ASIC3, a sensor of acidic and primary inflammatory pain
2008; Springer Nature; Volume: 27; Issue: 22 Linguagem: Inglês
10.1038/emboj.2008.213
ISSN1460-2075
AutoresEmmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski, Éric Lingueglia,
Tópico(s)Ion Channels and Receptors
ResumoArticle16 October 2008free access ASIC3, a sensor of acidic and primary inflammatory pain Emmanuel Deval Emmanuel Deval Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Jacques Noël Jacques Noël Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Nadège Lay Nadège Lay Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Abdelkrim Alloui Abdelkrim Alloui Laboratoire de Pharmacologie Médicale, UMR 766 INSERM/Faculté de Médecine -CHU, Clermont-Ferrand, France Search for more papers by this author Sylvie Diochot Sylvie Diochot Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Valérie Friend Valérie Friend Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Martine Jodar Martine Jodar Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Michel Lazdunski Michel Lazdunski Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Eric Lingueglia Corresponding Author Eric Lingueglia Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Emmanuel Deval Emmanuel Deval Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Jacques Noël Jacques Noël Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Nadège Lay Nadège Lay Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Abdelkrim Alloui Abdelkrim Alloui Laboratoire de Pharmacologie Médicale, UMR 766 INSERM/Faculté de Médecine -CHU, Clermont-Ferrand, France Search for more papers by this author Sylvie Diochot Sylvie Diochot Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Valérie Friend Valérie Friend Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Martine Jodar Martine Jodar Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Michel Lazdunski Michel Lazdunski Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Eric Lingueglia Corresponding Author Eric Lingueglia Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France Search for more papers by this author Author Information Emmanuel Deval1,‡, Jacques Noël1,‡, Nadège Lay1, Abdelkrim Alloui2, Sylvie Diochot1, Valérie Friend1, Martine Jodar1, Michel Lazdunski1 and Eric Lingueglia 1 1Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France 2Laboratoire de Pharmacologie Médicale, UMR 766 INSERM/Faculté de Médecine -CHU, Clermont-Ferrand, France ‡These authors contributed equally to this work *Corresponding author. Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS 6097, Université de Nice Sophia Antipolis, 660 Route des Lucioles, 06560 Valbonne, France. Tel.: +33 0 4 93 95 77 20; Fax: +33 0 4 93 95 77 04; E-mail: [email protected] The EMBO Journal (2008)27:3047-3055https://doi.org/10.1038/emboj.2008.213 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that ∼60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (∼pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia. Introduction Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons (Waldmann et al, 1997a, 1997b; Wemmie et al, 2006; Lingueglia, 2007). Four genes encoding seven subunits (ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3 and ASIC4) have been identified so far in mammals. Functional channels result from the association of the different ASIC subunits into trimers (Jasti et al, 2007) leading to homomeric or heteromeric channels (Lingueglia et al, 1997; Alvarez de la Rosa et al, 2002; Benson et al, 2002; Hesselager et al, 2004). ASICs are predominantly neuronal channels, expressed in central (CNS) and peripheral (PNS) nervous systems. Whereas ASIC1a and ASIC2 are widely present in both CNS and PNS, the expression of ASIC1b and ASIC3 is restricted to peripheral sensory neurons (Waldmann et al, 1997a; Chen et al, 1998; Bassler et al, 2001). As extracellular acidosis correlates with pain sensations (Steen et al, 1995a; Issberner et al, 1996; Reeh and Steen, 1996), ASICs have been proposed to sense extracellular acidifications occurring in pathological conditions such as inflammation, ischaemia, haematomas, fractures and lesions as well as in postoperative states (Krishtal and Pidoplichko, 1981; Waldmann et al, 1997a, 1997b; Waldmann and Lazdunski, 1998; Woo et al, 2004). Indeed, experiments performed in healthy human volunteers (Ugawa et al, 2002; Jones et al, 2004) using