (1 µM, n = 10 arterioles from 10 different animals, 15-min preincubation) in skeletal muscle arterioles (panel A, open symbols; id: 133±7 µm, asterisks denote significant differences from the control) and in coronary arterioles (panel B, open symbols; id: 108±12 µm). Panel C: The presence of functional TXA2 receptors was verified by the application of TXA2 receptor agonist U46619 (0.1 nM–10 µM) in skeletal muscle (closed symbols; id: 189±7 µm, n = 5 arterioles from 5 different animals) and coronary arterioles (open symbols; id: 119±12 µm, n = 5 arterioles from 5 different animals). Asterisks denote significant differences from the initial diameter.</p

H₂O₂-induced vasoconstrictions are mediated by the endothelium in skeletal muscle arterioles.

<p>H₂O₂ concentration-response relationships were determined (as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103858#pone-0103858-g001" target="_blank">Fig. 1A</a>) in intact (control, closed symbols, n = 6 from 6 different animals) and endothelium-denuded arterioles (id: 131±10 µm, open symbols, n = 5 arterioles from 5 different animals). The asterisk denotes a significant difference from the control.</p

Proposed molecular mechanisms of H₂O₂-evoked vasoconstriction, based on the present study.

<p>H₂O₂ may induce both vasodilation and vasoconstriction, depending on the applied H₂O₂ concentration, vessel type, species and experimental protocol (<i>e.g.</i> exposure time). Our data imply that H₂O₂ elicits vasoconstriction by activating Src kinase, which activates the phospholipase C (PLC), protein kinase (PKC), phospholipase A (PLA) and cyclooxygenase (COX) pathway, leading to the production of thromboxane A2 (TXA2), which increases the Ca²⁺ sensitivity of the vascular smooth muscle in skeletal muscle arterioles of the rat (DAG: diacylglycerol).</p

H₂O₂ increases the Ca²⁺ sensitivity of force production in vascular smooth muscle cells.

<p>The changes in intracellular Ca²⁺ levels and arteriolar diameters were studied in skeletal muscle arterioles under control conditions (panel A; n = 5 arterioles from 3 different animals), or after treatment with norepinephrine (panel B; n = 5 arterioles from 3 different animals), or by addition of the TXA2 receptor agonist U46619 (0.1 nM–10 µM; panel C; n = 5 arterioles from 4 different animals). Experiments were also performed in the presence of H₂O₂ together with Src kinase inhibitor, (Src inhibitor-1, 5 µM n = 4 arterioles from 3 different animals, 20-min preincubation; panel D). Asterisks denote significant differences from the initial values.</p

H₂O₂-induced vasoconstriction is mediated by COX-1.

<p>Arteriolar constrictions (control, as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103858#pone-0103858-g001" target="_blank">Fig. 1A</a>, closed symbols) were prevented in the presence of the non-specific COX inhibitor indomethacin (10 µM, n = 5 arterioles from 4 different animals, preincubation for 30 min, id: 111±3 µm, open symbols; panel A). Panel B: The roles of COX isoforms in H₂O₂-evoked responses were studied by comparing vascular diameters in the absence of COX inhibitors (dotted line) with those in the presence of COX-1 inhibitor SC-560 (1 µM, n = 5 arterioles from 3 different animals, id: 113±14 µm; open squares) or with COX-2 inhibitor celecoxib (3 µM, n = 4 arterioles from 4 different animals, id: 146±13 µm; open triangles). Asterisks denote significant differences from the control.</p

Effects of H₂O₂ on arterioles isolated from skeletal muscle and heart.

