Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

2007 ◽  
Vol 293 (1) ◽  
pp. C45-C54 ◽  
Author(s):  
Kirill Essin ◽  
Birgit Salanova ◽  
Ralph Kettritz ◽  
Matthias Sausbier ◽  
Friedrich C. Luft ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Patch Clamp ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Bk Channels ◽  
Bk Channel ◽  
Proton Channel ◽  
Nadph Oxidase Activity ◽  
Intracellular Ca ◽  
Channel Currents

Large-conductance Ca2+-activated K+(BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK−/−) mice, we stimulated NADPH oxidase activity with 12- O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2−and H2O2production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca2+and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+by N-formyl-l-methionyl-l-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK−/−and BK+/+mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

scholarly journals BK channels in innate immune functions of neutrophils and macrophages

Blood ◽  
2009 ◽  
Vol 113 (6) ◽  
pp. 1326-1331 ◽  
Author(s):  
Kirill Essin ◽  
Maik Gollasch ◽  
Susanne Rolle ◽  
Patrick Weissgerber ◽  
Matthias Sausbier ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Bk Channels ◽  
Bk Channel ◽  
Nadph Oxidase Activity ◽  
Factor Α ◽  
Necrosis Factor

Abstract Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK−/−) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-α secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK−/− and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-κB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-α release and signal transduction BMDMs.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

Blood ◽  
2014 ◽  
Vol 124 (4) ◽  
pp. 590-597 ◽  
Author(s):  
Roel P. Gazendam ◽  
John L. van Hamme ◽  
Anton T. J. Tool ◽  
Michel van Houdt ◽  
Paul J. J. H. Verkuijlen ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Candida Albicans ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Human Neutrophils ◽  
Nadph Oxidase Activity ◽  
Key Points

Key Points Human neutrophils use 2 independent mechanisms for the killing of unopsonized and serum-opsonized C albicans. Unopsonized Candida killing depends on CR3 and CARD9 but not dectin-1; opsonized Candida killing on FcγR, PKC, and NADPH oxidase activity.

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

2006 ◽  
Vol 290 (1) ◽  
pp. C77-C86 ◽  
Author(s):  
Hiroko Sade ◽  
Katsuhiko Muraki ◽  
Susumu Ohya ◽  
Noriyuki Hatano ◽  
Yuji Imaizumi
Keyword(s):  
Cell Voltage ◽  
Similar Efficacy ◽  
Bk Channels ◽  
Bk Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Α Subunit ◽  
Hek 293 ◽  
Intracellular Ca ◽  
Channel Currents

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the α-subunit of the BK channel (BK-α), both α- and β1-subunits (BK-αβ1), or both α- and β4-subunits (BK-αβ4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-αβ1 within a similar concentration range. Because methAEA could potentiate BK-α, BK-αβ1, and BK-αβ4 with similar efficacy, the β-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-α and HEK-BK-αβ1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.

Abstract WP274: Inhibition of Na+/H+ Exchanger Isoform 1 and/or Voltage Gated Proton Channel Improves Outcomes in an Embolic Stroke Model

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Weiguo Li ◽  
Rebecca Ward ◽  
Jingping Sun ◽  
Xinyue Guo ◽  
Adviye Ergul ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Infarct Size ◽  
Oxidase Activity ◽  
Embolic Stroke ◽  
Proton Channel ◽  
Experimental Stroke ◽  
Dependent Manner ◽  
Nadph Oxidase Activity ◽  
Voltage Gated ◽  
Significant Difference

