Accepted Article

Received Date : 24-Nov-2013 Revised Date

: 16-Jan-2014

Accepted Date : 09-Feb-2014 Article type

: Original Article

Inhibition of Acid Sensing Ion Channel Currents by Propofol in Rat Dorsal Root Ganglion Neurons

Zhen Leia, Xiaoyu Lic, Guizhi Wangb, Jianchun Feia, Tao Menga, Xinyu Zhanga, Jingya Yud, Jingui Yua, Jingxin Lic. a. Department of Anesthesiology, Qilu Hospital, Shandong University,107#, Wenhua Xi Road, Jinan, 250012 P.R. China b. Department of Physiology, Shandong Univerisity School of Medicine, 44#,Wenhua Xi Road,Jinan,Shandong, 250012 P.R. China

c. Department of Anesthesiology, Weifang Medical University, Weifang, 261053, China

d. Institute of Pathogenic biology, Shandong Univerisity School of Medicine, 44#,Wenhua Xi Road,Jinan,Shandong, 250012 P.R. China

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12215 This article is protected by copyright. All rights reserved.

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Corresponding Author: 1. Jingui Yu, M.D.,Ph.D. Department of Anesthesiology Mailing

address:

Department

of

Anesthesiology,

Qilu

Hospital,

Shandong

University,107#,Wenhua Xi Road, Jinan, 250012 P.R. China Phone : 86-53182166487 Fax :

86-531-86073074

Email : [email protected] 2. Jingxin Li, M.D.,Ph.D. Institute of Physiology Mailing address: Institute of Physiology, Shandong Univerisity School of Medicine, 44#,Wenhua Xi Road,Jinan,Shandong, 250012 P.R. China Phone : 0086-18663765076

Fax : 86-531-88382502

Email : [email protected] Short title: Propofol inhibits ASICs

The authors declare that they have no conflict of interest concerning this article.

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Abstract

Acid-sensing ion channels (ASICs), part of the epithelial sodium channel/degenerin (ENaC/DEG) family, are activated by extracellular protons. ASICs play a significant role in the acidosis-mediated perception of pain. The anesthetic agent, propofol, also exerts anti-nociceptive effects, but the underlying mechanisms for this effect are not clear. We used whole-cell patch clamping to investigate the effect of propofol on proton-gated currents in (i) rat dorsal root ganglion (DRG) neurons and (ii) human embryonic kidney (HEK) 293 cells transfected with either ASIC1a or ASIC3. Propofol inhibited the amplitude of proton-gated currents in DRG neurons, but did not change the sensitivity of ASICs to H+. Notably, propofol altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. In addition, we demonstrated that propofol inhibited ASICs by directly binding with these channels in HEK293 cells. These results suggested that propofol inhibits proton-gated currents in DRG neurons, and inhibition of proton-gated currents partly explains the anti-nociceptive effects of propofol in primary afferent neurons.

Keywords: Propofol; AISCs; Pain; Nociperception; DRG

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Introduction Acid-sensing ion channels (ASICs) belong to the epithelial sodium channel/degenerin (ENaC/DEG) family and are activated by extracellular protons [1]. They are widely distributed within both the central and peripheral nervous systems [2]. ASIC activation by protons induces sodium and/or calcium influx, giving rise to depolarization and evoking action potentials in neurons [3]. This process may be related to a local decrease in pH in pathologies associated with ischemia, inflammation, tissue injury, and cancer development [4,5]. In dorsal root ganglion (DRG) cells, ASICs play a significant role in the acidosis-mediated perception of pain [6]. Propofol (2, 6-diisopropylphenol) is one of the most widely used intravenous anesthetics for the induction and maintenance of general anesthesia. In addition to its anesthetic properties, several studies have shown that propofol has anti-nociceptive effects, including against visceral pain evoked by acetic acid, as well as the capacity to reduce inflammatory pain and depress nociceptive transmission at the spinal level [7-13]. However, the molecular mechanisms responsible for these anti-nociceptive effects of propofol remain unclear. As propofol is involved in various physiological processes that interact with ASICs, we hypothesized that ASICs might play a role in the analgesic properties of propofol.

