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The effect of imipramine and lithium on α- and β-receptor binding in rat brain
186
Brain Research, 160 (1979) 186-191
© Elsevier/North-Holland Biomedical Press
The effect of imipramine and lithium on a- and fl-receptor binding in rat brain
JACK E. ROSENBLATT*, CANDACE B. PERT, JOHN F. TALLMAN, AGU PERT** and WILLIAM E. BUNNEY, JR. Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Md. 20014 ( U.S..4. )
(Accepted September 7th, 1978)
Lithium is an efficacious prophylactic agent for preventing unipolar and bipolar mania and depression 9. Its mechanism of action is unknown, but it is assumed to interact at cellular sites normally designated for sodium 5. A recent proposal is that lithium's action is due to its ability to stabilize and, hence halt the development of receptor super- and subsensitivity which is postulated to accompany the various psychotic states 8. We have recently tested this hypothesis in an animal model in which chronic haloperidol treatment was used to induce dopamine receptor supersensitivity. In that study 6, we found that lithium administered concurrently with haloperidol blocked the development of haloperidol-induced dopamine receptor supersensitivity as measured both behaviorally and biochemically. Since the catecholamine theory of affective illness 2 postulates that functional activity of norepinephrine in brain may also be critical in mania and depression, we have examined the effects of lithium on changes in receptor binding in rat brain. Banerjee et al. 1 have recently demonstrated that chronic imipramine treatment is accompanied by the development of subsensitivity of fl-receptors in rats. U'Prichard 1~ has recently reported that a-receptor binding is increased by chronic imipramine treatment. Since imipramine affects two catecholamine receptors, we thought it would be a convenient model system for examining lithium's effect simultaneously on the development of receptor super- and subsensitivity. Male Sprague-Dawley rats (250-300 g) were treated with imipramine (10 mg/kg, i.p.) or saline injections for either 2 or 4 weeks. Some of the rats treated with imipramine for 4 weeks were fed a diet containing 1500 g powdered laboratory chow (Purina) mixed with 2000 ml water and 2266 mg lithium carbonate. We have previously found 6 that after 3-4 weeks of treatment this lithium diet induces blood levels comparable (0.7-0.9 mEq/1) to therapeutic levels in humans. While rats on such a lithium diet to not gain weight at the same rate as those on a normal diet (15 weight difference at 3-4 weeks after treatment), they do not appear unhealthy. The remaining rats were fed a regular diet. At the end of the imipramine or saline injection * Present Address: Laboratory of Clinical Psychopharmacology, NIMH, St. Elizabeth's Hospital, Washington, D.C. 20032, U.S.A. ** To whom reprint requests should be addressed.
1 2 3 4 1 2
* P < 0.025 c o m p a r e d to saline regular diet.
D H A (fl) binding (pmole/g wet weight) Spiroperidol (dopamine) binding (pmole/g wet weight) 1
WB4101 (a) binding (pmole/g wet weight)
Experiment
0 0 5 0 0 0 0
2
3
10 3 3 6 3 3
Number o]" Days off" imipramine washes when assayed
4 4 2 2 4 4
Weeks on imipramine
Effect of imipramine on a-, fl- and dopamine receptor binding
TABLE I
J: ± ± ± 44-
0.23 (8) 0.48 (5) 0.48 (6) 0.43 (4) 0.09* (8) 0.07* (9)
17.42 4- 0.99 (10)
1.79 3.12 3.46 3.04 0.51 0.95
Imipramine ( N)
--
----0.60 q- 0.07* (5) 0.84 ~- 0.05* (9)
lmipramine lithium (iV)
+ 0.13 + 0.25 4- 0.25 -4- 0.42 ± 0.04 4- 0.06
(9) (6) (6) (5) (9) (10) 18.51 ± 1.11 (10)
2.16 3.16 3.16 2.90 0.78 1.19
Saline regular diet ( N)
OO
