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Correlation of muscarinic receptor density and acetylcholinesterase activity in repeated DFP-treated rats after the termination of DFP administration
European Journal of Pharmacology, 123 (1986) 223-228
223
Elsevier
CORRELATION O F MUSCARINIC R E C E P T O R DENSITY AND A C E T Y L C H O L I N E S T E R A S E ACTIVITY IN REPEATED DFP-TREATED RATS AFTER T H E T E R M I N A T I O N O F D F P ADMINISTRATION D O N G KOO LIM, BETH H O S K I N S and I N G K A N G HO *
Department of Pharmacology of Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, U.S.A. Received 7 November 1985, accepted 21 January 1986
D.K. LIM, B. HOSKINS and I.K. HO, Correlation of muscarinic receptor density and acetylcholinesterase activity in repeated DFP-treated rats after the termination of DFP administration, European J. Pharmacol. 123 (1986) 223-228. Following chronic exposure to DFP, both AChE activity and muscarinic receptor density were markedly depressed in the rat striatum. The rate of recovery of AChE activity was 5.2% per day within 7 days, while the density of muscarinic receptor recovered at the rate of 1.94% per day. The correlation between 2 parameters was very high (R = 0.99). Thus, it is suggested that the msucarinic receptor density was intimately related to AChE activity. Correlation of recovery
Diisopropylfluorophosphate
1. Introduction
The development of tolerance to organophosphorus cholinesterase inhibitors, especially diisopropylfluorophosphate (DFP) and some organophosphorus insecticides, has been well established (Overstreet et al., 1974; Russell et al., 1971; 1975). Evidence suggests that this may result from subsensitivity to acetylcholine (Brodeur and Dubous, 1964; Costa and Murphy, 1982; Overstreet et al., 1974). Recent studies have demonstrated that following chronic treatment with anticholinesterase agents, central muscarinic receptors are down-regulated (Ben-Bark et al., 1980; Churchill et al., 1984; Gazit et al., 1979; Yamada et al., 1983a). This is evidenced by the fact that the density of muscarinic receptors was significantly decreased after chronic exposure to DFP or organophosphorus insecticides (Ben-Bark et al., 1980; Churchill et al., 1984; Costa et al., 1982; Ehlert et al., 1980; Levy, 1981; Sivam et al., 1983; Yamada et al., 1983b). It has further been shown that the dose-
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Muscarinic receptors
AChE
dependent rate of reduction in muscarinic receptors by chronic administration of D F P is closely related to the degree of cholinesterase inhibition (Sivam et al., 1983; Yamada et al., 1983a). However, the relationship between muscarinic receptor density and acetylcholinesterase activity after the termination of administration of organophosphorus cholinesterase inhibitors has not yet been investigated. The present communication reports on the rates of recovery of muscarinic receptor density and acetylcholinesterase activity after the cessation of chronic administration of DFP to rats.
2. Materials and methods 2.1. Materials
Male Sprague-Dawley rats (Charles River Lab., Wilmington, MA) weighing 175-200 g were used throughout the study. The animals were housed 4 to a cage with free access to food and water. [ 3H]Quinuclidinyl benzilate ([ 3H]QNB), specific activity 31.2 Ci/mmol, was purchased from New
224
England Nuclear Corporation, Boston, MA. DFP (lot No. 201781) was obtained from Calbiochem, La Jolla, CA; this preparation of DFP was found to have an IC50 (concentration required for 50% inhibition of AChE activity in the whole brain homogenate of rat) value in vitro of 7 /~g/ml. Other chemicals and reagents of analytical grade were obtained from commercial suppliers.
2.2. Administration of DFP Freshly prepared solutions of DFP in saline were administered subcutaneously (s.c.) in volumes of 0.1 ml/100 g body weight daily between 9 : 00 to 11 : 00 a.m. for 13 days. In accordance with our previous results (Lim et al., 1983; Sivam et al., 1983), the dosage schedule of D F P administration for 13 days was as follows: 1st through 3rd day, 1 mg/kg; 4th through 6th day, 0.5 mg/kg; 7th through 13th day, 1 m g / k g and on the 14th day, 2 mg/kg, s.c. All animals were then divided into 6 groups; they were decapitated at 6 h, 1, 3, 7, 14 or 21 days after the last injection of DFP. The brains were quickly removed and the striata were dissected out according to the procedure of Glowinski and Iversen (1966). Eight to ten rats were used in each group. The acute DFP-treated rats received daily injections of saline (0.1 ml/100 g) for 13 days. DFP, 2 mg/kg, s.c. was given to these saline-treated rats 24 h after the last saline injection. These rats were divided into 4 groups and decapitated at 6 h, 1, 3 or 7 days after the DFP administration. Control rats received daily injections of saline for 14 days. They were killed 6 h after the last injection.
