Nitric oxide does not modulate kainate receptor binding in human brain

Neuroscience Letters 233 (1997) 133–136

Nitric oxide does not modulate kainate receptor binding in human brain R.D. Lees*, P. Slater, S.W. D’Souza School of Biological Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, UK Received 9 July 1997; received in revised form 11 August 1997; accepted 27 August 1997

Abstract The ability of three nitric oxide (NO) donor compounds to modify ligand binding to kainate receptors was studied in tissue from human adult autopsy brains. Binding of [3H]kainic acid (5 nM) was measured in frontal cortex membranes made from Brodmann area 8 (BA 8) and autoradiographically using sections of frontal cortex (BA 8 and 9). None of the three donors, S-nitroso-N-acetyl-d,l-penicillamine (SNAP), S-nitrosocysteine (Cys-NO) and 3-morpholinosydnonimine chloride (SIN-1) altered the specific binding of [3H]kainic acid. Nitrite accumulation assays confirmed that adequate amounts of NO were released by the donors under the ligand binding conditions used. The findings show that binding to the kainate receptor, in contrast to the other ionotropic glutamate receptors, is not affected by NO and strongly suggest that endogenous NO produced by NO synthase (NOS) does not modulate kainate receptors in vivo. Mechanisms whereby NOS inhibitors potentiate kainic acid-induced seizures in animal models may include altered modulation of glutamate N-methyl-d-aspartate (NMDA) receptors.  1997 Elsevier Science Ireland Ltd. Keywords: Nitric oxide donors; Kainate receptor binding; Human cerebral cortex

Nitric oxide (NO) is produced by NO synthase (NOS) enzymes in brain where it functions as a regulator of cerebral blood flow and a second messenger produced by glutamate N-methyl-d-aspartate (NMDA) receptor activation. One of the main functions of NO produced by NMDA activation is stimulation of guanylate cyclase to produce cyclic guanosine 3′,5′-monophosphate [14]. NMDA receptors, and therefore NO, are involved in some forms of synaptic plasticity as well as in some pathological conditions such as hypoxia-ischaemia in which there is overstimulation of NMDA receptors [15,17]. Whilst new functions of NO are still being described, it has multiple influences on the functions of some neurotransmitters in brain, especially glutamate and, to a lesser extent, GABA systems. NO donors are reported to block rat brain NMDA receptors [12,13], possibly by modifying redox sites [10], and to upregulate a-amino-3-hydroxy-5-methylisoazole-4-propionic acid (AMPA) receptor binding [5]. The actions of NO in vivo are often investigated by administering a NOS inhibitor to reduce NO production in

* Corresponding author. Tel.: +44 161 2755354; fax: +44 161 2755363; e-mail: [email protected]

brain. One of the most frequently cited actions of NOS inhibitors is to potentiate seizure activity in rodents, including seizures evoked by kainic acid [11,16]. NO is described as an endogenous anticonvulsant compound [3]. Because of the ability of NO to affect the AMPA and NMDA types of ionotropic glutamate receptor, the alteration by NO of kainate-induced seizures may be caused by a direct modulatory influence on kainate receptors. In the present study, we have examined, using human brain membranes and three different NO donor compounds, whether NO modulates kainate receptor binding in vitro. Brains were removed at autopsy from two male and two female adults whose mean age (±SEM) was 62 ± 2 years and postmortem interval was 23 ± 3 h. Brains were quickfrozen within 2 h of removal at autopsy and were stored at −70°C. All four patients died from an acute cardiovascular event and none had a history of neurological or psychiatric illness. The pH of tissue samples taken from the brains was not below 6.61, indicating minimal acidosis resulting from prolonged agonal state. Kainate receptors were measured in membrane preparations and autoradiographically in brain sections. Tissue was dissected from Brodmann area 8 (BA 8) for preparing membranes. The ligand binding method was simi-

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940 (97 )0 0654- X

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Fig. 1. Effects of NO donors SIN-1 (X) and SNAP (W) on the specific binding of [3H]kainic acid in human cortical membranes. Each point is the mean binding ( + SEM) in fmol/mg protein, calculated using samples from four adult brains. Binding in the absence of NO donor is shown (C). Linear regression analysis showed that the slopes of the data curves were not significantly different from zero.

lar to a reported method [4]. Aliquots (100 mg) of tissue were thawed at room temperature for 15 min and homogenized in 50 vol. of ice-cold, pH 7.4, 50 mM Tris–acetate buffer and centrifuged at 19 000 × g and 4°C for 10 min. The supernatant was discarded and the pellet was resuspended in 50 mM Tris–acetate buffer and centrifuged as before. This procedure was repeated five times using 5 mM Tris–acetate buffer. The pellet was finally suspended in 75 vol. of 50 mM Tris–acetate buffer, pH 7.4, and equilibrated at 25°C prior to binding assays which were done in 96-well plates. Membranes (approximately 35 mg protein) and NO donor compound were incubated for 60 min at 25°C with 5 nM [vinylidene-3H]kainic acid (58 Ci/mmol). Non-

Fig. 2. Production of NO by the donor compounds Cys-NO (100 mM, B), SNAP (2 mM, O), SIN-1 (2 mM, W) measured as nitrite accumulation in solutions. Each point is the mean of triplicate measurements on three brains.

