Interaction of neuromuscular blocking drugs with recombinant human m1–m5 muscarinic receptors expressed in Chinese hamster ovary cells

Abstract

1 Neuromuscular blocking drugs (NMBD's) are known to produce cardiovascular side e ects manifesting as brady/tachycardias. In this study we have examined the interaction of a range of steroidal NMBD's with recombinant human m1 ± m5 muscarinic receptors expressed in Chinese hamster ovary cells. Our main hypothesis is that NMBD's may interact with m2 (cardiac) muscarinic receptors. 2 All binding studies were performed with cell membranes prepared from CHO m1 ± m5 cells in 1 ml volumes of 20 mM HEPES, 1 mM MgCl 2 at pH 7.4 for 1 h. Muscarinic receptors were labelled with [ 3 H]-NMS and displacement studies were performed with pancuronium, vecuronium, pipecuronium, rocuronium and gallamine. In addition a range of muscarinic receptor subtype selective reference compounds were included. In order to determine the nature of any interaction the e ects of pancuronium, rocuronium and vecuronium on methacholine inhibition of forskolin stimulated cyclic AMP formation in CHO m2 cells was examined. Cyclic AMP formation was assessed in whole cells using a radioreceptor assay. All data are mean+s.e.mean (n55). 50 ) by pirenzepine in CHO m1 membranes (7.97+0.04), methoctramine in CHO m2 membranes (8.55+0.1), 4-diphenylacetoxy-Nmethyl piperidine methiodide (4-DAMP) in CHO m3 membranes (9.38+0.03), tropicamide in CHO m4 membranes (6.98+0.01). 4-DAMP, pirenzepine, tropicamide and methoctramine displaced [ 3 H]NMS in CHO m5 membranes with pK 50 values of 9.20+0.14, 6.59+0.04, 6.89+0.05 and 7.22+0.01 respectively. These data con®rm homogenous subtype expression in CHO m1 ± m5 cells. 5 [ 3 H]NMS binding was displaced dose-dependently (pK 50 ) by pancuronium (m1, 6.43+0.12; m2, 7.68+0.02; m3, 6.53+0.06; m4, 6.56+0.03; m5, 5.79+0.10), vecuronium (m1, 6.14+0.04; m2, 6.90+0.05; m3, 6.17+0.04; m4, 7.31+0.02; m5, 6.20+0.07), pipecuronium (m1, 6.34+0.11; m2, 6.58+0.03; m3, 5.94+0.01; m4, 6.60+0.06; m5, 4.80+0.03), rocuronium (m1, 5.42+0.01; m2, 5.40+0.02; m3, 4.34+0.02; m4, 5.02+0.04; m5, 5.10+0.03) and gallamine (m1, 6.83+0.05; m2, 7.67+0.04; m3, 6.06+0.06; m4, 6.20+0.03; m5, 5.34+0.03). 6 Cyclic AMP formation was inhibited dose dependently by methacholine in CHO m2 cells pEC 50 for control and pancuronium (300 nM) treated cells were 6.18+0.34 and 3.57+0.36 respectively. Methacholine dose-response curves in the absence and presence of rocuronium (1 mM) and vecuronium (1 mM) did not di er signi®cantly. Pancuronium, vecuronium and rocuronium did not inhibit cyclic AMP formation alone indicating no agonist activity. 7 With the exception of rocuronium there was a signi®cant interaction with m2 muscarinic receptors with all NMBD's at clinically achievable concentrations suggesting that the brady/tachycardias associated with these agents may result from an interaction with cardiac muscarinic receptors. Furthermore pancuronium at clinically achievable concentrations antagonised methacholine inhibition of cyclic AMP formation in CHO m2 cells further suggesting that the tachycardia produced by this agent results from muscarinic antagonism. The mech...

Control of Glycinergic Input to Spinal Dorsal Horn Neurons by Distinct Muscarinic Receptor Subtypes Revealed Using Knockout Mice

“…We found that in M 2 /M 4 double-KO mice, the overall effect of oxotremorine-M was to increase glycinergic sIPSCs. Our observation that the M 3 subtype-preferring mAChR antagonist 4-DAMP (Cembala et al, 1998;Moriya et al, 1999;Zhang et al, 2005) abolished the potentiating effect of oxotremorine-M on sIPSCs in M 2 /M 4 double-KO mice provides further support for our conclusion that the M 3 subtype contributes to the potentiation of spinal glycine release. In M 2 /M 4 double-KO mice, oxotremorine-M significantly increased the frequency of mIPSCs, but significantly less than it increased the frequency of sIPSCs.…”

“…To confirm the role of the M 3 subtype in the potentiating effect of oxotremorine-M on synaptic glycine release in M 2 /M 4 double-KO mice, we further tested the effect of 5 M oxotremorine-M on glycinergic sIPSCs in the presence of 50 nM 4-DAMP, an M 3 subtype-preferring mAChR antagonist (Cembala et al, 1998;Moriya et al, 1999;Zhang et al, 2005). The potentiating effect of 5 M oxotremorine-M on sIPSCs was completely blocked by subsequent application of 50 nM 4-DAMP in all seven neurons examined (Fig.…”

Ligation of Toll-Like Receptor 3 Differentially Regulates M2 and M3 Muscarinic Receptor Expression and Function in Human Airway Smooth Muscle Cells

“…In untreated cells, cAMP accumulation was significantly reduced by ACh (100 µ M ), and this reduction was abrogated by the M2R antagonist gallamine (10 µ M ). The agent was applied in a concentration selective for M2R over M3R [28]. In poly I:C-treated cells, cAMP accumulation was to a lesser extent attenuated by ACh, and this attenuation was also abrogated by gallamine.…”

“…The responses to ACh, histamine, and LTD 4 in untreated and poly I:C-treated cells were completely blocked by the M3R antagonist 4- DAMP (1 µ M ), the H1R antagonist mepyramine (10 µ M ), and the CysLT1R antagonist MK571 (1 µ M ), respectively. 4-DAMP was applied in a concentration selective for M3R over M2R [28]. Complete inhibition of ACh-mediated calcium responses by 4-DAMP in untreated and poly I:C-treated cells implies that basal and upregulated M3R is functional.…”

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM5)

“…[6] For example, the M 5 mAChR is required for cholinergic dilation of central blood arteries and arterioles. For the pharmacological characterization of this binding site, receptor binding experiments with hM 5 stably expressed in CHO cells were for example, phosphate buffer, [19][20][21] Tris-HCl buffer, [22] Krebs-Henseleit buffer, [23] and HEPES buffer, [24][25][26][27] and beyond the incubation time (1-5 h) and temperature (20-37 C). Exposure to organophosphorus compounds has been associated with down-regulation of M 5 AChR related genes.…”