Quantitative comparisons of muscarinic and bradykinin receptor‐mediated Ins (1,4,5)P₃ accumulation and Ca²⁺ signalling in human neuroblastoma cells

Abstract

Road, Leicester LEI 9HN 1 Muscarinic and bradykinin receptor-mediated Ins(1,4,5)P3 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)P3 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)P3 accumulation but responses were smaller and not sustained. 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 receptormediated phosphoinositide and Ca24 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.

Differential Regulation of Muscarinic Acetylcholine Receptor-sensitive Polyphosphoinositide Pools and Consequences for Signaling in Human Neuroblastoma Cells

“…7b). This sustained phase of [Ca 2ϩ ] i elevation is dependent upon the influx of extracellular Ca 2ϩ via an as yet undefined mechanism but which most likely involves capacita- tive entry (20). Addition of 1 M thapsigargin to these cells also resulted in capacitative Ca 2ϩ entry and a sustained elevation of [Ca 2ϩ ] i of similar magnitude to that mediated by 1 mM carbachol, but this was unaffected by 10 M wortmannin (data not shown).…”

“…Materials-Reagents of analytical grade were obtained from suppliers listed previously (20,21,24 3 H]phosphoinositides and PtdIns(4,5)P 2 mass, duplicates were pooled at the lipid extraction stage. All data are presented as mean Ϯ S.E.…”

“…8). Desensitization is often reflected in a reduction in agonist potency rather than a reduction in the maximal response, and we therefore conducted an identical series of experiments in which cells were rechallenged with a concentration of carbachol (25 M) equivalent to the EC 50 for the peak Ins(1,4,5)P 3 response in these cells (20). Compared with rechallenge with 1 mM carba- chol, these experiments demonstrated a more prolonged delay in the recovery of the peak Ins(1,4,5)P 3 responses which took greater than 5 min to fully recover and clearly lagged behind the recovery of PtdIns(4,5)P 2 (Fig.…”

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor‐stimulated phosphoinositide turnover

“…Many studies, particularly those in neuronal preparations, have demonstrated that agonist-stimulated PIC activation is facilitated by increases in [Ca2+], (Eberhard & Holz, 1988;del Rio et al, 1994;Wojcikiewicz et al, 1994;Willars & Nahorski, 1995). Facilitation of agoniststimulated PIC activity may be caused by Ca2" mobilization from intracellular stores or Ca2+-entry via a multitude of influx pathways (Simpson et al, 1995).…”

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH‐SY5Y Human Neuroblastoma Cells

“…Inspection of Fig. 3 reveals that at the 12-min time point, a 100% recovery of functional response is elicited by only '--.50% of the original surface receptor number, indicative of a substantial receptor reserve for mobilization of intracellular Ca2~ (Willars and Nahorski, 1995).…”