scholarly journals Role of lysosomal and cytosolic pH in the regulation of macrophage lysosomal enzyme secretion

1990 ◽  
Vol 272 (2) ◽  
pp. 407-414 ◽  
Author(s):  
H Tapper ◽  
R Sundler
Keyword(s):  
Intracellular Ph ◽  
Fluorescent Probes ◽  
Lysosomal Enzyme ◽  
Enzyme Secretion ◽  
Relative Role ◽  
Ion Composition ◽  
Cytosolic Ph ◽  
Lysosomal Ph ◽  
Cytosolic Acidification

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca2+ overload and acidosis

2000 ◽  
Vol 279 (5) ◽  
pp. H2143-H2150 ◽  
Author(s):  
C. Schäfer ◽  
Y. V. Ladilov ◽  
M. Schäfer ◽  
H. M. Piper
Keyword(s):  
Intracellular Ph ◽  
Cytosolic Ph ◽  
Bicarbonate Buffer ◽  
Rat Cardiomyocytes ◽  
Hepes Buffer ◽  
Adult Rat ◽  
Reoxygenation Injury ◽  
Sodium Binding ◽  
Cytosolic Acidification

We investigated the question of whether inhibition of the Na+/H+ exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca2+ accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na+-HCO3 − symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca2+ (fura 2), Na+ [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 μmol/l HOE 642 or 1 μmol/l 5-( N-ethyl- N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Nai and Cai, respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 ± 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pHi), Nai, and Cai, but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 ± 1.4%, EIPA: 68.2 ± 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Cai overload.

scholarly journals Dynamic measurement of cytosolic pH and [NO3−] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

2020 ◽  
Vol 117 (26) ◽  
pp. 15343-15353 ◽  
Author(s):  
Elsa Demes ◽  
Laetitia Besse ◽  
Paloma Cubero-Font ◽  
Béatrice Satiat-Jeunemaitre ◽  
Sébastien Thomine ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Loss Of Function ◽  
Ion Transporters ◽  
Ph Homeostasis ◽  
Cytosolic Ph ◽  
Cellular Processes ◽  
Plasma Membrane Proton Pump ◽  
Cytosolic Acidification

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thalianaChloride Channel a) is a vacuolar NO3−/H+exchanger regulating stomata aperture inA.thaliana. Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo inArabidopsisguard cells. We first found that ClopHensor is not only a Cl−but also, an NO3−sensor. We were then able to quantify the variations of NO3−and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3−. In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3−and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.

scholarly journals Role of pH in a nitric oxide-dependent increase in cytosolic Cl− in retinal amacrine cells

2011 ◽  
Vol 106 (2) ◽  
pp. 641-651 ◽  
Author(s):  
Emily McMains ◽  
Evanna Gleason
Keyword(s):  
Nitric Oxide ◽  
Cell Voltage ◽  
Amacrine Cells ◽  
Excitatory Synapses ◽  
Cytosolic Ph ◽  
Ph Imaging ◽  
Gabaergic Synapses ◽  
The Relationship ◽  
Cytosolic Acidification

Nitric oxide (NO) synthase-expressing neurons are found throughout the vertebrate retina. Previous work by our laboratory has shown that NO can transiently convert inhibitory GABAergic synapses onto cultured retinal amacrine cells into excitatory synapses by releasing Cl− from an internal store in the postsynaptic cell. The mechanism underlying this Cl− release is currently unknown. Because transport of Cl− across internal membranes can be coupled to proton flux, we asked whether protons could be involved in the NO-dependent release of internal Cl−. Using pH imaging and whole cell voltage-clamp recording, we addressed the relationship between cytosolic pH and cytosolic Cl− in cultured retinal amacrine cells. We found that NO reliably produces a transient decrease in cytosolic pH. A physiological link between cytosolic pH and cytosolic Cl− was established by demonstrating that shifting cytosolic pH in the absence of NO altered cytosolic Cl− concentrations. Strong buffering of cytosolic pH limited the ability of NO to increase cytosolic Cl−, suggesting that cytosolic acidification is involved in generating the NO-dependent elevation in cytosolic Cl−. Furthermore, disruption of internal proton gradients also reduced the effects of NO on cytosolic Cl−. Taken together, these results suggest a cytosolic environment where proton and Cl− fluxes are coupled in a dynamic and physiologically meaningful way.

Intracellular pH modulates cytosolic free magnesium in cultured chicken heart cells

1992 ◽  
Vol 262 (4) ◽  
pp. C1024-C1030 ◽  
Author(s):  
C. C. Freudenrich ◽  
E. Murphy ◽  
L. A. Levy ◽  
R. E. London ◽  
M. Lieberman
Keyword(s):  
Intracellular Ph ◽  
Total Cell ◽  
Transient Increase ◽  
Heart Cells ◽  
Cytosolic Ph ◽  
Chicken Heart ◽  
Subcellular Organelles ◽  
Nh4cl Solution ◽  
Free Magnesium

