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Tricyclic antidepressants inhibit Ca2+-activated K+-efflux in cultured spinal cord neurons
Brain Research, 545 (1991) 59-65 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939116455T
59
BRES 16455
Tricyclic antidepressants inhibit Ca2+-activated K+-efflux in cultured spinal cord neurons Ganesan L. Kamatchi and Maharaj K. Ticku Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7764 (U.S.A.) (Accepted 23 October 1990) Key words: Tricyclic antidepressant; Ca2+-activated K + channel; ~-Aminobutyric acid-B receptor; Voltage-gated Ca 2+ channel; Spinal cord neuron
The effect of tricyclic antidepressants and monoamine oxidase inhibitors on the Ca2+-activated K+-efflux was studied using 86Rb-efflux assay in primary cultured mouse spinal cord neurons. Depolarization of the cultured cells with 100 mM KCI increased the 8~Rb-efflux significantly in Ca2÷-containing buffer, but not in Ca2+-free buffer. All the antidepressants examined, except the monoamine oxidase inhibitors, inhibited the 86Rb-efflux. Desipramine exhibited additivity with tetraethyl ammonium (TEA) and quinine sulfate (QSO4) , but not with GABA a receptor agonist baclofen. The inhibitory action of antidepressants was not mediated through the GABAB receptors, since GABA a receptor antagonist, phaclofen, was unable to antagonize this effect. The ability of tricyclic antidepressants to inhibit calcium ionophore (A 23187)-induced 86Rb-efflux suggests that these drugs do not act at the level of voltage-gated Ca2+-channels. Furthermore, this effect does not seem to involve the G-proteins, adenylate cyclase, or protein kinase C systems, since pertussis toxin (PTX) and the activators of adenylate cyclase and protein kinase C did not reverse the effect of tricyclics on 86Rb-efflux. Taken together, these results suggest that antidepressants inhibit Ca2+-aetivated K÷-channels at a stage subsequent to the voltage-gated Ca2+-channels. INTRODUCTION B a s e d on the clinical evidence that C S F and p l a s m a GABA concentrations are r e d u c e d in depressed patients 19'2°, attention has b e e n directed towards the changes that may occur in G A B A r e c e p t o r subtypes following r e p e a t e d antidepressant administration. G A B A B receptors have gained much importance, as its number has been reported to increase following chronic treatment of antidepressants 33'37. In contrast to these observations, a recent study by Cross and Horton 12 showed no changes in G A B A B receptor affinity or number after the chronic administration of antidepressants. The tricyclic antidepressants have been shown to reduce the inward calcium uptake and/or currents in several p r e p a r a t i o n s 2,18'24, and also acute t r e a t m e n t with antidepressants was r e p o r t e d to inhibit the turnover of neurotransmitters 3. Similarly, G A B A B r e c e p t o r stimulation has b e e n shown to inhibit both Ca 2÷ conductance and n e u r o t r a n s m i t t e r release ~7'29'42. In our previous study, we have shown that G A B A B r e c e p t o r stimulation inhibited voltage-gated Ca2+-activated (depolarizationinduced) 86Rb-efflux (an index of K+-efflux), an effect which was m e d i a t e d through the Gi/Go-proteins and
antagonized by the activatorg o f a d e n y l a t e cyclase and protein kinase C 25'26. Since t h e r e are similarities b e t w e e n their action, we have a t t e m p t e d to examine if G A B A B receptors are involved in the action of antidepressants. This investigation on d e p o l a r i z a t i o n - i n d u c e d 86Rb-efflux assay was executed in p r i m a r y cultured spinal cord neurons, choosing d e s i p r a m i n e as the p r o t o t y p e antidepressant. F u r t h e r m o r e , we have investigated the role of G-proteins, adenylate cyclase, and P K C in the action of antidepressants. M o r e o v e r , to differentiate the nature of the Ca 2+ mechanisms involved, the cells were depolarized by employing two methods. In o r d e r to study v o l t a g e - d e p e n d e n t Ca2+-activated 86Rb-efflux, depolarizing concentrations of KCI were used, and for voltagei n d e p e n d e n t Ca2+-channel-induced efflux, a calcium i o n o p h o r e , viz, A 23187 was e m p l o y e d .
MATERIALS AND METHODS Subjects Female and male C57BI/6J mice (8-10 weeks old) were purchased from Jackson Laboratories (Bar Harbor, MA, U.S.A.). They were housed 5 per cage at a constant room temperature of 25 °C and on a 12-h light/dark cycle (07.00-19.00 h). They had free access to food and water.
Correspondence: M.K. Ticku, Department of Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
60
Preparation of cell cultures Spinal cords were dissected from 13-14-day-old C57BI/6J mice embryos, as described by Ransom et al. 34. Briefy, the embryos, in their sacs, were removed and placed in a 60 mm culture dish containing ice-cold aerated (95% 0 2 and 5% CO2) Puck's buffer, pH 7.4 (100 ml of 10 x Puck's saline, 10 ml of 1 M N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 50 ml of 12% glucose/30% sucrose solution, <320-330 mOsm) and spinal cords were dissected out under a microscope fitted with a light source. The spinal cords were then minced with iridectomy scissors and the tissue was taken up in 1.5 ml of nutrient medium pH 7.4 (MEM 10/10), which contained 80% Eagle's minimal essential medium (MEM), glucose (33.3 mM), NaHCO 3 (44 raM), 10% heat-inactivated horse serum (56 °C for 30 min) and 10% fetal bovine serum, and transferred to a sterile 15 ml centrifuge tube. The tissue fragments were subjected to dissociation by trituration until supernatant volume of 0.75 ml/spinal cord was attained. Dissociated cells were plated on poly-L-lysine-coated sterile 25 mm round coverslips by adding 0.5 ml of the suspension to dishes containing 1 ml of MEM (10/10) which had been preincubated with 90% air and 10% CO2 for at least 1 h at 37 °C. The coverslips were prepared as described earlier 26. The plated cultures were incubated for 24 h. After this time, 1 ml of the growth medium was replaced with 1 ml of the medium containing 10% heat-inactivated horse serum (MEM 10) only. A mixture of sterile 5-fluoro-2"-deoxyuridine (FUDR) plus uridine (2 mg/ml FUDR and 5 mg/ml uridine) at a final concentration of 10 /~g/ml was added on the next day. After every 2-3 days, 1 ml of the medium was replaced with MEM 1°.
Efflux studies All the effux studies were conducted on 8-day-old intact primary cultured spinal cord neurons at room temperature. On the seventh day, the coverslips with cells were incubated overnight with 2/~Ci/ml of S6Rb in the tissue culture medium. In case of PTX studies, the cells were incubated with e'6Rb as well as PTX (140 ng/ml) overnight. In studies involving forskolin (10/~M) or phorbol ester (10/~M), the cultures were incubated with either of these agents 20 min before starting the efflux experiments. In studies with A 23187, the coverslips were incubated with 20/~M of A 23187 along with the test drugs. The experiment was started by washing the coverslips 4 times with 2 ml each of non-depolarizing buffer (in mM: NaCI 145, KCI 5, MgCI 2 2, CaCI 2 1.8, HEPES 10 and glucose 10, adjusted to pH 7.4 with Tris-base) kept in 4 separate petri dishes in order to remove the excess 86Rb. The depolarizing buffer was substituted with 100 mM KCI for an equal amount of NaCI. Both the non-depolarizing and depolarizing buffers also contained 2 mM ouabain to inhibit the Na+/K+-ATPase. In studies with Ca2+-free depolarizing-buffer, the same procedures mentioned above were followed. Before initiating the efflux, the coverslips with neurons were incubated for 4 min with the test drugs in non-depolarizing buffer. The efflux was initiated by incubating the coverslips for a period of 30 s in petri dishes with 2 ml of respective assay solution in which the test compounds were present. This step was preceded by taking duplicates of 100/~1 each from each of the assay solution (with the test compounds) for the determination of zero time cpm in order to get the background cpm. The efflux was terminated after the incubation by rapid transfer and immersion of the coverslips for 10 s in 1000 ml of an ice-cold stop solution which is continuously being stirred. The stop solution contained (in mM: TEA 145, RbCI 5, tetrabutyl ammonium chloride (TBA) 1, MgCI2 5, NiCI 2 10, and HEPES 20 adjusted to pH 7.4 with Tris-base). Following 10 s of immersion in the stop solution, each coverslip was drained on tissue paper and transferred to a scintillation vial containing 1.5 ml of 0:2 N NaOH. This was neutralized with 0.3 ml of 1 N HCI, mixed with 15 ml hydrofluor, and counted by liquid scintillation. In order to measure the effux, a 100/~1 sample in duplicate was taken from each petri dish and transferred to bio-viais. Along with this, 3 ml of hydrofluor was added, mixed well, and counted by
liquid scintillation. The results of all efflux assays were corrected for the background cpm present at time zero. The %86Rb-efflux was calculated as follows: %,6Rb_efflux = cpm x 100 cpm, Cpm t refers to the total count which includes the count present in coverslip and the cpm found at 30 s, i.e. from the duplicate samples taken from the assay solution.
