Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.     

Comparative study on sodium transport and Na+,K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine

The present study reports on the effects of dopamine on sodium transepithelial transport and Na+,K+-ATPase activity in Caco-2 cells, a human epithelial intestinal cell line which undergoes enterocyte differentiation in culture, and jejunal epithelial cells from 20 day old Wistar rats. Addition of amphotericin B to the mucosal side stimulated Isc in a concentration dependent manner (Caco-2 cells, EC50=0.9 [0.5, 1.7] microM; rat jejunum, EC50=7.4 [0.8; 70.1] microM). The presence of 1 microM dopamine did not change the effect of amphotericin B in Caco-2 cells, but produced a significant (P<0.05) decrease in the maximal effect of amphotericin B in the rat jejunum. Dopamine (1 microM), added to the serosal side, did not change the Isc profile in Caco-2 cells, but produced a significant increase in the rat jejunum. This effect was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). Basal Na+,K+-ATPase activity (in nmol Pi mg protein(-1) min(-1)) in Caco-2 cells (49.5+/-0.2) was similar to that observed in isolated rat jejunal epithelial cells (52.3+/-3.4). Dopamine (1 microM) significantly (P<0.05) decreased Na+,K+-ATPase activity in rat jejunal epithelial cells, but failed to inhibit Na+,K+-ATPase in Caco-2 cells. This effect of dopamine was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). The specific binding of [3H]-Sch 23390 to the rat intestinal mucosa was saturable with an apparent dissociation constant (KD) of 2.4 (0.4; 4.5) nM and maximum receptor density of 259.8+/-32.6 fmol/mg protein. No significant specific binding of [3H]-Sch 23390 was observed in membranes from Caco-2 cells. In conclusion, the results obtained show that D1-like receptor mediated effects of dopamine in the rat jejunum on sodium absorption are absent in Caco-2 cells, most probably because this cell line does not express D1-like dopamine receptors, which ultimately are responsible for the inhibitory effect of the amine upon intestinal Na+,K+-ATPase.     

Castanospermine blocks the hyperglycemic response to carbohydrates in vivo: a result of intestinal disaccharidase inhibition

Castanospermine is a potent inhibitor of rat intestinal glycohydrolases in vitro and prevents the hyperglycemic response to an oral sucrose challenge in vivo. Among the glycohydrolases tested, castanospermine was most effective against sucrase with an IC50 of 1.1 x 10(-7) M. In vivo, a significant effect was seen at doses less than 1 mg/kg in both normal and streptozotocin-treated rats. Castanospermine has a prolonged duration of activity in vivo with significant activity when administered 4 hours before sucrose.     

Time-dependent inhibition of sucrase and isomaltase from rat small intestine by castanospermine

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a potent time-dependent inhibitor of the sucrase-isomaltase complex purified from rat small intestine, in vitro. First-order kinetics for the inactivation of sucrase and isomaltase by castanospermine were observed. Protection studies showed that castanospermine competes for the glucosyl subsite with the substrates of sucrase and isomaltase. The second-order rate constants (k1) for the association reaction between castanospermine and the protein complex were calculated to be 6.5 X 10(3) and 0.3 X 10(3) M-1 s-1 for sucrase and isomaltase, respectively. Only barely detectable reactivation of the inhibited isomaltase was detectable over 24 h, whereas about 30% reactivation of the inhibited sucrase was observed in 24 h (k2 = 3.6 X 10(-6) s-1). These results suggest that castanospermine functions as a transition-state analog that binds extremely tightly to sucrase and isomaltase.     

Triiodothyronine Stimulates the Expression of Sucrase–Isomaltase in Caco-2 Cells Cultured in Serum-Free Medium

In a previous study we have shown that triiodothyronine (T₃) added to a serum-free medium supplemented with insulin, transferrin, and selenous acid (ITS) can stimulate Caco-2 cell differentiation. In this study we have focused on the effects of T₃on sucrase activity. The results obtained demonstrate that T₃(50 nM) does not change Caco-2 cell proliferation but enhances sucrase activity from 50 to 80%. Similar increases were observed whether or not insulin was present in the culture medium, showing that there was no synergistic effect between T₃and insulin on sucrase activity. Moreover, T₃acts specifically during the differentiation period since addition of T₃to the defined TS medium before confluency is reached does not stimulate sucrase activity. Sucrase kinetic parameters were evaluated for the first time in Caco-2 cells under various culture conditions. The presence of a single enzyme was verified, with aK_mof about 7 mMand aV_maxaround 20 nmol of substrate hydrolyzed min⁻¹mg⁻¹of protein. Our results showed that T₃did not change the enzyme's affinity for sucrose but doubled theV_max. Moreover, immunoblotting using anti-sucrase–isomaltase (SI) antibodies revealed an approximately twofold increase in the relative amount of SI immunoreactive protein in T₃-stimulated cells compared to untreated cells. Results obtained by both Northern hybridization and RT-PCR amplification showed a significant increase in SI mRNA contents. These results suggest that T₃acts primarily on sucrase expression at the mRNA level.     

Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis

We report data from two related assay systems (isolated enzyme assays and whole blood assays) that C-phycocyanin a biliprotein from Spirulina platensis is a selective inhibitor of cyclooxygenase-2 (COX-2) with a very low IC(50) COX-2/IC(50) COX-1 ratio (0.04). The extent of inhibition depends on the period of preincubation of phycocyanin with COX-2, but without any effect on the period of preincubation with COX-1. The IC(50) value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the well-known selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC(50) value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved.     