2.3. Determination of AChE activity Striatal AChE activity was determined according to the method of Ellman et al. (1961). The tissues were homogenized in ice-cold sodium phosphate buffer (0.1 M, pH 8.0) at a concentration of approximately 20 mg wet weight/ml buffer. The enzyme activities were expressed as nmol of acetylthiocholine hydrolyzed/min per mg protein. The protein content of tissue homogenate was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
2.4. [JH]QNB binding assay Membranes were prepared according to the method of Zukin et al. (1974) with slight modification as previously described (Sivam et al., 1983). The animals, after appropriate treatment, were decapitated, the brains were rapidly removed, and the striata were dissected out. The pooled samples were then homogenized in 15 volumes of ice-cold 0.32 M sucrose using a Brinkman Polytron PT-10 at low speed (setting 3). The homogenate was centrifuged at 1000 x g for 10 min; the pellet was discarded and the supernatant fluid was centrifuged at 20000 x g for 20 min to obtain a crude mitochondrial pellet. The crude mitochondrial pellet was resuspended in double-distilled deionized water and dispersed with a Brinkman PT-10 (setting 6) for 30 s. The suspension was centrifuged at 8000 x g for 20 min. The supernatant including the buffy layer was collected and centrifuged at 48 000 x g for 20 min to obtain a pellet. The pellet was resuspended in water and centrifuged at 48000 x g for 20 min. The final membrane preparation was suspended in 50 mM sodium phosphate buffer (pH 7.4). The binding of [3H]QNB was carried out according to the method of Yamamura and Snyder (1974) with minor modification. The binding assay was performed in 50 mM sodium phosphate buffer (pH 7.4), with different concentrations (0.01-2 nM) of [3H]QNB to generate saturation curves in a final volume of 1 ml. Specific binding was calculated as the total binding minus that occurring in the presence of 1 /~M atropine. The binding was initiated by addition of 0.2 ml of membrane preparation (0.2-0.4 mg protein/ml), and incubations were allowed to proceed for 1 h at 25°C in a shaking bath. The reaction was terminated by rapid filtering through Whatman G F / B glass fiber filters. Each filter was washed twice with 5 ml buffer, and the dried filter was transferred to scintillation vials containing 10 ml of Aquasol (New England Nuclear, Boston, MA). The radioactivity retained in each filter was determined by liquid scintillation spectrophotometry. The protein content of the membrane preparations was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
225
2.5. Analysis of data Scatchard analyses were used to o b t a i n values for dissociation c o n s t a n t s ( K d ) a n d m a x i m u m n u m b e r of b i n d i n g sites (Bmax). W h e n applicable, d a t a were analyzed for significance by Student's t-test a n d correlation analysis.
3. Results
3.1. Recovery of striatal acetylcholinesterase activity after acute and repeated administration of DFP Rats which received a single injection of D F P , 2 m g / k g , exhibited the usual parasympathetic overt toxicity within 6 h after the administration. Rats which received daily a d m i n i s t r a t i o n of D F P exhibited similar cholinergic overt activity for the period of 4-5 days; thereafter the symptoms gradually disappeared. After 13 injections, rats no longer revealed a n y of these symptoms. The striatal acetylcholinesterase activity at different time periods after the acute and daily adm i n i s t r a t i o n of D F P are presented in table 1. Six
TABLE 1 Effects of acute and daily administration of DFP on AChE activity in rat striatum. Values represent means_+S.E, of 4 samples. Numerals in parentheses denote % inhibition from the control values. Significant difference (P < 0.001) was found for all treatment groups when compared with the control value. * P < 0.001 compared to the value obtained for the 1 mg/kg for 13 days treatment group. Following repeated administration of DFP, s.c., for 13 days as described in the text, a challenge dose of DFP, 2 mg/kg, was administered on the 14th day. The animals were killed at the above indicated times after the administration of DFP. Treatment
Control 24 h (1 mg/kg for 13 days) 6h 24 h 3 days 7 days 14 days 21 days
AChE activity (nmol/min per mg protein) Acute
Daily
246 _+8
246 + 8 29 + 3 (88) 7 + 1 (97) * 18+1 (93) 48 + 3 (81) * 93 + 3 (62) * 142+ 3 (42) * 174+5 (29) *
25 + 3 (90) 29 + 2 (88) 58 + 4 (77) 96 + 7 (61)
a n d twenty-four hours after acute a d m i n i s t r a t i o n of D F P , 2 m g / k g , acetylcholinesterase activity was 90% inhibited in striatal regions. Even at 3 a n d 7 days after a n acute a d m i n i s t r a t i o n of D F P , the striatal acetylcholinesterase activity was still 77 a n d 61% inhibited, respectively. I n rats receiving 13 daily a d m i n i s t r a t i o n s of D F P , the striatal acetylcholinesterase activity 24 h after the last a d m i n i s t r a t i o n of D F P was only 12% of the control activity. W h e n rats treated daily with D F P received an additional dose of D F P , 2 m g / k g , 24 h after the 13th injection, the acetylcholinesterase activity in the striatum was less than 10% of that of the control group at 6 a n d 24 h after they had received an additional administration of D F P , 2 m g / k g . The striatal acetylcholinesterase activity gradually recovered after the t e r m i n a t i o n of D F P administration. In the group treated with D F P daily, 3, 7, 14 a n d 21 days after the additional a d m i n i s t r a t i o n of D F P , 2 m g / k g , the enzyme activity was 81, 62, 42 a n d 29% inhibited, respectively, as c o m p a r e d with control activity. The rate of recovery of acetylcholinesterase activity was estimated to be 5.2% per day within 7 days. I n rats treated with only one dose of D F P , the recovery rate of acetylcholinesterase activity was estimated to be 4.4% per
TABLE 2 Recovery of [3H]QNB binding sites in rat striatum after the cessation of daily administration of DFP. The values are the means+S.E, of 3-5 determinations, each done in duplicate. * P < 0.05 compared to the value obtained for 24 h. Significant difference (P < 0.01) in Bm~, was found for all treatment groups when compared with control group value. The experimental paradigm was the same as table 1. Treatment
Kd (nM)
Bmax
(pmol/g protein) Control 24 h after last administration 3 days after last administration 7 days after last administration 14 days after last administration 21 days after last administration
0.067 _+0.018
1 388 _+49
0.071 + 0.035
677 _+12.
