Reduced binding of (3H)-Quinuclidinyl benzilate associated with chronically low acetylcholinesterase activity

Life Sciences, Vol . 24, pp . 1159-1164 Printed in the U .S .A .

Pergamon Press

REDUCED BINDING OF ( 3H)-QUINUCLIDINYL BENZILATE ASSOCIATED WITH CHRONICALLY LOW ACETYLCHOLINESTERASE ACTIVITY Grant D . Schiller School of Biological Sciences The Flinders University of South Australia, Bedford Park, 5042 (Received in final form February 13, 1979)

Sumrnary ( 3H)-Quinuclidinyl benzilate (QNB) binding was examined in the cortex, striatum and hippocampus of rats repeatedly exposed to the anticholinesterase, diisopropyl fluorophosphate (DFP) . Compared to vehicle-treated controls, a reduction in maximal binding of 2530% was observed in these brain regions . The reduction in binding was associated with regional acetylcholinesterase (AChE) inhibition of 80-90% . A tendency of lower muscarinic acetylcholine receptor (m-AChR) affinity for ( H)-QNB in control preparations, compared to those from DFP-treated rats, was observed . However, only in the case of the striatal control homogenate was there a significant increase in apparent Kp . A concomitant feature of chronically low AChE activity is therefore a reduction, mainly in number, of m-AChR's . These findings support the hypothesis that in the central nervous system (CNS) DFP tolerance and cholinomimetic subsensitivity may involve the m-AChR . Both behavioral and physiological tolerance develops to repeated exposure of the organophosphate cholinesterase inhibitor, DFP . Tolerance is marked by an associated phenomenon of altered responsiveness to agonist and antagonist drugs which affect the central cholinergic system (1,2,3) . Russell et al (1) postulated that there was a reduction in the postsynaptic m-AChR sénsitivity as a result of chronic AChE inhibition . Although a combination of mechanisms may be involved in adaptation to chronic organophosphate exposure, recent evidence has suggested an involvement of the m-AChR . Reduced binding and affinity of (3H)-QNB for m-AChR's in longitudinal muscle of the rat ileum has been reported following repeated exposure to DFP (4) . The reduction in binding was associated with a diminished contractile responsiveness of ileum to muscarinic agonists . The purpose of the present study was to assess more directly possible CNS changes in the binding characteristics of m-AChR's following repeated ex sure to DFP . (H)-QNB, a potent muscarinic antagonist and an avid ligand (5~ was used in the present studies to investigate binding in the cortex, striatum and hippocampus of brain from control and DFP-treated ra is . Materials and Methods Male Hooded Wistar rats (250-35b g) received an initial dose of 1 mg/kg i .m . of DFP (Calbiochem), in anhydrous peanut oil as the vehicle . Subsequent 0 .5 mg/kg doses were administered every 72 h . Treatment was continued for 20 administrations . Initial treatment was marked by symptoms of parasympathetic 0024-3205/79/131159-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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over-activity and weight loss . Behavioral and physiological tolerance to this dose regimen has been well documented (1,3) . Controls received the vehicle . Animals were sacrificed by decapitation 24 h following the last injection ; brains were removed and rapidly dissected on ice into cortical (generalized sample), striatal and hippocampul regions . Three tissue samples were pooled per region, weighed and homogenized in 20 volumes of ice cold isotonic sucrose . The Sl fraction from a 1,000 