Cholinergic modulation of the Ca2+ response to bradykinin in neuroblastoma cells

Abstract

Fura 2 imaging was used to measure intracellular Ca2+ signals in N1E-115 mouse neuroblastoma cells during combined activation of bradykinin (BK) and cholinergic receptors. BK and carbachol (CCh) both activate phospholipase C (PLC) and cause Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores. The Ca2+ signal in response to CCh is prolonged by the activation of Ca2+ influx, but BK does not appear to activate the influx pathway. When BK and CCh are applied together (BK+CCh), the Ca2+ response is composed of both Ca2+ release and Ca2+ influx. Ca2+ influx is also activated by BK+CCh in a subset of cells that does not respond with a intracellular Ca2+ concentration increase when CCh is presented by itself. This suggests that CCh stimulates a Ca(2+)-silent cholinergic receptor that is not coupled to Ca2+ release but acts synergistically with BK receptors to activate Ca2+ influx. Pertussis toxin reduces influx without affecting release, indicating that the G protein that modulates the influx pathway is different from the G protein responsible for activating PLC. Cholinergic stimulation also causes progressive heterologous desensitization of BK-evoked Ca2+ release. Desensitization has the unique property of continuing to develop after the cholinergic agonist is removed and the cholinergic Ca2+ response has fully recovered. Heterologous desensitization is not the result of Ca2+ store depletion or a long-lasting inhibition of PLC or IP3-dependent Ca2+ release. Instead, it appears to involve an early step in the BK-signaling cascade, possibly at the level of the B2 receptor or associated G proteins.