Abstract 070: Superoxide And Hydrogen Peroxide Differentially Modulate Mouse Afferent Arteriolar Myogenic Responses Via Protein Kinase C And Protein Kinase G And Their Coupling To Membrane Potential

Abstract

Background: Although reactive oxygen species (ROS) mediate normal myogenic responses (MRs) of afferent arterioles (Affs), oxidative stress contribute to impaired MRs in chronic kidney disease (CKD) and thereby to barotrauma. The mechanisms underlying these apparently conflicting reports are unclear. MRs depend on reductions in membrane potential (Em) that activate voltage-operated calcium channels to raise intracellular [Ca 2+ ]. Therefore, we tested the hypothesis that MRs are increased by superoxide (O 2 .- ) and reduced by hydrogen peroxide (H 2 O 2 ) through differential signaling via protein kinase C (PKC) - and protein kinase G (PKG) - dependent changes in Em. Methods: MRs were assessed from changes in luminal diameter of mouse individual isolated perfused Affs (n = 5-6 per group) with change in perfusion pressure (PP) from 40 to 80 mmHg. The effects of O 2 .- and H 2 O 2 on the MR were assessed after incubation with 1 μM paraquat (PQ increases O 2 .- ) or incubation with 10 μM H 2 O 2 . O 2 .- with PP was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence, and H 2 O 2 from PEG-catalase (CAT) inhibitable H 2 DCFDA fluorescence and Em from DiBAC 4 (3) fluorescence. Results: 1. PQ increased O 2 .- with PP by 32 ± 4% (P<0.001) and MR by 140 ± 33% (P<0.01) (prevented by PEG-SOD), but H 2 O 2 reduced MR by 50 ± 2% (P<0.0001) (prevented by PEG-CAT). 2. The PP-induced fall in Em was increased by PQ (PQ -19 ± 2% vs vehicle -4 ± 1%, P<0.001), but was reversed by H 2 O 2 (H 2 O 2 +16 ± 1% vs vehicle -4 ± 1%, P<0.001). 3. Inhibition of PKC with 3 μM of Gö6983 blocked the reduction in Em with PQ (PQ -19 ± 2% vs PQ+ Gö6983 -2 ± 2%, P<0.01), while inhibition of PKG with 30 μM of Rp-8-Br-PET-cGMPS blocked the increase in Em with H 2 O 2 (H 2 O 2 +16 ± 2% vs H 2 O 2 +Rp-8-Br-PET-cGMPS -2 ± 1%, P<0.05). Conclusions: O 2 .- signals via PKC to depolarize the Em and thereby to activate the MR whereas H 2 O 2 signals via PKG to hyperpolarize the Em and thereby to inhibit the MR. Thus, O 2 .- and H 2 O 2 signaling via specific G-protein-coupled changes in Em may explain the differential effects of ROS on MRs.