Arch Toxicol (1992) 66:204-210

Archives of

Toxicologj 9 Springer-Verlag 1992

Mechanism of soman-induced contractions in canine tracheal smooth muscle* Michael Adler, David H. Moore, and Margaret G. Filbert NeurotoxicologyBranch, PathophysiologyDivision, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA Received 27 June 1991/Accepted 7 October 1991

Abstract. The actions of the irreversible organophosphorus cholinesterase (ChE) inhibitor soman were investigated on canine tracheal smooth muscle in vitro. Concentrations of soman > 1 nM increased the amplitude and decay of contractions elicited by electric field stimulation. The effect on decay showed a marked dependence on stimulation frequency, undergoing a 2.4-fold increase between 3 and 60 Hz. Soman also potentiated tensions due to bath applied acetylcholine (ACh). Little or no potentiation was observed for contractions elicited by carbamylcholine, an agonist that is not hydrolyzed by ChE. Concentration of soman _ 3 nM led to the appearance of sustained contractures. These contractures developed with a delayed onset and were well correlated with ChE activity. Alkylation of muscarinic receptors by propylbenzilylcholine mustard antagonized the actions of soman on both spontaneous and electrically-evoked muscle contractions. The results are consistent with a mechanism in which the toxic actions of soman are mediated by accumulation of neurally-released ACh secondary to inhibition of ChE activity. An important factor in this accumulation is suggested to be the buffering effect of the muscarinic receptors on the efflux of ACh from the neuroeffector junction.

t The term ChE as used here designates the combined activities of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8). This convention was adopted since in canine trachealis, butyrylcholinesterase constitutes an appreciable fraction of the total ChE activity and appears to participate in ACh hydrolysis. (Adler and Filbert 1990) * The opinions or assertions contained herein are the private views of the authors and are not to be construed as official views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals" as adopted and promulgated by the National Institutes of Health Offprint requests to: Dr. Michael Adler, Neurotoxicology Branch, USAMRICD, APG, MD 21010, USA

Key words: Tracheal smooth muscle - Cholinesterase inhibitors phosphate

Muscarinic receptor -

Soman -

Organo-

Introduction Exposure to organophosphorus cholinesterase 1 (ChE)inhibitors results in symptoms indicative of widespread overstimulation of both central and peripheral acetylcholine (ACh) receptors (Taylor 1990). These symptoms generally include bronchoconstriction, increases in tracheobronchial secretions, lacrimation, muscle fasciculations, weakness, convulsions, loss of consciousness and death (Ho and Hoskins 1987). Although the predominant signs of anti-ChE toxicity are mediated by persistent activation of muscarinic receptors, conceptual models for anti-ChE actions have been derived primarily from studies on skeletal muscle nicotinic receptors. From their findings that neostigmine prolonged the decay of the endplate potential and current but did not alter open channel lifetimes, Katz and Miledi (1973) suggested that prolongation of the macroscopic responses arose from multiple transmitter-receptor interactions. Each interaction delayed the diffusion of ACh by a duration equivalent to the lifetime of the transmitter-receptor complex, which in skeletal muscle is approximately 1 ms. At the neuromuscular junction, a key factor for the increased synaptic residence time of ACh following ChE inhibition is the high ACh receptor density (MatthewsBellinger and Salpeter 1978). This high density promotes large numbers of encounters between transmitter and receptor molecules. Other factors that appear to contribute to ACh persistence are close apposition and extensive overlap of the pre- and postsynaptic membranes (Eccles and Jaeger 1958). Inhibition of ChE in tracheal smooth muscle leads to alterations that are even more pronounced than those en-

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Fig. 1. Effect of soman on contractions elicited by 10 Hz EFS pulses for 20 s. The symbols represent the mean ___ SE of values obtained from 9 strips in 3 animals. The curves are arbitrary polynomial fits. The inset shows EFS tensions from one strip during the intial 20 min of soman addition; stimulations are indicated by dots

