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Brain Research 881 (2000) 208–211

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Short communication

Noncompetitive inhibition of the glycine receptor-mediated current by

melatonin in cultured neurons

a ,

_*

a b

Fong-Sen Wu

, Ying-Chen Yang , Jing-Jane Tsai

a

Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan

b

Department of Neurology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan

Accepted 2 August 2000

Abstract

The effect of melatonin on the glycine receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Melatonin rapidly and reversibly inhibited the glycine-induced current in a dose-dependent fashion, with an EC50 of 934 mM and a maximal inhibition of 100%. Furthermore, melatonin noncompetitively inhibited the glycine response by an agonist-independent mechanism that was distinct from that of an open-channel blocker.  2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Other neurotransmitters

Keywords: Melatonin; Glycine receptors; Cultured spinal neurons; Whole-cell recordings; Noncompetitive inhibition

The pineal hormone melatonin regulates many biologi- explore the mechanism of action of melatonin on the cal functions including circadian rhythm, sleep, mood, sex glycine receptor-mediated response in cultured chick spinal maturation, and immune responses [3]. In particular, cord neurons by using the whole-cell voltage-clamp re-melatonin has been shown to inhibit the excitability of the cording technique.

central nervous system [1]. Radioligand binding assays and Cultures of spinal cord neurons were prepared from behavioral studies suggest that melatonin may exert its 7-day chick embryos and were used 2–4 weeks after central effect by positively modulating the GABAA re- plating as described in detail [12]. Whole-cell currents ceptor in neuronal cells [2,6,9]. Indeed, this idea is were recorded by the whole-cell variant of the patch clamp confirmed by our recent electrophysiological studies, dem- technique [10]. Electrode resistance was 3–5 MV when onstrating that melatonin potentiates the GABA receptor-A filled with an intracellular solution containing (in mM): mediated current in cultured neurons [14]. Interestingly, 140 KCl, 3 NaCl, 1 MgCl , 4 MgATP, 11 EGTA, and 102 melatonin fails to enhance the glycine-induced current, HEPES (pH adjusted to 7.2 with KOH). In experiments in producing instead an inhibition of the glycine response. which high concentrations of glycine were used, the The mechanism underlying the effect of melatonin on the intracellular solution was replaced with a low chloride (20 GABAA receptor has been studied [14]. In contrast, mM) pipet solution. This solution contained the following relatively little is known of interaction of melatonin with (in mM): 130 potassium gluconate, 20 KCl, 3 sodium the glycine receptor. In the present study, we further gluconate, 4 MgATP, 11 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH). The bath solution contained (in mM): 150 NaCl, 4 KCl, 1 CaCl , 1 MgCl and 10 HEPES2 2 (pH adjusted to 7.2 with NaOH). All experiments were *Corresponding author. Tel.:1886-6-235-3535, ext. 5455; fax:1

886-performed at room temperature (23–258C). 6-236-2780.

E-mail address: fongsen@mail.ncku.edu.tw (F.-S. Wu). Recordings were made using an Axopatch-1D patch

F.-S. Wu et al. / Brain Research 881 (2000) 208 –211 209

clamp amplifier. Cells with series resistance greater than external buffer or melatonin solution, followed by a 10-s 10 MV were rejected. Only cells with resting membrane application of glycine or glycine plus melatonin, followed potential more negative than250 mV and input resistance by a 20-s pulse of external buffer solution. A period of 3–4 in excess of 150 MVwere used. All recordings were made min was allowed between successive applications of with the cell membrane potential clamped at 270 mV. glycine.

