4 research outputs found
A pH-sensitive chloride current in the chemoreceptor cell of rat carotid body
Cardiorespiratory response to acidosis is initiated by the carotid body.The direct effect of extracellular pH (pHo) on the chloride currents of isolated chemoreceptor cells of the rat carotid body was investigated using the whole-cell patch-clamp technique.On applying intra- and extracellular solutions with a symmetrical high-Cl− content and with the monovalent cations replaced with membrane-impermeant ones, an inwardly rectifying Cl− current was found.The current activated slowly and did not display any time-dependent inactivation. Current activation was present at membrane potentials negative to 0 mV (pHo = 7.0).The current was activated by extracellular acidosis and inhibited by alkalosis in the physiologically relevant pH range of 7.0-7.8.The current was reduced by 0.1 mM Cd2+ to the level of the leak current and by 1 mM anthracene-9-carboxylic acid (9-AC) to about 40 %, while 0.1 mM Ba2+ had no effect.Application of 1 mM 9-AC caused a slow but statistically significant increase in the resting pHi (from a mean of 7.29 to 7.37 in 5 min) in clusters of chemoreceptor cells in CO2/HCO3−-buffered media as measured with carboxy-SNARF-1.When membrane potential changes were estimated in the cell-attached mode, 1 mM 9-AC hyperpolarized three out of five tested cells (by 14 mV in average) incubated in CO2/HCO3−-buffered media.In summary, chemoreceptor cells express an inwardly rectifying Cl− current, which is directly regulated by pHo. The current may participate in intracellular acidification and membrane depolarization during acidic challenge
Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase
The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O(2). The activity of the oxidase at the plasma membrane can be measured as electron current (I(e)), and the voltage dependence of I(e) was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from −120 to 200 mV, were applied to obtain ‘steady-state’ current–voltage relationships (I–V). The amplitude of I(e) recorded at −40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (K(m)) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of I(e) is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, I(e) was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (−60 to 60 mV), the I–V curve strongly rectifying only below −100 mV. At lower NADPH concentrations the I–V curve was progressively shifted to more positive potentials and I(e) became voltage-independent also within the physiological range. Consequently, the K(m) of the oxidase decreased by approx. 40% (from 100 to 60 μM) when the membrane potential increased from −60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I(e)–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I(e) reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase
Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase
The phagocytic NADPH oxidase (phox) moves electrons across cell membranes to kill microbes. The activity of this lethal enzyme is tightly regulated, but the mechanisms that control phox inactivation are poorly understood for lack of appropriate assays. The phox generates measurable electron currents, I(e), that are associated with inward proton currents, I(H). To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize I(e) (induced by the addition of NADPH) and I(H) (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. I(e) decayed rapidly in the absence of nucleotides (τ≈6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 μM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5′-[γ-thio]triphosphate) (τ≈57 min), but not by either ATP or GTP[S] alone. I(H) also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of I(H) required further addition of 25 μM PI(3,4)P(2) (phosphoinositide 3,4-bisphosphate) to the cytosolic side of the patch. PI(3,4)P(2) had no effect on I(e) and its stabilizing effect on I(H) could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased I(H) stability, an effect that was not prevented by phosphatase inhibitors but by the non-hydrolysable ATP analogue ATP[S] (adenosine 5′-[γ-thio]triphosphate). Our data indicate that the assembled phox complex is very stable in eosinophil membranes if both ATP and GTP[S] are present, but inactivates within minutes if one of the nucleotides is removed. Stabilization of the phox-associated proton channel in a highly voltage-sensitive conformation does not appear to involve phosphorylation but ATP binding, and requires not only ATP and GTP[S] but also PI(3,4)P(2), a protein known to anchor the cytosolic phox subunit p47(phox) to the plasma membrane
Reactive oxygen species-mediated bacterial killing by B lymphocytes
Regulated production of ROS is mainly attributed to Nox family enzymes. In neutrophil granulocytes and macrophages, Nox2 has a crucial role in bacterial killing, and the absence of phagocytic ROS production leads to the development of CGD. Expression of Nox2 was also described in B lymphocytes, where the role of the enzyme is still poorly understood. Here, we show that peritoneal B cells, which were shown recently to possess phagocytic activity, have a high capacity to produce ROS in a Nox2-dependent manner. In phagocytosing B cells, intense intraphagosomal ROS production is detected. Finally, by studying 2 animal models of CGD, we demonstrate that phagocyte oxidase-deficient B cells have a reduced capacity to kill bacteria. Our observations extend the number of immune cell types that produce ROS to kill pathogens