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Results Effects of propofol on acid-evoked currents in rat DRG neurons An acid-evoked inward current was observed in the majority of DRG neurons (87.3%, 96/110). Three main current types were observed: 1) a rapidly inactivating current with a fast transient phase (40.6%, 39/96; Fig. 1A); 2) a slow-inactivating transient current followed by a small sustained component (38.5%, 37/96; Fig. 1 B); and, 3) a non-inactivating current with a small transient phase (20.8%, 20/96; Fig. 1C). Amiloride, a broad-spectrum proton-gated channel blocker, completely blocked all three types of transient currents (Fig. 1A, B and C), and treatment with propofol (30 μM) produced a significant reduction in the peak amplitude of acid-evoked currents (pH 6.0) in DRG neurons (17/25; Fig. 1). The peak amplitude of the fast-inactivating currents was reduced by 15.4% ± 2.3% (n = 6, P < 0.01); the low-inactivating currents reduced by 13.3% ± 2.3% (n = 6, P < 0.01); and the non-inactivating currents were reduced by 18.1 % ± 2.4% (n = 5, P < 0.01; Fig. 1 bar graphs in right panel). Under the current-clamp conditions, propofol decreased the number of action potentials induced by acid stimuli in rat DRG neurons (Fig. 1D). These effects of propofol were readily reversible upon washout. Together, these results indicate that propofol inhibited acid-evoked currents and excitability of rat DRG neurons.

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Dose-response relationship of propofol on acid-evoked currents in rat DRG neurons The peak amplitude of the currents were reduced in response to to 3, 30, and 300 μM of propofol (Fig. 2A), indicating that the propofol-induced inhibition was dose-dependent (Fig. 2B). Each dose of propofol was examined in 5-10 cells. The pH-dependent activation of ASIC currents in response to propofol in rat DRG neurons We next investigated whether propofol affects the sensitivity of ASICs to H+. The result showed no apparent shift in the H+ dose-response curve (Fig. 3A and B), and the half-maximum activation pH value (pH50) value was approximately 6 (n = 6).

The effects of propofol on acid-evoked currents in ASIC1a- and ASIC3-transfected HEK293 cells Since DRG neurons predominantly express ASIC1a and ASIC3 subunits [21], we investigated the effects of propofol (30 μM) on ASIC1a- and ASIC3-transfected HEK293 cells. As shown in Figure 4, propofol significantly inhibited both ASIC1a and ASIC3 currents (n = 5, P < 0.01; Fig. 4, A and B), indicating that propofol directly inhibits ASIC currents.

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Discussion The main findings of our study are that (1) propofol inhibits the peak current amplitude of proton-gated currents in primary sensory neurons and decreases the number of action potentials caused by acid stimulation; (2) propofol does not affect the sensitivity of ASICs to H+ in DRG neurons; and (3) propofol inhibits the acid-evoked currents in HEK293 cells transfected with either ASIC1a or ASIC3. ASICs are trimers found in a homomeric or heteromeric arrangement with seven subunits (1a, 1b1, 1b2, 2a, 2b, 3, and 4) [2]. Almost all ASIC subunits are expressed in primary sensory neurons of vagal, trigeminal, and dorsal root ganglia [6,14], and are activated by extracellular protons. In our studies, a pH 6.0 acid solution evoked three main types of current in DRG neurons: 1) a slow-inactivating transient current; 2) a rapidly-inactivating current; and 3) a sustained inward current. These findings are consistent with that reported in previous studies [15-17].

These proton-gated inward currents mainly belong to ASICs, and indeed the transient slow- and fast-inactivating currents observed in DRG neurons were largely blocked by amiloride, a broad-spectrum ASICs channel blocker, confirming these to be ASIC currents. The sustained currents were mostly associated with ASICs, however, TRPV1 may have also partially contributed to these currents [15-18]. In the present study, propofol mainly affected proton-gated transient currents and markedly inhibited the acid-evoked current in rat DRG neurons. Tissue acidosis is an This article is protected by copyright. All rights reserved.

