Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.

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Receptor-operated Ca2+ channels in gastric parietal cells: gastrin and carbachol induce Ca2+ influx in depleting intracellular Ca2+ stores

Biochemical Journal ◽

10.1042/bj2890117 ◽

1993 ◽

Vol 289 (1) ◽

pp. 117-124 ◽

Cited By ~ 10

Author(s):

S Roche ◽

J P Bali ◽

R Magous

Keyword(s):

Steady State ◽

Receptor Activation ◽

Parietal Cells ◽

The Other ◽

Bivalent Cation ◽

Gtp Binding ◽

Gastric Parietal Cells ◽

Type Receptor ◽

Ca2 Channels ◽

Stimulation Of

The mechanism whereby gastrin-type receptor and muscarinic M3-type receptor regulate free intracellular Ca2+ concentration ([Ca2+]i) was studied in rabbit gastric parietal cells stimulated by either gastrin or carbachol. Both agonists induced a biphasic [Ca2+]i response: a transient [Ca2+]i rise, followed by a sustained steady state depending on extracellular Ca2+. Gastrin and carbachol also caused a rapid and transient increase in Mn2+ influx (a tracer for bivalent-cation entry). Pre-stimulation of cells with one agonist drastically decreased both [Ca2+]i increase and Mn2+ influx induced by the other. Neither diltiazem nor pertussistoxin treatment had any effect on agonist-stimulated Mn2+ entry. Thapsigargin, a Ca(2+)-pump inhibitor, induced a biphasic [Ca2+]i increase, and enhanced the rate of Mn2+ entry. Preincubation of cells with thapsigargin inhibits the [Ca2+]i increase as well as Mn2+ entry stimulated by gastrin or by carbachol. Thapsigargin induced a weak but significant increase in Ins(1,4,5)P3 content, but this agent had no effect on the agonist-evoked Ins(1,4,5)P3 response. In permeabilized parietal cells, Ins(1,4,5)P3 and caffeine caused an immediate Ca2+ release from intracellular pools, followed by a reloading of Ca2+ pools which can be prevented in the presence of thapsigargin. We conclude that (i) gastrin and carbachol mobilize common Ca2+ intracellular stores, (ii) Ca2+ permeability secondary to receptor activation involves neither a voltage-sensitive Ca2+ channel nor a GTP-binding protein from the G1 family, and (iii) agonists regulate common Ca2+ channels in depleting intracellular Ca2+ stores.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.256.6.g1082 ◽

1989 ◽

Vol 256 (6) ◽

pp. G1082-G1089 ◽

Cited By ~ 17

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.

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Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells

Journal of Cell Science ◽

10.1242/jcs.115.6.1251 ◽

2002 ◽

Vol 115 (6) ◽

pp. 1251-1258 ◽

Cited By ~ 1

Author(s):

Joseph G. Duman ◽

Nimesh J. Pathak ◽

Mark S. Ladinsky ◽

Kent L. McDonald ◽

John G. Forte

Keyword(s):

Parietal Cell ◽

Three Dimensional ◽

Parietal Cells ◽

Apical Plasma Membrane ◽

Serial Sections ◽

Gastric Glands ◽

Dimensional Reconstruction ◽

Membrane Compartment ◽

Membrane Networks ◽

Gastric Parietal Cells

There is general agreement that stimulation and consequent secretion of gastric parietal cells result in a great expansion of the apical canalicular membrane at the expense of an extensive intracellular network of membranes rich in the gastric proton pump (H,K-ATPase). However, there is ongoing controversy as to the precise nature of the intracellular membrane network,conventionally called tubulovesicles. At the heart of this controversy lies the question of whether tubulovesicles are a distinct membrane compartment or whether they are continuous with the apical plasma membrane.To address this controversy we used high-pressure, rapid freezing techniques to fix non-stimulated (resting) rabbit gastric glands for electron microscopy. Ultra-thin (60-70 nm) serial sections were used for conventional TEM; 400-500 nm sections were used for tomography. Images were digitized and models constructed using Midas and Imod software(http://bio3d.colorado.edu). Images were aligned and contours drawn on specific cellular structures. The contours from a stack of serial sections were arranged into objects and meshed into 3D structures. For resting parietal cells our findings are as follows:(1) The apical canaliculus is a microvilli-decorated, branching membrane network that extends into and throughout the parietal cell. This agrees well with a host of previous studies. (2) The plentiful mitochondria form an extensive reticular network throughout the cytoplasm. This has not previously been reported for the parietal cell, and the significance of this observation and the dynamics of the mitochondrial network remain unknown. (3)H,K-ATPase-rich membranes do include membrane tubules and vesicles; however,the tubulovesicular compartment is chiefly comprised of small stacks of cisternae. Thus a designation of tubulocisternae seems appropriate; however,in the resting cell there are no continuities between the apical canaliculus and the tubulocisternae or between tubulocisternae. These data support the recruitment-recycling model of parietal cell stimulation.

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The gastric H, K-ATPase system also functions as the Na, K-ATPase and Ca-ATPase in altered states

F1000Research ◽

10.12688/f1000research.2-165.v1 ◽

2013 ◽

Vol 2 ◽

pp. 165

Author(s):

Tushar Ray

Keyword(s):

Atpase Activity ◽

Acid Secretion ◽

Proton Pump ◽

Intracellular Signaling ◽

Parietal Cells ◽

Apical Plasma Membrane ◽

Altered States ◽

Apparent Lack ◽

Ca Pump ◽

Na Pump

This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs. This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.3.c361 ◽

1999 ◽

Vol 277 (3) ◽

pp. C361-C372 ◽

Cited By ~ 74

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.

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