Cell volume regulation: osmolytes, osmolyte transport, and signal transduction.

In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.

Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney.

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.

Identification of a region critically involved in the interaction of phlorizin with the rabbit sodium-D-glucose cotransporter SGLT1.

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604-610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of alpha-methyl-D-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K(i) for phlorizin increased at least by a factor of 5 compared to the wild-type K(i) of 4.6 +/- 0.7 micromol/l; most striking changes were observed for Y604K (K(i) = 75.3 +/- 19.0 micromol/l) and C608K (K(i) = 83.6 +/- 13.9 micromol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (Km = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.

Different effects of cyclosporine a and FK506 on potassium transport systems in MDCK cells.

BACKGROUND: Hyperkalemia and metabolic acidosis are common manifestations in patients receiving the immunosuppressive agent cyclosporine A (CsA) and the recently introduced FK506. We compared the acute toxic and antiproliferative effects as well as the effects on the transport activity of Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter of CsA and FK506 in an established cell line of distal/collecting tubule origin (MDCK cells). METHODS: MDCK cells were exposed to various concentrations of CsA or FK506 and the effects on cell viability (MTT test and neutral red uptake), plasma membrane integrity (lactate dehydrogenase (LDH) release) and cell proliferation (bromodeoxyuridine (BrdU) incorporation) were compared. For transport studies, after confluence, MDCK cells were exposed to CsA or FK506 for 48 h in the presence and absence of aldosterone. Ouabain- and bumetanide-sensitive (86)Rubidium uptake measurements were used to study the activity of the Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter at the surface of intact cells. RESULTS: After 24 h of exposure CsA reduced the number of viable cells to 50% at 30 microM, whereas for FK506 2-3 times higher concentrations had to be employed. Similarly, LDH release was stimulated tenfold by 30 microM CsA but only fourfold by 70 microM FK506. In contrast, DNA synthesis was affected at lower concentrations of FK506 than of CsA. In cells treated for 24 h BrdU incorporation was significantly inhibited by 3 microM FK506, whereas a similar inhibition required 10 microM CsA. The transport activity of Na(+)/K(+)-ATPase and of Na(+)/K(+)/2Cl(-) cotransporter were significantly decreased (37 and 63%, respectively) on CsA administration (8 microM). In CsA-treated cells the K(+) channel blockers barium (1 mM), TEA (10 mM) and quinine (1 mM) did not further inhibit the transport activities suggesting that CsA might also act via inhibition of K(+) channels. FK506 at 8 microM had no effect on Na(+)/K(+)-ATPase transport activity but stimulated Na(+)/K(+)/2Cl(-) cotransporter activity by 59%. The stimulatory effect was abolished by K(+) channel blockers indicating that recycling of K(+) might increase by FK506. The simultaneous presence of aldosterone (5 microM) protected the cells from the inhibitory effect of CsA on Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter activity. The stimulatory effect of FK506 on the Na(+)/K(+)/2Cl(-)cotransporter activity was completely abolished in the presence of aldosterone. CONCLUSIONS: Both CsA and FK506 showed acute toxicity in MDCK cells in vitro with the effects of FK506 being less pronounced. CsA and FK506 had different effects on the in vivo transport rates of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter; CsA inhibited the activity of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter whereas FK506 stimulated the activity of Na(+)/K(+)/2Cl(-) cotransporter. These effects were abolished by the application of aldosterone.

Apoptosis induced in HepG2 cells by short exposure to millimolar concentrations of ethanol involves the Fas-receptor pathway.

