Nitric oxide as modulator of neuronal function.

The gas NO is a messenger that modulates neuronal function. The use of NO donors and NO synthase inhibitors as pharmacological tools revealed that this free radical is probably implicated in the regulation of excitability and firing, in long-term potentiation and long-term depression, as well as in memory processes. Moreover, NO modulates neurotransmitter release. In vivo and in vitro studies have shown that, in all brain structures investigated, endogenous NO modulates the release of several neurotransmitters, such as acetylcholine, catecholamines, excitatory and inhibitory amino acids, serotonin, histamine, and adenosine. In most cases, enhanced NO level in the tissue increases the release of neurotransmitters, although decreasing effects have also been observed. Cyclic 3'-5' guanosine monophosphate and glutamate mediate the modulation of transmitter release by NO. Recent observations suggest that the release of some transmitters is dually influenced by NO. Thus, besides modulation by presynaptically located auto- and heteroreceptors, NO released from nitrergic neurons seems to play a universal role in modulating the release of transmitters in the brain.

Nitric oxide modulates the release of serotonin in the rat hypothalamus.

To investigate the effect of nitric oxide (NO) on the release of serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), the posterior hypothalamus of the conscious rat was superfused through a push-pull cannula with drugs which either liberate NO, or inhibit NO synthase (NOS). The NO donors, linsidomine, diethylamine/nitric oxide (DEA/NO), S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-glutathione (SNOG) and sodium nitroprusside influenced the release of serotonin in a biphasic way. Low concentrations of drugs diminished, while higher concentrations of these compounds enhanced the outflow of serotonin. The NOS inhibitors N(G)-methyl-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NINA) enhanced the serotonin release. A high concentration of L-NAME slightly diminished the outflow of serotonin. Inhibition of the guanylyl cyclase by oxodiazolo[4, 3]quinoxaline-one (ODQ) abolished the changes in serotonin outflow induced by both low and high concentrations of linsidomine. The extracellular concentration of the 5-HIAA was not influenced by the compounds used. These data suggest that endogenous NO modulates the release of serotonin in a biphasic and cGMP-dependent way.

Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide.

The influence of nitric oxide (NO) on adenosine release was investigated by the push-pull technique in the ventral striatum of the urethane-anaesthetized rat. Superfusion with the NO donor diethylamine-NO enhanced, whereas superfusion with the NO synthase inhibitor L-NG-nitroarginine methyl ester decreased the output of adenosine. The effect of L-NG-nitroarginine methyl ester was abolished by L-arginine methyl ester. These findings indicate that, in the ventral striatum of the rat, NO modulates adenosine release.

Stereoselectivity of noradrenaline uptake into synaptic vesicles of the rat brain.

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 Km values of the ATP-Mg2+-dependent uptake were found to be different for the two isomers (mumol/l): 3H-(+/-)-noradrenaline 14.9 +/- 2.2 x 10(-1), 3H-(-)-noradrenaline 7.7 +/- 0.5 x 10(-1), 14C-(+)-noradrenaline 17.3 +/- 3.7 x 10(-1), whereas the Vmax 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 (Ki 19.2 +/- 1.0 x 10(-1) mumol/l) and 3H-(-)-noradrenaline (Ki 17.7 +/- 1.8 x 10(-1) mumol/l), the Ki values being nearly identical with the Km 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.

Competition of some biogenic amines for uptake into synaptic vesicles of the striatum.

Synaptic vesicles were isolated from the caudate nucleus of the pigs by differential centrifugation and incubated with labelled monoamines in the absence or in the presence of ATP-Mg(2+). Addition of ATP-Mg(2+) enhanced the uptake of (14)C-dopamine into the vesicles. Serotonin competitively inhibited the ATP-Mg(2+)-dependent uptake of (14)C-dopamine without influencing the uptake which took place in the absence of ATP-Mg(2+). Likewise, dopamine caused a dose-dependent inhibition of the ATP-Mg(2+)-dependent uptake of (14)C-serotonin without inhibiting the uptake in the absence of ATP-Mg(2+). Incubation of the vesicles with equal concentrations of(3)-dopamine and (14)C-serotonin revealed that the presence of the one amine competitively inhibited the ATP-Mg(2+)-dependent uptake of the other. Tyranimine competitively inhibited the ATP-Mg(2+)-dependent uptake of (14)C-dine, (14)C-serotonin and (14)C-noradrenaline into the vesicles; the uptake of the amines which took place in the absence of ATP-Mg(2+) was not impairedby tyramine. Analysis of the amine uptake by the ABC test showed that a mutual inhibition exists between dopamine and serotonin for the uptake into the synaptic vesicles. GABA did not influence the uptake of (14)C-dopamine either in the absence, or in the presence of ATP-Mg(2+)...

Uptake of dopamine into fractions of pig caudate nucleus homogenates.

Homogenates of the caudate nucleus of the pig were submitted to differential centrifugation. The 20000 g and 80000 g fractions were isolated and a part of them was osmotically shocked. The highest dopamine content per mg protein was found in the intact 80000 g fraction. Incubation experiments with the intact and the osmotically shocked fractions at 25 degrees C revealed that the particles of the intact 20000 g fraction took up dopamine, the influx of the amine was not enhanced by addition of ATP and magnesium to the incubation medium. On the other hand after osmotic shock the uptake of dopamine into the particles of this fraction was greatly enhanced by addition of ATP and magnesium. The uptake of dopamine into the particles of both intact and osmotically shocked 80000 g fractions was likewise enhanced by ATP and magnesium. The uptake in all fractions was not influenced by ouabain. The influx of dopamine into the particles of the intact 80000 g fraction was competitively inhibited by reserpine (Ki0.96 x 10-8M) and prenylamine (Ki1.74 x 10-8M). It is concluded that the intact 20000 g fraction contains intact synaptosomes; the uptake of dopamine is independent of the presence of ATP and magnesium. The shocked 20000 g fraction and the 80000 g fractions contain synaptic vesicles; the uptake of dopamine into these vesicles is enhanced by ATP and magnesium.

Uptake of serotonin, gamma-aminobutyric acid and histamine into synaptic vesicles of the pig caudate nucleus.

Dopamine-containing vesicles were isolated from the caudate nucleus of the pig by differential centrifugation and incubated with 14-C-serotonin (14-C-5-HT), 14-C-gamma-aminobutyric acid (14-C-GAGA), 14-C-dopamine or 14-C-histamine. At 25 degrees C the uptake of 14-C-5-HT and 14-C-GABA was enhanced by ATP and magnesium. The Km for the uptake of 14-C-5-HT in the presence of ATP and magnesium was 0.67 x 10-6M, that of 14-C-GABA 1.33 x 10-4M. 14-C-Histamine was also taken up into the vesicles but its uptake was not incluenced by ATP and magnesim. The ATP-magnesium-dependent uptake of 14-C-5-HT and 14-C-GABA was abolished at 0 degrees C. At 37 degrees C the accumulation of 14-C-GABA in the presence of ATP and magnesium reached a steady state after 20 min, while the accumulation of 14-C-5-HT reached a maximum after 2.5 min of incubation and then gradually declined. Osmotic lysis of the vesicles followed by sonication abolished the enhanced uptake of 14-C-GABA, 14-C-5-HT and 14-C-dopamine in the presence of ATP and magnesium. Determination of the endogenous contents of the vasicles revealed 14.6 pmoles per mg protein for 5-HT and 22.7 nmoles per mg protein for GABA. It is concluded that the caudate nucles preparation used in this study contains not only dopamine-storing vesicles able to take up biogenic amines but also GABA-containing vesicles which take up GABA by an ATP-magnesium-dependent process.