Materials 86Rb was purchased from Du Pont (Boston, MA, U.S.A.). Amitriptyline HCI, desipramine HCI, doxepin HCI, imipramine HCI, maprotiline HCI, nortriptyline HCI, phenelzine sulfate, tranylcypramine sulfate, trazodone HCI, trimipramine maleate, ouabain, TEA, TBA, QSO4, lanthanum, poly-L-lysine hydrobromide, FUDR, phorbol 12,13-dibutyrate, A 23187, and forskolin were obtained from Sigma (St. Louis, MO, U.S.A.), whereas uridine was purchased from Calbiochem, Boehring (La Jolla, CA, U.S.A.). Baclofen isomer, isocarboxazid, and protriptyline HCI were gifts from Ciba-Geigy (Basel, Switzerland), Hoffmann-La Roche, Inc. (Nutley, NJ, U.S.A.), and Merck Sharp and Dohme Research Lab (Rahway, NJ, U.S.A.), respectively. Phaclofen and PTX were purchased from Tocris Neuramin (Essex, U.K.) and List Biochemical Laboratories (Campbell, CA, U.S.A.), respectively. All the salts of antidepressants were dissolved in buffer, except isocarboxazid which was dissolved in dimethyl sulphoxide (DMSO). Baclofen, phaclofen, and phorbol 12,13-dibutyrate were dissolved in buffer. Forskolin and A 23187 were dissolved in DMSO.
Statistical analysis Statistical analysis was performed by one-way analysis of variance and with a multiple range test, using the Newman-Keuls procedure, or as described in the text. A value of P < 0.05 was considered statistically significant. RESULTS
Effects of baclofen, K+-channel blockers and antidepressants on Ca2+-activated 86Rb-efflux S6Rb-effiux significant
{20.
measured
increase
over
o Arnitriptyline
in o u r the
studies
basal
represents
values
with
a the
/J~
I00
80.
"6 6o-
J5 4020-
8 i /_
010~6
i~V5
Drug (M)
,(~-4
1(~-3
Fig. 1. Effects of amitryptyline and desipramine on the depolarization-induced 86Rb-efflux in primary cultured spinal cord cells. The indicated concentration of the drugs was added 4 min before and during the depolarization, as described in Materials and Methods. Two coverslips were used for each treatment. Each point represents mean + S.D. of 4 experiments.
61 TABLE I
TABLE II
Effect of (-)-baclofen, desipramine and the K÷-channel blockers on the depolarization-induced S6Rb-efflux
Effect of various antidepressants on the depolarization-induced S6Rb-efflux
The depolarization was studied in the presence of the agents mentioned above. The cells were preincubated with these agents for 4 min prior to the efflux determinations, as described in Materials and Methods. The results are mean ± S.D. of 4 experiments. Two coverslips were used for each treatment. This table compares the results of 9 treatments. First, a one-way ANOVA and StudentNewman-Keul's test were performed to compare all 9 treatments. The treatment effects have 8 degrees of freedom in one-way ANOVA. Next, the main effect of baclofen and desipramine and their interaction were tested. Four treatments, namely 100 mM KCI, baclofen, desipramine and baclofen + desipramine, were included in these tests. Similarly, the main effects of TEA and QSO 4 and their interaction were also tested, using 100 mM KCI, TEA, QSO 4 and TEA + QSO4. Each of these main effects and interactions is a contrast of single degree of freedom and is a part of the treatment effects with 8 degrees of freedom. In all these tests, the mean squared error with 27 degrees of freedom was used as the within treatment variance. Two sets of two-factor ANOVA were calculated. The first includes 100 mM KCI, baclofen, desipramine and baclofen + desipramine. The main effects of baclofen, desipramine and their interaction were tested in this analysis. The second set constitutes 100 mM KCI, TEA, QSO 4 and TEA + QSO4. In this set also, the main effects of TEA, QSO 4 and their interaction were tested.
The depolarization-induced 86Rb-efflux was obtained with 100 mM KCI for 30 s, as described in Materials and Methods. Two coverslips were used for each treatment and the results are mean + S.D. of the number of experiments shown in parentheses. The following drugs did not inhibit 86Rb-efflux at 10-~ M: carbamazepine, propranoloi, phenobarbital and flurazepam.
Treatment
% S6Rb-efflux (mean ± S. D.)
5 mM KCI 100 mM KC1 100 mM KC1 + 10-5 M baciofen 100 mM KC1 + 4 x 10-5 M desipramine 100 mM KCI + 10-5 M baclofen + 4 x 10-5 M desipramine 100 mM KCI + 10 -3 M TEA 100 mM KCi + 10-5 M QSO4 100 mM KCI + 10-3 M TEA + 10-s M QSO 4 100 mM KCI + 10 -3 M TEA + 10-s M QSO 4 + 4 x 10-s desipramine
11.7±0.8 27.4±0.9* 24.3±0.7** 18.0±2.0"*
Treatment
% S6Rb-efflux (mean + S.D.)
5 mM KCi 100 mM KCI 100 mM KC1 + 100 mM KCi + 100mMKCI + 100 mM KCI + 100 mM KC1 + 100 mM KCI + 100 mM KCI + 100 mM KCI + 100mMKCI + 100 mM KCI +
5.4 ± 14.2 ± 5.7 + 8.6 ± 6.5 ± 7.7 ± 7.6 + 8.6 ± 6.6 ± 8.7 ± 6.9 ± 10.7 ±
10-4 M amitriptyline 10-4 M amoxapine 10-4Mdesipramine 10-4 M doxepin 10-4 M imipramine 10-4 M maprotiline 10-4 M nortriptyline 10-4 M protriptyline 10-4Mtrimipramine 10-4 M trazodone
0.2 1.32 1.0"* 0.4"* 1.1"* 0.2"* 0.6"* 1.5"* 0.7"* 0.5"* 1.1"* 1.5"
(6) (6) (4) (4) (6) (4) (4) (5) (5) (5) (6) (4)
*P < 0.01; **P < 0.001 as compared to 100 mM KCI.
compared with desipramine alone. On the other hand, the combined use of TEA and QSO 4 showed additivity. Similarly, the combination
of TEA,
QSO4,
a n d desi-
pramine has been found to have additivity when com-
18.9±l.2**,n.s. 20.2±1.4"* 20.9±0.8** 15.7±0.7"**
pared to TEA
11.7±0.2 ~
desipramine, tertiary and secondary amine antidepres-
The Newman-Keuls tests to a two factor ANOVA are as follows: *P < 0.001, when compared to 5 mM KC1; **P < 0.001, when compared to 100 mM KCI; ***P < 0.001, when compared to TEA or QSO 4 alone; ~P < 0.001, when compared to TEA plus QSO4 combination. n.s., not significant, when compared to desipramine alone.
a n d Q S O 4 t o g e t h e r ( T a b l e I).
T a b l e II s h o w s t h a t all t h e a n t i d e p r e s s a n t s e x a m i n e d showed
i n h i b i t i o n o f C a 2 ÷ - a c t i v a t e d 86Rb-efflux. T h i s
effect was dose-dependent,
as b o t h a m i t r i p t y l i n e a n d
s a n t s , r e s p e c t i v e l y , i n h i b i t e d t h e e f f l u x in a c o n c e n t r a tion-dependent
manner
(Fig.
1). T h e IC5o v a l u e s f o r
a m i t r i p t y l i n e a n d d e s i p r a m i n e w e r e f o u n d t o b e 10 a n d 40 /tM, respectively. In contrast to the above, the antidepressants
of MAO
inhibitor-class did
not
show
any
i n h i b i t i o n o f t h e efflux ( T a b l e I I I ) . d e p o l a r i z i n g (100 m M K C I ) b u f f e r . I n c o n t r a s t , in s t u d i e s i n v o l v i n g t h e C a 2 + - f r e e d e p o l a r i z i n g - b u f f e r , n o signifi-
TABLE III
c a n t e f f l u x w a s f o u n d (5 m M K C I = 13.99 + 2.73; 100
Effect of MA 0 inhibitors on the depolarization-induced S6Rb-effiux
mM
The depolarization-induced 86Rb-efflux was obtained by incubating the coverslips for 30 s in buffer containing 100 mM KC1 and the test drugs. Two coverslips were used for each treatment and the results are mean ± S.D. of the number of experiments shown in parentheses.
KCI
=
16.21
+
0.57; n
=
4). T h e
basal and
d e p o l a r i z a t i o n - i n d u c e d S6Rb-efflux v a r i e d w i t h d i f f e r e n t b a t c h e s o f c u l t u r e d n e u r o n s . I n m o s t c a s e s , 100 m M K C I i n c r e a s e d 86Rb-efflux o v e r t h e b a s a l in t h e r a n g e o f 130-173%.
T h e d e p o l a r i z a t i o n - i n d u c e d 86Rb-efflux w a s
i n h i b i t e d b y G A B A B r e c e p t o r a g o n i s t b a c l o f e n , desi-
Treatment
%~Rb-e~ ~ean±S.D.)
