HETEROLOGOUS DESENSITIZATION OF BOTH PHOSPHOINOSITIDE AND CA2-SY5Y NEUROBLASTOMA-CELLS - A ROLE FOR INTRACELLULAR CA2+ STORE DEPLETION( SIGNALING IN SH)

Measurement of the intracellular Ca2+ concentration ([Ca2+](i)) in fur a-2-loaded single cells of the human neuroblastoma line SH-SY5Y indica ted coexpression of muscarinic and bradykinin receptors linked to acti vation of phosphoinositidase C (PIC). Both agonists elevated [Ca2+](i) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P-3] levels in population s of adherent cells, although in cells used directly upon attainment o f confluence the responses to carbachol were greater than those to bra dykinin and displayed additional sustained components. This model syst em was used to examine heterologous interactions when a second PIG-lin ked agonist was added 100-300 sec after but in the continued presence of the first. Maximal (1 mM) carbachol concentrations abolished the el evation of [Ca2+](i) produced by bradykinin but the muscarinic antagon ist atropine (10 mu M) restored the response, provided that extracellu lar Ca2+ was present throughout the experiment or was added before bra dykinin. Carbachol also abolished bradykinin-mediated Ins(1,4,5)P-3 el evation. In contrast, bradykinin did not influence [Ca2+](i) or Ins(1, 4,5)P-3 responses to carbachol in the presence of extracellular Ca2+. In cells maintained at confluence for 2 weeks, the rapid peak elevatio ns of [Ca2+](i) and Ins(1,4,5)P-3 levels induced by carbachol and brad ykinin were approximately equivalent in magnitude. In these cells carb achol again abolished bradykinin-mediated elevation of [Ca2+](i) but o nly attenuated, rather than abolished, the elevation of Ins(1,4,5)P-3 levels. The [Ca2+](i) and Ins(1,4,5)P-3 responses to bradykinin were f ully restored 100 sec after atropine only in the presence of extracell ular Ca2+. Thus, depletion of an intracellular Ins(1,4,5)P-3-sensitive Ca2+ store may underlie the ability of carbachol to produce not only heterologous desensitization of the [Ca2+](i) elevation induced by bra dykinin but also that of the Ins(1,4,5)P, response. This suggests a fe ed-forward activation of PIC by Ca2+ released from Ins(1,4,5)P-3-sensi tive stores. Furthermore, studies in which Ins(1,4,5)P-3-sensitive sto res were depleted with thapsigargin and cells were challenged in the p resence or absence of extracellular Ca2+ indicated that Ca2+, irrespec tive of its origin (intra- or extracellular), potentiated the Ins(1,4, 5)P-3 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)P-3 responses to carbachol in the presence of Ca2+. This lack of heterologous desensitization may be due to the rapid, full, h omologous desensitization of bradykinin receptors, compared with an in complete homologous desensitization of muscarinic receptors.