Regulation of renal organic anion transporter 3 (SLC22A8) expression and function by the integrity of lipid raft domains and their associated cytoskeleton.

BACKGROUND/AIMS: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. METHODS: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. RESULTS: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), ß-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MßCD. CONCLUSION: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

Vascular endothelial growth factor induces mRNA expression of pro-inflammatory factors in the uterine cervix of mice.

Inflammation is believed to play a role in uterine cervical remodeling and infection-induced preterm labor. One of the distinct features of remodeling uterine cervix is presence of prominent vascular events, such as angiogenesis, vasodilation, and vascular permeability. Although the functional significance of these features is not yet clear, we know that in most tissue types, vascular remodeling is intricately intertwined with inflammation. Since vascular endothelial growth factor (VEGF) is the major architect of vascular remodeling, we sought to examine and elucidate the potential relationship between VEGF and inflammation in the uterine cervix of non-pregnant mice. The animals used were divided into 4 treatment groups: A) negative control (vehicle only), B) positive control (lipopolysaccharide, LPS), C) recombinant VEGF-164 protein, and D) LPS + VEGF blocker (n = 3). After the appropriate treatments, the uterine cervices were harvested and analyzed using real-time PCR and confocal fluorescence microscopy. Results showed that exogenous VEGF upregulates expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α mRNAs, whereas VEGF blocker partially diminishes the LPS-induced expression of pro-inflammatory factors compared to the positive control group. We conclude that a positive feed-forward relationship likely exists between VEGF and inflammation in the uterine cervix, thus implicating VEGF in inflammation-induced preterm labor.

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs.

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Arg(87)respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368)and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Arg(368)and Asn(459)/Arg(87)pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.