title: Heterologous desensitization of both phosphoinositide and Ca2+ signaling in SH-SY5Y neuroblastoma cells: a role for intracellular Ca2+ store depletion?
abstract: Measurement of the intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded single cells of the human neuroblastoma line SH-SY5Y indicated coexpression of muscarinic and bradykinin receptors linked to activation of phosphoinositidase C (PIC). Both agonists elevated [Ca2+]i and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in populations of adherent cells, although in cells used directly upon attainment of confluence the responses to carbachol were greater than those to bradykinin and displayed additional sustained components. This model system was used to examine heterologous interactions when a second PIC-linked agonist was added 100-300 sec after but in the continued presence of the first. Maximal (1 mM) carbachol concentrations abolished the elevation of [Ca2+]i produced by bradykinin but the muscarinic antagonist atropine (10 microM) restored the response, provided that extracellular Ca2+ was present throughout the experiment or was added before bradykinin. Carbachol also abolished bradykinin-mediated Ins(1,4,5)P3 elevation. In contrast, bradykinin did not influence [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of extracellular Ca2+. In cells maintained at confluence for 2 weeks, the rapid peak elevations of [Ca2+]i and Ins(1,4,5)P3 levels induced by carbachol and bradykinin were approximately equivalent in magnitude. In these cells carbachol again abolished bradykinin-mediated elevation of [Ca2+]i but only attenuated, rather than abolished, the elevation of Ins(1,4,5)P3 levels. The [Ca2+]i and Ins(1,4,5)P3 responses to bradykinin were fully restored 100 sec after atropine only in the presence of extracellular Ca2+. Thus, depletion of an intracellular Ins(1,4,5)P3-sensitive Ca2+ store may underlie the ability of carbachol to produce not only heterologous desensitization of the [Ca2+]i elevation induced by bradykinin but also that of the Ins(1,4,5)P3 response. This suggests a feed-forward activation of PIC by Ca2+ released from Ins(1,4,5)P3-sensitive stores. Furthermore, studies in which Ins(1,4,5)P3-sensitive stores were depleted with thapsigargin and cells were challenged in the presence or absence of extracellular Ca2+ indicated that Ca2+, irrespective of its origin (intra- or extracellular), potentiated the Ins(1,4,5)P3 response to bradykinin alone. In cells maintained at confluence for 2 weeks, bradykinin was again unable to influence either [Ca2+]i or Ins(1,4,5)P3 responses to carbachol in the presence of Ca2+. This lack of heterologous desensitization may be due to the rapid, full, homologous desensitization of bradykinin receptors, compared with an incomplete homologous desensitization of muscarinic receptors.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, UK.
authors.forename: G B
authors.initials: GB
authors.lastname: Willars
authors.forename: S R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002448:Cell Adhesion; D004347:Drug Interactions; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000503:physiopathology / Q000648:ultrastructure; D010716:Phosphatidylinositols / Q000378:metabolism* / Q000502:physiology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D018002:Receptors, Bradykinin / Q000502:physiology; D011976:Receptors, Muscarinic / Q000502:physiology; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D010716:Phosphatidylinositols; D018002:Receptors, Bradykinin; D011976:Receptors, Muscarinic; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D001920:Bradykinin; D002118:Calcium