Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (K(M) = 5.3 microm), which is competitively inhibited by cyanidine 3-glucoside (Ki = 51.6 microm) and mainly noncompetitively by cyanidin (Ki = 88.3 microm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd = 1.7 microm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabeled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.     

The interaction of anthocyanins with bilitranslocase

Bilitranslocase (TC 2.A.65.1.1) is an organic anion membrane carrier expressed at the sinusoidal domain of the liver plasma membrane and in epithelial cells of the gastric mucosa. Its substrates are sulfobromophthalein, bilirubin, and nicotinic acid. This work reports on the identification of a new class of bilitranslocase substrates, i.e., anthocyanins. Seventeen out thes 20 compounds tested behaved as competitive inhibitors of bilitranslocase transport activity (K(I)=1.4-22 microM). Their structure-activity relationship reveals that mono- and di-glucosyl anthocyanins, the anthocyanin species occurring in food, are better ligands than the corresponding aglycones. Moreover, the first interaction of anthocyanins with the carrier occurs through hydrophilic moieties, such as the 3-glucosyl moiety and the B ring for the monoglucosides, through the 5-glucosyl moiety and the A ring for the diglucosides, and through either the B or the A ring for the aglycones. These findings suggest that bilitranslocase could play a role in the bioavailability of anthocyanins.     

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin-binding proteins was tested by searching for peptides containing the bilirubin-binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.     

The sulfhydryl groups responsible for bilitranslocase transport activity respond to the interaction of the carrier with bilirubin and functional analogues

Both inactivation of sulfobromophthalein transport in rat liver plasma membrane vesicles by sulfhydryl group reagents and subsequent reactivation by 2-mercaptoethanol are shown to be modulated by ligands to bilitranslocase. In particular, bilirubin, sulfobromophthalein and Thymol blue behave as negative effectors in the inactivation reaction and as positive effectors in the reactivation reaction. Kinetic data provide further evidence of the existence of two classes of sulfhydryl groups involved in transport activity. The effect brought about by remarkably low concentrations of bilirubin is in line with the physiological function of bilitranslocase as a bilirubin carrier.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono- and poly-clonal antibodies

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane.     

The role of sulfhydryl groups in sulfobromophthalein transport in rat liver plasma membrane vesicles

Sulfobromophthalein (BSP) electrogenic transport activity in a plasma membrane vesicle preparation from rat liver is shown to depend on free sulfhydryl groups. These are organized in two classes, one of which does not react with the sulfhydryl group reagent 5,5'-dithiobis(2-nitrobenzoate). The two classes appear to be involved in BSP transport independently. However, reactivity of one class can be shown to be affected by alkylation of the other. Hence, it is concluded that both classes are located on the same carrier system, which previous research has established to be the integral sinusoidal membrane protein bilitranslocase.     

Involvement of glutamate–cystine/glutamate transporter system in aspirin-induced acute gastric mucosa injury

Large-dose or long-term use of aspirin tends to cause gastric mucosa injury, which is recognized as the major side effect of aspirin. It has been demonstrated that glutamate exerts a protective effect on stomach, and the level of glutamate is critically controlled by cystine/glutamate transporter (Xc⁻). In the present study, we investigated the role of glutamate–cystine/glutamate transporter system in aspirin-induced acute gastric mucosa injury in vitro and in vivo. Results showed that in human gastric epithelial cells, aspirin incubation increased the activity of LDH and the number of apoptotic cells, meanwhile down-regulated the mRNA expression of Xc⁻ accompanied with decreased glutamate release. Similar results were seen in a rat model. In addition, exogenous l-glutamate attenuated the gastric mucosa injury and cell damage induced by aspirin both in vitro and in vivo. Taken together, our results demonstrated that acute gastric mucosa injury induced by aspirin is related to reduction of glutamate–cystine/glutamate transporter system activity.     

Study of localization of H3-pentagastrin in the fundal gland area of the rat gastric mucosa by means of light and electron autoradiography]

The most frequent localization of H-3-pentagasrine was found in the gastric mucosa of rats, a weak incorporation was observed in the cells of duodenum; liver and skeletal muscles showed no incorporation. In the acid-secreting region of gastric mucosa, the labeled pentagastrine was accumulated selectively in the main and endocrin cells, which allowed to suppose the presence of gastrine receptors in these cells. These data, together with the absence of the label over the oxyntic cells, amy evidence for the participation of endocrine histamine-containing cells in the control of hydrochloric acid secretion in the rat stomach.     

