Aims Excessive endothelial globotriaosylceramide (Gb3) accumulation is associated with endothelial dysfunction and impaired endothelium-dependent relaxation in Fabry disease. In endothelial cells, K_Ca3.1 channels contribute to endothelium-dependent relaxation. However, the effect of Gb3 on K_Ca3.1 channels and the underlying mechanisms of Gb3-induced dysfunction are unknown. Herein, we hypothesized that Gb3 accumulation induces K_Ca3.1 channel dysfunction and aimed to clarify the underlying mechanisms. Methods and results The animal model of Fabry disease, α-galactosidase A (Gla) knockout mice, displayed age-dependent K_Ca3.1 channel dysfunction. K_Ca3.1 current and the channel expression were significantly reduced in mouse aortic endothelial cells (MAECs) of aged Gla knockout mice, whereas they were not changed in MAECs of wild-type and young Gla knockout mice. In addition, K_Ca3.1 current and the channel expression were concentration-dependently reduced in Gb3-treated MAECs. In both Gb3-treated and aged Gla knockout MAECs, extracellular signal-regulated kinase (ERK) and activator protein-1 (AP-1) were down-regulated and repressor element-1 silencing transcription factor (REST) was up-regulated. Gb3 inhibited class III phosphoinositide 3-kinase and decreased intracellular levels of phosphatidylinositol 3-phosphate [PI(3)P]. In addition, endothelium-dependent relaxation was significantly attenuated in Gb3-treated mouse aortic rings. Conclusion Gb3 accumulation reduces K_Ca3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes K_Ca3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

Objective—: Globotriaosylceramide (Gb3) induces K _Ca 3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces K _Ca 3.1 endocytosis and degradation. Approach and Results—: K _Ca 3.1, especially plasma membrane–localized K _Ca 3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced K _Ca 3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress–inducing agents did not induce K _Ca 3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated K _Ca 3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered K _Ca 3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored K _Ca 3.1 expression, the current, and endothelium-dependent relaxation. Conclusions: —Gb3 accelerates the endocytosis and lysosomal degradation of endothelial K _Ca 3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.

Summary: K_Ca1.1 regulates smooth muscle contractility by modulating membrane potential, and age‐associated changes inK_Ca1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affectK_Ca1.1 expression. Thus, in this study, we examined whether alteredSLcomposition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) andSLlevels in theGSMof mice of varying ages and compared them with those in youngCerS2‐null mice. The levels ofC16‐ andC18‐ceramides, sphinganine, sphingosine, and sphingosine 1‐phosphate were increased, and levels ofC22,C24:1 and C24 ceramides were decreased in theGSMof both aged wild‐type and youngCerS2‐null mice. The alteredSLcomposition upregulatedK_Ca1.1 and increasedK_Ca1.1 currents, while no change was observed inK_Ca1.1 channel activity. The upregulation ofK_Ca1.1 impaired intracellular Ca²⁺mobilization and decreased phosphorylated myosin light chain levels, causingGSMcontractile dysfunction. Additionally, phosphoinositide 3‐kinase, protein kinaseC_ζ, c‐JunN‐terminal kinases, and nuclear factor kappa‐Bwere found to be involved inK_Ca1.1 upregulation. Our findings suggest that age‐associated changes inSLcomposition orCerS2 ablation upregulateK_Ca1.1 via the phosphoinositide 3‐kinase/protein kinaseC_ζ/c‐JunN‐terminal kinases/nuclear factor kappa‐B‐mediated pathway and impair Ca²⁺mobilization, which thereby induces the contractile dysfunction ofGSM.CerS2‐null mice exhibited similar effects to aged wild‐type mice; therefore,CerS2‐null mouse models may be utilized for investigating the pathogenesis of aging‐associated motility disorders.

Aims: Altered redox state has been related to the development of normal pregnancy (NP) and preeclampsia (PE). Endothelial K _Ca 2.3 and K _Ca 3.1 (K _Ca s) play an important role in vasodilation, and K _Ca s levels are affected by oxidative stress. We investigated the mechanisms of oxidative stress-mediated K _Ca s expression modulation during NP and PE. Results: Human uterine microvascular endothelial cells were incubated in serum from normal nonpregnant women ( n = 13) and women with NP ( n = 24) or PE ( n = 15), or in vascular endothelial growth factor (VEGF), oxidized low-density lipoprotein (ox-LDL), progesterone, or estradiol-17β (E ₂ )-containing medium for 24 h. NP serum elevated H ₂ O ₂ levels via reducing catalase and glutathione peroxidase 1 levels, thereby enhancing K _Ca s levels via a H ₂ O ₂ /fyn/extracellular signal-regulated kinase (ERK)-mediated pathway. VEGF enhanced H ₂ O ₂ and K _Ca s levels and K _Ca 3.1 currents. K _Ca s were upregulated and K _Ca s activation-induced endothelium-dependent relaxation (EDR) was augmented in vessels from pregnant mice and rats. Whereas PE serum, ox-LDL, progesterone, or soluble fms-like tyrosine kinase 1 (sFlt-1) elevated superoxide levels via elevating NADPH oxidase 2 (NOX2) and NOX4 levels and reducing superoxide dismutase (SOD) 1 levels, thereby downregulating K _Ca s. sFlt-1 inhibited EDR. PE serum- or progesterone-induced alterations in levels of K _Ca s were reversed by polyethylene glycol-SOD, NOX inhibition, or E ₂ . Innovation and Conclusions: This is the first study of how endothelial K _Ca s levels are modulated during NP and PE. K _Ca s were upregulated by soluble serum factors such as VEGF via H ₂ O ₂ generation in NP, and were downregulated by serum factors such as progesterone and ox-LDL via superoxide generation in PE, which may contribute to hemodynamic adaptations in NP or to the development of PE.

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium-dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial KCa 3.1, which contributes to EDR, is upregulated by H2 O2 . We investigated whether KCa 3.1 upregulation compensates for diminished EDR to NO during aging-related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1-phosphate were increased in aged wild-type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild-type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age-matched wild-type mice. Increased H2 O2 levels induced Fyn and extracellular signal-regulated kinases (ERKs) phosphorylation and KCa 3.1 upregulation. Catalase/GPX1 double knockout (catalase(-/-) /GPX1(-/-) ) upregulated KCa 3.1 in MAECs. NO production was decreased in aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) MAECs. However, KCa 3.1 activation-induced, N(G) -nitro-l-arginine-, and indomethacin-resistant EDR was increased without a change in acetylcholine-induced EDR in aortic rings from aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1-phosphate induced similar changes in levels of the antioxidant enzymes and upregulated KCa 3.1. Our findings suggest that, during aging-related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H2 O2 and thereby upregulate KCa 3.1 expression and function via a H2 O2 /Fyn-mediated pathway. Altogether, enhanced KCa 3.1 activity may compensate for decreased NO signaling during vascular aging.