Strong glucose dependence of electron current in human monocytes

Abstract

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.

Fig. 1.

Electron currents in human monocytes with and without glucose in the bath solution. Increased electron current at higher temperature and effects of glucose. A: electron current recorded in a peripheral blood monocyte (PBM) activated by 60 nM PMA without glucose in the bath solution. B: electron current elicited in a solution containing 5 mM glucose. Electron current was slowly reduced by 20 μM DPI. C: in this preactivated PBM, addition of glucose increased the electron current immediately. Subsequent addition of PMA did not increase the electron current, but 3 μM GF109203X (GFX) reduced the electron current. D: mean electron current in PBMs cultured for 1–3 days in the absence or presence of 5 mM glucose (GLC) at room temperature and with 5 mM glucose at 25°C. Data are displayed as means ± SE. Numbers of cells are given in each bar. No statistical differences could be detected at different times in culture. E: data from D with measurements at all times in culture pooled for cells studied without glucose, with 5 mM glucose, and with 5 mM glucose at 25°C. ***P < 0.01, significant differences between the third and the first two columns. Glucose vs. no glucose electron currents had a significant difference with P < 0.05. F: a spontaneously activated cell shows a sudden, drastic increase of electron current immediately upon addition of 5 mM glucose. PMA (60 nM) did not increase the electron current further. DPI (20 μM) decreased the electron current. G: electron current at 25°C in a PBM supplied with glucose and activated by PMA. The withdrawal of glucose reduced the electron current. Reapplication of glucose restored the electron current. The electron current was inhibited by 20 μM DPI.

Fig. 2.

Comparison of the properties of H⁺ currents before and after PMA and DPI in a single human monocyte. A: currents recorded in a control PBM during depolarizing pulses from holding potential (V_h) = −40 mV to −20 mV through +60 mV in 10-mV increments are superimposed. B: currents in the same cell after addition of 60 nM PMA. C: currents in the same cell directly after addition of 20 μM DPI and washout of PMA. Currents in A, B, and C are shown at the same current- and time base and were measured with the same protocol. D: chord conductance (g_H)-voltage relationship of the proton currents in this cell recorded (control) and in the presence of PMA or DPI. Conductance was calculated from the tail currents (see materials and methods). E: voltage dependence of activation time constant (τ_act) in this monocyte before stimulation (■), after PMA activation (○), and after DPI application (△). In control records during small depolarizations, τ_act was often longer than the pulse; hence in these cases, τ_act was not well determined.

Fig. 3.

H₂O₂ release measured with and without glucose (GLC) in human PBMs and neutrophils. Inhibition of proton currents and H₂O₂ release in human monocytes by Zn²⁺. A: H₂O₂ production measured in human monocytes (PBM) in the presence (■) or absence (○) of 5 mM glucose. B: glucose dependence of H₂O₂ production in human neutrophils (PMN). C: normalization of H₂O₂ production in neutrophils and monocytes from A and B to the same maximal rate. D: H₂O₂ production measured in PBMs with and without glucose. Glucose was added after 60 min to cells without glucose. E: proton current in a human PBM during pulses to +40 mV (inset). Zn²⁺ (5 μM) inhibited proton current immediately upon addition to the bath. Inset: proton current was restored by removal of Zn²⁺ with EGTA. F: H₂O₂ production in the absence or presence of Zn²⁺ at the indicated concentrations ranging from 30 μM to 3,000 μM.