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The toxic effect of an AChE-inhibitor on the cholinergic nervous system in airway smooth muscle
Toxicology, 49 (1988) 91--97 Elsevier Scientific Publishers Ireland Ltd.
T H E TOXIC E F F E C T OF A N ACHE-INHIBITOR ON T H E CHOLINERGIC NERVOUS SYSTEM IN AIRWAY SMOOTH MUSCLE
PAL AAS
Norwegian Defence Research Establishmen~ Division for Environmental Toxicology, P.O. Box 25, N.~O07 Kyeller {Norway/
SUMMARY
Excessive cholinergic stimulation of presynaptic muscarinic cholinergic receptors, due to complete inhibition of acetylcholinesterase (ACHE) by O(1,2,2-trimethylpropyl)-methyl-phosphonofluoridate (soman), reduced the release of acetylcholine (ACh) from cholinergic nerves in rat bronchi by almost 250/0. Furthermore, long-term (40 h) exposure by inhalation of soman (0.45--0.63 mg/m8) reduced the contraction of bronchi induced by ACh by approximately 700/0. This is probably due to reduction of the number of muscarinic cholinergic receptors, since there was a reduction in the binding capacity (Bm~) of [3H]QNB by 40%, without any changes in the dissociation constant (Kd).
Key words: Bronchi; Acetylcholine; Soman INTRODUCTON
The toxic effect of a large number of organophosphorus compounds is due to their inhibition of acetylcholinesterase (ACHE) activity. This enzyme, being responsible for the rapid inactivation of the neurotransmitter ACh, plays an important role in the regulation of cholinergic neurotransmission. The presence of cholinergic synapses both in the peripheral nervous system and in the central nervous system explains the characteristic symptoms of an organophosphate intoxication. The toxic symptoms following high acute intoxication include bronchoconstriction, increased bronchial secretion, salivation, lacrimation, muscle fasciculations, generalized tremor, excessive urination, defecation and finally death. Following exposure to low doses, the effects are mainly manifested in the autonomic nervous system. They include miosis, hypersalivation and bradycardia [1]. Previous studies have shown that the organophosphorus compound soman has a substantial effect on bronchial smooth muscle [2--4]. Inhibition of 0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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AChE-activity by soman exposure in vitro or by inhalation exposure increases the synaptic concentration of ACh and thereby stimulation of muscarinic cholinergic receptors with subsequent contraction of the bronchial smooth muscle. The primary objective of this study was to study both the presynaptic and post-synaptic effects of the AChE-inhibitor soman in the cholinergic nervous system in rat bronchi. MATERIAL AND METHODS
Animals Male Wistar rats (200--250 g) (from MOllegaard, Copenhagen) were used throughout the study. Chemicals Acetylcholine chloride and atropine sulphate were purchased from Norsk Medisinaldepot (Oslo, Norway). L-[Benzilic-4,4-3H]quinuclidinyl benzilate (1184.0 GBq/mmol), [methyl-3H]choline chloride (2960 GBq/mmol) and [114C]acetylcholine chloride (18.1 GBq/mmol) were purchased from New England Nuclear Corporation (Boston, MA). Soman (O-[1,2,2-trimethylpropyl]methylphosphonofluoridate) was synthesized in this laboratory and assessed to be more than 99% pure by nuclear magnetic resonance spectroscopy. All other chemicals were of analytical laboratory reagent grade. Physiological methods To determine contraction of bronchial smooth muscle the left and right bronchi were mounted in parallel as circular preparations as previously described [5]. Determination of [SH]A Ch release After decapitation and dissection the 2 main bronchi were opened ventrally and cut into small pieces (approx. 1 mg wet wt). Prior to superfusion, the tissue was incubated at 25°C for 60 min in 1.1 ~M [SH]choline chloride (370 GB/mmol). The pieces of bronchial smooth muscle tissue (4 x 1 mg wet wt) were stimulated by high potassium (51 mM), using a superfusion technique previously described by Aas and Fonnum [6]. Binding assay The airways (trachea from larynx and primary bronchi) were rapidly removed and transferred to a 50 mM sodium-potassium phosphate buffer (pH = 7.4, 4°C) according to the method described previously [3]. The tissue was homogenized (5%), centrifuged (50000 g, 15 rain, 4°C) and rehomogenized twice before the final pellets were resuspended in sodium-potassium phosphate buffer and used in the binding experiments. Binding assay was performed using a modification of the method of Yamamura and Snyder [7]. The homogenate was incubated for 60 rain at 25°C in sodium-potassium
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phosphate buffer in a total volume of 500 /~1 containing 0.004-12.8 nM [SH]QNB in the presence and absence of 6 ~M atropine [3].
Determination of cholinesterase activity The cholinesterase activity was determined by the radiochemical method of Sterri and Fonnum [8].
Inhalation method For receptor binding studies and measurement of muscle contraction rats were exposed to the acetylcholinesterase inhibitor soman in the dynamic inhalation instrument previously described by Aas et al. [9]. The experiments were carried out as whole body exposures to low concentrations of soman which did not induce any symptoms of poisoning.
