Reduction by sevoflurane of adenosine 5′-triphosphate-activated inward current of locus coeruleus neurons in pontine slices of rats

Brain Research 921 (2001) 226–232 www.elsevier.com / locate / bres

Research report

Reduction by sevoflurane of adenosine 59-triphosphate-activated inward current of locus coeruleus neurons in pontine slices of rats q a,b a a, Eiji Masaki , Masahito Kawamura , Fusao Kato * a b

Department of Pharmacology, Jikei University School of Medicine, 3 -25 -8 Nishi-shimbashi, Minato-ku, Tokyo 105 -8461, Japan Department of Anesthesiology, Jikei University School of Medicine, 3 -25 -8 Nishi-shimbashi, Minato-ku, Tokyo 105 -8461, Japan Accepted 13 September 2001

Abstract Increasing evidence indicates that volatile general anesthetics exert their effects by affecting various types of membrane conductance expressed in the central nervous system (CNS), such as ligand-gated receptor-channels. The most recently identified family of the receptor-channels in the CNS are the extracellular ATP-gated channels (P2X purinoceptors). In the present study, we tested whether volatile anesthetics can affect P2X receptor function in the CNS network. We recorded whole-cell currents of locus coeruleus (LC) neurons in pontine slices from young rats. Adenosine 59-triphosphate (ATP) sodium (0.03–3 mM) evoked a rapidly rising and moderately desensitizing inward current (50–200 pA) in a dose-dependent manner in LC neurons at a holding potential of 280 mV. Perfusion with clinically relevant concentration of sevoflurane (0.1–0.5 mM) reduced the ATP-induced inward current in a dose-dependent manner (to 56.865.9% of control with 0.5 mM sevoflurane; mean6S.E.M., n513). Estimated IC 50 of sevoflurane was 0.59 mM. We conclude that the attenuation of extracellular ATP-mediated signaling in the central nervous system might be one of the multiple actions of volatile anesthetics.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Sevoflurane; Adenosine 59-triphosphate; P2X receptor; Locus coeruleus; Patch-clamp; Pontine slice

1. Introduction Evidence accumulated over the past 10 years suggests that the volatile general anesthetics exert their effects by affecting membrane conductance expressed in the central nervous system (CNS) including ligand-gated and voltagegated ion channels [5,8]. We have recently demonstrated that intracerebroventricular (ICV) administration of P2purinergic receptor antagonists reduces the minimum

q This work was presented in part at the annual meeting of the American Society of Anesthesiologists, Orlando, FL, October, 1999 and The 72th Meeting of the Japanese Pharmacological Society, March, 1999, Sapporo. *Corresponding author. Tel.: 181-3-3433-1111, ext. 2256; fax: 181-35473-1428. E-mail address: [email protected] (F. Kato).

alveolar concentration (MAC) of volatile anesthetics, suggesting that anesthetic effect of sevoflurane involves modulation of intrinsic purinoceptor signaling in the CNS [14]. To examine this possibility, we examined whether volatile anesthetics affect extracellular ATP-mediated signaling in native CNS neurons in the present study. Extracellular ATP-gated channels (P2X receptors) are expressed widely in many structures in the CNS and underlie various central functions such as pain signal transmission and autonomic regulation [2,10,17]. To test whether the P2X receptors could be a target of volatile anesthetics, effects of sevoflurane at clinically relevant concentrations on ATP-activated P2X receptor currents in native CNS neurons were examined. We used pontine slice preparations to measure membrane currents in neurons in the locus coeruleus (LC). LC cells were chosen because (i) the LC plays an important role in determining the overall activity of higher centers through ascending and widely

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03125-0

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projecting noradrenergic pathways [1] and is involved in the expression of anesthetic effects [15], (ii) the mRNAs of the P2X2, 4 and 6 subunits are expressed in the LC [4], and (iii) exogenously applied ATP excites most LC cells in brainstem slices via activation of P2 purinoceptors [6,7,21]. Here we show that clinically relevant dose of sevoflurane significantly reduces ATP-activated inward currents of LC neurons in the acutely prepared brainstem slice of the rat.

