Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [14C]-alpha-methyl glucopyranoside ([14C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [14C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Characterization of JOK-1, a human gastric epithelial cell line

Summary Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line “JOK-1” These cells express conventional, epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherins, β-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epitheliallike barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line “MDCK” also expresses serveral epithelial markers (vimentin, cytokeratin-18, occludin, N-and E-cadherin, β-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.     

Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Characterization of WiDr: A human colon carcinoma cell line

Summary We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus oocytes, and transmembrane currents associated with this transporter were monitored using a two-electrode voltage clamp. Addition of D-glucose to a Na(+)-containing solution bathing these oocytes generated a current which was blocked by phlorizin. Water-injected control oocytes did not exhibit any currents under these conditions. The magnitude and shape of the currents were dependent on the extracellular glucose and Na+ concentrations and the membrane potential. At Vhold = -50 mV, the Km values for glucose and Na+ were 14 +/- 2 (N = 4) microM and 17 +/- 1 (N = 3) mM, respectively. These Km values and imax exhibited voltage dependence: increasing the membrane potential from -30 to -150 mV increased KGlcm and imax threefold and decreased KNam eightfold. The reversal potential (VR) of the phlorizin-sensitive, glucose-dependent current varied with log Nao+ (slope 46 +/- 6 [N = 9] mV). In the absence of sugar, a Na(+)-dependent, phlorizin-sensitive (Ki = 3 +/- 0.5 microM) current was detected only in RNA-injected oocytes. The amplitude of this current at -50 mV was 6 +/- 1% (N = 13) of the maximum current measured in the presence of D-glucose. The VR of this sugar-independent current varied with log Nao+ (slope 63 +/- 1 [N = 4] mV), indicating that the cotransporter may carry Na+ in the absence of sugar. We conclude that the Na+/glucose cotransporter is electrogenic and that investigations of currents associated with its operation can yield valuable insights into the mechanisms of solute translocation.     

Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells

Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK(1) cells, renal proximal epithelial cell line. ANGII inhibited alpha-[14C] methyl-D-glucopyranoside (AMG) uptake into LLC-PK(1) cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity.     

Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line

Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives.     

Calcium and vitamin D modulate mouse colon epithelial proliferation and growth characteristics of a human colon tumor cell line

The role of calcium and vitamin D in the prevention of colorectal cancer is only now being investigated at the organismal, cellular, and molecular biologic levels. Recent epidemiologic studies have supported the hypothesis that dietary calcium and vitamin D may be related to a reduced risk for colon cancer. The evidence from laboratory investigations in animals and in cell culture also indicate a possible preventative effect. Addition of calcium and vitamin D to the roster of developmental cancer chemopreventative agents for further research is warranted.     

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.     

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl(-) conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl(-) conductance by pCLCA1 protein expression. Cl(-) transport was measured as (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-sensitive Cl(-) conductance, endogenous cAMP-dependent Cl(-) conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl(-) conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl(-) conductance were retained through differentiation in Caco-2 cells, whereas Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.     

Characterization and antigen-presenting function of a murine thyroid-derived epithelial cell line

We have developed and evaluated the antigen-presenting function of a murine thyroid-derived epithelial cell line M.5 in order to further investigate the possible role of the thyroid follicular epithelium in the inductive phase of autoimmune thyroiditis. M.5 cells did not express class II major histocompatibility complex encoded (Ia) antigens constitutively, but these could be readily induced with interferon-gamma. We found that Ia expressing M.5 cells were ineffective in stimulating T cell proliferation when tested in a 4-day primary mixed leukocyte reaction (MLR). However, significant T cell stimulation was obtained when phorbol myristate acetate (PMA) was added either to the M.5-T cell co-cultures, or for a brief period to the M.5 cells prior to adding the responder T cells. Cytofluorographic analysis of M.5 cells disclosed that PMA did not significantly alter the expression of Ia antigens. Additional experiments indicated that interleukin 1 (IL-1) was unlikely to represent the co-stimulatory factor generated by PMA. This was based on the observations that M.5 cells failed to secrete significant IL-1 either spontaneously, or in the presence of various stimuli, and that murine recombinant IL-1 failed to substitute for PMA in the activation of T cells. The nature of the co-stimulatory signal is as yet unknown. We conclude from these experiments that a pure population of thyroid-derived epithelial cells may be able to function, under the described circumstances, as antigen presenting cells.