This study was designed to test the hypothesis that a 28-day tail suspension (SUS) could induce hypertrophy and enhanced myogenic and vasoconstrictor reactivity in middle cerebral arteries (MCAs), whereas atrophy and decreased myogenic and vasoconstrictor responses in mesenteric third-order arterioles (MSAs). Also, in addition to the functional enhancement in MCAs, structural changes in both kinds of arteries and functional decrement in MSAs could all be prevented by the intervention of daily 1-h dorsoventral (-G(x)) gravitation by restoring to standing posture. To test this hypothesis, vessel diameters to pressure alterations and nonreceptor- and receptor-mediated agonists were determined using a pressure arteriograph with a procedure to measure in vivo length and decrease hysteresis of vessel segments and longitudinal middlemost sections of vessels fixed at maximally dilated state were examined using electron microscopy and histomorphometry. Functional studies showed that 28-day tail-suspended, head-down tilt (SUS) resulted in enhanced and decreased myogenic tone and vasoconstrictor responses, respectively, in MCAs and MSAs. Histomorphometric data revealed that SUS-induced hypertrophic changes in MCAs characterized by increases in thickness (T) and cross-sectional area (CSA) of the media and the number of vascular smooth-muscle-cell layers (N(CL)), whereas in MSAs, it induced decreases in medial CSA and T and N(CL). Daily 1-h -G(x) over 28 days can fully prevent these differential structural changes in both kinds of small arteries and the functional decrement in MSAs, but not the augmented myogenic tone and increased vasoreactivity in the MCAs. These findings have revealed special features of small resistance arteries during adaptation to microgravity with and without gravity-based countermeasure.

This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O 2 ·À production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 11, 703-712.

Previous studies have demonstrated activation of the local renin-angiotensin system in hindlimb unweighting (HU) rat vasculature. The present study intended to identify the effects of blockade of angiotensin II (ANG II) type 1 (AT(1)) receptors with losartan on vascular reactivity, nitric oxide synthase (NOS) expression, and superoxide anion (O(2)(*-)) levels in 3-wk HU rat cerebral and carotid arteries. Three weeks later, vasoconstriction, vasodilatation, endothelial NOS (eNOS) and inducible NOS (iNOS) protein, as well as O(2)(*-) levels in rat cerebral and carotid arteries were examined. We found that HU enhanced maximal response to KCl/5-hydroxytryptamine (P < 0.01) in basilar arteries and KCl/phenylephrine (P < 0.05) in common carotid arteries from HU rats. Acetylcholine induced concentration-dependent vasodilatation in all the artery rings, but with significantly smaller amplitude in basilar (P < 0.01) and common carotid (P < 0.05) arteries from HU rats than those from control rats. Chronic treatment with losartan partially restored response to vasoconstrictors and acetylcholine-induced vasodilatation in basilar (P < 0.01) and common carotid (P < 0.05) arteries from losartan-treated HU rats. Furthermore, iNOS content in cerebral arteries and eNOS/iNOS content in carotid arteries were significantly (P < 0.01) increased in HU rats. Meanwhile, HU increased O(2)(*-) levels in all the layers of these arteries. However, losartan restored NOS content and O(2)(*-) levels toward normal. These results suggested that the HU-induced enhancement of vasoconstriction and reduction in endothelium-dependent relaxation involved alterations in O(2)(*-) and NOS content through an ANG II/AT(1) receptor signaling pathway.

The present study determined whether activation of acid sphingomyelinase (ASM) drives membrane proximal lysosomes to fuse to the cell surface, facilitating membrane lipid rafts (LRs) clustering in coronary arterial endothelial cells (CAECs) and leading to endothelial dysfunction. By confocal microscopy, the activators of ASM, phosphatidylinositol (PI), and bis (monoacylglyceryl) phosphate (Bis), and an inducer of ASM, butyrate, were found to increase LRs clustering in bovine CAECs, which was blocked by lysosome fusion inhibitor vacuolin-1. However, arsenic trioxide (Ars), an inducer of de novo synthesis of ceramide, had no such effect. Similarly, vacuolin-1-blockable effects were observed using fluorescence resonance energy transfer detection. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis demonstrated that all of these treatments, even Ars, increased ceramide production in CAECs. When ASM gene was silenced, all treatments except Ars no longer increased ceramide levels. Furthermore, dynamic fluorescence monitoring in live cells showed that PI and Bis stimulated lysosome-membrane fusion in CAECs. Functionally, PI and Bis impaired endothelium-dependent vasodilation in perfused coronary arteries, which was blocked by vacuolin-1 and a lysosome function inhibitor, bafilomycine. FasL (Fas ligand), a previously confirmed lysosome fusion stimulator as a comparison, also produced a similar effect. It is concluded that ASM activation serves as a triggering mechanism and driving force, leading to fusion of membrane proximal lysosomes into LR clusters on the cell membrane of CAECs, which represents a novel mechanism mediating endothelial dysfunction during death receptor activation or other pathological situation.

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