Fagocita NADPH-oxidáz és protoncsatorna aktivitását szabályozó fehérjék azonosítása RNS-interferencia felhasználásával = Identification of protiens regulating the activity of phagocyte NADPH-oxidase and proton channel using RNA-interference

Feszültségfüggő protonáramot (IHv) sok sejttípusban kimutattak, de a legtöbb vizsgálatot emberi granulocitákban végezték. Fagocitákban a protonáramért felelős protoncsatorna feltételezett szerepe az aktivált Nox2 (a fagocita NADPH-oxidáz komplex központi enzime) működésének támogatása a légzési robbanás során. 2006-ban klónozták a Hv1 fehérjét, mely feszültségfüggő proton csatornaként működik, és Hv1-re nézve génhiányos egerek fehérvérsejtjeiben az légzési robbanás csökkenését tapasztalták. Emberi fehérvérsejtekben azonban kevés ismerettel rendelkezünk a Hv1 fehérjéről. Vizsgálataink középpontjába ezért az emberi fehérvérsejtek által termelt Hv1 fehérje molekuláris és celluláris szintű jellemzését állítottuk. Megvizsgáltuk továbbá a Hv1 és Nox2 kifejeződés viszonyát. Eredményeink alapján a Hv1 a granulociták felszínén és intracelluláris membránjaiban található. A Hv1 molekula homodimer és monomer formában egyaránt kifejeződik fehérvérsejtekben. A Hv1 fehérje emberi fehérvérsejtekben is szükséges a protonáram létrejöttéhez, kifejeződésének gátlása ugyanis csökkenteti az IHv amplitúdóját különböző humán eredetű fehérvérsejt-vonalakban. Nyugvó granulocitákban a Hv1 és Nox2 kolokalizációt mutat, és aktivációkor a két fehérje azonos membránokban dúsul (pl. fagoszómák falában). Végezetül a Hv1 kifejeződés masszív gátlása a PLB-985-ös emberi granulocita sejtvonalban is a az légzési robbanás csökkenéséhez vezet. | Voltage-gated proton current (IHv) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of IHv in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently Hv1, a voltage-gated proton channel was cloned, and leukocytes from Hv1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about Hv1 in human granulocytes. Therefore, we set out to define the main characteristics of Hv1 expression in human granulocytes on the cellular, subcellular and molecular level and also its relationship to Nox2. Our results indicate that Hv1 is expressed in intracellular membranes and on the cell surface of granulocytes. Native Hv1 molecule is expressed in dimer and monomer form in different leukocytes and its expression is required for normal voltage-gated proton currents in different leukemia cell lines indicating that Hv1 is indispensable for IHv in human leukocytes as well. Additionally, Hv1 and Nox2 colocalize in granulocytes, and both accumulate in the same membrane compartment (e.g. in phagosomal wall) upon activation. Furthermore, severely impaired Hv1 expression can reduce the maximum rate of superoxide release in a human granulocyte cell line PLB-985

Elektron transzfer rendszerek élettani szerepe = The physiological role of electron transfer systems

Fagocitákban leírtuk a NADPH oxidázt szabályozó két különböző GTPáz aktiváló fehérje szabályozását és a kísérő K+ transzport baktérium ölő hatását. Agyi mitokondriumokban (mito) a légzési lánc I. komplexének szubsztrátjai membránpotenciál (Em) függően reaktív oxigénszármazékot (ROS) képeznek. Az alfa-glicerofoszfát (aGP) ROS-t képez az I. komplexen és az aGP-dehidrogenáz enzimen, utóbbit a Ca2+ aktivája. Idegvégződésekben a mito ROS képzését az Em nem befolyásolja. A mito-k elektromos szincíciumot képeznek, de a Ca2+ diffúziója korlátozott. Alacsony O2.- szint a Ca2+ -mobilizáló agonista Ca2+ jel képző hatását glomerulóza sejtben gátolja. A ROS támadáspontja a belső raktárból történő Ca2+ felszabadulás. UV hatására a mito Ca2+ felvétele is csökkent. Angiotenzin II -vel ingerelt H295R sejtben a mito Ca2+ jel képzés sebessége a mito és az endoplazmás retikulum (ER) közelségével korrelál. A p38 MAPK és az újtípusú PKC izoformák egyidejű gátlása a Ca2+ jelnek a citoszolból a mito-ba történő áttevődését gátolja és a fenti korrelációt megszünteti. Az ER lumenében a tiol/diszulfid rendszertől elkülönülő NAD(P)+/NAD(P)H rendszer működik. Redox állapotát a glukóz-6-foszfát transzporter és az intraluminális oxidoreduktázok határozzák meg. A redukált állapot fenntartása szükséges a glukokortikoidok prereceptoriális aktiválásához, s egyes sejtekben antiapoptotikus hatású. Jellemeztük az ER szulfát transzporterét, valamint a transzlokon peptid csatorna anion permeabilitását. | We described in phagocytes the regulation of two GTPase activating proteins, terminating the activity of plasmalemmal NADPH oxidase and the role of K+ movements in bacterial killing. In brain mitochondria complex I dependent substrates show a membrane potential (Em) dependent reactive oxygen species (ROS) formation. ROS production by alpha-glycerophosphate (aGP) occured at complex I and on the aGP-dehydrogenase enzyme. The latter is activited by Ca2+. Mitochondria form an electric syntitium but the diffusion of Ca2+ is limited. In glomerulosa cells, at low [O2.-] angiotensin-induced Ca2+ signalling is attenuated, the site of ROS action is Ca2+ release from the internal stores. The rate of mitochondrial Ca2+ uptake in angiotensin-stimulated cells correlates with the vicinity of the mitochondrion and the endoplasmic reticulum (ER). Simultaneous activation of p38 MAPK and the novel isoforms of PKC attenuates the transfer of cytosolic Ca2+ signal into the mitochondria and abolishes this correlation. In the ER we observed a novel NAD(P)+/NAD(P)H system different from the thiol/disulphide system. Its reduced state is tuned by the glucose-6-phosphate transporter and the luminal oxidoreductases and is required for the prereceptorial activation of glucocorticoids. We have characterized the sulphate transport in the ER, and the contribution of the translocon peptide channel to the membrane permeation of small anions

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