Feedback regulation of endothelial cell surface plasmin generation by PKC-dependent phosphorylation of annexin A2

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.     

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.     

Oxygen tension within the arterial wall: relationship to altered bioenergetic metabolism and lipid accumulation

Oxygen tension (pO2) was measured in upper thoracic arteries and in muscular foci at the celiac bifurcation from atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons at 6, 12, and 24 weeks of age. At each site the pO2 was measured within adventitial (outer), medial (middle), and subendothelial (inner) zones with polarographic oxygen microelectrodes while the animals were under dissociative anesthesia. The atherosclerosis-resistant Show Racer pigeons exhibited no significant age or site differences in pO2 levels. The medial pO2 was approximately 25 mm Hg, while the adventitial and subendothelial zones had pO2 levels of approximately 30 mm Hg. The pO2 profiles through the various zones of the arterial wall of 6-week-old atherosclerosis-susceptible White Carneau pigeons were similar to those of the Show Racers. However, significantly lower oxygen tension was found in the subendothelial zone from 12- and 24-week White Carneau celiac bifurcations (27 and 21 mm Hg) than in corresponding zones in Show Racer sites. Also, the medial zone in 24-week White Carneau celiac foci exhibited a significantly lower pO2 (20 mm Hg) than the medial zone of younger White Carneau. When correlated with previously described biochemical and morphological changes in White Carneau pigeons, the findings presented herein indicate that focal decreases in oxygen availability within arterial tissue occur after the onset of spontaneous atherogenesis, and probably result from decreased diffusion of oxygen into the arterial wall.     

Ecology and management of mahogany (Swietenia macrophylla King) in the Chimanes Forest, Bed, Bolivia

Mahogany ( Swietenia macrophylla King) regenerates in areas of erosion on high terraces and in forest killed by flooding and deposition of alluvial sediments in the Chimanes Forest, Bolivia. These hydrological disturbances are patchy, and only one of five stands of mahogany that we inventoried was regenerating. Mahogany survives these disturbances significantly better than the common tree species. The long time between disturbances appears to favour late maturation. Mahogany trees allocate little photosynthates to reproduction until they are very large emergents, at least 80 cm in diameter. The episodic nature of the regeneration sites means that mahogany stands are composed of one or a few cohorts, which are vulnerable to overharvesting, particularly with the current use of a minimum cutting diameter to regulate harvest. The delayed onset of fecundity means that the small trees that escape harvest are not very fecund, resulting in minimal seed input to logged forest. Only 7–9% of the gaps created by logging contain natural regeneration after 20 + yr. A successful management plan for mahogany would entail a monocyclic harvest, with a rotation age of 100 + years, the estimated time that it takes for trees to achieve commercial size in natural forest. Since the number of seed trees that will be left is small, they should be concentrated in sites that are likely to be conducive to natural regeneration, such as near rivers and flood damaged forest. Seed production will be maximized for a given basal area (opportunity cost to loggers) if trees c. 110 cm dbh are selected as seed trees. The mahogany stocks in the Chimanes Forest are nearly exhausted, but the findings of this study could be used to help rebuild the mahogany populations, or to design management plans for the commercial species that have similar ecologies to mahogany.     

Characteristics of the Amino Acid Transport System in the Mucosal Border of Rabbit Ileum

The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electron-withdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the α-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.     

The Sodium-Alanine Interaction in Rabbit Ileum : Effect of sodium on alanine fluxes

The model of the interaction between Na and alanine at the mucosal border of rabbit ileum has been tested further by examining the efflux of alanine from the cells toward the mucosal solution. Alanine efflux shows a tendency toward saturation as cellular alanine concentration increases and is influenced by cellular Na concentration. A decrease in cell Na concentration causes an increase in the apparent Michaelis constant with little change in maximal efflux rate. Studies on strips of mucosa treated with ouabain or cyanide showed that the direction of net alanine transfer between the cells and the medium is determined by the direction of the Na concentration difference. The cells extrude alanine against a concentration difference when cell [Na] exceeds medium [Na] and accumulate alanine when cell [Na] is less than medium [Na]. The observations are consistent with the model previously suggested involving a transport site that combines with and translocates both Na and alanine, and with the concept that the Na concentration difference between mucosal solution and cytoplasm provides at least part of the energy for active transport of alanine.