Dopamine and noradrenaline transport into subcellular vesicles of the striatum

Summary Dopamine storing vesicles were isolated from the caudate nucleus of the pig by differential centrifugation and incubated at various temperatures. The spontaneous release of endogenous dopamine was temperature-dependent. Incubation with ¹⁴C-dopamine or ¹⁴C(±)-noradrenaline revealed that the vesicles were able to take up catecholamines by two different transport mechanisms; one was dependent on ATP, magnesium and temperature, the other one was independent of ATP and magnesium, and partially dependent on temperature. The Km of the ATP-magnesium-dependent uptake was 1.52×10^–6 M for dopamine and 3.45×10^–6 M for noradrenaline. Incubation with dopamine increased the dopamine content of the vesicles and diminished the endogenous dopamine by approximately 90%. Addition of ATP and magnesium further increased the dopamine content without influencing the per cent exchange between endogenous and exogenous dopamine. The dopamine uptake at 37°C in the presence of ATP and magnesium was of short duration because of the thermo-lability of the vesicles. (+)-amphetamine competitively inhibited the ATP-magnesium-dependent uptake of dopamine and noradrenaline. Amantadine and desipramine influenced neither the ATP-magnesium-dependent nor the ATP-magnesium-independent uptake of the catecholamines.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

In vivo release of endogenous GABA in the cat hypothalamus

Summary The posterior hypothalamus of anaesthetized cats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of endogenous GABA from the hypothalamus into the superfusate was studied. The resting release of GABA varied rhythmically, since phases of high rate of release were separated from each other by phases of low rate of release. The time interval between two adjacent phases of high rate of release was about 70 min. Electrical stimulation of the posterior hypothalamus with the tip of the cannula enhanced the rate of release of GABA in a frequency-dependent way. Superfusion of the hypothalamus with CSF which contained a high concentration of potassium and a low concentration of sodium increased the rate of release of GABA; this effect was dependent on the presence of calcium ions in the superfusing fluid. Pretreatment of the cats with reserpine reduced the levels of GABA in hypothalamus and rest of brain and the concentration of GABA in the superfusate as well. Stimulation of the locus coeruleus with a bipolar electrode elicited an increased release of GABA in the hypothalamus.Part of the results was presented at the Spring Meeting of the German Pharmacological Society, Mainz, March 1978This work was supported by the Deutsche Forschungsgemeinschaft     

Expression of CD44 isoforms and β1,6-branched oligosaccharides in human malignant melanoma is correlated with tumor progression but not with mettastatisc potential

CD44, a family of closely related glycoproteins generated by alternative splicing, as well as the increased β1,6-branching of Asn-linked oligosaccharides (β1,6-branches), have been implicated in tumor progression and metastasis. We have investigated the expression of CD44 standard (CD44s), various CD44 splice variants (CD44v3,- v4,- v5,- v6 and- v9), and of β1,6-branches in a total of 37 paraffin-embedded human primary melanomas and metastases.
Out of the 28 studied primary melanomas, 27 were positive for CD44s, 21 for CD44v5 (cytoplasmic staining) and 26 for β1,6 branches. Furthermore, superficial spreading melanomas showed a significant (p=0.004) stronger staining for CD44s than the thick (> 1.5 mm) nodular melanomas, whereas no significant difference was found with regard to staining for CD44v5 and β1,6-branches. Eight of the 9 studied melanoma metastases were positive for CD44s, 6 for CD44v5 (cytoplasmic staining) and 7 for β1,6-branches. No CD44v3, -v4, -v6 and -v9 could be detected in any of the tumors. On average, metastases as compared to primary tumors, exhibited a significant (p=0.002) weaker staining for CD44s. However, metastasizing melanomas could not be distinguished from non-metastasizing ones based on CD44 immunostaining.     

Pattern of in vivo release of endogenous histamine in the mamillary body and the amygdala

Summary The mamillary body and the medial amygdaloid nucleus of cats anaesthetized with sodium pentobarbital were bilaterally and simultaneously superfused through push-pull cannulae with CSF and the release of endogenous histamine was determined in the superfusates. Collection of the superfusates in 10 min time periods revealed that histamine was rhythmically released in the two areas with frequencies of one cycle/90 min (mamillary body) or one cycle/135 min (medial amygdaloid nucleus). Collection of the superfusates in time periods of 2 min revealed the existence of an additional ultradian rhythm with a frequency of approximately one cycle/19 min in both areas. Bilateral lesions of the suprachiasmatic nucleus did not seem to influence the pattern of histamine release in the mamillary body and the medial amygdaloid nucleus, but the rate of histamine release was decreased in the mamillary body. It is concluded that the ultradian rhythms of histamine release in the mamillary body and the medial amygdaloid nucleus are not dependent on the integrity of the suprachiasmatic nucleus. The rate of histamine release in the mamillary body seems to be under the influence of excitatory neurons which originate from the suprachiasmatic nucleus.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (P5750) Send offprint requests to H. Prast     

