effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

Life Sciences, Vol. 29, pp. 833-841 Pergamon Press Printed in the U.S.A.

EFFECTS OF TRICYCLIC ANTIDEPRESSANTS ON MUSCARINIC CHOLINERGIC RECEPTOR BINDING IN MOUSE BRAIN

Bryan Bohman, Angelos Halaris* and Michael Karbowski

The University of Chicago, Department of Psychiatry, Chicago, IL. 60637

(Received in final form June 22, 1981)

Summary

Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to suppress oxotremorine-induced tremors in treated and in control animals. EDgO's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence Is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified.

Recent studies have demonstrated alterations in receptor binding following treatments which affect the activity of the neurotransmitter system under study (I). These receptor alterations correspond, in some cases, to the development of tolerance to the biochemical and/or behavioral effects of the administered agent (I). Tricyclic antidepressants (TADs) are potent muscarinic cholinergic receptor antagonists (2), and they produce a variety of clinically important antimuscarinic side effects. However, tolerance develops to most of these effects over a short period of time. To determine whether acetylcholine (ACh) receptor alterations might account for the development of tolerance, the effects of chronic administration of two representative TADs, amitriptyline (AMI) and imlpramine (IMI), on muscarinic receptor binding were examined in whole mouse brain.

Oxotremorine-induced tremors are mediated via central muscarinic mechanisms (3) and antagonism of these tremors is a measure of the in vivo antimuscarinic potency of various agents (4). This study reports ~n development of tolerance to this antimuscarinic effect of TADs. The question whether supersensitivity to the effects of the muscarinic agonist oxotremorine develops concomitantly was also investigated.

Materials and Methods

A. Animals Male Swiss-Webster mice, 20-28 g in weight, were obtained from Sprague-Dawley

*Address reprint requests to: Dept. of Psychiatry, UCLA, and VA Medical Brentwood, 11301Wilshire Blvd., Los Angeles, CA 90073

0024-3205/81/080833-09502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

Center

834 Tolerance to Antimuscarinic Effect of TADs Vol. 29, No. 8, 1981

(Madison, WI). They were housed in plastic cages with ad lib food and water. Lights were on a 12-hour light/dark cycle and temperature was kept constant at 24 ~ 1.0°C. Animals were allowed to acclimatize for a minimum of three days.

B. Antagonism of Oxotremorine-lnduced Tremors In initial experiments, mice were injected (i.p.) with various doses of

IMI or AMI. One min later they received i.p. injections of 0.2 mg/kg oxotremorine - a dose which, when given alone, produced tremors in virtually 100% of mice. A blinded observer noted the presence or absence of tremors over the following 30 min without assessing the degree of the tremor. Ten animals were injected with each dose of TAD used, and the ED50 was calculated as the dose necessary to abolish tremors in 50% of the oxotremorine-treated animals.

The control ED50's were compared with those calculated in the same manner but using animals pretreated with AMI 9r IMI (10 mg/kg/day, i.p.) for either 2 or 4 weeks. The experiments were performed 24-36 hours after the final pre-treatment injection.

C. Oxotremorine Supersensitivity Experiments Mice were injected (i.p.) with various doses of oxotremorine and

observed for 30 min to note the presence or absence of tremors. Ten animals were injected with each dose of oxotremorime used, and the ED50 was calculated as that dose necessary to induce tremors in 50% of the animals. A control ED50 was calculated for animals pretreated with saline and compared to the ED50's for animals pretreated for 3 weeks with either AMI or IMI (10 mg/kg/day, i.p.). The experiments were performed 48 hours after the final injection.

D. Receptor Binding Studies I. In Vitro Experiments The~method of Snyder and Yamamura (2) was used with minor modifications.

