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Abstract
Caveolae position CaV3.2 (T‐type Ca2+ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large‐conductance Ca2+‐activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Cav3.2 channel inhibition by Ni2+ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Cav3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Cav1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2‐RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 µM), but insensitive to CaV1.2 and CaV3.2 channel blockade. Our data demonstrate that the VSMC CaV3.2‐RyR axis is down‐regulated by aging. This defective CaV3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs.
Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10−6 mol L−1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10−6 mol L−1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.