INTERACTION OF NEUROMUSCULAR BLOCKING-DRUGS WITH RECOMBINANT HUMAN M1-M5 MUSCARINIC RECEPTORS EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

I Neuromuscular blocking drugs (NMBD's) are known to produce cardiovas cular side effects manifesting as brady/tachycardias. In this study we have examined the interaction of a range of steroidal NMBD's with rec ombinant 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 w ith cell membranes prepared from CHO m1 - m5 cells in I mi volumes of 20 mM HEPES, 1 mM MgCl2 at pH 7.4 for 1 h. Muscarinic receptors were l abelled with [H-3]-NMS and displacement studies were performed with pa ncuronium, vecuronium, pipecuronium, rocuronium and gallamine. In addi tion a range of muscarinic receptor subtype selective reference compou nds were included. In order to determine the nature of any interaction the effects of pancuronium, rocuronium and vecuronium on methacholine inhibition of forskolin stimulated cyclic AMP formation in CHO m2 cel ls was examined. Cyclic AMP formation was assessed in whole cells usin g a radioreceptor assay. All data are mean +/- s.e.mean (n greater tha n or equal to 5). 3 The binding of [H-3]-NMS was dose-dependent and sa turable in all cells tested. B-max and K-d values in m1-m5 cells were 2242+/-75, 165+/-13, 1877+/-33, 458+/-30 127+/-2 fmol mg(-1) protein a nd 0.11+/-0.02, 0.15+/-0.01, 0.12+/-0.01 0.12+/-0.01, 0.22+/-0.01 nM r espectively. 4 The binding of [H-3]-NMS was displaced dose dependently (PK50) by pirenzepine in CHO mi membranes (7.97+/-0.04), methoctramin e in CHO m2 membranes (8.55+/-0.1), 4-diphenylacetoxy-N-methyl piperid ine methiodide (4-DAMP) in CHO m3 membranes (9.38+/-0.03), tropicamide in CHO m4 membranes (6.98+/-0.01). 4-DAMP, pirenzepine, tropicamide a nd methoctramine displaced [H-3]NMS in CHO m5 membranes with PK50 valu es of 9.20+/-0.14, 6.59+/-0.04, 6.89+/-0.05 and 7.22+/-0.01 respective ly. These data confirm homogenous subtype expression in CHO m1-m5 cell s. 5 [H-3]NMS binding was displaced dose-dependently (pK(50)) by pancu ronium (ml, 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 (ml, 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 (ml, 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 (ml, 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 (ml, 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 i n CHO m2 cells pEC(50) for control and pancuronium (300 nM) treated ce lls were 6.18+/-0.34 and 3.57+/-0.36 respectively. Methacholine dose-r esponse curves in the absence and presence of rocuronium (1 mu M) and vecuronium (1 mu M) did not differ significantly. Pancuronium, vecuron ium and rocuronium did not inhibit cyclic AMP formation alone indicati ng no agonist activity. 7 With the exception of rocuronium there was a significant interaction with m2 muscarinic receptors with all NMBD's at clinically achievable concentrations suggesting that the brady/tach ycardias associated with these agents may result from an interaction w ith cardiac muscarinic receptors. Furthermore pancuronium at clinicall y achievable concentrations antagonised methacholine inhibition of cyc lic AMP formation in CHO m2 cells further suggesting that the tachycar dia produced by this agent results from muscarinic antagonism. The mec hanism of the bradycardia produced by vecuronium is unclear.