0.067 + 0.013
710 + 24
0.079 + 0.016
944 + 26 *
0.052 + 0.004
1044 + 25 *
0.068+0.010
1219+45 *
226
100
day. The recovery rates for acutely and repeatedly treated rats were not significantly different from each other.
3.2. Recovery of muscarinic receptors after termination of daily administration of DFP Repeated administration of D F P did not change the affinity of muscarinic receptors as measured by [3H]QNB binding (table 2). This confirms our previously reported results (Sivam et al., 1983). However, when animals had developed tolerance to DFP, the n u m b e r of muscarinic receptors as estimated by Q N B binding, was significantly reduced. These results substantiate the development of subsensitivity in muscarinic receptors in the rats which developed tolerance to D F P . As also shown in fig. 1, in striata from daily D F P treated rats, the receptor density was reduced to 50% of the control values at 24 h after the 14th injection. Twenty-one days after the cessation of D F P administration the n u m b e r of muscarinic receptors was still significantly lower than that of the controls (88% of the control level). However,
I-
rrC~ I-rr Zo 0~-(ja. u. I.u
•
•
°r=.99
,o ~ 50 I,--~
oz 0 (/)
s'0
1~o
PERCENT
O F CONTROL AChE ACTIVITY
Fig. 2.Correlation between muscarinic receptor density and AChE activity in the rat striatum. Each value represents the percentage of control value (tables 1 and 2). The points fit the linear equation, y = 0.61x+42.5 (r = 0.99), P < 0.001; y (ordinate) = number of muscarinic receptor (%), x (abscissa)= AChE activity (%).
100 the n u m b e r of receptors at 21 days after the termination of daily D F P administration was significantly recovered as compared to the n u m b e r of receptors found 1, 3 and 7 days after the termination of D F P administration. The density of muscarinic receptors recovered at the rate of 1.94% per day. The half life of the recovery of muscarinic receptors was estimated to be 13.3 days.
i+ s0
ZO
3.3. Correlation between recovery of acetylcholinesterase activity and muscarinic receptor density
O
t~
~ ~ ~ 1'I l'a l's l'r 1'9 ~I DAYS AFTER THE TERMINATION OF DAILY ADMINISTRATION OF DFP Fig. 1. The changes in muscarinic receptor density after the cessation of daily and challenge administration of DFP (2 mg/kg). All points fit the linear equation, y=l.94x+48.3 (r = 0.96), P < 0.05; y (ordinate)= the percentage of control muscarinic receptor density, x (abscissa)= days after last administration of DFP.
Our results reveal that there is a good correlation between the recovery of acetylcholinesterase activity and the density of muscarinic receptors after the termination of daily administration of D F P . As shown in fig. 2, the correlation coefficient between the acetylcholinesterase activity and muscarinic receptor density in the rats treated daily with D F P was 0.99.