188 periods, the animals were sacrificed either immediately or 5 days following cessation of the imipramine injections. Rats co-treated with lithium and imipramine received lithium for one week prior to the initiation of imipramine treatment and remained on both treatments until sacrifice. Rats treated with lithium alone were treated with the lithium diet until sacrifice at 3 or 5 weeks. Rats were sacrificed by decapitation, their brains quickly removed, and either assayed fresh or frozen for a-, fl-, and dopamine receptor binding. a- and fl-receptor assays. Brains were assayed for a-receptor binding using a modification of the method of U'Prichard et al. x3. Brains were assayed for fl-receptor binding using a modification of the method reported by Tallman et al. 1°. Brains were sagittally bisected and homogenized (Brinkmann Polytron setting 10 for 15 sec) in 50 vol. of 50 m M Tris buffer (pH 7.7 at 25 °C). The homogenates were centrifuged at 18,000 rpm for 10 min. The pellets were then washed by centrifugation 3, 6 or 10 times prior to final suspension in 50 m M Tris buffer. For the a-receptor assay, 1 ml aliquots of tissue suspension (equivalent of 20 mg wet brain weight) were incubated in triplicate or duplicate for 15 min at 25 °C with 0.3 n M [zH]WB-4101 (New England Nuclear, specific activity 20 Ci/mmole) either in the presence or absence of 10-z M phentolamine. For /3-receptor assays 0.15 ml aliquots of tissue suspension (15 mg) were incubated for 10 min at 37 °C with [3H]dihydroalprenolol ([aH]DHA) (New England Nuclear, 48.6 Ci/mmole) in the presence or absence of 10 -z M DLpropranolol. Following incubation, samples were quickly filtered (Whatman GF/B glass fiber filters) under vacuum using two 6-ml washes of cold buffer. Filter discs were counted in 10 ml Aquasol overnight by liquid scintillation spectroscopy. Stereospecific binding, taken as the difference between amount bound in the presence and absence of phentolamine (a-receptor assay) or DL-propranolol (fl-receptor assay) amounted to 75 ~ of the total amount of WB-4101 bound and 65 ~ of the total amount of D H A bound. D o p a m i n e receptor assay. Individual caudates (35 mg) were assayed for dopamine receptor binding by the method of Burt et al. 4 using [3H]spiroperidol. Following two 100-vol. washes in cold Tris 7.7 buffer, pellets were resuspended in 150 vols. of 50 m M Tris buffer containing 0.1 ~ ascorbic acid, 10 # M pargyline, 120 TABLE II Effect of chronic lithium on a-, fl- and dopamine receptor binding ExWeeks periment on lithium
WB-410I (c0 binding (pmole/g wet weight) DHA (fl) binding (pmole/g wet weight) Spiroperidol (dopamine) binding (pmole/g wet weight)
l 2 1 2 1 2 3
3 5 3 5 3 3 3
Regular diet ( N)
Lithium diet ( N)
Per cent control
2.17 i 0.17 (5) 2.25 :E 0.11 (8) 1.19 ± 0.06 (10) 1.12 :t- 0.02 (8) 11.67 4- 0.55 (9) 9.35 ± 0.54 (10) 13.05 ± 0.41 (13)
2.77 ± 0.11" (6) 2.64 ± 0.14" (6) 1.04 ± 0.03* (15) 1.01 4- 0.02* (6) 11199-t: 0.47 (10) 9.71 ~ 0.45 (9) 12.74 ~ 0.53 (13)
+27.6* +17.3" --12.7" - - 9.8* + 2.7 ÷ 3.9 - - 2.4
189 O.I "-
--
CONTROL LITHIUM
0.07 e
0.04.
0.01
"
20
60
ioo
i~o
18o
a H-DHA STEREOSPECIFICALL Y BOUND ( p M )
0.15
o •
° ,°
--
: CONTROL-
c
--LITHIUM
0.10 nn
O.O5
tO
20
30
40
5O
aH-WB-4101 STEREOSPECIFICALLY
60 BOUND ( p M )
Fig. 1. Scatchard analysis of [aH]dihydroalprenolol binding (above) and [aH]WB-4101 binding (below) in rats chronically treated with lithium and their control. Rats were treated for 3 weeks with lithium as described, their brains were pooled and triplicate aliquots as described were incubated with a range of concentrations of [aH]ligand. 'Blanks' as described were included in triplicate at every concentration and subtracted from the total binding values.
