scholarly journals Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles

1997 ◽  
Vol 325 (2) ◽  
pp. 559-564 ◽  
Author(s):  
Benjamin C. CALHOUN ◽  
James R. GOLDENRING
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Parietal Cell ◽  
Vesicle Fusion ◽  
Parietal Cells ◽  
Rab Proteins ◽  
Α Subunit ◽  
Gastric Parietal Cell ◽  
Secretory Carrier Membrane Proteins

The tubulovesicles of gastric parietal cells sequester H+/K+-ATPase molecules within resting parietal cells. Stimulation of parietal cell secretion elicits delivery of intracellular H+/K+-ATPase to the apically oriented secretory canaliculus. Previous investigations have suggested that this process requires the regulated fusion of intracellular tubulovesicles with the canalicular target membrane. We have sought to investigate the presence of critical putative regulators of vesicle fusion on immunoisolated gastric parietal cell tubulovesicles. Highly purified tubulovesicles were prepared by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the α subunit of H+/K+-ATPase. Western blot analysis revealed the presence of Rab11, Rab25, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs) on immunoisolated vesicles. The same cohort of proteins was recovered on vesicles immunoisolated with monoclonal antibodies against SCAMPs and VAMP-2. In contrast, whereas immunoreactivities for syntaxin 1A/1B and synaptosome-associated protein (SNAP-25) were present in gradient-isolated vesicles, none of the immunoreactivity was associated with immunoisolated vesicles. The observation of VAMP-2 and two Rab proteins on immunoisolated H+/K+-ATPase-containing tubulovesicles supports the role for tubulovesicles in a regulated vesicle fusion process. In addition, the presence of SCAMPs along with Rab11 and Rab25 implicates the tubulovesicles as a critical apical recycling vesicle population.

scholarly journals Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

1997 ◽  
Vol 8 (3) ◽  
pp. 399-407 ◽  
Author(s):  
X R Peng ◽  
X Yao ◽  
D C Chow ◽  
J G Forte ◽  
M K Bennett
Keyword(s):  
Membrane Protein ◽  
Acid Secretion ◽  
Parietal Cell ◽  
Membrane Trafficking ◽  
Parietal Cells ◽  
Gastric Parietal Cell ◽  
Protein Receptors ◽  
Alternative Function ◽  
Target Membrane ◽  
Syntaxin 3

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Functionally distinct pools of actin in secretory cells

2001 ◽  
Vol 281 (2) ◽  
pp. C407-C417 ◽  
Author(s):  
David A. Ammar ◽  
Phuong N. B. Nguyen ◽  
John G. Forte
Keyword(s):  
Parietal Cell ◽  
Cultured Cells ◽  
Parietal Cells ◽  
Secretory Cells ◽  
Resting Cells ◽  
Gastric Glands ◽  
Gastric Parietal Cell ◽  
Actin Organization ◽  
Isolated Gastric Glands ◽  
High Concentrations

Acid secretion by the gastric parietal cell is controlled through movement of vesicles containing the proton pump, the H+-K+-ATPase (HK). We have used latrunculin B (Lat B), which binds to monomeric actin, to investigate actin turnover in the stimulated parietal cell. In isolated gastric glands, relatively high concentrations of Lat B were required to inhibit acid accumulation (ED50∼70 μM). Cultured parietal cells stimulated in the presence of low Lat B (0.1–1 μM) have reduced lamellipodia formation and some aberrant punctate phalloidin-stained structures, but translocation of HK and vacuolar swelling appeared unaffected. High Lat B (10–50 μM) resulted in gross changes in actin organization (punctate phalloidin-stained structures throughout the cell and nucleus) and reduced translocation of HK and vacuolar swelling. Resting parietal cells treated with high Lat B showed minor effects on morphology and F-actin staining. If resting cells treated with high Lat B were washed immediately before stimulation, they exhibited a normal stimulated morphology. These data suggest distinct pools of parietal cell actin: a pool highly susceptible to Lat B primarily involved in motile function of cultured cells; and a Lat B-resistant pool, most likely microvillar filaments, that is essential for secretion. Furthermore, the stimulation process appears to accentuate the effects of Lat B, most likely through Lat B binding to monomer actin liberated by the turnover of the motile actin filament pool.

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.

Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa

2005 ◽  
Vol 289 (2) ◽  
pp. G320-G331 ◽  
Author(s):  
Catherine S. Chew ◽  
Curtis T. Okamoto ◽  
Xunsheng Chen ◽  
Ruby Thomas
Keyword(s):  
Gastric Mucosa ◽  
Actin Filament ◽  
Parietal Cell ◽  
Active Role ◽  
Cell Types ◽  
Parietal Cells ◽  
Dominant Negative ◽  
Filament Formation ◽  
Differential Distribution ◽  
Gastric Parietal Cell

Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels

2004 ◽  
Vol 286 (3) ◽  
pp. C495-C506 ◽  
Author(s):  
Danuta H. Malinowska ◽  
Ann M. Sherry ◽  
Kirti P. Tewari ◽  
John Cuppoletti
Keyword(s):  
Parietal Cell ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Extracellular Ph ◽  
Parietal Cells ◽  
In Vitro Translation ◽  
Rt Pcr ◽  
Open Probability ◽  
Gastric Parietal Cell ◽  
Cation Selectivity

Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to β-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at ∼50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+-K+-ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of ∼11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to ∼0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ >> Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.