Each mouse was decapitated and the brain quickly removed. The cerebellum was excised and the brain was homogenized in 10 vol sucrose in a glass tube fitted with a Teflon pestle. The homogenate was centrifuged at 1,000 g for 10 min, The resulting pellet (crude nuclear fraction) was discarded, and the nuclei-free homogenate (synaptosomal suspension) was rehomogenized. To determine the IC50 for antagonism of 3H-QNB binding by a given TAD, 30 ul aliquots of the brain preparation were incubated at 37°C in 2 ml of 0.05 M Na-K-P04 buffer containing 0.5 nM 3H-QNB and various concentrations of drug. After 60 min (preliminary experiments confirmed that 3H-QNB binding plateaued prior to this point), the incubation was terminated by adding 3 ml of ice-cold Na-K-P04 buffer to the tubes and vacuum filtering the contents through a Whatman GF/B glass microfibre filter. The tube was then rinsed once with 3 ml buffer and the filter itself was rinsed twice with 3 ml buffer. Dissociation of the QNB-receptor complex was negligible during the filtration procedure, which lasted less than 30 sec. The filter was subsequently placed in a glass vial and left on a warm surface for 20-30 min, until appreciable evaporation of moisture had ceased. Ten ml cf Aquasol were then added to the vial which was allowed to stand at room temperature overnight. Radioactivity was counted by liquid scintillation spectrometry at a counting efficiency of approximately 40%.

The brains of 3 mice were pooled for all experiments, and each determination of binding was performed in triplicate, along with duplicate tubes containing 100 uM oxotremorine or 1.0 uM atropine. Specific binding was calculated by subtracting non-specific binding (in the presence of atropine or oxotremorine) from total binding in the absence of these receptor blockers.

2. Chronic Experiments Mice were injected (i.p.) daily with 10 mg/kg of either AMI or IMI.

Control animals received an equivalent volume of physiological saline. After 2

Vol. 29, No. 8, 1981 Tolerance to Antimuscarinic Effect of TADs 835

to 7 weeks, and 24-36 hours following the final daily injection, animals were decapitated. Receptor binding was subsequently determined in the same manner as in the in vitro experiments, except that 10 ul aliquots of tissue were incubate--dr~rious concentrations of 3H-QNB. No TAD was added to the incubation mixture.

Preliminary experiments demonstrated that receptor blockade with either AMI or IMI could be completely overcome by saturating concentrations of QNB. Therefore, residual drug from pre-treatment injections would not be expected to affect the determination of Bmax in these chronic experiments. Conceivably, residual drug could alter the determination of K D through competitive interaction with the QNB ligand. However, animals were not sacrificed until at least 24 hours after the last injection. The effect of any residual drug would also be attenuated by the procedures used, i.e., brain homogenates were diluted several-fold before binding was actually measured. Finally, at the 2 week time point, the Sca~chard determinations of K D and Bmax were repeated with animai~ .... ~iTiced one week after the last pre-treatment injection. There were no significant differences between the results determined at one week, as opposed to 24-36 hours, following the final pre-treatment.

3. Ex Vivo Experiments To determine whether administration of TADs results in significant

muscarinic receptor blockade in the living animal, 25 mg/kg of IMI cr AMI (or an equivalent volume of saline) were injected (i.p.) and mice were decapitated 20 min later. The brains were homogenized and 50 ul aliquots were subsequently incubated for 60 min at 37°C in the presence of 0.5 nM 3H-QNB in 2 ml of Na-K-P04 buffer. Specific binding was calculated for both the treated and control animals; any decrease in the treated animals was assumed to result from TAD present in the brain of treated animals at the time of sacrifice.

E. Drugs The~owing drugs were used as their HCI salts dissolved in normal

saline: AMI (Merck, Sharp and Dohme); IMI (Ciba Geigy). Drug doses are expressed as the HCI salts in mg/kg. Atropine-S04 and oxotremorine-Br were obtained from Sigma (St. Louis, MO) and Aldrich (Milwaukee, Wl), respectively.

F. Statistical Analysis StatTstical significance (p<O.05) was evaluated by means

modification of Student's two-tailed t-tests. IC50 values were log-logit and ED50 values by log-probit analyses.

of the Welch determined by

Results

A. Antagonism of 3H-QNB Binding in Vitro Determination of the IC50's for TAD antagonism of 3H-qNB binding to

muscarinic receptors in vitro confirmed in mouse brain the data obtained by Snyder and Yamamura ( ~ t brain. The TADs were found to be potent blockers of central muscarinic receptors, with IC50's ranging from 39-380 nM (Table I). Although the actual values of the IC50's in this study are higher than those of Snyder and Yamamura, the rank order of potency for the TADs tested was identical (Table I).

The concentration of ligand used in a binding study affects the results of IC50 measurements (5). In the present experiments, the concentration of QNB used was higher than that used by Snyder and Yamamura (0.5 nM vs. 0.06 riM, respectively). This may explain the higher IC50 values obtained in the present experiments.


