To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.

The serum- and glucocorticoid-inducible kinase-1 (SGK1) is ubiquitously expressed and under genomic control by cell stress (including cell shrinkage) and hormones (including gluco- and mineralocorticoids). Similar to its isoforms SGK2 and SGK3, SGK1 is activated by insulin and growth factors via phosphatidylinositol 3-kinase and the 3-phosphoinositide-dependent kinase PDK1. SGKs activate ion channels (e.g., ENaC, TRPV5, ROMK, Kv1.3, KCNE1/KCNQ1, GluR1, GluR6), carriers (e.g., NHE3, GLUT1, SGLT1, EAAT1-5), and the Na+-K+-ATPase. They regulate the activity of enzymes (e.g., glycogen synthase kinase-3, ubiquitin ligase Nedd4-2, phosphomannose mutase-2) and transcription factors (e.g., forkhead transcription factor FKHRL1, beta-catenin, nuclear factor kappaB). SGKs participate in the regulation of transport, hormone release, neuroexcitability, cell proliferation, and apoptosis. SGK1 contributes to Na+ retention and K+ elimination of the kidney, mineralocorticoid stimulation of salt appetite, glucocorticoid stimulation of intestinal Na+/H+ exchanger and nutrient transport, insulin-dependent salt sensitivity of blood pressure and salt sensitivity of peripheral glucose uptake, memory consolidation, and cardiac repolarization. A common ( approximately 5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight. SGK1 may thus contribute to metabolic syndrome. SGK1 may further participate in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia. SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior. In conclusion, the SGKs cover a wide variety of physiological functions and may play an active role in a multitude of pathophysiological conditions. There is little doubt that further targets will be identified that are modulated by the SGK isoforms and that further SGK-dependent in vivo physiological functions and pathophysiological conditions will be defined.

To clarify the physiological role of Na+-d-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1−/− mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1−/− mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1−/− mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose–free diet. In wild-type mice, passage of d-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1−/− mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

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Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr(-/-)Smpd1(+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.-1 - infection. These data suggest inhibition of Asm as a novel treatment strategy in CF. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis-4 -

Previously we cloned a polyspecific transporter from rat (rOCT1) that is expressed in renal proximal tubules and hepatocytes and mediates electrogenic uptake of organic cations with different molecular structures. Recently a homologous transporter from rat kidney (rOCT2) was cloned but not characterized in detail. We report cloning and characterization of two homologous transporters from man (hOCT1 and hOCT2) displaying approximately 80% amino acid identity to rOCT1 and rOCT2, respectively. Northern blots showed that hOCT1 is mainly transcribed in liver, while hOCT2 is found in kidney. Using in situ hybridization and immunohistochemistry, expression of hOCT2 was mainly detected in the distal tubule where the transporter is localized at the luminal membrane. After expression in Xenopus laevis oocytes, hOCT1 and hOCT2 mediate tracer influx of N-1-methylnicotinamide (NMN), tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP). For cation transport by hOCT2 apparent K(m) and K(i) values were determined in tracer flux measurements. In addition, electrical measurements were performed with voltage-clamped oocytes. Similar to rOCT1, cation transport by hOCT2 was pH independent, electrogenic, and polyspecific; however, the cation specificity was different. In voltage-clamped hOCT2-expressing oocytes, inward currents were induced by superfusion with MPP, TEA, choline, quinine, d-tubocurarine, pancuronium, and cyanine863. Cation transport in distal tubules is indicated for the first time. Here hOCT2 mediates the first step in cation reabsorption. hOCT1 may participate in hepatic excretion of organic cations.

Exposure of erythrocytes to the Ca2+ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca2+-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca2+-sensitive Gardos K+ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 microM) induces activation of inwardly rectifying K+-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K+ concentration to 25 mM and exposure to the Gardos K+ channel blockers charybdotoxin (230 nM) and clotrimazole (5 microM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K+ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 microM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K+ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca2+-activated K+ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca2+-sensitive K+ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis.

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