Effects of Peroxynitrite on Vascular Smooth Muscle Cell BKCa Channels

Abstract

Oxidative stress impairs vascular function in the brain and has been linked to neurodegenerative diseases, such as Alzheimer’s Disease (AD). Peroxynitrite is an oxidant produced in the cerebral vasculature via the reaction between superoxide and nitric oxide. A major target of oxidants is the thiol-containing residues of ion channels in the vascular smooth muscle cells, leading to impairment in their function. One of such targets is Ca2+-activated, large-conductance K+ channel (BKCa), which plays a vital role in maintaining cerebral vascular control and is impaired in the 5x-FAD mouse model of AD. However, the direct effects of peroxynitrite on BKCa function remain unknown. We hypothesized that peroxynitrite will decrease BKCa currents in a reversible manner in wild-type and 5x-FAD mice. Cerebral arteries of 5-6 month-old C57BL/6J and 5x-FAD mice were isolated and underwent a mild enzymatic digestion to obtain single smooth muscle cells for patch-clamp electrophysiology experiments. Data are means ± SEM, all curves analyzed by 2-way ANOVA with Sidak correction for multiple comparisons, bar graphs by Student’s t-test. Using the inside-out patch clamp electrophysiology configuration, BKCa channel activity was assessed through single channel open probability (Po) after chemical intervention or stepwise increases in membrane voltage. We observed that peroxynitrite (10 µM) did not alter BKCa voltage sensitivity (PoVehicle = 0.0102 ± 0.0001 vs. PoPeroxynitrite = 0.0105 ± 0.0058, 60 mV, 1 µM Ca2+, P=0.7413). However, peroxynitrite significantly decreased BKCa sensitivity to Ca2+ (in 10 μM Ca2+: PoVehicle = 0.0082 ± 0.0033 vs. PoPeroxynitrite = 0.0013 ± 0.0004, 40 mV, 10 µM Ca2+, P=0.0312). Partial recovery of channel activity was achieved via reducing agent DTT (10 µM, PoPeroxynitrite = 0.0080 ± 0.0020 vs PoPeroxynitrite+DTT = 0.0169 ± 0.0062, 60 mV, 10 µM Ca2+, P=0.0197). Next, we assessed BKCa function in cerebral artery smooth muscle cells from male 5x-FAD mice, and observed that single channel activity is similar to WT littermates (PoWild-type =0.0430 ± 0.0143 vs Po5x-FAD = 0.0258 ± 0.0058, P = 0.217, 60 mV, 10 µM Ca2+), despite a reported vascular BKCa dysfunction (% Vasoconstriction following iberiotoxin application: Wild-type = 6.600 ± 1.300 vs 5x-FAD = 1.551 ± 0.4645, P=0.0051). Further, in male 5x-FAD mice, channel activity was not improved with DTT treatment (PoVehicle=0.0203 ± 0.0051 vs PoDTT=0.0204 ± 0.0062, 60 mV, 10 µM Ca2+, P=0.9865). We conclude that peroxynitrite reversibly oxidizes BKCa channels by decreasing the channel’s Ca2+-sensitivity, independent of voltage, and that reversible oxidation or other channel modifications are not the root cause of BKCa impairment in male 5x-FAD mice