non-specific blockers such as amiloride or non-steroidal anti-inflammatory drugs (NSAIDs) (Waldmann et al, 1997b; Voilley et al, 2001) and behavioural experiments in rats using a non-discriminative ASIC blocker (A-317567) (Dube et al, 2005) both support a function of ASICs in acid-induced cutaneous pain. However, data obtained from ASIC knockout mice have failed to demonstrate a clear function of these channels in acidic or primary inflammatory pain (Price et al, 2001; Chen et al, 2002; Ikeuchi et al, 2008; and our own unpublished results) but have shown an effect on secondary mechanical hyperalgesia (related to central sensitization in the spinal cord) in injured or inflamed muscle and joint (Price et al, 2001; Sluka et al, 2003; Sluka et al, 2007; Ikeuchi et al, 2008). Therefore, the participation of peripheral ASICs to acid-induced cutaneous pain and primary inflammatory hyperalgesia remains an open question. In this work, we show that rat cutaneous sensory neurons display a large amount of ASIC1a and ASIC3-like currents when stimulated by moderate acidosis (i.e., around pH 7.0). We then demonstrate the involvement of peripheral ASIC3 in sensing cutaneous acidic pain in normal and inflammatory conditions. Results DRG neurons innervating the skin exhibit a high level of ASIC3-like current We have investigated native ASIC currents activated by moderate acidifications in rat skin dorsal root ganglion (DRG) neurons stained by retrograde labelling with the fluorescent dye DiI (Figure 1A). The very moderate pH value used in these experiments (pH 6.6) was chosen to mainly activate ASIC1-like and ASIC3-like currents, as ASIC2-like and TRPV1 currents have been described to be activated by more drastic acidifications (Tominaga et al, 1998; Lingueglia, 2007). These neurons have a resting membrane potential of −55.0±1.8 mV and a membrane capacitance of 39.6±1.8 pF (n=42 and 43, respectively, data from four different cultures), corresponding to estimated neuron diameters ranging from 20 to 45 μm. We found that 65.8±6.3% of these skin DRG neurons exhibit a transient pH 6.6-induced ASIC-like current (4 of 7, 8 of 11, 8 of 10 and 8 of 15 neurons; Figure 1A), with a mean amplitude of −60.3±16.0 pA/pF (n=28). Within the remaining skin DRG neurons, pH 6.6 induces either no current (n=11) or only a small sustained current (−1.2±0.5 pA/pF, n=4). The ratio of ASIC-like current is not dramatically changed by the culture conditions; 70% (7 of 10 neurons) and 65.2% (15 of 23 neurons) of DRG neurons have a pH 6.6-evoked ASIC-like current after 24–48 h or 24–72 h of culture, respectively. Molliver et al (2005) have previously found that 11% of the skin afferent neurons have a functional pH 6.8-evoked ASIC-like current after 24–48 h of culture, whereas 28% are positively stained for ASIC3 by immunohistochemistry. The discrepancy with our data is most probably explained by differences in experimental procedure (rat upper back skin versus dorsal face of the hind paw, neurons of diameter 300 pA compared with 30 pA in our study). Figure 1.ASIC3 senses cutaneous acidic pain in rat. (A) Quantification and analysis of pH 6.6-evoked ASIC currents recorded at −80 mV from rat skin DRG neurons using the PcTx1 toxin. Skin DRG neurons in primary culture were identified using fluorescence microscopy after retrograde labelling with DiI (see the image on the top left). The respective percentages of the different current types are highlighted under each current trace and on the graph (data obtained from a total of 43 neurons). (B) Exemplar response of a CM-fibre to pH 6.9 (spikes) with the corresponding time plot of the spike-frequency shown below. The firing of action potential is maintained at pH 6.9, and application of APETx2 10 μM (bar) inhibits the response. The top trace shows the average action potential. Average spike frequency at pH 6.9 and pH 6.9 with APETx2 10 μM (n=9) is represented on the left. (C) Effect of moderate subcutaneous acidification on pain behaviour in rat (determined as the number of flinches of the injected hind paw; see Materials and methods). The injected solution (NaCl 0.9%+20 mM HEPES) was buffered at pH values ranging from 7.4 to 6.6. Condition in which APETx2 10 μM was added to the injected solution is represented in black (*P<0.05 and **P 90%). This current is identified as flowing through native ASIC1a homomers, and