title: Comparison of Ca2+puffs evoked by extracellular agonists and photoreleased IP3.
abstract: The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+signals (Ca2+puffs) that arise from the concerted openings of clustered IP3receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3into the cytosol. Photorelease of IP3from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+puffs evoked by photoreleased IP3fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1μm), suggesting that IP3from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3uniformly throughout the cytoplasm, our results imply that IP3generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, 92697, United States. Electronic address: lockj@uci.edu.
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Lock
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, 92697, United States. Electronic address: ismith@uci.edu.
authors.forename: Ian F
authors.initials: IF
authors.lastname: Smith
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, 92697, United States; Department of Physiology and Biophysics, University of California, Irvine, CA, 92697, United States. Electronic address: iparker@uci.edu.
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
mesh_terms: D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D007700:Kinetics; D008027:Light; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D018679:Cholinergic Agonists; D053496:Inositol 1,4,5-Trisphosphate Receptors; D014665:Vasodilator Agents; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D001920:Bradykinin; D002118:Calcium
keywords: Ca(2+) puffs; Calcium imaging; GPCRs; IP(3); Signaling nanodomains