Richard Heikkila

Studies on the distinction between uptake inhibition and release of [3H]dopamine in rat brain tissue slices

Abstract There exists some confusion over the classification of drugs as either uptake inhibitors or releasing agents for biogenic amines. We have evaluated this problem with rat brain tissue slices (neostriatum and cortex), using [3H]dopamine and the following compounds: potassium chloride, tyramine, d-and l-amphetamine, cocaine, amantacline, desipramine. amitriptyline, nortriptyline and protriptyline. Additional experiments were performed with l -dopa and [3H]serotonin in slices from whole rat brain. Potassium chloride and tyramine. both strong releasing agents, diminished the accumulation of [3H]dopamine during uptake studies. l -Dopa also caused release of [3H]serotonin and an inhibition of [3H]serotonin accumulation. Whenever a releasing action was observed, there was always a diminution in the amount of [3H]amine accumulation and this action (expressed as a per cent effect) was at least equal in magnitude to the per cent released. On the other hand, cocaine was an example of a pure uptake inhibitor; it did not evoke a releasing action at concentrations where a powerful uptake inhibition was seen. From these data, it was possible to conclude that, in the tissue slice system, an experimentally observed release was real (that is. not materially affected by blockade of reuptake), whereas a releasing action evoked an apparent inhibition of uptake equal in magnitude to the releasing action. Before a drug can be designated as an uptake inhibitor, the dose-response curve for inhibition of [3H]amine accumulation should be distinctly to the left of the dose-response curve for release. Our data indicate that, in the neostriatum, all of the drugs studied except cocaine were pure releasing agents and that drugs previously designated as uptake inhibitors were releasing agents. In the cortex, strong inhibition of uptake without significant releasing action was evident for many of the drugs.

6-Hydroxydopamine: Evidence for Superoxide Radical as an Oxidative Intermediate

Superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine as measured by the rate of formation of a quinone and the rate of oxygen consumption. These observations demonstrate the formation of the superoxide radical during the autoxidation process. This finding may be relevant to the mechanism of adrenergic nerve terminal degeneration caused by 6-hydroxydopamine.

The developing neostriatum of the rabbit: Correlation of fluorescence histochemistry, electron microscopy, endogenous dopamine levels, and [3H]dopamine uptake

Abstract A correlation has been made between the sequential variation of fluorescent profiles during early fetal development and the maturation of growth cones into more differentiated axons with synapses ‘en passage’. The initial ingrowth of fluorescent axons enters the ventromedial putamen, probably in the angle formed by the external capsule, anterior commissure, and internal capsule. Later in fetal life, a second ingrowth of axons running adjacent to the internal capsule enters both the caudate nucleus and putamen. Circumscribed islands of fluorescence which are visible in late fetal and early postnatal stages, may indicate an anatomical and functional organization not previously recognized. The attainment of maximum endogenous dopamine levels in the rabbit lags behind the maturation of the uptake system and the development of the mature number of vesicle-filled processes and synaptic profiles. The maturation of the uptake process and the development of adult numbers of vesicle-filled axonal profiles, on the other hand, appear to correlate quantitatively with one another. The latter observation and the linear character of the fluorescent profiles tend to support the notion that axons having synapses ‘en passage’ and vesicles along their length may correspond to the dopamine-containing axons.

Evaluation of amantadine as a releasing agent or uptake blocker for H3-dopamine in rat brain slices

Abstract Amantadine was studied for its ability to inhibit the uptake of H 3 -dopamine or H 3 -serotonin and to release H 3 -dopamine which had been previously accumulated by brain slices. Tissue slices were prepared from whole brain as well as from neostriatum and cortex of rats and rabbits. Amantadine was a very weak releasing agent for H 3 -dopamine in all brain areas. Amantadine exerted a stronger action as an inhibitor of the uptake of H 3 -dopamine by whole brain or cortex (50% inhibition at 10 −4 M). Similar inhibition was found for H 3 -serotonin in the whole brain. However, amantadine was relatively ineffective in inhibiting H 3 -dopamine uptake in the neostriatum. We conclude that neither direct release of endogenous dopamine nor blockade of reuptake, specifically in the stiatum, can account for the beneficial action of amantadine in patients with Parkinsons disease. Alternative mechanisms of action are discussed briefly.

6,7-dihydroxytetrahydroisoquinoline: Electron microscopic evidence for uptake into the amine-binding vesicles in sympathetic nerves of rat iris and pineal gland

Summary After incubation in a medium containing 6,7-dihydroxytetrahydroisoquinoline, slices of pineal gland and iris from reserpinized rats showed regranulation of a large number of small and large dense core vesicles in sympathetic boutons. The binding of 6,7-dihydroxy-TIQ to small vesicles that normally bind norepinephrine supports the suggestion that this alkaloid may be capable of functioning as a ‘false transmitter’.

