John Garthwaite

Glutamate, nitric oxide and cell-cell signalling in the nervous system

Nitric oxide (NO) is a recently discovered and highly unorthodox messenger molecule. Current evidence indicates that, in the CNS, NO is produced enzymatically in postsynaptic structures in response to activation of excitatory amino acid receptors. It then diffuses out to act on neighbouring cellular elements, probably presynaptic nerve endings and astrocyte processes. In several peripheral nerves, and quite possibly in parts of the CNS as well, NO might be formed presynaptically and thus act as a neurotransmitter. In both cases, a major action of NO is to activate soluble guanylate cyclase and so raise cGMP levels in target cells.

Excitatory amino acid neurotoxicity and neurodegenerative disease

The progress over the last 30 years in defining the role of excitatory amino acids in normal physiological function and in the abnormal neuronal activity of epilepsy has been reviewed in earlier articles in this series. In the last five years it has become clear that excitatory amino acids also play a role in a wide range of neurodegenerative processes. The evidence is clearest where the degenerative process is acute, but is more controversial for slow degenerative processes. In this article Brian Meldrum and John Garthwaite review in vivo and in vitro studies of the cytotoxicity of amino acids and summarize the contribution of such toxicity to acute and chronic neurodegenerative disorders.

NMDA RECEPTOR ACTIVATION INDUCES NITRIC-OXIDE SYNTHESIS FROM ARGININE IN RAT-BRAIN SLICES

Activation of N-methyl-D-aspartate (NMDA) receptors in rat cerebellum leads to the release of endothelium-derived relaxing factor, now identified as nitric oxide (NO), a stimulator of soluble guanylate cyclase. L-NG-monomethylarginine (L-NMMA), which blocks NO synthesis from L-arginine in several tissues, including a crude synaptosomal preparation from brain, inhibited the elevation of cyclic GMP induced by NMDA in rat cerebellar slices. D-NMMA was ineffective. L-Arginine, but not its D enantiomer, augmented the response to NMDA and reversed the inhibition by L-NMMA. The results indicate that stimulation of NMDA receptors results in the activation of the enzyme which catalyzes the formation of NO from L-arginine.

The nitric oxide-cyclic GMP signalling pathway in rat brain.

Nitric oxide is a novel signalling molecule in the brain and a potent activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. To determine if stimulation of cyclic GMP formation is a widespread mechanism of nitric oxide signal transduction, we have compared the distribution of the nitric oxide-generating enzyme (nitric oxide synthase) with that of nitric oxide-stimulated cyclic GMP accumulation, throughout the rat brain. The former was done using NADPH diaphorase histochemistry and the latter by cyclic GMP immunohistochemistry following perfusion of the nitric oxide donor, nitroprusside, in vivo. At a gross level, there was generally a good match when the two were compared in adjacent sections. Although the relative staining intensity varied from area to area, in no grey matter region did we observe cyclic GMP accumulation in the absence of nitric oxide synthase staining. In detail, the locations were complementary rather than identical. In some areas, nitric oxide synthase was found in postsynaptic structures and cyclic GMP accumulation in presynaptic elements and fibres; in others, the locations were reversed. Glial cells and their processes also accumulated cyclic GMP in the cerebellum. The results suggest that soluble guanylate cyclase is a major nitric oxide receptor throughout the brain. They also support the hypothesis that nitric oxide generated therein primarily functions as a mediator of cell-cell signaling rather than as a conventional second messenger acting within the cells in which it is produced. The types of communication subserved by nitric oxide appear to be extraordinarily diverse.

NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the l-arginine-nitric oxide pathway

When slices of young rat hippocampus were exposed briefly (2 min) to N-methyl-D-aspartate (NMDA), a rise in the levels of cyclic GMP took place. This response was dependent on NMDA concentration (EC50 approximately 30 microM) and the maximal elevations exceeded the unstimulated levels by 25-fold. The response to 100 microM NMDA was inhibited by two competitive antagonists of the conversion of arginine to nitric oxide, L-NG-methylarginine and L-NG-nitroarginine (IC50 approximately 6 microM and 100 nM respectively). The inhibitions produced by both antagonists were reduced or abolished when the incubation medium was supplemented with L-arginine (100-300 microM). Slices of adult hippocampus produced smaller increases (5-fold) in cyclic GMP levels in response to 100 microM NMDA than those found in the immature tissue, but the response could similarly be inhibited by NG-methylarginine. The results indicate that NMDA receptor activation in the hippocampus induces the generation of nitric oxide from arginine and that this novel intercellular messenger mediates the increases in cyclic GMP levels.

COMPARATIVE EFFECTS OF SOME NITRIC-OXIDE DONORS ON CYCLIC-GMP LEVELS IN RAT CEREBELLAR SLICES

In the central nervous system, glutamate receptor activation and other stimuli can lead to the cellular production of nitric oxide (NO), an activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. Four nitrovasodilators which yield NO were tested for their ability to elevate cGMP levels in rat cerebellar slices. Nitroprusside (NP), SIN-1, S-nitroso-N-penicillamine (SNAP) and hydroxylamine all caused very large (up to 300-fold) increments. Their threshold concentrations were between 1 and 30 microM. SNAP was the most potent (EC50 approximately 50 microM) followed by hydroxylamine (200 microM) and SIN-1 (1 mM), the latter compound having the highest efficacy. No maximal response to NP was evident at concentrations up to 10 mM. Slices could be challenged a second time with NP (300 microM) with no evidence of a change in sensitivity. The NO-donors are likely to be valuable for studying the functions of NO in brain tissue; however, the concentrations of NP, SNAP and SIN-1 required to elevate cGMP in the slices are orders of magnitude higher than those needed to stimulate guanylate cyclase activity in broken cell preparations, suggesting that rapid inactivation of NO takes place in the intact tissue.