227
4. Discussion The present study confirms our previous finding that muscarinic receptor density was significantly decreased in striata or rats repeatly treated with DFP indicating down regulation of muscarinic receptors. Furthermore, the results demonstrate that the recovery from such down regulation of muscarinic receptors is correlated closely with the recovery of acetylcholinesterase activity after the termination of DFP administration. In short, in striata from rats receiving 2 weeks of daily administration of DFP, the muscarinic receptor density was reduced to 50%, while acetylcholinesterase activity was only 10% of that of the control animals. Within 3 weeks after the termination of repeated DFP injections, the muscarinic receptor density returned to 88% of control values while the acetylcholinesterase activity returned to 70% of the control level. Our data suggest that the muscarinic receptor density is intimately related to acetylcholinesterase activity. It has been well demonstrated that behavioral tolerance to DFP develops when brain acetylcholinesterase activity is only 10% of control activity. It has been suggested that this behavioral tolerance may result from a decrease in density of muscarinic receptors (Brodeur and Dubous, 1964; Costa and Murphy, 1982; Overstreet et al., 1974). Yamada et al. (1983a) have suggested that a critical brain acetylcholinesterase activity of about 50% or less has to be maintained when muscarinic receptor density is significantly reduced. Yamada et al. (1983a) also reported that the reduction of muscarinic receptor density with repeated administration of DFP into guinea-pigs was dose-dependent. However, the recovery in muscarinic receptor density in 18 h and 1 week after the cessation of repeated DFP administration was not significantly different. The present results show that muscarinic receptor density in striata of rats which were repeatedly treated with DFP recovered gradually at a rate of about 2% per day after the cessation of DFP administration. The half-life of recovery was found to be approximately 13-14 days. Although the present study demonstrated a good correlation between the recovery of muscarinic
receptor density with the degree of recovery of acetylcholinesterase activity, correlations between other parameters of the cholinergic system (e.g. ACh level, uptake of choline, release of ACh, etc.) remain to be elucidated. It has been shown that repeated administration of organophosphorus acetylcholinesterase inhibitors increases ACh levels (Wecker et al., 1977) and decreases the release and synthesis of ACh (Bourdois et al., 1974; Saelens et al., 1974). Recently, it has also been reported that repeated administration of DFP significantly decreases high affinity uptake of choline (Yamada et al., 1983a,b). ACh synthesis is regulated by the availability of ACh, CoA and choline (Browing and Shulman, 1968; Richter and Marchbanks, 1971; Wecker et al., 1977). Furthermore, both choline acetyltransferase and acetylcholinesterase activities are involved in modulating ACh levels. During the course of the recovery of acetylcholinesterase activity after the cessation of repeated DFP administration, the elevated levels of ACh would also be expected to return to normal levels which would, in turn, modulate ACh receptors. Work is presently underway to correlate these different aspects of cholinergic activity with muscarinic receptor density after the termination of repeated injections of DFP.
Acknowledgement This study was supported by contract from the U.S. Army Medical Research and Development Command (contract DAMD 17-85-C-5036).
References Ben-Bark, J., H. Gazit and Y. Dubie, 1980, Fornix lesion prevents an organophosphate-induced decrease in muscarinic receptor level in rat hippocampus, Brain Res. 194, 240. Bourdois, P.S., J.F. Mitchell, G.T. Somogyi and J.C. Szerb, 1974, The output per stimulus of acetylcholine from cerebral cortical slices in the presence or absence of cholinesterase inhibition, Br. J. Pharmacol. 52, 509. Brodeur, J. and K.P. Dubois, 1964, Studies on the mechanism of acquired tolerance by rats. 0.0-diethyl-S-2-(ethylthio)ethyl phosphorodithioate (Disyston), Arch. Int. Pharmacodyn. Ther. 149, 560. Browing, E.T. and M.P. Shulman, 1968, [14C]-Acetylcholine
228 synthesis by cortex slices of rat brain, J. Neurochem. 15, 1391. Churchill, L., T.L. Pazdernik, F. Samson and S.R. Nelson, 1984, Topographic distribution of down-regulated muscarinic receptors in rat brains after repeated exposure to di-isopropylphosphofluoridate, Neuroscience 11,463. Costa, L.G. and S.D. Murphy, 1982, Passive avoidance retention in mice tolerant to the organophosphorus insecticide disulfoton, Toxicol. Appl. Pharmacol. 65, 451. Costa, L.G., B.W. Schwab and S.D. Murphy, 1982, Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticide, Biochem. Pharmacol. 31, 3407. Ehlert, F.J., N. Kokka and A.S. Fairhurst, 1980, Altered [3H]Quinuclidinyl benzilate binding in the striatum of rats following chronic cholinesterase inhibition with diisopropylfluorophosphate, Mol. Pharmacol. 17, 24. Ellman, G.L., L.D. Courtney, V. Andres, Jr. and R.M. Featherstone, 1961, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88. Gazit, H., 1. Silman and Y. Dubai, 1979, Administration of an organophosphate causes a decrease in muscarinic receptor level in rat brain, Brain Res. 174, 351. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamine in the rat brain. I. The disposition of [3H]norephinephrine, [3H]-dopamine and [3H]-DOPA in various regions of the brain, J. Neurochem. 13, 655. Levy, A., 1981, The effect of cholinesterase inhibition on the ontogenesis on central muscarinic receptors, Life Sci. 20, 1065. Lim, D.K., B. Hoskins and I.K. Ho, 1983, Assessment of diisopropylfluorophosphate (DFP) toxicity and tolerance in rats, Res. Commun. Chem. Pathol. Pharmacol. 39, 399. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. Overstreet, D.H., R.W. Russell, B.J. Vasquez and A.W. Dalglish, 1974, Involvement of muscarinic and nicotinic receptors ~n behavioral tolerance to DFP, Pharmacol. Biochem. Behav. 2, 45.
