Intracellular Ca2+ shift and signal transduction from the tubulovesicular portion of gastric parietal cells during gastrin stimulation or Ca2+ ionophore treatment: comparison between luminescent and fluorescent probes, and electron probe X-ray microanalyzer

1988 ◽  
Vol 66 (4) ◽  
pp. 279-287 ◽  
Author(s):  
Yasuhiro Tsunoda ◽  
Hiroshi Takeda ◽  
Toshihiro Otaki ◽  
Masahiro Asaka ◽  
Ikuko Nakagaki ◽  
...  
Keyword(s):  
Resting State ◽  
Electron Probe ◽  
Physiological Saline ◽  
Parietal Cells ◽  
X Ray ◽  
Fura 2 ◽  
Treatment Comparison ◽  
Apical Cytoplasm ◽  
Intracellular Ca ◽  
Gastric Parietal Cells

In gastrin-stimulated, aequorin-loaded parietal cells from guinea pig gastric mucosa, a rapid but transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), owing to Ca2+ released from the store(s), and a more prolonged Ca2+ entry from outside the cells were observed. However, there was a little increase in [Ca2+]i when similar measurements were assessed by quin 2 or fura-2 in physiological saline. However, depletion or elimination of Na+ from the incubation medium caused a significant increase in the [Ca2+]; response to gastrin as measured by quin 2. These findings suggest that aequorin and quin 2 (or fura-2) provide information about different aspects of Ca2+ homeostasis and that there is an inhomogeneity of [Ca2+]i in the cytoplasm during gastrin stimulation. By the gastrin stimulation, the intracellular Ca2+ gradients were shifted from the unidentified portion(s) to the restricted apical cytoplasm, as determined by electron probe X-ray microanalysis. Therefore, localization and identification of the source of intracellular Ca2+ as a pool were determined by an X-ray microanalyzer. In the resting state, the tubulovesicle had high Ca2+ concentration compared with the level in the apical cytoplasm. Cells treated with the Ca2+ ionophore ionomycin had a decreased tubulovesicular Ca2+ level, followed by a reciprocal increase in area of the canalicular membrane. The secretory canaliculus in stimulated cells had lower Ca2+ or higher K+ and Cl− concentrations than that of tubulovesicles or cytoplasm in the resting state, respectively. These findings suggest that the Ca2+ pool of the parietal cell is in the tubulovesicles and (or) luminal cell membrane and that the Ca2+ released from the store(s) may mediate a flow of K+ or Cl− into the secretory canaliculus.

Colocalization of the apical Cl−/HCO 3 − exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells

2002 ◽  
Vol 283 (5) ◽  
pp. G1207-G1216 ◽  
Author(s):  
Snezana Petrovic ◽  
Zhaohui Wang ◽  
Liyun Ma ◽  
Ursula Seidler ◽  
John G. Forte ◽  
...  
Keyword(s):  
Resting State ◽  
Parietal Cells ◽  
Immunocytochemical Staining ◽  
Cell Distribution ◽  
Sharp Reduction ◽  
Anion Transporter ◽  
Membrane Location ◽  
Immunofluorescence Labeling ◽  
Apical Membranes ◽  
Gastric Parietal Cells

The apical Cl−/HCO[Formula: see text] exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorusagglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled 36Cl flux studies demonstrated the functional presence of Cl−/HCO[Formula: see text] exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl−/HCO[Formula: see text]exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.

Tension and electrolyte changes with Na+-K+ pump inhibition in rat papillary muscle

1989 ◽  
Vol 257 (3) ◽  
pp. H942-H953
Author(s):  
D. Parsons ◽  
K. P. Burton ◽  
H. K. Hagler ◽  
J. T. Willerson ◽  
L. M. Buja
Keyword(s):  
Electron Probe ◽  
Cell Injury ◽  
Membrane Integrity ◽  
Bimodal Distribution ◽  
High Energy ◽  
Atp Depletion ◽  
X Ray ◽  
Elemental Concentrations ◽  
Intracellular Ca ◽  
The Mean

