RESUMEN
Voltage-gated proton channels (Hv1/VSOP), encoded by Hvcn1, are important regulator of reactive oxygen species (ROS) production in many types of immune cells. While in vitro studies indicate that Hv1/VSOP regulates ROS production by maintaining pH homeostasis, there are few studies investigating the functional importance of Hv1/VSOP in vivo. In the present study, we first show that Hv1/VSOP is functionally expressed in liver resident macrophage, Kupffer cells, regulating the hepatic oxidative stress in vivo. Our immunocytochemistry and electrophysiology data showed that Hvcn1 is specifically expressed in Kupffer cells, but not in hepatocytes. Furthermore, Hvcn1-deficiency drastically altered the hepatic oxidative stress. The Hvcn1-deficient mice showed high blood glucose and serum insulin but normal insulin sensitivity, indicating that these phenotypes were not linked to insulin resistance. Transcriptome analysis indicated that the gene expression of glycogen phosphorylase (Pygl) and Glucose-6-phosphatase, catalytic subunit (G6pc) were upregulated in Hvcn1-deficient liver tissues, and quantitative PCR confirmed the result for Pygl. Furthermore, we observed higher amount of glucose-6-phosphate, a key sugar intermediate for glucose in Hvcn1-deficient liver than WT, suggesting that glucose production in liver is accelerated in Hvcn1-deficient mice. The present study sheds light on the functional importance of Kupffer cells in hepatic oxidative stress and its potential relationship with glucose metabolism.
Asunto(s)
Glucosa/metabolismo , Canales Iónicos/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Estrés Oxidativo/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Protones , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The voltage-gated proton channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-gated proton channels exhibit a large variation of activation rate among different species. Here we identify an Hv1 ortholog from sea urchin, Strongylocentrotus purpuratus, SpHv1. SpHv1 retains most of key properties of Hv1 but exhibits 20-60 times more rapid activation kinetics than mammalian orthologs upon heterologous expression in HEK293T cells. Comparison between SpHv1 and mHv1 highlights novel roles of the third transmembrane segment S3 in activation gating of Hv1.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/fisiología , Erizos de Mar/metabolismo , Animales , Células HEK293 , Humanos , Canales Iónicos/química , RatonesRESUMEN
Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Canales Iónicos/metabolismo , Neutrófilos/metabolismo , Animales , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Membrana Celular/metabolismo , Exocitosis , Femenino , Inmunoglobulina G/inmunología , Canales Iónicos/genética , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Neutrófilos/inmunología , Peroxidasa/metabolismo , Unión Proteica , Transporte de Proteínas , Vesículas Secretoras/metabolismoRESUMEN
H(v) channels (voltage-gated proton channels) are expressed in blood cells, microglia and some types of epithelial cells. In neutrophils H(v) channels regulate the production of reactive oxygen species through regulation of membrane potential and intracellular pH. H(v) channels have also been suggested to play a role in sperm physiology in the human. However, the functions of the Hv channel at the whole-body level are not fully understood. In the present paper we show that Hvcn1 (voltage-gated hydrogen channel 1)-knockout mice show splenomegaly, autoantibodies and nephritis, that are reminiscent of human autoimmune diseases phenotypes. The number of activated T-cells was larger in Hvcn1-deficient mice than in the wild-type mice. Upon viral infection this was remarkably enhanced in Hvcn1-deficient mice. The production of superoxide anion in T-cells upon stimulation with PMA was significantly attenuated in the Hvcn1-deficient mice. These results suggest that H(v) channels regulate T-cell homoeostasis in vivo.