Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site.

Src-family tyrosine kinases are regulatory proteins that play a pivotal role in the disorganization of cadherin-dependent cell-cell contacts. We previously showed that Src was associated with vascular endothelial (VE)-cadherin and that tyrosine phosphorylation level of VE-cadherin was dramatically increased in angiogenic tissues as compared to quiescent tissues. Here, we examined whether VE-cadherin was a direct substrate for Src in vascular endothelial growth factor (VEGF)-induced VE-cadherin phosphorylation, and we identified the target tyrosine sites. Co-transfections of Chinese hamster ovary cells (CHO) cells with VE-cadherin and constitutively active Src (Y530F) resulted in a robust tyrosine phosphorylation of VE-cadherin that was not detected with kinase-dead Src (K298M). In an in vitro Src assay, the VE-cadherin cytoplasmic domain is directly phosphorylated by purified Src as well as the tyrosine residue 685 (Tyr)685-containing peptide RPSLY(685)AQVQ. VE-cadherin peptide mapping from human umbilical vein endothelial cells stimulated by VEGF and VE-cadherin-CHO cells transfected with active Src revealed that Y685 was the unique phosphorylated site. The presence of PhosphoY685 was confirmed by its ability to bind to C-terminal Src kinase-SH2 domain in a pull-down assay. Finally, we found that in a VEGF-induced wound-healing assay, cadherin adhesive activity was impaired by Src kinase inhibitors. These data identify that VEGF-induced-VE-cadherin tyrosine phosphorylation is mediated by Src on Y685, a process that appears to be critical for VEGF-induced endothelial cell migration.

Identification of membrane calcium channels essential for cytoplasmic and nuclear calcium elevations induced by vascular endothelial growth factor in human endothelial cells.

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the first stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

Stimulation of platelet-activating factor (PAF) receptors increases inositol phosphate production and cytosolic free Ca2+ concentrations in N1E-115 neuroblastoma cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, PAF) has recently been recognized as an important mediator in the pathophysiology of brain injury. This study demonstrates that, in suspended populations of N1E-115 cells loaded with Indo-1, biologically relevant concentrations of PAF produce a rapid and transient elevation in cytosolic free calcium concentration ([Ca2+]i). Moreover, nanomolar concentrations of PAF increase [3H]-inositol phosphate production. Using lyso-PAF and the specific PAF-receptor antagonists BN52021 and BN50739, we show that these effects were mediated by stimulation of PAF receptors. Experiments performed in Ca2+ free medium show that PAF-induced [Ca2+]i increase is the result of an influx of Ca2+ and of the release of intracellular Ca2+ stores. Studies of Mn2+ influx argue in favour of additional pathways for the PAF-induced Ca2+ influx other than the pathway for the thapsigargin-induced Ca2+ influx. Using the whole-cell voltage-clamp technique, we observe that PAF induces an increase of Ltype Ca2+ current. However, the effects of La3+, nifedipine and KCl-induced depolarization on the PAF-induced [Ca2+]i increase suggest a minor participation of these voltage-gated Ca2+ channels in the response to PAF. Altogether the results point to the existence of a PAF-induced Ca2+ influx through receptor-operated Ca2+ permeant channels.

An experimental model of hypoxia on isolated rat heart in recirculating system: study of fatty acid metabolism with an iodinated fatty acid.

An experimental model of hypoxia was developed on isolated rat heart to study the effects of hypoxia on cardiac performance and metabolism. Fatty acid (FA) metabolism was explored by external detection with a labelled FA, iodohexadecenoic acid (IHA). Hearts, after 30 min preperfusion in an open system, were transferred in a recirculating system for 40 min and perfused with oleate, glucose, lactate, pyruvate and IHA, either in normoxia (pO2 = 660 mmHg) or in hypoxia (pO2 = 220 mmHg). After 40 min hypoxic recirculation, oxygen uptake and dynamic parameters, except the heart rate, decreased respectively by 56% and 44%, and remained constant throughout the perfusion. Glucose utilization increased 2 fold, endogenous glycogen fell by 50% and lactate + pyruvate production increased 3 fold, showing a stimulation of glycolysis. Oleate uptake decreased by 28%, while triglycerides content remained higher. The ATP/ADP ratio decreased by 24%. Conversely to oleate, IHA uptake was not significantly modified, but its intracellular fate showed a higher radioactivity in all lipid fractions: polar lipids, diglycerides, free FAs and triglycerides. beta oxidation of IHA, evidenced by iodide production, decreased by 39%. The external detection of cardiac radioactivity allowed us to obtain time-activity curves that were analyzed with a 4-compartment mathematical model. The data evidenced an esterification ratio significantly higher in hypoxia. The metabolism of IHA as estimated by the intracellular analysis or, in a non-invasive way, by external detection, was similar to the metabolism of oleate. Thus, lipid metabolism, in hypoxia, can be explored by external detection with IHA.

