L. L. Iversen

Inhibition of catecholamine Uptake2 by steroids in the isolated rat heart

Various steroids (1 7‐fl‐oestradiol, cortico‐sterone, deoxycorticosterone, progesterone, testosterone and androsterone) produced a dose‐dependent inhibition of the uptake of 3H‐noradrenaline by the Uptake2 mechanism in the isolated perfused heart. It is suggested

The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process

In previous studies of the uptake of catechol amines in the rat isolated heart it was shown that adrenaline and noradrenaline were accumulated by a common mechanism (Iversen, 1963, 1965). In these experiments the uptake saturated at an external amine concentration of approximately 0.2 μg/ml. noradrenaline or 0.5 μg/ml. adrenaline. However, in subsequent experiments in which hearts were perfused with (±)-adrenaline at a concentration of 5.0 μg/ml. an unexpectedly large uptake of adrenaline was observed. This fortuitous observation led to an examination of the uptake of adrenaline and noradrenaline during perfusions at higher concentrations of each amine than had previously been studied. The results of these experiments led to the conclusion that a second type of uptake operates at high perfusion concentrations. The properties of this second process are sufficiently different from those described previously to justify a clear-cut distinction between the two processes.

The role of Uptake2 in the extraneuronal metabolism of catecholamines in the isolated rat heart

1 . (±)‐3H‐NA and labelled metabolites of NA were estimated in rat hearts after perfusion with various concentrations of NA in the range 0·01–50·0 μg/ml. Labelled metabolites of NA accounted for only a small proportion of the total uptake of radioactivity at low perfusion concentrations, but accounted for 50% of the total uptake at 1 μg NA/ml., thereafter declining to progressively smaller proportions at higher perfusion concentrations. 2 . If the formation of labelled metabolites of 3H‐NA was blocked by a combination of monoamine oxidase and catechol‐O‐methyl transferase inhibitors, the accumulation of unchanged 3H‐NA was doubled when hearts were perfused with 1 μg NA/ml. 3 . In hearts perfused with 0·5 μg NA/ml., an accumulation of unchanged 3H‐NA was demonstrated in the presence of a combination of metabolic inhibitors and metaraminol. This appeared to be due to Uptake2, since the accumulation of NA under these conditions could be prevented by a low concentration of normetanephrine. 4 . Phenoxybenzamine prevented extraneuronal uptake (Uptake2) and metabolism of 3H‐NA with an estimated ID50 of 2·5 μm. The inhibition of Uptake2 by phenoxybenzamine (2·0 μm) was diminished at very high NA concentrations, suggesting that the drug may act competitively with NA. 5 . It was concluded that Uptake2 operates at all catecholamine concentrations in the rat heart, but that in the lower range (less than 2·5 μg/ml. for NA and less than 0·75 μg/ml. for adrenaline) any catecholamine taken up by this process is rapidly metabolized. Thus the accumulation of unchanged amine is seen only at high perfusion concentrations. 6 . The relevance of these results to an understanding of the possible physiological and pharmacological importance of Uptake2 is discussed.

Time course of the effects of 6-hydroxydopamine on catecholamine-containing neurones in rat hypothalamus and striatum

1 . The effects of intraventricular injection of 6‐hydroxydopamine (6‐OHDA) on tyrosine hydroxylase activity, uptake of 3H‐noradrenaline and endogenous catecholamine concentration in rat hypothalamus and striatum were investigated at various times after the injection of 6‐OHDA. 2 . In the hypothalamus after the injection of 250 μg of 6‐OHDA there was a rapid decrease in tyrosine hydroxylase activity, 3H‐noradrenaline uptake and noradrenaline content, which was essentially complete within 2 hours. 3 . In the striatum after this dose of 6‐OHDA there was a much slower reduction in tyrosine hydroxylase activity and 3H‐noradrenaline uptake during the first 48 h after drug injection. For the first 24 h the dopamine concentration in this brain area was increased significantly above control values, but had fallen below control values by 48 hours. 4 . After the injection of a smaller dose of 6‐OHDA (25 μg) the only detectable change in the striatum was a rapid increase in the dopamine concentration. In the hypothalamus this dose induced a rapid depletion of noradrenaline, not accompanied initially by any significant reduction in tyrosine hydroxylase activity. 5 . These results are consistent with the hypothesis that 6‐OHDA causes a rapid degeneration of catecholamine‐containing nerve terminals in the central nervous system (CNS). These degenerative changes, indicated by the loss of tyrosine hydroxylase and noradrenaline uptake sites, did not appear to be preceded by an initial displacement of endogenous catecholamines by 6‐OHDA, except possibly at early times after the administration of small doses of the drug.