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important feature found in conditions associated with ischemia, inflammation, tissue injury, infection, and cancer development [4,5]. In such conditions, extracellular pH may decrease to as low as 5.4 in severe inflammation [19] and 6.8 in tumors [20]. It is well known that tissue acidosis can induce pain and a local reduction in pH is relevant to non-adapting pain in human volunteers [21]. A reduction in pH could activate ASICs to induce sodium and calcium influx, giving rise to membrane depolarization and neuronal excitation [3]. In this way, ASICs may play a significant role in the perception of acidosis-mediated pain. Importantly, this study showed that the action potentials triggered by a pH 6.0-induced membrane depolarization were significantly reduced by propofol, suggesting that propofol has an analgesic effect by inhibiting proton-gated ion channels. Paradoxically, 24~90% of all patients receiving propofol experienced pain at the site of injection [22], possibly as a result of directly activating TRPV1 and TRPA1 [23] or via bradykinin produced by the plasma kallikrein-kinin system initially activated by the lipid solvent for propofol [24]. However, several studies have shown that propofol has analgesic properties, such as reducing post-operative pain [25,26], anti-nociceptive and/or anti-hyperalgesic effects [27-29], and decreasing inflammatory pain [13]. Thus, the propofol-induced inhibition of ASICs as observed in this study could potentially mediate these anti-nociceptive properties. Propofol is well known to modulate GABAA receptor-mediated responses [30,31], and a recent study showed that the GABAA receptors modified ASICs [32]. In our study, the inhibitory effects of propofol on ASIC currents also existed in the This article is protected by copyright. All rights reserved.

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presence of picrotoxin, a GABAA receptor inhibitor (data not shown), suggesting that the GABAA receptors were not involved in the propofol-induced inhibitory effects on ASICs currents. To further investigate the underlying mechanisms of propofol-induced inhibition, we tested the effects of propofol in HEK 293 cells transfected with either ASIC1a or ASIC3, and found that it directly inhibited ASICs currents in the transfected HEK 293 cells. This result suggested that propofol directly inhibits channel activity by binding to the ASICs, but to the best of our knowledge no propofol binding site has been identified. Apart from acidosis-inducing pain [6], several studies have implicated ASICs in other physiological and pathological processes, including cone phototransduction and adaptation, learning/memory, and synaptic plasticity [33], suggesting the potential for additional, wide-ranging effects of propofol. Taken together, the present results suggested that propofol inhibits proton-gated currents in DRG neurons, and this effect could partly explain the anti-nociceptive effects of propofol in primary afferent neurons.

Methods Experimental animals Adult male Wistar rats (200–250g) were used. All experimental procedures were approved by the Medical Ethics Committee for Experimental Animals, Shandong University School of Medicine (number ECAESDUSM 2012029).

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DRG Cell Isolation and Culture Rats (n=7) were euthanized by cervical dislocation and the entire spinal columns were removed. Bilateral DRGs were collected and washed twice with L-15 medium (Gibco, Gaithersburg, MD), and then incubated in 10 ml L-15 medium containing 10 mg collagenase type 1 (Sigma, St. Louis, MO) and 0.25 ml 0.25%Trypsin (HyClone, Thermo scientific, USA) at 37°C for 50 min. DRGs were removed from the enzyme solution, centrifuged for 5 min at 1,000 revolutions/min, washed twice with L-15 medium, and transferred to 2 ml L-15 medium containing 10% FBS. The ganglia were triturated with a suction pipe for 3 min, and then centrifuged for 50 seconds at 1,000 revolutions/min. Supernatants were placed into 35-mm diameter Petri dishes and the cells were cultured at 37°C in a 5% CO2 incubator (Thermo Forma, Hamilton, NJ, USA). We used freshly isolated rat DRG neurons in the range of 15-30 μm diameter to test the effect of propofol on acid-evoked currents.

Transfection of HEK293 cells HEK 293 cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. Cells were transfected with pcDNA3.0 constructs encoding ASIC1a or ASIC3 and green fluorescent protein (GFP) using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. All recordings were made 24 to 48 h after transfection in GFP-positive cells. This article is protected by copyright. All rights reserved.

