Evidence for the involvement of presynaptic cholinergic functions in tolerance to diisopropylfluorophosphate

rOXICOLOCY

AND

APPLIED

PHARMACOLOGY

90,465-476

(1987)

Evidence for the Involvement of Presynaptic Cholinergic in Tolerance to Diisopropylfluorophosphate D. K. LIM, B. HOSKINS,

ANDI.

Functions

K. Ho’

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505

Received December 8, 1986; accepted June I, 1987 Evidence for the Involvement of Presynaptic Cholinergic Functions in Tolerance to Diisopropylfluorophosphate. LIM, D. K., HOSKINS, B., AND Ho, I. K. (1987). Toxicol. Appl. Pharmacol. 90,465-476. Rats were treated with diisopropylfluorophosphate (DFP) acutely or daily for 14 days. The involvement of various presynaptic and postsynaptic functions of the cholinergic system in the development of tolerance to DFP was studied. Receptor density and affinity of both muscarinic and nicotinic receptors, high-affinity choline uptake, and [K+]-evoked release of acetylcholine (ACh) by atropine were not changed after acute administration of 2 mg/kg DEP. Both muscarinic and nicotinic receptors were down-regulated to the same extent (40-50%) alter subacute administration of DFP (1 mg/kg) without changes in their affinities. Binding sites of muscarinic receptors were maximally decreased after 7 days of DFP administration. Thereafter, they remained constant throughout 14 days of administration. One hour after the last injection of 2 mg/kg DFP to subacutely treated rats, the maximum velocity of high-affinity choline uptake was significantly decreased in the striatum (33%) and hippocampus (53%) without changes in K,,, values. Twenty-four hours after the last injection of DFP, only a higher dose of DFP (2 mg/ kg) significantly inhibited choline uptake. Potassium-evoked release of ACh by slices of striatum was not different between acutely and subacutely treated rats. However, the release of ACh by slices of striatum and hippocampus was significantly increased by atropine in subacutely treated rats. It is suggested that along with the down-regulation of the postsynaptic receptors, subsensitivity of presynaptic functions of the cholinergic synapse also develops during subacute administration of DFP. 0 1987 Academic Press, Inc.

Subacute administration of sublethal doses of organophosphates has been reported to induce tolerance to toxicity with respect to behaviors and biochemical adaptations. One of the most extensively studied hypotheses is that muscarinic receptors become subsensitive to acetylcholine (ACh) after subacute treatment with organophosphates (Churchill et al., 1984a,b; Costa et al., 198 1, 1982; Costa and Murphy, 1982, 1983; Ehlert et al., 1980; Levy, 198 1; Sivam et al., 1983; Yamada et al., 1983a,b). Recently, numerous studies have reported that central nicotinic receptors ’ To whom requests for reprints should be addressed. 465

are also altered after subacute treatment with organophosphates (Costa and Murphy, 1983; Schwartz and Kellar, 1983, 1985). In addition to the alteration of receptors in organophosphate-tolerant animals, it has also been proposed that various presynaptic sites of the cholinergic synapse may also be involved in the development of tolerance to these compounds (Russell et al., 1985; Wecker et al., 1977; Yamada et al., 1983a,b). For example, it has been reported that the release of ACh from brains of tolerant animals is significantly higher than release of ACh from brains of acutely treated animals (Raiteri et al., 198 1; Russell et al., 1985). Therefore, it has OO41-008X/87 $3.00 Copyright 0 1987 by Academic Press. Inc. All right.3 of reproduction in any form reserved.

LIM, HOSKINS,

466

been suggested that the presynaptic cholinergic receptors also become less sensitive to presynaptic inhibition of acetylcholine release during subacute administration of organophosphates. Although several investigators (Costa and Murphy, 1982; Russell et al., 1979, 198 1) have reported that high-affinity choline uptake was not changed during subacute administration of organophosphates, Yamada et al. (1983a,b) observed a significant decrease in high-affinity choline uptake in tissue homogenates from subacutely treated guinea pigs. Acetylcholine content at presynaptic terminals was thus implicated in the phenomenon of tolerance development to organophosphates. Wecker et al. ( 1977) reported that the amount of bound ACh was significantly different after single and subacute treatment with paraoxon, even though the amount of free ACh was similar after the two treatment protocols. Since ACh synthesis is regulated by ACh, CoA, and choline as well as by the activity of choline acetyltransferase (Haga and Noda, 1973; Simon et al., 1976; Yamamura and Snyder, 1973), a change in the regulatory parameters of ACh synthesis could affect the ACh level in tolerant animals. Many aspects of cholinergic function may be involved in the development of tolerance to organophosphates. However, it is still largely unclear as to how such functions are altered during subacute administration of organophosphates. Since diisopropylfluorophosphate (DFP) has been one of the most extensively studied organophosphates, we have designed a study to determine the involvement of various presynaptic and postsynaptic functions of the cholinergic system in the development of tolerance to DFF. METHODS Animals and chemicals. Male Sprague-Dawley rats (Charles River Lab, Wilmington, MA) weighing 175200 g were used throughout the study. The animals were housed four to a cage with free accessto food and water. DFP (lot No. 84F-0248) was obtained from Sigma (St. Louis, MO). This preparation of DFP was found to have

