Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding

Abstract

Background/aims: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect.

Methods: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments.

Results: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent.

Conclusion: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.

Fig. 1

Inhibition of [³H]NMS binding following acute and longterm treatment with agonists in CHO cells expressing the human M3 receptor. a Cells were pretreated with increasing concentrations of carbachol for 1 h followed by washing and used immediately (▴) or after incubation for 23 h in control media (▵). Another group of cells was pretreated with carbachol for 24 h prior to washing (⋄). Subsequently, the specific binding of 0.2 nmol/l [³H]NMS was determined following carbachol pretreatments or using naive cells in the continuous presence of carbachol (▪) 1 h at 37°C. b Cells received the same treatments as above (a), except with pilocarpine instead of carbachol. Values represent the means ± SE of 3 experiments conducted in triplicate.

Fig. 2

Effects of carbachol pretreatment on [³H]NMS saturation binding in CHO cells expressing human M3 receptors. Cells were pretreated with 10 μmol/l carbachol for 1 h followed by washing and incubation in the absence of free carbachol for 23 h (▵) or treated with carbachol for 24 h prior to washing (♦). Subsequently, untreated cells (▪) or pretreated cells were incubated with increasing concentrations of [³H]NMS (0.01–7 nmol/l) for 1 h at 37°C. Non-specific binding was determined in the presence of 10 μmol/l atropine. Values represent the means ± SE of 3 experiments conducted in triplicate.

Fig. 3

Effects of long-term agonist exposure on the ability of various muscarinic agonists to stimulate inositol phosphates production. a Cells were pretreated with 10 μmol/l carbachol for 1 h followed by washing and waiting 23 h in control media (□) or treated for 24 h with carbachol prior to washing (⋄). Subsequently, treated or untreated (▪) cells were stimulated with increasing concentrations of carbachol and the accumulation of inositol phosphates was determined in the presence of 10 mmol/l LiCl over a 1 h incubation period at 37°C. b Cells were treated with pilocarpine similar to above (a), then stimulated with carbachol 10 μmol/l. c Cells were treated with pilocarpine as above (a), then stimulated with pilocarpine. d Cells were treated with carbachol similar to above (a), then stimulated with xanomeline. Values represent the means ± SE of 3–6 experiments conducted in triplicate.

Fig. 4

Comparison of the effects of xanomeline treatment on the specific binding of [³H]QNB and [³H]NMS in CHO cells expressing human M3 receptors. a Cells were pretreated with increasing concentrations of xanomeline for 1 h, washed and used immediately (▴) or after incubation in control medium for 23 h (▵). An additional group of cells was pretreated with xanomeline for 24 h prior to washing (♦). The binding of 1.4 nmol/l [³H]QNB was determined in untreated cells in the concurrent presence of xanomeline (▪) or following various xanomeline pretreatments. b Cells received the same treatments as above (a), except that the binding assay was conducted with 2.9 nmol/l [³H]NMS. Values represent the means ± SE of 3–4 experiments conducted in triplicate.

Fig. 5

Effects of atropine on long-term xanomeline-induced inhibition of [³H]NMS binding in CHO cells expressing human M3 receptors. a Cells were either untreated (▪) or treated with 10 μmol/l xanomeline for 1 h followed by washing and waiting for 23 h (▵). b Cells were treated with 10 μmol/l atropine for 1 h followed by washing and waiting 23 h in control medium (•) or treated simultaneously with atropine and xanomeline for 1 h followed by washing and waiting 23 h in control medium (○). c Cells were treated with atropine for 23 h prior to washing (♦) or with xanomeline for 1 h followed by washing and incubation with atropine for 23 h prior to washing (⋄). In all cases, saturation binding assays were conducted with increasing concentrations of [³H]NMS (0.01–14 nmol/l) for 1 h at 37°C. Values represent the means ± SE of 4 experiments conducted in duplicate.

Fig. 6

Effects of blocking receptor function using siRNA targeted against G_q and G₁₁ G-proteins on long-term xanomeline-induced inhibition of [³H]NMS binding. a Cells were left untreated (lanes 1 and 3) or treated with 50 nmol/l siRNA for G q and G 11 G-proteins (lanes 2 and 4). The level of G_q/11 G-protein expression was determined by Western blot. b Cells were untreated (open bars) or treated with 50 nmol/l siRNA for G_q and G₁₁ G-proteins (solid bars). Inositol phosphate production was determined following stimulation with two concentrations of carbachol (1 or 100 μmol/l) or xanomeline (100 nmol/l or 10 μmol/l). c Cells were treated with xanomeline (10 μmol/l) for 1 h followed by washing and used immediately (▴) or after 23 h incubation in control medium (▴). An additional group of cells was treated with xanomeline for 24 h prior to washing (♦). Subsequently, untreated cells (▪) or pretreated cells were incubated in suspension with increasing concentrations of [³H]NMS (0.01–7.5 nmol/l) for 1 h at 37°C. d Cells were treated with 50 nmol/l siRNA for G_q and G₁₁ G-proteins to inhibit receptor function and then subjected to various xanomeline treatment protocols as above (c). Values represent the means ± SE of 3 experiments conducted in triplicate.

Fig. 7

Effects of atropine on long-term modulation of inositol phosphates production by xanomeline in CHO cells expressing human M3 receptors. a Cells were either untreated (▪) or treated with 10 μmol/l xanomeline for 1 h followed by washing and waiting for 23 h in control medium (▵). b Cells were treated with atropine (10 μmol/l) for 1 h (•) or treated simultaneously with atropine and xanomeline for 1 h (○) followed by washing and waiting 23 h in control medium. c Cells were untreated (♦) or treated with xanomeline for 1 h (⋄) followed by washing and incubation with atropine for 23 h prior to washing. In all cases, the production of inositol phosphates was determined after the various pretreatments in the presence of increasing concentrations of carbachol for 1 h at 37°C in the presence of 10 mmol/l LiCl. Values represent the means ± SE of 3 experiments conducted in triplicate.

Fig. 8

Effects of wash-resistant binding in the absence of receptor activation on the ability of the propyl analog of xanomeline to initiate long-term effects on saturation binding parameters in CHO cells expressing human M3 receptors. Cells were pretreated with 100 μmol/l of the propyl xanomeline analog for 1 h followed by washing and incubation in the absence of free drug for 23 h (▵). An additional group of cells was treated for 24 h with the propyl analog prior to washing (♦). Subsequently, untreated cells (▪) or pretreated cells were incubated with increasing concentrations of [³H]NMS (0.02–15 nmol/l) for 1 h at 37°C. Non-specific binding was determined in the presence of 10 μmol/l atropine. Values represent the means ± SE of 3 experiments conducted in duplicate.

Fig. 9

Effects of blocking receptor activation on the ability of xanomeline to initiate long-term effects on [³H]NMS saturation binding parameters in CHO cells expressing human M3 receptors. Cells were pretreated with 10 μmol/l xanomeline for 1 h at 37°C followed by washing and incubation in the absence of free xanomeline for 23 h at 4°C (▵). Additional groups of cells were treated with 10 μmol/l (♦) or 100 μmol/l (⋄) xanomeline for 24 h at 4°C prior to washing. Subsequently, untreated cells incubated at 4°C (▪) or pretreated cells were incubated with increasing concentrations of [³H]NMS (0.02–16.5 nmol/l) for 1 h at 37°C. Nonspecific binding was determined in the presence of 10 μmol/l atropine. Values represent the means ± SE of 3–4 experiments conducted in duplicate.