The TiPS lecture: functional aspects of the neuronal uptake of noradrenaline.

For various amine transmitters (noradrenaline, dopamine, 5-HT) re-uptake into the releasing varicosity limits the transmitter's life span in the biophase. In the second TiPS Lecture, given at this year's FASEB meeting in Atlanta, Georgia, Ullrich Trendelenburg summarized the evidence relating to the function of the neuronal noradrenaline carrier (uptake1), why it is absolutely dependent on Na+ and Cl- and how it functions as a metabolizing system, hand in hand with intraneuronal monoamine oxidase and vesicular storage. This carrier clears noradrenaline from the extracellular space very efficiently. Hence, loading of incubated organs with exogenous substrates of uptake1 results in a very heterogeneous distribution of the amine. Moreover, under certain experimental and pathophysiological conditions the carrier is able to transport axoplasmic noradrenaline out of the varicosity, a 'release' mechanism operating, for instance, in cardiac ischaemia.     

Pharmacological characterization of presynaptic alpha 2-autoreceptors in rat submaxillary gland and heart atrium.

1. The pharmacological properties of presynaptic alpha 2-autoreceptors were studied in rat isolated submaxillary glands and atria. Tissue pieces were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine, and stimulated electrically. In one series of experiments, pEC30 values of 12 alpha-adrenoceptor antagonists were determined, i.e., negative logarithms of concentrations that increased the electrically evoked overflow of tritium by 30%. In another series, pKD values of 9 alpha-adrenoceptor antagonists against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), and of 3 antagonists against the release-inhibiting effect of methoxamine, were determined. 2. In submaxillary glands, the pEC30 values of the antagonists correlated well with their pKD values against UK 14304 (r = 0.93). The same was true for atria (r = 0.92). 3. In submaxillary glands, the pKD values of 3 antagonists against UK14304 were very similar to their pKD values against methoxamine, with a maximal difference of 0.4. The same was true for atria where the maximal difference was 0.3. 4. The pEC30 values obtained in submaxillary glands correlated significantly with those obtained in atria (r = 0.81). The same was true for the pKD values (r = 0.79). However, the pEC30 and pKD values also indicated consistent differences between the two tissues. 5. It is concluded that the sites of action of the imidazoline UK 14304 (alpha 2-selective), the phenylethylamine noradrenaline, and the phenylethylamine methoxamine (alpha 1-selective) are exclusively alpha 2-adrenoceptors. There is no indication for presynaptic alpha 1-adrenoceptors or for an effect of UK 14304 mediated by presynaptic imidazoline receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The handling of five amines by the extraneuronal deaminating system of the rat heart

Summary The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l ³H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (–)-adrenaline, (–)-noradrenaline, dopamine, tyramine and 5-HT. IC₅₀'s were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of ³H-noradrenaline. After correction for changes in the tissue/medium ratio for ³H-noradrenaline, half-saturating outside concentrations were obtained. They increased in the order (–)-adrenaline (15 mol/l) — tyramine — dopamine — noradrenaline —5-HT (53 mol/l). The V _max for extraneuronal deamination was determined for ³H-(–)-adrenaline, ³H-(–)-noradrenaline and ³H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (–)-adrenaline, intermediate for (–)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg     

The uptake and O-methylation of 3H-(±)-isoprenaline in rat cerebral cortex slices

Summary The O-methylation and accumulation of ³H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by mol/l U-0521 virtually abolished the O-methylation and increased the accumulation of ³H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake, (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake, and uptake2, although exhibiting some resemblance with uptake₂ (lack of dependence on Na⁺ and Cl^–, sensitivity to K⁺ and phenoxybenzamine, ability to transport ³H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC₃₀-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of ³H-isoprenaline. 5. IC₅₀-values (against initial rates of accumulation of ³H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake₂. There was no correlation with the IC₅₀-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984). 6. Unlabelled catecholamines half saturated the intracellular COMT when slices were incubated with 0.22 mol/l [(±)-dobutamine] to 4.9 mol/l [(–)-noradrenaline]. As the presence of unlabelled catecholamines increased tissue levels of ³H-isoprenaline, catecholamines are substrates of the central carrier. 7. The carrier of the central O-methylating system differs from uptake₂ of peripheral organs, although it resembles the peripheral carrier in some respects.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OMI 3-Omethyl-isoprenaline Supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176) and by a scholarship of the Royal Society for V. G. Wilson. Some of the results were presented to the British Pharmacological Society (Trendelenburg and Wilson 1986)     

Antibodies against C1q in patients with systemic lupus erythematosus

The first component of the classical pathway of complement (C1q) is considered to be involved in the pathogenesis of systemic lupus erythematosus (SLE). This view is based on the observation that a substantial number of patients with SLE develop hypocomplementemia with depletion of the classical pathway components, and C1q has been shown to play an important role in the clearance of immune complexes and apoptotic bodies. In addition, homozygous C1q deficiency is the strongest disease susceptibility gene for the development of SLE that has been characterised in humans. However, most SLE patients have no primary complement deficiency. Hypocomplementemia in SLE patients is a secondary event and often associated with antibodies against C1q (anti-C1q). Although anti-C1q have been found in a number of distinct autoimmune disorders, they are best described in patients with SLE where they strongly correlate with renal flares. Current data suggest that the occurrence of anti-C1q in SLE patients is necessary but not sufficient for the development of proliferative lupus nephritis, suggesting an interference with the normal function of the complement system.     