2. Materials and methods

2.1. Brain slice preparation The study protocol was approved by the Institutional Animal Use and Care Committee and conformed to the Guiding Principles in the Care and Use of Animals by the Japanese Physiological Society and the Japanese Pharmacological Society. Sprague–Dawley rats (1–3 weeks old) of either sex were anesthetized with ketamine (100–150 mg / kg, i.p.) and decapitated. Then two to three transverse slices of 350–450-mm thickness including the LC were made in ice-cold low-Ca 21 and high-Mg 21 artificial cerebrospinal fluid (aCSF) containing (in mM) NaCl 125, KCl 2.5, CaCl 2 0.1, MgCl 2 3, NaH 2 PO 4 1.25, D-glucose 12.5, L-ascorbic acid 0.4, NaHCO 3 25, saturated with 95% O 2 15% CO 2 (pH 7.4) with a vibrating slice cutter (DTK1000, Dosaka). The slices were incubated in aCSF with normal Ca 21 and Mg 21 concentration (in mM: CaCl 2 2 and MgCl 2 1.3) for 30–45 min at 378C then kept at room temperature until the recording. Patch electrodes were filled with the intracellular solution containing (in mM) potassium gluconate 120, NaCl 6, CaCl 2 5, MgCl 2 2, MgATP 2, NaGTP 0.3, EGTA 10, HEPES 10 (adjusted to pH 7.2 with KOH). The outer wall of the electrode tip was coated with Sigmacoat (Sigma). The tip resistance of the electrode was 4–6 MV. The slices were fixed in a recording chamber (|0.5 ml volume) and continuously perfused with aCSF at a flow rate of 2–3 ml / min. The neurons in the LC were visually identified under infra-red differential interference contrast videomicroscopy. The holding potential was kept at 280 mV in all recordings in order to isolate non-specific cationic currents activated by ATP and to minimize additional potassium currents [20,21]. The series resistance (8–20 MV) and electrode capacitance were compensated and checked before and after pharmacological manipulations. The membrane current was recorded with an AxoClamp 2B (Axon Instruments) or with a CEZ-2400 (Nihon-Kohden). All signals were recorded on digital audio tapes (RD-120TE, TEAC). After the experiments, the signals were filtered (500 kHz) and sampled at 1 kHz. The original traces in the figures and curve-fitting calculation were made with the Igor Pro graphic program (WaveMetrics).

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2.2. Drug application Adenosine 59-triphosphate disodium (ATP; Sigma), ATP 59-O-(3-thiotriphosphate) tetralithium (ATPgS; Sigma), a,b-methylene ATP (a,b-meATP; Sigma) and adenosine (Kohjin) were dissolved in aCSF and applied locally to the LC via a glass ‘puffing’ pipette (inner diameter 0.6 mm) placed |2–4 mm upstream of the tip of the recording electrode and 100–200 mm above the slice. The agonist solutions were applied by gravity to the slice with help of an electromagnetic valve. Tetrodotoxin citrate (TTX; RBI), pyridoxalphosphate-6-azophenyl-29,49-disulphonic acid (PPADS; RBI) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; Sigma) were dissolved in aCSF and applied via the perfusion line. As no apparent difference was observed in the ATP-induced currents and sevoflurane effects, the data with and without TTX were used equally in the present study. Sevoflurane (Maruishi) was vaporized (Sevotec 3; Ohmeda, West Yorkshire, UK) with carbogen and continuously bubbled into aCSF (resulting pH was 7.5). Sevoflurane-saturated aCSF was perfused for 10 min before measurements of ATP-induced current were commenced in order to avoid initial transient changes caused by sevoflurane so that the sevoflurane effect was stable during repeated ATP application. The actual aqueous concentration of sevoflurane in the recording chamber as analyzed by gas chromatography (GC-14B, Shimadzu) was 0.1260.02, 0.3360.03 and 0.4960.03 mM (mean6S.E.M.; n56), with the partial pressure of sevoflurane at 1, 3 and 5%, respectively. Based on this linear relation, the calibrated concentration in mM of sevoflurane in the recording chamber is shown in the present study.