5 mM 100 mM 100 mM 100 mM 100 mM
6.8±0.2 18.5±0.6 17.8±1.5 18.7±1.3 17.2±0.6
p r a m i n e , a n d t h e K ÷ - c h a n n e l b l o c k e r s ( T a b l e I). W h e n u s e d in c o m b i n a t i o n at s u b m a x i m a l c o n c e n t r a t i o n s , b a c l o f e n a n d d e s i p r a m i n e d i d n o t e x h i b i t s y n e r g i s t i c activity. Moreover,
a d d i t i v i t y w a s also n o t s e e n b e t w e e n t h e m
when
effects of baclofen
the
and
desipramine
were
KCI KCI KCI + 10 -4 M isocarboxazid KCI + 10-4 M phenelzine KCl + 10-4 M tranylcypramine
(4) (4) (5) (4) (5)
62 TABLE IV
TABLE VI
Effect of amitriptyline, desipramine and trazodone on the ~Rbeffiux-induced by A 23187
Effects of PTX, forskolin and phorbol 12,13-dibutyrate on the inhibition of SORb-effluxby desipramine
The coverslips were incubated with the ionophore A 23187 (20/zM) and the respective drugs for 4 min in non-depolarizing buffer. This was followed by the determination of 86Rb-efflux for 30 s, as described in Materials and Methods. Two coverslips were used for each treatment. The numbers in parentheses represent the number of experiments.
The coverslips were treated with PTX, forskolin, or phorbol 12,13-dibutyrate and ran in parallel with their respective control groups, as described in Materials and Methods. The results are mean _+ S.D. of 4 experiments. Two coverslips were used for each treatment.
% S6Rb-Effiux (mean -+S.D.)
Treatment Treatment
% ~Rb-efflux (mean _+S.D.)
5 mM KCI
5 mM KCI + 5 mM KCI + 5 mM KC1 + 5 mM KCi + 5 mMKCI +
A 23187 A 23187 A 23187 A 23187 A 23187
+ + + +
10-4 M baclofen I0 -4 M amitriptyline 10~ M desipramine 10-4 M trazodone
11.8 _+ 2.6 23.9 _+ 2.9* 25.8 _+ 3.6* 9.3_+2.1"* 11.4_+ 1.8"* 11.2_+ 1.6'*
(7) (4) (3) (4) (5) (4)
*P < 0.01 as compared to 5 mM KCI; **P < 0.01 as compared to 5 mM KC1 and A 23187.
T a b l e I V shows that w h e n spinal c o r d cultures w e r e t r e a t e d with C a 2 ÷ - i o n o p h o r e ( A 23187) to raise intracellular
Ca 2÷,
desipramine
inhibited
the
effiux,
while
b a c l o f e n failed to d o so. M o r e o v e r , p h a c l o f e n , a G A B A B receptor antagonist,
Without treatment
With treatment
PTX (140 ng/ml) 5mMKCI 100 mM KCI 100 mM KCI + 10-~ M desipramine
6.8 _+ 1.5 16.2 + 1.4 8.1 + 0.2*
9.4 + 1.8 17.4 -+ 1.0 9.2 -+ 0.6*
Forskolin (10/zM) 5 mM KCI 100mM KCI 100 mM KCI + 10-~ M desipramine
7.6 _+ 1.5 14.7_+0.6 8.4 _+ 1.7"
9.3 + 0.8 19.1 + 1.7 9.5 + 0.8*
Phorbo112,13-dibutyrate (10aM) 5 mM KCI 100mM KCI 100mM KCI + 10-~M desipramine
4.9 + 0.7 17.5_+0.8 8.0 _+0.5*
9.4 + 0.4 15.8+1.2 9.2 + 0.8*
*P < 0.001 as compared to 100 mM KCI of the respective treatment groups.
did n o t a n t a g o n i z e the effect of
d e s i p r a m i n e (Table V). ng/ml) s h o w e d that the effect o f d e s i p r a m i n e o n S6Rb-
Studies with pertussis toxin, forskolin and phorbol esters
efflux was not a n t a g o n i z e d by P T X (Table V I ) .
T h e effects o f pertussis toxin ( P T X ) , which is k n o w n
I n c u b a t i o n o f the c u l t u r e s for 20 m i n with an a c t i v a t o r
to i n a c t i v a t e s o m e G - p r o t e i n s ( G i and Go) on desip r a m i n e - i n d u c e d inhibition o f 86Rb-efflux, w e r e e x a m -
of a d e n y l a t e cyclase, f o r s k o l i n ( 1 0 / z M ) o r an a c t i v a t o r of protein
ined. O v e r n i g h t i n c u b a t i o n of t h e cultures with P T X (140
b e f o r e the efflux studies also failed to a n t a g o n i z e the
kinase
C, p h o r b o l
12,13-dibutyrate
(10 /zM)
effect of d e s i p r a m i n e on 86Rb-efflux (Table VI). TABLE V
DISCUSSION
Effect of phaclofen on the inhibition of S6Rb-efflux by desipramine The influence of the GABA B antagonist, phaclofen, was studied on the desipramine-induced inhibition of a6Rb-efflux. Buffer containing 100 mM KCI was used to depolarize the cells, as described in Materials and Methods. The numbers in parentheses represent the number of experiments. The results were analyzed by using a two-factor (desipramine and phaciofen) ANOVA, which includes 4 treatments viz., 100 mM KCI, desipramine, phaclofen and desipramine + phaclofen.
Chronic administration of antidepressants
has b e e n
s h o w n to p r o d u c e m a r k e d e n h a n c e m e n t of G A B A B r e c e p t o r b i n d i n g , and an i n t e r a c t i o n b e t w e e n m o n o a m i n e systems and G A B A B r e c e p t o r s m a y p r o v e to b e of i m p o r t a n c e in d e p r e s s i o n 9. Pilc and L l o y d 33 and S u z d a k and G i a n u t s o s 36'37 o b s e r v e d a m a r k e d i n c r e a s e in t h e n u m b e r of G A B A B r e c e p t o r s in t h e rat frontal c o r t e x
Treatment
% S6Rb-efflux (mean + S.D.)
5 mM KCI 100 mM KCI 100 mM KCI + 10-4 M desipramine 100 mM KCI + 10-4 M desipramine + 10-4 M phaclofen 100 mM KCI + 10-4 M desipramine + 5 × 10-4 M phaclofen 100 mM KCI + 5 x 10-4 M phaclofen
6.7 -+ 1.5 16.0 + 1.6 8.1 + 0.2*
(4) (4) (4)
9.1 + 1.7"
(4)
m a t e r i a l s o b t a i n e d f r o m t h e a n i m a l s t r e a t e d chronically
(4) (4)
with t h e a n t i d e p r e s s a n t s . O u r in vitro studies, using a f u n c t i o n a l assay, rule o u t an i n t e r a c t i o n b e t w e e n tricyclic
The Newman-Keuls test results are as follows: *P < 0.001, when compared to 100 mM KCI.
after c h r o n i c t r e a t m e n t with a n t i d e p r e s s a n t s . B u t , in contrast, a r e c e n t study by Cross and H o r t o n 12 s h o w e d n o changes in G A B A B r e c e p t o r affinity o r n u m b e r after the chronic administration
of a n t i d e p r e s s a n t s .
