American journal of physiology. Cell physiology
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Am. J. Physiol., Cell Physiol. · Aug 2004
Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow.
This study was undertaken to characterize the adhesion of LS174T colon adenocarcinoma cells to 4-h TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under flow in the presence and absence of platelets and erythrocytes. Cell binding to HUVECs was significantly enhanced by simultaneous perfusion of thrombin-activated, but not resting, platelets. This increase was achieved via a platelet bridging mechanism whereby a previously tethered LS174T cell (primary tether) captures a free-flowing cell (secondary tether) that subsequently attaches to the endothelium downstream of the already adherent cell. ⋯ Secondary tethering was dependent on both platelet P-selectin and alpha(IIb)beta(3)-integrin for LS174T cells and P-selectin alone for THP-1 cells. Furthermore, platelet-mediated secondary tethering of both cell types occurred in the presence of red blood cells. Altogether, these results reveal a novel role for platelets in promoting cell binding to endothelium through a secondary tethering mechanism.
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Am. J. Physiol., Cell Physiol. · May 2004
Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells.
The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. ⋯ Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.
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Am. J. Physiol., Cell Physiol. · Apr 2004
Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione.
In cultured rat cerebellar granule cells, glutamate or N-methyl-d-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca(2+) levels ([Ca(2+)](i)), reactive oxygen species (ROS) generation, and cell death (respective EC(50) values for glutamate were 12, 30, and 38 microM) but no increase in caspase-3 activity. Removal of extracellular Ca(2+) blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca(2+)](i) increase. This indicates that glutamate-induced cell death is attributable to [Ca(2+)](i) increase and ROS generation, and the [Ca(2+)](i) increase precedes ROS generation. ⋯ The transient [Ca(2+)](i) increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca(2+)](i) increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca(2+)](i) increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity.
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Am. J. Physiol., Cell Physiol. · Apr 2004
Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices.
Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH(i)). pH(i) was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH(i) in 270 of 436 (62%) SC neurons tested. ⋯ CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH(i). These results indicate that oxidative stress 1). decreases pH(i) in some SC neurons, 2). together with hypercapnia has an additive effect on pH(i), 3). partially inhibits NHE, and 4) directly affects excitability of CO(2)/H(+)-chemosensitive SC neurons independently of pH(i) changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction.
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Am. J. Physiol., Cell Physiol. · Mar 2004
A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells.
The choroid plexus epithelium of the brain ventricular system produces the majority of the cerebrospinal fluid and thereby defines the ionic composition of the interstitial fluid in the brain. The transepithelial movement of Na+ and water in the choroid plexus depend on a yet-unidentified basolateral stilbene-sensitive Na+-HCO3- uptake protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression in the choroid plexus of SLC4A10 mRNA, which encodes a stilbene-sensitive Na+-HCO3- transporter. ⋯ Bright-field immunohistochemistry and immunoelectron microscopy demonstrated strong labeling confined to the basolateral plasma membrane domain of the choroid plexus epithelium. Furthermore, the stilbene-insensitive Na+-HCO3- cotransporter, NBCn1, was also localized to the basolateral plasma membrane domain of the choroid plexus epithelium. Hence, we propose that the SLC4A10 gene product and NBCn1 both function as basolateral HCO3- entry pathways and that the SLC4A10 gene product may be responsible for the stilbene-sensitive Na+-HCO3- uptake that is essential for cerebrospinal fluid production.