2.3. Statistics Statistical comparisons were made by analysis of variance. Differences with probability (P) less than 0.05 were considered significant.

3. Results Recordings were made from 32 neurons in 32 slices from 28 rats. All LC neurons used in the present study had large (longitudinal diameter.25 mm) and multipolar soma and showed a strong afterhyperpolarization after an action potential, and an A-current like inactivating outward current upon depolarization. A total of 21 out of 29 LC cells showed spontaneous membrane current oscillation that was resistant to TTX. All these properties comply with previous studies of LC neurons [3,9,23,27]. Puff application of ATP for 30 s to LC neurons induced a rapidly rising inward current (IATP ) in 28 of 29 LC cells tested (Fig. 1). This current started to decay during application but was still present at the end of application,

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Fig. 1. Reduction of ATP-induced inward current by sevoflurane in LC neurons. (A) Effect of sevoflurane (0.5 mM) on the IATP of an LC neuron induced by ATP (30 mM–3 mM). Horizontal bars indicate the period of ATP application. (B) Above, effects of sevoflurane (0.5 mM) on inward currents evoked by ATP (100 mM) and ATPgS (100 mM); bottom, effects of adenosine (100 mM; left) in an LC neuron and effects of PPADS (50 mM) on the inward current activated by 100 mM ATP in another neuron (right). (C) Effect of sevoflurane (0.5 mM) on the concentration–response relation of IATP of LC neurons. Abscissa, concentration of puff-applied ATP. Ordinate, relative IATP normalized by the peak current induced by 1 mM ATP in each neuron. Vertical bars indicate standard error of the mean. Values are means of four to five neurons. The standard error of the mean estimated for the washout is not shown because of small sample size (n52–3). *P,0.05 compared with control. (D) Lineweaver-Burk plot of the relative IATP amplitude in the absence (open circles) and presence (filled circles) of sevoflurane (0.5 mM).

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indicating moderate desensitization. The amplitude of IATP was dose-dependent (Fig. 1A, top traces). Perfusion of sevoflurane-equilibrated aCSF reduced IATP (Fig. 1, middle traces). The larger IATP induced by higher doses of ATP was more sensitive to sevoflurane (Fig. 1A,C). Almost complete recovery of IATP was attained after |30-min perfusion with sevoflurane-free aCSF (Fig. 1A (bottom),C). An inward current highly similar to that evoked by ATP was also activated by ATPgS (Fig. 1B), suggesting that the inward current evoked by ATP does not involve activation of adenosine receptors by adenosine extracellularly produced from ATP [10]. Furthermore, application of adenosine did not activate any visible current or only activated a weak outward current (,10 pA; Fig. 1B) in three of three neurons tested at a holding potential of 280 mV. The inward current activated by ATPgS (100 mM) was also significantly reduced by sevoflurane (Fig. 1B; to 70.569.7% of the control; n53). The reduction of IATP was unchanged in the presence of DPCPX (1 mM; n52), further supporting that involvement of adenosine receptors in IATP is unlikely (data not shown). a,b-meATP did not activate any current in all three neurons that presented ATP-activated inward current (data not shown). The ATP-activated inward current sensitive to sevoflurane was almost completely blocked by 50 mM of PPADS (Fig. 1B), which is consistent with the previous report [6]. The Lineweaver-Burk plot of the ATP concentration and IATP with and without sevoflurane indicates that the antagonism by sevoflurane is non-competitive (Fig. 1D). Two lines, estimated with the least-squares method, crossed each other in the third quadrant of the Lineweaver-Burk plot (Fig. 1D), suggesting that sevoflurane affects P2X receptor activity more prominently when the ATP is bound to the receptor protein [26], which is consistent with the dependency of sevoflurane effect on the ATP concentration (see above). The reduction of IATP was dependent on the dose of sevoflurane (Fig. 2A,B). Due to the limits of the vaporizer used in this study which was specially made for sevoflurane in clinical practice, we could not test the concentration above 5% (0.5 mM in the extracellular solution). However preliminary estimation of IC 50 of sevoflurane based on the concentration–inhibition curve shown in Fig. 2B indicated that IC 50 should be higher than 0.5 mM. Therefore we made the Dixon plot analysis [26] to estimate the IC 50 (Fig. 2C). The IC 50 of sevoflurane on IATP was estimated to be 0.59 mM (Fig. 2C). To check whether this reduction of IATP by sevoflurane was due to changes in membrane properties, effects of sevoflurane on the electrical parameters of the membrane were analyzed (Fig. 3A). In all 15 cells to which sevoflurane (0.5 mM) was applied, a slow and sustained inward current of 40–80 pA was observed during the perfusion (Fig. 3A). This inward current is likely to result from the sevoflurane-activated transient and persistent chloride conductance [28] (estimated ECl is 250.3 mV in the present

