Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.

Expression of the bile salt export pump is maintained after chronic cholestasis in the rat.

BACKGROUND & AIMS: This study assessed the expression of the recently identified adenosine triphosphate-dependent bile salt export pump and the functional ability to excrete bile salts in cholestatic models in the rat. METHODS: The effects of common bile duct ligation, endotoxin, and ethinylestradiol on bile salt export pump messenger RNA levels, protein expression, and tissue localization were determined. Changes in the expression of 3 other hepatocyte membrane transporters (Na(+) taurocholate cotransporter, multispecific organic anion transporter, and P-glycoprotein) were also determined for comparison. Functional assessment of bile salt excretion was determined after bile duct ligation. RESULTS: Expression of the bile salt export pump was diminished but relatively preserved compared with other membrane transporters. Tissue localization of the bile salt export pump persisted at the canalicular domain in all 3 models. In contrast, expressions of the Na(+) taurocholate cotransporter and multispecific organic anion transporter were more profoundly diminished. P-glycoprotein levels increased severalfold with common bile duct ligation but were unchanged with either endotoxin or ethinylestradiol. The capacity to excrete bile salts was relatively maintained 3 and even 14 days after bile duct ligation. CONCLUSIONS: Alterations in expression of the bile salt export pump may account for the functional alterations of bile salt secretion observed in cholestasis. However, relative preservation of expression is associated with persistent bile salt excretion and may lessen the extent of liver injury produced by bile salt retention.

Canalicular export pumps traffic with polymeric immunoglobulin A receptor on the same microtubule-associated vesicle in rat liver.

Basolateral to apical vesicular transcytosis in the hepatocyte is an essential pathway for the delivery of compounds from the sinusoidal blood to the bile and to traffic newly synthesized resident apical membrane proteins to their site of function at the canalicular membrane front. To characterize this pathway better, microtubules in a hepatocyte homogenate were polymerized by addition of taxol, and associated membrane-bound vesicles were isolated. This fraction was enriched in polymeric immunoglobulin A receptor and contained apical membrane proteins. Immunoelectron microscopy demonstrated that polymeric immunoglobulin A receptor was localized predominantly on vesicles ranging from 100 to 160 nm and that the multidrug resistance protein 2 and the bile salt export pump co-localized on these vesicles. The minus-ended microtubule motor, dynein, was highly enriched in the fraction, and its intermediate chain could be released effectively by incubation with 1 mM ATP or GTP. However, the association of the transcytotic vesicles with the microtubules was not sensitive to hydrolyzable or non-hydrolyzable nucleotides. This study characterizes a fraction of microtubule-associated vesicles from rat hepatocytes and demonstrates that several resident apical membrane transport proteins and the polymeric immunoglobulin A receptor traffic on the same vesicle.

Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat.

In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.

Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells.

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 +/- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets.

The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions. This transport function is markedly diminished in mutant rats that lack the transport protein. To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity. The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted. The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein. The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane. Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane. The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference. These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane. The relevance of this phenomenon for the intact liver is discussed.

The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis.

BACKGROUND & AIMS: The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein (Mrp2). Mrp2 function is impaired in various experimental models of intrahepatic and obstructive cholestasis, but the underlying molecular mechanisms are unclear. The aim of this study was to investigate these molecular mechanisms. METHODS: The effects of endotoxin, ethinylestradiol, and common bile duct ligation (CBDL) on Mrp2 protein, messenger RNA (mRNA) expression, and Mrp2 tissue localization were determined in rat livers by Northern blotting, Western analysis, and tissue immunofluorescence. To assess whether changes were specific for Mrp2, we also examined the expression of canalicular ecto-adenosine triphosphatase (ecto-ATPase) and mdr P-glycoproteins (P-gp). RESULTS: All three cholestatic models resulted in a marked decrease in Mrp2 protein (P < 0.01) and its tissue localization at the canalicular membrane. Mrp2 mRNA levels diminished profoundly after endotoxin (P < 0.0005) and CBDL (P < 0.05), but did not change after ethinylestradiol. In contrast to Mrp2, protein expression of ecto-ATPase and P-gp remained unchanged in endotoxin- and ethinylestradiol-treated animals, whereas P-gp levels increased after CBDL (P < 0.05). CONCLUSIONS: Down-regulation of Mrp2 expression may explain impaired biliary excretion of amphiphilic anionic conjugates in these models of cholestasis.

EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.

