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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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Effects of NMS on formation of xanomeline wash-resistant activation at hM1 through hM4 receptors.Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in normalized fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: Control 300 nM carbachol (CBC) was applied for 5 s. Second stimulation: At 5 min receptors were blocked by 10 µM of the antagonist NMS (1 min), then a mixture of 10 µM xanomeline (Xano) and 10 µM NMS was applied for 1 min and then 10 µM NMS was applied for an additional 1 min. Cells were then washed with KHB for additional 3 min. Representative traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.033 at peaks. Results were confirmed in 5 to 7 additional independent experiments. Parameters of xanomeline effects are summarized in Table S7 in File S1.
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pone-0088910-g005: Effects of NMS on formation of xanomeline wash-resistant activation at hM1 through hM4 receptors.Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in normalized fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: Control 300 nM carbachol (CBC) was applied for 5 s. Second stimulation: At 5 min receptors were blocked by 10 µM of the antagonist NMS (1 min), then a mixture of 10 µM xanomeline (Xano) and 10 µM NMS was applied for 1 min and then 10 µM NMS was applied for an additional 1 min. Cells were then washed with KHB for additional 3 min. Representative traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.033 at peaks. Results were confirmed in 5 to 7 additional independent experiments. Parameters of xanomeline effects are summarized in Table S7 in File S1.

Mentions: In another set of experiments the effects of the antagonist NMS on the formation of xanomeline wash-resistant receptor activation were investigated. Five mins after 5-s control stimulation with 300 nM carbachol, cells were superfused for 3 min with 10 µM NMS. Xanomeline was applied for 1 min at 10 µM (together with NMS) during the second min of NMS superfusion (Fig. 5, Table S7 in File S1).


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)

Effects of NMS on formation of xanomeline wash-resistant activation at hM1 through hM4 receptors.Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in normalized fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: Control 300 nM carbachol (CBC) was applied for 5 s. Second stimulation: At 5 min receptors were blocked by 10 µM of the antagonist NMS (1 min), then a mixture of 10 µM xanomeline (Xano) and 10 µM NMS was applied for 1 min and then 10 µM NMS was applied for an additional 1 min. Cells were then washed with KHB for additional 3 min. Representative traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.033 at peaks. Results were confirmed in 5 to 7 additional independent experiments. Parameters of xanomeline effects are summarized in Table S7 in File S1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088910-g005: Effects of NMS on formation of xanomeline wash-resistant activation at hM1 through hM4 receptors.Changes in the concentration of intracellular calcium (ordinate) are expressed as changes in normalized fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. First (control) stimulation: Control 300 nM carbachol (CBC) was applied for 5 s. Second stimulation: At 5 min receptors were blocked by 10 µM of the antagonist NMS (1 min), then a mixture of 10 µM xanomeline (Xano) and 10 µM NMS was applied for 1 min and then 10 µM NMS was applied for an additional 1 min. Cells were then washed with KHB for additional 3 min. Representative traces are averages of 8 to 12 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.015 at the base line to ±0.033 at peaks. Results were confirmed in 5 to 7 additional independent experiments. Parameters of xanomeline effects are summarized in Table S7 in File S1.
Mentions: In another set of experiments the effects of the antagonist NMS on the formation of xanomeline wash-resistant receptor activation were investigated. Five mins after 5-s control stimulation with 300 nM carbachol, cells were superfused for 3 min with 10 µM NMS. Xanomeline was applied for 1 min at 10 µM (together with NMS) during the second min of NMS superfusion (Fig. 5, Table S7 in File S1).

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