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Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal
T Nordström1, S Grinstein, G F Brisseau
1Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract:
The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.
Insights
Protein kinase C activation significantly enhances vacuolar-type H(+)-ATPase (V-ATPase) activity in mouse macrophages. This boosts proton extrusion, demonstrating V-ATPase regulation by protein kinase C.
Area of Science:
- Cellular Physiology
- Biochemistry
- Immunology
Background:
- Vacuolar-type H(+)-ATPase (V-ATPase) plays a critical role in maintaining cellular pH homeostasis.
- Protein kinase C (PKC) is a family of enzymes involved in various cellular signaling pathways.
- The precise role of PKC in regulating V-ATPase activity, particularly in immune cells, requires further elucidation.
Purpose of the Study:
- To investigate the role of protein kinase C (PKC) in modulating the activity of vacuolar-type H(+)-ATPase (V-ATPase) in mouse peritoneal macrophages.
- To determine if PKC activation influences proton extrusion mediated by plasma membrane V-ATPases.
Main Methods:
- Thioglycolate-elicited mouse peritoneal macrophages were acid-loaded and their intracellular pH recovery was measured.
- Cells were treated with PKC agonists (TPA, mezerein) or inhibitors (staurosporine) and V-ATPase activity was assessed.
- Extracellular pH changes were monitored to confirm proton extrusion rates.
Main Results:
- PKC agonists (TPA, mezerein) markedly increased the rate of intracellular pH recovery by 82% in acid-loaded macrophages.
- This stimulatory effect was blocked by the V-ATPase inhibitor bafilomycin A1 and the PKC inhibitor staurosporine.
- Inactive TPA analogue did not stimulate pH recovery, confirming PKC involvement.
- Increased proton extrusion from cells was observed, indicating enhanced V-ATPase activity.
Conclusions:
- Protein kinase C activation significantly augments the proton pumping activity of plasma membrane V-ATPases in macrophages.
- These findings demonstrate a novel regulatory mechanism of V-ATPase-mediated proton extrusion by PKC signaling.
- This highlights a potential pathway for modulating macrophage function through PKC-V-ATPase interactions.