Large conductance, Ca²⁺/voltage-sensitive K⁺ channels (BK channels) are well characterized, but their physiological roles, often determined through pharmacological manipulation, are less clear. Iberiotoxin is considered the “gold standard” antagonist, but cost and membrane-impermeability limit its usefulness. Economical and membrane-permeable alternatives could facilitate the study of BK channels. Thus, we characterized the effect of penitrem A, a tremorigenic mycotoxin, on BK channels and demonstrate its utility for studying vascular function in vitro and in vivo. Whole-cell currents from human embryonic kidney 293 cells transfected with hSlo α or α + β1 were blocked >95% by penitrem A (IC₅₀ 6.4 versus 64.4 nM; p < 0.05). Furthermore, penitrem A inhibited BK channels in inside-out and cell-attached patches, whereas iberiotoxin could not. Inhibitory effects of penitrem A on whole-cell K⁺ currents were equivalent to iberiotoxin in canine coronary smooth muscle cells. As for specificity, penitrem A had no effect on native delayed rectifier K⁺ currents, cloned voltage-dependent Kv1.5 channels, or native ATP-dependent K_ATP current. Penitrem A enhanced the sensitivity to K⁺-induced contraction in canine coronary arteries by 23 ± 5% (p < 0.05) and increased the blood pressure response to phenylephrine in anesthetized mice by 36 ± 11% (p < 0.05). Our data indicate that penitrem A is a useful tool for studying the role of BK channels in vascular function and is practical for cell and tissue (in vitro) studies as well as anesthetized animal (in vivo) experiments.

Objective: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K ⁺ ) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type–Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. Conclusions: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.

Background: Hypercholesterolemia‐induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K⁺(Kir) channels and that Kir2.1 is an upstream mediator of flow‐inducedNOproduction. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐inducedNOproduction and flow‐induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions.Methods and Results: Kir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient (Apoe^−/−) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemicApoe^−/−mice,Kir2.1^+/−/Apoe^−/−exhibit the same bluntedFIVand flow‐inducedNOresponse asApoe^−/−orKir2.1^+/−alone, but while FIV inApoe^−/−mice can be rescued by cholesterol depletion, inKir2.1^+/−/Apoe^−/−mice cholesterol depletion has no effect onFIV. Endothelial‐specific overexpression of Kir2.1 in arteries fromApoe^−/−andKir2.1^+/−/Apoe^−/−mice results in full rescue ofFIVandNOproduction inApoe^−/−mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect.Kir2.1^+/−/Apoe^−/−mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta.Conclusions: We conclude that hypercholesterolemia‐induced reduction inFIVis largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐inducedNOproduction, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

Objective—: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. Approach and Results—: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. Conclusions—: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.