AND HO

an IC50 (concentration required for 50% inhibition of AChE activity in rat brain homogenate) value in vitro of 7 &ml. [‘HlQuinuclidinyl benzilate (QNB) (37.2 Ci/ mmol), [3H]nicotine (73.7 Ci/mmol), and [‘HIcholine (80 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). Other chemicals and reagents of analytical grade were obtained from commercial suppliers. Administration of DFP. Freshly prepared solutions of DFP in saline were administered subcutaneously in volumes of 0.1 ml/ 100 g body wt daily between 9:00 and 1l:OO AM for I3 days. In accordance with our previous studies (Lim et al., 1983; Sivam et al., 1983) the dosage schedule of DFP administration for 13 days was as follows: 1st through 3rd day, 1 mgjkg; 4th through 6th day, 0.5 mg/kg; 7th through 13th day, 1 mg/kg; and on the 14th day, 2 mg/kg, SC.The animals were decapitated for various studies at specified times (i.e., for receptor binding assaysand measurements of ACh release, they were decapitated 24 hr aher the last injection; for studies of choline uptake, they were decapitated 1 and 24 hr after the last administration of either 1 or 2 mg/kg of DFP). The brains were quickly removed and different brain areas were dissected out according to the procedure of Glowinski and Iversen ( 1966). Eight to ten rats were used in each group. The acutely treated rats received daily injections of saline (0.1 ml/ 100 g) for 13 days. DFP, either 1 or 2 mg/kg, sc, was given to these saline-treated rats 24 hr after the last saline injection. These rats were decapitated at the same times as were the subacutely treated rats. The control group received saline vehicle daily for 14 days. [3H]QNB binding assays. 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 frontal cortices and hippocampi were dissected out. The pooled samples were then homogenized in 15 vol of ice-cold 0.32 M sucrose using a Brinkman Polytron PT-10 at low speed (setting 3). The homogenate was centrifuged at 1OOOgfor 10 mitt; the pellet was discarded and the supernatant fluid was centrifuged at 20,OOOgfor 20 min to obtain a crude mitochondrial pellet. The crude mitochondrial pellet was resuspended in double-distilled deionized water and dispersed with a Brinkman Polytron PT-10 (setting 6) for 30 sec. The suspension was centrifuged at 8000g for 20 min. The supematant including the huffy layer was collected and centrifuged at 48,000g for 20 min to obtain a pellet. The pellet was resuspended in water and centrifuged at 48,000g for 20 min. The final membrane preparation was suspended in 50 mM sodium phosphate buffer (pH 7.4). The binding of [‘H]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

DFP AND PRESYNAPTIC [)H]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 PM atropine. The binding was initiated by addition of 0.2 ml of membrane preparation (0.2-0.4 mg protein/ml), and incubations were carried out for 1 hr at 25’C in a shaking water bath. The reaction was terminated by rapid filtering through Whatman GF/B glass fiber filters. Each titer was washed twice with 5 ml of buffer, and the dried filter was transferred to scintillation vials containing 10 ml of Aquasol (New England Nuclear, Boston, MA). The radioactivity retained in the filters was determined by liquid scintillation spectrophotometry. The change in presynaptic muscarinic receptors was determined by the method of Aquilar et al. ( 1979). Crude mitochondrial fractions were layered on a gradient of 0.8 and 1.2 M sucrose and centrifuged at 100,OOOg for 90 min. The bands which equilibrated at 1.2 M sucrose and contained the synaptosomal membranes were isolated and resuspended in 0.05 M phosphate buffer (pH 7.4). Aliquots were incubated with 0.34 X 10e9 M [3H]QNB for 60 min at 25°C. For determining nonspecific binding, parallel experiments were carried out in the presence of 1Om6M atropine. To separate the presynaptic and postsynaptic muscarinic receptor bindings, the membranes carrying the binding sites were treated for 10 min at 0°C with 0.2% Triton X-100 to destroy presynaptic receptors (Aquilar et al., 1979; Cohen et al., 1977; De Robertis et al., 1967) and centrifuged at 100,OOOgfor 60 min. The pellet was washed twice with 1 ml of buffer and solubilized with tissue solubilizer (Protosol). The radioactivity in the pellets was determined by liquid scintillation spectrophotometry. Nicotinic (I’H]nicotine) receptor binding assays. Membrane preparations and binding assays were performed according to the method of Roman0 and Goldstein (1980) with slight modification (Marks and Collins, 1982). After removal of the brains, the striata, hippocampi, and frontal cortices were dissected out. The pooled samples of each area were then homogenized at 4°C in 10 vol of buffer (w/v) using a Brinkman Polytron PT-10 at low speed. The buffer composition was as follows: 118 mM NaCl, 4.8 mM KCI, 2.5 mM CaCl,, 1.2 mM MgSO,, and 20 mM Hepe.s (pH 7.5). The homogenates were centrifuged at 48,000g for 30 min. The pellets were suspended in distilled water (5%, w/v) and allowed to lyse for 60 min. The suspensions were then centrifuged as previously described. The membrane pellets were then suspended in buffer ( 15%, w/v) for the assays.Between 400 and 600 pg of protein in a final incubation volume of 250 pl was used in the binding assays.Binding was initiated by the addition of [‘HInicotine (10 to 200 nM) to samples equilibrated at the final incubation temperature. Incubation was carried out for 2 hr in a shaking water bath at 4°C (in a cold room). Specific binding was calculated as