mM NaC1, 5 mM KC1, 2 mM CaCI~, and 1 mM MgCI~ (final pH 7.1 at 37 °C), warmed at 37 °C for 10 rain and returned to ice. Aliquots (0.8 ml, 5.3 mg tissue) of caudate membranes were incubated at 37 °C for 10 min in duplicate or triplicate with 0.5 nM [3H]spiroperidol (Amersham, specific activity 26 Ci/mmole) in the presence of either levobutaclamol (10 -6 M) or dextrobutaclamol (10-6 M). After incubation, samples were filtered (Whatman GF/B glass fiber filters) under vacuum with three 5-ml washes of cold buffer, and filters were counted overnight in 10 ml of Aquasol. Stereospecific binding, taken as the difference in amount of [3H]spiroperidol bound in the presence of levobutaclamol and dextrobutaclamol, amounted to 80 % of the total binding. Table I shows the results of the effect of imipramine on a- and fl-receptor binding in brain. We failed to detect an a-receptor supersensitivity induced by chronic imipramine treatment. Since imipramine binds to a-receptors 13, it is essential to remove all imipramine before assay. In 4 separate experiments we attempted different washing procedures. Using animals that had been maintained on imipramine for 2 or 4 weeks, washing the brain membranes 3, 6 or 10 times failed to reveal a significant
190 difference from saline-injected rats. In one experiment, rats that had been taken off chronic 2-week imipramine treatment 5 days previously also were no different from saline controls. U'Prichard 12 reports that while a-adrenergic receptor supersensitivity is routinely observed after chronic amitriptyline administration, chronic imipramine administration gives less reproducible alterations in a-receptor binding 12. Chronic treatment with imipramine for two weeks, moreover, failed to alter the binding of [3H]spiroperidol to dopamine receptors in rat striatum. However, we did consistently observe a decrease in binding of [3H]dihydroalprenolol to fl-adrenergic receptors in membranes derived from whole rat brain which ranged between 20 and 35 ~. Thus, we are able to confirm the observations of Banerjee et al. 1. A recent paper by Sarai et al. 8 also confirms this observation. Lithium failed to significantly alter the fl-receptor subsensitivity induced by chronic imipramine treatment. As a control we included receptor analysis of animals which had been maintained on the lithium diet only for a period of 3 weeks. We were surprised to find that lithium treatment alone increased a-receptor binding by 17-27 ~ and decreased fl-receptor binding 10-13 ~. We observed no significant effect on dopamine receptor binding (Table II) as previously reported 6. A Scatchard analysis of the alterations in aand fl-receptor binding is shown in Fig. 1. Chronic lithium treatment appears to increase the number of [3H]WB-4101 binding sites with no marked change in receptor affinity (Fig. 1, below). The decrease in fl-receptor binding induced by chronic lithium treatment seems to be attributable to a decreased number of specific binding sites (Fig. 1, above). Chronic treatment with lithium rather than acute exposure of rat brain membranes to lithium ion appears to be responsible for the effects we have observed. Acute exposure of rat brain membranes to lithium concentrations ranging between 0.03 and 3.0 mM, the concentrations of lithium expected to be carried over into the test tube from the brain levels which we have routinely noted, failed to alter the binding to either a-, fl- or dopamine receptors. Lithium failed to prevent the development of fl-receptor subsensitivity induced by imipramine. Thus, in one system, at least, lithium fails to stabilize against receptor alterations. In a very recent study 7, however, we have found that lithium is able to block the supersensitivity of a-and fl-receptors which develops after 6-hydroxydopamine lesions. This is the first report of lithium's ability to alter the sensitivity of adrenergic receptors in brain. Could this be due to a non-specific 'toxic' effect of lithium? Rats maintained on lithium for 3-5 weeks in our laboratory show a 15 ~ weight loss but no overt differences in behavior from controls. The effects of lithium on adrenergic receptors are highly reproducible, but it is impossible to rule out a 'stress' effect. Treiser et al. 11 failed to detect lithium-induced changes in fl-receptor binding for reasons that are unclear at this time but may involve their shorter (10-12 day) lithium treatment period. These alterations in receptor sensitivity may relate to lithium's therapeutic effect. It will be important to demonstrate concomitant behavioral alterations in receptor sensitivity accompanying the biochemical changes observed here.