While Na+/H+ exchanger isoform 1 inhibitor (NHEi) has been reported to be neuro- and cardio-protective in experimental stroke and myocardial infarction, respectively, clinical use of NHEi for cardioprotection has stalled due to increased cerebrovascular events. NHEi has been demonstrated to increase Hv1 activity and we recently showed that NHEi activates NADPH oxidase and results in amplified superoxide formation in a voltage gated proton channel Hv1-dependent manner. In the CNS, Hv1 is localized primarily to microglia and deletion of Hv1 is neuroprotective after permanent and transient middle cerebral artery occlusion (MCAO). In the current study, we hypothesized that beneficial effect of NHEi after MCAO will be greater in a rat model lacking Hv1 due to loss of a potentially deleterious increase in Hv1 activation and NADPH oxidase activity. The wild type (WT) and Hv1 knockout (KO) rats (n=4-6) were treated with vehicle or NHEi (KR-32568, 2 mg/kg, i.v.) at 30 min after embolic MCAO. The neurological deficiency, infarct size, HT index, and edema ratio were assessed 3 days after surgery (Table). Compare to WT rats, KO rats had smaller infarct, less edema, and better neurological outcomes as previously found in the suture model. NHEi decreased infarct size and edema in both strains. While there was no significant difference in HT between WT and KO rats, the HT was less in WT rats with NHEi. Functional outcomes were significantly improved with NHEi in WT group, while the KO groups had a trend for a better outcomes with NHEi. These data indicate that NHE inhibition in the acute stroke period is similarly effective in both WT and Hv1 KO animals in providing neurovascular protection. Our data do not support the hypothesis that a deleterious increase in Hv1 dependent NADPH oxidase activity limits the beneficial actions of NHEi in embolic stroke. Further studies are needed to explore the underlying mechanism of the interaction between NHEi and the Hv1 channel in ischemic stroke.

Number of KCa Channels Underlying Spontaneous Miniature Outward Currents (SMOCs) in Mudpuppy Cardiac Neurons

2001 ◽  
Vol 85 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Fabiana S. Scornik ◽  
Laura A. Merriam ◽  
Rodney L. Parsons
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Current Amplitude ◽  
Channel Current ◽  
Single Channel Current ◽  
Outward Currents ◽  
Intracellular Ca ◽  
Single Channel Currents ◽  
Channel Currents

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca2+-dependent K+(BK) channels. Previously we reported that SMOCs are activated by Ca2+-induced Ca2+ release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca2+ stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K+ concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca2+, external TEA, and caffeine. In cell-attached patch recordings, 166 μM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than −20 mV, suggesting that the transient rise in intracellular Ca2+from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 μM.

scholarly journals Neuroprotective Benefits of Exercise and MitoQ on Memory Function, Mitochondrial Dynamics, Oxidative Stress, and Neuroinflammation in D-Galactose-Induced Aging Rats

2021 ◽  
Vol 11 (2) ◽  
pp. 164
Author(s):  
Jae-Hoon Jeong ◽  
Jung-Hoon Koo ◽  
Jang Soo Yook ◽  
Joon-Yong Cho ◽  
Eun-Bum Kang
Keyword(s):  
Oxidative Stress ◽  
Antioxidant Activity ◽  
Cognitive Impairment ◽  
Nadph Oxidase ◽  
Learning And Memory ◽  
Oxidase Activity ◽  
Mitochondrial Dynamics ◽  
Nadph Oxidase Activity ◽  
Aging Rats ◽  
Positive Effects

Exercise and antioxidants have health benefits that improve cognitive impairment and may act synergistically. In this study, we examined the effects of treadmill exercise (TE) and mitochondria-targeted antioxidant mitoquinone (MitoQ), individually or combined, on learning and memory, mitochondrial dynamics, NADPH oxidase activity, and neuroinflammation and antioxidant activity in the hippocampus of D-galactose-induced aging rats. TE alone and TE combined with MitoQ in aging rats reduced mitochondrial fission factors (Drp1, Fis1) and increased mitochondrial fusion factors (Mfn1, Mfn2, Opa1). These groups also exhibited improved NADPH oxidase activity and antioxidant activity (SOD-2, catalase). TE or MitoQ alone decreased neuroinflammatory response (COX-2, TNF-α), but the suppression was greater with their combination. In addition, aging-increased neuroinflammation in the dentate gyrus was decreased in TE but not MitoQ treatment. Learning and memory tests showed that, contrarily, MitoQ alone demonstrated some similar effects to TE but not a definitive improvement. In conclusion, this study demonstrated that MitoQ exerted some positive effects on aging when used as an isolated treatment, but TE had a more effective role on cognitive impairment, oxidative stress, inflammation, and mitochondria dysfunction. Our findings suggest that the combination of TE and MitoQ exerted no synergistic effects and indicated regular exercise should be the first priority in neuroprotection of age-related cognitive decline.

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