Protein kinase-A affects sorting and conformation of the sodium-dependent glucose co-transporter SGLT1.

In Chinese hamster ovary cells expressing rabbit sodium-dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8-Br-cAMP) stimulated alpha-methyl D-glucopyranoside uptake. Kinetic analysis revealed an increase in both V(max) and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V(max) of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti-phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [(32)P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D-glucose. These results indicate that PKA-mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA-mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1-mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression.

A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains.

In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.

Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.

Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain.

The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.

Calcium transport in human salivary glands: a proposed model of calcium secretion into saliva.

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).

Sodium-phosphate cotransporter in human salivary glands: molecular evidence for the involvement of NPT2b in acinar phosphate secretion and ductal phosphate reabsorption.

OBJECTIVE: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated. DESIGN: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry. RESULTS: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell. CONCLUSIONS: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells.

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.

Different modes of sodium-D-glucose cotransporter-mediated D-glucose uptake regulation in Caco-2 cells.

We recently reported that a considerable amount of the sodium-d-glucose cotransporter SGLT1 present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments attached to microtubules. A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum, highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent d-glucose uptake into enterocytes, we compared SGLT1-mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 microM), a drug that enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (-60%). We also investigated the effect of altered extracellular D-glucose levels. Cells preincubated (1 h) with d-glucose-free medium exhibited significantly higher sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (+45%) than did cells preincubated with high d-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated d-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that, pharmacologically, d-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool; however, regulation by the physiological substrate d-glucose can be explained only by an alternative mechanism.

Ethanol treatment of hepatocellular carcinoma: high potentials of low concentrations.

Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, which is associated with a very poor prognosis. A curative treatment is difficult to achieve and is only possible in a low number of patients. Therefore, many different therapeutic strategies have been developed as alternative treatment. Among these, percutaneous injection of high concentrations of ethanol (>50 mM) has been proven to be effective for the treatment of small HCC (less than 3 cm in diameter). However, the principal problem with using ethanol is its toxic effects on non-tumor cells adjacent to the tumor area. The objective of this review is to juxtapose the therapeutic potential of high and low concentrations of ethanol in the treatment of HCC, based on experimental studies obtained with the human hepatocellular tumor cell line (HepG2). They have shown that high concentrations of ethanol lead to necrosis, while low concentrations induce apoptosis due to activation of Fas-receptors. Triggering of apoptosis through Fas-receptors represents a mechanism of action different from that observed with high concentrations of ethanol, thus, reducing the complications that follow the inflammatory process due to necrosis. Therefore, the use of low concentrations of ethanol could be an effective treatment for HCC.

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein.

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent D-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K(i) values of 22+/-5 microM for TIPDG and 12+/-2 microM for BzG. These K(i) values are comparable to those of their parent compounds arbutin (25+/-6 microM) and phlorizin (8+/-1 microM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure-function relationship of SGLT1 and other SGLT1 family transporter proteins.

Binding of phlorizin to the isolated C-terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence.

Phlorizin, a phloretin 2'-glucoside, is a potent inhibitor of the Na(+)/glucose cotransporter (SGLT1). On the basis of transport studies in intact cells, a binding site for phlorizin was suggested in the region between amino acids 604-610 of the C-terminal loop 13. To further investigate phlorizin binding titration experiments of the intrinsic Trp fluorescence of isolated wild-type loop 13 and two mutated loops (Y604K and G609K) were carried out. Phlorizin (135 microM) produced approximately 40% quenching of the fluorescence of wild-type loop 13; quenching could also be observed with the two mutated loops. The apparent K(d) was lowest for the wild-type loop 13 (K(d) approximately 23 microM), followed by mutant G609K (57 microM) and mutant Y604K (70 microM). Binding of phlorizin was further confirmed by a decrease of the accessibility of loop 13 to the collisional quencher acrylamide. The interaction involves the aromatic moiety of the aglucone since phloretin (the aglucone of phlorizin) showed almost the same effects as phlorizin, while d-glucose did not. MALDI-TOF experiments revealed that loop 13 contained a disulfide bond between Cys 560 and Cys 608 that is very important for phlorizin-dependent fluorescence quenching. These studies provide direct evidence that loop 13 is a site (important amino acids including 604-609) for the molecular interaction between SGLT1 and phlorizin. They confirm that the aglucone part of the glucoside is responsible for this interaction.

Omental graft improves functional recovery of transected peripheral nerve.

The omentum has several properties that are advantageous for neuronal sprouting and direction. We have therefore analyzed functional recovery following transection of rat sciatic nerve using omental graft to bridge the nerve defect. In group 1, a 25-30-mm nerve defect was produced and bridged with omental graft, whereas in group 2, an end-to-end repair was performed. The sciatic function index (SFI) was assessed at 2-week intervals until 8 weeks after surgery. Functional recovery was faster in group 1 than in group 2. After 8 weeks, SFI was improved significantly from -100% to -45% (+/- -4%) in group 1 (P < 0.001) compared to -72% +/- -2% in group 2 (n = 10). Histologically, the omental graft contained more newly developed nerve fibers and less scar tissue than the end-to-end repair. Thus, omental graft appears to improve directional growth of regenerating axon sprouts and may be a means of treating peripheral nerve injury.