Brain Research, 160 (1979) 186-191
© Elsevier/North-Holland Biomedical Press
The effect of imipramine and lithium on a- and fl-receptor binding in rat brain
JACK E. ROSENBLATT*, CANDACE B. PERT, JOHN F. TALLMAN, AGU PERT** and WILLIAM E. BUNNEY, JR. Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Md. 20014 ( U.S..4. )
(Accepted September 7th, 1978)
Lithium is an efficacious prophylactic agent for preventing unipolar and bipolar mania and depression 9. Its mechanism of action is unknown, but it is assumed to interact at cellular sites normally designated for sodium 5. A recent proposal is that lithium's action is due to its ability to stabilize and, hence halt the development of receptor super- and subsensitivity which is postulated to accompany the various psychotic states 8. We have recently tested this hypothesis in an animal model in which chronic haloperidol treatment was used to induce dopamine receptor supersensitivity. In that study 6, we found that lithium administered concurrently with haloperidol blocked the development of haloperidol-induced dopamine receptor supersensitivity as measured both behaviorally and biochemically. Since the catecholamine theory of affective illness 2 postulates that functional activity of norepinephrine in brain may also be critical in mania and depression, we have examined the effects of lithium on changes in receptor binding in rat brain. Banerjee et al. 1 have recently demonstrated that chronic imipramine treatment is accompanied by the development of subsensitivity of fl-receptors in rats. U'Prichard 1~ has recently reported that a-receptor binding is increased by chronic imipramine treatment. Since imipramine affects two catecholamine receptors, we thought it would be a convenient model system for examining lithium's effect simultaneously on the development of receptor super- and subsensitivity. Male Sprague-Dawley rats (250-300 g) were treated with imipramine (10 mg/kg, i.p.) or saline injections for either 2 or 4 weeks. Some of the rats treated with imipramine for 4 weeks were fed a diet containing 1500 g powdered laboratory chow (Purina) mixed with 2000 ml water and 2266 mg lithium carbonate. We have previously found 6 that after 3-4 weeks of treatment this lithium diet induces blood levels comparable (0.7-0.9 mEq/1) to therapeutic levels in humans. While rats on such a lithium diet to not gain weight at the same rate as those on a normal diet (15 weight difference at 3-4 weeks after treatment), they do not appear unhealthy. The remaining rats were fed a regular diet. At the end of the imipramine or saline injection * Present Address: Laboratory of Clinical Psychopharmacology, NIMH, St. Elizabeth's Hospital, Washington, D.C. 20032, U.S.A. ** To whom reprint requests should be addressed.
1 2 3 4 1 2
* P < 0.025 c o m p a r e d to saline regular diet.
D H A (fl) binding (pmole/g wet weight) Spiroperidol (dopamine) binding (pmole/g wet weight) 1
WB4101 (a) binding (pmole/g wet weight)
Experiment
0 0 5 0 0 0 0
2
3
10 3 3 6 3 3
Number o]" Days off" imipramine washes when assayed
4 4 2 2 4 4
Weeks on imipramine
Effect of imipramine on a-, fl- and dopamine receptor binding
TABLE I
J: ± ± ± 44-
0.23 (8) 0.48 (5) 0.48 (6) 0.43 (4) 0.09* (8) 0.07* (9)
17.42 4- 0.99 (10)
1.79 3.12 3.46 3.04 0.51 0.95
Imipramine ( N)
--
----0.60 q- 0.07* (5) 0.84 ~- 0.05* (9)
lmipramine lithium (iV)
+ 0.13 + 0.25 4- 0.25 -4- 0.42 ± 0.04 4- 0.06
(9) (6) (6) (5) (9) (10) 18.51 ± 1.11 (10)
2.16 3.16 3.16 2.90 0.78 1.19
Saline regular diet ( N)
OO