A l l o f the
a b o v e studies utilized r a d i o l i g a n d b i n d i n g studies with t h e
8.4 _+0.4" 15.7 _+0.6
antidepressants
and p r e s y n a p t i c
G A B A B r e c e p t o r s in
c u l t u r e d spinal c o r d n e u r o n s (see b e l o w ) . H o w e v e r , the
63 possibility that chronic tricyclic antidepressant treatment may affect G A B A B receptor function in cortical neurons cannot be ruled out. The stimulation of G A B A a receptors has been reported to inhibit the voltage-sensitive Ca 2÷ currents in dorsal root ganglia of chick and to also inhibit K ÷-evoked release of 5-HT in mouse cortical slices 8'15'23. Moreover, baclofen, a G A B A B agonist, has been shown to reduce the evoked release of neurotransmitters from peripheral sympathetic nerve terminals s'9'13'14 and from brain slices 15'23'3° which was mimicked by G A B A . Similarly, the acute treatment with the antidepressants has been reported to reduce the turnover of the central neurotransmitters 3, which may be related to its influence on calcium, as the tricyclic antidepressants have been reported to inhibit the inward calcium current in rat brain synaptosomes 2 and in several other preparations 18"24. These lines of evidence point out that the acute effects of antidepressants may be mediated through the G A B A n receptor activation. Recently, we have developed a functional assay for measuring G A B A a receptor (presynaptic) responses in vitro using 86Rb-efflux assay in primary cultured spinal cord neurons 25. The rationale for using 86Rb as a substitute for K + has already been well justified 5'6. The pharmacology of the Ca2+-dependent S6Rb-efflux is also consistent with the previous reports 5'6 and also the earlier reports from o u r laboratory 25'26'39. Thus Q S O 4 and T E A , which block CaE+-activated K+-channels 1'5"6'38, inhibited the CaE+-activated 86Rb-efflux. The inability of 100 mM KCI to induce a significant increase in 86Rb-efflux in the absence of C a 2+ suggests that the efflux is dependent o n C a 2+ influx. Thus, the inhibition of Ca2+-activated 86Rb-efflux by (-)-baclofen is consistent with the involvement of G A B A B receptors in this action. This effect of (-)-baclofen apparently is presynaptic and was blocked by phaclofen, a G A B A B receptor antagonist 27. Furthermore, our recent studies have indicated that pertussis toxin, phorbol ester and forskolin inhibit the effect of (-)-baclofen o n C a 2÷activated 86Rb-efflux26. In the present study, all the antidepressants, except the monoamine oxidase inhibitors, inhibited Ca2+-activated 86Rb-efflux. Moreover, this effect was concentration-dependent, as both amitriptyline and desipramine inhibited the K÷-efflux in a concentration-dependent manner (Fig. 1). The 1(75o value of desipramine (40 tiM) is higher than the reported blood concentrations in humans (4/aM) 4°. However, since the brain plasma ratio is greater than 2021 , higher concentrations of desipramine may be expected in the CNS. The absence of synergism between baclofen and desipramine may be explained by the possibility that these two agents might share the same K÷-channels,
albeit, via different mechanisms. This is also supported by other observations, since there are major differences between tricyclic antidepressants and G A B A B receptormediated inhibition of the efflux process. The ability of tricyclic antidepressants, but not baclofen, to inhibit Ca2÷-activated 86Rb-efflux following A 23187 (which increases intracellular Ca 2+ levels independent of voltage-gated Ca2+-channels) treatment indicates that the inhibition with tricyclics may occur at a stage subsequent to the voltage-gated Ca2+-channels. These results also indicate that a mechanism different than baclofen, which inhibits the efflux at the level of voltage-gated Ca 2÷channels, may be involved. Moreover, the observation that G A B A B antagonist, phaclofen, was not effective against desipramine also suggests that the acute effects of antidepressants are not mediated through the G A B A B receptors. Previous studies have shown the involvement of G-proteins in the action of neurotransmitters (including baclofen) that inhibit Ca 2+ current. In our previous study, PTX, which is known to uncouple G-proteins by ADP-ribosylation 7'28, blocked the action of baclofen on Ca 2+ activated 86Rb-efflux25. However, the action of desipramine on the K+-efflux was not reversed by the pretreatment with PTX, suggesting that the action of antidepressants may not be through the G-protein mechanism. It has been reported that G A B A B receptor activation leads to an inhibition of adenylate cyclase 32"41. Phorbol esters and forskolin, which are known to activate protein kinase C 4'11 and adenylate cyclase 35'41, respectively, have been shown to inhibit the action of baclofen T M . These mechanisms may not be applicable in the case of the antidepressants, in contrast to baclofen, since both forskolin and phorbol 12,13-dibutyrate were unable to influence their action. Taken together, this leads to the conclusion that even though the antidepressants inhibit the CaE+-activated K+-efflux like the G A B A B agonists, they do not mediate this effect via the G A B A B receptors. This effect of antidepressants to inhibit Ca2+-activated S6Rb-efflux m u s t be occurring at a step in the efflux process beyond voltage-gated Ca2+-channels and adenylate cyclase and G-protein coupled events. It is feasible that tricyclic may inhibit the 86Rb-efflux process by directly blocking the K+-channels. The pharmacological significance of this effect is not clear, but may be potentially relevant to neurotransmitter release. However, the possibility that this action of antidepressants may be due to an indirect effect via blockade of release of some excitatory neurotransmitter which opens calcium-gating channels or via blockade of such channels and/or their local anesthetic activities cannot be ruled out. The mechanism of local
64 a n e s t h e t i c p r o p e r t y of t h e s e agents m a y be due to t h e i r i n f l u e n c e on the v o l t a g e - g a t e d s o d i u m channels 31. Fi-
efflux, and so efforts are u n d e r w a y to see t h e i r effect on K + - e f f l u x after c h r o n i c t r e a t m e n t .
nally, since t h e a n t i d e p r e s s a n t s express t h e i r t h e r a p e u t i c effect o n l y after 2 w e e k s of t r e a t m e n t , it is n e c e s s a r y to e x a m i n e their effect after chronic t r e a t m e n t on 86Rb-
REFERENCES 1 Armstrong, C.M., Potassium pores of nerve and muscle membranes. In E. Eisenman (Ed.), Membranes -- A Series of Advances, Vol. 3, Dekker, New York, 1975, pp. 325-358. 2 Aronstam, R.S. and Hoss, W., Tricyclic antidepressant inhibition of depolarization-induced uptake of calcium by synaptosomes from rat brain, Biochem. Pharmacol., 34 (1985) 902-904. 3 Baldessarini, R.J., Drugs and the treatment of psychiatric disorders. In A.G. Gilman, L.S. Goodman, T.W. Wall and E Murad (Eds.), The Pharmacological Basis of Therapeutics, MacMillan, New York, 1985, pp. 387-445. 4 Baraban, J.M., Snyder, S.H. and Alger, B.E., Protein kinase C regulates ionic conductance in hippocampal neurons: electrophysiologicai effects of phorbol ester, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2538-2542. 5 Bartschat, D.R. and Blaustein, M.P., Potassium channels in isolated presynaptic nerve terminals from rat brain, J. Physiol., 361 (1985) 419-440. 6 Bartschat, D.R. and Blaustein, M.P., Calcium-activated potassium channels in isolated presynaptic nerve terminals from rat brain, J. Physiol., 361 (1985) 441-457. 7 Bokoch, G.M., Katada, T., Northup, J.K., Hewlett, E.L. and Gilman, A.G., Identification of the predominant substrate for ADP-ribosylation by islet activating protein, J. Biol. Chem., 258 (1983) 2072-2075. 8 Bowery, N.G., Hill, D.R., Hudson, A.L., Doble, A., Middlemiss, D.N., Shaw, J. and Turnbull, M.J., (-)-Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor, Nature, 283 (1980) 92-95. 9 Bowery, N.G., Price, G.W., Hudson, A.L., Hill, D.R., Wilkin, G.P. and Turnbull, M.J., GABA receptor multiplicity: visualization of different receptor types in the mammalian CNS, Neuropharmacology, 23 (1984) 219-231. 10 Bowery, N.G., Hill, D.R., Hudson, A.L. and Price, G.W., GABA a receptors. In R.E Squires (Ed.), G A B A and Benzodiazepine Receptors, Vol. 1, CRC, Florida, 1988, pp. 107-121. 11 Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y., Direct activation of calcium-activated phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem., 257 (1982) 7847-7851. 12 Cross, J.A. and Horton, R.W., Effects of chronic oral administration of the antidepressants, desmethylimipramine and zimelidine on rat cortical GABA B binding sites: a comparison with 5-HT 2 binding site changes, Br. J. Pharmacol., 93 (1988) 331-336. 13 Curtis, D.R., Lodge, D., Bornstein, J.C. and Peet, M.J., Selective effects of (-)-baclofen on spinal synaptic transmission in the cat, Exp. Brain Res., 42 (1981) 158-170. 14 Davies, J., Selective depression of synaptic excitation in cat spinal neurones by baclofen: an iontophoretic study, Br. J. Pharmacol., 72 (1981) 373-384. 15 Desarmenien, M., Feltz, P., Loeffler, J.P., Occhipinti, G. and Santangelo, F., Multiple GABA-receptors on A, 6 and C primary afferent neurones in the adult rat, Br. J. Pharmacol., 76 (1982) 289P. 16 Dolphin, A.C. and Scott, R.H., Inhibition of calcium currents in cultured rat dorsal root ganglion neurons by (-)-baclofen, Br. J. Pharmacol., 88 (1986) 213-220. 17 Dolphin, A.C. and Scott, R.H., Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurons: modulation by guanine nucleotides, J. Physiol., 386 (1987) 1-17.
Acknowledgement. We thank Mrs. Sadie Phillips for excellent secretarial help.
