Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2 alpha activity

Abstract

The prevailing hypothesis that a signalling pathway involving cPLA(2)alpha is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA(2)alpha as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (I(e)) and enhanced proton channel gating identically in the presence or absence of three specific cPLA(2)alpha inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF(3) (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA(2)alpha gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA(2)alpha is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised.

Figure 7. Staurosporine partially reverses the activation of proton currents by AA

A, a continuous record of currents during test pulses to +20 mV applied every 15 s from a holding potential of −60 mV. At the first arrow, 5 μm AA was added. I_e and H⁺ currents increased progressively. The interruptions (boxes) are due to recording a family of currents (shown in C) and then addition of 100 nm staurosporine (STR) directly into the bath followed by stirring. The horizontal line indicates zero current. Digitally filtered at 2 Hz to reduce capacity transients. B–D, currents in the same cell before AA (B), in the presence of 5 mm AA (C) (the blanked family in A), and after addition of 100 nm staurosporine to the bath, in the continued presence of AA (D). Currents in B–D are from −20 mV to +30 mV in 10 mV increments, from a holding potential of −60 mV.

Figure 1. cPLA₂α inhibitors have no effect on the activation of NADPH oxidase or H⁺ current by PMA

Records on the left show the current at −60 mV in human eosinophils in the perforated patch configuration. The traces on the right show proton currents in response to pulses to +60 mV applied at the time points indicated by lower case letters. Currents after PMA stimulation are shown as darker lines. Bath and pipette solutions consisted of TMA MeSO₃ at pH 7. Cells were pretreated with nothing (A), 10 μm Wyeth-1 (B), 5 μm pyrrolidine-2 (C), or 2 μm G109203FX (D) and then stimulated with 60 nm PMA.

Figure 3. Enhanced proton channel gating occurs despite cPLA₂α inhibition

A, summary of g_H–V (mean ±s.e.m) relationships before (▪, n = 9) or after stimulation with PMA alone (▴, n = 9) or following pretreatment with 10–20 μm pyrrolidine-2 (▾, n = 5), 1–10 μm Wyeth-1 (♦, n = 5), 10–20 μm AAOCOF₃ (▪, n = 5), or 0.2–3 μm GFX (○, n = 8). Data are fitted with Boltzmann curves constrained to limit to 0. Conductance was calculated from I_H measured at the end of the pulse, with leak subtracted. In most cases, V_rev was measured, or was assumed to be 0 mV. B, the time constant of activation (τ_act) of proton currents in human eosinophils before (▪, n = 20) or after stimulation with 60 nm PMA (▴, n = 9), in the presence of 10–20 μm pyrrolidine-2 (▾, n = 6), 10–20 μm Wyeth-1 (♦, n = 5) and 1 μm GFX (○, n = 6). The turn-on of proton current during depolarizing pulses was fitted by a single exponential, whose time constant is plotted here (mean ±s.e.m).

Figure 2. Proton channel ‘activation’ occurs despite cPLA₂α inhibition

Proton currents elicited by identical families of 8 s voltage pulses in 20 mV steps from −40 mV to 60 mV (inset) in human eosinophils before (left) or after (right) stimulation with 60 nm PMA. The eosinophil in A was untreated, that in B was pretreated with 10 μm pyrrolidine-2.

Figure 4. GFX reverses activation of both NADPH oxidase and proton channels in human eosinophils

Current at −60 mV (left records) in a human eosinophil in the perforated patch configuration stimulated with PMA and then treated with 3 μm GFX. The upper record shows currents during steps to +60 mV applied every 30 s, as well as the current at the holding potential, −60 mV. The lower record shows the same holding current at higher gain, with the currents during the test pulses blanked. On the right are proton currents during pulses to +60 mV recorded in this experiment at the times indicated by lower case letters.

Figure 5. Staurosporine reverses spontaneous activation of both NADPH oxidase and proton channels in human eosinophils

Current recorded shortly after establishing perforated-patch recording. Voltage pulses to +20 mV were applied from a holding potential of −60 mV every 15 s. At the arrow, 100 nm staurosporine was introduced into the bath. The time-dependent outward currents are proton currents; the inward current is presumably largely I_e. The horizontal line indicates zero current. It was evident that this cell was activated because a pulse to −20 mV elicited inward proton current (not shown), something never seen in resting cells. In addition, superposition of the inward current, presumed to be I_e, and the proton current during the test pulses results in a net inward current throughout the pulses to +20 mV, despite the activation of several picoamperes of outward proton current. Capacity transients have been truncated for clarity.

Figure 6. Okadaic acid inhibits the deactivation of proton current induced by GFX

Eosinophils were stimulated with PMA and then 2–3 μm GFX was introduced in the absence (▴) or presence of 100 nm okadaic acid (□) (n = 5 cells for both). Test pulses to +60 mV (+40 mV in one cell) were applied every 30 s. The mean (± s.e.m.) leak-corrected I_H (the time-dependent rising current) at the end of the 4 s test pulse is plotted, normalized to its value during the final test pulse before addition of GFX, which is indicated as a horizontal line. All I_H values obtained > 1.5 min after addition GFX in the presence of okadaic acid are significantly greater than those in its absence (P < 0.05, by Student's two-tailed t test). The I_H during the first few pulses after the bath change increased presumably due to a transient temperature increase. (Evaporation of the bath reduces its temperature below ambient.)

Figure 8. Similarity of the PMA responses of NADPH oxidase and H⁺ channels in granulocytes from control or cPLA₂α KO mice

Currents at −60 mV (left traces) in granulocytes in the perforated patch configuration from a control mouse (A) and a cPLA₂α KO mouse (B). Arrows indicate addition of 10 μm fMetLeuPhe, 60 nm PMA, or simple bath exchanges (‘wash’). Currents during 4 s depolarizing pulses to +60 mV (A) or +40 mV (B) (right traces) before (a) and after (b) addition of 60 nm PMA at the times indicated by lower case letters.

Figure 9. PMA enhances proton channel gating in murine granulocytes

Families of currents in a normal mouse granulocyte during identical pulses to −40 through +80 mV in 20 mV increments, before (left) and after stimulation with PMA (right). The dotted line shows zero current: PMA elicited inward electron current.

Figure 10. The g_H–V relationships before and after stimulation are similar in granulocytes from control and cPLA₂α KO mice

Mean ± s.e.m. values of g_H calculated from I_H at the end of 8 s pulses and measured V_rev values (in most cases) in control granulocytes (▪) and cPLA₂α KO (•) mice. The open symbols indicate data after stimulation with 60 nm PMA in control (□) or cPLA₂α KO cells (○). All data are from 10 to 12 cells.

Figure 11. Responses to fMetLeuPhe are similar in granulocytes from control and cPLA₂α KO mice

Families of currents before (A and D) and after stimulation with 10 μm fMetLeuPhe (B and E) or 100 nm PMA (C and F) in a normal mouse cell (A–C) and a cPLA₂α KO cell (D–F). All families illustrated are from −20 mV through +60 mV in 20 mV increments. The horizontal line indicates zero current. Calibration bars apply to all parts.