<p>H₂O₂ (1 µM–10 mM) was added to isolated, cannulated, skeletal muscle (initial diameter (id: 191±17 µm, n = 6 arterioles from 6 different animals) or coronary arterioles (id: 110±18 µm, n = 7 arterioles from 7 different animals) with intact endothelium. The arteriolar diameter was recorded and concentration-response (cumulative application) relationships were determined (panel A). Changes in relative arteriolar diameter are shown. Relative diameter changes during vasodilations were expressed as percentages of the difference between the maximum passive diameter (maximum dilation: 100%, determined in the absence of extracellular Ca²⁺) and initial diameter with positive values, while during constrictions they were expressed relative to the initial diameter (illustrated at 0% on the y axis) with negative values. Asterisks denote significant differences from the initial values. The kinetics of H₂O₂-evoked responses was studied in isolated skeletal muscle arterioles (panel B; means±SEM with solid and dashed lines, respectively). The effects of the indicated concentrations of H₂O₂ were recorded for 600 s in the continuous presence of H₂O₂ (n = 3–5 arterioles at each concentration from 11 different animals). The positions of maximum constrictions and dilations are illustrated by arrows.</p

Modeling cellular fitness to study cytotoxic effects.

<p>Calculation of cellular fitness by assuming a single toxic compound, X. Cytoplasmic concentration of the drug ([X_c]) was calculated using the time course simulation described in Methods. <i>Critical concentration</i> of X_c ([X_c,crit], dashed yellow line) was defined as the threshold concentration, which must be exceeded to cause cellular damage. <i>Chemical load</i> is defined as a nonlinear function of the [X_c]/[X_c,crit] ratio (magenta curve). The cell is assumed to have a constant <i>Regeneration capacity</i> (dashed black line). When <i>Chemical load</i> exceeds <i>Regeneration capacity (t0 < t < t1)</i>, the cell undergoes damage. Cellular damage is represented by the <i>Damage</i> variable (red curve), which has nonpositive values proportional to the light red shaded area. When <i>Chemical load</i> is below <i>Regeneration capacity</i> and <i>Fitness</i> is below of its maximal value (<i>t1 < t < t2</i>), the cell undergoes regeneration. Regeneration is represented by the <i>Regeneration</i> variable (green curve), which has nonnegative values proportional to the green shaded area. When <i>Chemical load</i> is below <i>Regeneration capacity</i> but <i>Fitness</i> is maximal (<i>t2 < t</i>), nor damage, neither regeneration occurs. <i>Fitness</i> (blue curve) is calculated by adding the (scaled) nonnegative values of <i>Regeneration</i> and the (scaled) nonpositive values of <i>Damage</i> to the maximal value of <i>Fitness</i>. See text for details.</p

Simplified wiring diagram of the chemoimmune network model.

<p>Modeled interactions of a single xenobiotic (X) with Phase 0-III effector enzymes and regulators. Solid arrows represent transport through membranes or biochemical reactions. Dashed arrows denote regulation including multi-step transcriptional and translational regulation (gray) and more direct interaction (black), such as binding of a drug to nuclear receptors. ABC₀ and ABC_III symbolize general Phase 0 and Phase III efflux transporters, respectively. CYP and GST represent a Phase I oxidase (a member of the cytochrome P450 superfamily) and a Phase II GSH transferase, respectively. NR symbolizes a general xenobiotic nuclear receptor, while Nrf2 denotes a specific transcription factor. (GST, ABC_III and Nrf2 are duplicated to increase clarity of the figure. Regulatory arrows are not duplicated.) Letters ‘c’ and ‘e’ indicate cytoplasmic and extracellular localization, respectively. X’_c is the CYP-oxidized cytoplasmic metabolite of X_c. X”_c is the glutathione-conjugated form of X’_c. X’_bc represents reactive species produced by normal cell metabolism. X’_bc is metabolized by the same pathway as X’_c. Negative feedback loops are X_c → NR → CYP —| X_c, X’_c (and X’_bc) → Nrf2 → GST —| X’_c (and X’_bc), X_c → NR → ABC₀ —| X_c, X_c → NR → Nrf2 → ABC₀ —| X_c, where → denotes activation and —| denotes inhibition. Feedforward loops are X_c → NR → GST —| X’_c, X_c → NR → ABC_III —| X”_c (‘direct’ regulation) and X_c → NR → Nrf2 → GST —| X’_c, X_c → NR → Nrf2 → ABC_III —| X”_c (‘indirect’ regulation). For the complete wiring diagram with all details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115533#pone.0115533.s001" target="_blank">S1 Fig.</a></p