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The Nociception Induced by Glutamate in Mice Is Potentiated by Protons Released into the Solution
The Journal of Pain, Vol 11, No 6 (June), 2010: pp 570-578 Available online at www.sciencedirect.com
The Nociception Induced by Glutamate in Mice Is Potentiated by Protons Released into the Solution Flavia Carla Meotti, Igor dos Santos Coelho, and Adair Roberto Soares Santos Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil.
Abstract: In this study we compare the effect of a glutamate solution with pH adjusted to 7 (3–30 mmol/paw), a non-pH-adjusted glutamate solution (.3–30 mmol/paw, pH range 2.24–1.14), and an acid solution (2% acetic acid, pH 1.4–7) in terms of causing licking behavior in mice. The sum of licking seconds was recorded in the first 15minutes following the intraplantar (i.pl.) injection of the solutions. Protons potentiated the nociception induced by glutamate. The ED50 values were 2.5 (1.5–4.2) and 15.1 (11.5–19.9) mmol/paw for the non-pH-adjusted and pH-adjusted glutamate solutions, respectively. The acid solutions at pH 1.4, 2 and 4 induced a similar nociception. The blocking of the acid-sensitive ion channels (ASICs) by amiloride and the antagonism of the transient receptor potential vanilloid subtype-1 (TRPV1) by capsazepine, injected via i.pl., significantly decreased the nociception mediated by acid and by non-pH-adjusted glutamate solutions, but did not affect the nociception caused by the pH-adjusted glutamate solution. The pretreatment with the NMDA-receptor antagonist (MK-801, i.pl.), with the cyclooxygenase inhibitor (indomethacin, i.pl.) or the disruption of the sensorial C fibers by capsaicin, decreased the nociceptive effect of the 3 algogen tested. In summary, the protons present in aqueous solution of glutamate can cause nociception per se or can potentiate the nociception caused by glutamate. These effects are related to the activation of ASICs, TRPV1 and NMDA receptors, inhibition of the synthesis of prostanoids, and disruption of the C fibers. Perspective: The nociception induced by glutamate is a useful method for investigation of the mechanisms of nociception and the effects of new analgesic drugs. Our findings showed that the protons released from glutamic acid have to be removed from the solution to avoid misinterpretation of results in the search for new analgesic drugs. ª 2010 by the American Pain Society Key words: Glutamate, protons, nociception, receptors.
E
xcitatory amino acids have received considerable attention in the study of the mechanisms that underlie nociception because these neurotransmitters lead to excitatory synapses in both afferent sensorial neurons and the central nervous system.6,7,18 The excitatory amino acids glutamate and aspartate are released in the spinal cord or in the hindpaw in response to noxious stimuli24,31,40 and inflammation.4,27,44 A direct injec-
Received May 12, 2009; Revised August 19, 2009; Accepted September 23, 2009. Supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Programa de Apoio aos Nu´cleos de Exceleˆncia (PRONEX), Fundac¸a˜o de Apoio a` Pesquisa Cientı´fica Tecnolo´gica do Estado de Santa Catarina (FAPESC) and Financiadora de Estudos e Projetos [FINEP, Rede Instituto Brasileiro de Neurocieˆncia (IBN-Net)], Brazil. Address reprint requests to Dr. Adair R. S. Santos, Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal de Santa Catarina, Campus Universita´rio – Trindade, 88040-900, Floriano´polis, SC, Brazil. E-mail: arssantos@ ccb.ufsc.br 1526-5900/$36.00 ª 2010 by the American Pain Society doi:10.1016/j.jpain.2009.09.012