CHOLINERGIC

FUNCTIONS

467

the total binding minus that occurring in the presence of 1 X low5 M L-nicotine. At the end of incubation, each sample was diluted with 4 ml of ice-cold wash buffer (composition identical to that of the incubation buffer, except that the Hepes concentration was reduced to 5 mM) and filtered under vacuum onto GF/C glass fiber filters which had been soaked in buffer containing 0.1% L-polylysine. The filters were subsequently washed three times with 4 ml ofwash buffer. All dried filters were transferred to scintillation vials containing 10 ml of Aquasol. The radioactivity retained in the filters was determined by liquid scintillation spectrophotometry. High-ajinity choline uptake assays. The method of Yamamura and Snyder (1973) for preparing crude synaptosomes was used with slight modification. Brains were rapidly removed and the discrete areas were dissected out. The pooled samples of each discrete area were then homogenized in 20 vol of ice-cold 0.32 M sucrose containing 100 FM eserine in a Potter glass homogenizer with a Teflon pestle. Since the Ki of eserine for choline transport is in the millimolar range (Diamond and Kennedy, 1969), this concentration was sufficient to prevent hydrolysis of ACh but not high enough to interfere with choline transport. The homogenate was centrifuged at 1OOOgfor 10 min. The supematant was then centrifuged at 17,OOOgfor 20 min. The resultant pellet was reconstituted to between 1 and 2 mg/ml of protein. One-tenth milliliter of the synaptosomal preparation was added to 0.4 ml of solution which consisted of 140 mM NaCl, 5 mM KCl, 1 mivt MgC12, 0.8 mM CaClr, 20 mM Trisbuffer (pH 7.4), 1 mM phosphate buffer (pH 7.4), 10 mM glucose, and varying concentrations of [3H]choline (0.075- 1.OPM). All procedures prior to and after incubation were carried out at 0-4°C. The choline uptake was carried out by transferring incubation tubes to a metabolic shaker, agitating the tubes at 37°C for 5 min, and then rapidly cooling to 4°C. The incubation mixtures were centrifuged at 27,000g for 10 min. “The paired samples” were kept in an ice bath. The pellets were superficially washed twice with 1 ml of ice-cold buffer and solubilized with tissue solubilizer (Protosol). The radioactivity of each sample was determined by liquid scintillation spectrometry. The specific uptake in the synaptosomes was calculated by subtracting the activity of the paired samples from the activity of the incubation samples. ACh release assays. Tissues were prepared according to the method of Nabeshima and Ho ( 1982). The discrete areas were cut into slices 0.5 mm thick with a McIlwain tissue chopper and were immersed in ice-cold buffer containing eserine. The slices, approximately 0.5 X 1 X 1 mm, were then transferred to nylon mesh round-bottom plastic vessels(1 cm in diameter and 1 cm in depth) and preincubated at 37’C for 30 min with 2 ml of Krebs medium containing 0.1 pM [3H]choline and 100 pM eserine. The medium which contained 125 mM NaCl, 3 mM KCl, 1.2 mM MgSO,, 2.6 mM CaClr, 1.2 mM NaHrP& 20 mM NaHC03, and 10 mM glucose was adjusted to pH