Analysis o/ data The binding c o n s t a n t s (Bmax and K d) were determined from the experimental data by non-linear regression analysis. Means and standard error of the mean (S.E.M.) were calculated for all data. The Student's t-test was applied to the results to determine significant differences between groups of data. RESULTS The release of [~H]ACh from cholinergic nerves in rat bronchi was dependent of the potassium concentration [4]. After 15 rain exposure to soman (1.0 and 100 /~I) in vitro by superfusion the AChE-activity in rat bronchi was completely inhibited. In the presence of soman (1.0 pM) the potassium (51 raM) evoked release of [3H]ACh was reduced by 23.1 _+ 7.9% to 64.0 _+ 6.4°/0 and in the presence of 100 i~I soman by 34.4 _+ 4.7% to 52.7 _+ 4.40/0 relative to control (Table I). There was no significant difference (P ~> 0.05) between potassium evoked release of [3H]ACh with 1.0 or 100 /~M soman present. Following washout with the low-potassium (4 mM) medium for 40 min potassium (51 mM) evoked release of [SH]ACh was still substantially reduced (23.8 _+ 15.3 and 32.7 _+ 14.6% respectively), although soman was not present (Table I). As shown in Table I, the effect of soman (1.0 /~M) on high potassium (51 raM) evoked release of [SH]ACh was antagonised by scopolamine (0.3 /~M). Scopolamine enhanced the potassium evoked release by 63.9 _+ 10.4°/0 relative to a control stimulation in the absence of soman. Rats exposed to a low concentration (0.45--0.63 mg/m 8) of soman by inhalation showed no symptoms of organophosphate intoxication during the 40 h of sub-acute exposure. Bronchi from rats exposed to soman by inhalation showed a substantial change in muscle contraction by an alteration in the concentration-response curves and an increase in the apparent affinity (pD2) to ACh (Table II). The increase in pD 2 correlated well with the inhibition of acetylcholinesterase
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TABLE I THE IN VITRO EFFECT OF SOMAN ON THE POTASSIUM EVOKED RELEASE OF [SH]ACh FROM CHOLINERGIC NERVE TERMINALS IN RAT BRONCHI Experiment
Stimulation I
II
A
100
87.1 ± 4.7....
B C
100
64.0 ± 6.4*
100 100
52.7 __. 4.4* 151.0 ± 9.3*
D
III 76.3 52.5 43.6 72.1
n
__. 13.8.... __ 6.5* ±
4.9*
± 5.2....
12 21 12 19
Experiment A: Three subsequent control stimulations with high potassium (51 mM). Stimulations were repeated with 45-min interval. Experiment B: Soman (1.0 ~M) was present 15 rain before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The AChE-activity was inhibited completely by exposure to soman (1.0 ~M) for 15 rain. Experiment C: Soman (100 i~I) was present 15 rain before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The AChE-activity was inhibited completely by exposure to soman (100 ~M) for 15 min. Experiment D: Soman (1.0 ~M) and scopolamine (0.3 ~M) were present 15 min before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The responses are given in percent ___ S.E.M. of the control stimulation with potassium (51 raM, stimulation I), and corrected for the unavoidable decline in 3H release with successive stimulations. Stimulation I and III are control stimulations with potassium (51 raM) only. The actual amount of ACh released in a control experiment during potassium (51 raM) stimulation is 72.2 -+ 8.10 pmol • mg protein -1 with a spontaneous release of 41.2 _+ 6.0 pmol •mg protein -1 (Ref. 6). *P < 0.01, "'P > 0.05.
(ACHE) a c t i v i t y in t h e r a t b r o n c h i w h i c h w a s s u b s t a n t i a l l y i n h i b i t e d b y i n h a l a t i o n of s o m a n (Table II). The maximal contraction induced by ACh was reduced substantially after s u b - a c u t e (40 h, 1080 m g m i n / m 3) e x p o s u r e t o s o m a n r e l a t i v e t o c o n t r o l . A s s h o w n in T a b l e I I t h e m a x i m a l c o n t r a c t i o n i n d u c e d b y A C h w a s r e d u c e d b y 70o/o. C o n c o m i t a n t l y t h e A C h E a c t i v i t y w a s r e d u c e d b y 950/o. N e i t h e r a t r o p i n e (7 ~M) n o r t h e s m o o t h m u s c l e r e l a x a n t p a p a v e r i n e (4 mM) h a d a n y effect on an u n s t i m u l a t e d b r o n c h i a l p r e p a r a t i o n , b u t t h e y r e l a x e d t o t a l l y t h e ACh stimulation preparations. I n a s e c o n d s e r i e s of e x p e r i m e n t s r a t s w e r e e x p o s e d to s i m i l a r c o n c e n t r a t i o n s of s o m a n b y s u b - a c u t e i n h a l a t i o n t o c o m p a r e t h e p h y s i o l o g i c a l e f f e c t s w i t h r e c e p t o r b i n d i n g . I n b r o n c h i f r o m c o n t r o l r a t s [3H]QNB b i n d i n g w a s s a t u r a b l e w i t h i n c r e a s i n g c o n c e n t r a t i