Taenia taeniaeformis: Increased cell growth and neutral mucus production in the gastric mucosa of the rat with a larval infection

Gross hyperplasia of the gastric mucosa and excessive mucus production in the stomach occur in rats heavily parasitized with larvae of Taenia taeniaeformis. In this study, a positive correlation between the number of larvae recovered from hepatic cysts and the weight of the stomachs of infected rats was found. By light microscopy, the hyperplasia was restricted to the glandular mucosa. Parietal and chief cells were very rare, and densely PAS-positive mucous cells were the major cell types in the hyperplastic stomach while, in comparison, alcian blue-positive cells were much fewer in number. The isolated gastric mucosa in organ culture had an increased [³H]thymidine incorporation rate in rats infected with T. taeniaeformis. The hexosamine concentration per milligram protein in the hyperplastic stomach mucosa was twice that in the control rat stomach mucosa. By electron microscopy, the apical cytoplasm of the mucous cells was found to be filled with small dark granules. These results indicate that the gastric hyperplasia is caused by stimulation of growth and major differentiation of stem cells to neutral mucus-producing cells.     

Distribution of 35S-taurine in rat neonates and adults

Summary The distribution of ³⁵S-taurine in rat neonates and adults was investigated by wholy-body autoradiography. The neonates (4-day-old) were frozen in dry-ice hexane at 30 min, 1, 3 and 6 h after an intraperitoneal injection of ³⁵S-taurine, whereas survival intervals for adult rats were 1 and 3 h. Whole-sagittal sections of the frozen rat, obtained by using a cryostat microtome were dried in situ and autoradiographed. In rat neonates and adults, ³⁵S-taurine was mainly accumulated in the renal cortex, urine, feces, liver, eye (lens, vitreous fluid, retina), hypophysis, thymus, adrenal glands, nasal mucous membrane, salivary glands, gastric mucosa, small and large intestinal mucosa, choroid plexus, myocardium and sebaceous glands. In the rat neonate, such regions as the olfactory bulb, cerebrum, and cerebellum showed relatively high optical density.     

Evidence for an intracellular somatostatin receptor in pancreas: a comparative study with reference to gastric mucosa

Specific binding sites for ¹²⁵I-Tyr₁somatostatin-14 were comparatively demonstrated in isolated rat pancreatic and gastric parietal cells. In both materials, the sites occurred mostly in cytosol, with apparent affinities of 1×10^?10M and 3×10^?11M, respectively, in pancreatic and gastric cells. Somatostatin-14 stimulated cytosolic phosphoprotein phosphatases (PPPases) in pancreas as well as in gastric mucosa with concentrations for half maximal effect consistent with binding affinities. Somatostatin 28 mimicked somatostatin 14 stimulation with a higher efficacy but an equivalent potency. Secretin and cholecystokinin C terminal octapeptide were ineffective. Furthermore, in intact isolated cells, somatostatinic stimulation of PPPases was blocked by 5×10^?4M dinitrophenol. We therefore suggest that in pancreas as in gastric mucosa, somatostatin's inhibitory effect on secretory functions could involve protein dephosphorylation mediated by an intracellular receptor.     

Immunohistochemical distribution of hepatic fatty acid-binding protein in rat and human alimentary tract

Tissues from rat and human alimentary tract were immunostained with rabbit antibodies to fatty acid-binding protein (FABP) isolated from rat liver, since the precise immunohistochemical localization of the protein in gut has not been determined. The results obtained indicated that FABP immunoreactivity was found almost exclusively in intestinal absorptive cells, the sole exception being its presence in the cytoplasm of a few goblet cells. In small bowel, FABP-positive cells were most often found in the upper and middle segments, and less frequently in the lower to terminal portion. Immunoreactive cells were also found in large bowel of rat and human, but with differing patterns of distribution. In rat, positive cells were found mainly in the lower portion of the large intestine, whereas in human positive cells were present in all portions. Immunoreactive cells were detected in rat and human cecum, in the upper half of human rectum, and in human vermiform appendix. No such cells were found in esophageal and nonmetaplastic gastric mucosa or in pancreatic tissue, whereas they were present in great numbers in metaplastic gastric mucosa. The results of this study therefore suggest that FABP is a useful marker for research into the physiology or pathology of absorptive cells in the gastrointestinal tracts of both species.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Contribution of carbon monoxide-producing cells in the gastric mucosa of rat and monkey

