Effects of nitric oxide donors on basal and K+-evoked release of [3H]noradrenaline from rat cerebral cortex synaptosomes

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Abstract

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 μM 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.

Introduction

Initially identified as a mediator used by macrophages and endothelial cells, nitric oxide (NO) is now recognized as a prominent neuronal messenger (Garthwaite, 1991; Bredt and Snyder, 1992). NO is thought to play a number of physiological roles within the central nervous system including long-term potentiation (Bliss and Collingridge, 1993), long-term depression (Zorumski and Izumi, 1993), and neuroprotection (Lipton et al., 1993). Several investigators have suggested that NO acts as a retrograde messenger produced by the postsynaptic neuron to control the release of neurotransmitters by presynaptic terminals (O'Dell et al., 1991; Schuman and Madison, 1991; Fazeli, 1992). More recent studies have in fact demonstrated NO-induced increases in the release of adenosine, acetylcholine, noradrenaline and glutamate from hippocampal slices (Fallahi et al., 1996; Lauth et al., 1995; Lonart and Johnson, 1995a, Lonart and Johnson, 1995b; Satoh et al., 1996), as well as that of glutamate from hippocampal synaptosomes (Meffert et al., 1994). Excitatory and inhibitory effects on neurotransmitter release in other areas of the brain have also been observed (Stewart et al., 1996; Prast and Philippu, 1992; Black et al., 1994; Bugnon et al., 1994; Guevara-Guzman et al., 1994; Lonart and Johnson, 1994; Seilicovich et al., 1995; Zhu and Luo, 1992).

The present study was conducted to assess the effects of NO on the release of noradrenaline by synaptosomes isolated from the cerebral cortex of the rat. Cortical tissue is also known to contain high levels of NO synthase, the enzyme that is responsible for the formation of NO. Investigation of cortical NO activity has been relatively limited, although the gas appears to play an important role in the development and function of this tissue (Vincent and Hope, 1992). The synaptosome model allowed us to focus our attention on the mechanisms that regulate presynaptic neurotransmitter release minimizing the interference by other brain structures.

The superfused synaptosomes were exposed to sodium nitroprusside and S-nitroso-N-acetylpenicillamine, which produce NO in biological systems (Feelisch, 1991), and the release of [3H]noradrenaline was measured under resting conditions and during K+-stimulated depolarization. Since many of the effects of NO are mediated by the guanosine 3′,5′-cyclic monophosphate (cGMP) system, the effects of the NO donors were also compared with those produced by the cell-permeable cGMP analogue, 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), and attempts were made to block their effects using a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (Garthwaite et al., 1995).

Section snippets

Preparation and superfusion of synaptosomes

Adult male Wistar rats (180–200 g) were sacrificed by decapitation and the brains rapidly removed. Crude cortical synaptosome fractions (P2) were prepared according to the method of Gray and Whittaker (1962). Using a glass-Teflon grinder, the tissue was homogenized in 40 volumes of 0.32 M sucrose that had been buffered to pH 7.4 with phosphate. The homogenate was centrifuged (5 min, 1000×g), and the resulting supernatant was recentrifuged (20 min, 12,000×g) to isolate the synaptosomes. The

Effects of nitric-oxide donors on basal release of [3H]noradrenaline from rat cortical synaptosomes

The synaptosomes were exposed for 5 min to sodium nitroprusside and S-nitroso-N-acetylpenicillamine (both at concentration of 0.01–10 mM) to identify the possible effects of NO on basal release of [3H]noradrenaline. As shown in Fig. 1, both drugs caused dose-dependent increases in the release of the labeled neurotransmitter. The maximum release induced by sodium nitroprusside was 2.37 times higher than that observed at baseline, and the ED50 was 300±15 μM; n=4. The ED50 for S-nitroso-N

Discussion

Because of its gaseous nature, NO diffuses rapidly across the cell membrane, and it can thus transmit a broad-range signal that complements the point-to-point signals of other transmitters. The present study was prompted by the suggestion that NO produced at the postsynaptic level might act as a retrograde messenger to modify presynaptic release of other neurotransmitters (O'Dell et al., 1991). The use of isolated superfused synaptosomes allowed us to focus on the mechanisms that regulate

Acknowledgements

This study was supported by grant 7020861 (60%) from the Italian Ministry of the University and of the Scientific and Technological Research. We would like to thank Sergio Campetella for his skilful technical assistance.

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