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study). Despite the |50% reduction in IATP during perfusion with 0.5 mM sevoflurane (Fig. 3B, right), the membrane input resistance and capacitance were only slightly modified by sevoflurane (Fig. 3B, left), suggesting that the change in amplitude of IATP was not fundamentally due to changes in membrane resistance and capacitance.

4. Discussion This study is the first direct demonstration indicating that a volatile anesthetic, sevoflurane, can affect the current induced by extracellular ATP in CNS neurons. The inhibition of IATP to |57% of control at 0.5 mM sevoflurane in the present study is comparable to the inhibitory effect of enflurane on the glutamate-induced current in nucleus tractus solitarii neurons [25], that of halothane on the current induced by N-methyl-D-aspartate (NMDA) on CA1 pyramidal neurons in the hippocampus [12] and that of ethanol on the ATP-induced current in dissociated dorsal root ganglion cells [13]. We have reported that ICV administration of P2 receptor antagonists, PPADS and suramin, significantly reduces the MAC of sevoflurane and isoflurane in the rat [14]. These results suggest that intrinsic ATP-signaling system participates in the conduction of noxious stimulus and / or the maintenance of wakefulness, an inhibition of which with specific antagonists may help anesthetic drugs to exert their anesthetic and analgesic effects at significantly lower doses. The results of the present study indicate that the ATP-activated current in the CNS neurons is indeed reduced by sevoflurane at clinically relevant concentrations. The MAC of sevoflurane in Sprague–Dawley rats is 2.4% [19]. Park et al. [18] estimated that 2.3% sevoflurane at 378C corresponds to 0.35 mM sevoflurane in bicarbonate-buffered modified Tyrode’s solution at room temperature (228C). In the present study, a significant reduction of IATP was readily observed with 0.5 mM sevoflurane and the estimated IC 50 was 0.59 mM. This effective concentration range of sevoflurane substantially overlaps that for sevoflurane-induced currents in CA1 pyramidal neurons of the rat [28]. Considering that concentration of sevoflurane 1.5–2.5 times larger than MAC is used in clinical practice to ensure sufficient depth of anesthesia, we conclude that sevoflurane significantly attenuates the IATP of LC neurons at clinically relevant anesthetic doses. The functional role of ATP-mediated signaling in the LC remains undetermined [6,21]. It is well known that ascending noradrenergic system arising from LC controls the activities of the upper structure networks such as thalamus, hippocampus and cortex [1], resulting in regulation of the conscious levels. As suppression of the LC noradrenergic system is involved in the expression of anesthetic effects [15], it is possible that a decrease by sevoflurane of excitatory influences of ATP co-released from axon termi-

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Fig. 2. Dose-dependent inhibition of ATP-induced currents by sevoflurane. (A) Effect of increasing sevoflurane concentration on IATP of an LC neuron. ATP (3 mM) was applied at the short horizontal bar above each trace. The shift in the holding current was compensated so that the pre-ATP current traces are on the same level (broken line). (B) Concentration–response curve of sevoflurane on the reduction of IATP evoked by 0.3 or 1 mM ATP. Abscissa, concentration of sevoflurane; ordinate, percent reduction of peak IATP during sevoflurane perfusion. Values are mean and the standard error of five to six neurons for 0.1–0.4 mM sevoflurane and 13 neurons for 0.5 mM sevoflurane. *P,0.05 compared either with control or with the effect of 0.1 mM sevoflurane. The curve indicates the estimated Hill plot. (C) The Dixon plot of the effect of sevoflurane on IATP . Based on the same data as in Fig. 2B. The line, estimated with least-squares method, crossed the abscissa at 20.59 mM [26].