Myocardial ischemic injury results in altered membrane integrity, energy depletion, and electrolyte shifts leading to accumulation of intracellular Ca. However, analysis of the direct effects of Ca accumulation is complicated by other concomitant cellular changes produced by ischemia. The purpose of this study was to examine the effects of Ca loading in rat papillary muscles produced by Na+-K+ pump inhibition in oxygenated K+-free buffer. Changes in contractile characteristics, high energy phosphate, and elemental concentrations of subcellular compartments were measured. Electron probe X-ray microanalysis was used to assess elemental concentrations in cryosections. After 3 h of Na+-K+ pump inhibition, resting tension (RT) increased to 164% and developed tension (DT) fell to 16.8% of control values. One hour after return to complete buffer, RT and DT partially recovered but remained significantly different from the 180 to 240-min values for the control muscles. Electron probe X-ray microanalysis showed increases in cytoplasmic and mitochondrial Na and Ca and a decrease in K during Na+-K+ pump inhibition. Mitochondrial Ca was greater than 100-fold greater than Ca in control mitochondria. Morphologically, the majority of cells showed ultrastructural damage. The mean ATP level was 20% of control. After 1 h of recovery, the cells appeared more heterogeneous, and the mean mitochondrial Ca decreased, whereas mean cytoplasmic Ca increased. Further statistical analysis showed a bimodal distribution for Na, Ca, K, Mg, and Cl, which coincided with the morphologically mixed population of cells. This suggests that replacement of extracellular K+ was associated with restored electrolyte gradients in some cells and the persistent or further alteration of electrolytes in others. These results suggest that variable Ca accumulation and associated ATP depletion without the compounding effects of ischemia lead to cell injury similar to reperfusion injury reported in ischemic myocardium.

Regulation of c-Jun NH2-terminal kinases in isolated canine gastric parietal cells

1998 ◽  
Vol 275 (4) ◽  
pp. G740-G748 ◽  
Author(s):  
A. Nagahara ◽  
L. Wang ◽  
J. Del Valle ◽  
A. Todisco
Keyword(s):  
Transcriptional Activation ◽  
Specific Protein ◽  
Parietal Cells ◽  
Stimulatory Action ◽  
Potent Inducer ◽  
Mrna Content ◽  
Intracellular Ca ◽  
Cytokine Tumor Necrosis Factor ◽  
Factor Α ◽  
Gastric Parietal Cells

c-Jun NH2-terminal kinases (JNKs) are protein kinases that are activated by a wide variety of extracellular signals. This study investigated the expression and regulation of JNKs in isolated gastric canine parietal cells. Western blot analysis of cell lysates from highly purified (>95%) parietal cells with an antibody recognizing JNK1 and to a lesser degree JNK2 revealed the presence of two bands of 46 and 54 kDa, respectively. JNK1 activity was quantitated by immunoprecipitation and in-gel kinase assays. Of the different agents tested, carbachol was the most potent inducer of JNK1 activity, whereas histamine and epidermal growth factor induced weaker responses. The proinflammatory cytokine tumor necrosis factor-α stimulated JNK1 but had no effect on extracellular signal-regulated kinase (ERK2) induction, suggesting that activation of JNK1 might represent an important event in mediation of the inflammatory response in the stomach. The action of carbachol was dose (0.1–100 μM) and time dependent, with a maximal stimulatory effect (fourfold) detected after 30 min of incubation and sustained for 2 h. Addition of the specific protein kinase C (PKC) inhibitor GF109203X did not affect the stimulatory action of carbachol. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM inhibited carbachol induction of JNK1 activity by 60%. Thapsigargin (1 μM), an intracellular Ca2+-rising agent, induced JNK1 activity more than threefold. Carbachol activation of JNK1 resulted in induction of c-Jun (protein) transcriptional activity and in stimulation of parietal cell mRNA content of c- jun. In conclusion, our data indicate that carbachol induces JNK activity in gastric parietal cells via intracellular Ca2+-dependent, PKC-independent pathways, leading to induction of c- jun gene expression via phosphorylation and transcriptional activation of c-Jun.

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

2002 ◽  
Vol 282 (6) ◽  
pp. G1069-G1078 ◽  
Author(s):  
Nonthalee Pausawasdi ◽  
Saravanan Ramamoorthy ◽  
Leslie J. Crofford ◽  
Frederick K. Askari ◽  
Andrea Todisco
Keyword(s):  
Gene Expression ◽  
Cdna Probe ◽  
Parietal Cells ◽  
P38 Kinase ◽  
Potent Inducer ◽  
Intracellular Ca ◽  
Cox 2 ◽  
Gastric Parietal Cells ◽  
Sb 203580 ◽  
Stimulation Of

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-κB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IκB, which expresses a repressor of NF-κB. Addition of SB-203580 with Ad.dom.neg.IκB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE2release by enzyme-linked immunoassay. Carbachol induced PGE2 release. Ad.dom.neg.IκB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE2 production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE2 release without affecting basal PGE2production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE2 release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca2+, PKC, p38 kinase, and activation of NF-κB. The functional significance of these effects seems to be stimulation of PGE2 release.

