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.

Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells

1993 ◽  
Vol 264 (3) ◽  
pp. F490-F495 ◽  
Author(s):  
A. W. Mangel ◽  
J. R. Raymond ◽  
J. G. Fitz
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Single Channel ◽  
Chinese Hamster ◽  
Channel Activity ◽  
Anion Channels ◽  
Open Probability ◽  
Single Channel Currents ◽  
High Conductance

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.

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.

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)

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.

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 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.

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.

scholarly journals The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

1995 ◽  
Vol 106 (1) ◽  
pp. 85-111 ◽  
Author(s):  
N Demaurex ◽  
J Orlowski ◽  
G Brisseau ◽  
M Woodside ◽  
S Grinstein
Keyword(s):  
Cho Cells ◽  
Time Course ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Outward Current ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Transmembrane Currents ◽  
Cytosolic Acidification

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

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