Gastrin release from the antral gastrin-expressing cell (G cell) is regulated by bombesin and luminal factors. Yet, these same extracellular regulators do not stimulate expression of the gene. Since the gastric mucosa expresses large quantities of EGF receptor ligands such as TGFalpha, we examined whether EGF receptor ligands stimulate gastrin gene expression in gastrin-expressing cell cultures. EGF receptor activation of primary cultures stimulated gastrin gene expression about twofold; whereas bombesin treatment of antral G cell cultures stimulated gastrin release but not gene expression. EGF and TGFalpha were weak stimulants of gastrin release. EGF receptor activation of AGS human gastric adenocarcinoma cell line stimulated gastrin gene expression nearly fourfold; and gastrin reporter constructs transfected into AGS cells were stimulated more than fourfold by EGF. EGF induction was conferred by the previously defined GC-rich gastrin EGF response element (gERE) element located at -68 to -53 bp upstream from the cap site since a mutation of the gERE element abolished both basal and EGF induction. Moreover, EGF treatment of AGS cells stimulated binding of the transcription factor Sp1 to this element. Collectively, these results demonstrate that gastrin gene expression and gastrin release are regulated by different signaling pathways: gene expression by EGF receptor activation and gastrin secretion by neuropeptides and luminal factors.

Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

Overexpression of transforming growth factor-alpha alters differentiation of gastric cell lineages.

Overexpression of transforming growth factor-alpha (TGF-alpha) in the gastric fundic mucosa of metallothionein promoter/enhancer-TGF-alpha(MT-TGF-alpha) transgenic mice produces a phenotype of foveolar hyperplasia similar to that observed in Ménétrier's disease. We have investigated the dynamics involved in the alterations of gastric mucosal morphology in the MT-TGF-alpha mouse model. The fundic mucosa of MT-TGF-alpha mice and nontransgenic littermates was evaluated in animals treated with cadmium sulfate. To mark the mucosal proliferative zone, 8-bromodeoxyuridine (BrdU) was administered 2 hr prior to killing. Gastric mucosa was examined by diastase-resistant, periodic acid-Schiff-positive (DR-PAS) staining and immunohistochemistry for H/K-ATPase an BrdU. MT-TGF-alpha mice demonstrated increased numbers of DR-PAS-staining mucous cells and lower parietal cell numbers per gland unit. While the proliferative zone in nontransgenic mice was located in the upper half of the gland, the zone in MT-TGF-alpha mice was located in the basal region. Overexpression of TGF-alpha in MT-TGF-alpha mice leads to an alteration in the development of mucosal lineages from the fundic progenitor zone, which is biased towards the predominant differentiation of foveolar mucous cells.

Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis.

BACKGROUND & AIMS: The molecular regulation of hepatic bile acid transporters during cholestasis is largely unknown. Cloning of complementary DNAs for the sinusoidal sodium-dependent taurocholate cotransporting polypeptide (ntcp), the cytosolic bile acid-binding protein 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), and a putative canalicular bile acid transporter Ca2+, Mg(2+)-ecto-adenosine triphosphatase, now facilitates such studies. METHODS: Protein mass, steady-state messenger RNA (mRNA) levels, and gene transcription were assessed in rat livers after common bile duct ligation (CBDL) from 1-7 days, and taurocholate uptake was determined in isolated hepatocytes. RESULTS: After CBDL, Na(+)-dependent taurocholate uptake (Vmax) declined by 70%. The levels of ntcp protein were reduced by more than 90%, and 3 alpha-HSD levels decreased by 66% by 7 days. Expression and canalicular localization of the ecto-adenosine triphosphatase remained unchanged. mRNA levels for both ntcp and 3 alpha-HSD diminished by about 60% 1 day after CBDL and remained unchanged up to 7 days. Transcriptional activity was decreased 1 day after CBDL only for ntcp. CONCLUSIONS: Extrahepatic cholestasis results in rapid down-regulation of Na(+)-dependent taurocholate uptake, ntcp transcription, and posttranscriptional regulation of both ntcp and 3 alpha-HSD mRNA. This selective decline of ntcp may represent a protective feedback mechanism in cholestasis to diminish uptake of potentially hepatotoxic bile acids.

Evidence for a regulatory role for histamine in gastric enterochromaffin-like cell proliferation induced by hypergastrinemia.