468

LIM, HOSKINS, AND HO TABLE EFFECTS OF ACUTE AND SUBACUTE IN THE RAT

I

ADMINISTRATION

OF DlT

Cortex Treatment

&

ON [‘H]QNB

BINDING

CORTEX AND H~PP~CAMPUS Hippocampus

B max

Control 0.073 + 0.014 1252 + 52 Acute 1 w/kg 0.067 + 0.012 1102 f 58 2 w/kg 0.059 f 0.011 1071 a51” Subacute 7 days 0.052 + 0.008 784 + 20’ 14 days 0.058 + 0.009 800 f 28’ 13 days, and then a challenge dose of 2 m/kg 0.056 k 0.007 763 + 13’ Subacute for 13 days, 3 days after the last dose of 1 w/kg 0.05 1 f 0.005 817f21’ 2 w/kg 0.056 + 0.010 893+ lib

Treatment Control Acute 2 &kg

Kd

B max

0.077 t- 0.008

1459 ” 52

0.076 + 0.004

1212 zk 42”

Subacute for 13 days, and then a challenge dose of 2 a/k 0.063 + 0.002 743 + 18b

Note. Kd, nM; B,, , pmol/g protein. The values are the mean f SE of four separate experiments done in duplicate. The Kd and B,,,, values were derived from Scatchard analysis of the binding data. The rats were killed 24 hr after designated dose. “p < 0.05, compared to the respective control values. bp < 0.00 1, compared to the respective control values. 7.4 by bubbling with 95% Oz-5% CO2 (Carbogen) immediately prior to use. At the end of the preloading period, the slices were washed twice with 2 ml of prewarmed medium and transferred to a fresh medium at 5-min intervals for 50 min for spontaneous release. The bottom of each vessel was blotted before each transfer. The slices were then transferred to 2 ml of medium containing 25 or 50 mM KC1 instead of NaCl and various concentrations ofatropine (1 O-* - 10m5M) at S-min intervals for 20 min. The slices were incubated at 37°C under a Carbogen atmosphere in a Dubnoff metabolic shaking incubator. An aliquot of medium from each time point was pipetted into a scintillation vial containing 10 ml of Safety-Solve. The radioactivity remaining in the slice was counted after solubilizing the tissue with tissue solubilizer (Protosol). The radioactivity of each sample was determined by liquid scintillation spectrophotometry. Percentage of release was defined as 100 X (dpm of out flow)/(dpm of accumulation in the slices). The accumulation oftritium within the slices was calculated as the sum of the outflow of [‘H]ACh during superfusion and the amount recovered from the tissue at the end of the experiment. The effect of atropine on [K+]-evoked release of ACh in each slice was expressed as 100 X (% of release with atropine)/ (% of release.without atropine). Determination of protein concentration. The protein content of the membrane preparations was determined by the method of Lowry et al. ( 195 1) using bovine serum albumin as a standard.

Statistics. The receptor binding of [3H]QNB was analyzed by the method of Scatchard (1949). The receptor binding of [3H]nicotine was analyzed by the graphic method of Scatchard ( 1949) and Thakur et al. ( 1980) to estimate the Kd and B,,,, values. The results of high-affinity choline uptake studies were analyzed by the double-reciprocal plot method to obtain K,,, and V,,,, Data for muscarinic receptor (cortex) and choline uptake (24 hr) were analyzed for significance by one-way analysis of variance and Dunnett’s test. Data for the K+-evoked release of ACh by atropine (striatum) were analyzed for significance by the analysis of covariante and the Newman-Keuls test. Other data were analyzed by Student’s t test.

RESULTS Eflects of DFP administration on muscarinic and nicotinic receptors. Similar to our previously reported results on rat striatum (Sivam et al., 1983), a single administration of DFP (1 mg/kg) failed to alter either the Kd or the B,, of muscarinic receptors in the cortex (Table I), but a high dose of DFP (2 mg/ kg) reduced the II,, to about 85% of the control value without a change in I& in either

DIP AND PRESYNAPTIC

CHOLINERGIC

FUNCTIONS

469

Eflects of DFP administration on highafinity choline uptake. The results of the dou-

Triton

X-100

%

FIG. 1. Change in specific binding of [‘H]QNB to the synaptosomal membranes of frontal cortex in the untreated controls (c)and after treatment with 0.2% Triton X-100. The results were calculated from the specific binding sites using 0.34 nM [‘H]QNB and the protein concentration after solubilization with detergent (n = 4).

cortex or hippocampus 24 hr after the administration. After subacute administration of DFP, the B,,,, was significantly decreased in both cortex (40%) and hippocampus (50%) without a change in & values. During repeated administration of DFP, binding sites of muscarinic receptors in cortex were maximally reduced after 7 days. Thereafter, the density of muscarinic receptors remained the same throughout the injection period of 14 days. After destruction of presynaptic muscarinic receptors with 0.2% Triton X-100, the remaining [3H]QNB binding sites (postsynaptic receptors) in all treated groups were decreased to the same extent (20%) (Fig. 1). Scatchard analysis of [3H]nicotine binding revealed the presence of two components over the range of concentrations tested. The results of analyses of [3H]nicotine saturation binding curves after a single and repeated administration of DFP are summarized in Table 2. Neither the Kd nor the B,, of high- and low-affinity binding sites was altered after a single administration of 2 mg/kg DFY. However, after repeated administration, the binding density of the high-affinity sites was significantly decreased in both striatum (40%) and cortex (60%) without changes in affinities. Neither the Kd nor the B,, of the lowaffinity sites was altered.

