Differential regulation of adenine nucleotide translocators by hypertonicity in the brain

To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up-regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT)2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up-regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of NaCl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.     

A novel glycosylphosphatidyl inositol-anchored protein on human leukocytes: a possible role for regulation of neutrophil adherence and migration

We report here a novel glycosylphosphatidyl-inositol (GPI)-anchored glycoprotein on human leukocytes. Treatment of neutrophils with a mAb (3H9) to this molecule sequentially up-regulates and down-regulates beta2 integrin-dependent adhesion of these cells as well as their transendothelial migration in vitro. In addition, this mAb simultaneously modulates the avidity of beta2 integrin for its ligand, iC3b, with kinetics similar to those observed in 3H9 modulation of neutrophil adherence. This mAb also induces beta2 integrin-dependent cytoskeletal remodeling. This novel GPI-anchored protein (GPI-80) is highly homologous with Vanin-1, a recently reported GPI-anchored protein that is expressed on perivascular thymic stromal cells and is involved in thymus homing in mice. The finding that both GPI-80 and Vanin-1 are 40% homologous with human biotinidase suggests the existence of a biotinidase superfamily of molecules that may be involved in the regulation of leukocyte trafficking.     

Differential distribution of 5-hydroxytryptamine3 receptor in the colon between human and guinea pig

Localization of 5-hydroxytryptamine3 (5-HT3) receptor in the human colon was examined by in vitro receptor autoradiography using [125I](S)iodozacopride, and compared with that in the guinea pig colon. [125I](S)iodozacopride binding sites were found with high densities around the myenteric plexus, but with low ones in the muscle layer and mucosa of the human colon, and the binding was abolished by granisetron, a specific 5-HT3 receptor antagonist. While in the guinea pig colon, specific [125I](S) iodozacopride binding was not detected in either the myenteric plexus or the muscle layers. Thus, the 5-HT3 receptors are present in the human colon, especially densely located in the myenteric plexus, but not in the guinea pig colon, and those may participate in the colonic motility. The results of functional studies of 5-HT3 receptor obtained from experiments using guinea pig are not always applying to the human.     

Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth

While the neurotrophin receptor p75NTR is expressed by many developing neurons, its function in cells escaping elimination by programmed cell death remains unclear. The lack of intrinsic enzymatic activity of p75NTR prompted a search for protein interactors expressed in the developing retina, which resulted in the identification of the GTPase RhoA. In transfected cells, p75NTR activated RhoA, and neurotrophin binding abolished RhoA activation. In cultured neurons, inactivation of Rho proteins mimicked the effect of neurotrophins by increasing the rate of neurite elongation. In vivo, axonal outgrowth was retarded in mice carrying a mutation in the p75NTR gene. These results indicate that p75NTR modulates in a ligand-dependent fashion the activity of intracellular proteins known to regulate actin assembly.     

Thr(207) of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP

We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood-brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway (Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr(207) in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr(207) (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr(207) of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.     

Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice

Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.