Influence of fatty acid backdiffusion on compartmental analysis of external detection curves obtained with 123-iodohexadecenoic acid in isolated rat heart.

In isolated rat hearts, we investigated a possible backdiffusion of fatty acids and tried to determine whether it impaired our compartmental analysis of myocardial time-radioactivity curves obtained with an iodinated fatty acid, 16-iodo-9-hexadecenoic acid (IHA). Backdiffusion was not observed directly in the coronary effluents but was estimated by analysis of the external detection curves. Furthermore, when backdiffusion was not taken into account in the mathematical analysis, we obtained similar data on IHA intramyocardial metabolism.

Effect of short-term fasting on [123I] iodohexadecenoic acid metabolism in the isolated perfused rat heart. Validation of mathematical model by comparison with experimental measurements.

In order to study metabolic modifications induced by short term fasting and their consequences on the uptake and intracellular fate of fatty acids iodine labelled in omega position, rats undergo a 36h fasting. Hearts are perfused in a Langendorff system with a glucose (11 mM) perfusion medium; [123I] hexadecenoic acid (IHA) is injected as a bolus. A comparison between time-activity curves p.i. demonstrates a much faster activity decrease for the hearts fasted animals. The intracellular analysis shows that short fasting did not significantly increase the myocardial uptake of fatty acids, but decreased the storage and increased the degradation of the fatty acids taken up. Mathematical analysis of the myocardial time-activity curves obtained by external detection provided results comparable to those of intracellular analysis. The coefficients of correlation between the values of the aqueous phases, organic phases and free fatty acids measured by intracellular analysis and calculated with the compartmental model are consistently higher than 0.97. Consequently, this experimental model combined with mathematical analysis of the time-course of myocardial radioactivity after 123IHA administration appears to be very promising method for studying the effects of drugs or variations of energy substrate availability on myocardial fatty-acid metabolism.

Age-dependent differences in energetic status, electrical and mechanical performance of rat myocardium.

Transmembrane action potential (AP), isotonic contraction and biochemical measurements were performed in 12-day, 1-, 3-, 14- and 24-month-old rat hearts. The major findings of this study are: (1) the AP and contraction duration decrease between 12 days and 1 month of age (growth period) and increase between 1 month and 24 months of age; (2) as compared with 1 month and 14 months, respectively, isotonic contraction peak shortening is lower at 12 days and 24 months of age; (3) the phosphorylation potential is higher during the postnatal period and decreases in an age-correlated manner; (4) the inorganic phosphate and glycogen contents are higher in the senescent heart. We conclude that, during the postnatal period the particular AP and the lower mechanical performances could be the result of immature sarcolemma and sarcoplasmic reticulum properties rather than modifications in the oxidative phosphorylation mechanism and by contrast, in senescent heart, that AP and contraction modifications could result from metabolic modifications.

Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats.

Glutathione peroxidase (GSh-Px), superoxide dismutase (SOD), catalase (CAT) activities and malon-dialdehyde (MDA) content were determined in heart, liver, kidney and brain of rats. Two different age groups (4 months; 24 months) were considered. GSH-Px and SOD activities decrease significantly for the aged liver and kidney. During aging, the activity of catalase increase in cardiac muscle and, in contrast, decrease in other organs. Lipids peroxidation, expressed in term of MDA formation, decrease in all the organs of the aged rats. The results indicate that: 1) the liver and kidney antioxidative defense decrease with age; 2) the enzymatic activities evolve in a different manner for different enzymes and organs. Furthermore, the results suggest that there is not any correlation between the SOD, CAT, and GSH-Px activities and the peroxidative status of the organs; thus, the age-related increase in the MDA content proposed as a criterion of aging process should be considered with caution.

Effects of clofibrate and tiadenol on the elimination of lipids and bile acids in rat bile.

The aim of the work presented here was to compare the biliary elimination of cholesterol and the different bile acids of rats that had been made hypolipidemic by short-term treatments with clofibrate or tiadenol. Both treatments induced a significant decrease in cholesterol output in the bile. The analysis of the different bile acids showed a decrease in dihydroxylated acids elimination (especially CDC acid) without any difference between the 2 sexes. This decrease was associated with an increase in cholic acid excretion. These results are directly correlated with the dose of the administered hypolipidemic drug. The drugs caused as significant increase in the ratio of trihydroxylated acids of the bile and on the output was obtained, for both drugs, with a treatment of 200 mg/kg/day. Clofibrate had a greater effect than tiadenol at this dose. Both drugs show a greater effect on lowering serum lipid levels in female animals when compared to males, whereas elimination of bile cholesterol and modifications of bile acids were greater in male animals than female animals.