o n s a n d half m a x i m a l b i n d i n g in t h e b r o n c h i o c c u r r e d at a p p r o x i m a t e l y 0.3 nM. T h e e x p e r i m e n t a l r e s u l t s s h o w t h a t t h e b i n d i n g c a p a c i t y (Bmax) in r a t b r o n c h i w a s a p p r o x i m a t e l y 200 f m o l / m g p r o t e i n [3]. F o l l o w i n g e x p o s u r e (40 h) to s o m a n (0.63 m g / m 3) w i t h a t o t a l d o s e of 1518 m g m i n / m 3 t h e r e w a s a 40°/o r e d u c t i o n in t h e b i n d i n g c a p a c i t y (Bmax) of [3H]QNB in r a t b r o n c h i (Table II). I n s p i t e of a l a r g e r e d u c t i o n in t h e specific b i n d i n g of [3H]QNB no c h a n g e s w e r e o b s e r v e d in t h e non-specific b i n d i n g . D u r i n g t h e s e e x p e r i m e n t s t h e A C h E - a c t i v i t y w a s r e d u c e d t o 50/o of c o n t r o l (Table II). 94
TABLE II THE EFFECT OF SUB-ACUTE EXPOSURE TO SOMAN BY INHALATION ON RAT BRONCHI Soman concentration
AChE (%)
pDz
Maximal contraction
B (%)
Kd (nM)
0
100
1.0 (5)
100
0.45 ± 0.08 (8) 0.63 ± 0.01 (6)
4.2 ± 1.0 (8)** 5.1 ± 1.3 (6)**
3.8 ± 0.3 (5) 5.9 ± 0.1 (8)**
0.3 ± 0.05 (8)**
-
0.16 ± 0.04 (5) --
--
-
58.6 ± 8.9 (5)*
0.13 _+ 0.03 (5)~*
( m g / m s)
Rats were exposed to soman by inhalation for 40 h. The atmospheric concentration of soman was 0.45 ± 0.08 and 0.63 ± 0.01 mg/ma and total dose 1080.0 ± 281 and 1518.7 ± 40 mg/min per m 8, respectively. The acetylcholinesterase (ACHE) activities and the binding capacity ( B ) are given in per cent of control. The control activity of AChE was 789 ± 94 nmol • hr -1 mg protein-1 and B u 177.8 ± 18.0 fmol •mg protein-1. The binding constants ( B and K d (dissociation constant)) were calculated from saturation binding curves. The apparent affinity (pD2) and maximal contraction to ACh in bronchi are shown. The results are mean ± S.E.M. of the number of experiments shown in brackets. **P < 0.01, *P < 0.05, n,.p ~ 0.05. The data are extracted from Ref. 3.
DISCUSSION These results show that the organophosphorus compound soman reduces t h e r e l e a s e o f A C h f r o m c h o l i n e r g i c n e r v e s in b r o n c h i . T h i s is d u e t o s t i m u l a t i o n o f m u s c a r i n i c r e c e p t o r s on t h e c h o l i n e r g i c n e r v e t e r m i n a l s f o l l o w i n g a n i n c r e a s e in t h e c o n c e n t r a t i o n of A C h a f t e r i n h i b i t i o n of A C h E w i t h s o m a n . F u r t h e r m o r e , t h e r e s u l t s a l s o d e m o n s t r a t e d o w n - r e g u l a t i o n of m u s c a r i n i c r e c e p t o r s in b r o n c h i a f t e r s u b - a c u t e i n h a l a t i o n e x p o s u r e t o s o m a n . I t is t h e r e f o r e e v i d e n c e f o r b o t h p r e s y n a p t i c a n d p o s t - s y n a p t i c a d a p t a t i o n in t h e c h o l i n e r g i c n e r v o u s s y s t e m in r e s p o n s e t o t h e t o x i c e f f e c t s of s o m a n . T h e f e e d b a c k r e g u l a t i o n of A C h r e l e a s e m a y b e o n e of s e v e r a l physiological mechanisms whereby neurotransmission is m o d u l a t e d [10]. W h e t h e r t h i s m o d u l a t i o n is of a n y p h y s i o l o g i c a l i m p o r t a n c e is s t i l l n o t c l e a r . During exposure to organophosphorus compounds, which inhibit ACHEa c t i v i t y , t h e r e is a s u b s t a n t i a l e l e v a t i o n of t h e A C h c o n c e n t r a t i o n a n d a s u b s e q u e n t i n c r e a s e in t h e c o n t r a c t i o n of t h e a i r w a y s m o o t h m u s c l e [2]. S t u d i e s h a v e s h o w n t h a t s t i m u l a t i o n of p r e s y n a p t i c m u s c a r i n i c r e c e p t o r s in t h e g u i n e a - p i g i l e u m [11,12] a n d in t h e a i r w a y s [6] a l s o r e d u c e t h e r e l e a s e of A C h a n d t h a t t h e r e l e a s e is e n h a n c e d b y m u s c a r i n i c a n t a g o n i s t s ( T a b l e I) [4,6,12,13]. T h e r e f o r e , t h e p r e s e n c e of m u s c a r i n i c a u t o r e c e p t o r s on t h e cholinergic nerve terminals may protect against organophosphate intoxication. 95