ble-reciprocal analysis of the accumulation of [3H]choline in each area of rat brain after various DFP treatments are summarized in Table 3 (1 hr after the last injection) and Table 4 (24 hr after the last injection). A single injection of DFE (1 or 2 mg/kg) failed to alter either the K, or the V,,,, except for a 25% decrease in the V,, in the hippocampus 1 hr after DFP treatment. In rats receiving subacute treatment with DFP for 7 and 14 days, the V,, was significantly decreased in the striatum and hippocampus 1 hr after the last treatment; however, there was no change in the cortex. The V,, of high-affinity choline uptake was decreased to 75% (in striatum) and 60% (in hippocampus) of the respective control values 1 hr after the additional dose of 1 mg/ kg DFP on the 14th day. When a higher dose of DFF’, 2 mg/kg, was administered on the 14th day, these values were further decreased to 67% (in striatum) and 46% (in hippocampus) of the control value 1 hr after the administration. Even 24 hr after the additional high dose of 2 mg/kg DFE, V,, was still significantly decreased to 76% (in striatum) of the controls. However, at 24 hr after the additional dose of 1 mg/kg DFP, and 72 hr after the additional high dose (2 mg/kg), the values were comparable to the control values. None of the above treatments affected the K, value of high-affinity choline uptake. The highaffinity choline uptake in the striatum was not changed by up to low5 M DFP, in vitro. The uptake of [3H]choline was markedly reduced by 0.1 PM hemicholinium-3 in the striatum and the hippocampus (Fig. 2). The reduction of choline uptake by hemicholinium-3 was concentration-dependent. However, the degree of inhibition by hemicholinium-3 was not different among the control, acute, and subacute treatments.

Eflect ofDFP administration on the release of13H]ACh. The release of ACh in the striatal slices was K+- and atropine-concentration dependent. Neither subacute nor acute treat-

470

LIM, HOSKINS, AND HO TABLE 2 EFFECTSOF ACUTE AND SUBACUTE ADMINISTRATION IN THE RAT STRIATUM,

Treatment

&R

Striatum Control 18.0 + 1.1 Acute, 2 mgjkg 16.4 f 0.1 Subacute for 13 days, and then 2 m/kg 16.1 f 0.8 Cortex Control 19.5 + 2.7 Acute, 2 mg/kg 21.3 + 2.2 Subacute for 13 days, and then 2 w/kg 17.3 + 1.4 Hippocampus Control 15.3, 13.2 Acute, 2 mg/kg 14.8, 12.8 Subacute for 13 days, and then 2 w/k 15.8, 13.8

CORTEX,

OF DFP ON [3H]N~~~~~~~ AND HIP~~CAMPUS

B maxtl

BINDING

B -I.

KdL

46.4 zk 3.6 34.0 + 4.0

180.1 f 9.1 182.3 + 11.3

488.3 + 13.7 503.7 f 27.9

28.0 + 5.4”

223.9 rt 17.9

532.1 rt 35.9

51.4 + 12.3 32.4 + 6.0

219.9? 7.5 228.8 + 5.4

486.3 f 18.6 453.9 + 14.6

23.8 k 3.8”

235.0 f 10.6

480.3 + 19.0

15.4, 19.6 13.1, 18.2

248.8,238.0 252.2,298.7

380.4,308.8 364.3,319.3

10.7,9.3

363.0, 131.9

323.3,300.4

Note. Km and KdL, nM; BmaxHand BmaxL,pmol/g protein. The values are the mean + SE of four determinations done in duplicate. The rats were killed 24 hr after designated dose. Similar results were also obtained in a separate experiment. Up < 0.05 compared to the respective control values.

TABLE 3 EFFECTS OF ACUTE AND SUBACUTE ADMINISTRATION OF DFT ON HIGH-AFFINITY CHOLINE UPTAKE STRIATUM, HIPPOCAMPUS, AND CORTEX AT 1 HR AFTER THE LAST INJECTION

IN THE RAT

Region Striatum Treatment

fGl

Acute Control 0.82 f 0.10 1 m/kg 0.84 +- 0.14 2 m/kg 0.85 +0.16 Subacute for I days Control 0.80 + 0.05 1 mtib 0.67 + 0.06 Subacute for 13 days, 1 mg/kg, Control 0.99+0.11 1 w/k 0.77 + 0.04 2 m/kg 0.87 f 0.05