18 Garcia de Jalon, P., Rodriguez, S. and Tamargo, J., Electrophysiological effects of imipramine in guinea-pig myocardium, Br. J. Pharmacol., 63 (1978) 373P. 19 Gerner, R.H. and Hare, T.A., CSF GABA in normal subjects and patients with depression, schizophrenia, mania and anorexia nervosa, Am. J. Psychiat., 138 (1981) 1089-1101. 20 Gold, B.I., Bowers, M.B., Roth, R.H. and Sweeney, D.W., GABA levels in CSF of patients with psychiatric disorders, Am. J. Psychiat., 137 (1980) 362-364. 21 Hrdina, P.D., Dubas, T.C. and Riva, E., Brain distribution and pharmacokinetics of psychotropic drugs: desipramine. In P. Deniker, C. Raduco-Thomas and A. Villenene (Eds.), Neuropsychopharmacology, Pergamon, New York, 1978, PI~. 841848. 22 Hill, D.R., GABA B receptor modulation of adenylate cyclase activity in rat brain slices, Br. J. Pharmacol., 84 (1985) 249-257. 23 Holz, G.G., Rane, S.G. and Dunlap, K., GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels, Nature, 319 (1986) 670-672. 24 Isenberg, G. and Tamargo, J., Effect of imipramine on calcium and potassium currents in isolated bovine ventricular myocytes, Eur. J. PharmacoL, 108 (1985) 121-131. 25 Kamatchi, G.L. and Ticku, M.K., G A B A B receptor activation inhibits Ca2÷-activated S6Rb-efflux in cultured spinal cord neurons via G-protein mechanism, Brain Research, 506 (1990) 181-186. 26 Kamatchi, G.L. and Ticku, M.K., Functional coupling of presynaptic GABA a receptors with voltage-gated Ca2÷-chan nels: regulation by protein kinase A and C in cultured spinal cord neurons, Mol. Pharmacol., 38 (1990) 342-347. 27 Kerr, D.I.B., Ong, J., Prager, R.H., Gynther, B.D. and Curtis, D.R., Phaclofen, a peripheral and central baclofen antagonist, Brain Research, 405 (1987) 150-154. 28 Kurose, H., Katada, T., Amano, T. and Ui, M., Specific coupling by islet-activating protein, pertussis toxin, of negative signal transduction via a-adrenergic, cholinergic and opiate receptors in neuroblastoma × glioma hybrid cells, J. Biol. Chem., 258 (1983) 2870-2875. 29 Levi, G. and Gailo, V., Release studies related to the neurotransmitter role of glutamate in the cerebellum: an overview, Neurochem. Res., 11 (1986) 1627-1642. 30 Lev-Tov, A., Meyers, D.E.R. and Burke, R.E., Activation of type B y-aminobutyric acid receptors in the intact mammalian spinal cord minus the effects of reduced presynaptic Ca 2÷ influx, Proc. Natl. Acad. Sci., U.S.A., 85 (1988) 5330-5334. 31 McNeal, E.T., Lewandowski, G.A., Daly, J.W. and Creveling, C.R., [3H]Batrachotoxinin A 20 a-benzoate binding to voltagesensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs, J. Med. Chem., 28 (1985) 381-388. 32 Nishikawa, M. and Kuriyama, K., Functional coupling of cerebral 7-aminobutyric acid (GABA)B receptor with adenylate cyclase system: effect of phaclofen, Neurochem. Int., 14 (1989) 85-90. 33 Pilc, A. and Lloyd, K.G., Chronic antidepressants and GABA B receptors: a GABA hypothesis of antidepressant drug action, Life Sci., 35 (1984) 2149-2154. 34 Ransom, B.R., Neale, E., Henkart, M., Bullock, P.N. and Nelson, P.A., Mouse spinal cord in cell culture. I. Morphological and intrinsic neuronal electrophysiological properties, J. Neurophysiol., 40 (1977) 1132-1150. 35 Seamon, K.B. and Daly, J.W., Forskolin, cyclic AMP and
65 cellular physiology, Trends Pharmacol. Sci., 4 (1983) 120-123. 36 Suzdak, P.D. and Gianutsos, G., Parallel changes in the sensitivity of ~-aminobutyric acid and noradrenergic receptors following chronic administration of antidepressant and GABAergic drugs, Neuropharmacology, 24 (1985) 217-222. 37 Suzdak, P.D. and Gianutsos, G., Effect of chronic imipramine or baciofen on GABA-B binding and cyclic AMP production in cerebral cortex, Eur. J. Pharmacol., 131 (1986) 129-133. 38 Thompson, S.H., Three pharmacologically distinct potassium channels in molluscan neurones, J. Physiol., 265 (1977) 465-488. 39 Ticku, M.K. and Delgado, A., GABA B receptor activation inhibits Ca2÷-activated potassium channels in synaptosomes:
involvement of G-proteins, Life Sci., 441 (1989) 271-276. 40 Winek, C.L., Tabulation of therapeutic, toxic and lethal concentrations of drugs and chemicals in blood, Clin. Chem., 22 (1976) 832-836. 41 Wojcik, W.J. and Neff, N.H., y-Aminobutyric acid B receptors are negatively coupled to adenylate cyclase in brain and in the cerebellum. These receptors may be associated with granule cells, Mol. Pharmacol., 25 (1984) 24-28. 42 Xing-Zu Zhu and De-Maw Chuang, Modulation of calcium uptake and D-aspartate release by GABA B receptors in cultured cerebellar granule cells, Eur. J. Pharmacol., 141 (1987) 401-408.
59
BRES 16455
Tricyclic antidepressants inhibit Ca2+-activated K+-efflux in cultured spinal cord neurons Ganesan L. Kamatchi and Maharaj K. Ticku Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284-7764 (U.S.A.) (Accepted 23 October 1990) Key words: Tricyclic antidepressant; Ca2+-activated K + channel; ~-Aminobutyric acid-B receptor; Voltage-gated Ca 2+ channel; Spinal cord neuron
The effect of tricyclic antidepressants and monoamine oxidase inhibitors on the Ca2+-activated K+-efflux was studied using 86Rb-efflux assay in primary cultured mouse spinal cord neurons. Depolarization of the cultured cells with 100 mM KCI increased the 8~Rb-efflux significantly in Ca2÷-containing buffer, but not in Ca2+-free buffer. All the antidepressants examined, except the monoamine oxidase inhibitors, inhibited the 86Rb-efflux. Desipramine exhibited additivity with tetraethyl ammonium (TEA) and quinine sulfate (QSO4) , but not with GABA a receptor agonist baclofen. The inhibitory action of antidepressants was not mediated through the GABAB receptors, since GABA a receptor antagonist, phaclofen, was unable to antagonize this effect. The ability of tricyclic antidepressants to inhibit calcium ionophore (A 23187)-induced 86Rb-efflux suggests that these drugs do not act at the level of voltage-gated Ca2+-channels. Furthermore, this effect does not seem to involve the G-proteins, adenylate cyclase, or protein kinase C systems, since pertussis toxin (PTX) and the activators of adenylate cyclase and protein kinase C did not reverse the effect of tricyclics on 86Rb-efflux. Taken together, these results suggest that antidepressants inhibit Ca2+-aetivated K÷-channels at a stage subsequent to the voltage-gated Ca2+-channels. INTRODUCTION B a s e d on the clinical evidence that C S F and p l a s m a GABA concentrations are r e d u c e d in depressed patients 19'2°, attention has b e e n directed towards the changes that may occur in G A B A r e c e p t o r subtypes following r e p e a t e d antidepressant administration. G A B A B receptors have gained much importance, as its number has been reported to increase following chronic treatment of antidepressants 33'37. In contrast to these observations, a recent study by Cross and Horton 12 showed no changes in G A B A B receptor affinity or number after the chronic administration of antidepressants. The tricyclic antidepressants have been shown to reduce the inward calcium uptake and/or currents in several p r e p a r a t i o n s 2,18'24, and also acute t r e a t m e n t with antidepressants was r e p o r t e d to inhibit the turnover of neurotransmitters 3. Similarly, G A B A B r e c e p t o r stimulation has b e e n shown to inhibit both Ca 2÷ conductance and n e u r o t r a n s m i t t e r release ~7'29'42. In our previous study, we have shown that G A B A B r e c e p t o r stimulation inhibited voltage-gated Ca2+-activated (depolarizationinduced) 86Rb-efflux (an index of K+-efflux), an effect which was m e d i a t e d through the Gi/Go-proteins and
antagonized by the activatorg o f a d e n y l a t e cyclase and protein kinase C 25'26. Since t h e r e are similarities b e t w e e n their action, we have a t t e m p t e d to examine if G A B A B receptors are involved in the action of antidepressants. This investigation on d e p o l a r i z a t i o n - i n d u c e d 86Rb-efflux assay was executed in p r i m a r y cultured spinal cord neurons, choosing d e s i p r a m i n e as the p r o t o t y p e antidepressant. F u r t h e r m o r e , we have investigated the role of G-proteins, adenylate cyclase, and P K C in the action of antidepressants. M o r e o v e r , to differentiate the nature of the Ca 2+ mechanisms involved, the cells were depolarized by employing two methods. In o r d e r to study v o l t a g e - d e p e n d e n t Ca2+-activated 86Rb-efflux, depolarizing concentrations of KCI were used, and for voltagei n d e p e n d e n t Ca2+-channel-induced efflux, a calcium i o n o p h o r e , viz, A 23187 was e m p l o y e d .
MATERIALS AND METHODS Subjects Female and male C57BI/6J mice (8-10 weeks old) were purchased from Jackson Laboratories (Bar Harbor, MA, U.S.A.). They were housed 5 per cage at a constant room temperature of 25 °C and on a 12-h light/dark cycle (07.00-19.00 h). They had free access to food and water.