Richter, J.A. and R.M. Marchbanks, 1971, Synthesis of radioactive acetylcholine from [3H]choline and its release from cerebral cortex slices in vitro, J. Neurochem. 18, 691. Russell, R.W., D.H. Overstreet, C.W. Cotman, V.G. Carson, L. Churchill, F.M. Dalglish and B.J. Vasquez, 1975, Experimental tests of hypothesis about neurochemical mechanisms underlying behavioral tolerance to the anticholinesterase, diisopropylfluorophosphate, J. Pharmacol. Exp. Ther. 192, 73. Russell, R.W., B.J. Vasquez, D.H. Overstreet and F.W. Dalglish, 1971, Effects of cholinolytic agents on behavior following development of tolerance to low cholinesterase activity, Psychopharmacologia 20, 32. Sealens, J.K., J.P. Simke, J. Schuman and M.P. Allens, 1974, Agents which influence acetylcholine metabolism in mouse brain, Arch. Int. Pharmacodyn. Ther. 209, 250. Sivam, S.P., J.C. Norris, D.K. Lim, B. Hoskins and I.K. Ho, 1983, Effect of acute and chronic cholinesterase inhibition with diisopropylfluorophosphate on muscarinic, dopamine and GABA receptors of the rat striatum, J. Neurochem. 40, 1414. Wecker, L., P.L. Mobley and W.D. Dettbarn, 1977, Central cholinergic mechanisms underlying adaptation to reduced cholinesterase activity, Biochem. Pharmacol. 26, 633. Yamada, S., M. Isogai, H. Okadaira and E. Hayashi, 1983a, Correlation between cholinesterase inhibition and reduction in muscarinic receptors and choline uptake by repeated diisopropylfluorophosphate administration antagonism by physostygmine and atropine, J. Pharmacol. Exp. Ther. 226, 519. Yamada, S., M. Isogai, H. Okudaira and E. Hayashi, 1983b, Regional adaptation of muscarinic receptors and choline uptake in brain following administration of diisopropylfluorophosphate and atropine, Brain Res. 268, 315. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Natl. Acad. Sci. U.S.A. 71, 1725. Zukin, S.R., A.E. Young and S.H. Snyder, 1974, Gammaaminobutyric acid binding to receptor sites in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A. 71', 4802.
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Elsevier
CORRELATION O F MUSCARINIC R E C E P T O R DENSITY AND A C E T Y L C H O L I N E S T E R A S E ACTIVITY IN REPEATED DFP-TREATED RATS AFTER T H E T E R M I N A T I O N O F D F P ADMINISTRATION D O N G KOO LIM, BETH H O S K I N S and I N G K A N G HO *
Department of Pharmacology of Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, U.S.A. Received 7 November 1985, accepted 21 January 1986
D.K. LIM, B. HOSKINS and I.K. HO, Correlation of muscarinic receptor density and acetylcholinesterase activity in repeated DFP-treated rats after the termination of DFP administration, European J. Pharmacol. 123 (1986) 223-228. Following chronic exposure to DFP, both AChE activity and muscarinic receptor density were markedly depressed in the rat striatum. The rate of recovery of AChE activity was 5.2% per day within 7 days, while the density of muscarinic receptor recovered at the rate of 1.94% per day. The correlation between 2 parameters was very high (R = 0.99). Thus, it is suggested that the msucarinic receptor density was intimately related to AChE activity. Correlation of recovery
Diisopropylfluorophosphate
1. Introduction
The development of tolerance to organophosphorus cholinesterase inhibitors, especially diisopropylfluorophosphate (DFP) and some organophosphorus insecticides, has been well established (Overstreet et al., 1974; Russell et al., 1971; 1975). Evidence suggests that this may result from subsensitivity to acetylcholine (Brodeur and Dubous, 1964; Costa and Murphy, 1982; Overstreet et al., 1974). Recent studies have demonstrated that following chronic treatment with anticholinesterase agents, central muscarinic receptors are down-regulated (Ben-Bark et al., 1980; Churchill et al., 1984; Gazit et al., 1979; Yamada et al., 1983a). This is evidenced by the fact that the density of muscarinic receptors was significantly decreased after chronic exposure to DFP or organophosphorus insecticides (Ben-Bark et al., 1980; Churchill et al., 1984; Costa et al., 1982; Ehlert et al., 1980; Levy, 1981; Sivam et al., 1983; Yamada et al., 1983b). It has further been shown that the dose-
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Muscarinic receptors
AChE
dependent rate of reduction in muscarinic receptors by chronic administration of D F P is closely related to the degree of cholinesterase inhibition (Sivam et al., 1983; Yamada et al., 1983a). However, the relationship between muscarinic receptor density and acetylcholinesterase activity after the termination of administration of organophosphorus cholinesterase inhibitors has not yet been investigated. The present communication reports on the rates of recovery of muscarinic receptor density and acetylcholinesterase activity after the cessation of chronic administration of DFP to rats.
