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.

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.

Na+/H+ exchangers in the human eccrine sweat duct

2003 ◽  
Vol 285 (5) ◽  
pp. C1047-C1058 ◽  
Author(s):  
D. Granger ◽  
M. Marsolais ◽  
J. Burry ◽  
R. Laprade
Keyword(s):  
Ph Regulation ◽  
Basolateral Membrane ◽  
Outer Cell ◽  
Sweat Duct ◽  
Sodium Removal ◽  
Luminal Membrane ◽  
Strong Staining ◽  
Outer Cell Layer ◽  
Eccrine Sweat

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3--free conditions. In the presence of basolateral 5-ethyl- N-isopropyl amiloride (EIPA), an acidification of 0.29 ± 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 ± 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.

Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule

1993 ◽  
Vol 264 (2) ◽  
pp. F259-F266 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt
Keyword(s):  
Cell Types ◽  
Intercalated Cells ◽  
Functional Identification ◽  
Proton Extrusion ◽  
Collecting Tubule ◽  
Initial Ph ◽  
Acid Load ◽  
Extrusion Mechanism ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

A K-dependent proton extrusion mechanism was investigated by means of fluorescence techniques in rabbit cortical collecting tubules. These experiments were performed in split opened tubules from normal animals exposed to the intracellular pH (pH(i)) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This preparation permitted the separate study of the intercalated cells (IC) from the principal cells (PC). In IC pH(i) recovery in response to an acute acid load was observed under Na-free conditions on addition of 5 mM K. This K-dependent recovery of pH(i) in the IC was only partial, but was Sch 28080 inhibitable (10(-5) M) and ouabain insensitive. This suggests the process is mediated by an H-K-adenosinetriphosphatase similar to that of gastric cells. The PC were capable of recovering from the acid load, but this Na-independent response was not blocked by the Sch 28080, suggesting some other mechanism for this result. In both cell types reintroduction of Na into the superfusate resulted in full recovery back to the initial pH(i), presumably the result of Na/H exchange.

Isolation, growth, and characterization of a gluconeogenic strain of renal cells

1987 ◽  
Vol 252 (2) ◽  
pp. C232-C238 ◽  
Author(s):  
G. Gstraunthaler ◽  
J. S. Handler
Keyword(s):  
Lactate Dehydrogenase ◽  
Proximal Tubule ◽  
Enzyme Activities ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Apical Membrane ◽  
Incidental Finding ◽  
Specific Inhibitor ◽  
Free Medium ◽  
Membrane Enzyme ◽  
Rna Biosynthesis

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.

Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells

1989 ◽  
Vol 256 (6) ◽  
pp. G1082-G1089 ◽  
Author(s):  
D. K. Hanzel ◽  
T. Urushidani ◽  
W. R. Usinger ◽  
A. Smolka ◽  
J. G. Forte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Secretion ◽  
Parietal Cell ◽  
Blood Cells ◽  
Apical Membrane ◽  
Staining Pattern ◽  
Parietal Cells ◽  
Entire Cell ◽  
Gastric Parietal Cells ◽  
Stimulation Of

Monoclonal antibodies were raised against an 80-kDa phosphoprotein (80K) that is phosphorylated upon stimulation of gastric acid secretion and that copurifies with the acid-forming H+-K+-ATPase isolated from stimulated tissue. These antibodies were used to demonstrate that in the gastric mucosa 80K is limited to parietal cells and not found in surface, mucous neck, or chief cells. 80K was also found in other transporting epithelia, including intestine and kidney, but was not found in brain, liver, red blood cells, or colon. Immunohistological localization of 80K in resting glands revealed a fine network, projecting from the gland lumen and anastomosing throughout the parietal cell. This network is quite similar to the staining pattern for F-actin contained in microvilli that line the apical membrane of parietal cells. Stimulation of acid secretion rearranges 80K to a more rugose pattern filling the entire cell. In stimulated cells the distribution pattern of 80K is indistinguishable from that stained with antibodies against the H+-K+-ATPase. These data strongly suggest that 80K is an apical membrane protein of the parietal cell.

In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii

Parasitology ◽  
1999 ◽  
Vol 119 (3) ◽  
pp. 259-265 ◽  
Author(s):  
D. J. P. FERGUSON ◽  
D. JACOBS ◽  
E. SAMAN ◽  
J-F. DUBREMETZ ◽  
S. E. WRIGHT
Keyword(s):  
Toxoplasma Gondii ◽  
Staining Pattern ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasite Development ◽  
Double Labelling ◽  
Granule Protein ◽  
Specific Variation ◽  
Strong Staining

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

DETECTION OF HUMAN T-LYMPHOTROPIC VIRUS TYPE I IN ECCRINE SWEAT DUCT EPITHELIUM

1999 ◽  
Vol 20 (4) ◽  
pp. A39
Author(s):  
Shimako Mizoguchi ◽  
Mitsuru Setoyama ◽  
Tamotsu Kanzaki ◽  
Yoshito Eizuru
Keyword(s):  
Virus Type ◽  
Type I ◽  
Sweat Duct ◽  
T Lymphotropic Virus ◽  
Duct Epithelium ◽  
Eccrine Sweat
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