Hippocampus Vma%

K,

Cortex VInax

Kfll

vmax

0.254 f 0.029 0.275 + 0.022 0.305 + 0.047

1.15 +0.15 1.08 + 0.40 1.06?0.10

0.108 -t 0.006 0.084 + 0.0 11 0.079 It 0.007”

1.54 + 0.09 1.39kO.14 1.54kO.17

0.074 + 0.004 0.066 + 0.008 0.066 * 0.007

0.216+0.010 0.160 f 0.010” and 0.284 + 0.018 0.210 + 0.017” 0.191 kO.021”

1.50 + 0.22 1.37 f 0.29

0.118*0.006 0.068 + 0.008”

2.09 + 0.38 2.17 + 0.29

0.043 f 0.007 0.043 f 0.004

1.37 f 0.46

0.122 kO.015 0.081 + 0.015” 0.057 + 0.012”

1.20 + 0.20 1.33 + 0.25 1.21 + 0.18

0.047 + 0.006 0.059 + 0.008 0.044 f 0.007

1.54 + 0.25 1.68 + 0.40

Note. K,,,, rmol; Vm, nmol/mg protein/5 min. The values are the mean rt SE of four separate experiments done in duplicate. Op < 0.05, compared to the respective control values.

DPP

AND

PRESYNAPTIC

CHOLINERGIC TABLE

471

FUNCTIONS

4

EFFE~TSOFACUTEANDSUBACWTEADMINISTRATION OFDPP ONHIGH-AFFINITY CHOLINEUPTAKEINTHERAT STRMTLJM, HIPPOCAMPUS, AND CORTEX AT 24 HR AFTER THE LAST INJECXON Region Striatum Treatment Control Acute 1 w&g 2 w&g Subacute for 1 w/k Subacute for 1 w/k 2 mg/k Subacute for 3 days after 1 mgjkg 2 w/k

N 9

V *ax

Kn 0.73kO.07

3 0.63 k 0.07 4 0.79 f 0.07 7 days 3 0.55+0.06 13 days, 1 mg/kg, and 3 0.58 + 0.03 5 0.62 kO.03 13 days, then the last dose of 3 0.92 f 0.06 3 0.77 + 0.02

Hippocampus

Cortex

V max

&

KIVI

vmax

0.160~0.010

0.88 -t 0.07

0.05 1 + 0.003

1.45 + 0.3 1

0.047

0.156 0.148

0.70 -t 0.02 1.26 r 0.40

0.044 + 0.003 0.053 + 0.016

1 .O 1 + 0.08 0.99 + 0.11

0.034 + 0.004 0.042 + 0.005

0.160~0.011

0.98 k 0.04

0.062

1.11 +0.19

0.042+0.004

0.140 k 0.005 0.122 +0.005’

0.90 + 0.10 0.72+0.06

0.043 AZ 0.008 0.041 +0.002

1.06 kO.15 1 .Ol f 0.14

0.033 + 0.004 0.044 + 0.006

0.193 + 0.011 0.129 + 0.012

0.97 + 0.10 1.20 + 0.10

0.058 + 0.006 0.055 + 0.009

1.72 ? 0.29 1.42 + 0.20

0.065 0.040

+ 0.003 + 0.009

+ 0.003

Note. K,,,, pmol; V,,,,; nmol/mg protein/5 min. The values are the mean + SE of separate by N. “p < 0.05, compared to the control values.

ment affected the K-evoked release of [3H]ACh. In striatal slices from subacutely treated rats, the K-evoked release of [3H]ACh was significantly increased by atropine ( 1Oe8, I Oe6 M). However, atropine was equally effective in increasing ACh release in striatal slices prepared from acutely treated and control rats (Fig. 3). Further analysis using the analysis of covariance among three response lines after the probit transformation revealed that the slopes of the three lines were not different (F(2, 64) = 1.264); however, the degrees of elevation of the three lines were significantly different (F(2,66) = 4.258;~ < 0.05). Further tests showed that the response curve after subacute treatment was significantly more elevated than were those of both controls and acute treatment. Atropine, at the concentration of 10e6 M, also enhanced the K+-evoked release of ACh in slices of hippocampus from subacutely treated rats as compared with that of the control rats. However, the enhance-

experiments

* 0.005

-c 0.012 + 0.007

as indicated

ment of ACh release by atropine was not observed in DFP acutely treated animals. The effect of atropine on release of ACh by slices of frontal cortex was not significantly different among all groups (Table 5). DISCUSSION The present results show that various biochemical changes, such as down-regulation of muscarinic and nicotinic receptors, decreased high-affinity choline uptake, and increased atropine stimulation of K+-evoked ACh release, take place after the development of tolerance to DFP. It has been well demonstrated that behavioral tolerance to DFP develops when brain acetylcholinesterase activity falls below 30% of control activity. It has also been suggested that this behavioral tolerance may result from a decrease in density of muscarinic receptors (Brodeur and Dubois, 1964; Costa and Murphy, 1982; Overstreet et

