Direct measurement of free Ca in organelles of gastric epithelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. G442-G451 ◽  
Author(s):  
A. M. Hofer ◽  
T. E. Machen
Keyword(s):  
Cell Types ◽  
Ruthenium Red ◽  
Acetoxymethyl Ester ◽  
Gastric Glands ◽  
Free Concentration ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mitochondrial Inhibitors ◽  
Carbonyl Cyanide ◽  
The Relationship

When loaded as the acetoxymethyl ester (AM) derivative, the fluorescent probe mag-fura 2 accumulates in both the cytoplasm and the subcellular compartments. The relatively high dissociation constant of this dye for Ca (53 microM) permits the measurement of changes in the free concentration of this ion in a variety of organelles where Ca concentration ([Ca]) is high. To characterize Ca stores in gastric cells, we used digitonin to permeabilize cells within isolated rabbit gastric glands loaded with mag-fura 2-AM. This allowed cytosolic dye to leak out, leaving only compartmentalized dye behind. It appears that mag-fura 2 monitors [Ca] changes in several ATP-dependent Ca-sequestering pools; an inositol 1,4,5-trisphosphate (IP3)-releasable and thapsigargin-sensitive store (which probably includes the endoplasmic reticulum), a pool that is released by the mitochondrial inhibitors oligomycin+azide, valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and ruthenium red (and therefore likely represents mitochondria), and a residual pool that was resistant to release by mitochondrial inhibitors and thapsigargin. Ca sequestration into all pools was sensitive to changes in [ATP], indicating that treatments that reduce cellular [ATP] will cause certain organelles to lose their Ca to the cytoplasm. Caffeine and ryanodine, which mobilize Ca from internal stores in many cell types, induced Ca sequestration into an IP3-insensitive store of gastric cells, and caffeine caused a reduction in cytoplasmic [Ca] (as measured with fura 2). We also show that the quantitation of free [Ca] in a given pool is complicated by a nonlinearity in the relationship between the mag-fura 2 ratio and [Ca]. This effect is likely a consequence of monitoring the fluorescence from multiple pools simultaneously. However, this limitation does not detract from the ability of this method to yield important qualitative information about the nature and number of Ca stores within single gastric cells.

Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells

1995 ◽  
Vol 269 (2) ◽  
pp. G287-G296 ◽  
Author(s):  
A. J. Dziki ◽  
S. Batzri ◽  
J. W. Harmon ◽  
M. Molloy
Keyword(s):  
Cell Death ◽  
Bile Salt ◽  
Cell Injury ◽  
Early Event ◽  
Acetoxymethyl Ester ◽  
Gastric Mucosal Cells ◽  
Maximal Increase ◽  
Fura 2 ◽  
Gastric Cells ◽  
Mucosal Cells

Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.

Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes

1991 ◽  
Vol 261 (4) ◽  
pp. H1123-H1134 ◽  
Author(s):  
H. Miyata ◽  
H. S. Silverman ◽  
S. J. Sollott ◽  
E. G. Lakatta ◽  
M. D. Stern ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Cardiac Myocyte ◽  
Continuous Measurement ◽  
Chelating Agent ◽  
Ruthenium Red ◽  
Mitochondrial Enzymes ◽  
Acetoxymethyl Ester ◽  
Carbonyl Cyanide ◽  
Rat Cardiac Myocytes ◽  
Single Living

A technique that allows the continuous measurement of mitochondrial free Ca2+ ([Ca2+]m) in a single living cardiac myocyte is described. It involves the introduction of the fluorescent chelating agent indo-1 into the cell by exposure to the acetoxymethyl ester, followed by selective quenching of the fluorescence of indo-1 in the cytosol by Mn2+. The identity of the remaining fluorescence due to intramitochondrial indo-1 is established by its resistance to treatment of the cell with digitonin at concentrations that release cytosolic but not mitochondrial enzymes and by the finding that ruthenium red and carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevent its response to elevated cytosolic free Ca2+ ([Ca2+]c). [Ca2+]m is found to be low (less than 100 nM) in unstimulated cells and to rise in procedures that chronically elevate [Ca2+]c, such as Na+ replacement. The gradient [Ca2+]m/[Ca2+]c is less than unity at values of [Ca2+]c of less than 500 nM but rapidly increases at higher values of [Ca2+]c. Although there is no detectable increase in [Ca2+]m during a single electrical stimulation, [Ca2+]m increases up to 600 nM as the pacing frequency is raised to 4 Hz in the presence of norepinephrine; this increase occurs over the course of many contractions. It is concluded that these findings are consistent with an increase in [Ca2+]m acting as a signal to increase dehydrogenase activity, and hence flux through oxidative phosphorylation, in response to increased work loads.