Correspondence: M.K. Ticku, Department of Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
60
Preparation of cell cultures Spinal cords were dissected from 13-14-day-old C57BI/6J mice embryos, as described by Ransom et al. 34. Briefy, the embryos, in their sacs, were removed and placed in a 60 mm culture dish containing ice-cold aerated (95% 0 2 and 5% CO2) Puck's buffer, pH 7.4 (100 ml of 10 x Puck's saline, 10 ml of 1 M N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 50 ml of 12% glucose/30% sucrose solution, <320-330 mOsm) and spinal cords were dissected out under a microscope fitted with a light source. The spinal cords were then minced with iridectomy scissors and the tissue was taken up in 1.5 ml of nutrient medium pH 7.4 (MEM 10/10), which contained 80% Eagle's minimal essential medium (MEM), glucose (33.3 mM), NaHCO 3 (44 raM), 10% heat-inactivated horse serum (56 °C for 30 min) and 10% fetal bovine serum, and transferred to a sterile 15 ml centrifuge tube. The tissue fragments were subjected to dissociation by trituration until supernatant volume of 0.75 ml/spinal cord was attained. Dissociated cells were plated on poly-L-lysine-coated sterile 25 mm round coverslips by adding 0.5 ml of the suspension to dishes containing 1 ml of MEM (10/10) which had been preincubated with 90% air and 10% CO2 for at least 1 h at 37 °C. The coverslips were prepared as described earlier 26. The plated cultures were incubated for 24 h. After this time, 1 ml of the growth medium was replaced with 1 ml of the medium containing 10% heat-inactivated horse serum (MEM 10) only. A mixture of sterile 5-fluoro-2"-deoxyuridine (FUDR) plus uridine (2 mg/ml FUDR and 5 mg/ml uridine) at a final concentration of 10 /~g/ml was added on the next day. After every 2-3 days, 1 ml of the medium was replaced with MEM 1°.
Efflux studies All the effux studies were conducted on 8-day-old intact primary cultured spinal cord neurons at room temperature. On the seventh day, the coverslips with cells were incubated overnight with 2/~Ci/ml of S6Rb in the tissue culture medium. In case of PTX studies, the cells were incubated with e'6Rb as well as PTX (140 ng/ml) overnight. In studies involving forskolin (10/~M) or phorbol ester (10/~M), the cultures were incubated with either of these agents 20 min before starting the efflux experiments. In studies with A 23187, the coverslips were incubated with 20/~M of A 23187 along with the test drugs. The experiment was started by washing the coverslips 4 times with 2 ml each of non-depolarizing buffer (in mM: NaCI 145, KCI 5, MgCI 2 2, CaCI 2 1.8, HEPES 10 and glucose 10, adjusted to pH 7.4 with Tris-base) kept in 4 separate petri dishes in order to remove the excess 86Rb. The depolarizing buffer was substituted with 100 mM KCI for an equal amount of NaCI. Both the non-depolarizing and depolarizing buffers also contained 2 mM ouabain to inhibit the Na+/K+-ATPase. In studies with Ca2+-free depolarizing-buffer, the same procedures mentioned above were followed. Before initiating the efflux, the coverslips with neurons were incubated for 4 min with the test drugs in non-depolarizing buffer. The efflux was initiated by incubating the coverslips for a period of 30 s in petri dishes with 2 ml of respective assay solution in which the test compounds were present. This step was preceded by taking duplicates of 100/~1 each from each of the assay solution (with the test compounds) for the determination of zero time cpm in order to get the background cpm. The efflux was terminated after the incubation by rapid transfer and immersion of the coverslips for 10 s in 1000 ml of an ice-cold stop solution which is continuously being stirred. The stop solution contained (in mM: TEA 145, RbCI 5, tetrabutyl ammonium chloride (TBA) 1, MgCI2 5, NiCI 2 10, and HEPES 20 adjusted to pH 7.4 with Tris-base). Following 10 s of immersion in the stop solution, each coverslip was drained on tissue paper and transferred to a scintillation vial containing 1.5 ml of 0:2 N NaOH. This was neutralized with 0.3 ml of 1 N HCI, mixed with 15 ml hydrofluor, and counted by liquid scintillation. In order to measure the effux, a 100/~1 sample in duplicate was taken from each petri dish and transferred to bio-viais. Along with this, 3 ml of hydrofluor was added, mixed well, and counted by
liquid scintillation. The results of all efflux assays were corrected for the background cpm present at time zero. The %86Rb-efflux was calculated as follows: %,6Rb_efflux = cpm x 100 cpm, Cpm t refers to the total count which includes the count present in coverslip and the cpm found at 30 s, i.e. from the duplicate samples taken from the assay solution.
Materials 86Rb was purchased from Du Pont (Boston, MA, U.S.A.). Amitriptyline HCI, desipramine HCI, doxepin HCI, imipramine HCI, maprotiline HCI, nortriptyline HCI, phenelzine sulfate, tranylcypramine sulfate, trazodone HCI, trimipramine maleate, ouabain, TEA, TBA, QSO4, lanthanum, poly-L-lysine hydrobromide, FUDR, phorbol 12,13-dibutyrate, A 23187, and forskolin were obtained from Sigma (St. Louis, MO, U.S.A.), whereas uridine was purchased from Calbiochem, Boehring (La Jolla, CA, U.S.A.). Baclofen isomer, isocarboxazid, and protriptyline HCI were gifts from Ciba-Geigy (Basel, Switzerland), Hoffmann-La Roche, Inc. (Nutley, NJ, U.S.A.), and Merck Sharp and Dohme Research Lab (Rahway, NJ, U.S.A.), respectively. Phaclofen and PTX were purchased from Tocris Neuramin (Essex, U.K.) and List Biochemical Laboratories (Campbell, CA, U.S.A.), respectively. All the salts of antidepressants were dissolved in buffer, except isocarboxazid which was dissolved in dimethyl sulphoxide (DMSO). Baclofen, phaclofen, and phorbol 12,13-dibutyrate were dissolved in buffer. Forskolin and A 23187 were dissolved in DMSO.
Statistical analysis Statistical analysis was performed by one-way analysis of variance and with a multiple range test, using the Newman-Keuls procedure, or as described in the text. A value of P < 0.05 was considered statistically significant. RESULTS
Effects of baclofen, K+-channel blockers and antidepressants on Ca2+-activated 86Rb-efflux S6Rb-effiux significant
{20.
measured
increase
over
o Arnitriptyline
in o u r the
studies
basal
represents
values
with
a the
/J~
I00
80.
"6 6o-
J5 4020-
8 i /_
010~6
i~V5
Drug (M)
,(~-4
1(~-3
Fig. 1. Effects of amitryptyline and desipramine on the depolarization-induced 86Rb-efflux in primary cultured spinal cord cells. The indicated concentration of the drugs was added 4 min before and during the depolarization, as described in Materials and Methods. Two coverslips were used for each treatment. Each point represents mean + S.D. of 4 experiments.
61 TABLE I
TABLE II
Effect of (-)-baclofen, desipramine and the K÷-channel blockers on the depolarization-induced S6Rb-efflux
Effect of various antidepressants on the depolarization-induced S6Rb-efflux
The depolarization was studied in the presence of the agents mentioned above. The cells were preincubated with these agents for 4 min prior to the efflux determinations, as described in Materials and Methods. The results are mean ± S.D. of 4 experiments. Two coverslips were used for each treatment. This table compares the results of 9 treatments. First, a one-way ANOVA and StudentNewman-Keul's test were performed to compare all 9 treatments. The treatment effects have 8 degrees of freedom in one-way ANOVA. Next, the main effect of baclofen and desipramine and their interaction were tested. Four treatments, namely 100 mM KCI, baclofen, desipramine and baclofen + desipramine, were included in these tests. Similarly, the main effects of TEA and QSO 4 and their interaction were also tested, using 100 mM KCI, TEA, QSO 4 and TEA + QSO4. Each of these main effects and interactions is a contrast of single degree of freedom and is a part of the treatment effects with 8 degrees of freedom. In all these tests, the mean squared error with 27 degrees of freedom was used as the within treatment variance. Two sets of two-factor ANOVA were calculated. The first includes 100 mM KCI, baclofen, desipramine and baclofen + desipramine. The main effects of baclofen, desipramine and their interaction were tested in this analysis. The second set constitutes 100 mM KCI, TEA, QSO 4 and TEA + QSO4. In this set also, the main effects of TEA, QSO 4 and their interaction were tested.
The depolarization-induced 86Rb-efflux was obtained with 100 mM KCI for 30 s, as described in Materials and Methods. Two coverslips were used for each treatment and the results are mean + S.D. of the number of experiments shown in parentheses. The following drugs did not inhibit 86Rb-efflux at 10-~ M: carbamazepine, propranoloi, phenobarbital and flurazepam.
Treatment
% S6Rb-efflux (mean ± S. D.)