T h e o t h e r a d a p t a t i o n in t h e cholinergic n e r v o u s s y s t e m in bronchi to high c o n c e n t r a t i o n s of t h e A C h E - i n h i b i t o r s o m a n w a s t h e r e d u c t i o n in t h e physiological r e s p o n s e to A C h due to r e d u c t i o n in t h e n u m b e r (Bm~x) of m u s c a r i n i c r e c e p t o r s (Table II). T h e bronchial s m o o t h m u s c l e f r o m r a t s e x p o s e d to s o m a n b y sub-acute e x p o s u r e r e s p o n d e d to c o n t r a c t i o n induced b y cholinergic s t i m u l a t i o n at l o w e r c o n c e n t r a t i o n s of A C h t h a n b r o n c h i f r o m control r a t s . A s s h o w n in T a b l e II, t h e r e w a s a significant i n c r e a s e in t h e a p p a r e n t affinity (pD 2) to ACh. This is due to t h e s u b s t a n t i a l inhibition (85O/o) of A C h E - a c t i v i t y (Table II) and is in a g r e e m e n t w i t h a p r e v i o u s r e p o r t w h e r e in v i t r o e x p o s u r e of r a t bronchi to s o m a n (10 nM, A C h E - a c t i v i t y = 35O/o of control) i n c r e a s e d t h e a p p a r e n t affinity to A C h f r o m 3.7 to 5.3 [2]. Following sub-acute e x p o s u r e to s o m a n a significant r e d u c t i o n in m a x i m a l muscle c o n t r a c t i o n induced b y cholinergic s t i m u l a t i o n w a s o b s e r v e d (Table II). T h e a l t e r a t i o n in t h e muscle c o n t r a c t i o n w a s c o m p a r e d w i t h c h a n g e s in a n t a g o n i s t binding. T h e a l t e r a t i o n of t h e n u m b e r of m u s c a r i n i c r e c e p t o r s in t h e b r o n c h i w a s 40o/o (Table II). T h e d a t a p r e s e n t e d a r e c o n s i s t e n t with r e p o r t e d d e c r e a s e s in [3H]QNB binding in s e v e r a l t i s s u e s f r o m m a m m a l s sub-chronically t r e a t e d w i t h d i i s o p r o p y l p h o s p h o r o - f l u o r i d a t e (DFP) b y injections [14,15]. T h e r e s u l t s t h e r e f o r e indicate t h a t t h e d e c r e a s e d r e s p o n s i v e n e s s of t h e bronchial s m o o t h m u s c l e to cholinergic s t i m u l a t i o n is due to a r e d u c t i o n in t h e n u m b e r of m u s c a r i n i c r e c e p t o r s . T h e d o w n - r e g u l a t i o n of r e c e p t o r s is a r e s u l t of t h e e n h a n c e d s t i m u l a t i o n b y A C h following inhibition of A C h E - a c t i v i t y b y soman. In conclusion, t h e results have shown that exposure to t h e o r g a n o p h o s p h o r u s c o m p o u n d s o m a n induce m o d u l a t i o n of A C h - r e l e a s e and d o w n - r e g u l a t i o n of m u s c a r i n i c r e c e p t o r s in r a t bronchi. REFERENCES 1
K.P. Dubois, in G.B. Koelle (Ed.), Handbueh der experimentellen Pharmacologie, SpringerVerlag, Berlin, 1963, p. 833. 2 P. Aas, T. Veiteberg and F. Fonnum, In vitro effects of soman on bronchial smooth muscle. Bioehem. Pharmacol., 35, 11 (1986) 1793. 3 P. Aas, T. Veiteberg and F. Fonnum, Acute and sub-acute inhalation of an organophosphate induced alteration of cholinergic muscarinic receptors. Biochem. Pharmacol., 36, 8 (1987) 1261. 4 P. Aas, T. Malmei and F. Fonnum, The effect of soman on potassium evoked [3H]acetyleholine release. Pharmaeol. Toxicol., 60 (1987) 206. 5 P. Aas and K.B. Helle, Neurotensin receptors in the rat bronchi. Regulatory Peptides 3 (1982) 405. 6 P. Aas and F. Fonnum, Presynaptic inhibition of acetylcholine release. Acta Physiol. Scand., 127 (1986) 335. 7 H.I. Yamamura and S. Snyder, Muscarinic cholinergic binding in rat brain. Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 1725. 8 S.H. Sterri and F. Fonnum, Isolation of organic anions by extraction with lipid anion exchangers and its application to micromethods for acetylcholinesterases and 4aminobutyrate aminotransferase. Eur. J. Biochem., 91 (1978) 215. 9 P. Aas, S.H. Sterri, H.P. Hjermstad and F. Fonnum, A method for generating toxic vapours of soman. Toxicity of soman by inhalation in rats. Toxicol. Appl. Pharmacol., 80 (1985) 437. 96
10
11
12
13
14
15
E.S. Vizi, Auto-modulation of transmitter/modulator release (negative and positive feedback modulation), in non-synaptic interactions between neurons: Modulation of neurochemical transmission. J. Wiley and Sons Ltd, New York, p. 126, 1984. H. Kilbinger, R. Kruel and I. Wessler, Modulation by oxotremorine and atropine of acetylcholine release evoked by electrical stimulation of the myenteric plexus of the guineapig ileum. Naunyn-Schmiedeberg's Arch. Pharmacol., 300 (1977) 145. P. Alberts, T. Bartfal and L. Stj~rne, The effects of atropine on [3H]acetylcholine secretion from guinea-pig myenteric plexus evoked electrically or by high potassium. J. Physiol., 329 (1982) 93. H. Kilbinger, R. Kruel and I. Wessler, Modulation by scopolamine, acetylcholine and choline of the evoked release of acetylcholine from guinea-pig myenteric plexus: evidence for a muscarinic feedback inhibition of acetylcholine secretion, in G. Pepeu and H. Ladinsky (Eds.), Cholinergic Mechanisms. Plenum Press, London pp. 169, 1981. F.J. Ehlert, N.K. Kokka and A.S. Fairhurst, Muscarinic receptor subsensitivity in the longitudinal muscle of the rat ileum following chronic anticholinesterase treatment with diisopropylfluoropbosphate. Biochem. Pharmacol., 29 (1980) 1391. F.J. Ehlert, N.K. Kokka and A.S. Fairhurst, Altered [SH]QuinuclidinylBenzilate Binding in the striatum of rats following chronic cholinesterase inhibition with diisopropylfluorophosphate. Mol. Pharmacol., 17 (1980) 24.