NEUROTOXICITY OF EXCITATORY AMINO-ACID RECEPTOR AGONISTS IN RAT CEREBELLAR SLICES - DEPENDENCE ON CALCIUM-CONCENTRATION

In slices of developing rat cerebellum, a 30-min application of the excitatory amino acid receptor agonist, N-methyl-D-aspartate (NMDA), led to the necrosis of differentiating granule cells and deep nuclear neurones. The corresponding effect of another agonist, kainate, was the death of Golgi cells. The toxic effects of both agonists were prevented if the concentration of calcium in the exposing solution was reduced to 0.3 mM from the control level of 2.5 mM. A lesser reduction (to 1 mM) was enough to prevent 90% of the NMDA-induced necrosis of granule cells. The results indicate that an important component of the acute neurotoxic effects of excitatory amino acids is calcium-dependent and suggest reasons why this may not have been revealed in some previous studies.

Cellular uptake disguises action of L‐glutamate on N‐methyl‐D‐aspartate receptors: With an appendix: Diffusion of transported amino acids into brain slices

1 Pharmacological properties of the guanosine 3′5′‐cyclic monophosphate (cyclic GMP) responses to excitatory amino acids and their analogues were compared in slices and dissociated cells from the developing rat cerebellum maintained in vitro. The intention was to determine the extent to which cellular uptake might influence the apparent properties of receptor‐mediated actions of these compounds. 2 In slices, the potencies of the weakly (or non‐) transported analogues, N‐methyl‐D‐aspartate (NMDA) and kainate (KA) (EC50 = 40 μM each) were higher than those of the transported amino acids, D‐ and L‐aspartate (EC50 = 250 μM and 300 μM) and D‐ and L‐glutamate (EC50 = 540 μM and 480 μM). Quisqualate (up to 300 μM) failed to increase cyclic GMP levels significantly. The sensitivity of agonist responses to the NMDA receptor antagonist, DL‐2‐amino‐5‐phosphonovalerate (APV), was in the order NMDA > L‐aspartate > L‐glutamate, KA. 3 In dissociated cells, L‐glutamate was 280 fold more potent (calculated EC50 = 1.7 μM). L‐ and D‐aspartate (calculated EC50 = 13 μM) and D‐glutamate (EC50 = 130 μM) were also more effective than in slices. The potencies of NMDA and KA were essentially unchanged. Responses to NMDA, L‐glutamate and L‐aspartate under these conditions were equally sensitive to inhibition by APV but the response to KA remained relatively resistant to this antagonist. 4 The implications of these results are that, in slices, cellular uptake is responsible for (i) the dose‐ response curves to L‐glutamate, L‐ and D‐aspartate bearing little or no relationship to the true (or relative) potencies of these amino acids; (ii) the potency of APV towards the actions of transported agonists acting at NMDA receptors being reduced and (iii) a differential sensitivity to APV of responses to L‐glutamate and L‐aspartate being created, the consequence being that a potent action of L‐glutamate on NMDA receptors is disguised. 5 These conclusions are supported by theoretical considerations relating to the diffusion of transported amino acids into brain slices, as elaborated in the Appendix.

Sources and targets of nitric oxide in rat cerebellum

Nitric oxide (NO) mediates cell-cell signalling in the brain and stimulates cyclic GMP (cGMP) production in target cells. We have used NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) histochemistry to identify NO-producing neurones and cGMP immunohistochemistry to locate the targets of NO in rat cerebellum. NADPH-diaphorase staining was prominent in granule cells and in the molecular layer. cGMP immunostaining in cerebellar slices stimulated with the NO donors, nitroprusside and SIN-1, was found in granule cells, glomeruli, fibres, Bergmann glia and in other astrocytes. The results provide visible evidence that NO mediates neuron-neuron and neuron-glia communication.

The Nitric Oxide - Cyclic GMP Pathway and Synaptic Depression in Rat Hippocampal Slices

The ability of exogenous nitric oxide (NO) to modify synaptic transmission was investigated in area CA1 of the rat hippocampal slice. The NO donors S‐nitroso‐N‐acetylpenicillamine (SNAP) and S‐nitrosoglutathione (SNOG) depressed field excitatory postsynaptic potentials evoked by low frequency stimulation of the Schaffer collateral ‐ commissural pathway. Upon washout of the NO donors, synaptic transmission rapidly returned to control levels. A similar reversible synaptic depression was produced by SNAP when tetanic stimulation (100 Hz; 1 s) was delivered in its presence. The effect of SNAP was not mimicked by its precursor or breakdown product and was blocked by haemoglobin, indicating that the effect involved NO. Roussins black salt, a photolabile NO donor, also depressed transiently field excitatory postsynaptic potentials following photolysis. The depression was induced rapidly following a flash of UV light (20 s duration) focused onto the slice using a confocal microscope. The depressant effect of the NO donors on synaptic transmission was mimicked by zaprinast, a specific cGMP ‐ phosphodiesterase inhibitor. Zaprinast depressed to a similar extent both the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate and N‐methyl‐d‐aspartate receptor‐mediated components of excitatory postsynaptic currents without affecting passive membrane properties, indicating a presynaptic locus of action. SNAP, SNOG and zaprinast all elevated cGMP levels in rat hippocampal slices. Immunocytochemical staining revealed that the cGMP accumulation was mainly in a network of varicose fibres running throughout the CA1 region, consistent with a presynaptic site of action of NO. We conclude that NO, possibly through activation of guanylate cyclase, may be involved in transient presynaptic depression in the CA1 region of the hippocampus.