SAP 97 is a potential candidate for basolateral fixation of ezrin in parietal cells

 Acid secretion in gastric parietal cells is preceded by a dramatic increase in surface area of the apical membrane compartment, due to fusion of the H⁺/K⁺-ATPase-containing tubulovesicles. The resulting canaliculi must be fixed for a period of minutes by cytoskeletal elements to sustain acid secretion. Using immunofluorescence microscopy, the cytoskeletal linker molecule, ezrin, localizes to the apical canalicular membrane of parietal cells. Antibodies against ezrin precipitate H⁺/K⁺-ATPase and β-actin. In addition to its apical localization, ezrin is found to be colocalized at the basolateral compartment with synapse-associated protein (SAP) 97. Immunoprecipitation confirms a direct binding of SAP 97 and ezrin. We conclude that ezrin is fixed to the basolateral compartment by SAP 97. Upon stimulation of acid secretion, ezrin moves to the apical surface where it might stabilize the canalicular microvilli by connecting to β-actin and H⁺/K⁺-ATPase, thereby sustaining acid secretion. Accepted: 14 January 1999     

Demonstration of a pH gradient in the gastric gland of the acid-secreting guinea pig mucosa

The gastric mucosa is covered by a continuous layer of mucus. Although important for understanding the mechanism of this protective function, only scarce information exists about the pH inside the gastric gland and its outlet. pH in the lumen of the gastric glands, in the outlet of gastric crypts, and in the adjacent cells was measured in the isolated acid-secreting mucosa of the guinea pig. Ultrafine double-barreled pH microelectrodes were advanced at high acceleration rates through the gastric mucus and the tissue to ensure precise intracellular and gland lumen pH measurements. A pH gradient was found to exist along the gastric gland, where the pH is 3.0 at parietal cells, i.e., in the deepest regions, and increases to 4.6 at the crypt outlet. Intracellular pH (pH(i)) of epithelial cells bordering a crypt outlet, and of neck cells bordering a gland, was acidic, averaging 6.0 and 6.5, respectively. pH(i) of deep cells bordering a gland was nearly neutral, averaging 7.1, and the secreting parietal cells were characterized by a slightly alkaline pH(i) of 7.5. This gland pH gradient is in general agreement with a model that we recently proposed for proton transport in the gastric mucus, in which protons secreted by the parietal cells are buffered to and transported with the simultaneously secreted mucus toward the gastric lumen, where they are liberated from the degraded mucus.     

Morphometric analysis of parietal cell membrane transformations in isolated gastric glands

Summary Changes in parietal cell membranous structures that accompany the onset of acid secretion were studied with electron microscopy using isolated gastric glands from rabbit. A stereological analysis was performed to quantitate the morphological changes occurring within 5 min following histamine stimulation. These changes were compared to the changes resulting from osmotic expansion of parietal cell components following addition of 1mm aminopyrine (AP) to glands incubated in medium containing 108mm K⁺ (high-K⁺). Morphometric analyses, together with measurements of glandular water content, indicated that parietal cells swell in high-K⁺ medium. Addition of 1mm AP to glands incubated in high-K⁺ medium resulted in massive distention of the secretory canaliculus but no difference was observed in the amount of tubulovesicular membrane or the relative size of these cytoplasmic structures. In the histamine-treated glands the parietal cells displayed a rapid loss of tubulovesicular membrane and a reciprocal increase in canalicular membrane. These morphological changes were complete long before a maximum level of acid formation was achieved. Taken together, these results indicate that; (i) the morphological change accompanying stimulation does not require acid formationper se; (ii) the site of acid secretion is the intracellular canaliculus and not the tubulovesicles; (iii) there is no preexisting actual or potential continuity between the tubulovesicular space and the canalicular space; and (iv) the AP-induced expansion of the canaliculus in high-K⁺ medium, while yielding some valuable information, is not an appropriate model for studying the normal stimulus-induced morphological transition, despite a superficial similarity of appearance.     

Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells

Previous investigations in several systems have demonstratedthat Rab3 family members redistribute to soluble fractions on fusion ofsecretory granules with target plasma membranes. Rab proteins are thenrecycled back onto mature secretory vesicles after reinternalization ofthe membrane. Although this cycle is well established for Rab3, farless is known about redistribution of other Rab proteins during vesiclefusion and recycling. In the gastric parietal cell, Rab11a isassociated with H-K-ATPase-containing tubulovesicles, which fuse withthe apical plasma membrane (secretory canaliculus) in response toagonists such as histamine. We have analyzed distribution of Rab11a andother tubulovesicle proteins in resting and histamine-stimulated rabbitparietal cells. Stimulation of isolated gastric glands in the presenceof 100 µM histamine and 100 µM 3-isobutyl-1-methylxanthine did notcause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a,Rab25, syntaxin 3, and SCAMPs increased immunoreactivity instimulus-associated vesicles prepared from rabbits treated withhistamine compared with those from ranitidine-treated animals. Thelarge GTPase dynamin was found in both vesicle preparations, but therewas no change in amount of immunoreactivity. Immunofluorescencestaining of resting and histamine-stimulated primary cultures ofparietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate thatRab11a does not cycle off the membrane during the process oftubulovesicle fusion with the secretory canaliculus. Thus Rab11a mayremain associated with recycling apical membrane vesicle populations.

     

Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.     

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2^-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2^-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H⁺] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2^-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84–95%, P<0.005) in ClC-2^-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG, RANA CATESBIANA : II. The Acid-Secreting Gastric Mucosa

The oxyntic cell in the gastric glands of the bullfrog was examined in lead hydroxide-stained sections of gastric mucosae fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. During gastric acid secretion (pH 1–2) induced by histamine administration in cannulated frogs, the pattern of fine structure in the oxyntic cell differs strikingly from that in the oxyntic cell of the non-acid-secreting stomach. The relative number of smooth surfaced profiles decreases and a greater concentration of these elements is associated with the apical region of the oxyntic cell facing the lumen of the gastric gland. Similar concentrations of these elements are found in those regions of cytoplasm surrounding intercellular canaliculi which lie between adjacent cells and communicate with the lumen of a gastric gland. In these regions, the smooth surfaced profiles (35 to 65 mµ in width) characteristically form a tubular network. The membrane-bounded contents appear to be continuous with the extracellular medium, both on the capillary side and at the apical surface of the cell adjoining the lumen of the gastric gland. Mitochondria are distributed randomly in the cytoplasmic matrix of the oxyntic cell.     

Detection of BrdU-label retaining cells in the lacrimal gland: implications for tissue repair

The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2′-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half days post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 weeks of chase period, a substantial number of lacrimal gland cells were BrdU⁺ (11.98 ± 1.84 and 7.95 ± 1.83 BrdU⁺ cells/mm², respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU⁺ cells (0.38 ± 0.06 BrdU⁺ cells/mm²), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU⁺ cells/mm²; injured: 2.91 ± 0.62 BrdU⁺ cells/mm²). Furthermore, during repair, among BrdU⁺ cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Visualization of the secretory canaliculi of human parietal cells with a peroxidase-labelled peanut lectin

Summary Peanut lectin reactivity was examined in normal fundic glands from human gastric samples, both at light- and electron-microscopic levels, using a peroxidase conjugate. Positive reaction was observed in the glycocalyx of parietal cell secretory canaliculi as well as in the mucous globules of mucous cells and in the luminal cell coat of chief cells. The presence of terminal galactose in the canalicular glycocalyx may be connected with the peculiar function of hydrochloric acid secretion. Peroxidase-labelled peanut lectin is proposed as a marker for visualizing the secretory canaliculus of parietal cells.     

Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry

Summary A cytochemical study of gastric K⁺-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K⁺-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method.Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method.These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.In honour of Prof. P. van DuijnPart of this paper was presented at the 24th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Nagoya, October 27–28, 1983 (Ogawa KS, Fujimoto K, Ogawa K (1983) A new lead citrate method for the cytochemical demonstration of the H⁺–K⁺-ATPase with p-NPP as a substrate. Acta Histochem Cytochem 16:662)This study was supported by Grants-in-Aid for Encouragement of Young Scientists No. 60770019 to K. Fujimoto from the Ministry of Education, Science and Culture, the Japanese Government     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.     

Modulation of H,K-ATPase activity by the molecular chaperone ERp57 highly expressed in gastric parietal cells

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H⁺,K⁺-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H⁺,K⁺-ATPase significantly increased the ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H⁺,K⁺-ATPase activity. In contrast, knockdown of endogenous ERp57 in H⁺,K⁺-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na⁺,K⁺-ATPase. These results suggest that ERp57 positively regulates H⁺,K⁺-ATPase activity apart from its chaperoning function.     

Rab proteins in gastric parietal cells: evidence for the membrane recycling hypothesis.

The gastric parietal cell secretes large quantities of HCl into the lumen of the gastric gland in response to secretagogues such as histamine. In the membrane recycling hypothesis, this secretory activity requires the trafficking of the gastric H+/K(+)-ATPase to the cell surface from intracellular tubulovesicles. The Rab subclass of small GTP-binding proteins is thought to confer specificity to vesicle transport throughout the secretory pathway, and previous investigations established that Rab11 is highly expressed in gastric parietal cells. Recent discoveries in intra-Golgi transport and neuronal synaptic vesicle fusion have fortuitously converged on an evolutionarily conserved protein complex involved in vesicle docking and fusion. Recent results indicate that Rab11 is involved in the apical targeting of vesicles in parietal cells and other epithelial cells throughout the gastrointestinal tract. In support of the membrane recycling hypothesis, Rab co-segregates with H+/K(+)-ATPase in parietal cells. The presence of Rab11 on tubulovesicles supports a role for this Rab protein in recycling vesicle trafficking.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

Localization of ClC-2 Cl- channels in rabbit gastric mucosa

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.     

Differential pH restoration after ammonia-elicited vacuolar alkalisation in rice and maize root hairs as measured by fluorescence ratio

Changes of vacuolar pH in hair cells of young rice (Oryza sativa L.) and maize (Zea mays L.) roots were measured after ammonia application at various levels of external pH. After loading the pH-sensitive, fluorescent dye Oregon green 488 carboxylic acid 6-isomer into the vacuoles of root hairs, ratiometric pH data of high statistical significance were obtained from root hair populations comprising hundreds of cells. The pH of the vacuole at external pH 5.0 was 5.32 ± 0.08 (±SD, n= 15) and 5.41 ± 0.13 (±SD, n= 15) in rice and maize, respectively. A moderate external ammonia concentration of 2 mM led to vacuolar alkalisation at both, low (pH 5.0) and high (pH 7.0–9.0) external pH, presumably due to NH₃ permeation into the vacuole. With increasing external pH, ammonia application did not cumulatively increase vacuolar pH. In rice, the increase in vacuolar pH ranged from 0.1–0.8 pH units; in maize a more constant increase of 0.5 pH units was observed. The vacuolar pH increase was efficiently depressed in rice (especially at high external pH), but not in maize. Inhibition of the tonoplast H⁺-ATPase by concanamycin A raised vacuolar pH and increased the ammonia-elicited vacuolar alkalisation in both species, proving that vacuolar H⁺-ATPase activity counters the ammonia-elicited alkalisation effect. However, even under conditions of vacuolar H⁺-ATPase inhibition, rice was still able to restore an ammonia-elicited pH increase. High vacuolar pH levels as found in maize under conditions of high NH₃ influx may derive from inefficient cytosolic ammonia assimilation and tonoplast proton pumping. Thus, in maize, prolonged reduction of the proton gradient between the cytosol and the vacuole may play an important role in NH₃ toxicity. Received: 12 September 1997 / Accepted: 19 January 1998     

Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.