TABLE I

In Vitro Inhibition of Muscarinic Binding by Tricyclic Antidepressants

IC50 (nM)

Tricyclic Antidepressant Rat Brain* Mouse Brain

AMITRIPTYLINE 10 35 PROTRIPTYLINE -- 51 DOXEPINE 44 107 CHLORIMIPRAMINE -- 123 NORTRIPTYLINE 57 135 IMIPRAMINE 78 173 DESMETHYLIMIPRAMINE 170 381

Potency of the tricyclic antidepressants in blocking binding of 3H-QNB to mouse and rat brain muscarinic cholinergic receptors. Values are expressed as the concentration required to displace 50% of the specific binding of a given concentration of 3H-QNB (IC50). *From Snyder and Yamamura, 1977 (2).

B. Ex vivo Experiments Twenty min following i.p. injection of 25 mg/kg of AMI or IMI,

sufficient drug was present in mouse brain to inhibit ex vivo binding of 0.5 nM 3H-QNB to muscarinic receptors by 19 and 34%, respecti~-ely. Since these binding measurements were made only after the brain tissue was homogenized (entailing a 10-fold dilution) and added to an incubation mixture (resulting in a 40-fold dilution), the degree of blockade in vivo was undoubtedly higher than that which was measured ex vivo. Thus, it is clear that i.p. injections of moderate doses of TADs produce su-Eb-stantial blockade of central muscarinic receptors.

C. Inhibition of Oxotremorine-lnduced Tremors Since no experiments were performed to determine the time course or

point of maximum antitremorogenic activity, the acute experiments do not provide a valid comparison of the in vivo antimuscarinic activity of the two TADs tested - those data have previously been obtained (4). These experiments do, however, provide a valid baseline with which to assess the development of antimuscarinic tolerance following chronic administration of the agents tested. The dose of TAD required to block tremors in 50% of oxotremorine-treated animals (ED50) increased after chronic treatment both with IMI and with AMI (Table II). The increases were nearly 2-fold after 2 weeks of treatment (from 18 to 33 mg/kg for IMI; from 15 to 28 mg/kg for AMI) and even larger at 4 weeks (41 mg/kg for IMI; 43 mg/kg for AMI). These results clearly demonstrate the development of tolerance to this central antimuscarinic effect of the two TADs

tested.

D. Receptor Binding After Chronic Treatment In the first experiment, 10 mg/kg/day of IMI (or an equivalent volume of

saline) was injected for 2 to 7 weeks. The number of muscarinic receptors (Bmax) was determined at 2, 3, 4, 5 and 7 weeks by determining specific binding of 3H-QNB at a concentration (2.0 nM, approximately 25-30 times the apparent K n) which in preliminary studies had produced complete receptor saturation (Eaturation binding). No significant difference in Bmax was noted at any of the time points examined (Figure I). At 2 weeks, Scatchard analyses of the binding data obtained with 3H-QNB concentrations ranging from 0.05 to 0.40 nM were performed. These revealed no significant difference between the two groups in


90-

~80- <

70-

60- ~ IMIPRAMINE

H SALINE

H I I I I I I

2 3 4 5 6 7

W E E K S OF T R E A T M E N T

FIG. I

The effect of chronic administration of imipramine (10 mg/kg/day, i.p.), on the number of muscarinic binding sites (Bmax) in whole mouse brain, as determined by saturation binding experiments (see text). Points represent the mean of 4-8 animals; bars represent the S.E.M.

700- x~ N ~ X- "X IMPRAMINE

600- ~ H SALINE

\ \

_~.~ 6oo- \ ~ \

~ 400-

~ ~oo- "~ TREATMENT BMAXlp~d/fl K DCp

IM NE -- ~\ X \

150- - \ i--// 2'0 3'0 4'0 6'0 ~0 7'0

BOUND Ip~na/B ,

FIG. 2

Composite Scatchard plot of 3H-QNB binding to brain homogenates of mice treated with either saline or imipramine (10 mg/kg/, i.p.) for 14 days. Each point represents the mean of six animals. The lines were determined by linear regression analysis. The values given in the chart were obtained by performing independent Scatchard analyses of the binding data for each animal and then calculating the mean + S.E.M. of the resulting KD and B max values.