PURPOSE: We previously found that ethanol-induced apoptosis is associated with an activation of caspase-3. However, the initial triggering of this process is yet unknown. Therefore, the present study was designed to determine whether the Fas-receptor pathway plays a role in the initiation by ethanol of human hepatocellular carcinoma (HepG2) cell apoptosis. METHODS: HepG2 cells were incubated with or without 1 mM ethanol for 24 h. Apoptosis was assessed by DNA fragmentation and caspase-8 activity. Selective inhibitors of caspase-8 and caspase-9 were used to analyze the role of both caspases on apoptosis. Soluble human Fas ligand (Fas-L) was determined by enzyme-linked immunosorbent assay (ELISA). A fluorescent dye was used to investigate the permeability of the mitochondrial outer membrane. A recombinant Fas fusion protein was used to inhibit the activation of Fas receptors. Human anti-Fas-L antibody was employed to neutralize Fas-L released from the cells. RESULTS: Caspase-8 activity increased significantly threefold (P < 0.005) after 12 h incubation of HepG2 cells with 1 mM ethanol whereas no change was observed in control cells. Incubation with caspase-8 inhibitor completely prevented apoptosis induced by ethanol (P < 0.001). In contrast, a caspase-9 inhibitor did not significantly reduce apoptosis. The permeability of the outer mitochondrial membrane was not altered. Neutralization of Fas-receptors by Fas fusion proteins completely attenuated ethanol-induced apoptosis in HepG2 cells treated with ethanol. CONCLUSIONS: These findings show that apoptosis induced by low concentrations of ethanol in human HepG2 cells is associated with Fas-receptor activation and subsequent caspase-8 activation. Triggering of apoptosis through Fas-receptors represents a mechanism of action different from that observed with high concentrations of ethanol.

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias.

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

Non-invasive single cell pH measurements in the isolated perfused pancreas.

1. A new method was developed for non-invasive investigations of intracellular pH (pHi) regulation in different cell types of the isolated perfused pancreas using a confocal laser scanning technique. 2. After removal of the rat pancreas the coeliac artery was cannulated and the splenic segment of the pancreas was perfused with dextran (5%)-Ringer solution at a constant flow rate of 2 mL/min. In a temperature-controlled (37 degrees C) chamber, pH regulation was studied using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein (BCECF) with a confocal microscope (MRC-600; Bio-Rad, Hercules, CA, USA). 3. Image analysis permitted the identification and comparison of different cell types with a pHi of 7.26+/-0.1 in acinar cells and of 7.02+/-0.1 in endothelial cells. Increasing PCO2 from 5 to 20% resulted in a rapid decrease in pHi. Omission of sodium from the perfusate resulted in a smooth decline in pHi. Both decreases were found to be fully reversible. Increasing PCO2 under sodium-free conditions also resulted in a drop of pHi that was, however, not fully reversible, suggesting involvement of the Na+/H+ exchanger in the regulation of pHi in the intact organ. 4. The above method completely preserves tissue integrity and, therefore, allows the study of pH regulation in different cell types of the pancreas simultaneously and without interference with their functional arrangement. The technique should be of specific value to investigate experimental disease states of the pancreas.

Evolution of the Na-P(i) cotransport systems.

Membrane transport systems for P(i) transport are key elements in maintaining homeostasis of P(i) in organisms as diverse as bacteria and human. Two Na-P(i) cotransporter families with well-described functional properties in vertebrates, namely NaPi-II and NaPi-III, show conserved structural features with prokaryotic origin. A clear vertical relationship can be established among the mammalian protein family NaPi-III, a homologous system in C. elegans, the yeast system Pho89, and the bacterial P(i) transporter Pit. An alternative lineage connects the mammalian NaPi-II-related transporters with homologous proteins from Caenorhabditis elegans and Vibrio cholerae. The present review focuses on the molecular evolution of the NaPi-II protein family. Preliminary results indicate that the NaPi-II homologue cloned from V. cholerae is indeed a functional P(i) transporter when expressed in Xenopus oocytes. The closely related NaPi-II isoforms NaPi-IIa and NaPi-IIb are responsible for regulated epithelial Na-dependent P(i) transport in all vertebrates. Most species express two different NaPi-II proteins with the exception of the flounder and Xenopus laevis, which rely on only a single isoform. Using an RT-PCR-based approach with degenerate primers, we were able to identify NaPi-II-related mRNAs in a variety of vertebrates from different families. We hypothesize that the original NaPi-IIb-related gene was duplicated early in vertebrate development. The appearance of NaPi-IIa correlates with the development of the mammalian nephron.

Functional asymmetry of the sodium-D-glucose cotransporter expressed in yeast secretory vesicles.

The sodium-D-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane. By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H+-ATPase and invertase. In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis. In addition, higher molecular weight protein bands probably representing dimers and aggregates were found. In transport studies with the microsomes D-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence. Influx of D-glucose showed no dependence on sodium and was not inhibited by phlorizin. Furthermore, the transporter exhibited a striking asymmetry with regard to the D-glucose affinity and the sugar specificity. These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells. Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter.