it has the same inactivation kinetics as the recombinant ASIC1a current expressed in the F-11 cell line (τinactivation=1.6±0.4 s, n=2 versus τinactivation=1.7±0.09 s, n=10; see Supplementary Figure 1). Then, 23.3% of the neurons (10 of 43) exhibit a PcTx1-insensitive current (inhibition <10%). This current is identified as an ASIC3-like current, and its inactivation kinetics are similar to that of recombinant ASIC3 expressed in F-11 cells (τinactivation=0.2±0.01 s, n=14 versus τinactivation=0.4±0.09 s, n=9; see Supplementary Figure 1). Finally, 37.2% of the neurons (16 of 43) have partially PcTx1-sensitive currents (10%⩽inhibition⩽90%). This native mix current has an inactivation time constant (τinactivation=1.2±0.2 s, n=16) that is reduced by the treatment with PcTx1 to a value (τinactivation=0.5±0.1 s, n=16) not significantly different from that of recombinant ASIC3 and native ASIC3-like currents (see Supplementary Figure 1). This current is therefore considered as resulting from a mix of ASIC1a homomeric and ASIC3-like currents. Taken together, these data demonstrate that ASIC3-like currents are the most highly expressed ASIC currents activated by moderate acidifications in skin DRG neurons. They are present in 60.5% (26 of 43) of the skin sensory neurons. Inhibition of ASIC3 removes cutaneous pain produced by moderate acidosis To measure the contribution of the ASIC3 channel to nociceptor activation in response to moderate acidification, we have recorded unmyelinated single C-fibre activity with the nerve–skin preparation (Reeh, 1986; Alloui et al, 2006). When challenged with a moderate acidification to pH 6.9, a total of 51% of skin rat C-fibres show significant activation (n=17 of 33 fibres, P<0.001, Wilcoxon test) and 41% are activated by an exposure to pH 6.6 (n=9 of 22 fibres, P<0.01, Wilcoxon test). The spike discharge is irregular with bursts of activity, and some fibres show a delayed onset, but the activity is maintained as long as the pH is kept acidic (Figure 1B). In all the fibres tested (n=9), the sea anemone toxin APETx2 (a specific ASIC3 blocker; Diochot et al, 2004) at 10 μM suppresses the pH 6.9-induced spike activity (P<0.01, Wilcoxon test), confirming ASIC3 as the major pH-sensor of nociceptive fibres for moderate acidosis (Figure 1B). Investigation of the pain behaviour of rats following subcutaneous injections of moderately acid solutions (pH 7.4, 7.2, 6.9 and 6.6) in one of the hind paws (Figure 1C) shows a significant pain behaviour at pH 6.9 (flinching score increasing from 1.9±0.6 at pH 7.4 to 10.4±2.3 at pH 6.9, n=16 and 25 respectively, P<0.05, Kruskal–Wallis test followed by a Dunn's post hoc test). The pain behaviour in rats fails to develop when APETx2 is co-injected together with the pH 6.9 solution (Figure 1C). Together, these results demonstrate that ASIC3 is a key sensor of cutaneous pain produced by moderate acidification. Hypertonicity increases neuronal excitability in skin DRG neurons through an effect on ASIC3 In inflamed or injured tissue, several potent mediators meet in the interstitial fluid and form an inflammatory exudate (Steen et al, 1995b), the content of which is acidic and hyperosmotic (Vakili et al, 1970). We have thus investigated the effect of hyperosmolarity on the ASIC currents in skin DRG neurons evoked by moderate acidification (i.e., pH 7.0). We show that hyperosmolarity (600 mosmol kg−1 with mannitol) strongly enhances the pH 7.0-evoked ASIC current in skin DRG neurons (increased by 95±35%, n=8, P<0.01, paired Student's t-test; Figure 2A). This leads to an increase of neuronal excitability by triggering more action potentials in current-clamped neurons (Figure 2B). The percentage of neurons in which APs are triggered following a pH 7.0 application is 37.5% (3 of 8 neurons), and all of them display an increase of the firing rate when hyperosmolarity or arachidonic acid (AA) was combined with pH 7.0. ASIC3 and ASIC1a are responsible for most of the currents activated by moderate acidification in rat cutaneous DRG neurons (Figure 1). To precisely determine which of these ASIC isoform(s) is involved in this effect, the ASIC3 and ASIC1a channels were heterologously expressed in the F-11 DRG cell line (Francel et al, 1987; Deval et al, 2006). Figure 2C shows that hyperosmolarity (600 mosmol kg−1 with mannitol) significantly potentiates the ASIC3 