QUANTITATIVE COMPARISONS OF MUSCARINIC AND BRADYKININ RECEPTOR-MEDIATED INS (1,4,5)P-3 ACCUMULATION AND CA2+ SIGNALING IN HUMAN NEUROBLASTOMA-CELLS

1 Muscarinic and bradykinin receptor-mediated Ins(1,4,5)P-3 accumulation, Ca2+ mobilization and Ca2+ entry have been examined in human SH-SY5Y neuroblastoma cells. This has allowed both direct comparison of signalling events by two receptor types potentially linked to the same transduction pathway and an investigation of the interactions between the components of this pathway. 2 Stimulation of muscarinic receptors with carbachol produced biphasic accumulations of Ins(1,4,5)P-3 consisting of a rapid peak followed by a lower sustained phase. Both phases were dose-dependent but the potency of elevation at peak was significantly less than that of the sustained phase. Bradykinin also dose-dependently stimulated Ins(1,4,5)P-3 accumulation but responses were smaller and not sustained. 3 Lowering of [Ca2+](e) reduced basal Ins(1,4,5)P-3 levels. Peak Ins(1,4,5)P-3 elevation in response to carbachol and bradykinin were lowered by an amount approximating this reduction over the entire dose-response curves. Sustained Ins(1,4,5)P-3 elevation in response to carbachol showed a more marked absolute reduction. Agonist potencies were unaffected by lowering [Ca2+](i). Thus, a consistent but small 21 amount of PLC activity during rapid activation appears to be sensitive to lowered[Ca2+](e), whilst activity during sustained stimulation is greatly facilitated by external Ca2+, probably through Ca2+ entry. 4 The temporal- and dose-dependency of carbachol-mediated Ins(1,4,5)P-3 accumulations were unaffected by loading cells with fura-2, thus allowing direct comparison of Ins(1,4,5)P-3 and [Ca2+](i) changes monitored by fura-2. 5 Changes in [Ca2+](i) by both agonists revealed temporal patterns that were accumulations. Only carbachol stimulated a marked sustained [Ca2+](i) signal and this was fully dependent on external Ca2+. 6. All agonist-mediated [Ca2+](i) elevations occurred with significantly greater potency than that of the respective Ins(1,4,5)P-3 accumulations. Further examination of peak elevations in response to carbachol indicated that this was independent of Ca2+ entry. Thus, a major site for amplification of the potency of rapid agonist-mediated responses lies at the level of the Ins(1,4,5)P-3 receptor. 7 The transient nature of Ins(1,4,5)P-3 and [Ca2+](i) peaks followed by either lower but sustained levels with carbachol or a return to basal levels with bradykinin suggests rapid but partial desensitization of the muscarinic receptor and complete desensitization of the bradykinin receptor. This indicates receptor-specific desensitization. Further analysis of this was provided by detecting accumulations of [H-3]-inositol phosphates ([H-3]-InsPs) in Li+-blocked, myo-[H-3]-inositol labelled cells. Carbachol produced a rapid accumulation over the first minute, followed by a slower linear accumulation for at least 29 min. At this point accumulations were dose-related with a potency similar to that of sustained Ins(1,4,5)P-3 accumulation. However, bradykinin produced a minor accumulation of [H-3]-InsPs, maximal by 1 min. Thus, analysis of PLC activation by measurement of [H-3]-InsPs over relatively long time frames will indicate the ability of agonists for predominantly sustained PLC activation, potentially driven by a partially desensitized receptor, as opposed to rapid activation by a fully sensitized receptor. 8 These data provide quantitative comparisons between and within components of the receptor-mediated phosphoinositide and Ca2+ signalling pathway, provide mechanistic insights into regulation of these components and characterize a model system in which heterologous interaction between two receptors linked to the same transduction pathway may be examined.