472

LIM, HOSKINS, AND HO

s E

~~~“I~~~ $

-..--....9 -t

10

E a

.

0

9

Hemicholinlum-3

It has been reported that the treatment of synaptosomes with a mild concentration of Triton X- 100 destroys presynaptic muscarinic sites of synaptic membranes (Cohen et al., 1977; De Robertis et al., 1967). The present results reveal that the decrease in muscarinic receptor density in the synaptic preparation which had been treated with Triton X100 was the same among the acute, subacute, and control groups. The presynaptic sites which corresponded with the difference before and after Triton X-100 treatment was smaller for the subacutely treated group than for the other groups (Fig. 1). These results suggest that down-regulation of muscarinic sites occurred at presynaptic sites as well as at postsynaptic sites after subacute administration of DFP. Although the characteristics of the CNS nicotinic receptors are the subject of debate (Abood et al., 1980; Lippiello and Fernandes, 1986; Marks and Collins, 1982; Schwartz et al., 1982), our study reveals that there are two nicotinic binding sites. Recently, several in-

8 Concentration

7

(-log

M)

FIG. 2. Effect of hemicholinium-3 on high-affinity choline uptake in striatum and hippocampus of each treatment group. The synaptosomes in each treatment group 1 hr after DFP administration were incubated with 0.1 PM choline and various concentrations of hemicholinium-3 for 5 min. The values are the mean + SE of four determinations performed in duplicate.

al., 1974). In the present studies, the downregulation of muscarinic receptors was monitored during the course of DFP treatment. In cortex and hippocampus from rats treated with DFP daily for 2 weeks, the muscarinic receptor density was significantly decreased without changes in Kd values as has been previously reported in rat striatum (Sivam et al., 1983). Interestingly, the value of muscarinic receptor density in cortex after the 7th injection was the same as that after the 14th administration. These results seem to suggest that there is a threshold density of muscarinic receptors which maintains cholinergic activities.

k 8 Atroplw

7 Concentretlon

6

6 (-log

Y)

RG. 3. Effect of atropine on evoked release of ACh in the striatal slices of each treatment group. The slices were incubated with 0.1 pM [3H]choline at 37°C for 30 min. The slices were transferred to a fresh medium every 5 min for 50 min for spontaneous release. The slices were then incubated in 2 ml of medium containing 25 mM KC1 and various concentrations of atropine at 5-min intervals for 20 min. The results were calculated as 100 x (b of release with atropine)/(% of release without atropine). The values are the mean & SE of 3 to 10 determinations performed in duplicate.

DFF AND PRESYNAPTIC

CHOLINERGIC

473

FUNCTIONS

TABLE 5 EFFECTSOF ATROPINE ON THE [K+]-EVOKED RELEASE OF ACh AT 24 HR AFTER THE LAST ADMINISTRATION OF DFP IN THE SLICE OF EACH AREA OF RAT BRAIN Atropine (M)

0

Striatum (25 mM KCl)” Control 100 Acute 100 (2adk) Subacuteb 100 (2w/kg) Hippocampus (50 mM KCl)” Control 100 Acute 100 (2w&9 Subacute’ 100 (2w/k) Cortex (50 mM KCl)’ Control 100 Acute 100 (2 mg/W Subacute b 100 (2 w&g)

10-a

lo-’

1o-6

1o-5

103 + 3

114f

3

121 f 3

130+

101+ 5

118&

7

124?5

133-c 12

118*6’

119*

7

137 + 5’

1362

6

8

118+4 124?3 139 f 7’ 112*

3

119+3

108&

9

126+8

133* 13

155+s

’ The concentration of [K+] for the evoked release in the slice. The results were calculated as 100 X (% of release with atropine)/(% of release without atropine). b Treated daily for 13 days according to the dosing schedule (Methods) followed by an injection of 2 mg/kg DFP. ‘p < 0.05, compared to the control values. The values are the means -C SE of 3 to 10 determinations done in duplicate.

vestigators have reported that the density of nicotinic receptors was also down-regulated after subacute treatment with organophosphates (Costa and Murphy, 1983; Schwartz and Keller, 1983, 1985). Our results substantiate these findings in that nicotinic binding sites were reduced to the same extent as were muscarinic binding sites. Although the functional role of the low-affinity nicotinic binding sites is unknown, the high-affinity binding sites of nicotinic receptors were only affected by the subacute treatment with DFP. Thus, the reduction in the number of both muscarinic and high-affinity sites of nicotinic receptors appears to be an adaptive response to develop subsensitivity to an increase in synaptic ACh levels during subacute treatment with DFP. Yamamura and Snyder (1973) reported that the high-affinity choline uptake process