97
T H E TOXIC E F F E C T OF A N ACHE-INHIBITOR ON T H E CHOLINERGIC NERVOUS SYSTEM IN AIRWAY SMOOTH MUSCLE
PAL AAS
Norwegian Defence Research Establishmen~ Division for Environmental Toxicology, P.O. Box 25, N.~O07 Kyeller {Norway/
SUMMARY
Excessive cholinergic stimulation of presynaptic muscarinic cholinergic receptors, due to complete inhibition of acetylcholinesterase (ACHE) by O(1,2,2-trimethylpropyl)-methyl-phosphonofluoridate (soman), reduced the release of acetylcholine (ACh) from cholinergic nerves in rat bronchi by almost 250/0. Furthermore, long-term (40 h) exposure by inhalation of soman (0.45--0.63 mg/m8) reduced the contraction of bronchi induced by ACh by approximately 700/0. This is probably due to reduction of the number of muscarinic cholinergic receptors, since there was a reduction in the binding capacity (Bm~) of [3H]QNB by 40%, without any changes in the dissociation constant (Kd).
Key words: Bronchi; Acetylcholine; Soman INTRODUCTON
The toxic effect of a large number of organophosphorus compounds is due to their inhibition of acetylcholinesterase (ACHE) activity. This enzyme, being responsible for the rapid inactivation of the neurotransmitter ACh, plays an important role in the regulation of cholinergic neurotransmission. The presence of cholinergic synapses both in the peripheral nervous system and in the central nervous system explains the characteristic symptoms of an organophosphate intoxication. The toxic symptoms following high acute intoxication include bronchoconstriction, increased bronchial secretion, salivation, lacrimation, muscle fasciculations, generalized tremor, excessive urination, defecation and finally death. Following exposure to low doses, the effects are mainly manifested in the autonomic nervous system. They include miosis, hypersalivation and bradycardia [1]. Previous studies have shown that the organophosphorus compound soman has a substantial effect on bronchial smooth muscle [2--4]. Inhibition of 0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
91
AChE-activity by soman exposure in vitro or by inhalation exposure increases the synaptic concentration of ACh and thereby stimulation of muscarinic cholinergic receptors with subsequent contraction of the bronchial smooth muscle. The primary objective of this study was to study both the presynaptic and post-synaptic effects of the AChE-inhibitor soman in the cholinergic nervous system in rat bronchi. MATERIAL AND METHODS
Animals Male Wistar rats (200--250 g) (from MOllegaard, Copenhagen) were used throughout the study. Chemicals Acetylcholine chloride and atropine sulphate were purchased from Norsk Medisinaldepot (Oslo, Norway). L-[Benzilic-4,4-3H]quinuclidinyl benzilate (1184.0 GBq/mmol), [methyl-3H]choline chloride (2960 GBq/mmol) and [114C]acetylcholine chloride (18.1 GBq/mmol) were purchased from New England Nuclear Corporation (Boston, MA). Soman (O-[1,2,2-trimethylpropyl]methylphosphonofluoridate) was synthesized in this laboratory and assessed to be more than 99% pure by nuclear magnetic resonance spectroscopy. All other chemicals were of analytical laboratory reagent grade. Physiological methods To determine contraction of bronchial smooth muscle the left and right bronchi were mounted in parallel as circular preparations as previously described [5]. Determination of [SH]A Ch release After decapitation and dissection the 2 main bronchi were opened ventrally and cut into small pieces (approx. 1 mg wet wt). Prior to superfusion, the tissue was incubated at 25°C for 60 min in 1.1 ~M [SH]choline chloride (370 GB/mmol). The pieces of bronchial smooth muscle tissue (4 x 1 mg wet wt) were stimulated by high potassium (51 mM), using a superfusion technique previously described by Aas and Fonnum [6]. Binding assay The airways (trachea from larynx and primary bronchi) were rapidly removed and transferred to a 50 mM sodium-potassium phosphate buffer (pH = 7.4, 4°C) according to the method described previously [3]. The tissue was homogenized (5%), centrifuged (50000 g, 15 rain, 4°C) and rehomogenized twice before the final pellets were resuspended in sodium-potassium phosphate buffer and used in the binding experiments. Binding assay was performed using a modification of the method of Yamamura and Snyder [7]. The homogenate was incubated for 60 rain at 25°C in sodium-potassium
92
phosphate buffer in a total volume of 500 /~1 containing 0.004-12.8 nM [SH]QNB in the presence and absence of 6 ~M atropine [3].
