Eric S Kim | PubMed Health

PubMed2003

Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function.

Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC alpha (PKC alpha EC), PKC delta (PKC delta EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC alpha EC. Conversely, the basal level of permeability was significantly blunted in PKC delta EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC alpha and PKC delta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC alpha EC, an effect blocked by pretreatment with Gö6976. PKC delta EC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKC delta EC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKC delta EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC alpha promotes endothelial barrier dysfunction and PKC delta enhances basal endothelial barrier function.

Acetophenones↗

PubMed2002

Arp2/3 complex is required for actin polymerization during platelet shape change.

Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown. The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (alpha Arp2). Intact and Fab fragments of alpha Arp2 inhibited TRAP-stimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with alpha Arp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that is required for all subsequent actin-dependent events.

Actin Cytoskeleton↗