SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The Na+-retaining hormone aldosterone increases the cell-surface expression of the luminal epithelial sodium channel (ENaC) and the basolateral Na+ pump (Na,K-ATPase) in aldosterone-sensitive distal nephron cells in a coordinated fashion. To address the question of whether aldosterone-induced serum and glucocorticoid-regulated kinase-1 (SGK1) might be involved in mediating this regulation of Na,K-ATPase subcellular localization, similar to that of the epithelial Na+ channel (ENaC), we co-expressed the Na,K-ATPase (rat α1- and Xenopus laevis β1-subunits) and Xenopus SGK1 in Xenopus oocytes. Measurements of the Na+ pump current showed that wild-type SGK1 increases the function of exogenous Na,K-ATPase at the surface of Xenopus oocytes. This appeared to be secondary to an increase in Na,K-ATPase cell-surface expression as visualized by Western blotting of surface-biotinylated proteins. In contrast, the functional surface expression of two other exogenous transporters, the heterodimeric amino acid transporter LAT1-4F2hc and the Na+/phosphate cotransporter NaPi-IIa, was not increased by SGK1 co-expression. The total pool of exogenous Na,K-ATPase was increased by the co-expression of SGK1, and similarly also by ENaC co-expression. This latter effect depended on the [Na+] of the buffer and was not additive to that of SGK1. When the total Na,K-ATPase was increased by ENaC co-expression, SGK1 still increased Na,K-ATPase cell-surface expression. These observations in Xenopus oocytes suggest the possibility that SGK1 induction and/or activation could participate in the coordinated regulation of Na,K-ATPase and ENaC cell-surface expression in the aldosterone-sensitive distal nephro

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation. Fibroblast-like synoviocytes are important mediators of joint pathology in rheumatoid arthritis (RA). Here the authors show that Lasp1 is epigenetically regulated and highly expressed by these cells in RA and its deletion can limit joint pathology in a mouse model of inflammatory arthritis

Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts

Mit der vorliegenden Arbeit wurde die Regulation des Na+2Cl-K+-Cotransporters in der basolateralen Membran der Rattenkolonkrypte untersucht. Dazu wurde die NH3/NH4+-Puls Technik zusammen mit der BCECF-Fluoreszenzmessung verwendet. Dabei stellt die langsame Azidifizierung des Zytosols in der zweiten Phase des NH3/NH4+-Pulses ein Maß für den NH4+-Einstrom in die Zelle dar. Vorexperimente ergaben, daß neben dem Na+2Cl-K+-Cotransporter auch Ba2+-hemmbare K+-Kanäle und die Ouabain-hemmbare Na+/K+-ATPase zum NH4+-Einstrom beitragen. Außerdem beeinflussen zelleigene pH-Regulationssysteme die zweite Phase des NH3/NH4+-Pulses. Der HOE 694-hemmbare Na+/H+-Austauscher und der DIDS-hemmbare, Na+-abhängige HCO3-/Cl--Austauscher führen durch pH-Gegenregulation unter NH3/NH4+-Gabe zu einer Verminderung der langsamen Azidifizierungsrate. Aufgrund dieser Befunde wurde der Azosemid-hemmbare Anteil der langsamen Azidifizierung in der zweiten Phase des NH3/NH4+-Pulses als spezifisches, qualitatives Maß für die Transportrate des Na+2Cl-K+-Cotransporters herangezogen (Azosemid hemmt diesen Transporter spezifisch und vollständig). Der Na+2Cl-K+-Cotransporter wurde erstens durch einen Abfall von [Cl-]i, zweitens durch Zellschrumpfung, drittens durch [Ca2+]i-Erhöhung und viertens durch Zunahme von [cAMP]i in seiner Aktivität gesteigert. Zellvolumenmessungen an der Rektaldrüse des Dornhais und an der Kolonkrypte der Ratte machen es sehr wahrscheinlich, daß die Zellschrumpfung die gemeinsame Endstrecke aller Aktivierungswege des Cotransporters darstellt und damit das entscheidende Signal für die Na+2Cl-K+-Cotransporter Aktivierung ist. The Na+2Cl-K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units ( delta fluorescence ratio units/1000 s). In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70±0.11 to 2.54±0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93±0.17 to 2.84±0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 µmol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06±0.34 to 2.40±0.30 U/1000 s (n=11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 µmol/l and 5 µmol/l, respectively) was from 2.39±0.24 to 3.06±0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na+2Cl-K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume

Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia

Epithelial cells of the gastrointestinal tract are an important barrier between the "milieu interne" and the luminal content of the gut. They perform transport of nutrients, salts, and water, which is essential for the maintenance of body homeostasis. In these epithelia, a variety of K+ channels are expressed, allowing adaptation to different needs. This review provides an overview of the current literature that has led to a better understanding of the multifaceted function of gastrointestinal K+ channels, thereby shedding light on pathophysiological implications of impaired channel function. For instance, in gastric mucosa, K+ channel function is a prerequisite for acid secretion of parietal cells. In epithelial cells of small intestine, K+ channels provide the driving force for electrogenic transport processes across the plasma membrane, and they are involved in cell volume regulation. Fine tuning of salt and water transport and of K+ homeostasis occurs in colonic epithelia cells, where K+ channels are involved in secretory and reabsorptive processes. Furthermore, there is growing evidence for changes in epithelial K+ channel expression during cell proliferation, differentiation, apoptosis, and, under pathological conditions, carcinogenesis. In the future, integrative approaches using functional and postgenomic/proteomic techniques will help us to gain comprehensive insights into the role of K+ channels of the gastrointestinal tract

No Potassium, No Acid: K+Channels and Gastric Acid Secretion

The gastric H+-K+-ATPase pumps H+ into the lumen and takes up K+ in parallel. In the acid-producing parietal cells, luminal KCNE2/KCNQ1 K+ channels play a pivotal role in replenishing K+ in the luminal fluid. Inactivation of KCNE2/KCNQ1 channels abrogates gastric acid secretion and dramatically modifies the architecture of gastric mucosa

SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The Na+-retaining hormone aldosterone increases the cell-surface expression of the luminal epithelial sodium channel (ENaC) and the basolateral Na+ pump (Na,K-ATPase) in aldosterone-sensitive distal nephron cells in a coordinated fashion. To address the question of whether aldosterone-induced serum and glucocorticoid-regulated kinase-1 (SGK1) might be involved in mediating this regulation of Na,K-ATPase subcellular localization, similar to that of the epithelial Na+ channel (ENaC), we co-expressed the Na,K-ATPase (rat α1- and Xenopus laevis β1-subunits) and Xenopus SGK1 in Xenopus oocytes. Measurements of the Na+ pump current showed that wild-type SGK1 increases the function of exogenous Na,K-ATPase at the surface of Xenopus oocytes. This appeared to be secondary to an increase in Na,K-ATPase cell-surface expression as visualized by Western blotting of surface-biotinylated proteins. In contrast, the functional surface expression of two other exogenous transporters, the heterodimeric amino acid transporter LAT1-4F2hc and the Na+/phosphate cotransporter NaPi-IIa, was not increased by SGK1 co-expression. The total pool of exogenous Na,K-ATPase was increased by the co-expression of SGK1, and similarly also by ENaC co-expression. This latter effect depended on the [Na+] of the buffer and was not additive to that of SGK1. When the total Na,K-ATPase was increased by ENaC co-expression, SGK1 still increased Na,K-ATPase cell-surface expression. These observations in Xenopus oocytes suggest the possibility that SGK1 induction and/or activation could participate in the coordinated regulation of Na,K-ATPase and ENaC cell-surface expression in the aldosterone-sensitive distal nephro