Calcium signalling during RVD of kidney cells.

The balance of a high extracellular osmolarity in the kidney medulla is mainly based on an accumulation of organic osmolytes in the cells. The regulation of cell volume during hypotonic conditions results in a release of organic osmolytes - a process that is partly calcium-dependent. Using calcium-sensitive fluorescent dye and confocal laser scanning microscopy, we have investigated calcium signalling during regulatory volume decrease (RVD) in kidney cells. In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induced a calcium release from intracellular stores that preceded calcium entry from the extracellular milieu. Hyposmotic stress had no effect on the cellular IP(3) content. Preincubation with 100 micromol/l ETYA (a non-metabolizible derivative of arachidonic acid), however, reduced the calcium response to hypotonic stress as well as the RVD. Blocker of voltage-dependent calcium channels (verapamil, diltiazem, and nifedipine) in the concentration of 40 micromol/l reduced partly the calcium response. SKF-96365, an inhibitor of receptor-mediatedcalcium channels, also attenuated the calcium influx. In conclusion, swelling of IMCD cells increases intracellular calcium by release from intracellular stores and entry across the cell membranes. The signalling involves arachidonic acid metabolism.

Regulation of sorbitol efflux in different renal medullary cells: similarities and diversities.

It is well accepted that organic osmolytes, including sorbitol, play a major role in the volume regulation of renal medullary cells. The signal leading to an activation of release channels during RVD is, however, poorly understood. Hypotonicity induced sorbitol efflux was investigated in freshly isolated rat inner medullary collecting duct (IMCD) cells and in rabbit medullary thick ascending limb of Henle's loop (TALH) cells biochemically or using labeled sorbitol. The time course of release was compared with changes in cell volume, measured by confocal microscopy, and alterations in cell calcium (Ca(i)) determined by Fura 2 technology. In IMCD cells sorbitol release, volume decrease and Ca(i) transients show a close temporal correlation. In addition increases in Ca(i) without volume changes stimulate sorbitol efflux. In TALH cells sorbitol release starts after a significant lag time and reaches a maximum when cell volume is already partially restored. The same discrepancy is observed with regard to changes in Ca(i) and sorbitol efflux. These studies suggest that in IMCD cells changes in Ca(i) are the main regulator for the sorbitol permeability of the plasma membrane. The sorbitol channel present in TALH cells seems to operate predominantly independently of Ca(i). Despite this diversity in signal transduction the sorbitol channels in both renal cell types appear, however, not to be stretch-activated.

Cytotoxicity of millimolar concentrations of ethanol on HepG2 human tumor cell line compared to normal rat hepatocytes in vitro.

PURPOSE: The antiproliferative effect of high concentrations of ethanol (80-100 mmol) on liver carcinoma is well known. However, the high concentrations of ethanol affect both tumor cells and normal hepatocytes. The present study was designed to determine the effect of low ethanol concentrations (0-10 mmol) on cell proliferation and cell death (apoptosis and necrosis) in a human tumor cell line HepG2 and in normal rat hepatocytes. METHODS: Primary cultures of normal rat hepatocytes and HepG2 cells cultures were used. Cells were incubated with increasing ethanol concentrations or without ethanol (control group) for 24 h and analyzed immediately (group I) or after an additional incubation time of 48 h without additional ethanol application (group II). Cell proliferation was determined by assessing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Apoptosis was assessed by means of DNA fragmentation and cysteine aspartate-specific protease (caspase-3) activity. Necrosis was analyzed by quantification of lactate dehydrogenase (LDH) release into culture medium. RESULTS: Twenty-four h exposure to 1 mmol ethanol inhibited cell proliferation in HepG2 cells by 75% (P < 0.05), while it remained unaltered in rat hepatocytes. The effect of ethanol persisted for another 48 h where cell proliferation was 5% of control in HepG2 cells and 70% of control in rat hepatocytes (P < 0.005). After 24 h incubation with 1 mmol ethanol 28% of HepG2 cells and 12% of rat hepatocytes showed DNA fragmentation as sign of apoptosis (P < 0.001). In group II 39% of HepG2 cells and 26% of rat hepatocytes were apoptotic (P < 0.001). Caspase-3 activation progressively increased after ethanol treatment in HepG2 cells and rat hepatocytes. The first significant difference was observed after 4 h (activity in HepG2 was 68% higher than in rat hepatocytes) and was maximum after 10 to 12 h where the activity in HepG2 was 180% of the activity in rat hepatocytes. Lactate dehydrogenase release into culture medium as an indicator of necrosis in HepG2 cells, increased from 0.5% in group I to 12% in group II, and from 0.1% to 8% in rat hepatocytes (P < 0.005). Increasing ethanol concentration to 10 mmol increased necrosis to 75% in HepG2 cells, and to 45% in rat hepatocytes (P < 0.05) whereas the effects on cell proliferation and apoptosis were not significantly different. CONCLUSIONS: Small ethanol concentrations (equivalent to 1 mmol) inhibit cell proliferation and increase apoptosis more strongly in HepG2 cells than in normal rat hepatocytes. These findings suggest the use of 1 mmol ethanol as a treatment for hepatocellular carcinoma because this mainly affects tumor cells but not surrounding normal tissue.