Fig. 3. Effect of NO donor compound cys-NO on the specific binding of [3H]kainic acid in human cortical membranes. Each point is the mean binding (+SEM) in fmol/mg protein, calculated using samples from four adult brains. Linear regression analysis showed that the slope of the data curve was not significantly different from zero.

specific binding was measured by adding 100 mM l-glutamate to assays. Assays were ended by vacuum filtration and washing. Protein was measured in membrane aliquots. Frozen sections (20 mm) were cut from coronal brain blocks containing BA 8 and 9 and were thaw-mounted on glass slides. Autoradiographic measurements of kainate receptors were made on sections from three brains, using a published method [1]. Sections were covered with 50 mM Tris–acetate buffer containing 20 nM [3H]kainic acid and were incubated at 25°C for 60 min with NO donor compound. Non-specific binding was measured by adding 1 mM l-glutamate to incubations. Sections were washed three times in fresh buffer (1 l), dipped in ice-cold distilled water and dried. Exposure to tritium film with Microscales (Amersham) 3H-Standards were for 5 weeks. Films were developed in Kodak D19 developer. Binding data were taken from autoradiographs using an image analyzer. Three NO donors used were S-nitroso-N-acetyl-d,l-penicillamine (SNAP), S-nitrosocysteine (Cys-NO) and 3-morpholinosydnonimine chloride (SIN-1). SNAP was synthesized by reacting N-acetyl-d,l-penicillamine with excess NaNO2 at acidic pH [6]. Product formation was monitored by microscopic identification of crystals and melting point determination [6]. SNAP was first dissolved in dimethyl sulphoxide and diluted with 50 mM Tris–acetate buffer containing 2 mM CuSO4 to encourage decomposition to NO [2]. Cys-NO, synthesized by reacting equimolar amounts of l-cysteine and NaNO2 at acidic pH [9], was dissolved in Tris–acetate buffer containing 2 mM CuSO4. SIN-1, purchased from Tocris Cookson (Bristol, UK), produces both NO and superoxide which, together, may yield potentially damaging (to membranes) peroxynitrite. Thus, superoxide was scavenged by adding superoxide dismutase (50 U/ml) to assays, and hydrogen peroxide formed was removed using catalase (50 U/ml).

R.D. Lees et al. / Neuroscience Letters 233 (1997) 133–136 Table 1 3

Influence of SNAP and SIN-1 on autoradiographic binding of [ H]kainic acid in sections of cortex from three human brains Ligand bound, fmol/mg tissue Brain

Control

SNAP

SIN-1

1 2 3

71.5 (61.7–80.2) 44.5 (34.3–54.0) 82.6 (81.0–84.7)

90.8 (74.8–98.8) 48.0 (32.8–70.8) 90.7 (66.7–119.0)

90.8 (73.8–107.9) 56.6 (55.2–58.9) 87.4 (78.7–95.8)

Data for each brain are the mean (and range) obtained from triplicate autoradiographs. Non-specific binding values obtained from separate autoradiographs have been subtracted from total binding.

