- Email: [email protected]
Intact 5Ht neuroterminals are not required for 5HT2 receptor down-regulation by amitriptyline
OO28-3908/82/070725-03 W3.00/0 Copyright 0 1982 PergamonPress Ltd
Neurophormacoloyy Vol. 21, pp. 125 to 721, 1982 Printed in Great Britain. All rights reserved
INTACT 5HT NEUROTERMINALSARE NOT REQUIRED FOR 5HTz RECEPTOR DOWN-REGULATION BY AMITRIPTYLINE.
S.
Clements-Jewery
and P.A.
Robson
Biochemical Pharmacology Section, Research Department, Roussel Laboratories Ltd., Swindon, Wilts., England.
(Accephd
26 May
1982)
Summary - Chronic treatment of rats with the antidepressant, amitriptyline, resulted in a reduced number of cerebral cortical 5HT2 receptors measured as cinanserin displaceable [3H] spiperone binding. Pretreatment of rats with the serotonergic neurotoxin, p-chloroamphetamine, did not inhibit the effects of subsequent chronic amitriptyline treatment on 5HT2 receptor density whilst- still inducing severe ncurotermtnal destruction. It is suggested that such data indicate a post-synaptic locus of action for amltriptyline and have implications in regard to current concepts of antidepressant action. INTRODUCTION Monoamine theories of depression (see Carver and Davis, 1979) suggest that the efficacies of antidepressant agents in the treatment of affective disorder are directly associated with the facilitation of monoaminergic neurotransmission, typically by re-uptake blockade or inhibition of monoamine catabolism at presynaptic sites. Indeed, the down-regulation of cortical B-adrenoceptors induced by the chronic treatment of rats with the potent noradrenaline (NA) uptake blocker, desipramine, is abolished after 6-hydroxydopamine degeneration of noradrenergic neurotermlnals (Schweitzer, Schwartz and Friedhoff, 1979). However , WCnow report that an extensive destruction of cortical serotonergic neurotermlnals with p-chloroamphetamine (pCA) (Sanders-Bush, Bushing and Sulser, 1975) does not lessen the ability of chronic amitriptyline treatment to reduce the numbers of cortical 5HT2 receptors (Peroutka and Snyder, 1980). METHODS Four groups of 5 male CFHB rats (180-22Og) were dosed with either saline or 10 mg/kg i.p. pCA, 24 hours before a 7 day series of either dail saline or 20 mg/kg i..p. amitriptyline Xl. Twenty-four hours after the last injection [ ] 5-hydroxytryptamine (5HT) uptake and [3H] spiperone binding were measured in, respe>iYely cortical synaptosomes or cortical P2 membranes. The uptake of 1OnM [3H] 5HT (Ameriham International) by 5ml volumes of crude cortical synaptosomes (approx. 2 mg original brain wt per ml) was assayed over 15 minute incubations in triplicate at 37’. Non-specific uptake was accounted for by parallel incubations at O”. The synaptosomes were separated by filtration through Whatman CF/B filters under vacuum, followed by 2 x 10 ml rinses with cold assay buffer containing Na$POk 40 mM, NaCl 100 mM, KC1 4 u&l, glucose 200 mM, pargyline 0.1 mM, l-ascorbic acid 1mM and EDTA 0.1 mM, pH 7.0. For the measurement of [3H] spiperone binding to cortical 5HT receptors, Pk membranes (approx. 12 mg original brain wt per assay) were Incubated in f ml volumes of 50 mM Trls HCl, 3mM MgC12, pH 7.6 buffer for 15 minutes at 37O. Nonspecific binding was accounted for by the Inclusion of 1 pM cinanserin HCl in blank binding assays. All assays were run in triplicate. Membranes prepared from individual rats were incubated with 1.0 nM L3H] spiperone (Amersham International) whereas pooled membranes were subjected to saturation analysis using 5 concentrations, 0.05 - 1.0 nM, of radioligand. Bound radio-activity was separated by filtrations through Whatman GF/B filters, followed by 2 x 10 ml rinses of cold buffer, drying at 80” and counting in ECONOFLUOR(NEN) scintillation cocktail.
