Prostaglandin E2 stimulates glutamate release from synaptosomes of rat spinal cord

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Neuroscience Letters 196 (1995) 57-60

NfUROSCIHC[ LETTERS

Prostaglandin E 2 stimulates glutamate release from synaptosomes of rat spinal cord Isao N i s h i h a r a a,h, T o s h i a k i M i n a m i a,b, Y a s u y o s h i W a t a n a b e c, Seiji Ito b,d,*, O s a m u H a y a i s h i b aDepartment of Anesthesiology, Osaka Medical College, Takatsuki 569, Japan bDepartment of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita 565, Japan CDepartment of Neuroscience, Osaka Bioscience Institute, Suita 565, Japan dDepartment of Medical Chemistry, Kansai Medical University, 10-15 Fumizono, Moriguchi 570, Japan Received 3 July 1995; accepted 11 July 1995

Abstract

We recently reported that intrathecal administration of prostaglandin E2 induced hyperalgesic and allodynic effects through the glutamate receptor system. Here we examined whether prostaglandin E2 could evoke amino acid release from nerve terminals using rat spinal cord synaptosomes Exposure in superfusion to prostaglandin Ez significantly increased endogenous glutamate and aspartate release and dose dependencies showed bell-shaped patterns with a peak at 1 nM. Both releases were almost absolutely Ca2+-dependent. These results demonstrate that prostaglandin E2 may stimulate the release of excitatory amino acids presynapticaUyin the spinal cord.

Keywords: Prostaglandin E2; Spinal cord; Synaptosomes; Superfusion; Glutamate; Presynaptic regulation

Prostaglandins (PGs)have diverse biological effects on a variety of physiological and pharmacological activities in the central and peripheral nervous systems [23]. Recent evidence indicates that PGs are critical for the processing of pain information not only by sensitizing the peripheral terminals of primary afferent nociceptors but also by acting in the central nervous system, especially at the spinal level. For example, exposure of noxious stimuli increases the level of PGs detected in the spinal cord [3,21], and intrathecal (i.t.) administration of PGs increases activity in the spinal cord neurons [2]. In addition, i.t. administration of non-steroidal anti-inflammatory agents relieves hyperalgesia [11,12,26]. Finally, i.t. administration of PGs produces hyperalgesia [16,27,28] and allodynia [16,17] in response to noxious and innocuous stimuli, respectively. Recently we have demonstrated that i.t. administration of NMDA receptor antagonists blocked the hyperalgesic and allodynic effects by i.t. PGE2 and suggested that PGE2 may produce hyperalgesia and allodynia mediated by the glutamate (Glu) receptor system [18,20]. * Corresponding author, Tel.: +81 6 9921001, ext. 2450; Fax: +81 6 9921781.

It is well established that release of neurotransmitters is controlled by presynaptic autoreceptors [9,24]. In addition, the heteroreceptors have been suggested to be located on the presynaptic terminals, which are acted upon by the other transmitters released from adjacent nerve terminals [15]. Experiments using synaptosomal preparations revealed the existence of heteroreceptors that regulate the release of excitatory amino acids in various brain regions [6-8,14]. Therefore, perfusion of synaptosomes is a suitable procedure for investigating the mechanism of regulation of neurotransmitter release, and especially for examining the location of modulating receptors. In the present study, in order to evaluate the above-mentioned hypothesis that PGE2 produces hyperalgesia and allodynia mediated through the Glu receptor system, we examined whether PGE2 could evoke amino acid release from synaptosomes prepared from the rat spinal cord. Preparation of crude synaptosomes and experiments on amino acid release were carried out according to the method of Kamisaki et al. [7,8]. Briefly, adult male Wistar rats weighing 170-200g obtained from SLC Inc. (Shizuoka, Japan) were killed by decapitation and the spinal cord was removed quickly. After careful removal of pia-arachnoid membranes, the thoracolumbar region