To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained were as follows: pHi = 7.21 +/- 0.10 (n = 7), [Mg]i = 0.51 +/- 0.08 mM (n = 9), [Ca]i = 126 +/- 15 nM (n = 7). When cells were perfused with 10 mM NH4Cl solution for 5 min, a transient alkalinization (0.53 U) of the cytosol was accompanied by a transient decrease (0.12 mM) in [Mg]i and a transient increase (59 nM) in [Ca]i; these changes approached control levels within 5 min. Upon removal of NH4Cl, a transient acidification (0.89 U) of the cytosol was accompanied by a transient increase (0.10 mM) in [Mg]i and a transient increase (125 nM) in [Ca]i; again, these changes returned toward control levels within 5 min. No significant changes in total cell Mg or Ca were observed during these manipulations. NH4Cl-evoked changes in [Mg]i were not altered significantly by either Mg-free or Ca-free conditions. Changes in [Mg]i were inversely correlated with changes in pHi and were not secondary to changes in [Ca]i. The results suggest that pHi modulates Mgi, probably by affecting cytosolic Mg binding and/or the transport of Mg across subcellular organelles.

scholarly journals Regulation of macrophage lysosomal enzyme secretion: role of arachidonate metabolites, divalent cations and cyclic AMP

1980 ◽  
Vol 44 (1) ◽  
pp. 299-315
Author(s):  
R.M. McMillan ◽  
D.E. Macintyre ◽  
J.E. Beesley ◽  
J.L. Gordon
Keyword(s):  
Cyclic Amp ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Divalent Cations ◽  
Cell Types ◽  
Enzyme Secretion ◽  
Ionophore A23187 ◽  
Enzyme Release ◽  
Arachidonate Metabolites

We have investigated the role in macrophage lysosomal enzyme release of arachidonate metabolites, extracellular divalent cations and cyclic AMP (cAMP) which modulate secretion in other cell types. Lysosomal enzyme secretion induced by zymosan was accompanied by release of malondialdehyde (MDA), which is derived from arachidonic acid via prostaglandin synthase. Blockade of MDA formation, by aspirin or indomethacin, was associated with only a small inhibitory effect on lysosomal enzyme release by zymosan: arachidonate metabolites thus play only a minor role in mediating macrophage lysosomal enzyme release. Zymosan-induced secretion of lysosomal enzymes from macrophages did not require extracellular magnesium or calcium although release was enhanced by magnesium and inhibited by calcium. These effects may be related to an influence of the ions on phagocytosis. Elevation of intracellular divalent cation concentrations, by ionophore A23187, induced release of lysosomal enzymes but this was a result of cell lysis. Adenylate cyclase stimulants and dibutyryl cAMP produced slight inhibition of zymosan-induced lysosomal enzyme release. Aminophylline and papaverine caused more marked inhibition but their effects may be due to actions independent of phosphodiesterase inhibition. Our data indicate that arachidonate metabolites and cAMP do not play a major role in regulating zymosan-induced enzyme release from macrophages. Extracellular calcium and magnesium may modulate secretion but the role of intracellular divalent cations remains to be established. We conclude that macrophage lysosomal enzyme secretion is controlled by regulatory mechanisms different from those which control similar degranulation processes in other cell types.

scholarly journals Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation.

1991 ◽  
Vol 98 (1) ◽  
pp. 183-196 ◽  
Author(s):  
D Chang ◽  
N L Kushman ◽  
D C Dawson
Keyword(s):  
Epithelial Cells ◽  
Intracellular Ph ◽  
Gain Control ◽  
Activation Process ◽  
Cytosolic Ph ◽  
Variable Gain ◽  
Ionic Conductances ◽  
Cell Layers ◽  
Conduction Properties

The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control."

Effect of osmolarity on LDL binding and internalization in hepatocytes

1997 ◽  
Vol 273 (4) ◽  
pp. C1409-C1415 ◽  
Author(s):  
Annette Krämer-Guth ◽  
Gillian L. Busch ◽  
Nubia Kristen Kaba ◽  
Susanne Schwedler ◽  
Christoph Wanner ◽  
...  
Keyword(s):  
Cell Volume ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Partial Replacement ◽  
Coated Pits ◽  
Cytosolic Ph ◽  
Lysosomal Ph ◽  
Hypertonic Glucose ◽  
Ldl Binding

The present study has been performed to elucidate a possible role of cell volume in low-density lipoprotein (LDL) binding and internalization (LDLb+i). As shown previously, increase of extracellular osmolarity (OSMe) and K+ depletion, both known to shrink cells, interfere with the formation of clathrin-coated pits and thus with LDLb+i. On the other hand, alterations of cell volume have been shown to modify lysosomal pH, which is a determinant of LDLb+i. LDLb+i have been estimated from heparin-releasable (binding) or heparin-insensitive (internalization) uptake of 125I-labeled LDL. OSMe was modified by alterations of extracellular concentrations of ions, glucose, urea, or raffinose. When OSMe was altered by varying NaCl concentrations, LDLb+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe and LDLb+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to altered affinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 μg/ml at 219, 293, and 435 mosM, respectively. A 25% increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreased lysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMe decreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by 0.17 ± 0.02) pH. Partial replacement of extracellular Na+ with K+ had little effect on LDLb+i, although it swelled hepatocytes and increased lysosomal and cytosolic pH. Hypertonic glucose, urea, or raffinose did not exert similar effects despite a shrinking effect of hypertonic raffinose. Monensin, which completely dissipates lysosomal acidity, virtually abolished LDLb+i. In conclusion, the observations reveal a significant effect of ionic strength on LDLb+i. The effect is, however, not likely to be mediated by alterations of cell volume or alterations of lysosomal pH.

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