Ca2+ mobilization is usually central to many cellular processes including stimulated exocytosis and cytokine ARRY-520 R enantiomer production in mast cells. shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response. Keywords: IgE receptors Ca2+ puffs Ca2+ oscillations GCaMP2 TRPC channels INTRODUCTION Changes in intracellular Ca2+ play significant functions in numerous cellular responses such as secretion gene expression and cell migration. These cellular functions require spatial and temporal regulation of cytosolic Ca2+ (1 2 Among these regulated events are Ca2+ puffs waves and regenerative oscillations that mediate localized cellular responses and support transfer of information across the cell and organelles (3). Ca2+ ARRY-520 R enantiomer waves were first characterized in Xenopus oocyte fertilization (4) and they have since been identified in excitable (5) and nonexcitable cell types including hepatocytes (6) HeLa cells (7) and neutrophils (8). In myocytes Ca2+ waves were shown to initiate from elementary Ca2+ events called “Ca2+ sparks” (9) and are thought to propagate through the cytosol by calcium-induced calcium release from ER stores (10). Ca2+ waves are frequently initiated by activation of plasma membrane receptors that stimulate Ca2+-dependent signaling within the cell (11). Comparable mechanisms may be involved in stimulating Ca2+ puffs and maintaining the propagation of Ca2+ waves in non-excitable cells (3 12 However Ca2+ waves in response to immunoreceptor signaling have not been previously reported. Ca2+ oscillations have been characterized in many cell types including RBL-2H3 mast cells (13 14 where they have been temporally correlated with degranulation events (15 16 Ca2+ oscillations are sustained by store-operated Ca2+ entry (SOCE) other ion channels as well as membrane potential ARRY-520 R enantiomer (17). Mast cells play key functions in the inflammatory process in both innate and adaptive immune responses (18). In the latter binding of multivalent antigen to receptor-associated IgE aggregates this receptor FcεRI which causes mast cell activation resulting in Ca2+ mobilization and consequent exocytotic release of mediators of allergy and inflammation (19). RBL-2H3 cells are immortalized mucosal mast cells that have been utilized for extensive biochemical and cell biological investigations of mast cell function (20-22). In the present study we used high-speed confocal imaging to ARRY-520 R enantiomer investigate cytoplasmic Ca2+ dynamics activated via FcεRI in RBL cells and in rat bone marrow-derived mast cells (BMMCs) which are also mucosal in character (23). We find that Ca2+ responses to soluble antigen initiate in the form of a wave that begins most frequently at the tip of an extended cell protrusion and propagates throughout the entire cell in several seconds. In contrast localized delivery of antigen attached to the tip of a micropipette results in repetitive localized Ca2+ puffs that infrequently develop into propagated waves. Our results provide evidence that Ca2+ wave initiation from extended protrusions depends Mouse Monoclonal to Goat IgG. on Ca2+ influx via TRPC channels leading to the onset of SOCE-dependent Ca2+ oscillations and mast cell activation. MATERIALS AND METHODS cDNA plasmids The GCaMP2 construct (24) was provided by Dr. M. Kotlikoff Cornell University College of Veterinary Medicine. shRNA plasmids targeting TRPC channels (TRPC1 TRPC3 TRPC5 TRPC7 and GFP control) were characterized in RBL cells as previously described (25). Chemicals and reagents Fluo4AM and Fluo5FAM were purchased from Invitrogen/Molecular Probes (Eugene OR). “type”:”entrez-nucleotide” attrs :”text”:”U73122″ term_id :”4098075″ term_text :”U73122″U73122 ARRY-520 R enantiomer D-sphingosine thapsigargin “type”:”entrez-nucleotide” attrs :”text”:”A23187″ term_id :”833253″ term_text :”A23187″A23187 2 borate (2-APB) and GdCl3 were from Sigma-Aldrich (St. Louis MO). N N’-dimethylsphingosine (DM-sphingosine) was from Avanti Polar Lipids (Alabaster AL). Cells RBL-2H3 cells (26) were maintained in monolayer culture in Minimum Essential Medium supplemented with 20% fetal bovine serum (Atlanta Biologicals Norcross GA USA) and 10 μg/ml.