EVIDENCE FOR DEGENERATION OF SYMPATHETIC NERVE TERMINALS CAUSED BY THE ORTHO‐ AND PARA‐QUINONES OF 6‐HYDROXYDOPAMINE

6‐Hydroxydopamine and its corresponding ortho‐ and para‐quinones were injected intraperitoneally into male Swiss‐Webster mice. Measurements made 72 h after injection showed that all three compounds caused a decrease in the uptake of [3H]norepinephrine into slices of mouse heart tissue in vitro, a decrease in the endogenous content of heart norepine‐phrine, and a disappearance of the adrenergic nerve plexus of the mouse iris as viewed by fluorescence histochemistry. These data suggest that both the ortho‐ and para‐quinones of 6‐hydroxydopamine are capable of producing a chemical sympathectomy similar to that caused by 6‐hydroxydopamine.

The inhibition of 3H-biogenic amine uptake by 5,6-dihydroxytryptamine: A comparison with the effects of 6-hydroxydopamine

5,6-Dihydroxytryptamine was a competitive inhibitor of both 3H-serotonin and 3H-dopamine uptake into rat brain tissue slices. In contrast, 6-hydroxydopamine inhibited 3H-dopamine uptake but did not inhibit 3H-serotonin uptake even at a 6-Hydroxydopamine concentration of 10−4 M. In these experiments, catalase was present in the incubation medium to decompose hydrogen peroxide which is a product of the autoxidation of 6-hydroxydopamine and an inhibitor of biogenic amine uptake systems. These above data may help to explain the relative specificity of 6-hydroxydopamine for adrenergic nerve terminals in contrast to the effects of 5,6-dihydroxytryptamine on both adrenergic and serotonergic nerve terminals. Kinetic analyses showed that 5,6-dihydroxytryptamine had almost the same affinity for the 3H-serotonin transport system as did 3H-serotonin itself. In contrast, 5,6-dihydroxytryptamine had an affinity for the 3H-dopamine transport system approximately 1/30 that of 3H-dopamine.

An evaluation of various antiparkinsonian agents as releasing agents and uptake inhibitors for 3H-dopamine in slices of rat neostriatum

Various antiparkinsonian agents (trihexyphenidyl, diphenhydramine, benztropine, orphenadrine, diphenylpyraline and phenindamine) produced a dose dependent inhibition of accumulation of 3H-catecholamines in tissue slices of rat neostriatum and cerebral cortex. In the neostriatum, trihexyphenidyl, orphenadrine and phenindamine were almost equipotent in causing an inhibition of accumulation and in causing release of 3H-dopamine. Benztropine, diphenylpyraline and diphenhydramine were better inhibitors of accumulation than releasing agents. In the cortex, all of the compounds studied, except trihexyphenidyl, were better inhibitors of 3H-norepinephrine accumulation than releasing agents. Trihexyphenidyl was equally effective both as an inhibitor of accumulation and as a releasing agent. Inhibition of 3H-catecholamine accumulation as measured experimentally consists of two components: an inhibition of uptake and an apparent inhibition of uptake caused by release. When the observed inhibition of accumulation equals the observed release, we conclude that both phenomena are due to release and that there is not actual inhibition of uptake (see discussion). With this in mind, we conclude that three of the compounds (trihexyphenidyl, orphenadrine and phenindamine) are striatal releasing agents. The other three drugs are both uptake inhibitors and releasing agents for 3H-dopamine in the neostriatum. In the cortex, trihexyphenidyl appearstto be the sole releasing agent for 3H-norepinephrine. The remaining compounds appear to be considerably better cortical uptake inhibitors than releasing agents.

THE BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF 6-AMINODOPAMINE: SIMILARITY WITH 6-HYDROXYDOPAMINE

6‐aminodopamine was injected intraperitoneally into male Swiss–Webster mice. At 72 h post injection 6‐aminodopamine had caused a reduction in the endogenous content of heart norepinephrine, a decrease in the capacity of heart slices to accumulate [3H]‐norepinephrine in vitro, and a virtual disappearance of the adrenergic plexus of the mouse iris as viewed by fluorescence histochemistry. Similar data were obtained with the same dose of 6‐hydroxydopamine. These data suggest that 6‐aminodopamine causes a destruction of sympathetic nerve terminals. Model experiments showed that 6‐aminodopamine, like 6‐hydroxydopamine, generated H2o2both in vitro and in vivo. 6‐Aminodopamine, like 6‐hydroxydopamine, also blocked the accumulation of [3H]dopamine into slices of rat brain in vitro.