ABSTRACT
Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store-operated Ca(2+) entry (SOCE), the prevalent Ca(2+) influx mechanism in non-excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca(2+) sensors and Orai Ca(2+) channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down-regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca(2+) entry induced by cyclopiazonic acid, ATP, the P2Y6 receptor agonist UDP, or the P2Y12 receptor agonist 2-methylthio-ADP (2-MeSADP) was clearly affected in microglia from Stim1(-/-) , Stim2(-/-) , and Orai1(-/-) mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide-induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia.
Subject(s)
Calcium Channels/metabolism , Calcium Signaling/immunology , Calcium/metabolism , Membrane Glycoproteins/metabolism , Microglia/immunology , Microglia/metabolism , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Calcium Channels/deficiency , Calcium Channels/genetics , Cell Culture Techniques , Indoles/metabolism , Lipopolysaccharides/immunology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Microglia/cytology , ORAI1 Protein , Phagocytosis/immunology , Stromal Interaction Molecule 1 , Stromal Interaction Molecule 2 , Thionucleotides/metabolism , Uridine Diphosphate/metabolismABSTRACT
Glasses with the mol% composition 30ZnO, 10Na(2)O, 10BaO and 50SiO(2) were annealed at 700°C and 760°C ex situ as well as in situ using a hot stage scanning electron microscope (SEM). The formed crystal phase (BaZn(2)Si(2)O(7)) was proved ex situ by X-ray diffraction. Annealing the glass samples in the SEM resulted in a strong surface crystallization, which was monitored for 1 h and 21 min and 2 h and 2 min at the temperatures 700°C and 760°C, respectively. The crystal growth velocities for these two temperatures were determined from a series of micrographs. It is experimentally shown that during the course of the crystallization in this non-isochemical system, the residual glassy phase changes its composition, and hence, the crystal growth velocity depends on time. Furthermore, it is shown that the electron beam irradiation directly affects the nucleation and crystal growth velocities.