X g ; 10 min centrifugation (Sorval RC-5) was stored frozen at -20 oC . Immediately before use the samples were thawed and polytronned (Brinkman polytron, PT-10) at a setting of 5 for 60 sec . ( 3H)-QNB (16 .4 Ci/mmole ; Radiochemical Center, Amersham) binding assays were performed using a rapid filtration technique, essentially as described by Yamamura and Snyder (5) . Tissue homogenate (=0 .5 mg pr~tein) was incubated at 37 oC for 40 min in a final volume of 2 ml containing ( H)-QNB in 0 .05 M (Na-K) phosphate buffer, pH 7 .4 . Nonspecific binding was determined using parallel tubes containing 10 -4 oxotremorine (Aldrich Chem . Co .) . The incubation was terminated by the addition of 3 ml of ice cold 0 .05 M phosphate buffer, pH 7 .4 . and immediate filtration with a 3 X 3 ml buffer rinse . Binding to filter papers (Whatman GF/B) was examined in the absence of tissue homogenate . All binding determinations were performed in duplicate . Radioactivity was determined in a Serle MKIII scintillation counter at an efficiency of 45% . AChE activity was assayed according to Ellman et al (5) and protein was determined by the method of Lowry et al (7) using bovineserum albumin as a standard . Four independent binding studies were performed . Results The amount of (3H)- QNB bound in the presence of 10 -4 M oxotrenarine contained a saturating component over the concentrations of labelled ligand used . Therefore, the usual definition of specific binding ; namely, the total bound ligand minus that bound in the presence of an excess of competitor, could not be used . More rgcent observations (Sc~iller, in preparation) have indicated that 10-3 to 10 - oxotremoring, or 10 - M atropine sulfate, is required to maximally displace (3H)-QNB binding and yield a linear, nonspecific binding function . At these latter concentrations of competitive displacing drug, the ratio of specific to nonspecific binding is greatly increased, and (3H)-QNB bound approximates the background binding to filter papers . This indicates that QNB is a highly specific ligand . For purposes of the present comparison, binding has been defined as that totally bound minus filter bound . In cortex, striatum and hippocampus of DFP-treated and control rats ( 3 H)-QNB binding was saturable with increasing concentrations and half maximal binding occurred around 0 .4 nM . The data was subjected to Scatchard analysis and binding constants (Kp ; B ,ax) were determined by linear regression . For control tissued preparations the binding capacity (Bmax) was highest in the protein), marginally lower in the l cortex (1800 fmoles stn atom (1857 fmoles mgmg - protein) and lowest in the hippocampus (1451 fmoles mg - protein) . There were no significant differences in apparent KD values between control brain areas (striatum KD = 0 .51 nM ; cortex Kp = 0 .54 nM ; hippocampus Kp = 0 .37nM ; p > 0 .05) . Con~rol ~ChE activity (expressed as umoles acetylthiocholine iodide hydrolozed min - mg - protein) was greatest in the striatum, 107±9 (z ; s .e .m . ; n = 4) ; followed by the hippocampus, 22 .8±1 .8, and cortex, 16±1 .8 . Compared to controls, DFP treatment reduced AChE activity to 11 .7±1 .7% ; 17 .1±2 .8% and 14 .8±1 .6%, (z ; s .e .m . ; n's = 4), in striatum, hippocampus and cortex, respectively . Associated with the dramatic red~ction of AChE activity by DFP, there was a reduction in the concentration of ( H)-QNB binding sites (Tables I-III) . The loss of binding capacity does not appear to be attributable to DFP binding to the m-AChR . When control homogenates were preincubated with 10 -5 M DFP