5 mM KCI 100 mM KC1 100 mM KC1 + 10-5 M baciofen 100 mM KC1 + 4 x 10-5 M desipramine 100 mM KCI + 10-5 M baclofen + 4 x 10-5 M desipramine 100 mM KCI + 10 -3 M TEA 100 mM KCi + 10-5 M QSO4 100 mM KCI + 10-3 M TEA + 10-s M QSO 4 100 mM KCI + 10 -3 M TEA + 10-s M QSO 4 + 4 x 10-s desipramine
11.7±0.8 27.4±0.9* 24.3±0.7** 18.0±2.0"*
Treatment
% S6Rb-efflux (mean + S.D.)
5 mM KCi 100 mM KCI 100 mM KC1 + 100 mM KCi + 100mMKCI + 100 mM KCI + 100 mM KC1 + 100 mM KCI + 100 mM KCI + 100 mM KCI + 100mMKCI + 100 mM KCI +
5.4 ± 14.2 ± 5.7 + 8.6 ± 6.5 ± 7.7 ± 7.6 + 8.6 ± 6.6 ± 8.7 ± 6.9 ± 10.7 ±
10-4 M amitriptyline 10-4 M amoxapine 10-4Mdesipramine 10-4 M doxepin 10-4 M imipramine 10-4 M maprotiline 10-4 M nortriptyline 10-4 M protriptyline 10-4Mtrimipramine 10-4 M trazodone
0.2 1.32 1.0"* 0.4"* 1.1"* 0.2"* 0.6"* 1.5"* 0.7"* 0.5"* 1.1"* 1.5"
(6) (6) (4) (4) (6) (4) (4) (5) (5) (5) (6) (4)
*P < 0.01; **P < 0.001 as compared to 100 mM KCI.
compared with desipramine alone. On the other hand, the combined use of TEA and QSO 4 showed additivity. Similarly, the combination
of TEA,
QSO4,
a n d desi-
pramine has been found to have additivity when com-
18.9±l.2**,n.s. 20.2±1.4"* 20.9±0.8** 15.7±0.7"**
pared to TEA
11.7±0.2 ~
desipramine, tertiary and secondary amine antidepres-
The Newman-Keuls tests to a two factor ANOVA are as follows: *P < 0.001, when compared to 5 mM KC1; **P < 0.001, when compared to 100 mM KCI; ***P < 0.001, when compared to TEA or QSO 4 alone; ~P < 0.001, when compared to TEA plus QSO4 combination. n.s., not significant, when compared to desipramine alone.
a n d Q S O 4 t o g e t h e r ( T a b l e I).
T a b l e II s h o w s t h a t all t h e a n t i d e p r e s s a n t s e x a m i n e d showed
i n h i b i t i o n o f C a 2 ÷ - a c t i v a t e d 86Rb-efflux. T h i s
effect was dose-dependent,
as b o t h a m i t r i p t y l i n e a n d
s a n t s , r e s p e c t i v e l y , i n h i b i t e d t h e e f f l u x in a c o n c e n t r a tion-dependent
manner
(Fig.
1). T h e IC5o v a l u e s f o r
a m i t r i p t y l i n e a n d d e s i p r a m i n e w e r e f o u n d t o b e 10 a n d 40 /tM, respectively. In contrast to the above, the antidepressants
of MAO
inhibitor-class did
not
show
any
i n h i b i t i o n o f t h e efflux ( T a b l e I I I ) . d e p o l a r i z i n g (100 m M K C I ) b u f f e r . I n c o n t r a s t , in s t u d i e s i n v o l v i n g t h e C a 2 + - f r e e d e p o l a r i z i n g - b u f f e r , n o signifi-
TABLE III
c a n t e f f l u x w a s f o u n d (5 m M K C I = 13.99 + 2.73; 100
Effect of MA 0 inhibitors on the depolarization-induced S6Rb-effiux
mM
The depolarization-induced 86Rb-efflux was obtained by incubating the coverslips for 30 s in buffer containing 100 mM KC1 and the test drugs. Two coverslips were used for each treatment and the results are mean ± S.D. of the number of experiments shown in parentheses.
KCI
=
16.21
+
0.57; n
=
4). T h e
basal and
d e p o l a r i z a t i o n - i n d u c e d S6Rb-efflux v a r i e d w i t h d i f f e r e n t b a t c h e s o f c u l t u r e d n e u r o n s . I n m o s t c a s e s , 100 m M K C I i n c r e a s e d 86Rb-efflux o v e r t h e b a s a l in t h e r a n g e o f 130-173%.
T h e d e p o l a r i z a t i o n - i n d u c e d 86Rb-efflux w a s
i n h i b i t e d b y G A B A B r e c e p t o r a g o n i s t b a c l o f e n , desi-
Treatment
%~Rb-e~ ~ean±S.D.)
5 mM 100 mM 100 mM 100 mM 100 mM
6.8±0.2 18.5±0.6 17.8±1.5 18.7±1.3 17.2±0.6
p r a m i n e , a n d t h e K ÷ - c h a n n e l b l o c k e r s ( T a b l e I). W h e n u s e d in c o m b i n a t i o n at s u b m a x i m a l c o n c e n t r a t i o n s , b a c l o f e n a n d d e s i p r a m i n e d i d n o t e x h i b i t s y n e r g i s t i c activity. Moreover,
a d d i t i v i t y w a s also n o t s e e n b e t w e e n t h e m
when
effects of baclofen
the
and
desipramine
were
KCI KCI KCI + 10 -4 M isocarboxazid KCI + 10-4 M phenelzine KCl + 10-4 M tranylcypramine
(4) (4) (5) (4) (5)
62 TABLE IV
TABLE VI
Effect of amitriptyline, desipramine and trazodone on the ~Rbeffiux-induced by A 23187
Effects of PTX, forskolin and phorbol 12,13-dibutyrate on the inhibition of SORb-effluxby desipramine
The coverslips were incubated with the ionophore A 23187 (20/zM) and the respective drugs for 4 min in non-depolarizing buffer. This was followed by the determination of 86Rb-efflux for 30 s, as described in Materials and Methods. Two coverslips were used for each treatment. The numbers in parentheses represent the number of experiments.
The coverslips were treated with PTX, forskolin, or phorbol 12,13-dibutyrate and ran in parallel with their respective control groups, as described in Materials and Methods. The results are mean _+ S.D. of 4 experiments. Two coverslips were used for each treatment.
% S6Rb-Effiux (mean -+S.D.)
Treatment Treatment
% ~Rb-efflux (mean _+S.D.)
5 mM KCI
5 mM KCI + 5 mM KCI + 5 mM KC1 + 5 mM KCi + 5 mMKCI +
A 23187 A 23187 A 23187 A 23187 A 23187
+ + + +
10-4 M baclofen I0 -4 M amitriptyline 10~ M desipramine 10-4 M trazodone
11.8 _+ 2.6 23.9 _+ 2.9* 25.8 _+ 3.6* 9.3_+2.1"* 11.4_+ 1.8"* 11.2_+ 1.6'*
(7) (4) (3) (4) (5) (4)
*P < 0.01 as compared to 5 mM KCI; **P < 0.01 as compared to 5 mM KC1 and A 23187.
T a b l e I V shows that w h e n spinal c o r d cultures w e r e t r e a t e d with C a 2 ÷ - i o n o p h o r e ( A 23187) to raise intracellular
Ca 2÷,
desipramine
inhibited
the
effiux,
while
b a c l o f e n failed to d o so. M o r e o v e r , p h a c l o f e n , a G A B A B receptor antagonist,
Without treatment
With treatment
PTX (140 ng/ml) 5mMKCI 100 mM KCI 100 mM KCI + 10-~ M desipramine
6.8 _+ 1.5 16.2 + 1.4 8.1 + 0.2*
9.4 + 1.8 17.4 -+ 1.0 9.2 -+ 0.6*
Forskolin (10/zM) 5 mM KCI 100mM KCI 100 mM KCI + 10-~ M desipramine
7.6 _+ 1.5 14.7_+0.6 8.4 _+ 1.7"
9.3 + 0.8 19.1 + 1.7 9.5 + 0.8*
Phorbo112,13-dibutyrate (10aM) 5 mM KCI 100mM KCI 100mM KCI + 10-~M desipramine
4.9 + 0.7 17.5_+0.8 8.0 _+0.5*
9.4 + 0.4 15.8+1.2 9.2 + 0.8*
*P < 0.001 as compared to 100 mM KCI of the respective treatment groups.
did n o t a n t a g o n i z e the effect of
d e s i p r a m i n e (Table V). ng/ml) s h o w e d that the effect o f d e s i p r a m i n e o n S6Rb-
Studies with pertussis toxin, forskolin and phorbol esters
efflux was not a n t a g o n i z e d by P T X (Table V I ) .