Determination of cholinesterase activity The cholinesterase activity was determined by the radiochemical method of Sterri and Fonnum [8].
Inhalation method For receptor binding studies and measurement of muscle contraction rats were exposed to the acetylcholinesterase inhibitor soman in the dynamic inhalation instrument previously described by Aas et al. [9]. The experiments were carried out as whole body exposures to low concentrations of soman which did not induce any symptoms of poisoning.
Analysis o/ data The binding c o n s t a n t s (Bmax and K d) were determined from the experimental data by non-linear regression analysis. Means and standard error of the mean (S.E.M.) were calculated for all data. The Student's t-test was applied to the results to determine significant differences between groups of data. RESULTS The release of [~H]ACh from cholinergic nerves in rat bronchi was dependent of the potassium concentration [4]. After 15 rain exposure to soman (1.0 and 100 /~I) in vitro by superfusion the AChE-activity in rat bronchi was completely inhibited. In the presence of soman (1.0 pM) the potassium (51 raM) evoked release of [3H]ACh was reduced by 23.1 _+ 7.9% to 64.0 _+ 6.4°/0 and in the presence of 100 i~I soman by 34.4 _+ 4.7% to 52.7 _+ 4.40/0 relative to control (Table I). There was no significant difference (P ~> 0.05) between potassium evoked release of [3H]ACh with 1.0 or 100 /~M soman present. Following washout with the low-potassium (4 mM) medium for 40 min potassium (51 mM) evoked release of [SH]ACh was still substantially reduced (23.8 _+ 15.3 and 32.7 _+ 14.6% respectively), although soman was not present (Table I). As shown in Table I, the effect of soman (1.0 /~M) on high potassium (51 raM) evoked release of [SH]ACh was antagonised by scopolamine (0.3 /~M). Scopolamine enhanced the potassium evoked release by 63.9 _+ 10.4°/0 relative to a control stimulation in the absence of soman. Rats exposed to a low concentration (0.45--0.63 mg/m 8) of soman by inhalation showed no symptoms of organophosphate intoxication during the 40 h of sub-acute exposure. Bronchi from rats exposed to soman by inhalation showed a substantial change in muscle contraction by an alteration in the concentration-response curves and an increase in the apparent affinity (pD2) to ACh (Table II). The increase in pD 2 correlated well with the inhibition of acetylcholinesterase
93
TABLE I THE IN VITRO EFFECT OF SOMAN ON THE POTASSIUM EVOKED RELEASE OF [SH]ACh FROM CHOLINERGIC NERVE TERMINALS IN RAT BRONCHI Experiment
Stimulation I
II
A
100
87.1 ± 4.7....
B C
100
64.0 ± 6.4*
100 100
52.7 __. 4.4* 151.0 ± 9.3*
D
III 76.3 52.5 43.6 72.1
n
__. 13.8.... __ 6.5* ±
4.9*
± 5.2....
12 21 12 19
Experiment A: Three subsequent control stimulations with high potassium (51 mM). Stimulations were repeated with 45-min interval. Experiment B: Soman (1.0 ~M) was present 15 rain before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The AChE-activity was inhibited completely by exposure to soman (1.0 ~M) for 15 rain. Experiment C: Soman (100 i~I) was present 15 rain before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The AChE-activity was inhibited completely by exposure to soman (100 ~M) for 15 min. Experiment D: Soman (1.0 ~M) and scopolamine (0.3 ~M) were present 15 min before and during (5 rain) stimulation with high potassium (51 raM) (stimulation II). The responses are given in percent ___ S.E.M. of the control stimulation with potassium (51 raM, stimulation I), and corrected for the unavoidable decline in 3H release with successive stimulations. Stimulation I and III are control stimulations with potassium (51 raM) only. The actual amount of ACh released in a control experiment during potassium (51 raM) stimulation is 72.2 -+ 8.10 pmol • mg protein -1 with a spontaneous release of 41.2 _+ 6.0 pmol •mg protein -1 (Ref. 6). *P < 0.01, "'P > 0.05.
