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Cytosolic pH regulation in mouse macrophages. Characteristics of HCO3(-)-dependent mechanisms
Abstract:
Mechanisms regulating cytosolic pH (pHi) in adherent resident mouse macrophages have been characterized by use of the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Na+/H+ exchange was activated after an acid load of the macrophage cytosol. However, when Na+/H+ exchange was the only pHi-regulatory mechanism operative, recovery did not proceed beyond a pHi of approx. 6.6. The mechanisms found to be operative at physiological pHi levels were alkalinizing Na(+)-dependent and acidifying Na(+)-independent Cl-/HCO3- exchangers and a H(+)-ATPase further characterized in the accompanying paper [Tapper & Sundler (1992) Biochem. J. 281, 245-250]. Acid extrusion via Na+/Cl-/HCO3- exchange was demonstrated by the dependence on external Na+ and HCO3- and on internal Cl- and by the sensitivity to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). By monitoring pHi changes upon Cl- removal and re-addition, the pH-dependence and sensitivity to SITS were found to differ for the alkalinizing and the acidifying Cl-/HCO3- exchangers.
Insights
Mouse macrophages regulate cytosolic pH using multiple mechanisms. While Na+/H+ exchange is active, it
Area of Science:
- Cellular Physiology
- Immunology
- Biochemistry
Background:
- Cytosolic pH (pHi) regulation is crucial for macrophage function.
- Adherent resident mouse macrophages possess complex pHi regulatory systems.
- Previous studies indicated the involvement of Na+/H+ exchange.
Purpose of the Study:
- To characterize the mechanisms regulating cytosolic pH in mouse macrophages.
- To identify additional pHi regulatory systems beyond Na+/H+ exchange.
- To elucidate the roles of Cl-/HCO3- exchangers and H+-ATPase at physiological pHi.
Main Methods:
- Utilized the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
- Investigated Na+/H+ exchange activity following acid loading.
- Examined Na(+)-dependent and Na(+)-independent Cl-/HCO3- exchangers and H+-ATPase.
- Assessed acid extrusion via Na+/Cl-/HCO3- exchange by manipulating ion concentrations and using stilbene disulfonic acids (SITS, DIDS).
Main Results:
- Na+/H+ exchange alone could not restore pHi beyond approximately 6.6.
- Identified alkalinizing Na(+)-dependent and acidifying Na(+)-independent Cl-/HCO3- exchangers.
- Demonstrated acid extrusion via Na+/Cl-/HCO3- exchange, dependent on external Na+, HCO3-, and internal Cl-, and sensitive to SITS/DIDS.
- Observed differing pH-dependence and SITS sensitivity between alkalinizing and acidifying Cl-/HCO3- exchangers.
Conclusions:
- Mouse macrophages employ multiple pHi regulatory mechanisms, including Na+/H+ exchange and distinct Cl-/HCO3- exchangers.
- These exchangers, along with H+-ATPase, are essential for maintaining physiological pHi levels.
- The identified mechanisms highlight the complexity of cellular pH homeostasis in macrophages.