T h e effects o f pertussis toxin ( P T X ) , which is k n o w n
I n c u b a t i o n o f the c u l t u r e s for 20 m i n with an a c t i v a t o r
to i n a c t i v a t e s o m e G - p r o t e i n s ( G i and Go) on desip r a m i n e - i n d u c e d inhibition o f 86Rb-efflux, w e r e e x a m -
of a d e n y l a t e cyclase, f o r s k o l i n ( 1 0 / z M ) o r an a c t i v a t o r of protein
ined. O v e r n i g h t i n c u b a t i o n of t h e cultures with P T X (140
b e f o r e the efflux studies also failed to a n t a g o n i z e the
kinase
C, p h o r b o l
12,13-dibutyrate
(10 /zM)
effect of d e s i p r a m i n e on 86Rb-efflux (Table VI). TABLE V
DISCUSSION
Effect of phaclofen on the inhibition of S6Rb-efflux by desipramine The influence of the GABA B antagonist, phaclofen, was studied on the desipramine-induced inhibition of a6Rb-efflux. Buffer containing 100 mM KCI was used to depolarize the cells, as described in Materials and Methods. The numbers in parentheses represent the number of experiments. The results were analyzed by using a two-factor (desipramine and phaciofen) ANOVA, which includes 4 treatments viz., 100 mM KCI, desipramine, phaclofen and desipramine + phaclofen.
Chronic administration of antidepressants
has b e e n
s h o w n to p r o d u c e m a r k e d e n h a n c e m e n t of G A B A B r e c e p t o r b i n d i n g , and an i n t e r a c t i o n b e t w e e n m o n o a m i n e systems and G A B A B r e c e p t o r s m a y p r o v e to b e of i m p o r t a n c e in d e p r e s s i o n 9. Pilc and L l o y d 33 and S u z d a k and G i a n u t s o s 36'37 o b s e r v e d a m a r k e d i n c r e a s e in t h e n u m b e r of G A B A B r e c e p t o r s in t h e rat frontal c o r t e x
Treatment
% S6Rb-efflux (mean + S.D.)
5 mM KCI 100 mM KCI 100 mM KCI + 10-4 M desipramine 100 mM KCI + 10-4 M desipramine + 10-4 M phaclofen 100 mM KCI + 10-4 M desipramine + 5 × 10-4 M phaclofen 100 mM KCI + 5 x 10-4 M phaclofen
6.7 -+ 1.5 16.0 + 1.6 8.1 + 0.2*
(4) (4) (4)
9.1 + 1.7"
(4)
m a t e r i a l s o b t a i n e d f r o m t h e a n i m a l s t r e a t e d chronically
(4) (4)
with t h e a n t i d e p r e s s a n t s . O u r in vitro studies, using a f u n c t i o n a l assay, rule o u t an i n t e r a c t i o n b e t w e e n tricyclic
The Newman-Keuls test results are as follows: *P < 0.001, when compared to 100 mM KCI.
after c h r o n i c t r e a t m e n t with a n t i d e p r e s s a n t s . B u t , in contrast, a r e c e n t study by Cross and H o r t o n 12 s h o w e d n o changes in G A B A B r e c e p t o r affinity o r n u m b e r after the chronic administration
of a n t i d e p r e s s a n t s .
A l l o f the
a b o v e studies utilized r a d i o l i g a n d b i n d i n g studies with t h e
8.4 _+0.4" 15.7 _+0.6
antidepressants
and p r e s y n a p t i c
G A B A B r e c e p t o r s in
c u l t u r e d spinal c o r d n e u r o n s (see b e l o w ) . H o w e v e r , the
63 possibility that chronic tricyclic antidepressant treatment may affect G A B A B receptor function in cortical neurons cannot be ruled out. The stimulation of G A B A a receptors has been reported to inhibit the voltage-sensitive Ca 2÷ currents in dorsal root ganglia of chick and to also inhibit K ÷-evoked release of 5-HT in mouse cortical slices 8'15'23. Moreover, baclofen, a G A B A B agonist, has been shown to reduce the evoked release of neurotransmitters from peripheral sympathetic nerve terminals s'9'13'14 and from brain slices 15'23'3° which was mimicked by G A B A . Similarly, the acute treatment with the antidepressants has been reported to reduce the turnover of the central neurotransmitters 3, which may be related to its influence on calcium, as the tricyclic antidepressants have been reported to inhibit the inward calcium current in rat brain synaptosomes 2 and in several other preparations 18"24. These lines of evidence point out that the acute effects of antidepressants may be mediated through the G A B A n receptor activation. Recently, we have developed a functional assay for measuring G A B A a receptor (presynaptic) responses in vitro using 86Rb-efflux assay in primary cultured spinal cord neurons 25. The rationale for using 86Rb as a substitute for K + has already been well justified 5'6. The pharmacology of the Ca2+-dependent S6Rb-efflux is also consistent with the previous reports 5'6 and also the earlier reports from o u r laboratory 25'26'39. Thus Q S O 4 and T E A , which block CaE+-activated K+-channels 1'5"6'38, inhibited the CaE+-activated 86Rb-efflux. The inability of 100 mM KCI to induce a significant increase in 86Rb-efflux in the absence of C a 2+ suggests that the efflux is dependent o n C a 2+ influx. Thus, the inhibition of Ca2+-activated 86Rb-efflux by (-)-baclofen is consistent with the involvement of G A B A B receptors in this action. This effect of (-)-baclofen apparently is presynaptic and was blocked by phaclofen, a G A B A B receptor antagonist 27. Furthermore, our recent studies have indicated that pertussis toxin, phorbol ester and forskolin inhibit the effect of (-)-baclofen o n C a 2÷activated 86Rb-efflux26. In the present study, all the antidepressants, except the monoamine oxidase inhibitors, inhibited Ca2+-activated 86Rb-efflux. Moreover, this effect was concentration-dependent, as both amitriptyline and desipramine inhibited the K÷-efflux in a concentration-dependent manner (Fig. 1). The 1(75o value of desipramine (40 tiM) is higher than the reported blood concentrations in humans (4/aM) 4°. However, since the brain plasma ratio is greater than 2021 , higher concentrations of desipramine may be expected in the CNS. The absence of synergism between baclofen and desipramine may be explained by the possibility that these two agents might share the same K÷-channels,
albeit, via different mechanisms. This is also supported by other observations, since there are major differences between tricyclic antidepressants and G A B A B receptormediated inhibition of the efflux process. The ability of tricyclic antidepressants, but not baclofen, to inhibit Ca2÷-activated 86Rb-efflux following A 23187 (which increases intracellular Ca 2+ levels independent of voltage-gated Ca2+-channels) treatment indicates that the inhibition with tricyclics may occur at a stage subsequent to the voltage-gated Ca2+-channels. These results also indicate that a mechanism different than baclofen, which inhibits the efflux at the level of voltage-gated Ca 2÷channels, may be involved. Moreover, the observation that G A B A B antagonist, phaclofen, was not effective against desipramine also suggests that the acute effects of antidepressants are not mediated through the G A B A B receptors. Previous studies have shown the involvement of G-proteins in the action of neurotransmitters (including baclofen) that inhibit Ca 2+ current. In our previous study, PTX, which is known to uncouple G-proteins by ADP-ribosylation 7'28, blocked the action of baclofen on Ca 2+ activated 86Rb-efflux25. However, the action of desipramine on the K+-efflux was not reversed by the pretreatment with PTX, suggesting that the action of antidepressants may not be through the G-protein mechanism. It has been reported that G A B A B receptor activation leads to an inhibition of adenylate cyclase 32"41. Phorbol esters and forskolin, which are known to activate protein kinase C 4'11 and adenylate cyclase 35'41, respectively, have been shown to inhibit the action of baclofen T M . These mechanisms may not be applicable in the case of the antidepressants, in contrast to baclofen, since both forskolin and phorbol 12,13-dibutyrate were unable to influence their action. Taken together, this leads to the conclusion that even though the antidepressants inhibit the CaE+-activated K+-efflux like the G A B A B agonists, they do not mediate this effect via the G A B A B receptors. This effect of antidepressants to inhibit Ca2+-activated S6Rb-efflux m u s t be occurring at a step in the efflux process beyond voltage-gated Ca2+-channels and adenylate cyclase and G-protein coupled events. It is feasible that tricyclic may inhibit the 86Rb-efflux process by directly blocking the K+-channels. The pharmacological significance of this effect is not clear, but may be potentially relevant to neurotransmitter release. However, the possibility that this action of antidepressants may be due to an indirect effect via blockade of release of some excitatory neurotransmitter which opens calcium-gating channels or via blockade of such channels and/or their local anesthetic activities cannot be ruled out. The mechanism of local
64 a n e s t h e t i c p r o p e r t y of t h e s e agents m a y be due to t h e i r i n f l u e n c e on the v o l t a g e - g a t e d s o d i u m channels 31. Fi-
efflux, and so efforts are u n d e r w a y to see t h e i r effect on K + - e f f l u x after c h r o n i c t r e a t m e n t .
nally, since t h e a n t i d e p r e s s a n t s express t h e i r t h e r a p e u t i c effect o n l y after 2 w e e k s of t r e a t m e n t , it is n e c e s s a r y to e x a m i n e their effect after chronic t r e a t m e n t on 86Rb-
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