Lochner M and Thompson AJ (2016), The muscarinic antagonists scopolamine and atro...

XB-ART-52055

Neuropharmacology 2016 Sep 01;108:220-8. doi: 10.1016/j.neuropharm.2016.04.027.

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The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors.

Lochner M , Thompson AJ .

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Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [(3)H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects.

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Species referenced: Xenopus laevis

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	Graphical Abstract
	Fig. 1. Chemical structures of endogenous agonist 5-HT, 5-HT3 receptor antagonists granisetron, tropisetron and SDZ-ICT 322, scopolamine, atropine and the radioligand [3H]N-methylscopolamine. Note that scopolamine is a single enantiomer whereas atropine is a mixture of epimers at the indicated (asterisk) carbon atom.
	Fig. 2. The effect of scopolamine on 5-HT3 receptor currents. (A) Concentration-response curve for 5-HT. (B) Concentration-inhibition of the 2 μM 5-HT response by co-applied scopolamine. The data in 2A are normalised to the maximal peak current response for each oocyte and represented as the mean ± S.E.M. for a series of oocytes. In Fig. 2B, inhibition by scopolamine is shown relative to the peak current response to 2 μM 5-HT alone. For 5-HT curve fitting yielded a pEC50 of 5.65 ± 0.02 (EC50 = 2.24 μM, n = 6) and Hill slope of 2.06 ± 0.14. The pIC50 value for scopolamine was 5.68 ± 0.05 (IC50 = 2.09 μM, n = 6) with a Hill Slope of 1.06 ± 0.05. (C) Sample traces showing the onset (τon) and recovery (τoff) of scopolamine inhibition (grey bar) during a 2 μM 5-HT application (filled bar). (D) Onset of inhibition was well fitted by mono-exponential functions to give kobs (n = 17). A plot of the reciprocal of these time constants versus the scopolamine concentration showed a linear relationship where the slope = kon (2.60 × 104 M−1 s−1) and the y-axis intercept = koff (0.32 s−1).
	Fig. 3. The mechanism of 5-HT3 receptor inhibition by scopolamine. (A) Concentration-response curves were performed in the absence or presence of the indicated concentrations of scopolamine. The curves showed parallel dextral shifts with maximal currents restored by increasing concentrations of 5-HT. Parameters derived from these curves can be seen in Table 1. (B) A Schild plot was created from the dose ratios of the curves shown in 3A and fitted with Eq. (3) to yield a slope of 1.06 ± 0.10 (R2 = 0.97) and a pA2 of 5.03 ± 0.43 (Kb, 9.33 μM).
	Fig. 4. Competition of scopolamine with an established 5-HT3 receptor antagonist. (A) Radioligand binding curves for the competition of 0.6 nM [3H]granisetron and varying concentrations of scopolamine at crude membrane extracts of 5-HT3 receptors from stably expressing HEK 293 cells. Data was normalised to [3H]granisetron binding in the absence of antagonist and fitted with Eq. (10). The curve is representative of 3 similar experiments, which gave an average pKi of 5.17 ± 0.24 (Ki = 6.76 μM, n = 3). (B) Flow cytometry, showing the competition of 10 nM G-FL (a fluorescent derivative of granisetron; Jack et al., 2015) and varying concentrations of scopolamine at 5-HT3 receptors expressed on the surface of live HEK 293 cells. The average pKi of these experiments was similar to values from radioligand competition (5.31 ± 0.09, Ki = 4.90 μM, n = 8).
	Fig. 5. Effects of atropine on the electrophysiological responses to 5-HT and binding of G-FL. (A) Concentration-inhibition of the 2 μM 5-HT response by co-applied atropine. For each oocyte the responses in the presence of antagonist are normalised to the peak current response to 5-HT alone and data represented as the mean ± S.E.M. for a series of oocytes. Curve fitting yielded a pIC50 of 5.76 ± 0.14 (IC50 = 1.74 μM, n = 5) and Hill Slope of 1.06 ± 0.05. (B) Flow cytometry, showing the competition of 10 nM G-FL (a fluorescent derivative of granisetron; Jack et al., 2015) and varying concentrations of atropine at 5-HT3 receptors expressed on the surface of live HEK 293 cells. The affinity (pKi = 5.10 ± 0.16, Ki = 7.94 μM, n = 5) of atropine calculated from these experiments was similar to that measured using electrophysiology.
	Fig. 6. Representative examples of 5-HT3 receptor antagonists (ball-and-stick representation) docked into a 5-HT3 receptor orthosteric binding site model (PDB ID: 2YME; a co-crystal of granisetron bound to a mutant AChBP that contains residues from the 5-HT3 receptor binding site (termed 5HTBP; Kesters et al., 2013) and important binding site residues (stick representation). Principle face (left-hand side, light grey), complementary face (right-hand side, dark grey). (A) 2YME from the side (y-axis) showing the location of granisetron (green) in the orthosteric binding site at the interface of two adjacent subunits. (B) Proposed binding pose for tropisetron (blue) overlaying granisetron (green) from the co-crystal structure 2YME. (C) The proposed binding pose for scopolamine (orange) showing its orientation in the 5-HT3 binding site. (D) A surface representation of 5HTBP bound with granisetron and an overlay of docked scopolamine showing the hydroxyl of the carbonyl linker that, owing to steric constraints, is located within a cavity at the rear of the binding site. It can be seen that while the scopine head of scopolamine (orange) is at the same location as the azabicyclic rings of granisetron (green), the steric bulk, flexibility and presence of a hydroxyl in the linker region results in the aromatic ring being orientated away from loops D and F. (D) In contrast, the proposed binding pose for SDZ-ICT 322 (yellow) is more similar to that of granisetron. For chemical structures of the described ligands see Fig. 1.

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