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

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1−/− mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1−/− neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils

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

Complement MASP-1 enhances adhesion between endothelial cellsand neutrophils by up-regulating E-selectin expression

The complement system and neutrophil granulocytes are indispensable in the immune response against extracellular pathogens such as bacteria and fungi. Endothelial cells also participate in antimicrobial immunity largely by regulating the homing of leukocytes through their cytokine production and their pattern of cell surface adhesion molecules. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), a complement lectin pathway enzyme, is able to activate endothelial cells by cleaving protease activated receptors, which leads to cytokine production and enables neutrophil chemotaxis. Therefore, we aimed to investigate how recombinant MASP-1 (rMASP-1) can modify the pattern of P-selectin, E-selectin, ICAM-1, ICAM-2, and VCAM-1 adhesion molecules in human umbilical vein endothelial cells (HUVEC), and whether these changes can enhance the adherence between endothelial cells and neutrophil granulocyte model cells (differentiated PLB-985). We found that HUVECs activated by rMASP-1 decreased the expression of ICAM-2 and increased that of E-selectin, whereas ICAM-1, VCAM-1 and P-selectin expression remained unchanged. Furthermore, these changes resulted in increased adherence between differentiated PLB-985 cells and endothelial cells. Our finding suggests that complement MASP-1 can increase adhesion between neutrophils and endothelial cells in a direct fashion. This is in agreement with our previous finding that MASP-1 increases the production of pro-inflammatory cytokines (such as IL-6 and IL-8) and chemotaxis, and may thereby boost neutrophil functions. This newly described cooperation between complement lectin pathway and neutrophils via endothelial cells may be an effective tool to enhance the antimicrobial immune response

Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes.

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was approximately 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was approximately 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only approximately 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface

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

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

Recent work on animal models has revealed the important role played by the voltage-gated proton channel Hv1 during bacterial killing by innate immune cells. Studies from mice lacking Hv1 channels showed that Hv1 proton channels are required for high-level production of reactive oxygen species (ROS) by the NADPH oxidase of phagocytes (NOX2) in two ways. First, Hv1 channels maintain a physiological membrane potential during the respiratory burst of neutrophils by providing a compensating charge for the electrons transferred by NOX2 from NADPH to superoxide. Second, Hv1 channels maintain a physiological cytosolic pH by extruding the acid generated by the NOX2-dependent consumption of NADPH. The two mechanisms directly sustain the activity of the NOX2 enzyme and indirectly sustain other neutrophil functions by enhancing the driving force for the entry of calcium into cells, thereby boosting cellular calcium signals. The increased depolarization of Hv1-deficient neutrophils aborted calcium responses to chemoattractants and revealed adhesion and migration defects that were associated with an impaired depolymerization of the cortical actin cytoskeleton. Current research aims to transpose these findings to phagosomes, the phagocytic vacuoles where bacterial killing takes place. However, the mechanisms that control the phagosomal pH appear to vary greatly between phagocytes: phagosomes rapidly acidify in macrophages but remain neutral for several minutes in neutrophils following ingestion of solid particles, whereas in dendritic cells phagosomes alkalinize, a mechanism thought to promote antigen cross-presentation. In this review, we discuss how the knowledge gained on the role of Hv1 channels at the plasma membrane of neutrophils can be used to study the regulation of the phagosomal pH, ROS, membrane potential, and calcium fluxes in different phagocytic cells

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

Physiological roles of voltage-gated proton channels in leukocytes

Voltage-gated proton channels are designed to extrude large quantities of cytosolic acid in response to depolarising voltages. The discovery of the Hvcn1 gene and the generation of mice lacking the channel molecule have confirmed several postulated functions of proton channels in leukocytes. In neutrophils and macrophages, proton channels are required for high-level production of superoxide anions by the phagocytic NADPH oxidase, a bactericidal enzyme essential for host defence against infections. In B lymphocytes, proton channels are required for low-level production of superoxide that boosts the production of antibodies. Proton channels sustain the activity of immune cells in several ways. By extruding excess cytosolic acid, proton channels prevent deleterious acidification of the cytosol and at the same time deliver protons required for chemical conversion of the superoxide secreted by membrane oxidases. By moving positive charges across membranes, proton channels limit the depolarisation of the plasma membrane, promoting the electrogenic activity of NADPH oxidases and the entry of calcium ions into cells. Acid extrusion by proton channels is not restricted to leukocytes but also mediates the intracellular alkalinisation required for the activation of spermatozoids. Proton channels are therefore multitalented channels that control male fertility as well as our innate and adaptive immunity