Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells.

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the alpha and beta PKC isoforms. gamma PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC alpha immunofluorescence redistributed to the perinuclear region whereas PKC beta staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.     

Effects of ATP-MgCl2 and adenosine-MgCl2 administration on intracellular ATP levels in the kidney

The effects of exogenous administration of 1 mM [8-14C]ATP-MgCl2 and adenosine-MgCl2 on intracellular accumulation of adenine nucleotides were examined in isolated, perfused rat kidneys. The kidneys were made filtering or non-filtering by increasing the colloid oncotic pressure of the perfusate solution in order to assess the relative contributions of the glomerular and peritubular routes in the uptake of the nucleotides. The results indicate that: although labeled ATP is undetectable in the perfusate after 20 min, there is a significant accumulation of labeled ATP in the tissue and although labeled adenosine-MgCl2 administration also leads to labeled intracellular ATP, the total intracellular ATP is much less than with ATP-MgCl2 administration.     

Crayfish,catfish and snails: the perils of uncontrolled biological control

A recent proposal that the Australian redclaw crayfish Cherax quadricarinatus and hybrid catfish could potentially control the snail hosts of schistosomiasis has been criticised on the grounds that crayfish pose a severe threat to aquatic ecosystems into which it might be introduced. This note examines the issue further, pointing out that both lack the host-specificity requirement to be a successful biological control agent. The catfish Clarias gariepinus is an omnivore and snails form only a small proportion of its diet; there is no evidence to suggest that it controls snail populations anywhere in Africa. The same applies to other species that have been proposed as biological control agents. Simple laboratory experiments are not an adequate guide to the efficiency of an animal as a biological control agent and detailed ecological investigations would usually demonstrate that few African fish species have this capability.     

Pulsatile to-fro flow induces greater and sustained expression of tissue factor RNA in HUVEC than unidirectional laminar flow

Tissue factor (TF) is expressed in atherosclerotic lesions. Since mechanical forces influence endothelial cell (EC) function and are thought to account for the unique distribution of atherosclerosis in areas exposed to disturbed flow, we hypothesized that disturbed to-fro flow (TFF) and unidirectional pulsatile forward flow (PFF) would have different effects on TF expression in EC. TF RNA expression in HUVEC exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin was determined. TFF induced a significantly higher TF expression than PFF that was sustained for 8 h. Combination of mechanical and chemical stimuli induced significantly higher TF expression than only mechanical stresses, and this effect was synergistic in both TFF and PFF. The MAPK p38 inhibitor SB-203580 significantly inhibited TF expression induced by mechanical and chemical stimulations, but the MEK inhibitor PD-98059 did not inhibit TF induced by TFF. Immunoblotting revealed that ERK1/2 phosphorylation induced by TFF was sustained for 120 min, whereas that induced by PFF was not. We conclude that disturbed flow induced greater and sustained amplification of TF expression, and this synergistic effect may be regulated by p38 MAPK and ERK1/2. These results provide added insight into the mechanism of atherosclerosis in areas of disturbed flow.     

Cells in focus: endothelial cell

The endothelial cell is thought to arise from the splanchnopleuric mesoderm. Endothelial cells form the inner lining of a blood vessel and provides an anticoagulant barrier between the vessel wall and blood. In addition to its role as a selective permeability barrier, the endothelial cell is a unique multifunctional cell with critical basal and inducible metabolic and synthetic functions. The endothelial cell reacts with physical and chemical stimuli within the circulation and regulates hemostasis, vasomotor tone, and immune and inflammatory responses. In addition, the endothelial cell is pivotal in angiogenesis and vasculogenesis. Endothelial cell injury, activation or dysfunction is a hallmark of many pathologic states including atherosclerosis, loss of semi-permeable membrane function, and thrombosis.

Cell facts: (1) Endothelium consists of approximately (1–6)×10¹³ endothelial cells forming an almost 1 kg organ. (2) They uniquely contain Weibel–Palade bodies, 0.1 μm wide, 3 μm long membrane-bound structures that represent the storage organelle for von Willebrand factor (vWF). (3) The endothelial cell is not only a permeability barrier but also a multifunctional paracrine and endocrine organ. It is involved in the immune response, coagulation, growth regulation, production of extracellular matrix components, and is a modulator of blood flow and blood vessel tone.     

MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells

PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK(1/2), p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals.