Biomedical subjects

Whaseon Lee-Kwon

Publications and source records attributed to Whaseon Lee-Kwon.

9 recordsLinked to original sources

PubMed2004

Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta.

The PDZ domain adaptor protein Na+/H+ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca2+, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca2+ signaling.

Animals↗

PubMed2004

Chronic ANG II infusion increases NO generation by rat descending vasa recta.

We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng.kg(-1).min(-1) for 11-13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N(omega)-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca(2+) response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.

Acetylcholine↗

PubMed2004

Lysophosphatidic acid induces exocytic trafficking of Na(+)/H(+) exchanger 3 by E3KARP-dependent activation of phospholipase C.

Lysophosphatidic acid (LPA) stimulates Na(+)/H(+) exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca(2+) in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca(2+) leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca(2+) chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca(2+) but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.

Animals↗

PubMed2003

Akt as a mediator of cell death.

Protein kinase B/Akt possesses prosurvival and antiapoptotic activities and is involved in growth factor-mediated neuronal protection. In this study we establish Akt deactivation as a causal mediator of cell death. Akt deactivation occurs in multiple models of cell death including N-methyl-d-aspartate excitotoxicity, vascular stroke, and nitric oxide (NO)- and hydrogen peroxide (H2O2)-elicited death of HeLa, PC12, and Jurkat T cells. Akt deactivation characterizes both caspase-dependent and -independent cell death. Conditions rescuing cell death, such as treatment with poly(ADP-ribose) polymerase or NO synthase inhibitors and preconditioning with sublethal concentrations of N-methyl-d-aspartate, restore Akt activity. Infection of neurons with adenovirus expressing constitutively active Akt prevents excitotoxicity, whereas phosphatidylinositol 3-kinase inhibitors or infection with dominant negative Akt induce death of untreated neuronal cells.

Animals↗

PubMed2003

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Animals↗

PubMed2003

Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse.

In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.

Animals↗

PubMed2003

The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines.

Epithelial-mesenchymal transition (EMT) is an important process during development and oncogenesis by which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. Squamous cell carcinoma lines engineered to express constitutively active Akt underwent EMT, characterized by down-regulation of the epithelial markers desmoplakin, E-cadherin, and beta-catenin and up-regulation of the mesenchymal marker vimentin. The cells lost epithelial cell morphology and acquired fibroblast-like properties. Additionally, E-cadherin was down-regulated transcriptionally. The cells expressing constitutively active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. AKT is activated in many human carcinomas, and the AKT-driven EMT may confer the motility required for tissue invasion and metastasis. These findings suggest that future therapies based on AKT inhibition may complement conventional treatments by controlling tumor cell invasion and metastasis.

Cadherins↗

PubMed2003

Lysophosphatidic acid stimulates brush border Na+/H+ exchanger 3 (NHE3) activity by increasing its exocytosis by an NHE3 kinase A regulatory protein-dependent mechanism.

Na(+)/H(+) exchanger 3 (NHE3) kinase A regulatory protein (E3KARP) has been implicated in cAMP- and Ca(2+)-dependent inhibition of NHE3. In the current study, a new role of E3KARP is demonstrated in the stimulation of NHE3 activity. Lysophosphatidic acid (LPA) is a mediator of the restitution phase of inflammation but has not been studied for effects on sodium absorption. LPA has no effect on NHE3 activity in opossum kidney (OK) proximal tubule cells, which lack expression of endogenous E3KARP. However, in OK cells exogenously expressing E3KARP, LPA stimulated NHE3 activity. Consistent with the stimulatory effect on NHE3 activity, LPA treatment increased the surface NHE3 amount, which occurred by accelerating exocytic trafficking (endocytic recycling) to the apical plasma membrane. These LPA effects only occurred in OK cells transfected with E3KARP. The LPA-induced increases of NHE3 activity, surface NHE3 amounts, and exocytosis were completely inhibited by pretreatment with the PI 3-kinase inhibitor, LY294002. LPA stimulation of the phosphorylation of Akt was used as an assay for PI 3-kinase activity. LY294002 completely prevented the LPA-induced increase in Akt phosphorylation, which is consistent with the inhibitory effect of LY294002 on the LPA stimulation of NHE3 activity. The LPA-induced phosphorylation of Akt was the same in OK cells with and without E3KARP. These results show that LPA stimulates NHE3 in the apical surface of OK cells by a mechanism that is dependent on both E3KARP and PI 3-kinase. This is the first demonstration that rapid stimulation of NHE3 activity is dependent on an apical membrane PDZ domain protein.

Animals↗

PubMed2002

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

Actinin↗