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...

The precise cellular target site(s) for intravenous anaesthetic induction agents remains to be determined, but a reduction in neurotransmission is believed to be important [l]. We have examined the effects of the induction agent 2,6 diisopropylphenol (propofol) on neuronal stimulus secretion coupling. Specifically what are the effects of propofol on the activity of adenylyl cyclase and phospholipase C and noradrenaline uptake and release. All studies were performed with whole SH-SY5Y human neuroblastoma cells in Krebs/HEPES buffer pH 7.4 at 37OC. Measurements of CAMP and Ins(1,4,5)P3 mass formation were made in suspensions of cells as described previously [ Hlnoradrenaline was measured in confluent monolayers of cells grown in 12 well plates. Cglls were incubated with -50nM [ Hlnoradrenaline in Krebs buffer further supplemented with pargyline (0.2mM) and ascorbic acid (0.2mM) for up to 60mins. For release studies, after a fixed 60min loading period, the monolayers were washed 3xlmlxl5mins with fresh buffer. The cells were then challenged with Kt (lOOmM, Na+ adjusted) or cgrbachol (CCh, 1mM) for 3mins. Released [3H]noradrenaline in the buffer was quan ified by liquid scintillation the monolayer was extracted with perchloric acid (0.4M) for a further 30mins and counted. Release is expressed as % of content [41. Propofol stock was made in DMSO at 1OOmM. At 100pM propofol the DMSO amounted to 0.1% and was used in all controls. Where stimulated cAMP/Ins(l,4,5)P3 formation and evoked release were studied, propofol was preincubated for 15mins. All data are expressed as meaniSEM of at least 3 experiments. [3r31 Uptake and release of spectroscopy. [ s Hlnoradrenaline remaining in Figure 1. Effect of propoffl (100pM) and imipramine (10pM) on [ Hlnoradrenaline uptake. *p

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