Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

2003 ◽  
Vol 285 (6) ◽  
pp. G1242-G1248 ◽  
Author(s):  
Philipp Kirchhoff ◽  
Carsten A. Wagner ◽  
Florian Gaetzschmann ◽  
Klaus Radebold ◽  
John P. Geibel
Keyword(s):  
Apical Membrane ◽  
Specific Inhibitor ◽  
Parietal Cells ◽  
Proton Extrusion ◽  
Gastric Glands ◽  
Functional Studies ◽  
Sodium Hydrogen ◽  
Low Concentrations ◽  
Sodium Hydrogen Exchanger ◽  
Nhe Isoforms

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the β-subunit of the H+-K+-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H+-K+-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H+-K+-ATPase activity.

scholarly journals Acid secretion-associated translocation of KCNJ15 in gastric parietal cells

2011 ◽  
Vol 301 (4) ◽  
pp. G591-G600 ◽  
Author(s):  
Wenjun He ◽  
Wensheng Liu ◽  
Catherine S. Chew ◽  
Susan S. Baker ◽  
Robert D. Baker ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Western Blot ◽  
Acid Secretion ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Apical Membrane ◽  
Parietal Cells ◽  
Immunofluorescence Staining ◽  
Gastric Glands ◽  
Gastric Parietal Cells

Potassium ions are required for gastric acid secretion. Several potassium channels have been implicated in providing K+ at the apical membrane of parietal cells. In examining the mRNA expression levels between gastric mucosa and liver tissue, KCNJ15 stood out as the most highly specific K+ channel in the gastric mucosa. Western blot analysis confirmed that KCNJ15 is abundant in the stomach. Immunofluorescence staining of isolated gastric glands indicated that KCNJ15 was expressed in parietal cells and chief cells, but not in mucous neck cells. In resting parietal cells, KCNJ15 was mainly found in puncta throughout the cytoplasm but was distinct from H+-K+-ATPase. Upon stimulation, KCNJ15 and H+-K+-ATPase become colocalized on the apical membranes, as suggested by immunofluorescence staining. Western blot analysis of the resting and the stimulated membrane fractions confirmed this observation. From nonsecreting preparations, KCNJ15-containing vesicles sedimented after a 4-h centrifugation at 100,000 g, but not after a 30-min spin, which did sediment most of the H+-K+-ATPase-containing tubulovesicles. Most of the KCNJ15 containing small vesicle population was depleted upon stimulation of parietal cells, as indicated by the fact that the KCNJ15 signal was shifted to a large membrane fraction that sedimented at 4,000 g. Our results demonstrate that, in nonsecreting parietal cells, KCNJ15 is stored in vesicles distinct from the H+-K+-ATPase-enriched tubulovesicles. Furthermore, upon stimulation, KCNJ15 and H+-K+-ATPase both translocate to the apical membrane for active acid secretion. Thus KCNJ15 can be added to the family of apical K+ channels in gastric parietal cells.

scholarly journals ETB receptor activation causes exocytic insertion of NHE3 in OKP cells

2001 ◽  
Vol 280 (1) ◽  
pp. F34-F42 ◽  
Author(s):  
Yan Peng ◽  
Morimasa Amemiya ◽  
Xiaojing Yang ◽  
Lingzhi Fan ◽  
Orson W. Moe ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Receptor Activation ◽  
Cytochalasin D ◽  
Maximal Effect ◽  
Latrunculin B ◽  
Opossum Kidney ◽  
Sodium Hydrogen ◽  
Threefold Increase ◽  
Sodium Hydrogen Exchanger ◽  
Etb Receptor

Endothelin-1 (ET-1) activates sodium/hydrogen exchanger 3 (NHE3) in opossum kidney clone P (OKP) cells expressing ETB receptors. ET-1 (10−8 M) caused a two- to threefold increase in apical membrane NHE3 (assessed by surface biotinylation), in the absence of a change in total cellular NHE3. A maximal effect was achieved within 15 min. The increase in apical NHE3 was not blocked by cytochalasin D but was blocked by latrunculin B, which also prevented the ET-1-induced increase in NHE3 activity. Endocytic internalization of NHE3, measured as protection of biotinylated NHE3 from the membrane-impermeant, sulfhydryl-reducing agent MesNa was minimal within 35 min and was not regulated by ET-1. Exocytic insertion of NHE3, measured as the appearance of biotinylated NHE3 after the blockade of reactive sites with sulfo-NHS-acetate, was increased in response to ET-1. These studies demonstrate that ET-1 induces net trafficking of NHE3 to the apical membrane that is mediated by enhanced exocytic insertion and is required for increased NHE3 activity.

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes

2001 ◽  
Vol 280 (2) ◽  
pp. G247-G254 ◽  
Author(s):  
Albert Mennone ◽  
Daniel Biemesderfer ◽  
Daniel Negoianu ◽  
Chao-Ling Yang ◽  
Thecla Abbiati ◽  
...  
Keyword(s):  
Bile Duct ◽  
Ph Regulation ◽  
Fluid Secretion ◽  
Wild Type ◽  
Western Blots ◽  
Functional Studies ◽  
Sodium Hydrogen Exchanger ◽  
Apical Membranes ◽  
Nhe Isoforms

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3 (−/−) mice after stimulation with forskolin, using videomicroscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3 (−/−) mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the Na+/H+ exchanger inhibitor 5-( N-ethyl- N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations.