nals projecting to LC neurons [1,10,21] may help to lower the level of consciousness of animal together with other anesthetic effects of sevoflurane. Pharmacological identification indicates that the LC neurons express three distinct types of P2 purinoceptors [6,24]. LC expresses P2X2, P2X4 and P2X6 mRNAs and is rich in P2X2 subunit protein [4,16]. We assume that the ATP-induced inward current shown to be sensitive to sevoflurane in the present study is likely to be mediated by

P2X receptors in the LC neurons because (i) its rise time was rapid, (ii) P2Y receptor- and / or adenosine receptormediated potassium currents [6] were minimized at the holding potential near the equilibrium potential of potassium ions [21], and (iii) it was almost completely blocked by pre-perfusion of 50 mM PPADS, a relatively selective P2X receptor antagonist [10] known to block P2X receptors in the LC [6]. Tomioka et al. [22] reported that an intravenous anes-

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Fig. 3. Effect of sevoflurane on the transmembrane current of an LC neuron. (A) Effect of sevoflurane on spontaneous membrane current of an LC neuron. (B) Left, membrane current responses to various potentials from holding potential of 280 mV before (bold lines) and after (narrow lines) sevoflurane perfusion; right, responses to ATP (1 mM) of the same neuron as in the left before and after sevoflurane perfusion (0.5 mM). Note that the scales for the membrane current amplitude in the left and right traces are identical.

thetic, 2,6-diisopropylphenol (propofol), does not reduce, but rather enhances the ATP- and a,b-meATP-activated currents in the human embryonic kidney cell lines expressing recombinant P2X4 receptors [22] at clinically relevant concentrations. They described that propofol had little effect on ATP- or a,b-meATP-activated currents in cells expressing P2X2 and P2X213 receptors. The present results that (i) IATP did not show rapid desensitization, (ii) ATPgS, but not a,b-meATP, induced a very similar inward current to IATP , and (iii) complete blockade by PPADS, indicate that the ATP-activated current suppressed by sevoflurane is mainly caused by P2X2 receptor activation in LC neurons [17]. Though it still remains to be tested whether propofol reduces ATP-activated current in the native LC cells, it will be an interesting issue to clarify whether reduction of native P2X receptor current is a common feature or not of general anesthesia as P2X2 subunits are abundant in the central nervous system [4,16,17]. The mechanism of the inhibitory effect of sevoflurane on the P2X receptor cannot be determined at this stage. Interestingly, the reduction was more manifest with the

IATP induced by larger doses of ATP, which is also the case with the effect of halothane on the current induced by a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) in hippocampal CA1 pyramidal neurons [12]. This means that sevoflurane affects the P2X receptors more prominently when there are more P2X receptors in active state in agreement with the result of the LineweaverBurk analysis (Fig. 1D). It is possible that (i) the interaction of sevoflurane with P2X receptor is non-competitive in nature, (ii) P2X receptors expressed in an LC neuron are not homogeneous probably due to expression of diverse subunit compositions [4] resulting in distinct sensitivity to ATP and sevoflurane, and / or (iii) sevoflurane also affected systems other than the P2X receptors, such as a series of ecto-enzymes that hydrolyze ATP and convert to adenosine [11,21] expressed in the LC. The molecular target of sevoflurane in the LC purinergic signaling systems should be identified in future studies. As a conclusion, these findings provide a potential new mechanism operating during clinical anesthesia with general anesthetics, i.e. suppression of ATP-mediated signaling, in addition to those that have been proposed [5].

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Acknowledgements This reseach was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan to F.K. (No. 13680902) and to E.M. (No. 11671525) and Grants for the Research on Health Sciences focusing on Drug Innovation from The Japan Health Sciences Foundation (KH21014), and a Research Grant from the Takeda Science Foundation to F.K. The encouragement of Professors M. Kawamura and S. Kurihara is acknowledged. The technical assistance given by Takako Matsuo was invaluable.

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