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was homogenized in 20 vols. of 0.32 M sucrose buffered with 20 mM Tris-HCl (pH 7.5 at 4°C) in a glass homogenizer by six strokes. The homogenate was centrifuged at 1000 x g for 5 min. Synaptosomes were isolated from the supernatant by centrifugation at 12000 x g for 20 min. The pellet was resuspended in a Krebs-Ringer buffer (KRB; 135mM NaCI, 4 . 8 m M KC1, 2 m M CaC12, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 12.5 mM N-(2-hydroxyethyl)-piperazine-N'-2ethanesulfonic acid, 10 mM glucose, pH 7.4 at 37°C) previously equilibrated with O2/CO 2 (95:5). Synaptosomes (approximately 0.8 mg of protein) were layered on Whatman GF/C filters and superfusion was conducted at a flow rate of 0.5 ml/min with a standard medium of KRB aerated continuously with O2/CO2 (95:5) at 37°C. After a 30-min equilibration period, fractions were collected at 5-min intervals into an ice-cold plastic tube and kept frozen at -80°C until assayed for amino acid concentrations. Forty minutes after the start of superfusion, the synaptosomes were stimulated with various doses of PGE 2 or 30 mM KC1 for 2 min. Basal release was defined as the average content of amino acids in 5-min fractions just before the stimulation (35-40 min after the start of superfusion) and is taken as 100%. Secretagogue-evoked release was defined as the release in the 40-45-rain fraction just after stimulation. In Ca2÷-free experiments, superfusion was started with a Ca2+-free KRB to which 0.5 mM ethyleneglycol-bis(fl-aminoethyl ether)-N,N'tetraacetic acid (EGTA) was added. The contents of Glu and aspartate (Asp) in 5-min fractions of superfusates were measured by HPLC with a fluorescence detector following o-phthaldialdehyde reagent derivatization [ 13]. PGE 2 was a generous gift from Ono Central Research Institute (Osaka, Japan). For superfusion, an aliquot of the desired PGE 2 solution was put into a borosilicate tube and the ethanol was removed by evaporation to dryness under nitrogen gas. Other reagents were of HPLC grade or of the highest purity, and were used without further purification. The protein content was determined by the method of Lowry et al. [10], using bovine serum albumin as a standard. Statistical analysis was carried out using Student's t-test or Duncan's test. Differences were considered to be significant when P < 0.05. Fig. 1 illustrates the patterns of endogenous Glu and Asp release from rat spinal cord synaptosomes in superfusion to 1 nM PGE2. After 35-min superfusion the releases of Glu and Asp were almost constant, but still gradually decreased (Fig. 1A). The basal releases for Glu and Asp in 35-40-min fractions were 68.3 _+2.7 and 72.4 __.5.3 pmol/min per mg protein (mean _+ SEM, n = 26), respectively. The outputs of Glu and Asp in the 4 0 45-min fraction were 92.9 _+ 1.3% and 93.7 -+ 1.0% of the basal release, respectively. When the synaptosomal preparations were exposed to 1 nM PGE2 at 40 min for 2 min, significant increases in Glu and Asp release were observed in 40-45-min fractions, being 117.7 +_8.7% and

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Time (min) Fig. 1. Effects of PGE 2 on releases of endogenous Glu and Asp from rat spinal cord synaptosomes and their Ca 2+ dependency. Crude synaptosomes were superfused in the standard KRB medium (A and B) or a Ca2+-free medium (C) as described in the text. After 40-min perfusion, the synaptosomal preparations were exposed to the same buffer (A) or 1 nM PGE 2 for 2 min (B,C). The outputs of Glu (hatched column) and Asp (open column) are expressed as % of the basal release. Basal releases for Glu and Asp were 68.3 ± 2.7 and 72.4 ± 5.3 pmol/minper mg protein (mean _+SEM, n = 26), respectively. -kp < 0.05, compared with the basal release.

113.2 +_6.6% of the basal release, respectively (Fig. 1B). The KCl-evoked release of Glu and Asp were 162.2 _+ 8.1% and 128.0 _+6.4% of the basal release, respectively (data not shown). Both PGE2- and KCl-evoked outputs of Glu and Asp were transient and back to the basal levels in the following fractions.

