Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy: Evidence for a primary proton pump in the plasmalemma of a marine fungus

1990 ◽  
Vol 96 (4) ◽  
pp. 731-736
Author(s):  
JULIA M. DAVIES ◽  
C. BROWNLEE ◽  
D. H. JENNINGS
Keyword(s):  
Intracellular Ph ◽  
Proton Pump ◽  
Marine Organism ◽  
Marine Fungus ◽  
Primary Proton ◽  
Acetoxymethyl Ester ◽  
Fluorescence Ratio Imaging ◽  
Negative Membrane Potential ◽  
Ratio Imaging

The facultative marine fungus, Dendryphiella salina, has the most negative membrane potential yet recorded for a marine organism. The ionic basis for this is thought to be through the action of a primary proton pump, though there exists the possibility of electrogenic pumping of Na+ or Cl−, given the high ambient concentration of these ions. Fluorescence ratio imaging microscopy with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) has been used to estimate intracellular pH. Hyphae loaded readily with BCECF after incubation with the acetoxymethyl ester (BCECF/AM). Mean resting intracellular pH (pH1) was 7.3, calculated by comparing 490/450 nm fluorescence ratios with in vivo calibration curves obtained by pH equilibration using nigericin. Distinct pH compartments could be observed, corresponding to cytoplasmic and smaller vacuolar compartments. Sodium azide reversibly reduced pH1 by an average of 0.51 of a pH unit, though the response varied between individual hyphae. Inhibiting the plasmalemma ATPase with orthovanadate also reversibly decreased pH|. The results support the presence of a proton pump in the plasmamembrane. The energetic and evolutionary implications are discussed.

scholarly journals Intracellular pH distribution as a cell health indicator in Saccharomyces cerevisiae

2011 ◽  
Vol 8 (64) ◽  
pp. 1635-1643 ◽  
Author(s):  
Thomas Aabo ◽  
Jesper Glückstad ◽  
Henrik Siegumfeldt ◽  
Nils Arneborg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Metabolic Enzyme ◽  
Transport Mechanisms ◽  
Cell Functions ◽  
Internal Ph ◽  
Ratio Imaging

Internal pH regulation is vital for many cell functions, including transport mechanisms and metabolic enzyme activity. More specifically, transport mechanisms are to a wide degree governed by internal pH distributions. We introduce the term standard deviation of the intracellular pH (s.d.(pH int )) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pH i ) and the s.d.(pH int ) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using fluorescence ratio imaging microscopy (FRIM). An exponential decline in pH i was observed after an initial small decline. For the first time, we report the use of FRIM for determining in vivo plasma membrane proton permeability coefficients in yeast. Furthermore, the exponential decay of pH i and the rupture of the cell plasma membrane, as measured by propidium iodide staining, at 70°C were not simultaneous but were separated by a significant temporal difference. Finally, a nonlinear relationship between the pH i and s.d.(pH int ) was found; i.e. the s.d.(pH int ) was significantly more sensitive to supralethal temperatures than pH i . s.d.(pH int ) is therefore proposed as an early health/vitality indicator in S. cerevisiae cells exposed to heat stress.

Determination of apoplastic Na+ in intact leaves of cotton by in vivo fluorescence ratio-imaging

2002 ◽  
Vol 29 (12) ◽  
pp. 1491 ◽  
Author(s):  
Karl H. Mühling ◽  
André Läuchli
Keyword(s):  
Mature Leaves ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Compatible Osmolytes ◽  
Concentration Changes ◽  
Sodium Binding ◽  
Leaf Apoplast

Salinity may reduce plant growth via Na+-toxicity symptoms in mature leaves after long-term exposure. It has been suggested by other authors that Na+ accumulates in the leaf apoplast and leads to dehydration of leaves, wilting, and finally to death of these leaves. Two methods were employed to determine the Na+ concentration in the leaf apoplast of salt-tolerant cotton plants under salinity. The ratio imaging of sodium-binding benzofuran isophthalate (SBFI) fluorescence was used to detect in vivo concentration changes and gradients of Na+ within the leaf apoplast under salinity stress, and results were compared with the infiltration–centrifugation method. A�significant increase in Na+ concentration was found in the leaf apoplast under salinity (75 mM NaCl), but no further significant increase was determined when NaCl supply was increased from 75 to 150 mM. Both methods revealed that Na+ concentrations remained relatively low, and thus could not be responsible for the decline in yield under salinity. The ratio images showed changes in Na+ concentration and gradients within the leaf apoplast under salt stress, and demonstrated the validity of the method. However, SBFI fluorescence was also influenced by pH, proteins and salt-induced compatible osmolytes.

