Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.
The current study demonstrates a differential structure of the adventitial VV in different vascular beds. This intra- and intervessel heterogeneity in VV anatomy is a phenotypic variability that might determine a differential local response to systemic risk factors and, thereby, variable propensity for vascular disease among different vascular beds.
Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.
The ubiquitin‐proteasome system (UPS) is involved in the removal of damaged proteins and the activation of transcription factors, such as nuclear‐factor‐κB. Recent reports, however, questioned the functional activity of the UPS under conditions of increased oxidative stress, such as experimental hypercholesterolemia, which was the objective of our study. Pigs were placed on a normal chow diet (N) or on a hypercholesterolemic diet without (HC) or with vitamin C and E supplementation (HC+VIT) for 12 weeks. Compared with N, plasma concentration of total cholesterol increased in both HC and HC+VIT [76 ± 21 vs. 400 ± 148 (P<0.05) and 329 ± 102 (P<0.05) mg/dL], whereas increase in lipid peroxidation, as assessed by LDL‐malondialdehyde plasma concentration, was found in HC but not in HC+VIT [6.6 ± 0.7 vs. 8.5 ± 0.3 (P<0.05) and 6.8 ± 0.7 nmol/mg protein]. In comparison with N, the level of ubiquitin conjugates in the coronary artery, as assessed by immunoblotting, increased by 42% in HC but not in HC+VIT and was localized predominantly to media vascular smooth muscle cells by immunostaining. There was no difference in proteasome proteolytic activity among the study groups. These results demonstrate that the UPS is functionally active in early atherogenesis despite increase in oxidative stress with important repercussions in the pathophysiology and therapy of cardiovascular diseases.
Background and Purpose-The local renin-angiotensin system (RAS) and cyclooxygenase-2 contribute to the activation of nuclear factor B (NFB) and C-reactive protein (CRP
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This study demonstrated that low vasa vasorum spatial density and higher lumen area observed in the left internal thoracic artery compared with that seen in the native coronary artery can be the structural background for the low incidence of atherosclerosis in this vessel.
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