scholarly journals Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

2012 ◽  
Vol 302 (10) ◽  
pp. R1134-R1142 ◽  
Author(s):  
Sergey I. Dikalov ◽  
Wei Li ◽  
Abdulrahman K. Doughan ◽  
Raul R. Blanco ◽  
A. Maziar Zafari
Keyword(s):  
Nadph Oxidase ◽  
Mitochondrial Dysfunction ◽  
Oxidase Activity ◽  
Cytosolic Calcium ◽  
Ruthenium Red ◽  
Antimycin A ◽  
Nadph Oxidases ◽  
Nadph Oxidase Activity ◽  
Fura 2 ◽  
Mitochondrial Inhibitors

Production of superoxide (O2·−) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca2+]iwas measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca2+uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca2+-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca2+measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca2+]i, while BAPTA-AM abolished the increase in [Ca2+]i. Furthermore, depletion of cellular Ca2+with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O2·−and impaired buffering of cytosolic Ca2+by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.

Mechanism of cholinergic stimulation of glucose oxidation in isolated gastric glands

1994 ◽  
Vol 267 (2) ◽  
pp. G227-G234
Author(s):  
I. Hernandez ◽  
J. Chacin
Keyword(s):  
Glucose Oxidation ◽  
Ruthenium Red ◽  
Mitochondrial Enzymes ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Acetoxymethyl Ester ◽  
Gastric Glands ◽  
Mitochondrial Uncouplers ◽  
Dose Dependent ◽  
Stimulation Of

The mechanisms of cholinergic activation of carbohydrate metabolism were investigated in isolated rabbit gastric glands. Carbachol stimulated the rate of glucose oxidation in a dose-dependent fashion with a half-maximal effect occurring at approximately 9 microM. Atropine and omeprazole, but not cimetidine, completely blocked the stimulation induced by carbachol. Direct activation of the H(+)-K(+)-adenosinetriphosphatase by NH+4 caused a significant stimulation of glucose oxidation that was totally abolished by oligomycin and by the mitochondrial uncouplers dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These latter agents did not abolish the stimulating effect of carbachol on glucose oxidation. Ionomycin increased the rate of glucose oxidation in a dose-dependent manner, and this effect was not blocked by oligomycin. The metabolic effect of ionomycin was reduced but not abolished by omeprazole. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester eliminated the carbachol-induced stimulation of glucose oxidation and partially inhibited the effect of NH+4. The mitochondrial enzymes pyruvate dehydrogenase and oxoglutarate dehydrogenase were activated by physiological concentrations of calcium in the isolated mitochondria. This effect was blocked by incubation with ruthenium red.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.

1990 ◽  
Vol 1 (3) ◽  
pp. 259-268 ◽  
Author(s):  
P A Negulescu ◽  
A Harootunian ◽  
R Y Tsien ◽  
T E Machen
Keyword(s):  
Cell Types ◽  
Parietal Cells ◽  
Acetoxymethyl Ester ◽  
Gastric Glands ◽  
Great Ability ◽  
Chief Cells ◽  
Fluorescence Measurements ◽  
Na Efflux ◽  
Rapid Perfusion ◽  
Acid Secreting

Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.

scholarly journals Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi

1995 ◽  
Vol 310 (3) ◽  
pp. 1005-1012 ◽  
Author(s):  
R Docampo ◽  
D A Scott ◽  
A E Vercesi ◽  
S N J Moreno
Keyword(s):  
Trypanosoma Cruzi ◽  
Acridine Orange ◽  
Bafilomycin A1 ◽  
Uptake Mechanism ◽  
Acetoxymethyl Ester ◽  
Permeabilized Cells ◽  
Fura 2 ◽  
Carbonyl Cyanide ◽  
Acidic Compartment ◽  
Proton Uptake

The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the ‘acidocalcisome’ and which appears to be a unique organelle present in trypanosomatids.

Inhibition of pepsinogen secretion in isolated gastric glands by amphotericin B is secretagogue specific

1984 ◽  
Vol 62 (12) ◽  
pp. 1518-1524 ◽  
Author(s):  
S. H. Norris ◽  
S. J. Hersey
Keyword(s):  
Amphotericin B ◽  
Chloride Content ◽  
Potassium Content ◽  
Gastric Glands ◽  
Intracellular Chloride ◽  
Mitochondrial Inhibitors ◽  
Mechanism Of Inhibition ◽  
Isolated Gastric Glands ◽  
Pepsinogen Secretion ◽  
Carbonyl Cyanide

