Inducible Nitric Oxide Synthase Underlies BKCa Channel Dysfunction and Microvascular Deficits in Female 5x-FAD Mice

Approximately 30-50% of cases of Alzheimer’s Disease (AD) overlap with vascular dementia. Previous work from our laboratory showed that cerebral arteries of 5x-FAD mice are hyper-contractile due to decreased activity of large conductance, calcium-activated potassium channels (BKCa) in smooth muscle cells. In female mice, this was due to exacerbated S-nitrosylation of the channel, likely due to expression of inducible nitric oxide synthase (iNOS, gene Nos2). In the pro-oxidative environment of the AD brain, iNOS-dependent uncontrolled nitric oxide (NO) production may result in accumulation of peroxynitrite, a strong oxidant. We hypothesize that increased iNOS expression leads to BKCa dysfunction due to nitro-oxidation of the channel, resulting in cerebral vascular hypercontractility in female 5x-FAD mice. Eight-month-old female 5x-FAD, 5x-FAD:Nos2+/-, and wild-type (WT) mice were used. Pial arteries were isolated and either canulated for pressure myography or underwent a mild enzymatic digestion to isolate smooth muscle cells. BKCa channel activity, assessed via single-channel and perforated patch clamp electrophysiology, was lower in 5x-FAD than in WT, but was recovered in 5x-FAD:Nos2+/- mice. Dithiothreitol (DTT, 10 μM), a potent reducing agent, restored BKCa channel activity in 5x-FAD mice, without additive effects in the 5x-FAD: Nos2+/- mice, suggesting that removal of one copy of the Nos2 gene partially reduces nitro-oxidation of the channel. Although spontaneous myogenic tone of pressurized pial arteries was not different between 5x-FAD and 5x-FAD: Nos2+/- mice, vasoconstriction after BKCa blockade with iberiotoxin (300 nM), was larger in 5x-FAD: Nos2+/- mice, suggesting improved BKCa basal activity. DTT incubation increased pial artery constriction to iberiotoxin in 5x-FAD but had no additive effects in 5x-FAD: Nos2+/- mice. In summary, our data show iNOS expression underlies BKCa dysfunction in cerebral arteries of female 5x-FAD mice. Our findings are the first to identify iNOS as a central mechanism of microvascular deficits in AD mice and a potential therapeutic target to improve cerebral vascular function in AD Funding Sources: National Institutes of Health (RO1 AG073230) and the Alzheimer’s Association (AARGD-21-850835) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.