725
Preliminary
126
Notes
RESULTS The results for both [3H] 5HT uptake and [3H] spiperone binding are shown in the figure. In this experiment, as previously reported by Peroutka and Snyder (1980), chronic treatment induced a significant (7, but not I,2 or 3 days - not shown) of rats with amitriptyline Scatchard analysis reduction of [3H] spiperone binding to rat cortical 5HTr, receptors. indicated that this effect was due only to a receptor density change and not a change in affinity. Chronic amitriptyline treatment did not significantly effect the uptake Treatment of [3H] 5HT into cortical synaptosomes assayed 24 hours after the last dose. with 10 mg/kg i.p. pCA led to an extensive destructlon of 5HT neuroterminals, measured Amitriptyline treatment initiated as specific [3H] 5HT uptake, assayed 9 days after. 24 hours after pCA administration did not significantly change the neurotoxic effect. The reduction in (3H] spiperone binding induced by amitriptyline was also demonstrated in pCA treated animals and also shown to be solely a receptor density change. PCA Throughout this treatment alone did not significantly alter [3H] spiperone binding. experiment, no effects were shown on the non-specific binding components by any treatment.
Atnitriptyline
-
PCA
-
The effects of 7 day amitriptyline treatment (20 mg/kg/d) on cortical Figure. binding sites and 5HT uptake in saline and pCA (10 mg/kg) 5HT2 receptor Shaded columns indicate cinserin specific [3H] spiperone pretreated rats. binding and open columns indicate [3H] 5HT uptake. Mean values (+ SEM) pCA was ad%inistered 1 obtained from groups of 5 rats assayed in triplicate. day before initiation of amitriptyline or saline treatment. Animals were Stars indicate significant differences sacrificed 1 day after last injection. from saline/saline controls, P< 0.01. (Student’s “t” test) DISCUSSION
The assumption that an extended increase in the availability of 5HT at postsynaptic receptors leads to a subsensitivity or down-regulation of the numbers of these receptors is brought into doubt by previous observations. The potent 5HT uptake blocker, whereas the established antidepressant fluoxetine, does not down-regulate 5HTz receptors, which has minimal monamine uptake blocking properties, is effective agent, iprindole, The experiment reportd here also indicates that presynaptic (Peroutka and Snyder, 1980). The interactions of antidepressant drugs may not mediate postsynaptic receptor changes. ability of chronic amitriptyline treatment to down-regulate cortical 5HTz receptors is maintained in the presence of extensive degeneration of serotonergic neuroterminals. that 5HT2 receptor binding sites are post-synaptic to a small It is conceivable, however, Indeed pCA treatment number of terminals which are insensitive to pCA neurotoxicfty. alone did not cause a significant change in receptor number. However, electrolytic raphe lesions have also been reported not to effect 5HT, or 5HTz cortical binding sites However, these data do suggest that greater (Blackshear, Steranka and Sanders-Bush, 1981). attention should be payed to postsynaptic actions of antidepressant drugs and have general implications in regard to current concepts of antidepressant action and monoamine theories of depression.
727
Preliminary Notes REFERENCES
and E. Sanders-Bush, 1981. Multiple Blackshear, M. A. , L.R. Steranka regional distribution and effect of raphe lesions. European .I. 76 : 325-334.
Garver, D.L. and J.M. Davis, Sci. 24 : 383-394.
Peroutka, S.J. and S.H. spiroperidol-labeled
1979.
Biogenic
amine
Snyder, 1980. Long-term serotonin receptors.
hypotheses
affective
antidepressant treatment Science 210 : 88 - 90.
Sanders-Bush, E., J.A. Bushing and F. Sulser, 1975. tamine and related drugs on central serotonergic Therap. 192 : 33 - 41.
Schweitzer, J.W., required for : 377 - 379.
of
serotonin Pharmacol.
Long-term effects mechanisms. J.
receptors:
disorders.
Life
lowers
of p-chloroamphePharmacol. Exp.