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(producing a 100% inhibition of AChE activity), there were no losses of binding capacity . For "DFP" striatal tissue there was a significant reduction of about 30% in Box compared to control, as determined by Scatchard analysis (Table I) . Significant differences in binding between control and DFP fractions were observed abôve ( 3 H)-QNB concentrations of 0.5 nM . Since binding at lower concentrations was not significantly different, it suggests the change in binding characteristics are predominantly towards a reduction in receptor number rather than a change in affinity . However, the difference in the apparent Kp's for striatun were marginally significant (control Kp = 0 .51 nM ; DFP KD = 0 .36 nM ; p < 0 .05) . TABLE I ( 3 H)-QNB Binding in Striatum of Control and DFP Treated Rats Ligand Concentration nM 0 .05 0 .25 0 .50 0 .80 1 .Q0 2 .00 4 .00

( 3 H)-QNB Bound : Mean (fmoles mg -1 protein) t s .e .m . -~ -- 4~ Control DFP 82 398 152 1071 1196 1408 1495

B~x

apparent Kp(nM)

± 6 ± 27 t 47 t 57 t 68 t 58 t 50

84 383 653 889 950 1037 1073

1857 t 54

± 4 ± 18 t 27 ± 441 t 41 1 t 39 3 ± 583

1308 t 54 4

0.51 t 0 .06

0 .36 ± 0.021

1 Significantly different from control, p < 0 .05 ;

t-2 tailed 3 Significantly different from control, p < 0 .01 ; t-2 tailed 4 Significantly different from control, p < 0 .001 ; t-2 tailed TABLE II ( 3H)-QNB Binding in Cortex of Control and DFP Treated Rats Ligand Concentration nM 0 .05 0 .25 0 .50 0.80 1 .00 2 .00 4 .00

( 3 H)-QNB Bound : Mean (fmoles mg -1 protein) t s .e .m . 78 368 687 992 1156 1342 1406

Bmax apparent Kp(nM)

~~_4)

Control t ± t t ± t

11 42 75 97 100 88 86

1800 t

91

t

0 .54 t

0 .05

1 Significantly different from control, p < 0.05 ; 2 Significantly different from control, p < 0.02 ;

74 336 597 786 898 1065 1099

DFP

t 12 ± 46 t 76 ± 80 ± 83 t 74 ± 751

1358 t 862

0.46 t 0 .07 t-2 tailed t-2 tailed

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In "DFP" cortex and hippocampus a reduction in B ox of about 25% compared to controls was observed . Control hippocampul fractions bound significantly more ( 3 H)-QNB than DFP fractions above 0 .5 nM, while this was only evident at 4 .0 nM for cortex . There were no significant differences in the apparent KD values between control and DFP in cortex nor hippocampus (Tables II-III) . TABLE III ( 3 H)-QNB Binding in Hippocampus of Control and DFP Treated Rats Ligand Concentration nM

-1 protein) ± s .e .m . ( 3 H)-QNB Bound : Mean (fmoles mg (n = 4) Control 6 68 87 77 75 70 54

DFP ± ± ± ± ± ± ±

7 22 34 401 321 402 333

78 423 764 986 1044 1178 1198

B~ x

1451 ± 61

1070 ± 36

0 .37 ± 0 .07

0 .31 ± 0 .01

apparent Kp(nM)

± ± ± ± ± ± ±

81 385 614 766 827 882 890

0 .05 0 .25 0 .50 0 .80 1 .00 2 .00 4 .00

1 Significantly different from control, p < 0 .05 ; 2 Significantly different from control, p < 0 .02 ; 3 Significantly different from control, p < 0 .01 ;

t-2 tailed t-2 tailed t-2 tailed

Discussion The present findings indicate that following chronic reduction of AChE activity there is a decrease in the number of m-AChR's . This is demonstrated by reductions in ( 3 H)-QNB Box of up to 30% in some of the most cholinergic rich areas of the brain . The reduction in binding capacity within the CNS appears to be a generalized phenomenon since the decrease was of similar magnitude in the cortex, striatum and hippocampus . With the exception of the striatum, where a marginally significant difference in Kp was noted between DFP and control, there were no appreciable changes in affinity of the m-AChR for ( 3 H)-QNB . Chang et aZ . (8) point out that when receptor concentrations exceed 0 .1 Kp, (as in the present studies), apparent K increases as a function of the receptor concentration . This may account for tRe higher Kp values observed for control fractions compared to those for "DFP" . Ideally apparent affinities should be determined at equal concentrations of receptor when comparing between tissues . The observed distribution of m-AChR's in control brain, striatum > cortex > hippocampus, parallels that reported by Kobayashi et aZ . (9) . The ~ B x (=0 .4 nM ( 3 H)-QNB) and the apparent K values (0 .31 0 .54 nM) are compara~e to those previously reported for the ~ammalian CNS (4,10,11) . Functionally, receptor sensitivity may be altered through changes in either their number and or affinity (12 . The observed reduction in m-AChR number may therefore account for the reported behavioral subsensitivity to cholinomimetics following repeated DFP treatment (1) . Further, the reduction in m-AChR's may, in part, represent a mechanism underlying tolerance to