Effects of phenoxybenzamine on the uptake and metabolism of noradrenaline in the rat heart and vas deferens

1 . In the isolated rat heart perfused with various concentrations of (±)‐3H‐noradrenaline (3H‐NA) the addition of phenoxybenzamine, cocaine or desipramine to the perfusion medium resulted in an inhibition of 3H‐NA uptake which appeared on kinetic analysis to be of a competitive nature. 2 . Phenoxybenzamine also blocked the formation of labelled metabolites of 3H‐NA in the heart at all perfusion concentrations of 3H‐NA; this effect appeared to be unrelated to the inhibition of the neuronal uptake of NA produced by phenoxybenzamine, since no blockade of 3H‐NA metabolism was produced by cocaine in similar experiments. 3 . In slices of rat vas deferens incubated with various concentrations of 3H‐NA, cocaine and desipramine and phenoxybenzamine were also shown to act as competitive inhibitors of NA uptake. Cocaine and phenoxybenzamine were less potent inhibitors of uptake in the vas deferens than they were in the heart; desipramine was equally potent in both tissues. 4 . When vas deferens slices were incubated in a medium containing phenoxybenzamine for 30 min before the addition of 3H‐NA, the resulting inhibition of 3H‐NA uptake was increased and changed to a non‐competitive type of interaction. 5 . In hearts or vasa deferentia taken from animals pretreated in vivo with phenoxybenzamine (20 mg/kg), a significant inhibition of 3H‐NA uptake was found when the tissues were exposed to low concentrations of 3H‐NA but not when higher concentrations of 3H‐NA were used.

Inhibition of catecholamine uptake in the isolated rat heart by haloalkylamines related to phenoxybenzamine

1 . Twenty‐one haloalkylamine derivatives were tested as inhibitors of both the neuronal uptake of 3H‐noradrenaline (NA) by the Uptake1 mechanism and the extraneuronal uptake of 3H‐NA by the Uptake2 mechanism in the isolated rat heart. 2 . At a concentration of 50 μm most of the compounds tested caused a significant inhibition of both uptake processes, although there were wide differences in the relative effects on Uptake1 and Uptake2. Some tentative structure activity relationships for uptake inhibition were formulated from these results. 3 . Phenoxybenzamine was confirmed to be a potent inhibitor of both the Uptake2 and Uptake1 mechanisms, with IC50 values for these two systems of 2·8 μm and 0·9 μm respectively. 4 . The substances N‐(9‐fluorenyl)‐N‐methyl‐β‐chloroethylamine (SKF 550), N‐(3,4‐dimethoxyphenylisopropyl)‐N‐benzyl‐β‐chloroethylamine (SKF 625A) and N‐(4‐methoxyphenoxyisopropyl)‐N‐benzyl‐β‐chloroethylamine (SKF 784A) were significantly more potent than phenoxybenzamine as Uptake2 inhibitors, and were all less potent than phenoxybenzamine as Uptake1 inhibitors. The compound SKF 550 is the most potent and selective inhibitor of Uptake2 so far described. It has an IC50 for Uptake2 of 008 μm, and an IC50 for Uptake1 of approximately 400 μm. 5 . Comparison of the present results with the known activities of these blocking agents suggests that no correlation exists between adrenoceptor blocking activity and ability of the substances to act as inhibitors of Uptake2 or Uptake1.

Differences in the uptake, storage and metabolism of (+)- and (—)-noradrenaline

1 The rate of uptake of (+)‐ and (—)‐noradrenaline was measured in isolated perfused hearts of reserpine treated rats, mice and guinea‐pigs by fluorimetric analysis of the removal of catecholamine from the perfusion medium. In rat and mouse heart (—)‐noradrenaline was taken up significantly more rapidly than (+)‐noradrenaline, but no stereochemical specificity was found for noradrenaline uptake in guinea‐pig hearts. 2 Using radioactively labelled (+)‐14C‐noradrenaline and (—)‐3H‐noradrenaline, the kinetic constants for uptake into noradrenaline‐containing and dopamine‐containing synaptosomes from rat brain were determined. The uptake by noradrenaline terminals in the hypothalamus had a higher affinity for (—)‐noradrenaline than for the (+)‐isomer, but no differences in affinity were found for uptake into dopamine terminals. 3 When equal amounts of labelled (+)‐ and (—)‐noradrenaline were injected in vivo in double isotope experiments, the (+)‐isomer disappeared more rapidly than the (—)‐isomer from rat heart and spleen, but no significant differences were found between the rates of disappearance of the two isomers from rat brain or in the whole mouse. 4 Analysis of the radioactive metabolites of the two isomers of noradrenaline after administration of mixed doses of the labelled substances showed that a significantly higher proportion of (+)‐noradrenaline was metabolized to normetanephrine than of (—)‐noradrenaline, in the whole mouse, rat heart and rat brain.