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,
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,
