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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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Saturation binding of [3H]NMS and [3H]Ach. Specific binding of [3H]NMS (circles) and [3H]ACh (squares) to membranes from CHO cells expressing individual subtypes of muscarinic receptors is plotted against the concentration of free radioligand. Binding of radioligand in the absence (closed symbols) and presence of 10 μM (open symbols) or 100 μM (hatched symbols) rapacuronium, respectively. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 1 to data. Binding parameters are summarized in Table 1.
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Figure 1: Saturation binding of [3H]NMS and [3H]Ach. Specific binding of [3H]NMS (circles) and [3H]ACh (squares) to membranes from CHO cells expressing individual subtypes of muscarinic receptors is plotted against the concentration of free radioligand. Binding of radioligand in the absence (closed symbols) and presence of 10 μM (open symbols) or 100 μM (hatched symbols) rapacuronium, respectively. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 1 to data. Binding parameters are summarized in Table 1.

Mentions: Saturation binding experiments (Figure 1; Table 1) with 68 pM to 2 nM [3H]NMS in cell membranes showed similar binding capacity (1 to 2 pmol of binding sites per mg of protein) and affinity (equilibrium dissociation constant (KD) ranging from 205 pM at M4 to 320 pM at M2 receptors) for all receptor subtypes (Figure. 1; Table 1). Significant depletion (up to 34% at M1 for 68 pM [3H]NMS) occurred despite the use of 0.8 ml incubation volume in the binding assays. Thus, free concentrations of [3H]NMS were calculated and used in Eq. 1. Saturation binding experiments with 3.4 nM to 100 nM [3H]ACh showed similar high affinity binding among all subtypes with KD around 20 nM. Rapacuronium concentration dependently decreased affinity for [3H]NMS and [3H]ACh at all subtypes without change in maximum binding capacity (BMAX). Competition experiments of unlabeled ACh vs. [3H]NMS displayed high and low binding sites for ACh at all subtypes with higher proportion of high affinity binding sites at even-numbered subtypes (Figure 2). Equilibrium dissociation constants (KI) of ACh high-affinity binding derived from competition experiments with [3H]NMS (pKD = 7.32 ± 0.06, 7.59 ± 0.03, 7.79 ± 0.05, 7.69 ± 0.04, 7.68 ± 0.05, mean ± SE for M1 to M5 receptor) correspond to those measured in [3H]ACh saturation experiments (Table 1). In the presence of 10 μM GTPγS to uncouple receptors and G-proteins ACh low affinity binding was similar at all five subtypes with equilibrium dissociation constant (KI) ranging from 25.5 μM at M4 to 46.8 μM at M1.


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)

Saturation binding of [3H]NMS and [3H]Ach. Specific binding of [3H]NMS (circles) and [3H]ACh (squares) to membranes from CHO cells expressing individual subtypes of muscarinic receptors is plotted against the concentration of free radioligand. Binding of radioligand in the absence (closed symbols) and presence of 10 μM (open symbols) or 100 μM (hatched symbols) rapacuronium, respectively. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 1 to data. Binding parameters are summarized in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Saturation binding of [3H]NMS and [3H]Ach. Specific binding of [3H]NMS (circles) and [3H]ACh (squares) to membranes from CHO cells expressing individual subtypes of muscarinic receptors is plotted against the concentration of free radioligand. Binding of radioligand in the absence (closed symbols) and presence of 10 μM (open symbols) or 100 μM (hatched symbols) rapacuronium, respectively. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 1 to data. Binding parameters are summarized in Table 1.
Mentions: Saturation binding experiments (Figure 1; Table 1) with 68 pM to 2 nM [3H]NMS in cell membranes showed similar binding capacity (1 to 2 pmol of binding sites per mg of protein) and affinity (equilibrium dissociation constant (KD) ranging from 205 pM at M4 to 320 pM at M2 receptors) for all receptor subtypes (Figure. 1; Table 1). Significant depletion (up to 34% at M1 for 68 pM [3H]NMS) occurred despite the use of 0.8 ml incubation volume in the binding assays. Thus, free concentrations of [3H]NMS were calculated and used in Eq. 1. Saturation binding experiments with 3.4 nM to 100 nM [3H]ACh showed similar high affinity binding among all subtypes with KD around 20 nM. Rapacuronium concentration dependently decreased affinity for [3H]NMS and [3H]ACh at all subtypes without change in maximum binding capacity (BMAX). Competition experiments of unlabeled ACh vs. [3H]NMS displayed high and low binding sites for ACh at all subtypes with higher proportion of high affinity binding sites at even-numbered subtypes (Figure 2). Equilibrium dissociation constants (KI) of ACh high-affinity binding derived from competition experiments with [3H]NMS (pKD = 7.32 ± 0.06, 7.59 ± 0.03, 7.79 ± 0.05, 7.69 ± 0.04, 7.68 ± 0.05, mean ± SE for M1 to M5 receptor) correspond to those measured in [3H]ACh saturation experiments (Table 1). In the presence of 10 μM GTPγS to uncouple receptors and G-proteins ACh low affinity binding was similar at all five subtypes with equilibrium dissociation constant (KI) ranging from 25.5 μM at M4 to 46.8 μM at M1.

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