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Normal vascular homeostasis is essential for normal vascular homeostasis, as shown by the following: nitric oxide synthase in 2014. For maximum eNOS production, the conversion of eNOS to specialized plasma membrane invaginations termed caveolae has been suggested. We first investigated the effects of cholesterol reduction in caveolae and hypercholesterolemia is linked to increased NO production, and hypocholesterolemia is associated with elevated NO production. The treatment of BAECs with 30 bcg/mL cholesterol for 24 hours resulted in significant rises in total eNOS protein expression, eNOS associated with caveolae-enriched membranes, and calcium ionophore-stimulated NO production boosted by calcium ionophore-stimulated NO production. In vitro exposure of caveolae-enriched membranes to ROS dissociated caveolin more quickly than eNOS from the membranes. These results show that cholesterol therapy improves eNOS expression, while ROS treatment reduces eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes.
Source link: https://doi.org/10.1161/01.res.85.1.29
Methods and Resultsu2014 Rat tail artery devoid of endothelium was treated with cholesterol acceptor methyl-u03b2-cyclodextrin, and the effects on force and Ca 2+ handling were investigated. The rise in global intracellular free Ca 2+ concentrations in reaction to 5-HT was reduced, as was the emergence of Ca 2+ waves at the cellular level. By electron microscopy, cholesterol depletion was discovered to destabilize caveolae. The contents of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction, according to Western blots. In the caveolin-1 u2013containing fractions, enrichment of 5-HT2A receptors but not u03b1 1 -adrenergic receptors was found enrichment, but not u03b1 1 -adrenergic receptors, suggesting localization of the former to caveolae. The results, according to these studies, indicate that a subset of signaling pathways that control smooth muscle contraction is specific to cholesterol. In addition, the cholesterol-dependent step in serotonergic signaling occurs early in the path and depends on the strength of caveolae.
Source link: https://doi.org/10.1161/01.atv.0000023438.32585.a1
When varying fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein, chimeric GFPs were localized to various membranes in a fatty acylation-dependent manner. In the Golgi area, a dipalmitoylated GFP chimera colocalized with cholesterol and GM 1 at the plasma membrane and with caveolin. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts made with Triton X-100 or detergent-free methods, and did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. These results show that, although N-terminal acylation will bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. N-terminal fatty acylation may be a cost-effective way to increase the number of signaling proteins in the vicinity of detergent-resistant membranes and promote protein-protein interactions mediating transfer to a detergent-resistant lipid raft core in comparison to limiting acceptor membrane localization.
Source link: https://doi.org/10.1091/mbc.12.11.3601
However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear, although PACSIN2-mediated membrane deformation remains unclear. The amount of cholesterol in the membrane could influence PACSIN2's binding to the membrane, according to our results. Probably due to a decrease in electrostatic charge density, the reconstituted membrane with cholesterol had a smaller link to PACSIN2's F-BAR domain than the membrane without cholesterol, owing to a decrease in electrostatic charge density. The depletion of cholesterol from the plasma membrane resulted in the emergence of PACSIN2-localized tubules with caveolin-1 at their tips, suggesting that cholesterol blocking inhibited the prominent membrane tubulation by PACSIN2. PACSIN2 deficient in caveolae removal on cholesterol depletion was reduced, consistently, as the removal of caveolae from the plasma membrane during cholesterol depletion was slowed. These findings revealed that PACSIN2 mediated the caveolae internalization dependently on the amount of cholesterol at the plasma membrane, indicating a potential explanation for caveolae's cholesterol-dependent regulation.
Source link: https://doi.org/10.1101/2020.03.25.008854
Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost no information is known about the physical properties, cholesterol dynamics, or intracellular factors that influence caveolar cholesterol dynamics. In summary, caveolae/raft lipids were less elastic than those of plasma membranes, sterol domains in caveolae/rafts, were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes.
Source link: https://doi.org/10.1042/bj20031562
We have investigated the effect of lipid-free human plasma apolipoprotein A-I on the movement of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which are mainly represented by caveolae. After labelling cells with [14C]acetate at 15°C and warming cells to enable cholesterol transfer, as well as selective oxidation of cholesterol in cholesterol-rich domains in the plasma membrane prior to partial purification, changes in the transport of newly synthesized cholesterol were evaluated. apoA-I's effect on cholesterol transport was greater than that on cholesterol absorption, resulting in an increase of intracellular cholesterol in caveolae. The delivery of freshly synthesized cholesterol to the caveolae was blocked by Progesterone. The expression of caveolin was stimulated by apoA-I treatment of cells, increasing the amount of caveolin protein and mRNA by approx. We conclude that apoA-I causes intracellular cholesterol transfer to cell-surface caveolae, perhaps in part through caveolin expression stimulation.
Source link: https://doi.org/10.1042/bj3580079
BC 0398 binders in fixed cells were particularly prominent in the plasma membrane, particularly at the base of apical microvilli and in lateral processes. If living cultured cells were treated with BCu0398 and later with fluorescein D or colloidal goldu2013streptavidin D or colloidal goldu2013streptavidin D or colloidal goldu2013streptavidin D was applied to the cell surface in fine dots, providing the labeling in fine dots as long as cells were kept on ice. The probe can be used not only for cytochemical analysis of fixed cells but also for investigating crosslinked cholesterol molecules in living cells because the plasma membrane maintains its integrity even after binding of BC-0198.
Source link: https://doi.org/10.1177/002215549704500903
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