Binding and competitive inhibition of amine uptake at postsynaptic neurones (transport‐P) by tricyclic antidepressants

Abstract

1 We have provided evidence for a novel amine uptake process for which prazosin is a substrate in postsynaptic neurones, characterized by a paradoxical increase in accumulation of the radioligand when the concentration of the unlabelled drug is increased above 10' M. This increase is due to activation of a proton-dependent, vacuolar type-ATPase-linked uptake process which is blocked by desipramine but is resistant to reserpine. We have now examined the effects of tricyclic antidepressants on this uptake system in a cell line derived from hypothalamic peptidergic neurones, known to be innervated by noradrenergic nerve terminals in vivo.2 [3H]-imipramine bound to the cells and was displaced by unlabelled imipramine, desipramine, amitriptyline and nortriptyline. The data fitted a single binding site model. This is the first demonstration of antidepressant binding sites in postsynaptic neurones. 3 There was no increase in the binding of [3H]-imipramine at high concentrations of unlabelled imipramine, suggesting that antidepressants inhibit uptake but are not themselves accumulated by peptidergic gonadotrophin releasing hormone neurones. 4 Accumulation of prazosin was competitively inhibited by antidepressants. Tertiary amines were slightly more potent than secondary amines and the presence of a nitrogen atom in the heterocyclic ring enhanced blocking activity. 5 The affinities of the antidepressants for the uptake process are within the range of plasma concentrations that are observed during therapeutic use of these compounds. Since it is likely that this uptake process has a physiological function, its inhibition by antidepressants may provide a new avenue for investigating the mechanism of action of these compounds.

α1B adrenergic receptors in gonadotrophin‐releasing hormone neurones: relation to Transport‐P

“…A considerable amount of evidence supported this second part of the hypothesis. Thus: (a) the paradoxical increase in binding of [ 3 H]‐prazosin is inhibited competitively by antidepressants which are known to inhibit the pre‐synaptic re‐uptake of amines ( Al‐Damluji et al ., 1993 ; Al‐Damluji & Kopin, 1996b ); (b) the paradoxical increase in accumulation of [ 3 H]‐prazosin was not seen in membrane preparations, indicating that it requires intact cells or storage organelles ( Al‐Damluji et al ., 1993 ); (c) the prazosin paradox is an energy‐dependent process, the source of energy being the electrochemical proton gradient which is generated by vacuolar‐type ATPase (V‐ATPase; Al‐Damluji & Kopin, 1996a ); (d) ligands for Transport‐P must possess a specific chemical structure, consisting of a hydrophobic phenyl group, an alkyl side chain and a basic amine ( Al‐Damluji & Kopin, 1998 ); (e) a fluorescent analogue of prazosin accumulated in intracellular vesicles, providing visual evidence for uptake ( Al‐Damluji et al ., 1997 ); (f) following accumulation by Transport‐P, the amines can be released by an energy‐dependent process ( Shen & Al‐Damluji, 1997 ).…”

Visual detection of transport‐P in peptidergic neurones

“…Uptake of prazosin in peptidergic neurones (transport‐P) was distinguishable from presynaptic plasma membrane transporters by its independence of sodium and its reliance on protons for a source of energy (Al‐Damluji & Kopin, 1996a). Although transport‐P resembles the presynaptic vesicular amine transporters by its dependence on protons and V‐ATPase, it differs from these transporters by its insensitivity to reserpine and blockade by antidepressants (Al‐Damluji & Kopin, 1996a,b). The lack of effects of steroid hormones and sensitivity to antidepressants also differentiated transport‐P from the uptake processes in non‐neuronal cells (Al‐Damluji & Kopin, 1996a,b).…”

Structural properties of phenylethylamine derivatives which inhibit transport‐P in peptidergic neurones

“…Efficacy was expressed as % of the effect of a maximal inhibitory concentration of desipramine (10 −5 m). Typically, desipramine 10 −5 M inhibited the accumulation of prazosin 10 −6 M by 80% (Figure 1; Al‐Damluji & Kopin, 1996b); the remaining 20% was regarded as non‐specific uptake for the purposes of this study. Half‐maximal inhibitory concentrations (IC 50 values) were calculated from the concentration‐response curves.…”

“…Drugs were dissolved in uptake buffer consisting of DMEM with 25 mM HEPES and 0.5 × 10 −3 m sodium ascorbate, pH 7.4. The cells were washed twice with 1 ml of buffer and then incubated for 60 min at 37°C in buffer containing [ 3 H]‐prazosin 2 × 10 −9 M and unlabelled prazosin 10 −6 M, with or without phenylethylamine derivatives in concentrations of 10 −6 M to 10 −3 M. Accumulation of prazosin and antidepressants reaches equilibrium within 60 min (Al‐Damluji & Kopin, 1996b). At the end of the incubation period, the wells were placed on ice and the cells were washed twice with 1 ml volumes of ice‐cold buffer.…”