BACKGROUND/AIMS: Hypergastrinemia, induced by sustained suppression of gastric acid secretion, is associated with gastric enterochromaffin-like (ECL) cell hyperplasia and carcinoid tumor formation. We examined the effect of a selective H1-histamine antagonist, terfenadine, on gastric mucosal cell proliferation to determine whether histamine might modulate ECL cell generation. METHODS: The rodent mastomys received the H2-antagonist loxtidine (2 g/l drinking water) alone or in combination with terfenadine (0.5 g/l or 35 mg/l drinking water) for 120 days. Controls received water or terfenadine alone. Serum gastrin levels and tissue histamine content were assayed by radioimmunoassays, and tissue chromogranin levels determined (Western blot analysis). In vivo cell proliferation was measured by bromodeoxyuridine (BrdU, 200 mg/kg/day, 3 days) incorporation. Gastric mucosal thickness was determined, ECL cell number was assessed, and the percentage of proliferating ECL cells quantitated. To evaluate the direct action on ECL cells we then studied the effect of terfenadine on histamine secretion and DNA synthesis (BrdU uptake) in an isolated preparation (approximately 90% pure) of ECL cells. RESULTS: Loxtidine increased serum gastrin levels, mucosal thickness, tissue chromogranin levels, tissue histamine content, BrdU incorporation, ECL cell number, and proliferating ECL cells (all parameters p < 0.05). Terfenadine alone, irrespective of dosage, had no significant effect. The high dose in combination with loxtidine significantly inhibited the increase in tissue chromogranin levels, tissue histamine content, ECL cell number and proliferating ECL cells (p < 0.05), but did not alter other parameters, compared to loxtidine alone. The low does did not alter the loxtidine-induced changes. In pure isolated ECL cells, terfenadine did not alter histamine secretion either alone or in combination with gastrin (10 nM). DNA synthesis was significantly inhibited by terfenadine (IC50 10(-10) M). CONCLUSIONS: Terfenadine specifically inhibited the effect of loxtidine-induced ECL cell proliferation in vivo and significantly inhibited ECL cell DNA synthesis in vitro. We postulate that histamine, through an H1 receptor, positively modulates gastric ECL cell proliferation.

Vesicle targeting to the apical domain regulates bile excretory function in isolated rat hepatocyte couplets.

BACKGROUND & AIMS: Plasma membrane solute transport may be regulated in many epithelial cells by vesicle traffic to and from the site of residence of the transporter. The aim of this study was to determine if this phenomenon may also play a role in the regulation of canalicular transport of bile acids. METHODS: Confocal microscopy and image analysis were performed to quantitatively assess changes in secretory capacity and vesicle targeting in isolated rat hepatocyte couplets that had been exposed to fluorescent bile acid after pretreatment with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and/or nocodazole. RESULTS: DBcAMP stimulated bile acid secretion by 240% while significantly increasing canalicular circumference. Nocodazole decreased secretion by 410% and significantly decreased canalicular circumference. When DBcAMP was added to nocodazole-treated couplets, a slight but significant increase was found in both fluorescent bile acid secretion and canalicular circumference as compared with nocodazole alone. Finally, DBcAMP stimulated translocation of vesicles to the canalicular membrane as determined by immunocytochemical localization of a putative bile acid transporter, Ca2+, Mg2+-ecto-adenosine triphosphatase. CONCLUSIONS: The findings support the view that apical membrane transport activity in the rat hepatocyte is highly regulated by the insertion of vesicles into this domain and that this process involves both microtubule-dependent and -independent mechanisms.

Enrichment of rab11, a small GTP-binding protein, in gastric parietal cells.

Parietal cell secretion of acid requires the coordinated fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing tubulovesicles with a secretory canalicular target membrane. We have previously reported the presence of rab2 on parietal cell tubulovesicles (L. H. Tang, S. A. Stoch, I. M. Modlin, and J. R. Goldenring. Biochem. J. 285: 715-719, 1992). Since 60% of the small GTP-binding protein sequences obtained from parietal cells were > 95% homologous with human rab11 (J. R. Goldenring, K. R. Shen, H. D. Vaughan, and I.M. Modlin. J. Biol. Chem. 268: 18419-18422, 1993), we sought to study rab11 in gastric parietal cells. A complete rab11 sequence was obtained, and the deduced amino acid sequence of rabbit rab11 was identical to that for human. Rab11 mRNA was present throughout the gastrointestinal mucosa. mRNA for both rab11 and rab2 were enriched in isolated parietal cells compared with chief cells. A polyclonal antiserum against rab11 labeled a single 25-kDa band in isolated parietal cells. Immunostaining of rat fundic tissue demonstrated prominent staining of parietal cells. Rab11 staining cosegregated with alpha-H(+)-K(+)-ATPase staining in enriched preparations of rabbit parietal cell tubulovesicles. These results suggest that rab11 is enriched in parietal cells and is associated with intracellular tubulovesicles.

Histamine as an intermediate growth factor in genesis of gastric ECLomas associated with hypergastrinemia in mastomys.