Intracellular Ca2+ responses induced by acetylcholine in the submucosal nasal gland acinar cells in guinea pigs

1995 ◽  
Vol 268 (3) ◽  
pp. L361-L367 ◽  
Author(s):  
K. Ikeda ◽  
M. Ishigaki ◽  
D. Wu ◽  
H. Sunose ◽  
M. Suzuki ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Acinar Cells ◽  
Transient Increase ◽  
Acetoxymethyl Ester ◽  
Molecular Events ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Muscarinic Cholinergic ◽  
Initial Peak ◽  
External Ph

We examined intracellular Ca2+ responses of the nasal gland acinar cells to clarify cellular responses and molecular events with regard to the regulatory mechanism of the nasal secretion. The acinar cells of the serous gland in the nasal septum of guinea pigs were obtained by meticulous and selective dissection with minimal contamination by epithelial lining cells after collagenase treatment. The dispersed acini were incubated in the oxygenated solution supplemented with fura 2 acetoxymethyl ester, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by fluorescence ratio imaging microscopy. The application of acetylcholine (ACh) to the cells induced an initially rapid increased [Ca2+]i followed by a sustained plateau. The increase in [Ca2+]i induced by ACh was concentration dependent, ranging between 10(-8) and 10(-4) M. The [Ca2+]i response was completely inhibited by atropine, further indicating the involvement of muscarinic cholinergic receptors. Removal of external Ca2+ with addition of EGTA resulted in a transient increase without a sustained phase, and the transient increase was abolished by the intracellular Ca2+ antagonist 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, indicating that this increase in [Ca2+]i was due to release from internal stores. The initial peak was not altered by changes in external pH, addition of adenosine 3',5'-cyclic monophosphate (cAMP), nor addition of phorbol 12-myristate 13-acetate (PMA) but was augmented by external K(+)-induced depolarization, suggesting that the transient increase was due to a changing in the binding affinity to inositol 1,4,5-trisphosphate. The sustained Ca2+ entry induced by ACh was inhibited by Ni2+, but not by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Osmotic Stress Leads to Decreased Intracellular pH of Listeria monocytogenes as Determined by Fluorescence Ratio-Imaging Microscopy

2004 ◽  
Vol 70 (5) ◽  
pp. 3176-3179 ◽  
Author(s):  
Weihuan Fang ◽  
Henrik Siegumfeldt ◽  
Birgitte Bjørn Budde ◽  
Mogens Jakobsen
Keyword(s):  
Listeria Monocytogenes ◽  
Osmotic Stress ◽  
Liquid Medium ◽  
Intracellular Ph ◽  
Solid Medium ◽  
Fluorescence Ratio ◽  
Solid Media ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging

ABSTRACT Intracellular pH (pHi) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pHi, and recovery on solid medium was impaired compared to that in liquid medium. N,N′-dicyclohexylcarbodiimide abolished pHi recovery, and lowering aw with glycerol showed no effect on pHi.

Altered Na+/H+ exchanger isoform 1 activity in immortalized lymphoblasts from women with pre-eclampsia: evidence for an intermediate phenotype

2002 ◽  
Vol 103 (5) ◽  
pp. 503-509 ◽  
Author(s):  
V.M. LEE ◽  
P.A. QUINN ◽  
S.C. JENNINGS ◽  
A.W.F. HALLIGAN ◽  
L.L. NG
Keyword(s):  
Intracellular Ph ◽  
Blood Cells ◽  
Post Partum ◽  
White Blood Cells ◽  
Intermediate Phenotype ◽  
Protein Abundance ◽  
Acetoxymethyl Ester ◽  
Abnormal Volume ◽  
In Cells