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chronically low AChE levels . What triggers an adaptive change in receptors remains uncertain . It has been suggested that in the peripheral nervous system increased levels of ACh may lead to a decrease in the number of AChR's (13) . However, in the central nervous system Russell et al . (submitted for publication) were unable to find arty significant changes in the concentration of acetylcholine (ACh), choline, high affinity transport of choline .or rate of synthesis of ACh as a result of acute or repeated DFP administration . Other aspects of ACh release/metabolism/half life warrant investigation . It is likely for example, that under conditions of low AChE activity there is an "overstimulation" of AChR's . For catecholamine receptors at least, there is is a correlation of transmitter overstimulation with induced receptor hyposensitivity (14) . Regardless of the mechanisms leading to receptor desensitization, the present findings provide evidence for central m-AChR chânges which may contribute to our understanding of tolerance to low AChE activities . Açknowle dgem ents The author would like to thank Dr . D .H . Overstreet for his support and interest in these studies . References 1 . R .W . RUSSELL, D .H . OVERSTREET, C .W . COTMAN, V .G . CARSON, L . CHURCHILL, F .W . DALGLISH and B .J . VASQUEZ, J . Pharmacol . Exp . Ther . 19 2 1 73-85 (1975) . 2 . T .J . CHIPPENDALE, G .A . ZAWOLKOW, R .W . RUSSELL and D .H . OVERSTREET, Psychopharmacologia 26 127-139 (1972) . 3 . D .H . OVERSTREET, R .W.RUSSELL, B .J . VASQUEZ and F .W . DALGLISH, Pharmacol . Biochem . Behav . 2 45-54 (1974) . 4 . F .J . EHLERT and N. KOKKA, Proc . West . Pharnwcol . Soc . 2 0 1-7 (1977) . 5 . H .I . YAMAMURA and S .H . SNYDER, Proc . Nat . Acad . Sci . U S~1 71 5 1725-1729 (1974) . 6 . G .L . ELLMAN, K .O . COURTNEY, V . ANDRES and R .M . FEATHERSTONE, Biochem . Pharmacol . 7 88-95 (1961) . 7 . O .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR and R .J . RANDALL, J . Biol . Chem . 193 265-275 (1951) . 8 . .J K . CHUAG, S . JACOBS and P . CUATRECASAS, Biochim . Biophys . Acta 406 294-303 (1975) . 9 . G .M . KOBAYASHI, M . PALKOVITS, R .E . HRUSKA, R . ROTHSCHILD and H .I . YAMAMURA, Brain Res . 154 13-23 (1978) . 10 . Y . KLOOG and. SOKOLOVSKY, Brain Res . 134 167-172 (1977) . 11 . H .I . YAMAMURA, G . WASTEK, K .J . CHUAG an~ .H . SNYDER, Proc . West . Pharmacol . Soc . 1 9 13-18 (1976) . 12 . W .W . FLEMING, In : Reviews of Neuroscience Yol . 2, S . EHRENPREIS and I .J . KOPIN, Eds ., p .43-90, Raven Press, New York 1976 . 13 . C .C . CHUAG, T .F . CHEN and S .T . CHUAN, J . Physiol . (London) 230 613-618 (1973) . 14 . J .C . SCHWARTZ, J . COSTENTIN, M .P . MARTRES, P . PROTAIS and M . BAUDRY, Neuropharmacology 17, 665-685 (1978) .