 Recent studies have shown that carbon monoxide (CO) may function as a gaseous signaling molecule in a similar way to nitric oxide. In the gastrointestinal tract, immunoreactivity against a CO-producing enzyme, heme oxygenase-2 (HO-2), was reported in epithelial cells and neurons of submucosal and myenteric plexus. However, details of the epithelial cells in the gastric mucosa remain unknown. The aim of this study was to clarify if mRNA for HO-2 is expressed in the rat stomach, if HO-2 protein is present in the mucosa, and to define the cell types of the HO-2-immunoreactive cells. HO-2 mRNA and protein were detected in fundic and pyloric mucosa of rat stomach using an RNA protection assay and western blot analysis. Immunohistochemical study showed that HO-2 was localized in parietal cells of the fundic glands and gastrin cells of the pyloric glands of both rat and monkey. The results suggest that HO-2 enzyme is produced in the gastric mucosa, and that CO is released from parietal cells and gastrin cells. Accepted: 12 November 1997     

Mast cells in the rat gastric mucosa are not primarily responsible for PGD2 generation

The present study investigates the contribution of gastric mast cells on PGD2 generation in rat gastric mucosa. Cold-restraint induced stress or i.v. carbachol injection methods were used for gastric mast cell degranulation. In 19 stressed, 15 carbachol-infused and 14 control rats, gastric mast cell counts and gastric mucosa PGD2 assay were performed. Gastric mucosal content of PGF2 alpha was also determined in carbachol infused and control rats. The mean number of gastric mast cells was significantly lower in stressed and carbachol infused than in control rats. Despite these differences in gastric mast cell counts, neither PGD2 or PGF2 alpha contents in the gastric mucosa were significantly different in mast cells degranulated rats than in control animals. These results suggest another source of PGD2 in the rat gastric mucosa other than mast cells.     

Cellular localization of dopamine receptors in the gastric mucosa of rats

Dopamine (DA) plays a critical role in the protection of gastric mucosa and is mediated through corresponding receptors. However, the details of the expression of DA receptors (D1-D5) in the gastric mucosa are lacking. The present study investigated the expression and cellular localization of DA receptors in rat gastric mucosa by means of real-time PCR and immunofluorescent techniques. The results indicated that the mRNA expressions of all five subtypes of DA receptors were found in the gastric mucosa, among which the D2 level was the highest. The immunopositive cells of D1-D3 and D5 were primarily localized to the basilar gland of the epithelial layer in gastric corpus, but D4 immunoreactivity (IR) was only observed in the enteric nerve plexus. The D1, D2, and D5 IR were found in pepsin C-IR cells except D3. No IR of any DA receptor was detected in the H(+)/K(+)-ATPase- or mucin 6-IR cells. In conclusion, for the first time, this study demonstrates the predominant distribution of DA receptors in the chief cells, not the parietal and mucous neck cells, in rat gastric mucosa, thus suggesting that DA may not directly regulate the function of parietal cells or mucous neck cells, but it may modulate the function of chief cells through the D1, D2, and D5 receptors.     

Remodeling of the Residual Gastric Mucosa after Roux-En-Y Gastric Bypass or Vertical Sleeve Gastrectomy in Diet-Induced Obese Rats

Whereas the remodeling of intestinal mucosa after bariatric surgeries has been the matter of numerous studies to our knowledge, very few reported on the remodeling of the residual gastric mucosa. In this study, we analyzed remodeling of gastric mucosa after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) in rats. Diet-induced obese rats were subjected to RYGB, VSG or sham surgical procedures. All animals were assessed for food intake, body-weight, fasting blood, metabolites and hormones profiling, as well as insulin and glucose tolerance tests before and up to 5 weeks post-surgery. Remodeling of gastric tissues was analyzed by routine histology and immunohistochemistry studies, and qRT-PCR analyses of ghrelin and gastrin mRNA levels. In obese rats with impaired glucose tolerance, VSG and RYGB caused substantial weight loss and rats greatly improved their oral glucose tolerance. The remaining gastric mucosa after VSG and gastric pouch (GP) after RYGB revealed a hyperplasia of the mucous neck cells that displayed a strong immunoreactivity for parietal cell H⁺/K⁺-ATPase. Ghrelin mRNA levels were reduced by 2-fold in remaining fundic mucosa after VSG and 10-fold in GP after RYGB. In the antrum, gastrin mRNA levels were reduced after VSG in line with the reduced number of gastrin positive cells. This study reports novel and important observations dealing with the remaining gastric mucosa after RYGB and VSG. The data demonstrate, for the first time, a hyperplasia of the mucous neck cells, a transit cell population of the stomach bearing differentiating capacities into zymogenic and peptic cells.