Preparation and characterization of radioactive castanospermine

A procedure for the preparation of tritiated castanospermine is described. The tritiated alkaloid was shown to be chromatographically identical to the native material and exhibited the same inhibitory properties. Radiolabeled castanospermine tightly bound to purified intestinal sucrase. Following gel chromatography, each mole of enzyme was shown to have bound 1 mol of the radioactive alkaloid. Cultured MDCK cells were also shown to take up the labeled castanospermine. This compound should be a useful tool in the investigation of enzymes that are responsible for the processing of glycoprotein oligosaccharides.     

Studies on the mechanism of castanospermine inhibition of alpha- and beta-glucosidases

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine alkaloid that was isolated from the Australian plant, Castanospermum australe. This alkaloid was found to be a potent inhibitor of lysosomal alpha- and beta-glucosidases. In this report, the mechanism of inhibition of amyloglucosidase (an exo-1,4-alpha-glucosidase) and almond emulsin beta-glucosidase was examined. Castanospermine proved to be a competitive inhibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the p-nitrophenyl-alpha-D-glucoside. It was also a competitive inhibitor of almond emulsin beta-glucosidase at pH 6.5, but in this case previous studies had shown that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation mixture had a marked effect on the inhibition. Thus, in all cases, castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower pH values. The pK for castanospermine was found to be 6.09, indicating that the alkaloid was probably more active in the unprotonated form. This was also suggested by the fact that the N-oxide of castanospermine, while still a competitive inhibitor, was 50 to 100 times less active than was castanospermine, and its activity was not markedly altered by pH. These results probably explain why castanospermine is a good inhibitor of the glycoprotein processing enzyme, glucosidase I, since this is a neutral enzyme.     

Development of vasoactive intestinal peptide-responsive adenylate cyclase during enterocytic differentiation of Caco-2 cells in culture. Evidence for an increased receptor level

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.     

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.     

Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.     

Mechanistic studies on protopanaxadiol, Rh2, and ginseng (Panax quinquefolius) extract induced cytotoxicity in intestinal Caco-2 cells

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.     

Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.     

A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.     

A cyclic AMP protein kinase A-dependent mechanism by which rotavirus impairs the expression and enzyme activity of brush border-associated sucrase-isomaltase in differentiated intestinal Caco-2 cells

We undertook a study of the mechanism by which rhesus monkey rotavirus (RRV) impairs the expression and enzyme activity of brush border-associated sucrase isomaltase (SI) in cultured, human, fully differentiated, intestinal Caco-2 cells. We provide evidence that the RRV-induced defects in the expression and enzyme activity of SI are not related to the previously observed, RRV-induced, Ca2+ -dependent, disassembly of the F-actin cytoskeleton. This conclusion is based on the facts that: (i) the intracellular Ca2+ blocker, BAPTA/AM, which antagonizes the RRV-induced increase in [Ca2+](i), fails to inhibit the RRV-induced decrease in SI expression and enzyme activity; and (ii) Jasplakinolide (JAS) treatment, known to stabilize actin filaments, had no effect on the RRV-induced decrease in SI expression. Results reported here demonstrate that the RRV-induced impairment in the expression and enzyme activity of brush border-associated SI results from a hitherto unknown mechanism involving PKA signalling. This conclusion is based on the observations that (i) intracellular cAMP was increased in RRV-infected cells and (ii) treatment of RRV-infected cells with PKA blockers resulted in the reappearance of apical SI expression, accompanied by the restoration of the enzyme activity at the brush border. In addition, in RRV-infected cells a twofold increase of phosphorylated form of cytokeratin 18 was observed after immunopurification and Western Blot analysis, which was antagonized by exposing the RRV-infected cells to the PKA blockers.     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Ranitidine induces inhibition and structural changes in sucrase

Ranitidine is an antagonist of histamine-2 (H(2)) receptor. It is employed to treat peptic ulcer and other conditions in which gastric acidity must be reduced. Sucrase is a hydrolytic enzyme that catalyzes the breakdown of sucrose to its monomer content. A liquid of yeast sucrase was developed for treatment of congenital sucrase-isomaltase deficiency (CSID) in human. In this study, the effect of ranitidine on yeast sucrase activity was investigated. Our results showed that ranitidine binds to sucrase and inhibits the enzyme in a noncompetitive manner. The K(i) and IC(50) values were measured to be about 2.3 and 2.2 mM, respectively. Fluorescence measurement showed conformational changes after binding of ranitidine to the enzyme. The fluorescence spectra showed that ranitidine could bind to both free enzyme and enzyme-substrate complex, which was accompanied with reduction of emission intensity and red shift production.     

Stimulatory effect of regucalcin on mitochondrial ATP-dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca(2+) signaling, on Ca(2+)-ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-6) M) or lanthunum chloride (10(-6) M), an inhibitor of mitochondrial Ca(2+) uptake, markedly inhibited regucalcin (100 nM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (100 nM) in elevating Ca(2+)-ATPase activity was completely prevented by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca(2+)-ATPase activity was not further enhanced by calmodulin (0.30 microM) or dibutyryl cyclic AMP (10(-4) M), which could increase Ca(2+)-ATPase activity. Trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, significantly decreased Ca(2+)-ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca(2+)-pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.     

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.