R. Schwartz and A.J. Friedhoff, 1979. Intact presynaptic beta-adrenergic receptor regulation by desipramine. J.
terminals Neurochem.
33
Neurophormacoloyy Vol. 21, pp. 125 to 721, 1982 Printed in Great Britain. All rights reserved
INTACT 5HT NEUROTERMINALSARE NOT REQUIRED FOR 5HTz RECEPTOR DOWN-REGULATION BY AMITRIPTYLINE.
S.
Clements-Jewery
and P.A.
Robson
Biochemical Pharmacology Section, Research Department, Roussel Laboratories Ltd., Swindon, Wilts., England.
(Accephd
26 May
1982)
Summary - Chronic treatment of rats with the antidepressant, amitriptyline, resulted in a reduced number of cerebral cortical 5HT2 receptors measured as cinanserin displaceable [3H] spiperone binding. Pretreatment of rats with the serotonergic neurotoxin, p-chloroamphetamine, did not inhibit the effects of subsequent chronic amitriptyline treatment on 5HT2 receptor density whilst- still inducing severe ncurotermtnal destruction. It is suggested that such data indicate a post-synaptic locus of action for amltriptyline and have implications in regard to current concepts of antidepressant action. INTRODUCTION Monoamine theories of depression (see Carver and Davis, 1979) suggest that the efficacies of antidepressant agents in the treatment of affective disorder are directly associated with the facilitation of monoaminergic neurotransmission, typically by re-uptake blockade or inhibition of monoamine catabolism at presynaptic sites. Indeed, the down-regulation of cortical B-adrenoceptors induced by the chronic treatment of rats with the potent noradrenaline (NA) uptake blocker, desipramine, is abolished after 6-hydroxydopamine degeneration of noradrenergic neurotermlnals (Schweitzer, Schwartz and Friedhoff, 1979). However , WCnow report that an extensive destruction of cortical serotonergic neurotermlnals with p-chloroamphetamine (pCA) (Sanders-Bush, Bushing and Sulser, 1975) does not lessen the ability of chronic amitriptyline treatment to reduce the numbers of cortical 5HT2 receptors (Peroutka and Snyder, 1980). METHODS Four groups of 5 male CFHB rats (180-22Og) were dosed with either saline or 10 mg/kg i.p. pCA, 24 hours before a 7 day series of either dail saline or 20 mg/kg i..p. amitriptyline Xl. Twenty-four hours after the last injection [ ] 5-hydroxytryptamine (5HT) uptake and [3H] spiperone binding were measured in, respe>iYely cortical synaptosomes or cortical P2 membranes. The uptake of 1OnM [3H] 5HT (Ameriham International) by 5ml volumes of crude cortical synaptosomes (approx. 2 mg original brain wt per ml) was assayed over 15 minute incubations in triplicate at 37’. Non-specific uptake was accounted for by parallel incubations at O”. The synaptosomes were separated by filtration through Whatman CF/B filters under vacuum, followed by 2 x 10 ml rinses with cold assay buffer containing Na$POk 40 mM, NaCl 100 mM, KC1 4 u&l, glucose 200 mM, pargyline 0.1 mM, l-ascorbic acid 1mM and EDTA 0.1 mM, pH 7.0. For the measurement of [3H] spiperone binding to cortical 5HT receptors, Pk membranes (approx. 12 mg original brain wt per assay) were Incubated in f ml volumes of 50 mM Trls HCl, 3mM MgC12, pH 7.6 buffer for 15 minutes at 37O. Nonspecific binding was accounted for by the Inclusion of 1 pM cinanserin HCl in blank binding assays. All assays were run in triplicate. Membranes prepared from individual rats were incubated with 1.0 nM L3H] spiperone (Amersham International) whereas pooled membranes were subjected to saturation analysis using 5 concentrations, 0.05 - 1.0 nM, of radioligand. Bound radio-activity was separated by filtrations through Whatman GF/B filters, followed by 2 x 10 ml rinses of cold buffer, drying at 80” and counting in ECONOFLUOR(NEN) scintillation cocktail.