current evoked by a shift from pH 7.4 to 7.2 (increase of 148±20%, n=20, P<0.001, paired Student's t-test). Conversely, the same external acidification to pH 7.2 failed to produce any significant ASIC1a current from ASIC1a-transfected cells and hyperosmolarity was without effect (Figure 2C, lower panel). The control (measured in isotonic conditions) and the enhanced (measured in 600 mosmol kg−1 hypertonic conditions) pH 7.2-induced currents recorded from ASIC3-transfected cells have the same reversal potential (49.4±0.7 and 45.9±3.4 mV respectively, n=3, P=0.48, paired t-test; see Figure 2D), confirming the specificity of the effect on ASIC3. Interestingly, hyperosmolarity (600 mosmol kg−1) had no effect on pH 6.6-evoked ASIC3 current and was also without effect on the pH 6.6-evoked ASIC1a current (Figure 2E). Hyperosmolarity therefore seems to affect preferentially the persistent, non-inactivating ASIC3 window current (Yagi et al, 2006). The osmotic activation of pH 7.2-induced ASIC3 current was almost maximal when external osmolarity reached 600 mosmol kg−1 (Figure 2F). These results indicate that hyperosmolarity potentiates ASIC3 current, but not ASIC1a, at pH 7.2 probably through an effect on the window current. Figure 2.The potentiating effect of hypertonicity on native pH 7.0-evoked ASIC current is mainly mediated by ASIC3. (A) Typical ASIC current, recorded from a skin DRG neuron under voltage clamp at −80 mV, induced by a shift from pH 7.4 to 7.0. This current was strongly potentiated when a hypertonic solution (600 mosmol kg−1 with mannitol; see Materials and methods) was co-applied with pH 7.0. (B) Current clamp experiment performed on the same neuron as in A. The depolarization induced by the pH 7.0-evoked ASIC current was sufficient to trigger three action potentials (APs). Co-application of the hypertonic solution together with the pH drop led to an increase of the number of APs triggered. Time-scale magnifications of these APs are shown within the dotted rectangles. (C) Effect of hyperosmolarity (600 mosmol kg−1 with mannitol; see Materials and methods) on pH 7.2-induced ASIC1a and ASIC3 currents recorded at −50 mV from F-11-transfected cells. The number of experiments (n) is indicated above each bar (***P<0.001, paired Student's t-test). (D) Current–voltage relationship of the pH 7.2-induced ASIC3 current obtained from F-11-transfected cells before (control, isotonic) and during hyperosmotic shocks. Typical current traces are shown above the I/V curve. (E) Hyperosmolarity (600 mosmol kg−1 with mannitol) was without effect on both pH 6.6-induced ASIC3 and ASIC1a currents. (F) The increase in the percentage of the pH 7.2-induced ASIC3 current is represented as a function of external osmolarity. The hyperosmotic solutions were obtained by the addition of mannitol (n=5–20). Download figure Download PowerPoint Moderate acidosis, hypertonicity and AA synergistically affect ASIC3 to produce cutaneous pain Arachidonic acid is known to positively affect ASIC currents (Allen and Attwell, 2002; Smith et al, 2007). We show here that AA increases the native ASIC current induced by a shift from pH 7.4 to 7.0 (+172±65%, n=6, P=0.06, paired Student's t-test). This effect leads to an increased excitability of the skin DRG neurons by increasing the triggering of AP (Figure 3A). We have further explored the pH sensitivity of the AA effect on ASIC1a and ASIC3 channels transfected in F-11 cells. As observed for the effect of hypertonicity, AA potentiates the pH 7.2-evoked ASIC3 current (+547±103%, P<0.0001, n=23, Wilcoxon test), whereas it has no effect on ASIC1a at the same pH (Supplementary Figure 2A). However, the kinetics of the two effects (i.e., hypertonicity and AA) are different. The effect of hypertonicity (co-application with the pH drop) is immediate, whereas the effect of AA needs a few minutes to be fully established (Supplementary Figure 2A). We have observed that AA also increases ASIC1a. The activation is larger at pH 7.0 (+183±54%, n=5, P=0.06, Wilcoxon test) than at pH 6.6 (+21±15%, n=2), but remains much lower than that observed for ASIC3 (increase of 493±84%, n=9, P<0.001, Wilcoxon test, and of 40±22%, n=7, P=0.45, paired Student's t-test at pH 7.0 and 6.6, respectively; see Supplementary Figure 2B). The potent effect of AA on the ASIC3 current essentially results from a shift of the pH dependence