TABLE II

Effect of Chronic Pretreatment on Inhibition of Oxotremorine-lnduced Tremors by Tricyclic Antidepressants

Duration of Pretreatment

ED50 (mg/kg)

IMIPRAMINE AMITRIPTYLINE

NAIVE 18 15 2 WEEKS 33 28 4 WEEKS 41 43

The dose (ED50) of tricylic antidepressant necessary to prevent tremors from occurring in 50% of animals treated simultaneously with 0.2 mg/kg oxotremorine. For both drugs tested, the ED50 was determined in naive animals and also following 2 or 4 weeks of pretreatment (10 mg/kg/day) with that same agent.

either K D or Bmax (Figure 2). The Bmax determined by Scatchard analysis corresponded well to that determined by saturation binding.

In a second experiment, 10mg/kg/day of AMI or an equivalent volume of saline was injected for 2 to 4 weeks. Bmax was determined by saturation binding (as above) at 2, 3, and 4 weeks (Figure 3). Bmax and K were determined independently at 2 weeks by Scatchard analyses (Figure ~). There was no statistically significant difference between treated and control animals in either Bmax or K~ at any of the time points examined. Bmax calculated by Scatchard analys~s corresponded well to that determined by saturation binding.

No adverse effects were noted in the treated as compared to the control animals in either study. Weight gains in the two groups were similar. Wet weight and protein content of the brains from the two groups did not differ significantly.

Control values for Bmax and K_ in the present study are quite similar to those obtained by previous investigators using similar methods (6).

E. Sensitivity to Oxotremorine after Chronic TAD Treatment Three weeks of treatment with 10 mg/kg/day of either IMI or AMI resulted

in increased sensitivity to the tremorogenic (i.e., muscarinic) effect of oxotremorine, as compared to saline controls. The decrease in the ED50 was significant (p<O.05) and was similar for both agents; 33% for AMI (0.080 mg/kg vs. 0.119 mg/kg, control) and 31% for IMI (0.082 vs. 0.119 mg/kg). Thus, during chronic treatment with TADs, marked tolerance to the antimuscarinic effects of these agents is accompanied by a substantially increased sensitivity to the muscarinic agonist oxotremorine.

Discussion

Recent studies have demonstrated changes in cholinergic receptor binding following pharmacological treatments which alter CNS cholinergic activity. Agents which increase cholinergic activity (e.g., organophosphate cholinesterase inhibitors, striatal treatment with 6-OHDA) cause a decrease in the number of cholinergic receptors (7,8). Conversely, decreased cholinergic activity (e.g., following treatment with atropine or barbiturates) produces increases in receptor density (9,10). Changes in dissociation constants have been reported less frequently (I). Simon and Klein (11) have demonstrated


90-

~80 -

70- X'- ~ IMIPRAMINE

~_ ~ SALINE

WEEKS OF TREATMENT

FIG. 3

The effect of chronic administration of amitriptyline (10 mg/kg/day, i.p.) on the number of muscarinic binding sites (Bmax) in whole mouse brain, as determined by saturation binding experiments (see text). Points represent the mean of 8 animals; bars represent the S.E.M.

70()~

600-

500-

400-

300=

2O0-

15O-

N~\ X" -X A MITRIPTYLINE

~k ~--. SAUNE

TREATMENT BMAX,~d~ KD' XpMINX~N

SALINE 83.4 ~ 4.5 03.0_.+4.2 \ - x \

AMITRIPTYLINE 78.4 .~* 4.2 ~1.2 +_. 4.3 X \ ~ " 4 K N

\ k

4 / 2'o :~'. 4'0 do do 1 7 0 B () U N Dl~t¢~l

FIG. 4

Composite Scatchard plot of 3H-QNB binding to brain homogenates of mice treated with either saline or amitriptyline (I0 mg/kg/day, i.p.) for 14 days. Each point represents the mean of six animals. The lines were determined by linear regression analysis. The values given in the chart were obtained by performing independent Scatchard analyses of the binding data for each animal and then calculating the mean + S.E.M. of the resulting KD and Bmax values.


regulation of muscarinic receptor density by cholinergic activity in cultured chicken cerebrum and neuroblastoma cell lines. Studies of catecholaminergic receptor binding have demonstrated a similar relationship between the level of activity of a given neurotransmitter system and the density or conformation of its receptors (I). It is clear that alteration of receptors is a significant and widespread method of CNS regulation and homeostasis.

