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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 long-term application of classic agonists on the time-course of changes in intracellular calcium concentration in CHO cells expressing individual subtypes of muscarinic receptors.The time-course of changes in intracellular calcium concentration (abscissa) after stimulation of hM1 to hM5 muscarinic receptor subtypes with the agonists carbachol (CBC), oxotremorine and pilocarpine was measured as described in Methods. First stimulation: After 10 s of initial (resting) period 300 nM carbachol was applied for 10 s and then washed. Second stimulation: Three min after the first stimulation either 1 µM carbachol (black curve) or 1 µM oxotremorine (red curve) or 3 µM pilocarpine was applied for 1 hour followed by 30-min washing. Third stimulation: After washing following the second stimulation 300 nM carbachol was applied for 10 s followed by washing. Intracellular calcium concentration (ordinate) is expressed as fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. Representative traces are averages of 12 to 16 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.018 at the base line to ±0.067 at peaks. Results were confirmed in 2 additional independent experiments. Parameters of agonist effects are summarized in Table S5 in File S1.
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pone-0088910-g003: Effects of long-term application of classic agonists on the time-course of changes in intracellular calcium concentration in CHO cells expressing individual subtypes of muscarinic receptors.The time-course of changes in intracellular calcium concentration (abscissa) after stimulation of hM1 to hM5 muscarinic receptor subtypes with the agonists carbachol (CBC), oxotremorine and pilocarpine was measured as described in Methods. First stimulation: After 10 s of initial (resting) period 300 nM carbachol was applied for 10 s and then washed. Second stimulation: Three min after the first stimulation either 1 µM carbachol (black curve) or 1 µM oxotremorine (red curve) or 3 µM pilocarpine was applied for 1 hour followed by 30-min washing. Third stimulation: After washing following the second stimulation 300 nM carbachol was applied for 10 s followed by washing. Intracellular calcium concentration (ordinate) is expressed as fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. Representative traces are averages of 12 to 16 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.018 at the base line to ±0.067 at peaks. Results were confirmed in 2 additional independent experiments. Parameters of agonist effects are summarized in Table S5 in File S1.

Mentions: In microflourometric experiments measuring effects of long exposure to the agonists carbachol, oxotremorine and pilocarpine on the level of intracellular calcium (Fig. 3) CHO cells expressing individual subtypes of muscarinic receptors were exposed to 1 µM carbachol, 1 µM oxotremorine or 3 µM pilocarpine for 1 hour. Intracellular calcium levels were measured during agonist exposure and following 30-min of continuous superfusion with KHB. Control 5-s stimulation with 300 nM carbachol was done before agonist application and at the end of measurements.


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 long-term application of classic agonists on the time-course of changes in intracellular calcium concentration in CHO cells expressing individual subtypes of muscarinic receptors.The time-course of changes in intracellular calcium concentration (abscissa) after stimulation of hM1 to hM5 muscarinic receptor subtypes with the agonists carbachol (CBC), oxotremorine and pilocarpine was measured as described in Methods. First stimulation: After 10 s of initial (resting) period 300 nM carbachol was applied for 10 s and then washed. Second stimulation: Three min after the first stimulation either 1 µM carbachol (black curve) or 1 µM oxotremorine (red curve) or 3 µM pilocarpine was applied for 1 hour followed by 30-min washing. Third stimulation: After washing following the second stimulation 300 nM carbachol was applied for 10 s followed by washing. Intracellular calcium concentration (ordinate) is expressed as fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. Representative traces are averages of 12 to 16 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.018 at the base line to ±0.067 at peaks. Results were confirmed in 2 additional independent experiments. Parameters of agonist effects are summarized in Table S5 in File S1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088910-g003: Effects of long-term application of classic agonists on the time-course of changes in intracellular calcium concentration in CHO cells expressing individual subtypes of muscarinic receptors.The time-course of changes in intracellular calcium concentration (abscissa) after stimulation of hM1 to hM5 muscarinic receptor subtypes with the agonists carbachol (CBC), oxotremorine and pilocarpine was measured as described in Methods. First stimulation: After 10 s of initial (resting) period 300 nM carbachol was applied for 10 s and then washed. Second stimulation: Three min after the first stimulation either 1 µM carbachol (black curve) or 1 µM oxotremorine (red curve) or 3 µM pilocarpine was applied for 1 hour followed by 30-min washing. Third stimulation: After washing following the second stimulation 300 nM carbachol was applied for 10 s followed by washing. Intracellular calcium concentration (ordinate) is expressed as fluorescence intensity (340 nm/380 nm) ratio normalized to basal calcium level. Representative traces are averages of 12 to 16 best responding cells from one experiment. Signal variation (SD) among cells ranges from ±0.018 at the base line to ±0.067 at peaks. Results were confirmed in 2 additional independent experiments. Parameters of agonist effects are summarized in Table S5 in File S1.
Mentions: In microflourometric experiments measuring effects of long exposure to the agonists carbachol, oxotremorine and pilocarpine on the level of intracellular calcium (Fig. 3) CHO cells expressing individual subtypes of muscarinic receptors were exposed to 1 µM carbachol, 1 µM oxotremorine or 3 µM pilocarpine for 1 hour. Intracellular calcium levels were measured during agonist exposure and following 30-min of continuous superfusion with KHB. Control 5-s stimulation with 300 nM carbachol was done before agonist application and at the end of measurements.

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