of activation towards less acidic values (pH1/2 for activation shifted from 6.68±0.01 to 6.84±0.01, n=5 and 3 cells, respectively, Figure 3B). No significant effect of AA is observed on the pH dependence of the inactivation curve. As a consequence of these differential effects of AA on the pH dependence of activation and inactivation, the non-inactivating ASIC3 window current is strongly enhanced in the presence of AA (Figure 3C, upper panel). This leads to an activation of the ASIC3 channel at resting physiological pH (i.e., pH 7.4; see Figure 3C, lower panel). Figure 3D shows that the effects of AA and hypertonicity on ASIC3 currents are synergistic, demonstrating that this channel is built to integrate different signals such as moderate acidification, hypertonicity and AA that are found in inflammatory conditions. Figure 3.ASIC3 senses different inflammatory signals to produce cutaneous pain. (A) Current clamp experiment showing that arachidonic acid (AA) increased the excitability of a skin DRG neuron in response to a depolarization induced by a pH 7.0-evoked ASIC current. Time-scale magnifications of the APs triggered are shown within the dotted rectangle. Note that pH 7.0-induced ASIC current leads to a membrane depolarization near the AP threshold, which is not always sufficient to produce firing (left panel). (B) pH-dependent activation and inactivation curves were obtained from F-11 cells transfected with ASIC3, at −80 mV, according to the protocol shown in inset (n=3–5). The framed rectangle indicates a part of the graph where the activation and inactivation curves overlap (window current). (C) Magnification of the framed zone shown in B indicating that the non-inactivating ASIC3 window current is strongly enhanced by AA (upper panel). The effect of AA on ASIC3 current induced by moderate acidifications is also represented (lower panel). (D) Representative whole-cell recording from transfected F-11 cells of a pH 7.2-induced ASIC3 current at −80 mV showing the synergistic effect of AA (10 μM) and hypertonicity (600 mosmol kg−1 with sucrose). (E) The effect on pain behaviour in rat of subcutaneous injections of acid (pH 6.9), hyperosmolarity and AA 10 μM together are compared with the effect of pH 6.9 alone. Conditions in which APETx2 10 μM or PcTx1 60 nM were added to the injected inflammatory cocktail are indicated on the bargraph. The number of experiments (n) is indicated above each bar (*P<0.05, Kruskal–Wallis test followed by a Dunn's post hoc test). Download figure Download PowerPoint In good agreement with the latter results, combining hypertonicity (NaCl 2%, ∼600 mOsm kg−1) and AA (10 μM) significantly increases the flinching score of rats (Figure 3E) induced by moderate acidosis (i.e., pH 6.9; from 10.4±2.3, n=25 to 20.4±2.5, n=25, P<0.05, Kruskal–Wallis test followed by a Dunn's post hoc test). This increase in pain behaviour is largely prevented by co-injection of the ASIC3 blocker APETx2, whereas the homomeric ASIC1a blocker PcTx1 has no significant effect (Figure 3E). Taken together, all these results strongly suggest that the activation of peripheral ASIC3, but not homomeric ASIC1a, by inflammatory mediators contributes to inflammatory pain. ASIC3 contributes to the development of CFA-induced primary inflammatory pain To demonstrate more directly the function of ASIC3 in cutaneous inflammatory pain, the effect of APETx2 and PcTx1 were tested on a rat model of inflammatory pain. Four hours after the induction of inflammation by CFA injection in the hind paw, a significant heat hyperalgesia appears (Figure 4A). The heat hyperalgesia does not develop when the ASIC3 blocker APETx2 is co-injected with CFA, whereas PcTx1 has no significant effect. The implication of ASIC3 in inflammatory thermal hyperalgesia is further confirmed by intrathecal injections of an siRNA targeting the ASIC3 channel before the induction of inflammation. A marked and specific knockdown of ASIC3 expression at the mRNA level has been demonstrated in lumbar DRGs after intrathecal injections of this siRNA (Supplementary Figure 3). In pain behaviour experiments, these injections prevent CFA-induced heat hyperalgesia, whereas the corresponding scramble siRNA used as a control is without effect (Figure 4B). These results directly demonstrate that ASIC3, but not homomeric ASIC1a, has an important function in primary