Platelet Agglutinating Protein p37 Causes Platelet Agglutination through its Binding to Membrane Glycoprotein IV

1991 ◽  
Vol 65 (01) ◽  
pp. 102-106 ◽  
Author(s):  
Eric C-Y Lian ◽  
Farooq A Siddiqui ◽  
G A Jamieson ◽  
Narendra N Tandon
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Receptor Site ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Normal Platelet ◽  
Reducing Conditions ◽  
Rabbit Igg

SummaryA 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that r2sI-p37 bound to a membrane protein of 97 kDa (red/unred). Furthernore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti- GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti- GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.

Parietal cell MAP kinases: multiple activation pathways

1996 ◽  
Vol 271 (4) ◽  
pp. G640-G649 ◽  
Author(s):  
K. Nakamura ◽  
C. J. Zhou ◽  
J. Parente ◽  
C. S. Chew
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Parietal Cell ◽  
Intracellular Signaling ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Cell Types ◽  
Parietal Cells ◽  
Gastric Parietal Cell ◽  
Cell Acid

Epidermal growth factor (EGF) is a potent mitogen for many cell types; however, the best known effect of EGF on gastric parietal cell HCl secretion is inhibition of this response. Using rabbit parietal cells in primary culture, we recently showed that the effect of EGF is biphasic with acute inhibition followed by sustained enhancement of acid secretory-related responses. We hypothesized that EGF might activate a mitogen-activated protein (MAP) kinase signaling pathway in parietal cells, and this pathway might play a role in mediating sustained and/or acute effects of EGF on parietal cell acid secretory-related functions [C. S. Chew, K. Nakamura, and A. C. Petropolous. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G818-G826, 1994]. We used several methodological approaches to demonstrate the presence of MAP kinase (MAPK) isoforms, extracellular signal-regulated kinases (ERKs) 1 and 2, in parietal cells and to begin to characterize their mechanisms of activation in this highly differentiated cell type. In acutely isolated, 90-98% enriched parietal cells, EGF biphasically activated ERK-1 and ERK-2, with peak response occurring at approximately 5 min followed by a sustained lower level of activation for at least 2 h. The EC50 for EGF (1.2 +/- 0.4 nM) was similar to the previously determined EC50 for the stimulatory effect of EGF on acid secretory responses. In contrast to EGF, the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a sustained activation of ERK-1 and ERK-2 for at least 2 h. Carbachol also activated ERK-1 and ERK-2; however, this response was weaker and monophasic. Neither the Ca2+ ionophore ionomycin nor the adenylyl cyclase activator forskolin altered basal or stimulated ERK activity. Carbachol, but not EGF or TPA, also activated an unidentified 70-kDa protein kinase as detected with in-gel myelin basic protein (MBP) kinase renaturation assays. Parietal cell MAPK activation was not correlated to a shift in apparent relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that basal phosphorylation of ERK isoforms may be higher in parietal cells compared with actively proliferating cell lines. Also, in contrast to observations in neutrophils, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor, wortmannin (0.3-3 microM), failed to inhibit ERK activation in response to EGF, carbachol, or TPA. The combined data indicate that 1) EGF, TPA, and carbachol activate overlapping as well as distinct intracellular signaling pathways in gastric parietal cells, 2) EGF activates ERKs and enhances parietal cell acid secretory related functions via receptors with similar affinities, and 3) in contrast to some cell types, the parietal cell ERK-signaling cascade does not appear to be directly modulated by the PtdIns 3-kinase pathway or by elevated intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate concentrations.

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase

1999 ◽  
Vol 277 (3) ◽  
pp. C361-C372 ◽  
Author(s):  
Joseph G. Duman ◽  
Kamala Tyagarajan ◽  
Michelle S. Kolsi ◽  
Hsiao-Ping H. Moore ◽  
John G. Forte
Keyword(s):  
Parietal Cell ◽  
Small Gtpase ◽  
Parietal Cells ◽  
Apical Plasma Membrane ◽  
Dominant Negative Mutant ◽  
Resting Cells ◽  
Laser Desorption Mass Spectrometry ◽  
Gastric Parietal Cell ◽  
Ap Index ◽  
Stimulation Of

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 ± 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 ± 0.9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 ± 3.1 and 9.6 ± 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody

1993 ◽  
Vol 264 (4) ◽  
pp. G637-G644 ◽  
Author(s):  
J. Cuppoletti ◽  
P. Huang ◽  
M. A. Kaetzel ◽  
D. H. Malinowska
Keyword(s):  
Parietal Cell ◽  
Parietal Cells ◽  
Bee Venom ◽  
Gastric Parietal Cell ◽  
Functional Changes ◽  
Hcl Secretion ◽  
Stimulation Of

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

Production of monoclonal antibodies against gastric parietal cell antigens

1992 ◽  
Vol 144 (3) ◽  
pp. 369-378 ◽  
Author(s):  
J. L. CABERO ◽  
T. SASAKI ◽  
Y.-H. SONG ◽  
R. HOLMDAHL ◽  
S. MÅRDH
Keyword(s):  
Monoclonal Antibodies ◽  
Parietal Cell ◽  
Gastric Parietal Cell
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