725
Preliminary
126
Notes
RESULTS The results for both [3H] 5HT uptake and [3H] spiperone binding are shown in the figure. In this experiment, as previously reported by Peroutka and Snyder (1980), chronic treatment induced a significant (7, but not I,2 or 3 days - not shown) of rats with amitriptyline Scatchard analysis reduction of [3H] spiperone binding to rat cortical 5HTr, receptors. indicated that this effect was due only to a receptor density change and not a change in affinity. Chronic amitriptyline treatment did not significantly effect the uptake Treatment of [3H] 5HT into cortical synaptosomes assayed 24 hours after the last dose. with 10 mg/kg i.p. pCA led to an extensive destructlon of 5HT neuroterminals, measured Amitriptyline treatment initiated as specific [3H] 5HT uptake, assayed 9 days after. 24 hours after pCA administration did not significantly change the neurotoxic effect. The reduction in (3H] spiperone binding induced by amitriptyline was also demonstrated in pCA treated animals and also shown to be solely a receptor density change. PCA Throughout this treatment alone did not significantly alter [3H] spiperone binding. experiment, no effects were shown on the non-specific binding components by any treatment.
Atnitriptyline
-
PCA
-
The effects of 7 day amitriptyline treatment (20 mg/kg/d) on cortical Figure. binding sites and 5HT uptake in saline and pCA (10 mg/kg) 5HT2 receptor Shaded columns indicate cinserin specific [3H] spiperone pretreated rats. binding and open columns indicate [3H] 5HT uptake. Mean values (+ SEM) pCA was ad%inistered 1 obtained from groups of 5 rats assayed in triplicate. day before initiation of amitriptyline or saline treatment. Animals were Stars indicate significant differences sacrificed 1 day after last injection. from saline/saline controls, P< 0.01. (Student’s “t” test) DISCUSSION
The assumption that an extended increase in the availability of 5HT at postsynaptic receptors leads to a subsensitivity or down-regulation of the numbers of these receptors is brought into doubt by previous observations. The potent 5HT uptake blocker, whereas the established antidepressant fluoxetine, does not down-regulate 5HTz receptors, which has minimal monamine uptake blocking properties, is effective agent, iprindole, The experiment reportd here also indicates that presynaptic (Peroutka and Snyder, 1980). The interactions of antidepressant drugs may not mediate postsynaptic receptor changes. ability of chronic amitriptyline treatment to down-regulate cortical 5HTz receptors is maintained in the presence of extensive degeneration of serotonergic neuroterminals. that 5HT2 receptor binding sites are post-synaptic to a small It is conceivable, however, Indeed pCA treatment number of terminals which are insensitive to pCA neurotoxicfty. alone did not cause a significant change in receptor number. However, electrolytic raphe lesions have also been reported not to effect 5HT, or 5HTz cortical binding sites However, these data do suggest that greater (Blackshear, Steranka and Sanders-Bush, 1981). attention should be payed to postsynaptic actions of antidepressant drugs and have general implications in regard to current concepts of antidepressant action and monoamine theories of depression.
727
Preliminary Notes REFERENCES
and E. Sanders-Bush, 1981. Multiple Blackshear, M. A. , L.R. Steranka regional distribution and effect of raphe lesions. European .I. 76 : 325-334.
Garver, D.L. and J.M. Davis, Sci. 24 : 383-394.
Peroutka, S.J. and S.H. spiroperidol-labeled
1979.
Biogenic
amine
Snyder, 1980. Long-term serotonin receptors.
hypotheses
affective
antidepressant treatment Science 210 : 88 - 90.
Sanders-Bush, E., J.A. Bushing and F. Sulser, 1975. tamine and related drugs on central serotonergic Therap. 192 : 33 - 41.
Schweitzer, J.W., required for : 377 - 379.
of
serotonin Pharmacol.
Long-term effects mechanisms. J.
receptors:
disorders.
Life
lowers
of p-chloroamphePharmacol. Exp.
R. Schwartz and A.J. Friedhoff, 1979. Intact presynaptic beta-adrenergic receptor regulation by desipramine. J.
terminals Neurochem.
33