inflammatory heat hyperalgesia at the peripheral level in rats. Figure 4.ASIC3 is a detector of cutaneous inflammatory pain in rat. (A) Effect of APETx2 20 μM and PcTx1 120 nM on CFA-induced thermal hyperalgesia in rat. Hind paw withdrawal latencies were measured at 50°C (see Materials and methods), and the time at which inflammation was induced (t0) is indicated by the arrow (**P<0.01; ♦♦♦P<0.001, significantly different from control, Kruskal–Wallis test followed by a Dunn's post hoc test). (B) Effect of intrathecal injections (see the inset for the protocol) of ASIC3 siRNA on the CFA-induced heat hyperalgesia described in A (* and P<0.05; ♦♦P<0.01; P<0.001, significantly different from scramble and siRNA control, respectively; one-way ANOVA followed by a Tukey's post hoc test). Download figure Download PowerPoint Discussion Protons are direct activators of nociceptors (Steen et al, 1992). Studies conducted both in humans and animals have shown a positive correlation between pain and tissue acidity. Perfusion of acidic solutions or iontophoresis of protons into the skin produces pain in humans (Steen et al, 1995a; Ugawa et al, 2002; Jones et al, 2004), and ASIC channels seem to be the best candidates to sense this cutaneous acidic pain (Ugawa et al, 2002; Jones et al, 2004). Recent results with transcutaneous iontophoresis, a non-invasive method, are particularly illustrative (Jones et al, 2004). They have shown that amiloride, a blocker of ASIC channels (Waldmann et al, 1997b), and NSAIDs, which also inhibit this class of channels (Voilley et al, 2001), significantly decrease acidic pain. They have also demonstrated that skin desensitized by repeated capsaicin application shows no significant reduction in acid-induced pain. This latter result strongly suggests that the acid detection is not through the capsaicin receptor TRPV1 (Jones et al, 2004). This conclusion is fully consistent with previous observations using direct perfusion of acidic solutions into human skin (Ugawa et al, 2002), which strongly suggested that acidic pain elicited by pH values between 7.4 and 6 was not significantly associated with the TRPV1 channel but was blockable by amiloride. The only limit of this interesting series of papers is that neither amiloride nor NSAIDs are specific inhibitors of ASIC channels, and that besides ASIC and TRPV1 channels, other types of ionic channels could be involved. Results obtained in humans have yet had no parallel in mice. Deletion of ASIC3 channels in this animal species has failed to indicate a clear function of this channel in pain behaviour associated with cutaneous acidosis or inflammation (Price et al, 2001; Chen et al, 2002). Silencing of ASIC3 using a dominant-negative subunit has even led to an increased sensitivity to inflammatory stimuli (Mogil et al, 2005). Interpretation of these results is complicated by the fact that (i) mice express relatively low levels of ASIC channels in their DRGs (Leffler et al, 2006; Lin et al, 2008; and unpublished data from this laboratory) as compared with other animal species such as rats (this article), (ii) deleting or silencing ASIC genes might be associated with the appearance of compensatory mechanisms. We report here that subcutaneous injections of moderately acidic solutions elicit pain in rats within the same pH range (pH ∼6.9) as in humans (Steen et al, 1995a), and that this effect depends on ASIC3, but not homomeric ASIC1a. Consistent with this in vivo observation, we have shown that DRG neurons innervating rat skin display a high level of ASIC-like currents, which, when they are activated by slight acidification (pH 7.0), depolarize the neurons and trigger APs. We also show that very moderate acidifications also induce a significant increase in skin C-fibres firing, which is totally inhibited by the ASIC3-specific toxin APETx2. This demonstrates that ASIC3 is the leading receptor for moderate acidosis in skin nociceptors and participates in the signalling of acidic pain in rat. Inflammation is one of the pain conditions that produce local tissue acidosis which, in principle, can be detected by ASIC channels. Inflammation also induces a large increase of ASIC channel expression in rat sensory neurons, particularly ASIC3. The nociceptor level of ASIC3 mRNA is increased by more than 15 times in CFA-e
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