Pepsinogen secretion from isolated gastric glands, stimulated by 8-bromoadcnosine 3′,5′-cyclic monophosphate (8BrcAMP), forskolin, or cholecystokinin octapeptide, was inhibited by the presence of amphotericin B in the incubation medium. However, amphotericin had no effect, or only a slight effect (<10% inhibition), on pepsinogen secretion stimulated by crude secretin. Incubation of glands with either of the mitochondrial inhibitors, rotenone or carbonyl cyanide m-chlorophenylhydrazone, reduced pepsinogen secretory responses both to 8BrcAMP and to crude secretin. This suggests that amphotericin inhibition, which is secretagogue specific, was not the result of a general metabolic inhibition. Amphotericin caused an increase in sodium and chloride content and a decrease in potassium content of glands. Experiments in which the medium content of either sodium, potassium, or chloride was varied, suggested that part of the amphotericin inhibition could be attributed to a rise in intracellular chloride content. Results did not support the involvement of changes in intracellular sodium or potassium content in the inhibitory mechanism of amphotericin. It was concluded that amphotericin caused a rapid and secretagogue-specific inhibition of pepsinogen secretion in isolated gastric glands, and that the mechanism of inhibition may, to some extent, involve changes in intracellular chloride content.

Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i

2008 ◽  
Vol 295 (4) ◽  
pp. G671-G681 ◽  
Author(s):  
Jose Francisco Perez-Zoghbi ◽  
Adriana Mayora ◽  
Marie Christine Ruiz ◽  
Fabian Michelangeli
Keyword(s):  
Acid Secretion ◽  
Slow Component ◽  
Gastric Gland ◽  
Cell Types ◽  
Gastric Fluid ◽  
Cholinergic Stimulation ◽  
Gastric Glands ◽  
Alkali Ions ◽  
Different Cell Types ◽  
The Relationship

The gastric glands of the mammalian fundic mucosa are constituted by different cell types. Gastric fluid is a mixture of acid, alkali, ions, enzymes, and mucins secreted by parietal, chief, and mucous cells. We studied activation of acid secretion using LysoSensor Yellow/Blue in conjunction with fluo 3 to measure changes in pH and Ca2+ in isolated rabbit gastric glands. We evidenced a spatial heterogeneity in the amplitude of acid response along the gland axis under histamine and cholinergic stimulation. Carbachol induced a transitory pH increase before acidification. This relative alkalinization may be related to granule release from other cell types. Omeprazole inhibited the acid component but not the rise in pH. Histamine stimulated acid secretion without increase of lumen pH. We studied the relationship between Ca2+ release and/or entry and H+ secretion in glands stimulated by carbachol. Ca2+ release was associated with a fast and transient components of H+ secretion. We found a linear relationship between Ca2+ release and H+ secretion. Ca2+ entry was associated with a second slow and larger component of acid secretion. The fast component may be the result of activation of Cl− and K+ channels and hence H+/K+ pumps already present in the membrane, whereas the slow component might be associated with translocation of H+/K+ pumps to the canaliculi. In conclusion, with cholinergic stimulation, gastric glands secrete a mixture of acid and other product(s) with a pH above 4.2, both triggered by Ca2+ release. Maintenance of acid secretion depends on Ca2+ entry and perhaps membrane fusion.

Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt

1993 ◽  
Vol 264 (4) ◽  
pp. H1098-H1110 ◽  
Author(s):  
P. H. Backx ◽  
H. E. Ter Keurs
Keyword(s):  
Time Course ◽  
Temporal Relationship ◽  
Sarcomere Length ◽  
Muscle Length ◽  
Papillary Muscles ◽  
Fluorescent Properties ◽  
Acetoxymethyl Ester ◽  
Fura 2 ◽  
Relaxation Period ◽  
The Relationship

We have measured force, sarcomere length, and Ca2+ during twitches in rat cardiac trabeculae. To avoid the difficulties associated with fura-2/acetoxymethyl ester (AM), fura-2 salt was iontophoretically microinjected into the preparation using a single impalement site; this is possible because fura-2 diffuses through the gap junctions between cells. By use of this method, the estimated peak of the [Ca2+] transient during a twitch was not statistically different at different sarcomere lengths: 875 +/- 92 nM at a sarcomere length of 2.15 microns vs. 905 +/- 67 nM at a sarcomere length of 1.65 microns (means +/- SD, n = 10). When trabeculae were loaded using fura-2/AM, the estimated peak of the [Ca2+] transient at a sarcomere length of 2.15 microns was 540 +/- 180 nM (means +/- SD, n = 5). The time course of the Ca2+ transients at different sarcomere lengths is qualitatively similar, but small systematic differences were observed during the relaxation period. On the other hand, the duration of twitch force increases dramatically as the muscle length is increased. As a result, when the trabeculae were held at short muscle lengths, the temporal relationship between force and the Ca2+ transient resembled the relationship between cell shortening and the Ca2+ transient measured in isolated myocytes. At longer lengths the temporal relationship between force and the Ca2+ transient more closely resembles that obtained in papillary muscles using aequorin.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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