(ACHE) a c t i v i t y in t h e r a t b r o n c h i w h i c h w a s s u b s t a n t i a l l y i n h i b i t e d b y i n h a l a t i o n of s o m a n (Table II). The maximal contraction induced by ACh was reduced substantially after s u b - a c u t e (40 h, 1080 m g m i n / m 3) e x p o s u r e t o s o m a n r e l a t i v e t o c o n t r o l . A s s h o w n in T a b l e I I t h e m a x i m a l c o n t r a c t i o n i n d u c e d b y A C h w a s r e d u c e d b y 70o/o. C o n c o m i t a n t l y t h e A C h E a c t i v i t y w a s r e d u c e d b y 950/o. N e i t h e r a t r o p i n e (7 ~M) n o r t h e s m o o t h m u s c l e r e l a x a n t p a p a v e r i n e (4 mM) h a d a n y effect on an u n s t i m u l a t e d b r o n c h i a l p r e p a r a t i o n , b u t t h e y r e l a x e d t o t a l l y t h e ACh stimulation preparations. I n a s e c o n d s e r i e s of e x p e r i m e n t s r a t s w e r e e x p o s e d to s i m i l a r c o n c e n t r a t i o n s of s o m a n b y s u b - a c u t e i n h a l a t i o n t o c o m p a r e t h e p h y s i o l o g i c a l e f f e c t s w i t h r e c e p t o r b i n d i n g . I n b r o n c h i f r o m c o n t r o l r a t s [3H]QNB b i n d i n g w a s s a t u r a b l e w i t h i n c r e a s i n g c o n c e n t r a t i o n s a n d half m a x i m a l b i n d i n g in t h e b r o n c h i o c c u r r e d at a p p r o x i m a t e l y 0.3 nM. T h e e x p e r i m e n t a l r e s u l t s s h o w t h a t t h e b i n d i n g c a p a c i t y (Bmax) in r a t b r o n c h i w a s a p p r o x i m a t e l y 200 f m o l / m g p r o t e i n [3]. F o l l o w i n g e x p o s u r e (40 h) to s o m a n (0.63 m g / m 3) w i t h a t o t a l d o s e of 1518 m g m i n / m 3 t h e r e w a s a 40°/o r e d u c t i o n in t h e b i n d i n g c a p a c i t y (Bmax) of [3H]QNB in r a t b r o n c h i (Table II). I n s p i t e of a l a r g e r e d u c t i o n in t h e specific b i n d i n g of [3H]QNB no c h a n g e s w e r e o b s e r v e d in t h e non-specific b i n d i n g . D u r i n g t h e s e e x p e r i m e n t s t h e A C h E - a c t i v i t y w a s r e d u c e d t o 50/o of c o n t r o l (Table II). 94
TABLE II THE EFFECT OF SUB-ACUTE EXPOSURE TO SOMAN BY INHALATION ON RAT BRONCHI Soman concentration
AChE (%)
pDz
Maximal contraction
B (%)
Kd (nM)
0
100
1.0 (5)
100
0.45 ± 0.08 (8) 0.63 ± 0.01 (6)
4.2 ± 1.0 (8)** 5.1 ± 1.3 (6)**
3.8 ± 0.3 (5) 5.9 ± 0.1 (8)**
0.3 ± 0.05 (8)**
-
0.16 ± 0.04 (5) --
--
-
58.6 ± 8.9 (5)*
0.13 _+ 0.03 (5)~*
( m g / m s)
Rats were exposed to soman by inhalation for 40 h. The atmospheric concentration of soman was 0.45 ± 0.08 and 0.63 ± 0.01 mg/ma and total dose 1080.0 ± 281 and 1518.7 ± 40 mg/min per m 8, respectively. The acetylcholinesterase (ACHE) activities and the binding capacity ( B ) are given in per cent of control. The control activity of AChE was 789 ± 94 nmol • hr -1 mg protein-1 and B u 177.8 ± 18.0 fmol •mg protein-1. The binding constants ( B and K d (dissociation constant)) were calculated from saturation binding curves. The apparent affinity (pD2) and maximal contraction to ACh in bronchi are shown. The results are mean ± S.E.M. of the number of experiments shown in brackets. **P < 0.01, *P < 0.05, n,.p ~ 0.05. The data are extracted from Ref. 3.