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration

2002 ◽  
Vol 282 (6) ◽  
pp. C1454-C1460 ◽  
Author(s):  
D. Granger ◽  
M. Marsolais ◽  
J. Burry ◽  
R. Laprade
Keyword(s):  
Apical Membrane ◽  
Staining Pattern ◽  
Specific Inhibitor ◽  
Sweat Duct ◽  
Proton Extrusion ◽  
Cold Preservation ◽  
Concanamycin A ◽  
Acid Load ◽  
Strong Staining ◽  
Eccrine Sweat

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO[Formula: see text]-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpHi/d t) following an acid load. Oligomycin (25 μM), an inhibitor of F-type ATPases, decreased dpHi/d t by 88 ± 6%, suggesting a role for an ATP-dependent process involved in pHi recovery. Moreover, dpHi/d t was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 μM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpHi/d t by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.

Regulation of intracellular pH in resting and in stimulated parietal cells

1989 ◽  
Vol 257 (3) ◽  
pp. C554-C561 ◽  
Author(s):  
A. M. Paradiso ◽  
M. C. Townsley ◽  
E. Wenzl ◽  
T. E. Machen
Keyword(s):  
Intracellular Ph ◽  
Apical Membrane ◽  
Parietal Cells ◽  
The Other ◽  
Disulfonic Acid ◽  
Gastric Glands ◽  
Maximum Decrease ◽  
Intact Rabbit ◽  
Specific Inhibitors ◽  
Cellular Acidification

Microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in single parietal cells (PC) of intact rabbit gastric glands during resting and stimulated conditions. In 61% of PC, histamine plus isobutylmethylxanthine (IBMX) (both 100 microM) caused a small increase in pHi, ranging from 0.04 to 0.21 pH units (average delta pHi = 0.09 +/- 0.04 units over a 6-min period). In the other 39% of PC, pHi remained constant or decreased slightly (maximum decrease was 0.10 unit). The specific inhibitors omeprazole (50 microM, blocks H+- K+-ATPase), 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (H2DIDS; 100-200 microM, blocks Cl-HCO3 exchange) and amiloride (1 mM, blocks Na-H exchange) were added to both resting and stimulated PC. In stimulated PC, omeprazole caused pHi to decrease by 0.08 unit, but this inhibitor had no effect on pHi of resting PC. H2DIDS caused pHi to increase in stimulated PC five times faster compared with resting PC. Amiloride or Na-free solution (which should reverse the Na-H exchanger and cause cellular acidification) caused pHi to decrease 2.5 or 5 times, respectively, more slowly in stimulated PC compared with resting PC. Also, recovery from NH4-induced acidification (due primarily to Na-H exchange) was 1.8 times faster (measured at pHi 6.7) in resting vs. in stimulated PC. During histamine plus IBMX-induced stimulation, increased H secretion by the H+-K+-ATPase at the apical membrane is accompanied by an increase in activity of the Cl-HCO3 exchanger at the serosal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of augmented duodenal HCO3−secretion after elevation of luminal CO2

2005 ◽  
Vol 288 (3) ◽  
pp. G557-G563 ◽  
Author(s):  
Osamu Furukawa ◽  
Masahiko Hirokawa ◽  
Lening Zhang ◽  
Tetsu Takeuchi ◽  
Luke C. Bi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Carbonic Anhydrase ◽  
Acid Stress ◽  
Specific Inhibitor ◽  
Anion Channel ◽  
Continuous Exposure ◽  
Uptake Mechanism ◽  
Sodium Hydrogen ◽  
Sodium Hydrogen Exchanger ◽  
Dimethyl Amiloride

The proximal duodenum is exposed to extreme elevations of Pco2because of the continuous mixture of secreted HCO3−with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO2for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal Pco2on duodenal HCO3−secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO2challenge, the duodenum was superfused with a high Pco2solution. Intracellular pH (pHi) of duodenal epithelial cells was measured by ratio microfluorometry. CO2challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO2-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO2-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO3−cotransporter, also inhibited CO2-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO2decreased epithelial cell pHi, followed by an overshoot after removal of the CO2solution. We conclude that luminal CO2diffused in the duodenal epithelial cells and was converted to H+and HCO3−by carbonic anhydrase. H+initially exited the cell, followed by secretion of HCO3−. Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO3−uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO2into HCO3−and H+.

Platelet-Bound Prekallikrein Promotes Pro-Urokinase-Induced Clot Lysis: A Mechanism for Targeting the Factor XII Dependent Intrinsic Pathway of Fibrinolysis

1994 ◽  
Vol 71 (03) ◽  
pp. 347-352 ◽  
Author(s):  
Jean-Pierre Loza ◽  
Victor Gurewich ◽  
Michael Johnstone ◽  
Ralph Pannell
Keyword(s):  
Platelet Rich Plasma ◽  
Specific Inhibitor ◽  
Specific Effect ◽  
Clot Lysis ◽  
Factor Xii ◽  
Intrinsic Pathway ◽  
Clot Retraction ◽  
Enzymatic Mechanism ◽  
Low Concentrations ◽  
Factor Xiia

SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.

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