Fig. 2. Effect of 1 nM soman on ChE activities. Intact trachealis strips (n = 12) were exposed to soman for 6.4, 60 or 240 min. Tissues were then homogenized and assayed for total ChE activity, b Differs significantly from mean activity at 6.4 min. c Differs significantly from mean activity at 6.4 and 60 min

countered in s k e l e t a l m u s c l e . C o m p l e t e i n h i b i t i o n o f C h E prolongs t h e r e l a x a t i o n o f c o n t r a c t i o n s e v o k e d b y e l e c t r i c field s t i m u l a t i o n ( E F S ) b y o v e r 3 0 - f o l d and l e a d s to a l a r g e sustained c o n t r a c t u r e ( A a s et al. 1986; A d l e r et al. 1987; Adler and F i l b e r t 1990). The p a r a t r a c h e a l n e u r o e f f e c t o r j u n c t i o n l a c k s t h e h i g h receptor d e n s i t y a n d t h e m o r p h o l o g i c a l f e a t u r e s that r e n d e r the v e r t e b r a t e n e u r o m u s c u l a r j u n c t i o n s u s c e p t i b l e to C h E inhibitors ( S u z u k i et al. 1976; B a s b a u m et al. 1984), C o n sequently, it is n o t c l e a r w h e t h e r the b u f f e r e d t r a n s m i t t e r diffusion m o d e l is v a l i d f o r this s y n a p s e . T h e p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m i n e t h e b a s i s f o r the u n d e r l y i n g sensitivity o f c a n i n e t r a c h e a l s m o o t h m u s c l e to a n t i - C h E agents u s i n g s o m a n as a m o d e l C h E i n h i b i t o r .

pulse parameters of 50 V and 1 ms. Frequency-response curves were obtained over the range 0.3 - 6 0 Hz.

Materials a n d m e t h o d s Materials. ACh chloride and carbamylcholine chloride (carbachol) were purchased from the Sigma Chemical Co, St Louis, MO. []4C]-ACh was obtained from Dupont NEN Research Products, Boston, MA. Propylbenzilylcholine mustard (PrBCM) was synthesized by Ash, Stevens Co., Detroit Research Park, MI. Soman and VX were provided by the US Army CRDEC, APG, MD.

Isometriccontractions. Tracheae were excised from adult mongrel dogs under pentobarbital anesthesia (30 mg/kg i.v.), freed of mucosa and surface connective tissue and cut into rectangular strips approximately 2 mm wide, 1 mm thick and 10 mm long, weighing 23-86 mg (mean +SE = 52.9 ___3.7 mg). The strips were placed in 10 ml tissue chambers and attached by silk sutures to Grass FT 0.03 force-displacement transducers, the outputs of which were connected to an 8 channel Gould polygraph and a Honeywell 101 FM tape recorder. The tissues were bathed in Tyrode's solution of the following composition (mM): NaC1, 137; KC1, 2.7; CaC12, 1.8; MgC12, 0.5; NaH2PO4, 0.3; NaHCO3, 11.9 and glucose, 5.6 (pH 7.3-7.4). The solution was maintained at 37~ and bubbled with 95% 02/5% CO2. Preparations were equilibrated for 6 0 90 min until they sustained a resting tension of 4 g without decrement. Most experiments were repeated on bronchial smooth muscle with similar results. For EFS, current was passed between two platinum electrodes, one in physical contact with the muscle and the other located 2 cm above. Pulse trains of 20 s duration were delivered by a Grass S-88 stimulator using

Muscarinic receptor alkylation. Muscarinic receptors were progressively alkylated by incubating muscles in 0.1 laM PrBCM for periods of 3 46 min followed by a 60 rain wash to remove unbound inhibitor. ACh concentration-response relationships were determined before and after PrBCM treatment and their ratios were used to estimate the fraction of functional (nonalkylated) receptors according to the method of Furchgott (1966). Soman ( 1/aM) was then added to inhibit ChE completely and the resulting contracture and response to 10 Hz EFS pulses were recorded.

ChE assay. ChE activity was determined by the radiometric method of Siakotos et al. (1969). The tissues were homogenized in phosphate buffered saline (pH 7.4, 10% w/v) and incubated for 10 min at 37~ with 1 mM [14C]-ACh (specific activity 1 laCi/mmol). At the end of the incubation period, unhydrolyzed substrate was removed by the addition of Amberlite CG- 102 suspended in dioxane. After centrifugation, the supernatant was transferred to a vial containing scintillation cocktail for monitoring of radioactivity. Protein was measured by the bicinchoninic technique of Smith et al. (1985) using bovine serum as a standard. Statistical analysis. Analysis of data was performed using a one way analysis of variance combined with the Newman-Keuls test (Tallarida and Murray 1987). P values