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Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors.

Jakubík J, Randáková A, El-Fakahany EE, Dolezal V - BMC Pharmacol. (2009)

Bottom Line: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors.Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions.These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. jakubik@biomed.cas.cz

ABSTRACT

Background: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results: At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 microM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPgammaS binding by rapacuronium.

Conclusions: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 microM) but not at high concentrations (10 microM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction.

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Effects of rapacuronium on ACh-induced [35S]GTPγS binding to membranes. Membranes from CHO cells expressing individual subtypes of muscarinic receptors or nontransfected CHO-K1 cells were preincubated for 60 min with buffer (closed symbols) or with rapacuronium (open symbols). [35S]GTPγS binding was induced by rapacuronium alone (closed squares), ACh alone (closed circles), or by ACh in the presence of rapacuronium (0.1 μM (open circles), 1 μM (open squares), or 10 μM (open triangles)). Data are expressed as fold increase of basal [35S]GTPγS binding and are presented as means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 5 to data. Parameters are summarized in Tables 4 and 5.
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Figure 6: Effects of rapacuronium on ACh-induced [35S]GTPγS binding to membranes. Membranes from CHO cells expressing individual subtypes of muscarinic receptors or nontransfected CHO-K1 cells were preincubated for 60 min with buffer (closed symbols) or with rapacuronium (open symbols). [35S]GTPγS binding was induced by rapacuronium alone (closed squares), ACh alone (closed circles), or by ACh in the presence of rapacuronium (0.1 μM (open circles), 1 μM (open squares), or 10 μM (open triangles)). Data are expressed as fold increase of basal [35S]GTPγS binding and are presented as means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 5 to data. Parameters are summarized in Tables 4 and 5.

Mentions: Rapacuronium alone concentration dependently lowered [35S]GTPγS binding to membranes (Figure 6, closed squares; Table 4) with a maximal effect of approximately 25% at odd-numbered subtypes and 15% at even-numbered subtypes, with similar half-effective concentrations (EC50) ranging from 28 μM at M2 receptors to 76 μM at M3 receptors. While the EC50 values of rapacuronium in inhibiting [35S]GTPγS binding at individual subtypes correlated with affinities measured in binding experiments with [3H]ACh (R2 = 0.76) they were lower (4- to 12-fold) at all subtypes. We could not test the involvement of muscarinic receptors in the effects of rapacuronium on [35S]GTPγS binding using orthosteric antagonists, since 10 μM NMS or 10 μM atropine by themselves decreased [35S]GTPγS binding by more than 30% at all receptor subtypes. Inhibitory effects of rapacuronium were not additive to those of NMS or atropine (not shown). However, rapacuronium did not decrease [35S]GTPγS binding in membranes from nontransfected CHO cells (Figure 6, bottom row right).


Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors.

Jakubík J, Randáková A, El-Fakahany EE, Dolezal V - BMC Pharmacol. (2009)

Effects of rapacuronium on ACh-induced [35S]GTPγS binding to membranes. Membranes from CHO cells expressing individual subtypes of muscarinic receptors or nontransfected CHO-K1 cells were preincubated for 60 min with buffer (closed symbols) or with rapacuronium (open symbols). [35S]GTPγS binding was induced by rapacuronium alone (closed squares), ACh alone (closed circles), or by ACh in the presence of rapacuronium (0.1 μM (open circles), 1 μM (open squares), or 10 μM (open triangles)). Data are expressed as fold increase of basal [35S]GTPγS binding and are presented as means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 5 to data. Parameters are summarized in Tables 4 and 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806265&req=5

Figure 6: Effects of rapacuronium on ACh-induced [35S]GTPγS binding to membranes. Membranes from CHO cells expressing individual subtypes of muscarinic receptors or nontransfected CHO-K1 cells were preincubated for 60 min with buffer (closed symbols) or with rapacuronium (open symbols). [35S]GTPγS binding was induced by rapacuronium alone (closed squares), ACh alone (closed circles), or by ACh in the presence of rapacuronium (0.1 μM (open circles), 1 μM (open squares), or 10 μM (open triangles)). Data are expressed as fold increase of basal [35S]GTPγS binding and are presented as means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 5 to data. Parameters are summarized in Tables 4 and 5.
Mentions: Rapacuronium alone concentration dependently lowered [35S]GTPγS binding to membranes (Figure 6, closed squares; Table 4) with a maximal effect of approximately 25% at odd-numbered subtypes and 15% at even-numbered subtypes, with similar half-effective concentrations (EC50) ranging from 28 μM at M2 receptors to 76 μM at M3 receptors. While the EC50 values of rapacuronium in inhibiting [35S]GTPγS binding at individual subtypes correlated with affinities measured in binding experiments with [3H]ACh (R2 = 0.76) they were lower (4- to 12-fold) at all subtypes. We could not test the involvement of muscarinic receptors in the effects of rapacuronium on [35S]GTPγS binding using orthosteric antagonists, since 10 μM NMS or 10 μM atropine by themselves decreased [35S]GTPγS binding by more than 30% at all receptor subtypes. Inhibitory effects of rapacuronium were not additive to those of NMS or atropine (not shown). However, rapacuronium did not decrease [35S]GTPγS binding in membranes from nontransfected CHO cells (Figure 6, bottom row right).

Bottom Line: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors.Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions.These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. jakubik@biomed.cas.cz

ABSTRACT

Background: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results: At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 microM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPgammaS binding by rapacuronium.

Conclusions: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 microM) but not at high concentrations (10 microM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction.

Show MeSH
Related in: MedlinePlus