Bildung und Speicherung von α-Methylnoradrenalin

Zusammenfassung An Meerschweinchen wurde die Wirkung von Reserpin auf die Bildung von -Methylnoradrenalin aus -Methyldopa untersucht. Im Herzen und Vas deferens verursacht Reserpin eine starke Verminderung der -Methylnoradrenalin-Bildung, im Gehirn hat es nur einen geringen Einfluß.Bei der Perfusion isolierter Meerschweinchenherzen mit Dopamin bzw. -Methyldopamin wird wesentlich mehr -Methyldopamin in das Herz aufgenommen als Dopamin. Reserpin-Vorbehandlung vermag weder die Dopamin- noch die -Methyldopamin-Aufnahme zu hemmen; es vermindert jedoch die Bildung von Noradrenalin bzw. -Methylnoradrenalin aus ihren Vorstufen, wobei die Bildung von Noradrenalin am stärksten reduziert wird.Bei der Inkubation isolierter Meerschweinchenherz-Granula mit -Methyldopamin oder -Methylnoradrenalin vermindert Reserpin die Aufnahme der beiden Amine in die Granula.Nach Perfusion von Kaninchenherzen mit Dopamin bzw. -Methyldopamin wurde die subcelluläre Verteilung dieser beiden Amine untersucht. Während -Methyldopamin hauptsächlich partikulär gebunden vorliegt, befindet sich Dopamin vorwiegend im Cytoplasma.

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Summary Experiments have been carried out in order to study the influence of reserpine pretreatment on the storage of -methylnoradrenaline and its synthesis from -methyldopa (3×400 mg/kg).Reserpine diminishes in heart and vas deferens of guinea-pigs the synthesis of -methylnoradrenaline from -methyldopa while its effect in brain is not significant.During infusion of isolated guinea-pig hearts with -methyldopamine or dopamine (0.6 moles/min, 30 min) much more -methyldopamine is taken up into the hearts than dopamine. Pretreatment with reserpine (4×25 g/kg) has no effect on the uptake of these amines; it causes, however, a decrease of synthesis of noradrenaline and -methylnoradrenaline from their precursors. The inhibitory effect on the synthesis of noradrenaline is much more pronounced than that on the synthesis of -methylnoradrenaline.In incubation experiments with storage granules isolated from guinea-pig hearts the uptake of -methyldopamine and -methylnoradrenaline (0.25 moles/ml) is significantly reduced by reserpine (40 g/ml).After infusion of rabbit hearts with -methyldopamine or dopamine (1.5 moles/min, 60 min) the subcellular distribution of these amines was studied. The main part of -methyldopamine is located in the particulate fractions whereas dopamine is predominantly found in the cytoplasma.

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Herrn Prof. Dr. Peter Holtz zum 65. Geburtstag am 6.2.1967 gewidmet.

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Ausgeführt mit der Unterstützung der Deutschen Forschungsgemeinschaft.     

The release of endogenous histamine in distinct brain areas is modified by electrical stimulation

Summary In anaesthetized cats, mamillary bodies, hypothalamic areas and medial amygdaloid nuclei were bilaterally superfused through push-pull cannulae and the effects of the electrical stimulation on the release of endogenous histamine were investigated.Electrical stimulation of the mamillary body increased the release of histamine in the stimulated area, as well as in the contralateral mamillary body. Electrical stimulation of the lateral hypothalamic area enhanced the histamine release in the contralateral hypothalamic area. Stimulation of the posterior hypothalamic area led to a delayed increase in the histamine release in the stimulated area. Stimulation of the medial amygdaloid nucleus reduced the release if histamine in the ipsilateral posterior hypothalamic area, while the histamine release in the contralateral lateral hypothalamic area was enhanced.The results demonstrate that electrical stimulation of distinct brain areas rich in histaminergic neurons may either increase, or decrease the release rate of histamine in the stimulated area and/or in remote brain areas.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (P5750). Part of the results has been presented at the 26th Spring Meeting of the German Society for Pharmacology and Toxicology, Mainz 1986 Send offprint requests to H. Prast at the above address     

Histaminergic neurons modulate acetylcholine release in the ventral striatum: role of H1 and H2 histamine receptors

