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.

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.

Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa

1994 ◽  
Vol 266 (2) ◽  
pp. C559-C568 ◽  
Author(s):  
A. Stuart-Tilley ◽  
C. Sardet ◽  
J. Pouyssegur ◽  
M. A. Schwartz ◽  
D. Brown ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Parietal Cell ◽  
Anion Exchanger ◽  
Cell Types ◽  
Parietal Cells ◽  
Morphological Evidence ◽  
Mucous Cells ◽  
Gastric Glands ◽  
Basolateral Membranes ◽  
Mucous Neck Cells

The gastric mucosa secretes both protons and bicarbonate. The molecular identity of the H(+)-K(+)-ATPase, which mediates acid secretion, has long been known, but the other components of the secretory machinery and their cellular disposition are less well characterized. This study identifies and localizes in rat and rabbit gastric mucosa a chloride-bicarbonate exchanger protein and a Na(+)-H+ exchanger protein. The previously described band 3-related protein of the parietal cell has been identified by isoform-specific antibodies as anion exchanger (AE) 2 and localized to the basolateral membranes of the parietal cells. The Na(+)-H+ exchanger protein NHE-1 was located in the basolateral membranes of the mucous neck cells, interdigitated between the parietal cells of the gastric glands and in the basolateral membranes of the surface mucous cells. Neither transporter protein was abundantly expressed deep in the gland, where most of the pepsinogen cells reside. Carbonic anhydrase II (CA II) was expressed at higher abundance in the surface mucous cells and mucous neck cells, which expressed NHE-1, than in the parietal cells, which expressed AE2. The morphological evidence identified AE2 as a major parietal cell anion exchanger, whereas NHE-1 and CA II colocalized in mucous neck, chief, and surface mucous cells. We propose that all three of these cell types contribute to gastric bicarbonate secretion.

Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell

1998 ◽  
Vol 275 (1) ◽  
pp. C56-C67 ◽  
Author(s):  
C. S. Chew ◽  
J. A. Parente ◽  
C.-J. Zhou ◽  
E. Baranco ◽  
X. Chen
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Signaling Pathway ◽  
Parietal Cell ◽  
Cell Types ◽  
Parietal Cells ◽  
Camp Signaling ◽  
Gastric Parietal Cell ◽  
Putative Protein ◽  
Camp Signaling Pathway

Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.

scholarly journals Purification of Gastric Mucosal ECL Cells from a Crude Elutriation Fraction

2002 ◽  
Vol 2 ◽  
pp. 1643-1645 ◽  
Author(s):  
John Graham
Keyword(s):  
Gastric Mucosa ◽  
Ion Transport ◽  
Parietal Cell ◽  
Cell Function ◽  
Parietal Cells ◽  
Centrifugal Elutriation ◽  
Ecl Cells ◽  
Acid Secreting ◽  
Density Barrier

Acid-secreting parietal cells from the gastric mucosa are widely studied as a model in studies on ion transport and the endocrine/paracrine ECL cells effectively control parietal cell function. Discontinuous gradients of iodixanol for the purification of ECL cells were subsequently simplified to the use of a density barrier. This technique is now commonly used following initial centrifugal elutriation.

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.

scholarly journals Localization of ClC-2 Cl− channels in rabbit gastric mucosa

2001 ◽  
Vol 280 (6) ◽  
pp. C1599-C1606 ◽  
Author(s):  
Ann M. Sherry ◽  
Danuta H. Malinowska ◽  
Randal E. Morris ◽  
Georgianne M. Ciraolo ◽  
John Cuppoletti
Keyword(s):  
Gastric Mucosa ◽  
Parietal Cell ◽  
Functional Characterization ◽  
Parietal Cells ◽  
Immunogold Electron Microscopy ◽  
Gastric Glands ◽  
Canalicular Membrane ◽  
Isolated Gastric Glands ◽  
Hcl Secretion

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.

Cl- channels of the gastric parietal cell that are active at low pH

1993 ◽  
Vol 264 (6) ◽  
pp. C1609-C1618 ◽  
Author(s):  
J. Cuppoletti ◽  
A. M. Baker ◽  
D. H. Malinowska
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Parietal Cell ◽  
Asymmetric Reduction ◽  
Ion Selectivity ◽  
Channel Activity ◽  
Open Probability ◽  
Gastric Parietal Cell ◽  
Cl Channel ◽  
Cl Channels

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

scholarly journals Modulatory role of phosphoinositide 3-kinase in gastric acid secretion

2007 ◽  
Vol 293 (3) ◽  
pp. G532-G543 ◽  
Author(s):  
S. E. Mettler ◽  
S. Ghayouri ◽  
G. P. Christensen ◽  
J. G. Forte
Keyword(s):  
Acid Secretion ◽  
Parietal Cell ◽  
Phosphodiesterase Inhibitors ◽  
Dominant Negative ◽  
Gastric Glands ◽  
Gastric Parietal Cell ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Phosphoinositide 3 Kinase

The gastric parietal cell is responsible for the secretion of HCl into the lumen of the stomach mainly due to stimulation by histamine via the cAMP pathway. However, the participation of several other receptors and pathways have been discovered to influence both stimulation and inhibition of acid secretion (e.g., cholinergic). Here we examine the role of phosphoinositide 3-kinase (PI3K) in the modulation of acid secretion. Treatment of isolated gastric glands and parietal cells with the PI3K inhibitor, LY294002 (LY), potentiated acid secretion in response to histamine to nearly the maximal secretion obtained with histamine plus phosphodiesterase inhibitors. As cAMP levels were elevated in response to histamine plus LY, but other means of elevating cAMP (e.g., forskolin, dbcAMP) were not influenced by LY, we posited that the effect might require activation of G-protein-coupled histamine H2 receptors, possibly through the protein kinase B pathway (also known as Akt). Study of downstream effectors of PI3K showed that histaminergic stimulation increased Akt phosphorylation, which in turn was blocked by inhibition of PI3K. Expression studies showed that high expression of active Akt decreased acid secretion, whereas dominant-negative Akt increased acid secretion. Taken together, these data suggest stimulation with histamine increases the activity of PI3K leading to increased activity of Akt and decreased levels of cAMP in the parietal cell.

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