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tion of glutamate in the mouse footpad or lumbar area causes an immediate nociceptive response identified by licking and biting behavior.2,14,43 These models of nociception have been broadly used to understand the mechanisms of nociceptive transmission and the mechanism of action for analgesic drugs.2,14,29,33,42,43 The nociception triggered by the intraplantar injection of glutamate has been mainly attributed to the activation of both ionotropic and metabotropic glutamate receptors.2,29 However, the chemical profile of glutamate confers to this molecule an acidic property by releasing its protons from the carboxyl group in aqueous solution. Thus, the nociception caused by glutamate could result from the release of protons in solution and not only from the action of glutamate on its receptors. Protons that are released in the interstitial fluid can directly activate the receptors TRPV1 (transient receptor potential vanilloid subtype-1) and ASICs (acid-sensing ion channels).23,39 The ASIC functional channel results from an association of the ASIC subunits (ASIC1a, ASIC1b,
Meotti, Coelho, and Santos ASIC1b2, ASIC2a, ASIC2b, ASIC3 and ASIC4) forming trimers with homomeric or heteromeric structures.16,28 ASICs are predominantly, but not exclusively, expressed in neurons at both central and peripheral levels.25 Together with TRPV1, they rapidly respond to a fall in pH, leading to cation influx. The consequences are changes in the membrane potential and nociceptive transmission by the sensory neurons.25,47,51 The main difference in the activation of ASICs and of TRPV1 is that ASICs are sensitive to small changes in pH, whereas TRPV1 responds to a more significant change (pH less than 6).5,48 TRPV1 can also respond to endogenous products of inflammation, phospholipase C, protein kinase A and C and both, TRPV1 and ASICs, are sensitive to products of arachidonic acid metabolism.9,19,50 Since the intraplantar injection of glutamate is a broadly applied and useful method to evaluate the mechanisms of nociception and analgesic drugs,2,14,29,33,42,43 and considering that the acidification of the solution could lead to misinterpretation of the results, this study was designed to investigate the mechanisms of nociception induced by a glutamate solution at neutral pH (solution adjusted to pH 7) by a glutamate solution with no pH adjustment and by an acid solution. For this purpose, we compared the nociceptive effect of these solutions and evaluated their effects using an antagonist of the ionotropic glutamate receptor N-methyl-Daspartate (NMDA), a blocker of ASICs, and a TRPV1 antagonist.
Methods Animals Experiments were conducted using adult female Swiss mice (25–35 g) housed at 22 6 2 C under a 12-hour light/ 12-hour dark cycle (lights on at 6:00) with access to food and water ad libitum. The animals were from our own breeding colony. They were acclimatized to the laboratory for at least 1 hour before testing and were used only once during the experiments. The experiments were performed after approval of the protocol by the Institutional Ethics Committee of the Universidade Federal de Santa Catarina (UFSC) (protocol number 23080.0011700/2005-03/UFSC). All experiments were carried out in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations on experimental pain in conscious animals as previously specified.53 The number of animals and intensity of noxious stimuli used were the minimum necessary to demonstrate consistent effects of the drug treatments.
Reagents The following substances were used: glutamic acid, was purchased from Sigma Aldrich (St. Louis, MO); amiloride, capsazepine, capsaicin, indomethacin and (5S,10R)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] (MK-801) were purchased from Tocris Cookson Inc (Ellisville, MO); acetic acid, sodium chloride and sodium hydroxide were purchased from Merck (Darmstadt,
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Germany). All other chemicals were of analytical grade and obtained from standard commercial suppliers.
