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Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Šantrůčková E, Doležal V, El-Fakahany EE, Jakubík J - PLoS ONE (2014)

Bottom Line: Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes.However long-term receptor activation takes place in full only at hM1 and hM4 receptors.These findings suggest the existence of particular activation mechanisms specific to these two receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, United States of America.

ABSTRACT
Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

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Concentration response to acute treatment with xanomeline.Cells were seeded, handled and loaded with Fura-2 as described in Methods. After an initial 10-s period cells were stimulated with 300 nM carbachol (CBC) for 5 s, washed with KHB for 5 min, then stimulated with 0.1 (black), 1 (red) or 10 µM (green) xanomeline (Xano) for 20 s and washed with KBH for 7 min. Traces are averages from 10 to 12 cells from representative experiment confirmed by 3 independent experiments. Signal variation (SD) among cells ranges from ±0.019 at the base line to ±0.035 at peaks. Parameters of calcium response are summarized in Table S2 in File S1. Calculated pEC50 and EMAX of response to xanomeline are in Table 1.
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pone-0088910-g001: Concentration response to acute treatment with xanomeline.Cells were seeded, handled and loaded with Fura-2 as described in Methods. After an initial 10-s period cells were stimulated with 300 nM carbachol (CBC) for 5 s, washed with KHB for 5 min, then stimulated with 0.1 (black), 1 (red) or 10 µM (green) xanomeline (Xano) for 20 s and washed with KBH for 7 min. Traces are averages from 10 to 12 cells from representative experiment confirmed by 3 independent experiments. Signal variation (SD) among cells ranges from ±0.019 at the base line to ±0.035 at peaks. Parameters of calcium response are summarized in Table S2 in File S1. Calculated pEC50 and EMAX of response to xanomeline are in Table 1.

Mentions: Brief exposure (20 s) to xanomeline elicited a transient increase in intracellular calcium level (Fig. 1). At hM2, hM3 and hM5 receptors intracellular calcium level returned to basal but remained elevated at hM1 and hM4 receptors (Fig. 1). EMAX effect elicited by 10 µM xanomeline was close to the maximal at all subtypes (Table S2 in File S1). Xanomeline had the same potency at all five receptor subtypes (Table 1). However, there was marked difference in xanomeline EMAX among receptor subtypes. Calculated EMAX is highest at hM1 and lowest at hM5 receptors (Table 1). Order of EMAX values taken as per cent of full agonists carbachol EMAX is M1>M4 = M3>M5>M2 and ranges from 90% to 44%. In control experiments (Fig. S1 in File S1) selectivity in efficacy of agonists oxotremorine and pilocarpine was much smaller and ranged from 56% at hM2 to 73% at hM5 to and from 52% at hM2 to 66% at hM5, respectively (Fig. S2 in File S1, Table 1). The order of apparent affinity constants of G-protein for agonist-receptor complex (KG) based on membrane expression level (Table 2) and calculated according Eq. 3 was M1>M4>M3>M5>M3 (Table 1).


Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Šantrůčková E, Doležal V, El-Fakahany EE, Jakubík J - PLoS ONE (2014)

Concentration response to acute treatment with xanomeline.Cells were seeded, handled and loaded with Fura-2 as described in Methods. After an initial 10-s period cells were stimulated with 300 nM carbachol (CBC) for 5 s, washed with KHB for 5 min, then stimulated with 0.1 (black), 1 (red) or 10 µM (green) xanomeline (Xano) for 20 s and washed with KBH for 7 min. Traces are averages from 10 to 12 cells from representative experiment confirmed by 3 independent experiments. Signal variation (SD) among cells ranges from ±0.019 at the base line to ±0.035 at peaks. Parameters of calcium response are summarized in Table S2 in File S1. Calculated pEC50 and EMAX of response to xanomeline are in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928307&req=5

pone-0088910-g001: Concentration response to acute treatment with xanomeline.Cells were seeded, handled and loaded with Fura-2 as described in Methods. After an initial 10-s period cells were stimulated with 300 nM carbachol (CBC) for 5 s, washed with KHB for 5 min, then stimulated with 0.1 (black), 1 (red) or 10 µM (green) xanomeline (Xano) for 20 s and washed with KBH for 7 min. Traces are averages from 10 to 12 cells from representative experiment confirmed by 3 independent experiments. Signal variation (SD) among cells ranges from ±0.019 at the base line to ±0.035 at peaks. Parameters of calcium response are summarized in Table S2 in File S1. Calculated pEC50 and EMAX of response to xanomeline are in Table 1.
Mentions: Brief exposure (20 s) to xanomeline elicited a transient increase in intracellular calcium level (Fig. 1). At hM2, hM3 and hM5 receptors intracellular calcium level returned to basal but remained elevated at hM1 and hM4 receptors (Fig. 1). EMAX effect elicited by 10 µM xanomeline was close to the maximal at all subtypes (Table S2 in File S1). Xanomeline had the same potency at all five receptor subtypes (Table 1). However, there was marked difference in xanomeline EMAX among receptor subtypes. Calculated EMAX is highest at hM1 and lowest at hM5 receptors (Table 1). Order of EMAX values taken as per cent of full agonists carbachol EMAX is M1>M4 = M3>M5>M2 and ranges from 90% to 44%. In control experiments (Fig. S1 in File S1) selectivity in efficacy of agonists oxotremorine and pilocarpine was much smaller and ranged from 56% at hM2 to 73% at hM5 to and from 52% at hM2 to 66% at hM5, respectively (Fig. S2 in File S1, Table 1). The order of apparent affinity constants of G-protein for agonist-receptor complex (KG) based on membrane expression level (Table 2) and calculated according Eq. 3 was M1>M4>M3>M5>M3 (Table 1).

Bottom Line: Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes.However long-term receptor activation takes place in full only at hM1 and hM4 receptors.These findings suggest the existence of particular activation mechanisms specific to these two receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, United States of America.

ABSTRACT
Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Show MeSH
Related in: MedlinePlus