Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

Abstract

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

Formyl Peptide Receptor Signaling in HL-60 Cells through Sphingosine Kinase

“…Assay of PLC-Measurement of PLC-catalyzed inositol phosphate formation was performed as described in detail previously (19). In brief, after labeling for 48 h with myo-[ 3 H]inositol (2.5 Ci/ml) in inositol-free medium, differentiated HL-60 cells were equilibrated for 10 min at 37°C in Hanks' balanced salt solution/bovine serum albumin with or without the indicated agents.…”

“…In brief, after labeling for 48 h with myo-[ 3 H]inositol (2.5 Ci/ml) in inositol-free medium, differentiated HL-60 cells were equilibrated for 10 min at 37°C in Hanks' balanced salt solution/bovine serum albumin with or without the indicated agents. Cells were then incubated with 10 M fMLP at 37°C in the presence of 10 mM LiCl for 20 s and 10 min to measure formation of [ 3 H]inositol 1,4,5-trisphosphate (IP 3 ) and total [ 3 H]inositol phosphates, respectively, as reported before (19).…”

G Protein-coupled Receptor-induced Sensitization of Phospholipase C Stimulation by Receptor Tyrosine Kinases

“…Thereafter, the cells were incubated for 2 min at 37°C in Hanks' balanced salt solution with and without the indicated receptor agonist in the absence of LiCl, followed by thorough washout of the agonist and further incubation of the cells for 30 min or the indicated periods of time without agonist as reported before (8,9). Then the adherent cells were incubated for 10 min at 37°C with 10 mM LiCl in Hanks' balanced salt solution, immediately followed by the addition of stimulatory agents in the presence of 10 mM LiCl to measure the formation of total [ 3 H]inositol phosphates (usually for 30 min at 37°C) as described before (16). To study the effects of cycloheximide, Gö 6976, and BAPTA/AM on PLC stimulation, the cells were pretreated for 60 min (cycloheximide) or 30 min (Gö 6976, BAPTA/AM) with these agents or their solvent, dimethyl sulfoxide (0.1 or 0.2%).…”

“…Since the GPCRs that induced sensitization of PLC stimulation also markedly increase cytosolic Ca 2ϩ concentration in HEK-293 cells (16,25), we finally examined whether this increase is involved in sensitization of PLC stimulation. For this, the cells were treated before carbachol treatment with the intracellular Ca 2ϩ chelator, BAPTA/AM (20 M, 30 min), which completely prevented the agonist-induced increase in cytosolic Ca 2ϩ concentration (data not shown).…”

Signaling Diversity Mediated by Muscarinic Acetylcholine Receptor Subtypes and Evidence for Functional Selectivity

“…For example, M 1 /M 3 /M 5 mACh receptors were also reported to positively or negatively modulate adenylyl cyclase (20,21) and to regulate phospholipases A 2 and D via mechanisms apparently independent of phospholipase C (22,23) . Similarly, it was reported that M 2 /M 4 mACh receptors can stimulate phospholipase C activity via mechanisms involving either pertussis toxin-sensitive or insensitive G proteins (24,25) . Although some of these early reports were subsequently shown to be brought about by indirect mechanisms (e.g., activation of adenylate cyclase might be brought about indirectly through the M 1 /M 3 /M 5 mACh receptor-G q/11 protein-phospholipase C proximal signaling pathway resulting in increases in cytoplasmic Ca 2+ and the modulation of Ca…”