188 periods, the animals were sacrificed either immediately or 5 days following cessation of the imipramine injections. Rats co-treated with lithium and imipramine received lithium for one week prior to the initiation of imipramine treatment and remained on both treatments until sacrifice. Rats treated with lithium alone were treated with the lithium diet until sacrifice at 3 or 5 weeks. Rats were sacrificed by decapitation, their brains quickly removed, and either assayed fresh or frozen for a-, fl-, and dopamine receptor binding. a- and fl-receptor assays. Brains were assayed for a-receptor binding using a modification of the method of U'Prichard et al. x3. Brains were assayed for fl-receptor binding using a modification of the method reported by Tallman et al. 1°. Brains were sagittally bisected and homogenized (Brinkmann Polytron setting 10 for 15 sec) in 50 vol. of 50 m M Tris buffer (pH 7.7 at 25 °C). The homogenates were centrifuged at 18,000 rpm for 10 min. The pellets were then washed by centrifugation 3, 6 or 10 times prior to final suspension in 50 m M Tris buffer. For the a-receptor assay, 1 ml aliquots of tissue suspension (equivalent of 20 mg wet brain weight) were incubated in triplicate or duplicate for 15 min at 25 °C with 0.3 n M [zH]WB-4101 (New England Nuclear, specific activity 20 Ci/mmole) either in the presence or absence of 10-z M phentolamine. For /3-receptor assays 0.15 ml aliquots of tissue suspension (15 mg) were incubated for 10 min at 37 °C with [3H]dihydroalprenolol ([aH]DHA) (New England Nuclear, 48.6 Ci/mmole) in the presence or absence of 10 -z M DLpropranolol. Following incubation, samples were quickly filtered (Whatman GF/B glass fiber filters) under vacuum using two 6-ml washes of cold buffer. Filter discs were counted in 10 ml Aquasol overnight by liquid scintillation spectroscopy. Stereospecific binding, taken as the difference between amount bound in the presence and absence of phentolamine (a-receptor assay) or DL-propranolol (fl-receptor assay) amounted to 75 ~ of the total amount of WB-4101 bound and 65 ~ of the total amount of D H A bound. D o p a m i n e receptor assay. Individual caudates (35 mg) were assayed for dopamine receptor binding by the method of Burt et al. 4 using [3H]spiroperidol. Following two 100-vol. washes in cold Tris 7.7 buffer, pellets were resuspended in 150 vols. of 50 m M Tris buffer containing 0.1 ~ ascorbic acid, 10 # M pargyline, 120 TABLE II Effect of chronic lithium on a-, fl- and dopamine receptor binding ExWeeks periment on lithium
WB-410I (c0 binding (pmole/g wet weight) DHA (fl) binding (pmole/g wet weight) Spiroperidol (dopamine) binding (pmole/g wet weight)
l 2 1 2 1 2 3
3 5 3 5 3 3 3
Regular diet ( N)
Lithium diet ( N)
Per cent control
2.17 i 0.17 (5) 2.25 :E 0.11 (8) 1.19 ± 0.06 (10) 1.12 :t- 0.02 (8) 11.67 4- 0.55 (9) 9.35 ± 0.54 (10) 13.05 ± 0.41 (13)
2.77 ± 0.11" (6) 2.64 ± 0.14" (6) 1.04 ± 0.03* (15) 1.01 4- 0.02* (6) 11199-t: 0.47 (10) 9.71 ~ 0.45 (9) 12.74 ~ 0.53 (13)
+27.6* +17.3" --12.7" - - 9.8* + 2.7 ÷ 3.9 - - 2.4
189 O.I "-
--
CONTROL LITHIUM
0.07 e
0.04.
0.01
"
20
60
ioo
i~o
18o
a H-DHA STEREOSPECIFICALL Y BOUND ( p M )
0.15
o •
° ,°
--
: CONTROL-
c
--LITHIUM
0.10 nn
O.O5
tO
20
30
40
5O
aH-WB-4101 STEREOSPECIFICALLY
60 BOUND ( p M )
Fig. 1. Scatchard analysis of [aH]dihydroalprenolol binding (above) and [aH]WB-4101 binding (below) in rats chronically treated with lithium and their control. Rats were treated for 3 weeks with lithium as described, their brains were pooled and triplicate aliquots as described were incubated with a range of concentrations of [aH]ligand. 'Blanks' as described were included in triplicate at every concentration and subtracted from the total binding values.