Previous studies have demonstrated a raised Na+ content in leucocytes isolated from women with pre-eclampsia. Increased Na+/H+ exchanger activity is one membrane transport abnormality that may contribute to this phenomenon and may be implicated in the abnormal volume homoeostasis and hypertension associated with the disease. Increased Na+/H+ exchanger activity has been documented in nucleated white blood cells from both pre-eclamptic and post-partum pre-eclamptic women, and may suggest the importance of genetic influences on exchanger activity. In the present study, we used lymphoblasts from women with pre-eclampsia and from age- and gestation-matched normotensive pregnant controls to determine Na+/H+ exchanger activity and intracellular resting pH using fluorimetry and the pH-sensitive dye BCECF-AM [bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester]. Determination of Na+/H+ exchanger protein abundance was performed by Western blotting. Intracellular pH was not significantly different in cells from pre-eclamptic women compared with those from normotensive controls. Na+/H+ exchanger activity was measured when the intracellular pH was clamped at 6.0, and was found to be significantly higher in cells from pre-eclamptic women (20.77±0.92mmol·min-1·l-1) compared with those from normotensive controls (15.22±0.92mmol·min-1·l-1; P = 0.001). Na+/H+ exchanger protein abundance was established to be similar in the two subject groups, suggesting that the turnover number for the Na+/H+ exchanger is increased in the women with pre-eclampsia. These changes in Na+/H+ exchanger activity indicate the importance of genetic factors in determining this particular phenotype, since in this cell culture model of pre-eclampsia it is likely that environmental or hormonal influences present in vivo would have declined. Overactivity of the Na+/H+ exchanger may contribute to the raised intracellular Na+ concentration reported previously in white blood cells from women with pre-eclampsia.

Evaluation of BCECF fluorescence ratio imaging to properly measure gastric intramucosal pH variations in vivo

2007 ◽  
Vol 12 (6) ◽  
pp. 064014 ◽  
Author(s):  
Philippe Rochon ◽  
Mercé Jourdain ◽  
Jacques Mangalaboyi ◽  
François Fourrier ◽  
Sylvie Soulié-Bégu ◽  
...  
Keyword(s):  
Fluorescence Ratio ◽  
Intramucosal Ph ◽  
Gastric Intramucosal Ph ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Ph Variations

Effects of in vivo and in vitro alkali treatment on intracellular pH regulation of OMCDis cells

1993 ◽  
Vol 265 (5) ◽  
pp. F729-F735
Author(s):  
M. Hayashi ◽  
M. Iyori ◽  
Y. Yamaji ◽  
T. Saruta
Keyword(s):  
Intracellular Ph ◽  
Cell Function ◽  
Ph Regulation ◽  
Alkali Treatment ◽  
Acid Base ◽  
Acetoxymethyl Ester ◽  
Functional Changes ◽  
Significant Difference

To examine functional changes of the transporters in the inner stripe of the outer medullary collecting ducts (OMCDis) by the peritubular acid-base status, in vitro microperfusion using the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was performed. Cell alkalinization systems were assessed by the recovery rate (dpHi/dt) of intracellular pH (pHi) after intracellular acid loading by NH(4+)-NH3 prepulse with bath amiloride. In alkali-loaded rabbits (0.15 M NaHCO3 drinking for 14 days), dpHi/dt showed a significant decrease (1.80 +/- 0.29 pH units/s x 10(3)) compared with either control (3.30 +/- 0.59) or acid-loaded rabbits (0.15 M NH4Cl drinking for 14 days, 3.05 +/- 0.46). The difference of dpHi/dt between control and alkali-loaded rabbits was eliminated by lumen N-ethylmaleimide (NEM), suggesting that H+ pump activity was decreased. The effect of in vitro alkali treatment (50 mM HCO3-, pH 7.7) for 3-4 h was also examined. This incubation significantly decreased the dpHi/dt (1.83 +/- 0.35) compared with the time control experiments (3.18 +/- 0.28), whereas no significant difference was seen in the presence of lumen NEM. Anion exchanger activity, as determined from the pHi changes after Cl- addition to the bath, showed no significant change with in vivo or in vitro alkali treatment. The results indicate that cell function of the OMCDis is regulated in response to the peritubular acid-base environment via changes in the H(+)-adenosinetriphosphatase.

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