Endothelium of the cerebral blood microvessels, which constitutes the major component of the bloodbrain barrier, controls leukocyte and metastatic cancer cell adhesion and trafficking into the brain parenchyma. In this study, using rat primary brain microvascular endothelial cells (BMEC), we demonstrate that the vascular endothelial growth factor (VEGF), a potent promoter of angiogenesis, up-regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) through a novel pathway that includes phosphatidylinositol 3 OH-kinase (PI3K), AKT, and nitric oxide (NO), resulting in the migration of BMEC. Upon VEGF treatment, AKT is phosphorylated in a PI3K-dependent manner. AKT activation leads to NO production and release and activation-deficient AKT attenuates NO production stimulated by VEGF. Transfection of the constitutive myr-AKT construct significantly increased basal NO release in BMEC. In these cells, VEGF and the endothelium-derived NO synergistically up-regulated the expression of ICAM-1, which was mediated by the PI3K pathway. This activity was blocked by the PI3K-specific inhibitor, wortmannin. Furthermore, VEGF and NO significantly increased BMEC migration, which was mediated by the up-regulation of ICAM-1 expression and was dependent on the integrity of the PI3K/AKT/NO pathway. This effect was abolished by wortmannin, by the specific ICAM-1 antibody, by the specific inhibitor of NO synthase, N G -L-monomethyl-arginine (L-NMMA) or by a combination of wortmannin, ICAM-1 antibody, and L-NMMA. These findings demonstrate that the angiogenic factor VEGF up-regulates ICAM-1 expression and signals to ICAM-1 as an effector molecule through the PI3K/ AKT/NO pathway, which leads to brain microvessel endothelial cell migration. These observations may contribute to a better understanding of BMEC angiogenesis and the physiological as well as pathophysiological function of the blood-brain barrier, whose integrity is crucial for normal brain function.The BBB 1 is involved in the maintenance of the microenvironment of the central nervous system. The BBB system consists of endothelial cells, pericytes, perivascular microglia, astrocytes, and basal lamina. Whereas the endothelial cell microvascular monolayer forms the barrier proper, the interaction between these various cell types seems to be necessary for the induction or maintenance of the specialized function of the BBB (1). The permeability of the BBB is important for its normal functioning, and changes in this permeability may lead to brain infection (2) and cancer metastasis to the brain (3).VEGF is a potent angiogenic factor that induces NO formation (4), as well as endothelial cell proliferation and migration (5). Angiogenesis occurs after tissue ischemia (6, 7) or in tumor growth and metastasis (8,9). VEGF functions as a survival factor for newly formed vessels during developmental neovascularization but is not required for the maintenance of mature vessels (8). Formation of blood vessels occurs via two distinct processes, vasculogenesis and angiogenesis....

The responses of human neocortical neurons to iontophoretic application of excitatory amino acids and their modulation by dopamine (DA) were studied in vitro. Brain slices were obtained from children undergoing surgery for intractable epilepsy. Application of N-methyl-D-aspartate (NMDA) to the slices induced slow depolarizations accompanied by decreased input conductances and sustained action potentials in cortical neurons. Glutamate produced rapid depolarizations and firing with few changes in input conductances. Quisqualate also induced depolarization and firing, but input conductances increased during the rising phase of the membrane depolarization. Iontophoretic application of DA alone produced no change in membrane potential or input conductance. However, when DA was applied in conjunction with the excitatory amino acids, it produced contrasting effects. With either bath application of DA or when iontophoresis of DA preceded application of NMDA, the amplitude of the membrane depolarizations and the number of action potentials were increased, whereas the latency of these responses decreased. In contrast, DA decreased the amplitude of the depolarizations and the number of action potentials evoked by glutamate or quisqualate. The fact that DA affects responses to NMDA and glutamate or quisqualate in opposite directions is of considerable importance to the understanding of cellular mechanisms of neuromodulation and the role of DA in cognitive processing and in epilepsy.

BACKGROUNDThe winged helix/forkhead transcriptional factor FKHRL1 (FOXO3a) triggers apoptosis, but its mode of action is not well understood. ROCK kinase is an effector molecule in human breast carcinoma cell apoptosis, but its relation to FKHRL1 is unknown. Because the human breast carcinoma T47D cell line releases a great amount of nitric oxide (NO), I investigated signaling of FKHRL1 to ROCK kinase during NO suppression.METHODSExpression of phosphorylated FKHRL1 in T47D cells was analyzed using Western blotting. Apoptosis was evaluated by flow cytometry. Transfection of FKHRL1‐HA wild‐type and mutant FKHRL1‐HA T32A constructs were performed by lipofectamine plus reagent. Measurement of NO generation was performed by Griess reaction.RESULTSNitric oxide suppression promotes FKHRL1 thr‐32‐enhanced phosphorylation, which was significantly (P < 0.005) sensitive to Y‐27632, a specific inhibitor of the ROCK kinase, but not to capase‐3 inhibitor or wortnannin, a specific inhibitor of phosphoinositol‐3‐OH kinase (PI3K). Nitric oxide suppression by N‐G‐monomethyl‐L‐arginine, an inhibitor of NO synthase, causes a significant (P < 0.001) increase in the apoptosis of T47D cells. However, a significant decrease (P < 0.01) in NO generation and a significant (P < 0.01) increase in apoptosis were observed when FKHRL1‐HA wild‐type cells were transfected, which caused increased FKHRL1 thr‐32 phosphorylation.CONCLUSIONSThis novel unknown phenomenon of breast carcinoma cell apoptosis was triggered by NO suppression, which promotes FKHRL1 thr‐32‐enhanced phosphorylation and initiates signaling of FKHRL1 to ROCK kinase as an effector molecule. This apoptotic signalling process is caspase‐3 as well as PI3K/Akt independent. Cancer 2003;97:1358–63. © 2003 American Cancer Society.DOI 10.1002/cncr.10081