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. 2016 Jun 1;310(11):F1197-205.
doi: 10.1152/ajprenal.00575.2015. Epub 2016 Apr 6.

Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles

Lingli Li  1 , En Yin Lai  2 , Anton Wellstein  3 , William J Welch  1 , Christopher S Wilcox  4
Affiliations

Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles

Lingli Li et al. Am J Physiol Renal Physiol. .

Abstract

Myogenic contraction is the principal component of renal autoregulation that protects the kidney from hypertensive barotrauma. Contractions are initiated by a rise in perfusion pressure that signals a reduction in membrane potential (Em) of vascular smooth muscle cells to activate voltage-operated Ca(2+) channels. Since ROS have variable effects on myogenic tone, we investigated the hypothesis that superoxide (O2 (·-)) and H2O2 differentially impact myogenic contractions. The myogenic contractions of mouse isolated and perfused single afferent arterioles were assessed from changes in luminal diameter with increasing perfusion pressure (40-80 mmHg). O2 (·-), H2O2, and Em were assessed by fluorescence microscopy during incubation with paraquat to increase O2 (·-) or with H2O2 Paraquat enhanced O2 (·-) generation and myogenic contractions (-42 ± 4% vs. -19 ± 4%, P < 0.005) that were blocked by SOD but not by catalase and signaled via PKC. In contrast, H2O2 inhibited the effects of paraquat and reduced myogenic contractions (-10 ± 1% vs. -19 ± 2%, P < 0.005) and signaled via PKG. O2 (·-) activated Ca(2+)-activated Cl(-) channels that reduced Em, whereas H2O2 activated Ca(2+)-activated and voltage-gated K(+) channels that increased Em Blockade of voltage-operated Ca(2+) channels prevented the enhanced myogenic contractions with paraquat without preventing the reduction in Em Myogenic contractions were independent of the endothelium and largely independent of nitric oxide. We conclude that O2 (·-) and H2O2 activate different signaling pathways in vascular smooth muscle cells linked to discreet membrane channels with opposite effects on Em and voltage-operated Ca(2+) channels and therefore have opposite effects on myogenic contractions.

Keywords: chloride channels; potassium channels; protein kinase C; protein kinase G; reactive oxygen species; renal autoregulation.

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Figures

Fig. 1.
Fig. 1.
Paraquat (PQ) enhances afferent arteriolar contractions during increases in perfusion pressure (PP) from 40 to 80 mmHg, whereas H2O2 has opposite effects. A: effects of PQ (10−6 mol/l) alone or with pegalated (PEG)-SOD (200 U/ml) or with PEG-catalase (1,000 U/ml) on myogenic contractions. B: effects of H2O2 (10−5 mol/l) alone or with PEG-SOD (200 U/ml) or with PEG-catalase (1,000 U/ml) on myogenic contractions. C: effects of PQ (10−6 mol/l) alone or with H2O2 (10−5 mol/l). D: changes in the ethidium-to-dihydroethidium (E:DHE) fluorescence ratio of afferent arterioles with PQ (10−6 mol/l) alone or with PEG-SOD (200 U/ml) or with PEG-catalase (1,000 U/ml). E: changes in 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence of afferent arterioles by H2O2 (10−5 mol/l) alone or with PEG-SOD (200 U/ml) or with PEG-catalase (1,000 U/ml). F: changes in H2DCFDA fluorescence of afferent arterioles by PQ (10−6 mol/l) alone or with PEG-SOD (200 U/ml) or with PEG-catalase (1,000 U/ml). MR, myogenic response. *P < 0.05, **P < 0.01, and ***P < 0.005 compared with vehicle; ††P < 0.01 compared with PQ or H2O2 alone.
Fig. 2.
Fig. 2.
PKC mediates the effects of PQ on myogenic contractions and membrane depolarization and PKG mediates the effects of H2O2. A and C: effects of PQ (10−6 mol/l) and/or Gö6983 (3 × 10−6 mol/l). B and D: effects of H2O2 (10−5 mol/l) and/or Rp-cGMPs (3 × 10−5 mol/l). DiBAC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol. ***P < 0.005 compared with vehicle; ††P < 0.01 and †††P < 0.005 compared with PQ or H2O2 alone.
Fig. 3.
Fig. 3.
Membrane potential (Em) is reduced by activation of Ca2+-activated Cl− channels (CaCCs) with PP or PQ. DiBAC4(3) fluorescence (A) was increased with PP and PQ (10−6 mol/l) (implying depolarization), but this was blunted by inhibition of CaCCs with CaCCinh-A01 (10−5 mol/l). Luminal diameter (B) was reduced by PP, and this was enhanced by PQ but blunted by CaCCinh-A01. *P < 0.05 and ***P < 0.005 compared with vehicle; †††P < 0.005 compared with PQ alone.
Fig. 4.
Fig. 4.
H2O2 activates small-conductance Ca2+-activated K+ (SKCa) channels, large-conductance Ca2+-activated K+ (BKCa) channels, and voltage-activated K+ channel 7 (Kv7) to blunt myogenic contractions. The reduction in luminal diameter with PP was inhibited by H2O2, but the effect of H2O2 was lessened by blockade of SKCa and BKCa channels with apamin (10−7 mol/l) and charybdotoxin (10−8 mol/l) (A) or blockade of Kv7 by linopirdine (10−5 mol/l; B) and was prevented by blockade of all channels by a combination of all three blockers (C). The increase in Em by H2O2 was prevented by blockade of K+ channels. DiBAC4(3) fluorescence was decreased with H2O2 (10−5 mol/l) (implying hyperpolarization), but this effect of H2O2 was lessened by apamin (10−7 mol/l) and charybdotoxin (10−8 mol/l; D) or linopirdine (10−5 mol/l; E) and prevented by a combination of all three blockers (F). *P < 0.05, **P < 0.01, and ***P < 0.005 compared with vehicle; †P < 0.05, ††P < 0.01, and †††P < 0.005 compared with H2O2 alone; ‡P < 0.05 and ‡‡‡P < 0.005 compared with antagonists alone.
Fig. 5.
Fig. 5.
Changes in Em are upstream from activation of voltage-operated Ca2+ channels. Luminal diameter (A) was reduced by PP and reduced further by PQ (10−6 mol/l), but this was largely prevented by nifedipine (10−6 mol/l). Em (B) was reduced by PP or PQ (10−6 mol/l) (implying depolarization), but these effects were unchanged by nifedipine (10−6 mol/l). ***P < 0.005 compared with vehicle; †††P < 0.005 compared with PQ alone.
Fig. 6.
Fig. 6.
Changes in DiBAC4(3) fluorescence by PP alone or with saponin (0.125 mg/ml; A), changes in luminal diameter by by PP alone or with saponin (0.125 mg/ml; B), by PQ (10−6 mol/l) alone or with Nω-nitro-l-arginine methyl ester (l-NAME; 10−6 mol/l; C), or by H2O2 (10−5 mol/l) alone or with l-NAME (10−6 mol/l; D) during increasing PP from 40 to 80 mmHg. *P < 0.05 and **P < 0.01 compared with vehicle (by two-way ANOVA); ††P < 0.01.
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