L Nishihara et al. / Neuroscience Letters 196 (1995) 57-60

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Fig. 2. Dose dependencies of PGE2 for Glu and Asp release from synaptosornes. Amino acid release by PGE2 was examined as described in the text. The release of Glu (0) or Asp (O) evoked by PGE2 is expressed as % of the control value in the 40-45-min fraction without exposure to PGE2. "k0.01< P < 0.05, .k,kp < 0.01, compared with the control. Next we examined the effect of extracellular Ca 2÷ on the PGE2-evoked release of Glu and Asp. When Ca 2÷ was omitted and the Ca 2÷ chelator EGTA was added in the superfusion system, basal releases were not affected, but the PGE2-evoked outputs of Glu and Asp were negated completely (Fig. 1C). "[hese results demonstrate that the PGE2-induced amino acid release is dependent on Ca 2+ in the medium. Fig, 2 shows dose dependencies of PGE2 for amino acid release from rat spinal cord synaptosomes in 4045-min fractions. Releases of Glu and Asp were significantly increased at the concentrations of 0.1-10 nM and 1-10 nM of PGE2, respectively, as compared with the control and both showed bell-shaped patterns with a peak at 1 nM. Previous studies on pain transmission suggested mutual interactions among neurotransmitter and PG receptor systems. The extracellular level of PGs in spinal perfusates increased following increasing neuronal activity elicited by high-threshold afferent stimulation [21], high potassium [31], and noxious thermal stimulation [3]. Recently it has been reported that non-steroidal antiinflammatory drugs administered intrathecally blocked the excessive sensitivity to pain induced by the activation of spinal Glu and subs~Iance P receptors [11] or by the subcutaneous injection of formalin [12]. These observations suggested that tl:~e effects of non-steroidal antiinflammatory drugs are likely mediated by inhibition of PG synthesis in the spinal cord. Direct evidence was provided by the demonstration that i.t. injection of PGD 2 and PGE2 induced hyperalgesic effects [26-28]. Conversely, the PGD 2- and PGE2-induced hyperalgesia were blocked by the substance P receptor antagonist [28] and NMDA

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receptor antagonists [20], respectively. Furthermore we recently demonstrated that i.t. PGE 2 and PGF2~ induced allodynia in response to innocuous stimuli, which were also blocked by Glu receptor antagonists [16-18]. It was reported that PGE2 could enhance the release of the sensory transmitter substance P from isolated neurons of the avian dorsal ganglion, suggesting that the capacity of PGE 2 to facilitate the Ca 2÷ current may be one mechanism to account for the sensitization of sensory neurons by PGE 2 [19]. In the present study, we first demonstrate that PGE2 increased the release of excitatory amino acids from rat spinal cord synaptosomes (Fig. 1B). Dose dependencies of PGE 2 showed bell-shaped patterns for both Glu and Asp release with a peak at 1 nM (Fig. 2). We previously reported that PGE2 produced bell-shaped dose dependency for hyperalgesia at the dosage of 1 pg to I0 ng/ 5/zl per mouse [16,20,28]. When simply calculated without considering dilution factors after i.t. injection, 10 pg/ 5/zl corresponds to 5.68 nM. Because Kd values of cloned mouse PGE receptor subtypes are between 2.9 and 21 nM, the 1-10 nM concentration employed here (Fig. 2) might be sufficient to allow the PG to bind to and activate PGE receptors. The present study supports the possibility that PGE2 may induce hyperalgesia and/or allodynia by increasing Glu release. The excitatory amino acids such as Glu and Asp are found in high concentrations in the dorsal horn of the spinal cord [1,29] and known to be neurotransmitters of primary afferents and descending projections from the brain and may also be one of some intrinsic spinal neurons [4,5,22,30]. The PGE receptor subtype EP 3 mRNA is highly expressed in dorsal root ganglion [25] and binding sites of PGE 2 are dense in the substantia gelatinosa of the dorsal horn. Thus the use of neurotransmitter release studies, in conjugation with in situ hybridization and the receptor binding technique, provides a powerful tool for the characterization of PGE receptor subtypes with effects at the spinal cord. This work was supported in part by grants-in-aid for Scientific Research on Priority Areas, Scientific Research (B) (06454171) and for Encouragement of Young Scientists (06671243) from the Ministry of Education, Science, and Culture of Japan and by grants from the Japan Medical Association, Ono Medical Research Foundation, and Takeda Scientific Foundation. [1] Besson,J.-M. and Chaouch, A., Peripheral and spinal mechanisms of noeiception, Physiol. Rev., 67 (1987)67-186. [2] Coceani,F. and Viti, A., Response of spinal neurons to iontophoretically applied prostaglandin El, Can. J. Physiol. Pharmacol.,53 (1975) 273-284. [31 Coderre,T.J., Gonzales, R., Goldyne, M.E., West, J. and Levine, J.D., Noxious stimulus-induced increase in spinal prostaglandin E2 is noradrenergic terminal-dependent, Neurosci. Lett., 115 (1990) 253-258. [4] De Biasi, S. and Rustioni, A., Glutamate and substance P coexist

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