Currents were filtered at 1 kHz using an eight-pole Bessel At a holding potential of 270 mV, glycine produced filter and digitized (4 ms / point) using an on-line data inward currents, which reversed at or near 0 mV in

2 2

acquisition system (pClamp, Axon Instruments). symmetrical Cl solutions, as expected for Cl -mediated Drug solutions were applied to single neurons by currents (data not shown). In agreement with the previous pressure ejection (15 psi) from seven-barrel pipets [4,5]. work [13], the current induced by 50 mM glycine slowly All drugs were obtained from Sigma, with the exception of desensitized and did not decrease with repeated glycine melatonin (Acros). Stock solution of melatonin was pre- applications. The effect of melatonin on the current pared in dimethyl sulfoxide (final concentration, 0.5%, induced by 50mM glycine is illustrated in Fig. 1. Pressure v / v). To obviate the possible effect of dimethyl sulfoxide application of 1 mM melatonin reversibly inhibited the on the glycine-induced current, all other drug solutions, glycine response by 5562.0% (n54) (Fig. 1A). To including glycine and external buffer (in the pressure quantitatively evaluate the potency and efficacy of pipet), also contained 0.5% dimethyl sulfoxide. In all melatonin for glycine receptors, pooled data were used to experiments, neurons received a 10-s prepulse of either construct the dose–response curve for inhibition of the 50

mM glycine response by melatonin. As shown in Fig. 1B, melatonin produced a dose-dependent inhibition of the glycine-induced current, and curve-fit analysis revealed an EC50 of 934 mM and a maximal inhibition of 100%. Inhibition of the glycine response by melatonin was evident over roughly the same concentration range as enhancement of the GABA response, although melatonin was somewhat less potent at the glycine receptor than at the GABA receptor (ECA 505766mM) [14]. In both cases, Hill slopes were close to one, consistent with a single class of non-interacting sites.

Fig. 1. Melatonin inhibits the whole-cell current induced by glycine. (A) Melatonin (Mel) (1 mM) inhibits the current induced by 50mM glycine. Horizontal bar above each trace, period of drug application. (B) Dose– response curve for inhibition of the glycine (50 mM) response by melatonin. Data points, percentage change in peak current in the presence of melatonin (mean of 4–6 experiments). Error bars, standard errors. Error bars are not indicated when smaller than the size of the circle.

Fig. 2. Antagonism of the glycine response by melatonin is noncompeti-Melatonin dose–response curve is fitted with the logistic equation [8]. (%

nH nH nH tive. Inhibition of the glycine-induced current by 1 mM melatonin is not

inhibition) /(% inhibition)max5[Mel] /([Mel] 1EC50) where [Mel] is

significantly reduced by increasing glycine from 100mM to 300mM or to the concentration of melatonin, and nH is the Hill coefficient. Curve-fit

1 mM. Error bars, standard errors. Number of cells is indicated in analysis reveals an EC50of 934mM, a maximal inhibition of 100%, and

210 F.-S. Wu et al. / Brain Research 881 (2000) 208 –211

Fig. 3. Inhibition of the glycine response by melatonin is not agonist-dependent. Melatonin (1 mM) inhibits rapidly and reversibly the current induced by 50mM glycine in the absence of melatonin prepulse.

To investigate the mechanism of melatonin action on the latter three compounds is competitive [7,11,13], indicating glycine receptor, we examined the effect of 1 mM that melatonin and these drugs do not act through a melatonin on currents induced by higher concentrations of common site. Like melatonin, antagonism of the glycine

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glycine. In this experiment, a low Cl (20 mM) pipet response by progesterone is noncompetitive [12]. Whether solution was used to reduce the amplitude of the glycine- melatonin acts through the progesterone modulatory site to induced current when the cell was held at 270 mV. As inhibit the glycine response needs further investigation. illustrated in Fig. 2, the percentage of inhibition by

melatonin of the glycine-induced current was not sig-nificantly attenuated by increasing glycine from 100 mM

Acknowledgements (5163.2%, n54) to 300mM (4666.1%, n54) or to 1 mM

(5466.9%, n55) (P.0.05, unpaired t-test). Because 1

Research support was provided by the National Health mM is a saturating concentration of glycine [13], this

Research Institutes (DOH88-HR-830) of Taiwan. suggests that antagonism of the glycine response by

melatonin is noncompetitive in nature and that melatonin inhibits the glycine response by acting through a site

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