High-pressure Freezing of Isolated Gastric Glands Provides New Insight into the Fine Structure and Subcellular Localization of H+/K+-ATPase in Gastric Parietal Cells

The effects of osmotically induced changes in cell volume on cytoplasmic free Ca (Cai) were studied in parietal cells from intact rabbit gastric glands using digital image processing of fura-2 fluorescence. In resting unstimulated cells, Cai was unaffected by either cell swelling or shrinking when osmolarity was varied between 200 and 400 mosM (isotonicity 290 mosM). However, when cells were swelled in a 165 mosM solution (55% tonicity), a biphasic Ca increased was observed. On average, Cai increased transiently from 80 to 218 nM before stabilizing at approximately 140 nM. The peak was due to release from intracellular pools because it was present in Ca-free solutions while the sustained elevation was dependent on external Ca. In carbachol-stimulated cells, Ca influx was most sensitive to cell shrinkage. For example, addition of 25 mM sucrose (108% tonicity) caused a 30% decrease in the sustained carbachol-stimulated Cai increase (plateau). In contrast, carbachol-stimulated cells were relatively insensitive to cell swelling, with a 30% decrease in tonicity causing only a 15% increase in the plateau. However, as in the unstimulated cells, extreme (55% tonicity) swelling caused additional increases in Cai levels. The carbachol-dependent effects of changes in cell volume on Cai could be mimicked by treating cells with thapsigargin, an inhibitor of Ca pumps of intracellular membranes that also has been shown to stimulate Ca entry. Thus, although extreme swelling conditions (55% tonicity) could elicit Cai increases in either the presence or absence of agonist, agonist was required to observe Cai decreases due to cell shrinkage.(ABSTRACT TRUNCATED AT 250 WORDS)

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Functionally distinct pools of actin in secretory cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.2.c407 ◽

2001 ◽

Vol 281 (2) ◽

pp. C407-C417 ◽

Cited By ~ 26

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.6.c1599 ◽

2001 ◽

Vol 280 (6) ◽

pp. C1599-C1606 ◽

Cited By ~ 48

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.

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Inhibitory action of somatostatin on isolated gastric glands and parietal cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1983.245.2.g221 ◽

1983 ◽

Vol 245 (2) ◽

pp. G221-G229 ◽

Cited By ~ 7

Author(s):

C. S. Chew

Keyword(s):

Parietal Cell ◽

Inhibitory Action ◽

Parietal Cells ◽

Acid Formation ◽

Gastric Glands ◽

Isolated Gastric Glands ◽

Cell Acid ◽

Kinetics Of ◽

Apparent Ic50

The action of somatostatin in vitro was characterized using glands and parietal cells isolated from rabbit gastric mucosa. In the presence of the reducing agent dithiothreitol, somatostatin was found to inhibit gastrin- and histamine-stimulated acid formation in glands as measured by [14C]aminopyrine (AP) accumulation and oxygen consumption, both measurements that appear to be reliable indexes of parietal cell acid formation. In glands the inhibition of the secretory response to gastrin was more potent (60-80%) than that to histamine (15-25%). The kinetics of somatostatin inhibition of responses to both agents were noncompetitive. The apparent IC50 for the partial somatostatin inhibition of histamine-stimulated AP accumulation was similar to that for gastrin (approx 3 X 10(-9) M) when maximum concentrations of histamine (10(-4) M) or gastrin (10(-7) M) were used. The inhibitory action of somatostatin appeared to be specific, inasmuch as this peptide had no significant effect on basal secretion or secretion stimulated by carbachol, dibutyryl cAMP, cholera toxin, or elevated extracellular K+. In purified parietal cell preparations, somatostatin inhibited histamine- but not gastrin-stimulated AP accumulation. Moreover, the inhibition of histamine-stimulated AP accumulation in parietal cells was more pronounced than in glands. These results suggest that somatostatin acts directly on parietal cells to inhibit histamine activation of H+ secretion. Somatostatin also acts indirectly to inhibit gastrin, perhaps by blocking the release of histamine from paracrine- or endocrinelike cells present in the glands.

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The fine structure of human gastric parietal cells

The American Journal of Digestive Diseases ◽

10.1007/bf02235070 ◽

1965 ◽

Vol 10 (1) ◽

pp. 13-21 ◽

Cited By ~ 21

Author(s):

G. Victor Rohrer ◽

J. Randolph Scott ◽

Walter Joel ◽

Stewart Wolf

Keyword(s):

Fine Structure ◽

Parietal Cells ◽

Gastric Parietal Cells

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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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