DISCUSSION These results show that the organophosphorus compound soman reduces t h e r e l e a s e o f A C h f r o m c h o l i n e r g i c n e r v e s in b r o n c h i . T h i s is d u e t o s t i m u l a t i o n o f m u s c a r i n i c r e c e p t o r s on t h e c h o l i n e r g i c n e r v e t e r m i n a l s f o l l o w i n g a n i n c r e a s e in t h e c o n c e n t r a t i o n of A C h a f t e r i n h i b i t i o n of A C h E w i t h s o m a n . F u r t h e r m o r e , t h e r e s u l t s a l s o d e m o n s t r a t e d o w n - r e g u l a t i o n of m u s c a r i n i c r e c e p t o r s in b r o n c h i a f t e r s u b - a c u t e i n h a l a t i o n e x p o s u r e t o s o m a n . I t is t h e r e f o r e e v i d e n c e f o r b o t h p r e s y n a p t i c a n d p o s t - s y n a p t i c a d a p t a t i o n in t h e c h o l i n e r g i c n e r v o u s s y s t e m in r e s p o n s e t o t h e t o x i c e f f e c t s of s o m a n . T h e f e e d b a c k r e g u l a t i o n of A C h r e l e a s e m a y b e o n e of s e v e r a l physiological mechanisms whereby neurotransmission is m o d u l a t e d [10]. W h e t h e r t h i s m o d u l a t i o n is of a n y p h y s i o l o g i c a l i m p o r t a n c e is s t i l l n o t c l e a r . During exposure to organophosphorus compounds, which inhibit ACHEa c t i v i t y , t h e r e is a s u b s t a n t i a l e l e v a t i o n of t h e A C h c o n c e n t r a t i o n a n d a s u b s e q u e n t i n c r e a s e in t h e c o n t r a c t i o n of t h e a i r w a y s m o o t h m u s c l e [2]. S t u d i e s h a v e s h o w n t h a t s t i m u l a t i o n of p r e s y n a p t i c m u s c a r i n i c r e c e p t o r s in t h e g u i n e a - p i g i l e u m [11,12] a n d in t h e a i r w a y s [6] a l s o r e d u c e t h e r e l e a s e of A C h a n d t h a t t h e r e l e a s e is e n h a n c e d b y m u s c a r i n i c a n t a g o n i s t s ( T a b l e I) [4,6,12,13]. T h e r e f o r e , t h e p r e s e n c e of m u s c a r i n i c a u t o r e c e p t o r s on t h e cholinergic nerve terminals may protect against organophosphate intoxication. 95
T h e o t h e r a d a p t a t i o n in t h e cholinergic n e r v o u s s y s t e m in bronchi to high c o n c e n t r a t i o n s of t h e A C h E - i n h i b i t o r s o m a n w a s t h e r e d u c t i o n in t h e physiological r e s p o n s e to A C h due to r e d u c t i o n in t h e n u m b e r (Bm~x) of m u s c a r i n i c r e c e p t o r s (Table II). T h e bronchial s m o o t h m u s c l e f r o m r a t s e x p o s e d to s o m a n b y sub-acute e x p o s u r e r e s p o n d e d to c o n t r a c t i o n induced b y cholinergic s t i m u l a t i o n at l o w e r c o n c e n t r a t i o n s of A C h t h a n b r o n c h i f r o m control r a t s . A s s h o w n in T a b l e II, t h e r e w a s a significant i n c r e a s e in t h e a p p a r e n t affinity (pD 2) to ACh. This is due to t h e s u b s t a n t i a l inhibition (85O/o) of A C h E - a c t i v i t y (Table II) and is in a g r e e m e n t w i t h a p r e v i o u s r e p o r t w h e r e in v i t r o e x p o s u r e of r a t bronchi to s o m a n (10 nM, A C h E - a c t i v i t y = 35O/o of control) i n c r e a s e d t h e a p p a r e n t affinity to A C h f r o m 3.7 to 5.3 [2]. Following sub-acute e x p o s u r e to s o m a n a significant r e d u c t i o n in m a x i m a l muscle c o n t r a c t i o n induced b y cholinergic s t i m u l a t i o n w a s o b s e r v e d (Table II). T h e a l t e r a t i o n in t h e muscle c o n t r a c t i o n w a s c o m p a r e d w i t h c h a n g e s in a n t a g o n i s t binding. T h e a l t e r a t i o n of t h e n u m b e r of m u s c a r i n i c r e c e p t o r s in t h e b r o n c h i w a s 40o/o (Table II). T h e d a t a p r e s e n t e d a r e c o n s i s t e n t with r e p o r t e d d e c r e a s e s in [3H]QNB binding in s e v e r a l t i s s u e s f r o m m a m m a l s sub-chronically t r e a t e d w i t h d i i s o p r o p y l p h o s p h o r o - f l u o r i d a t e (DFP) b y injections [14,15]. T h e r e s u l t s t h e r e f o r e indicate t h a t t h e d e c r e a s e d r e s p o n s i v e n e s s of t h e bronchial s m o o t h m u s c l e to cholinergic s t i m u l a t i o n is due to a r e d u c t i o n in t h e n u m b e r of m u s c a r i n i c r e c e p t o r s . T h e d o w n - r e g u l a t i o n of r e c e p t o r s is a r e s u l t of t h e e n h a n c e d s t i m u l a t i o n b y A C h following inhibition of A C h E - a c t i v i t y b y soman. In conclusion, t h e results have shown that exposure to t h e o r g a n o p h o s p h o r u s c o m p o u n d s o m a n induce m o d u l a t i o n of A C h - r e l e a s e and d o w n - r e g u l a t i o n of m u s c a r i n i c r e c e p t o r s in r a t bronchi. REFERENCES 1
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97