Amantadine, in vitro, produced dose-dependent blockade of dopamine uptake into a synaptosome-rich particulate fraction of an homogenate of the basal ganglia of rats. A concentration of 3.6 x 1 0 -6~ was required to inhibit uptake by 50%. Diethazine was less potent in blocking dopamine uptake, the equiactive concentration being 8.0 x lO-'j~. Amantadine and (+)-amphetamine in concentrations in excess of 1 0 -5~ caused the release of small quantities of added dopamine from the particulate fraction.The findings of Vernier, Harmon & others (1969) and of Heimans, Rand & Fennessy (1 972) suggest that amantadine potentiates the peripheral pharmacological effects of dopamine by blocking uptake of amines in a variety of adrenergically innervated preparations. We have investigated the effect of amantadine on dopamine uptake and release by the particulate fraction of an homogenate obtained from basal ganglia, the presumed site of pathology in Parkinson's disease (Hornykiewicz, 1963). METHODS Preparation of homogenate from rat basal gangliaThe preparation was modified from that described by Horn, Coyle & Snyder (1971). Rats, 200-300 g, were stunned and decapitated and the basal ganglia, consisting of corpus striatum, caudate and putamen were rapidly dissected and removed to ice, according to Glowinski & Iverson (1966). The tissue was weighed (100-125 mg) and homogenized in 4 ml of 0 . 2 5~ sucrose at 0" before centrifuging for 10 min at 100 g in an M.S.E. refrigerated centrifuge. The supernatant fluid was then carefully decanted from the pellet of tissue debris which was discarded. The resulting fluid containing particulate fraction was gently stirred to produce a homogeneous suspension. Dopamine uptake by the synaptosome-containing homogenateAn aliquot of the homogenate (0.1 ml) was transferred to a 20 ml vial containing 3.9 ml of modified Krebs-Henseleit solution of the following composition: NaCI, 118 mM; KCI, 4.7 mM; NaHCO,, 25 mM; MgSO,, 0.45 m~; KH,P04, 1.03 mM; CaCI,, 1.25 m~; D-(+)-glucose, 11.1 mM; together with nialamide 1.25 x 1 0 -5~ and ascorbic acid, 0.2 mg/ml. Disodium EDTA, 0.1 mg/ml, and various concentrations of amantadine or diethazine were added to this solution. The solution was kept at 37" and continuously aerated with a stream of 5 % carbon dioxide in oxygen. The preparation was incubated for 5 min, 5 to 40 p1 of 30 mM dopamine (side-chain l,WH; specific activity 1.3 Ci/mmol) was added and incubation continued for 5 min.
1. Morphine-like analgesic drugs caused depression of twitches of the isolated guinea-pig ileum in response to transmural electrical stimulation. The drugs tested were the narcotic analgesics codeine, diamorphine, fentanyl, morphine, morphine-N-oxide, normorphine, oxymorphone, pethidine, phenazocine and phenoperidine and the analgesic narcotic antagonists nalorphine and pentazocine. 2. With the first application of one of these drugs the extent of depression of twitches was proportional to concentration. Except in the case of pethidine, there was no further depression when additional drug was added to the organ bath. With the second application of a drug after washing out the first dose, the depressant effect was less; that is, tolerance developed. With pethidine, the depression of twitches was proportional to concentration and tolerance could not be observed. 3. When tolerance had been produced by cumulative addition of these drugs, a concentration was reached at which further addition resulted in increased activity of the ileum. 4. With codeine, morphine and normorphine, the twitches were increased in height and regular. 5. With diamorphine, fentanyl, oxymorphone, pentazocine, phenazocine and phenoperidine there were increased but irregular responses to transmural stimulation. 6. Having reached the concentration at which these effects were observed, washout of the drug resulted in reduction of activity; the twitches became smaller or the irregular responses ceased. 7. Readministration of a drug after activity of the ileum had been depressed by withdrawal of that drug resulted in restoration of activity, the ileum being dependent on the presence of the drug for its activity. 8. Codeine and nalorphine did not produce as great an increase in activity on readministration to a dependent ileum as did morphine: they seem to act as partial agonists in producing this effect. 9. In similar experiments with the isolated urinary bladder of the rat and guinea-pig, morphine was less active in depressing responses to stimulation than it was on the ileum, and tolerance to the drug and dependence on it did not occur.
Amantadine has been tested on several peripheral preparations. It depressed responses of the chick isolated oesophagus to nicotine and preganglionic parasympathetic nerve stimulation without depressing responses to acetylcholine. It did not affect the amplitude or duration of action potentials in toad sciatic nerve. It enhanced the effects of noradrenaline and dopamine while reducing the effect of tyramine on the coaxially stimulated guinea‐pig ileum, isolated central artery of the rabbit ear and spontaneously beating guinea‐pig isolated atria preparations. Amantadine enhanced the depressor effect of dopamine in the rabbit.
After administration of morphine‐N‐oxide (MNO) to rats the opiates appearing in the urine were morphine (61%) and MNO (39%). After administration of morphine, the urinary opiates were morphine (80%) and normorphine (20%). When tacrine was given with morphine the urine also contained MNO (46% of total urinary opiates) and the amount of normorphine was much decreased (to 1%), the remainder being morphine (53%). Tacrine and amiphenazole inhibited demethylation of morphine and codeine by a rat liver fraction in vitro. MNO had weak inhibitory activity. Neither MNO nor codeine‐N‐oxide were demethylated in vitro.
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