To investigate whether H1 and H2 histamine receptors are implicated in the modulation of acetylcholine release by endogenous histamine, the ventral striatum of the conscious, freely moving rat was superfused by the push-pull superfusion technique with drugs and the release of acetylcholine was determined in the superfusate. Superfusion with the H1 receptor agonist 2-thiazolylethylamine (TEA, 50 micromol/l) enhanced the release of acetylcholine, while the H1 receptor antagonist triprolidine (50 micromol/l) reduced acetylcholine outflow and abolished the TEA-evoked release of the neurotransmitter. The inhibitory effect of triprolidine was not influenced either on simultaneous superfusion with 10 micromol/l (+/-)-7-bromo-1-(fluoresceinylthioureido)phenyl-8-hydroxy-3-methyl -2,3,4,5-tetrahydro-1H-benzazepine (SKF-83566, D1 dopamine receptor antagonist) and 50 micromol/l quinpirole (D2/D3 dopamine receptor agonist) or on superfusion with the GABAA receptor antagonist bicuculline (50 micromol/l). The H2 receptor antagonists ranitidine or famotidine (50 micromol/l each) greatly enhanced acetylcholine release rate in the ventral striatum. Presuperfusion with alpha-fluoromethylhistidine (FMH, 1 mmol/l), which inhibits neuronal synthesis of histamine, abolished the famotidine-induced release of acetylcholine. The releasing effect of famotidine was also abolished on simultaneous superfusion with 10 micromol/l SKF-83566 and 50 micromol/l quinpirole. The release of acetylcholine elicited by famotidine was reversed to a decreased acetylcholine outflow when the striatum was superfused with the GABA(A) receptor antagonist bicuculline (50 micromol/l) prior to famotidine. Superfusion with the H2 receptor agonist impromidine (1 micromol/l) decreased acetylcholine outflow, while the H2 agonist dimaprit (50 micromol/l) exerted the opposite effect. The releasing effect of dimaprit was not influenced by FMH (1 mmol/l), but it was abolished in the presence of SKF-83566 (10 micromol/l) and quinpirole (50 micromol/l). In the presence of bicuculline the release of acetylcholine by dimaprit was enhanced and prolonged. It seems possible that dimaprit and impromidine stimulate different subtypes of H2 receptors. The findings suggest that the release of acetylcholine in the striatum is modulated by neighbouring histaminergic neurons in a complex way. Stimulation of H1 histamine receptors, probably located on cholinergic neurons, enhances acetylcholine release. Stimulation by histamine of H2 receptors located on cholinergic or GABAergic neurons enhances the release of acetylcholine, while stimulation of H2 receptors located on dopaminergic neurons exerts the opposite effect.     

Stereoselectivity of noradrenaline uptake into synaptic vesicles of the rat brain

Summary To investigate the stereoselectivity of the ATP-Mg²⁺-dependent uptake of noradrenaline, synaptic vesicles were isolated from the rat brain by differential centrifugation and incubated with ³H-(±)-, ³H-(–)- or ¹⁴C-(+)-noradrenaline in the absence and in the presence of ATP-Mg²⁺. The K _m values of the ATP-Mg²⁺-dependent uptake were found to be different for the two isomers (mol/l): ³H(±)-noradrenaline 14.9 ± 2.2 × 10^–1, ³H-(–)-noradrenaline 7.7 ± 0.5 × 10^–1, ¹⁴C-(+)-noradrenaline 17.3 ± 3.7 × 10^–1, whereas the V _maX of the racemate was identical with those of the two isomers (pmol/mg protein/min): ³H-(±)-noradrenaline 5.5 ± 0.4, ³H-(–)-noradrenaline 4.9 + 0.1, ¹⁴C-(+)-noradrenaline 5.1 ± 0.4. Moreover, (+)-noradrenaline inhibited competitively the ATP-Mg²⁺-dependent uptake of ³H-(±)-noradrenaline (K_i 19.2 + 1.0 × 10^–1 mol/l) and ³H-(–)-noradrenaline (Ki 17.7 ± 1.8 × 10^–1 mol/l), the K_i values being nearly identical with the K _m of the ATP-Mg²⁺-dependent uptake of ¹⁴C-(+)-noradrenaline. It is concluded that the ATP-Mg²⁺-dependent uptake of noradrenaline into synaptic vesicles of the rat brain is stereoselective and that both isomers share the same transport system.The experiments were carried out at the Institut für Pharmakologie and Toxikologie der Universitat Würzburg, FRG. This work was supported by the Deutsche Forschungsgemeinschaft Send offprint requests to A. Philippu at the above address     

Hyperforin enhances the extracellular concentrations of catecholamines, serotonin and glutamate in the rat locus coeruleus

Hyperforin is the main antidepressant component of hypericum perforatum (St. John's Wort). Using the push-pull superfusion technique we tested whether hyperforin influences extracellular concentrations of neurotransmitters in the rat locus coeruleus. Hyperforin (10 mg/kg, i.p.) not only enhanced the extracellular levels of the monoamines dopamine, noradrenaline and serotonin, but also that of the excitatory amino acid glutamate. The levels of the main serotonin metabolite 5-hydroxyindolacetic acid, as well as those of the amino acids GABA, taurine, aspartate, serine and arginine, were not influenced. Together with in vitro studies, our findings suggest that the antidepressant property of hyperforin is due to enhanced concentrations of monoamines and glutamate in the synaptic cleft, probably as a consequence of uptake inhibition.