2. Materials and methods 2.1. Materials
Male Sprague-Dawley rats (Charles River Lab., Wilmington, MA) weighing 175-200 g were used throughout the study. The animals were housed 4 to a cage with free access to food and water. [ 3H]Quinuclidinyl benzilate ([ 3H]QNB), specific activity 31.2 Ci/mmol, was purchased from New
224
England Nuclear Corporation, Boston, MA. DFP (lot No. 201781) was obtained from Calbiochem, La Jolla, CA; this preparation of DFP was found to have an IC50 (concentration required for 50% inhibition of AChE activity in the whole brain homogenate of rat) value in vitro of 7 /~g/ml. Other chemicals and reagents of analytical grade were obtained from commercial suppliers.
2.2. Administration of DFP Freshly prepared solutions of DFP in saline were administered subcutaneously (s.c.) in volumes of 0.1 ml/100 g body weight daily between 9 : 00 to 11 : 00 a.m. for 13 days. In accordance with our previous results (Lim et al., 1983; Sivam et al., 1983), the dosage schedule of D F P administration for 13 days was as follows: 1st through 3rd day, 1 mg/kg; 4th through 6th day, 0.5 mg/kg; 7th through 13th day, 1 m g / k g and on the 14th day, 2 mg/kg, s.c. All animals were then divided into 6 groups; they were decapitated at 6 h, 1, 3, 7, 14 or 21 days after the last injection of DFP. The brains were quickly removed and the striata were dissected out according to the procedure of Glowinski and Iversen (1966). Eight to ten rats were used in each group. The acute DFP-treated rats received daily injections of saline (0.1 ml/100 g) for 13 days. DFP, 2 mg/kg, s.c. was given to these saline-treated rats 24 h after the last saline injection. These rats were divided into 4 groups and decapitated at 6 h, 1, 3 or 7 days after the DFP administration. Control rats received daily injections of saline for 14 days. They were killed 6 h after the last injection.
2.3. Determination of AChE activity Striatal AChE activity was determined according to the method of Ellman et al. (1961). The tissues were homogenized in ice-cold sodium phosphate buffer (0.1 M, pH 8.0) at a concentration of approximately 20 mg wet weight/ml buffer. The enzyme activities were expressed as nmol of acetylthiocholine hydrolyzed/min per mg protein. The protein content of tissue homogenate was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
2.4. [JH]QNB binding assay Membranes were prepared according to the method of Zukin et al. (1974) with slight modification as previously described (Sivam et al., 1983). The animals, after appropriate treatment, were decapitated, the brains were rapidly removed, and the striata were dissected out. The pooled samples were then homogenized in 15 volumes of ice-cold 0.32 M sucrose using a Brinkman Polytron PT-10 at low speed (setting 3). The homogenate was centrifuged at 1000 x g for 10 min; the pellet was discarded and the supernatant fluid was centrifuged at 20000 x g for 20 min to obtain a crude mitochondrial pellet. The crude mitochondrial pellet was resuspended in double-distilled deionized water and dispersed with a Brinkman PT-10 (setting 6) for 30 s. The suspension was centrifuged at 8000 x g for 20 min. The supernatant including the buffy layer was collected and centrifuged at 48 000 x g for 20 min to obtain a pellet. The pellet was resuspended in water and centrifuged at 48000 x g for 20 min. The final membrane preparation was suspended in 50 mM sodium phosphate buffer (pH 7.4). The binding of [3H]QNB was carried out according to the method of Yamamura and Snyder (1974) with minor modification. The binding assay was performed in 50 mM sodium phosphate buffer (pH 7.4), with different concentrations (0.01-2 nM) of [3H]QNB to generate saturation curves in a final volume of 1 ml. Specific binding was calculated as the total binding minus that occurring in the presence of 1 /~M atropine. The binding was initiated by addition of 0.2 ml of membrane preparation (0.2-0.4 mg protein/ml), and incubations were allowed to proceed for 1 h at 25°C in a shaking bath. The reaction was terminated by rapid filtering through Whatman G F / B glass fiber filters. Each filter was washed twice with 5 ml buffer, and the dried filter was transferred to scintillation vials containing 10 ml of Aquasol (New England Nuclear, Boston, MA). The radioactivity retained in each filter was determined by liquid scintillation spectrophotometry. The protein content of the membrane preparations was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
225
2.5. Analysis of data Scatchard analyses were used to o b t a i n values for dissociation c o n s t a n t s ( K d ) a n d m a x i m u m n u m b e r of b i n d i n g sites (Bmax). W h e n applicable, d a t a were analyzed for significance by Student's t-test a n d correlation analysis.