Electron-probe x-ray microanalysis studies on the intracellular calcium translocation during the contraction-relaxation cycle in the fish swimbladder muscle

1990 ◽  
Vol 48 (2) ◽  
pp. 168-169
Author(s):  
Suechika Suzuki ◽  
Naoki Hino ◽  
Haruo Sugi
Keyword(s):  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Teleost Fish ◽  
Electron Probe ◽  
Middle Portion ◽  
Relaxation Cycle ◽  
X Ray ◽  
Thin Carbon ◽  
Freeze Dried ◽  
Intracellular Ca

The swimbladder muscle of a teleost fish, Sebasticus marmoratus, contracts rapidly to produce sounds for communication and contains extremely well developed sarcoplasmic reticulum (SR), providing a material suitable for studying the intracellular Ca translocation during the contraction-relaxation cycle. We examined the change of Ca distribution along the sarcomere at various states of the fibers during the contractionrelaxation cycle in the swimbladder muscle by means of quantitative electron probe X-ray microanalysis of cryosections.The muscle fibers were rapidly frozen at rest, during sustained contraction and at 0.1 and 1.0 sec after the onset of relaxation with a dual-jet liquid propane freezing device. Cryosections (200 nm thick) were cut from the middle portion of the frozen fibers at -110°C on a LKB NOVA cryoultramicrotome, and placed on thin carbon supporting films on Nigrids. Then the cryosections were freeze-dried at 10-6 torr and below -80°C. Electron probe X-ray microanalysis was performed on a liquid N2-cooled Be-stage at -130°C in a JEOL 2000FX electron microscope operated at 80 kV and equipped with a Tracor Northern TN5500 energy dispersive X-ray microanalyzer.

scholarly journals Heterogenous distribution of free calcium and propagation of calcium transient in gastric parietal cells revealed by digital imaging microscopy.

1989 ◽  
Vol 37 (7) ◽  
pp. 999-1005 ◽  
Author(s):  
Y Tsunoda ◽  
S Yodozawa ◽  
Y Tashiro
Keyword(s):  
Digital Imaging ◽  
Resting State ◽  
Calcium Concentration ◽  
Gastric Gland ◽  
Calcium Transient ◽  
Parietal Cells ◽  
Free Calcium ◽  
Marked Contrast ◽  
Free Calcium Concentration ◽  
Gastric Parietal Cells

Spatial and temporal changes of cytoplasmic free calcium concentration ([Ca2+]i) in single parietal cells of guinea pig were investigated with a digital imaging microscope equipped with a microspectrofluorometer, using a Ca2+-sensitive dye, fura-2. Intracellular distribution of [Ca2+]i was not homogeneous, but there were two kinds of [Ca2+]i gradient in the resting parietal cells, one a continuous gradient increasing towards the plasma membrane and a second discontinuous gradient (Ca2+ plateau) in some restricted regions of the cytoplasm. When treated with gastrin, only about 40% of parietal cells in the gastric gland responded with an almost twofold increase in the average resting [Ca2+]i of 52.4 +/- 7.1 nM. In the responding cells, the discontinuous plateaus transiently enlarged to the entire cytoplasm. In marked contrast, all of these cells responded to Ca2+ ionophore ionomycin. We also found that when provoked by gastrin Ca2+ transient in the parietal cells in the gastric gland often propagated to some adjacent cells, and occasionally spontaneous Ca2+ transient and oscillation were observed even in the resting state.

Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells

1989 ◽  
Vol 139 (3) ◽  
pp. 632-640 ◽  
Author(s):  
E. W. Black ◽  
T. L. Cornwell ◽  
T. M. Lincoln ◽  
S. J. Strada ◽  
W. J. Thompson
Keyword(s):  
Calcium Regulation ◽  
Cytosolic Calcium ◽  
Parietal Cells ◽  
Fura 2 ◽  
Gastric Parietal Cells

Sem Studies of Co-Cr-Mo Surgical Implant Alloy Corrosion Behavior

1982 ◽  
Vol 40 ◽  
pp. 520-521
Author(s):  
Ann Chidester Van Orden ◽  
John L. Chidester ◽  
Anna C. Fraker ◽  
Pei Sung
Keyword(s):  
Electron Microscopy ◽  
Corrosion Behavior ◽  
Anodic Polarization ◽  
Saline Solution ◽  
Saturated Calomel Electrode ◽  
Physiological Saline ◽  
X Ray ◽  
Physiological Saline Solution ◽  
Scanning Electron

The influence of small variations in the composition on the corrosion behavior of Co-Cr-Mo alloys has been studied using scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX), and electrochemical measurements. SEM and EDX data were correlated with data from in vitro corrosion measurements involving repassivation and also potentiostatic anodic polarization measurements. Specimens studied included the four alloys shown in Table 1. Corrosion tests were conducted in Hanks' physiological saline solution which has a pH of 7.4 and was held at a temperature of 37°C. Specimens were mechanically polished to a surface finish with 0.05 µm A1203, then exposed to the solution and anodically polarized at a rate of 0.006 v/min. All voltages were measured vs. the saturated calomel electrode (s.c.e.).. Specimens had breakdown potentials near 0.47V vs. s.c.e.

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