Because the release of NO by donors in biological systems is influenced by temperature, pH and other factors, it was important to measure the rates of production of NO by the donors, using nitrite formation [7], under the assay conditions. Membranes, as used in binding assays, were incubated with NO donor and transferred at intervals to the reaction mixture (100 ml) in 96-well plates consisting of 1 part N-(1-naphthyl)-ethylenediamine hydrochloride (1 mg/ ml), 1 part sulfanilic acid (2 mg/ml), 1 part 0.4 M HCl, 2 parts water. Initial concentrations of donor compounds were 100 mM Cys-NO, and 2 mM SNAP and SIN-1. Optical densities (540 nm) were read at intervals with a microplate reader (Bio-Tek Instruments, Model EL11) which also read NaNO2 standards. Binding of [3H]kainic acid to human brain membranes was displaced by 1 mM l-glutamate and in this study the mean specific binding (n = 4) was 77 ± 1% of the total binding. Incubation of membranes with 5 nM [3H]kainic acid and SIN-1 or SNAP (1 mM–10 mM) did not alter the specific [3H]kainic acid binding (Fig. 1). The rate of NO release by the donor compounds was monitored as nitrite accumulation under identical conditions to the binding assays, although there are potential problems with the nitrite assay. Any NO which may be converted to nitrate will not be measured and the nitrite assay may therefore slightly underestimate NO production. Also, the S-nitrosothiols Cys-NO and SNAP may absorb at similar wavelengths to nitrite, although the curves showing the rate of apparent NO production suggest little or no interference by the donors themselves (Fig. 2). The data show that NO produced by SNAP exceeded the production by SIN-1 and that both donors released NO continuously throughout the 60 min period of the [3H]kainic acid binding assays. [3H]Kainic acid binding was also unaffected by 25–200 mM concentrations of Cys-NO (Fig. 3). Monitoring of nitrite accumulation showed that Cys-NO (100 mM) released NO for up to 20 min of the 60 min assay incubation with [3H]kainic acid (Fig. 2). The lack of effect of NO donors on [3H]kainic acid binding was not due to insufficient release of NO in the assay solutions. NO donors were also tested on kainate receptor binding in brain sections, a far more intact tissue preparation than homogenates. Specific binding of [3]kainic acid in forebrain

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sections, measured autoradiographically, was 77% of the total binding. In cortex, kainate receptor binding was measured with the image analyzer in all laminae, including laminae I and II which had the highest density of receptors. Incubation of sections with the NO donors SNAP (2 mM) and SIN-1 (2 mM) had no effect on the specific binding of [3H]kainic acid (Table 1). A reduction in the synthesis of NO in rat brain, achieved by administering NOS inhibitors in vivo, potentiated the seizures which were induced by systemic administration of either kainic acid or pilocarpine [8]. It is proposed that NO functions as an endogenous anticonvulsant in brain [3]. Because the present study shows that NO has no direct action in vitro on the kainate subtype of glutamate receptor, it is unlikely that the anti-seizure action of NO in vivo is produced via an inhibitory action upon kainate receptors. Instead, NO may be involved in the progression and severity of chemically-induced seizures, although the mechanisms are uncertain. GABAA receptor mechanisms do not explain why reducing NO production with a NOS inhibitor is proconvulsant. NO donors inhibit GABAA receptor function in cerebellar granule cells [18]. If this effect occurs elsewhere in brain, NOS inhibitors will reduce NO production and thus increase GABAA function which will act to counter the spread of seizures. The anti-seizure property of NO [3] may arise from an interaction between NO and the subtypes of ionotropic glutamate receptor. The present findings tend to rule out any effect of NO at kainate receptors. NO was reported to increase ligand binding to AMPA receptors [5] which is not consistent with NO having anti-seizure properties. In contrast, because NO reduces NMDA receptor function [12,13], a reduction in NO production by NOS inhibitors could, in theory, produce a proconvulsant effect by enhancing NMDA function. It remains to be shown whether the release of glutamate by kainic acid is sufficient to activate NMDA receptors and thus promote seizure activity [11]. This work was funded by the Little Foundation. [1] Ball, E.F., Shaw, P.J. and Johnson, M., The distribution of excitatory amino acid receptors in the normal human midbrain and basal ganglia with implications for Parkinson’s disease: a quantitative autoradiographic study using [3H]MK-801, [3H]glycine, [3H]CNQX and [3H]kainate, Brain Res., 658 (1994) 209–218. [2] Boulton, C.L., Irving, A.J., Southam, E., Potier, B., Garthwaite, J. and Collingridge, G.L., The nitric oxide-cyclic GMP pathway and synaptic depression in rat hippocampal slices, Eur. J. Neurosci., 6 (1994) 1528–1535. [3] Buisson, A., Lakhmeche, N., Verrecchia, C., Plotkine, M. and Boulu, R.G., Nitric oxide: an endogenous anticonvulsant substance, NeuroReport, 4 (1993) 444–447. [4] Deakin, J.F.W., Slater, P., Simpson, M.D.C., Gilchrist, A.C., Skan, W.J., Royston, M.C., Reynolds, G.P. and Cross, A.J., Frontal cortical and left temporal glutamatergic dysfunction in schizophrenia, J. Neurochem., 52 (1989) 1781–1786. [5] Dev, K.K. and Morris, B.J., Modulation of a-amino-3-hydroxy-5methylisoazole-4-propionic acid (AMPA) binding sites by nitric oxide, J. Neurochem., 63 (1994) 946–952.

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