3. Results
3.1. Recovery of striatal acetylcholinesterase activity after acute and repeated administration of DFP Rats which received a single injection of D F P , 2 m g / k g , exhibited the usual parasympathetic overt toxicity within 6 h after the administration. Rats which received daily a d m i n i s t r a t i o n of D F P exhibited similar cholinergic overt activity for the period of 4-5 days; thereafter the symptoms gradually disappeared. After 13 injections, rats no longer revealed a n y of these symptoms. The striatal acetylcholinesterase activity at different time periods after the acute and daily adm i n i s t r a t i o n of D F P are presented in table 1. Six
TABLE 1 Effects of acute and daily administration of DFP on AChE activity in rat striatum. Values represent means_+S.E, of 4 samples. Numerals in parentheses denote % inhibition from the control values. Significant difference (P < 0.001) was found for all treatment groups when compared with the control value. * P < 0.001 compared to the value obtained for the 1 mg/kg for 13 days treatment group. Following repeated administration of DFP, s.c., for 13 days as described in the text, a challenge dose of DFP, 2 mg/kg, was administered on the 14th day. The animals were killed at the above indicated times after the administration of DFP. Treatment
Control 24 h (1 mg/kg for 13 days) 6h 24 h 3 days 7 days 14 days 21 days
AChE activity (nmol/min per mg protein) Acute
Daily
246 _+8
246 + 8 29 + 3 (88) 7 + 1 (97) * 18+1 (93) 48 + 3 (81) * 93 + 3 (62) * 142+ 3 (42) * 174+5 (29) *
25 + 3 (90) 29 + 2 (88) 58 + 4 (77) 96 + 7 (61)
a n d twenty-four hours after acute a d m i n i s t r a t i o n of D F P , 2 m g / k g , acetylcholinesterase activity was 90% inhibited in striatal regions. Even at 3 a n d 7 days after a n acute a d m i n i s t r a t i o n of D F P , the striatal acetylcholinesterase activity was still 77 a n d 61% inhibited, respectively. I n rats receiving 13 daily a d m i n i s t r a t i o n s of D F P , the striatal acetylcholinesterase activity 24 h after the last a d m i n i s t r a t i o n of D F P was only 12% of the control activity. W h e n rats treated daily with D F P received an additional dose of D F P , 2 m g / k g , 24 h after the 13th injection, the acetylcholinesterase activity in the striatum was less than 10% of that of the control group at 6 a n d 24 h after they had received an additional administration of D F P , 2 m g / k g . The striatal acetylcholinesterase activity gradually recovered after the t e r m i n a t i o n of D F P administration. In the group treated with D F P daily, 3, 7, 14 a n d 21 days after the additional a d m i n i s t r a t i o n of D F P , 2 m g / k g , the enzyme activity was 81, 62, 42 a n d 29% inhibited, respectively, as c o m p a r e d with control activity. The rate of recovery of acetylcholinesterase activity was estimated to be 5.2% per day within 7 days. I n rats treated with only one dose of D F P , the recovery rate of acetylcholinesterase activity was estimated to be 4.4% per
TABLE 2 Recovery of [3H]QNB binding sites in rat striatum after the cessation of daily administration of DFP. The values are the means+S.E, of 3-5 determinations, each done in duplicate. * P < 0.05 compared to the value obtained for 24 h. Significant difference (P < 0.01) in Bm~, was found for all treatment groups when compared with control group value. The experimental paradigm was the same as table 1. Treatment
Kd (nM)
Bmax
(pmol/g protein) Control 24 h after last administration 3 days after last administration 7 days after last administration 14 days after last administration 21 days after last administration
0.067 _+0.018
1 388 _+49
0.071 + 0.035
677 _+12.
0.067 + 0.013
710 + 24
0.079 + 0.016
944 + 26 *
0.052 + 0.004
1044 + 25 *
0.068+0.010
1219+45 *
226
100
day. The recovery rates for acutely and repeatedly treated rats were not significantly different from each other.
3.2. Recovery of muscarinic receptors after termination of daily administration of DFP Repeated administration of D F P did not change the affinity of muscarinic receptors as measured by [3H]QNB binding (table 2). This confirms our previously reported results (Sivam et al., 1983). However, when animals had developed tolerance to DFP, the n u m b e r of muscarinic receptors as estimated by Q N B binding, was significantly reduced. These results substantiate the development of subsensitivity in muscarinic receptors in the rats which developed tolerance to D F P . As also shown in fig. 1, in striata from daily D F P treated rats, the receptor density was reduced to 50% of the control values at 24 h after the 14th injection. Twenty-one days after the cessation of D F P administration the n u m b e r of muscarinic receptors was still significantly lower than that of the controls (88% of the control level). However,
I-
rrC~ I-rr Zo 0~-(ja. u. I.u
•
•
°r=.99
,o ~ 50 I,--~
oz 0 (/)
s'0
1~o
PERCENT
O F CONTROL AChE ACTIVITY
Fig. 2.Correlation between muscarinic receptor density and AChE activity in the rat striatum. Each value represents the percentage of control value (tables 1 and 2). The points fit the linear equation, y = 0.61x+42.5 (r = 0.99), P < 0.001; y (ordinate) = number of muscarinic receptor (%), x (abscissa)= AChE activity (%).