Cellular uptake of dietary flavonoid quercetin 4'-beta-glucoside by sodium-dependent glucose transporter SGLT1.

Although it has been suggested that the intestinal glucose transporter may actively absorb dietary flavonoid glucosides, there is a lack of direct evidence for their transport by this system. In fact, our previous studies with the human Caco-2 cell model of intestinal absorption demonstrated that a major dietary flavonoid, quercetin 4'-beta-glucoside, is effluxed by apically expressed multidrug resistance-associated protein-2, potentially masking evidence for active absorption. The objective of this study was to test the hypothesis that quercetin 4'-beta-glucoside is a substrate for the intestinal sodium-dependent D-glucose cotransporter SGLT1. Cellular uptake of quercetin 4'-beta-glucoside was examined with Caco-2 cells and SGLT1 stably transfected Chinese hamster ovary cells (G6D3 cells). Although quercetin 4'-beta-glucoside is not absorbed across Caco-2 cell monolayers, examination of the cells by indirect fluorescent microscopy as well as by HPLC analysis of cellular content revealed cellular accumulation of this glucoside after apical loading. Consistent with previous observations, the accumulation of quercetin 4'-beta-glucoside in both Caco-2 and G6D3 cells was markedly enhanced in the presence of multidrug resistance-associated protein inhibition. Uptake of quercetin 4'-beta-glucoside was greater in SGLT1-transfected cells than in parental Chinese hamster ovary cells. Uptake of the glucoside by Caco-2 and G6D3 cells was sodium-dependent and was inhibited by the monovalent ionophore nystatin. In both Caco-2 and G6D3 cells, quercetin 4'-beta-glucoside uptake was inhibited by 30 mM glucose and 0.5 mM phloridzin. These results demonstrate for the first time that quercetin 4'-beta-glucoside is transported by SGLT1 across the apical membrane of enterocytes.

Short exposure to millimolar concentrations of ethanol induces apoptotic cell death in multicellular HepG2 spheroids.

PURPOSE: We have shown previously that 1 mM ethanol reduces cell proliferation and increases apoptosis in monolayers of human hepatocellular carcinoma (HepG2) cells. However, in vivo liver tumors are usually three-dimensional and multicellular. The purpose of this study was therefore to determine the effect of ethanol in multicellular tumor spheroids (MCTS) as a model system in vitro. METHODS: After the application of 1 mM ethanol for 24 h and 48 h, viable, apoptotic and necrotic cells within MCTS were stained with specific fluorescent dyes, and their amount and distribution within the MCTS were assessed by confocal laser scanning microscopy. To evaluate the effect on HepG2 cell migration and cell proliferation, the outgrowth potential after 1 week in culture was evaluated. RESULTS: As assessed by YO-PRO-1 staining, ethanol increased the number of apoptotic cells from 21.5 units (U) in control spheroids to 364 U and 482.2 U after 24 h and 48 h in ethanol-treated spheroids, respectively (P < 0.001). Merocyanine staining fluorescence increased from 10.7 U in the control to 122 U after 24 h and 293.2 U after 48 h (P < 0.001). Cell viability, as determined by staining with the acetoxymethyl ester of calcein, decreased from 578.5 U in the control to 236 U and 73.4 U after 24 h and 48 h of ethanol exposure respectively (P < 0.001). Necrosis showed an increase from 2 U in control to 24.9 after 24 h and 54 U after 48 h. MCTS treated with ethanol showed almost complete inhibition of outgrowth potential after 1 week in culture, compared to controls (P < 0.005). CONCLUSIONS: Small concentrations of ethanol (1 mM) induced apoptosis in HepG2 MCTS with a concomitant inhibition on outgrowth potential, accompanied with a low degree of necrosis. These findings suggest that low concentrations of ethanol may already be sufficient for the treatment of hepatocellular carcinoma.