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Electrophysiological Recordings Whole-cell voltage-clamp and current-clamp recordings were performed at room temperature (22–25°C) using a computer amplifier (Multiclamp 700B; Axon, New York, NY, USA) and a Digidata (1440A; Axon). Patch pipettes were filled with intracellular solution comprising: 140 mM KCl, 2.5 mM MgCl2, 10 mM HEPES, 11 mM EGTA and 5 mM Na2ATP, with pH adjusted to 7.2 using KOH. Cells were bathed in extracellular saline made up of: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2, 10 mM HEPES and 10 mM D-glucose, with pH adjusted to 7.4 using NaOH. The resistance of the recording pipettes was in the range of 5-8 MΩ. The series resistance was compensated for 70–80% after establishing a whole-cell configuration. The membrane potential was held at -60 mV throughout the recordings unless otherwise specified. Current-clamp recordings were obtained by switching to current-clamp mode after a stable whole-cell configuration was formed in the voltage-clamp mode. In this experiment, only cells with a stable resting membrane potential (less than -50 mV) were used. Signals were filtered at 4 kHz and then digitized at 10 kHz. The data were analyzed with the pCLAMP 10 acquisition software (Axon Instruments, CA, USA).

Drug Application All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). Propofol and amiloride were initially made up as stock solutions in dimethyl sulfoxide (DMSO) and This article is protected by copyright. All rights reserved.

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subsequently diluted in the external solution of the cells at a maximum of 1:1000 to achieve their final working concentrations. The extracellular solution was added with equal concentrations of DMSO used as a control.

Data Analysis Data were expressed as mean ± SEM and compared statistically using SigmaPlot 10.0 paired t tests, with data considered statistically significant when P < 0.05. The pH50 value for H+ dose–response was fitted using the following equation: I = a/(1+(C50/pH)n) [34], where a is the normalized amplitude of the ASIC current, C50 is the pH at which a half-maximal response occurs, and n is the Hill coefficient.

Conflict of interest statement None declared.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant no. 31171108 and 81270174). We thank Ph.D. Xu TL from the Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China, for providing the plasmids of ASIC1a and ASIC3 plasmids.

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Figure legends Figure 1. Inhibition of propofol on proton-gated currents and membrane excitability in rat DRG neurons. Representative traces of a fast-inactivating current (A), a slow-inactivating current (B), a non-inactivating current (C), and action potentials (D). Pre-application of propofol (30 μM) decreased the peak phases of all three types of acid-induced currents and the number of action potentials in DRG (original current traces on left). Bar graphs in right panel show currents normalized to control (100%, white column). Data in all bar graphs are shown relative to controls. *P < 0.05. **P < 0.01; n = 5-6. Figure 2. Propofol inhibited proton-gated currents in DRG neurons in a concentration-dependent manner. A) Representative current traces showing the effect of different concentration of propofol on the proton-gated currents. B) Data summary of traces showing concentration-dependent inhibition of proton-gated currents by propofol. ASIC currents were elicited by application of pH 6.0 and the membrane potential was clamped at -60 mV. Each point represents the mean ± SEM; n = 5-10. Figure 3. Propofol did not change the sensitivity of ASICs to H+ in DRG neurons. A) Representative current traces showing pH-dependent activation of the ASIC currents in the absence or presence of propofol. B) Summary data showing H+ dose–response curves for ASICs with or without propofol (30 μM). Each point represents the mean ± SEM. All current values were normalized to the current induced by pH 5.0; n = 6. Figure 4. Propofol inhibited acid-induced currents in HEK293 cells transfected with either ASIC1a or ASIC3. The representative current traces (left) and summary bar

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graph (right) show that propofol inhibited the acid-evoked proton-gated currents in HEK293 cells transfected with either ASIC1a (A) or ASIC3 (B) ; n = 5, ** P < 0.01.

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Accepted Article This article is protected by copyright. All rights reserved.

Inhibition of acid-sensing ion channel currents by propofol in rat dorsal root ganglion neurons.

Acid-sensing ion channels (ASICs), part of the epithelial sodium channel/degenerin family, are activated by extracellular protons. The ASICs play a si...
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