100 the n u m b e r of receptors at 21 days after the termination of daily D F P administration was significantly recovered as compared to the n u m b e r of receptors found 1, 3 and 7 days after the termination of D F P administration. The density of muscarinic receptors recovered at the rate of 1.94% per day. The half life of the recovery of muscarinic receptors was estimated to be 13.3 days.
i+ s0
ZO
3.3. Correlation between recovery of acetylcholinesterase activity and muscarinic receptor density
O
t~
~ ~ ~ 1'I l'a l's l'r 1'9 ~I DAYS AFTER THE TERMINATION OF DAILY ADMINISTRATION OF DFP Fig. 1. The changes in muscarinic receptor density after the cessation of daily and challenge administration of DFP (2 mg/kg). All points fit the linear equation, y=l.94x+48.3 (r = 0.96), P < 0.05; y (ordinate)= the percentage of control muscarinic receptor density, x (abscissa)= days after last administration of DFP.
Our results reveal that there is a good correlation between the recovery of acetylcholinesterase activity and the density of muscarinic receptors after the termination of daily administration of D F P . As shown in fig. 2, the correlation coefficient between the acetylcholinesterase activity and muscarinic receptor density in the rats treated daily with D F P was 0.99.
227
4. Discussion The present study confirms our previous finding that muscarinic receptor density was significantly decreased in striata or rats repeatly treated with DFP indicating down regulation of muscarinic receptors. Furthermore, the results demonstrate that the recovery from such down regulation of muscarinic receptors is correlated closely with the recovery of acetylcholinesterase activity after the termination of DFP administration. In short, in striata from rats receiving 2 weeks of daily administration of DFP, the muscarinic receptor density was reduced to 50%, while acetylcholinesterase activity was only 10% of that of the control animals. Within 3 weeks after the termination of repeated DFP injections, the muscarinic receptor density returned to 88% of control values while the acetylcholinesterase activity returned to 70% of the control level. Our data suggest that the muscarinic receptor density is intimately related to acetylcholinesterase activity. It has been well demonstrated that behavioral tolerance to DFP develops when brain acetylcholinesterase activity is only 10% of control activity. It has been suggested that this behavioral tolerance may result from a decrease in density of muscarinic receptors (Brodeur and Dubous, 1964; Costa and Murphy, 1982; Overstreet et al., 1974). Yamada et al. (1983a) have suggested that a critical brain acetylcholinesterase activity of about 50% or less has to be maintained when muscarinic receptor density is significantly reduced. Yamada et al. (1983a) also reported that the reduction of muscarinic receptor density with repeated administration of DFP into guinea-pigs was dose-dependent. However, the recovery in muscarinic receptor density in 18 h and 1 week after the cessation of repeated DFP administration was not significantly different. The present results show that muscarinic receptor density in striata of rats which were repeatedly treated with DFP recovered gradually at a rate of about 2% per day after the cessation of DFP administration. The half-life of recovery was found to be approximately 13-14 days. Although the present study demonstrated a good correlation between the recovery of muscarinic
receptor density with the degree of recovery of acetylcholinesterase activity, correlations between other parameters of the cholinergic system (e.g. ACh level, uptake of choline, release of ACh, etc.) remain to be elucidated. It has been shown that repeated administration of organophosphorus acetylcholinesterase inhibitors increases ACh levels (Wecker et al., 1977) and decreases the release and synthesis of ACh (Bourdois et al., 1974; Saelens et al., 1974). Recently, it has also been reported that repeated administration of DFP significantly decreases high affinity uptake of choline (Yamada et al., 1983a,b). ACh synthesis is regulated by the availability of ACh, CoA and choline (Browing and Shulman, 1968; Richter and Marchbanks, 1971; Wecker et al., 1977). Furthermore, both choline acetyltransferase and acetylcholinesterase activities are involved in modulating ACh levels. During the course of the recovery of acetylcholinesterase activity after the cessation of repeated DFP administration, the elevated levels of ACh would also be expected to return to normal levels which would, in turn, modulate ACh receptors. Work is presently underway to correlate these different aspects of cholinergic activity with muscarinic receptor density after the termination of repeated injections of DFP.
Acknowledgement This study was supported by contract from the U.S. Army Medical Research and Development Command (contract DAMD 17-85-C-5036).
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