The membrane potential of vascular smooth muscle appears to modulate uptake2 of 3H-isoprenaline

Summary Segments of the rabbit main pulmonary artery and of its two branches were exposed for 10 min to 50 nmol/l ³H-(±)-isoprenaline, and the accumulation of tritium in the tissue was determined; COMT was inhibited in all experiments. 1. The accumulation of the tritium label was sensitive to 3-O-methyl-isoprenaline (OMI), showing that this vascular smooth muscle possesses uptake₂. 2. In the presence of 0.01 to 1 mol/l (–)-noradrenaline, the accumulation of tritium was depressed (in a concentration-dependent manner). This decline involved the OMI-sensitive accumulation of ³H-isoprenaline. 3. The presence of any one of three selective alpha₁-adrenoceptor antagonists (1 gmol/l prazosin, 1 mol/l WB4101, 10 gmol/l corynanthine) prevented the effect of 1 mol/l (–)-noradrenaline on the accumulation of tritium. However, in the absence of (–)-noradrenaline, the three antagonists failed to affect the accumulation of tritium. 4. 10 mol/l nicorandil caused the accumulation of tritium to increase. 5. As stimulation of alpha₁-adrenoceptors is known to result in depolarization, and as nicorandil is known to hyperpolarize this smooth muscle, it is concluded that the resting membrane potential modulates uptake₂. Send offprint requests to U. Trendelenburg at the above address     

The effect of partial inhibition of monoamine oxidase on the steady-state rate of deamination of 3H-catecholamines in two metabolizing systems

Two different "deaminating systems" were compared (i.e., intact tissues in which an uptake process translocates the 3H-catecholamine from the extracellular space to the intracellular MAO): the adrenergic nerve endings of the rat vas deferens exposed to 10 nmol/l 3H-(-)-noradrenaline, and the extraneuronal deaminating system of the rat heart perfused with 50 nmol/l 3H-(-)-adrenaline. Vesicular uptake and COMT were inhibited. In both systems MAO was partially inhibited by pargyline, and the steady-state tissue content of the 3H-catecholamine was determined as well as the steady-state rate of deamination. Rat vas deferens (preincubated with 10-40 nmol/l pargyline for 30 min). Inhibition of neuronal MAO caused not more than a moderate decrease of the steady-state rate of deamination of 3H-(-)-noradrenaline, but the steady-state tissue content was greatly increased. Determinations of the activity of MAO in homogenates of vasa deferentia showed that preincubation with 10 and 20 nmol/l pargyline inhibited the enzyme by 80 to 95%. Rat heart (of animals pretreated with 1 to 30 mg/kg pargyline). Inhibition of extraneuronal MAO caused a steep decline of the steady-state rate of deamination of 3H-(-)-adrenaline, but only a small rise in the steady-state tissue content. The decisive difference between the two deaminating systems lies in the fact that the ratio "kmao/kout" (where the two k-values characterize the activity of the unsaturated intracellular MAO and the ability of the 3H-catecholamine to leave the relevant cells, respectively) is much higher for the neuronal deaminating system exposed to 3H-(-)-noradrenaline than for the extraneuronal deaminating system exposed to 3H-(-)-adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(−)-noradrenaline in the rat heart

Summary The extraneuronal metabolism of ³H-(–)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mol/l cocaine).Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity.These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for ³H-(–)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of ³H-(–)-noradrenaline which-by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio-actually decreases the net removal of ³H-(–)-noradrenaline from the perfusion fluid.The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.The following abbreviations are used here NMN normetanephrine - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid - OMDA MOPEG+VMA Supported by the Deutsche Forschungsgemeinschaft     

Presynaptic α2A-adrenoceptors inhibit the release of endogenous dopamine in rabbit caudate nucleus slices

₂-Adrenoceptors modulating the release of dopamine were identified and characterized in slices of the head of the rabbit caudate nucleus. Release of endogenous dopamine was measured by fast cyclic voltammetry as the increase in the extracellular concentration of dopamine elicited by electrical stimulation. The electrochemical signal was identified as dopamine by means of the oxidation potential, the voltammogram and the fact that the signal was not changed by desipramine, which inhibits the high affinity uptake of noradrenaline, but was greatly increased by nomifensine, which in addition inhibits the high affinity uptake of dopamine.Stimulation by 6 pulses/100 Hz increased the extracellular concentration of dopamine by about 85 nM. The selective ₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced this release with an EC₅₀ of 173 nM and by maximally 75%. The ₂-adrenoceptor agonists clonidine and oxymetazoline only tended to cause a decrease. Six drugs, including oxymetazoline, were tested as antagonists against UK 14,304. Their order of antagonist potency (pK_D values in brackets) was rauwolscine (8.0) > oxymetazoline (7.5) > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; 7.3) > phentolamine (7.1) > corynanthine (5.1) prazosin (< 6). Given alone, the antagonists did not change the release of dopamine elicited by 6 pulses/100 Hz, and the same was true for the dopamine receptor antagonist sulpiride. When caudate slices were stimulated by 10 pulses/1 Hz, sulpiride increased the release of dopamine. Desipramine and rauwolscine, in contrast, again caused no change.It is concluded that dopaminergic axons in the rabbit caudate nucleus possess release-inhibiting ₂-adrenoceptors. The antagonist affinities indicate that they belong to the _2A subtype. In this, they agree with all presynaptic ₂-autoreceptors studied so far in rabbits as well as with the ₂-heteroreceptors modulating the release of serotonin in rabbit brain cortex, suggesting that at least the majority of presynaptic ₂-adrenoceptors in the rabbit are _2A. The agonist sensitivity of the caudate presynaptic ₂-adrenoceptors is low in comparison with cerebrocortical presynaptic ₂-autoreceptors, possibly due to absence of a receptor reserve. Correspondence to: N. Limberger at the above address