represents a selective accumulation of choline by cholinergic neurons in rat brain. Since then, this high-affinity choline uptake system has been suggested as one of the sites which regulate ACh synthesis (Haga and Noda, 1973). It has been reported that high-affinity choline uptake in organophosphate-tolerant animals was not changed (Costa and Murphy, 1982; Russell et al., 1979, 1981). However, Yamada et al. (1983a,b) have reported that subacute administration of DFP to guinea pigs caused a significant decrease in choline uptake at the higher dosing schedule. The observed dissimilarity in the high-affinity choline uptake as reported by other investigators (Russell et al., 1979) might be due to the brain areas used. Discrete brain areas were used in our study, while whole brains were used in those studies. Our results show that although acute administration of DFP did

474

LIM, HOSKINS,

not change choline uptake, uptake in the tolerant rats was significantly decreased at an early hour (1 hr) after an additional dose of 1 mg/kg DFP. The reduced high-affinity choline uptake was also substantiated by the addition of hemicholinium. This uptake process returned to the control level 24 hr later except when a higher additional dose of DFP (2 mg/kg) was administered. Thus, the DFPinduced alteration in high-affinity choline uptake was short and transient. Possible explanations for this include (i) the highly elevated free ACh produced after each administration may inhibit the choline uptake (Yamamura and Snyder, 1974); (ii) rapid conformational changes in the choline uptake complex may have occurred, resulting in changes of exo-endocellular concentrations of choline during subacute administration of DFP. The exact role of the transient change of high-affinity choline uptake remains to be investigated. The reduced high-affinity choline uptake in the early hours could attenuate the early toxic symptoms by decreasing the synthesis of ACh. However, at later times, the recovered choline uptake might result in the prevention of the underactivity of choline& neurons whose muscarinic and nicotinic receptors are already down-regulated. Wecker et al. ( 1977) reported that the total increase of ACh in chronic paraoxon-treated rats was less than half of that in acutely treated rats. Furthermore, bound ACh levels were higher after acute treatment than after chronic treatment. However, the free ACh levels were the same after both treatments. It has been demonstrated that synthesis of existing ACh is regulated by the concentration of ACh, CoA, and choline as well as by choline acetyltransferase activity (Haga and Noda, 1973; Simon et al., 1976; Yamamura and Snyder, 1973). Our results suggest that the transient, decreased high-affinity choline uptake in tolerant rats might play a role in the development of tolerance by decreasing the synthesis of ACh in the early hours. This decreased high-affinity choline uptake in rats receiving subacute administration of DFP

AND HO

could be an adaptive change which would regulate ACh availability at presynaptic sites. It has been reported that the release of ACh was inhibited less in brains from rats treated subacutely with paraoxon (Raiteri et al., 198 1) and DFP (Russell et al., 1985) than in brains from control animals. The presynaptic autoreceptor in the cholinergic nerve terminal was proposed to regulate the release of bound ACh in order to maintain an adequate amount of free ACh in synapse (Kilbinger, 1984; Molenaar and Polak, 1980). Also, it has been reported that animals tolerant to organophosphate are subsensitive to muscarinic agonists (Brodeur and Dubois, 1964; Costa et al., 198 1; Costa and Murphy, 1982) and supersensitive to muscarinic antagonists (McPhillips, 1969; Modrow and McDonough, 1986; Overstreet, 1973). Therefore, the decrease in presynaptic muscarinic receptor densities could explain the increase in ACh release; that is, the increased release of ACh might result from down-regulation of presynaptic muscarinic receptors which show supersensitivity to muscarinic antagonists and serve to further regulate the ACh content in synaptic terminals of tolerant animals. The present results obtained on various biochemical parameters of the choline& system suggest that the adaptation in cholinergic function during tolerance development to DFP might be sequential to the daily administration with DFP as follows: the ACh level in brain was significantly increased by the inhibition of AChE activity. The initially elevated free ACh induced an increase in bound ACh content by acting on the presynaptic muscarinic autoreceptors. Meanwhile, continuous elevation of free ACh during daily administration also caused down-regulation of both muscarinic and nicotinic receptors which reached a peak after the seventh administration. The decrease in postsynaptic neuronal activity by down-regulation would be countered by the abnormally released ACh to maintain the normal cholinergic relationships within the synapse. Finally, the reduced high-affinity choline up-

DFP

AND

take after each additional elevation in bound ACh.

PRESYNAPTIC

dose lessened the

ACKNOWLEDGMENTS This work was supported by Contract DAMD17-85C-5036 from U.S. Army Medical Research and Develop mental Command and NIH Biomedical Research Sup port Grant Program 2S07RR05386.

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