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.