Probing the conformation of the sugar transport inhibitor phlorizin by 2D-NMR, molecular dynamics studies, and pharmacophore analysis.

Sodium/D-glucose cotransport, one of the prototypes for sodium gradient-driven symport systems in kidney and intestine, is known to be inhibited by aromatic and aliphatic glucosides (Diedrich, D. F. Biochim. Biophys. Acta 1963, 71, 688-700; Diedrich, D. F. Arch. Biochem. Biophys. 1966, 117, 248-256; Kipp, H.; et al. Biochim. Biophys. Acta 1996, 1282, 124-130; Ramaswamy, K.; et al. Biochim. Biophys. Acta 1976, 433, 32-38). The conformation in which the most potent inhibitor, phlorizin, interacts with the transport protein was investigated with different approaches. Phlorizin consists of the glucose moiety and two aromatic rings (A and B) joined by an alkyl spacer. First the interaction of these various parts of the molecule was determined by two-dimensional (2D) solution NMR. From the 2D-NOESY (nuclear Overhauser effect) measurements spatial distances (up to 5 A) between various interacting H atoms could be detected. Using these values as distance constraints, conformations of phlorizin were calculated and analyzed by the valence force-field method. As a result, a set of conformations could be obtained. The most probable phlorizin conformation shows a nearly perpendicular arrangement of the two aromatic rings (A and B) with the ring B situated above the sugar ring. A very similar conformation could be found by using molecular dynamics simulations when water was chosen as the solvent. This phlorizin conformation in aqueous solution then served as a template for conformational analysis of various phlorizin derivatives. The resulting conformations of derivatives were taken as input to establish a pharmacophore model using the DISCO calculation. As a result, the essential elements of phlorizin for interaction with its binding pocket could be deduced: namely hydrogen bonding via hydroxyl groups of the pyranoside at C(2), C(3), C(4), and C(6) and at C(4) and C(6) of aromatic ring A and hydrophobic interactions via the pyranoside ring and aromatic ring A. Finally, from these conformational features of the pharmacophore the dimension of the phlorizin binding site on the sodium/D-glucose cotransporter was estimated to be 17 x 10 x 7 A(3).

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1.

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.

Probing transmembrane topology of the high-affinity Sodium/Glucose cotransporter (SGLT1) with histidine-tagged mutants.

To reexamine the existing predictions about the general membrane topology of the high-affinity Na+/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 x Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na+/glucose cotransport activity at 0.1 mM D-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541-aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition.

Vitalism and synthesis of urea. From Friedrich Wöhler to Hans A. Krebs.

In 1828, Friedrich Wöhler, a German physician and chemist by training, published a paper that describes the formation of urea, known since 1773 to be a major component of mammalian urine, by combining cyanic acid and ammonium in vitro. In these experiments the synthesis of an organic compound from two inorganic molecules was achieved for the first time. These results weakened significantly the vitalistic hypothesis on the functioning of living cells, although Wöhler, at that time, was more interested in the chemical consequences of isomerism than in the philosophical implications of his finding. However, the chemical synthesis observed by Wöhler does not represent the reaction which is employed in the mammalian liver for urea synthesis. The mechanism of this process was elucidated by the German physician Hans A. Krebs and his medical student Kurt Henseleit in 1932 and was shown to include the ornithine cycle. This 'urea cycle' is only observed in living cells; this apparently vitalistic phenomenon is caused by the compartmentalization of the various enzymatic reactions in mitochondria and cytosol, respectively.