Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2

Abstract

The potential role of cytosolic phospholipase A2 (cPLA2) in the regulation of the electrogenic arachidonic acid (AA)-activatable H+ translocator of neutrophils was investigated. (1) The trifluoromethyl ketone analogue of arachidonate (AACOCF3), a newly developed selective blocker of cPLA2, inhibited both the N-formylmethionyl-leucylphenylalanine (fMLP)- and the phorbol-ester-induced rheogenic H+ efflux (K0.5 ≈ 5 μM) and abrogated the stimulus-triggered release of AA from these cells. The drug failed to reduce the fMLP-evoked Ca2+ signal or protein tyrosine phosphorylation and did not affect the activity of protein kinase C. By using the patch-clamp technique we verified that the agent did not interfere with the voltage- and the pH-dependent activation of the H+ conductance of the peritoneal macrophages and therefore is not a direct blocker of the H+ channel itself. AACOCF3, however, slightly decreased the AA-induced stimulation of the H+ currents. We conclude that AA, liberated by the agonist-induced stimulation of cPLA2, is a direct activator of H+ conductance. (2) AACOCF3 did not inhibit superoxide generation, indicating that activation of cPLA2 may not be a prerequisite for turning on NADPH oxidase. (3) Since neither acid generation by the oxidase, nor the basal or stimulated Na+/H+ exchange (the predominant acid-eliminating mechanism) were influenced by the drug, we could use AACOCF3 to address whether the H+ channel in fact opens and plays any physiological role during activation of neutrophils. Stimulus-induced cytosolic alkalinization was smaller, whereas depolarization became larger, in the presence of AACOCF3. Stimulated H+ conductance therefore does contribute to intracellular pH (pHi) homoeostasis and membrane potential changes of intact neutrophils.

Regulation of the NADPH oxidase activity and anti-microbial function of neutrophils by arachidonic acid

“…It appears, therefore, that AA is required for the production of superoxide and efflux of H + . A role for cPLA 2 in opening the H + channel has been reported [65,67,99].…”

Cell Volume-dependent Regulation of L-selectin Shedding in Neutrophils

“…The intracellular pH was monitored spectrofluorimetrically using a Perkin-Elmer fluorimeter as in Ref. 33. After obtaining a baseline reading, neutrophils were challenged with hypertonicity by the addition of 100 mM NaCl.…”

Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers

“…Human neutrophils were prepared from healthy volunteers as described previously (22) except that lysis of red blood cells was carried out using NH 4 Cl. Prior to use, cells (10 6 /ml) were kept in Hepesbuffered RPMI medium at 37°C.…”

“…Analysis of the data was carried out using Felix ® software. In neutrophils, pH i was monitored in cell suspension (0.5 ϫ 10 6 /ml) using a Beckman fluorimeter (22).…”