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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 xanomeline on the accumulation of cyclic AMP.Cells expressing hM2 (left) or hM4 (right) receptors were treated with 10 µM xanomeline for 3 min followed by washing for 10 min or 1 hour (coordinate) prior to 20-min incubation with 5 µM (white bars) or 20 µM (black bars) forskolin. Accumulation of [3H]cAMP (ordinate) is expressed as per cent of control accumulation of [3H]cAMP in xanomeline sham treated cells (corrected for content of protein). Data are means ± S.E.M. from 3 experiments performed in triplicates. *, different from control (sham treated) cells by t-test, P<0.05.
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pone-0088910-g008: Effects of xanomeline on the accumulation of cyclic AMP.Cells expressing hM2 (left) or hM4 (right) receptors were treated with 10 µM xanomeline for 3 min followed by washing for 10 min or 1 hour (coordinate) prior to 20-min incubation with 5 µM (white bars) or 20 µM (black bars) forskolin. Accumulation of [3H]cAMP (ordinate) is expressed as per cent of control accumulation of [3H]cAMP in xanomeline sham treated cells (corrected for content of protein). Data are means ± S.E.M. from 3 experiments performed in triplicates. *, different from control (sham treated) cells by t-test, P<0.05.

Mentions: Accumulation of [3H]cAMP stimulated by 5 or 20 µM forskolin in cells expressing M2 or M4 receptors was measured after treatment of the cells with 10 µM xanomeline for 3 min followed by 10-min or 1-hour washing (Fig. 8). Xanomeline treatment had minimal effects on accumulation of [3H]cAMP in cells expressing M2 receptors under this experimental setup. After 10 min of washing xanomeline slightly (8%) inhibited [3H]cAMP accumulation (stimulated by 20 µM forskolin) but it had no effect on [3H]cAMP accumulation after 1-hour washing. In cells expressing M4 receptors xanomeline inhibited [3H]cAMP accumulation by almost 40% after 10-min washing and by more than 20% after 1-hour washing (Fig. 8).


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 xanomeline on the accumulation of cyclic AMP.Cells expressing hM2 (left) or hM4 (right) receptors were treated with 10 µM xanomeline for 3 min followed by washing for 10 min or 1 hour (coordinate) prior to 20-min incubation with 5 µM (white bars) or 20 µM (black bars) forskolin. Accumulation of [3H]cAMP (ordinate) is expressed as per cent of control accumulation of [3H]cAMP in xanomeline sham treated cells (corrected for content of protein). Data are means ± S.E.M. from 3 experiments performed in triplicates. *, different from control (sham treated) cells by t-test, P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088910-g008: Effects of xanomeline on the accumulation of cyclic AMP.Cells expressing hM2 (left) or hM4 (right) receptors were treated with 10 µM xanomeline for 3 min followed by washing for 10 min or 1 hour (coordinate) prior to 20-min incubation with 5 µM (white bars) or 20 µM (black bars) forskolin. Accumulation of [3H]cAMP (ordinate) is expressed as per cent of control accumulation of [3H]cAMP in xanomeline sham treated cells (corrected for content of protein). Data are means ± S.E.M. from 3 experiments performed in triplicates. *, different from control (sham treated) cells by t-test, P<0.05.
Mentions: Accumulation of [3H]cAMP stimulated by 5 or 20 µM forskolin in cells expressing M2 or M4 receptors was measured after treatment of the cells with 10 µM xanomeline for 3 min followed by 10-min or 1-hour washing (Fig. 8). Xanomeline treatment had minimal effects on accumulation of [3H]cAMP in cells expressing M2 receptors under this experimental setup. After 10 min of washing xanomeline slightly (8%) inhibited [3H]cAMP accumulation (stimulated by 20 µM forskolin) but it had no effect on [3H]cAMP accumulation after 1-hour washing. In cells expressing M4 receptors xanomeline inhibited [3H]cAMP accumulation by almost 40% after 10-min washing and by more than 20% after 1-hour washing (Fig. 8).

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