Algogen-Induced Overt Nociception in Mice The nociceptive behavior was induced by an i.pl. injection of 20 mL non-pH-adjusted glutamate solution (.3, 1, 3, 10, 20 or 30 mmol/paw), a pH 7 glutamate solution (3, 10, 20 or 30 mmol/paw) or 2% acetic acid at different pH values (1.4, 2, 4, 6 or 7). The glutamate was prepared in a sterile isotonic saline solution (.9% NaCl). The corresponding pH values for the non-pH-adjusted glutamate solution were 2.24, 2.1, 2, 1.56, 1.34 and 1.14 for the .3, 1, 3, 10, 20 or 30 mmol/paw solutions, respectively. The neutral glutamate solution was prepared by adding 1 M sodium hydroxide to obtain a solution at pH 7. The acetic acid was diluted to 2% and the different values of pH were obtained by adding 1 M chloridric acid or 1 M sodium hydroxide. The solutions were injected into the ventral surface of the right hind paw. After the injection, mice were observed individually for 60 minutes and the amount of time that they spent licking or biting the injected paw was timed in intervals of 5 minutes. The sum of licking seconds accumulated in the first 15 minutes was considered representative of the nociception induced by all solutions. The glutamate concentration was chosen based in previous experiments.2
Analysis of the Mechanisms Involved in the Nociception Induced by pH-Adjusted, Non-pH-Adjusted Glutamate and Acetic Acid Solutions To evaluate the mechanisms involved in the nociceptive effect of pH-adjusted glutamate, non-pH-adjusted glutamate or acetic acid solutions, the mice were treated intraplantary (i.pl.) with 10 mL/paw amiloride10 (an antagonist of the acid-sensitive ion channel); MK-8012 (an antagonist of the glutamate receptor subtype NMDA, N-methyl-D-aspartate); capsazepine41 (an antagonist of the vanilloid receptor) or indomethacin12 (an inhibitor of cyclooxigenase) immediately before an injection of 20 mL/paw of pH-adjusted glutamate (20 mmol/paw, pH 7), non-pH-adjusted glutamate (3 mmol/paw, pH 2) or acetic acid (2%, pH 2). The concentrations of 3 mmol/ paw non-pH-adjusted glutamate and 20 mmol/paw pHadjusted glutamate were chosen for the subsequent tests because these concentrations had a similar response (seconds of licking) when injected into the hindpaw. A stock solution of indomethacin, capsazepine or amiloride was made up in DMSO plus ethanol. The final work solution was prepared immediately before use and contained 5% DMSO and 5% ethanol in saline. MK-801 was dissolved in saline solution. After the injection, the mice were observed individually for 60 minutes and the amount of time that they spent licking or biting the injected paw was timed in intervals of 5 minutes. The sum of licking seconds accumulated in the first 15 minutes was chosen as representative of nociception induced by all solutions.
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Involvement of Capsaicin-Sensitive Fibers
Table 1.
To investigate the role of capsaicin-sensitive fibers in the nociceptive response induced by pH-adjusted, nonpH-adjusted glutamate, and acid solutions, newborn mice were treated by the subcutaneous (sc) route with 50 mg/kg capsaicin on the second day of life. This treatment induces an irreversible degeneration of unmyelinated afferent neurons, especially C fibers.17 Control animals received a subcutaneous injection of the vehicle used to dissolve the capsaicin (10% ethanol, 10% Tween 80, and 80% saline). The nociceptive response caused by pH-adjusted glutamate, non-pH-adjusted glutamate or acid solutions was analyzed 6 to 7 weeks after the neonatal capsaicin or vehicle treatment. The efficiency of the treatment of mice with capsaicin was confirmed by a topical application of 50 mL capsaicin (.01%) on the right eye. The number of wiping motions occurred during the subsequent 1-minute period was counted as described previously.20 In the group treated with capsaicin neonatal, only mice that had a number of wiping motions less than 6 were considered for the experiment.
GLUTAMATE SOLUTION
Statistical Analyses The results are presented as mean 1 S.E.M. The ED50 or ID50 values (ie, the dose that induced or decreased the nociceptive response by 50% relative to the control value) were reported as geometric mean accompanied by their respective 95% confidence limits. The ED50 or ID50 values were determined by linear regression from individual experiments using linear regression GraphPad software (GraphPad software, San Diego, CA). The percentage of inhibition was calculated taking each individual response and compared to the average of the respective control group, providing a standard error for each calculated percentage. Comparisons between experimental and control groups were performed by ANOVA followed by the Newman-Keuls or Student t test when appropriate. P-values less than .05 (P < .05) were considered as indicative of significance.
