RESUMEN
Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.
Asunto(s)
Potenciales de Acción , Anoctamina-1 , Canales de Potasio de Gran Conductancia Activados por el Calcio , Vasos Linfáticos , Animales , Anoctamina-1/metabolismo , Anoctamina-1/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Ratones , Ratas , Potenciales de Acción/fisiología , Masculino , Vasos Linfáticos/fisiología , Vasos Linfáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/fisiología , Ratas Sprague-Dawley , Femenino , Miocitos del Músculo Liso/fisiología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacosRESUMEN
Endothelial dysfunction, underlying the vascular complications of diabetes and other cardiovascular disorders, may result from uncoupling of endothelial nitric oxide synthase (eNOS) activity due to decreased levels of tetrahydrobiopterin (BH4), a critical co-factor for eNOS. Some clinical trials attempting to deliver exogenous BH4 as a potential therapeutic strategy in vascular disease states have failed due to oxidation of BH4 in the circulation. We sought to develop a means of protecting BH4 from oxidation while delivering it to dysfunctional endothelial cells. Polymeric and solid lipid nanoparticles (NPs) loaded with BH4 were delivered by injection or oral gavage, respectively, to streptozotocin-induced diabetic rats. BH4 was measured in coronary endothelial cells and endothelium-dependent vascular reactivity was assessed in vascular rings. Lymphatic uptake of orally delivered lipid NPs was verified by sampling mesenteric lymph. BH4-loaded polymeric NPs maintained nitric oxide production by cultured endothelial cells under conditions of oxidative stress. BH4-loaded NPs, delivered via injection or ingestion, increased coronary endothelial BH4 concentration and improved endothelium-dependent vasorelaxation in diabetic rats. Pharmacodynamics assessment indicated peak concentration of solid lipid NPs in the systemic bloodstream 6 hours after ingestion, with disappearance noted by 48 hours. These studies support the feasibility of utilizing NPs to deliver BH4 to dysfunctional endothelial cells to increase nitric oxide bioavailability. BH4-loaded NPs could provide an innovative tool to restore redox balance in blood vessels and modulate eNOS-mediated vascular function to reverse or retard vascular disease in diabetes.
Asunto(s)
Biopterinas , Diabetes Mellitus Experimental , Endotelio Vascular , Nanopartículas , Animales , Biopterinas/análogos & derivados , Biopterinas/farmacología , Biopterinas/administración & dosificación , Biopterinas/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Masculino , Nanopartículas/química , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Ratas , Ratas Sprague-Dawley , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismoRESUMEN
BACKGROUND: Coronary artery disease (CAD) is a leading cause of death in women. Epicardial adipose tissue (EAT) secretes cytokines to modulate coronary artery function, and the release of fatty acids from EAT serves as a readily available energy source for cardiomyocytes. However, despite having beneficial functions, excessive amounts of EAT can cause the secretion of proinflammatory molecules that increase the instability of atherosclerotic plaques and contribute to CAD progression. Although exercise mitigates CAD, the mechanisms by which exercise impacts EAT are unknown. The Yucatan pig is an excellent translational model for the effects of exercise on cardiac function. Therefore, we sought to determine if chronic aerobic exercise promotes an anti-inflammatory microenvironment in EAT from female Yucatan pigs. METHODS: Sexually mature, female Yucatan pigs (n = 7 total) were assigned to sedentary (Sed, n = 3) or exercise (Ex, n = 4) treatments, and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk (n = 7 total) and single nucleus transcriptomic sequencing (n = 2 total, 1 per exercise treatment). RESULTS: Based on the bulk transcriptomic analysis, exercise upregulated S100 family, G-protein coupled receptor, and CREB signaling in neurons canonical pathways in EAT. The top networks in EAT affected by exercise as measured by bulk RNA sequencing were SRC kinase family, fibroblast growth factor receptor, Jak-Stat, and vascular endothelial growth factor. Single nucleus transcriptomic analysis revealed that exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT, with fibroblast and macrophage populations predominant in O-Ex EAT and T cell populations predominant in N-Ex EAT. Unlike the findings for exercise alone as a treatment, there were not increased interactions between endothelial and mesenchymal cells in O-Ex EAT. Coronary artery occlusion impacted the most genes in T cells and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. CONCLUSIONS: According to bulk transcriptomics, exercise upregulated pathways and networks related to growth factors and immune cell communication. Based on single nucleus transcriptomics, aerobic exercise increased cell-to-cell interaction amongst immune, mesenchymal, and endothelial cells in female EAT. Yet, exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.