Profound and sustained inhibition of gastric acid secretion has been associated with development of carcinoid tumors of the fundic enterochromaffin-like (ECL) cells in rodents. While ECL cell hyperplasia has been recognized in humans, the development of carcinoid tumors is rare and often confined to patients under treatment for gastrinoma related to the multiple endocrine neoplasia type I (MEN1) syndrome. The Mastomys was utilized as a model for the rapid induction of ECLomas by insurmountable acid secretory blockade induced by the pharmacologically irreversible H2-receptor antagonist, loxtidine. Loxtidine-induced ECL cell hyperplasia and neoplasia were compared in the absence of presence of cyproheptadine (0.5 mg/kg), an H1-receptor antagonist. Loxtidine administration resulted in a significant increase in ECL cell hyperplasia and neoplasia as well as an increase in ECL cell number, mucosal thickness, plasma gastrin levels, and stomach weight. Cyproheptadine ameliorated loxtidine-induced ECL cell hyperplasia and neoplasia and significantly decreased loxtidine-stimulated increases in ECL cell number. Nevertheless, cyproheptadine failed to alter the loxtidine-induced increase in plasma gastrin, stomach weight or mucosal height. The results indicate that cyproheptadine, an H1-receptor antagonist, inhibits loxtidine-induced ECL cell hyperplasia independent of any effects on serum gastrin.

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

Gastrin-dependent inhibitory effects of octreotide on the genesis of gastric ECLomas.

BACKGROUND: The efficacy of octreotide in the regulation of endocrine tumor secretion and symptomatology has been well documented. Its effects on neuroendocrine tumor generation and cell proliferation are less well understood. The purpose of this study was to determine if blockade of somatostatin receptors by octreotide would alter gastrin levels and influence enterochromaffin-like (ECL) cell proliferation. METHODS: The well-established gastric ECLoma model of the rodent, mastomys, was used. Animals received loxtidine (1 mg/kg/day), an irreversible H2 blocker, and subcutaneous slow release, octreotide pellet implants (150 or 300 micrograms/kg/day) or placebo pellets for a 4-month period. RESULTS: Control parameters for gastric mucosal thickness, plasma gastrin level, ECL cell density, and bromodeoxyuridine-positive cells were 517 +/- 20 microns, 46.1 +/- 11.4 pmol/L, 7.4 +/- 0.9 cells/visual field, and 13.8 +/- 2.6 cells/visual field, respectively. After loxtidine-placebo treatment all values were significantly increased (p < 0.05; 883 +/- 70 microns, 192.8 +/- 10.6 pmol/L, 97 +/- 16.2 cells/visual field, and 51.7 +/- 19.2 cells/visual field, respectively). High dose octreotide significantly inhibited all parameters (668 +/- 3.5 microns, 66.2 +/- 20.5 pmol/L, 37.0 +/- 8.0 cells/visual field, and 10.9 +/- 2.2 cells/visual field; p < 0.05). Low dose octreotide failed to significantly inhibit ECL cell density mucosal thickness, or cell proliferation. CONCLUSIONS: Irreversible H2 receptor blockade results in hypergastrinemia and ECL cell tumor generation. Hypergastrinemia, ECL cell hyperplasia, and cell proliferation are significantly inhibited by in vivo blockade of somatostatin receptors by administration of octreotide.

Possible role of transforming growth factor alpha in the pathogenesis of Ménétrier's disease: supportive evidence form humans and transgenic mice.

Ménétrier's disease is an uncommon disorder of unknown etiology characterized by enlarged gastric folds with foveolar hyperplasia and cystic dilatation of gastric glands. Biochemical features that are seen frequently include hypoproteinemia, hypochlorhydria, and increased gastric mucus. Because transforming growth factor alpha (TGF alpha) is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin content, we hypothesized that its altered expression might be involved in the pathogenesis of this disease. Therefore, we characterized TGF alpha immunoreactivity in the gastric mucosa of 4 patients with Ménétrier's disease. In contrast to the normal pattern of TGF alpha immunostaining in which TGF alpha appears most concentrated in parietal cells, there was intense staining in the majority of mucous cells in the gastric mucosa of patients with Ménétrier's disease. In one patient from whom sufficient fresh tissue was obtained to isolate RNA, expression of TGF alpha and the epidermal growth factor receptor was higher in the gastric mucosa relative to a normal control. In addition, metallothionein-TGF alpha transgenic mice, which overexpress TGF alpha in gastric mucosa, show a number of features characteristic of Ménétrier's disease. These include foveolar hyperplasia and glandular cystic dilatation, increased gastric neutral mucin staining, and reduced basal and histamine-stimulated rates of acid production. Taken together, observations derived from the human material and correlation with data from a transgenic mouse model support an important role for TGF alpha in the pathogenesis of Ménétrier's disease.