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

1 Muscarinic and bradykinin receptor-mediated Ins(1,4,5)P-3 accumulati on, Ca2+ mobilization and Ca2+ entry have been examined in human SH-SY 5Y neuroblastoma cells. This has allowed both direct comparison of sig nalling events by two receptor types potentially linked to the same tr ansduction pathway and an investigation of the interactions between th e components of this pathway. 2 Stimulation of muscarinic receptors wi th carbachol produced biphasic accumulations of Ins(1,4,5)P-3 consisti ng of a rapid peak followed by a lower sustained phase. Both phases we re dose-dependent but the potency of elevation at peak was significant ly less than that of the sustained phase. Bradykinin also dose-depende ntly 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 bra dykinin were lowered by an amount approximating this reduction over th e entire dose-response curves. Sustained Ins(1,4,5)P-3 elevation in re sponse to carbachol showed a more marked absolute reduction. Agonist p otencies 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 stimu lation is greatly facilitated by external Ca2+, probably through Ca2entry. 4 The temporal- and dose-dependency of carbachol-mediated Ins(1 ,4,5)P-3 accumulations were unaffected by loading cells with fura-2, t hus 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 ext ernal 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 t o 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 tran sient nature of Ins(1,4,5)P-3 and [Ca2+](i) peaks followed by either l ower but sustained levels with carbachol or a return to basal levels w ith bradykinin suggests rapid but partial desensitization of the musca rinic receptor and complete desensitization of the bradykinin receptor . This indicates receptor-specific desensitization. Further analysis o f this was provided by detecting accumulations of [H-3]-inositol phosp hates ([H-3]-InsPs) in Li+-blocked, myo-[H-3]-inositol labelled cells. Carbachol produced a rapid accumulation over the first minute, follow ed by a slower linear accumulation for at least 29 min. At this point accumulations were dose-related with a potency similar to that of sust ained 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 fra mes will indicate the ability of agonists for predominantly sustained PLC activation, potentially driven by a partially desensitized recepto r, as opposed to rapid activation by a fully sensitized receptor. 8 Th ese data provide quantitative comparisons between and within component s of the receptor-mediated phosphoinositide and Ca2+ signalling pathwa y, provide mechanistic insights into regulation of these components an d characterize a model system in which heterologous interaction betwee n two receptors linked to the same transduction pathway may be examine d.