Cytoplasmic pH regulation in phorbol ester-activated human neutrophils

Area of Science:

• Cellular Physiology
• Immunology
• Biochemistry

Background:

• Neutrophil activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) involves initial cytoplasmic acidification followed by alkalinization.
• The source of this acidification, particularly in conditions limiting Na+-H+ exchange, requires investigation.

Purpose of the Study:

• To elucidate the origin of cytoplasmic acidification during neutrophil activation by phorbol esters.
• To correlate pH changes with specific metabolic pathways activated in neutrophils.

Main Methods:

• Investigated TPA-induced pH changes in neutrophils using Na+-free and amiloride-containing media.
• Utilized degranulated and enucleated cytoplasts to assess the role of organelles.
• Monitored cytoplasmic and extracellular pH changes.
• Assessed the impact of inhibiting NADPH-oxidase and stimulating the hexose monophosphate shunt.
• Examined the effects of various phorbol ester derivatives.

Main Results:

• Cytoplasmic acidification was observed independently of the nucleus and secretory vesicles.
• Acidification correlated with extracellular pH changes, suggesting metabolic H+ generation.
• Superoxide production inhibition prevented TPA-induced acidification.
• Activation of the hexose monophosphate shunt also induced cytoplasmic acidification.
• Beta-phorbol diesters, but not inactive phorbol derivatives, elicited acidification.

Conclusions:

• Phorbol ester-induced cytoplasmic acidification in neutrophils is primarily due to H+ accumulation from metabolic activity.
• The metabolic burst, involving NADPH-oxidase and the hexose monophosphate shunt, is the source of this acidification.