In the present study, TADs were shown to be potent blockers of mouse brain muscarinic receptors in vitro and to produce a substantial blockade in ex vivo experiments. Following chronic administration, substantial tolerance developed to the central antimuscarinic effect (blockade of oxotremorine-induced tremors) of both IMI and AMI. According to the principles suggested by the studies mentioned above, it was hypothesized that blockade of central muscarinic receptors produced by chronic administration of the TADs would result in an increase in the density and/or affinity of those receptors. These changes could in turn explain the observed development of tolerance. However, chronic administration of the two TADs, at the same dose which produced tolerance to their antimuscarinic effects, produced no alteralions in either Bmax or K D of 3H-QNB binding to muscarinic receptors.

The present results do not rule out the possibility that receptor alterations were responsible for the observed tolerance. It is possible that changes occurred which were of too small a magnitude to be detected by our methods. However, since the standar~ ~h ru~s uf thc ~easur~ments were quite small, it is unlikely i hat any receptor alteration rot detecied in these experinlents could account for the large degrce of tolerance produced. Another possibility is that in examining receptor binding in whole brain, we failed to detect changes which may have occurred in the specific brain region(s) responsible for the development of tolerance to the particular antimuscarinic effect studied. However, since inhibition of muscarinic binding was demostrated in whole brain both in vitro and ex vivo, it is reasonable to expect ihat whatever changes in muscarinic binding resulted from that blockade should also be demonstrable in whole brain. A third consideration is that increased metabolism of the TADs after chronic administration may have accounted for the observed tolerance. This possibility cannot be ruled out since drug levels were not measured over the course of chronic administration or after the acute TAD dose used to determine the ED50 for inhibition of oxotremorine-induced binding. However, we are unaware of any studies suggesting that the induction of metabolic enzymes following chronic exposure to the TADs is of sufficient magnitude to account for the substantial degree of tolerance demonstrated in

the present study.

Another possible mechanism for the development of tolerance to the antimuscarinic effects of TADs is alteration of acetylcholinesterase (ACHE) activity. A decrease in AChE activity would result in a higher concentration of ACh in the synapse, which would overcome a competitive blockade of the muscarinic receptor. Although the TADs appear to have little effect on AChE acutely (IC50 for inhibition is in the 100 uM range) (12), we are aware of no data regarding their effect during chronic administration.

An increase in ACh turnover rate could also account for tolerance, again by raising the concentration of ACh in the synapse and overcoming competitive receptor blockade. However, TADs actually decrease turnover acutely (unpublished results); again the effects of chronic administration are unknown.

A final possibility to explain the development of tolerance is that although the actual number of receptors is unchanged, the effect of a given ACh-ACh receptor interaction may be magnified. There is evidence, for example, that the post synaptic effects of an activated ACh receptor are mediated through an inhibition of the production of a "second messenger" in the


postsynaptic neuron. Although a correlation has been demonstrated between the density of receptors and cellular response to transmitter stimulation (11), it is conceivable that tolerance could be produced by an alteration of the magnitude of postsynaptic response produced per receptor-transmitter interaction. Thus, although a certain proportion of receptors might be blocked by an antagonist (e.g., a TAD), tolerance to that agent would result if the remaining available receptors were more "potent" in their postsynaptic effects.

This study has shown that chronic treatment with TADs results in both tolerance to their antimuscarinic effects and supersensitivity to the muscarinic agonist effects of oxotremorine, with no observable concomitant changes in muscarinic receptors. With no alteration of receptors, a given dose of oxotremorine should result in the same number of receptor-agonist interactions in both control and chronically treated animals. Therefore, the increased sensitivity to oxotremorine could only be explained by phenomena occurring distal to the recognition/binding site of the muscarinic receptor. Such phenomena may include a change in the production of second messengers (such as cAMP), alterations in the direct ionic forces exerted per transmitter-receptor interaction (13), or changes in the electrical properties of the post-synaptic cell membrane. Further investigation will be necessary to elucidate the precise mechanism.

Overstreet and Schiller (14) have obtained results which are, in some respects, comparable to ours. They found no alteration in CMS muscarinic receptor binding after chronic treatment with physostigmine despite demonstration of tolerance to that agent. They did not report, however, whether such tolerance was accompanied by supersensitivity to a direct muscarinic agonist (e.g., oxotremorine). Therefore, it is unclear whether the mechanism of tolerance in their study is similar to that observed in the present experiments.

Acknowledgements

This work was supported by grant 8037-23 from the lllinois Department of Mental Health. The authors are thankful to Mrs. Margarita Tayag for her excellent technical assistance.

References

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effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

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