mM NaC1, 5 mM KC1, 2 mM CaCI~, and 1 mM MgCI~ (final pH 7.1 at 37 °C), warmed at 37 °C for 10 rain and returned to ice. Aliquots (0.8 ml, 5.3 mg tissue) of caudate membranes were incubated at 37 °C for 10 min in duplicate or triplicate with 0.5 nM [3H]spiroperidol (Amersham, specific activity 26 Ci/mmole) in the presence of either levobutaclamol (10 -6 M) or dextrobutaclamol (10-6 M). After incubation, samples were filtered (Whatman GF/B glass fiber filters) under vacuum with three 5-ml washes of cold buffer, and filters were counted overnight in 10 ml of Aquasol. Stereospecific binding, taken as the difference in amount of [3H]spiroperidol bound in the presence of levobutaclamol and dextrobutaclamol, amounted to 80 % of the total binding. Table I shows the results of the effect of imipramine on a- and fl-receptor binding in brain. We failed to detect an a-receptor supersensitivity induced by chronic imipramine treatment. Since imipramine binds to a-receptors 13, it is essential to remove all imipramine before assay. In 4 separate experiments we attempted different washing procedures. Using animals that had been maintained on imipramine for 2 or 4 weeks, washing the brain membranes 3, 6 or 10 times failed to reveal a significant
190 difference from saline-injected rats. In one experiment, rats that had been taken off chronic 2-week imipramine treatment 5 days previously also were no different from saline controls. U'Prichard 12 reports that while a-adrenergic receptor supersensitivity is routinely observed after chronic amitriptyline administration, chronic imipramine administration gives less reproducible alterations in a-receptor binding 12. Chronic treatment with imipramine for two weeks, moreover, failed to alter the binding of [3H]spiroperidol to dopamine receptors in rat striatum. However, we did consistently observe a decrease in binding of [3H]dihydroalprenolol to fl-adrenergic receptors in membranes derived from whole rat brain which ranged between 20 and 35 ~. Thus, we are able to confirm the observations of Banerjee et al. 1. A recent paper by Sarai et al. 8 also confirms this observation. Lithium failed to significantly alter the fl-receptor subsensitivity induced by chronic imipramine treatment. As a control we included receptor analysis of animals which had been maintained on the lithium diet only for a period of 3 weeks. We were surprised to find that lithium treatment alone increased a-receptor binding by 17-27 ~ and decreased fl-receptor binding 10-13 ~. We observed no significant effect on dopamine receptor binding (Table II) as previously reported 6. A Scatchard analysis of the alterations in aand fl-receptor binding is shown in Fig. 1. Chronic lithium treatment appears to increase the number of [3H]WB-4101 binding sites with no marked change in receptor affinity (Fig. 1, below). The decrease in fl-receptor binding induced by chronic lithium treatment seems to be attributable to a decreased number of specific binding sites (Fig. 1, above). Chronic treatment with lithium rather than acute exposure of rat brain membranes to lithium ion appears to be responsible for the effects we have observed. Acute exposure of rat brain membranes to lithium concentrations ranging between 0.03 and 3.0 mM, the concentrations of lithium expected to be carried over into the test tube from the brain levels which we have routinely noted, failed to alter the binding to either a-, fl- or dopamine receptors. Lithium failed to prevent the development of fl-receptor subsensitivity induced by imipramine. Thus, in one system, at least, lithium fails to stabilize against receptor alterations. In a very recent study 7, however, we have found that lithium is able to block the supersensitivity of a-and fl-receptors which develops after 6-hydroxydopamine lesions. This is the first report of lithium's ability to alter the sensitivity of adrenergic receptors in brain. Could this be due to a non-specific 'toxic' effect of lithium? Rats maintained on lithium for 3-5 weeks in our laboratory show a 15 ~ weight loss but no overt differences in behavior from controls. The effects of lithium on adrenergic receptors are highly reproducible, but it is impossible to rule out a 'stress' effect. Treiser et al. 11 failed to detect lithium-induced changes in fl-receptor binding for reasons that are unclear at this time but may involve their shorter (10-12 day) lithium treatment period. These alterations in receptor sensitivity may relate to lithium's therapeutic effect. It will be important to demonstrate concomitant behavioral alterations in receptor sensitivity accompanying the biochemical changes observed here.