Results The nociceptive effect of glutamate was measured using a non-pH-adjusted and a pH-adjusted glutamate solution. The pH of the non-pH-adjusted glutamate solution decreased with increasing glutamate concentration (Table 1). Besides, the nociceptive action of glutamate was directly increased raising the glutamate concentration (Table 1). A comparative study with the non-pH-adjusted glutamate, pH-adjusted glutamate and acid solution showed that the nociceptive effect of glutamate is more potent in an acid condition. The ED50 values were 2.5 (1.5–4.2) mmol/paw and 15.1 (11.5–19.9) mmol/paw for the non-pH-adjusted and pHadjusted glutamate, respectively. The licking behavior was initiated from 1 mmol/paw in mice injected with a non-pH-adjusted glutamate solution. However, a solution of 10 mmol/paw was necessary to initiate the licking behavior in mice injected with a pH-adjusted glutamate
Licking Behavior and Correspondent pH in Different Concentrations of Glutamate
.3 mmol/paw 1 mmol/paw 3 mmol/paw 10 mmol/paw 20 mmol/paw 30 mmol/paw
PH
LICKING (S)
2.24 2.1 2 1.56 1.34 1.14
762 63 6 14 136 6 26 200 6 17.2 212 6 30.3 240 6 30.1
NOTE. The nociception was evaluated within 15 minutes after glutamate injection. The data are mean 6 S.E.M. of 6 mice.
solution (Fig 1). This demonstrated that the protons present in the glutamate solution potentiate the algesic effect of this neurotransmitter. Taking this into account, the mice were injected with an acetic acid solution at different pH values to evaluate the sensitization caused by the protons. The mice presented a nociceptive behavior from the lowest pH used (pH 1.4). The same nociceptive behavior was observed at pH 2 and 4, but it was absent at pH 6 and 7 (Fig 1). To investigate the activation of the acid-sensitive ion channel in the nociception caused by the acid glutamate, neutral glutamate, and acid solutions, the mice received an i.pl. injection of amiloride prior to the algogens. The nociception caused by the non-pH-adjusted glutamate (3 mmol/paw, pH 2) and by the acid solution (2% acetic acid, pH 2) was inhibited by amiloride at the very low doses of 10 and 30 nmol/paw, respectively. As expected, amiloride was unable to inhibit the nociception induced by the pHadjusted glutamate solution (Fig 2). The calculated doses of amiloride that inhibited 50% (ID50) of the response to the non-pH-adjusted glutamate and acid solution were 18.5 (13.9–24.5) and 25.64 (23.6–28.5) nmol/paw, respectively. The maximal inhibition caused by amiloride (100 nmol/paw) was 66 6 6% for the non-pH-adjusted glutamate and 66 6 8% for the acid solution. The treatment with the NMDA-receptor antagonist, MK-801, blocked the nociceptive effect of the 3 algogens tested (Fig 3). MK-801 had a similar potency to inhibit the nociception induced by the pH-adjusted glutamate solution (20 mmol/paw, pH 7) and acid solution (2% acetic acid, pH 2) with ID50 values of 6.6 (3.3–13.3) and 7.7 (4.4–13.6) nmol/paw, respectively. In contrast, a dose of 18.7 (15–23.2) nmol/paw was necessary to inhibit 50% of the response induced by the non-pH-adjusted glutamate (3 mmol/paw, pH 2). MK-801 (50 nmol/paw) was more efficacious in inhibiting the nociception caused by the acid solution, achieving an inhibition of 89 6 8%. When tested against pH-adjusted and non-pH-adjusted glutamate solution, MK-801 (50 nmol/paw) inhibited 62 6 6 and 68 6 11% of the nociception, respectively. The TRPV1 antagonist, capsazepine, greatly inhibited the nociception induced by the acid solution (2% acetic acid, pH 2). A higher dose of capsazepine was necessary to inhibit significantly the nociception induced by nonpH-adjusted glutamate (3 mmol/paw, pH 2). Capsazepine was ineffective against the nociception induced by pHadjusted glutamate (20 mmol/paw, pH 7) (Fig 4). The
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Figure 1. Nociceptive effect of different concentrations of non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 20 ml/paw was injected into the right hindpaw and the licking behavior was monitored for 15 minutes. The control group (C) received 20 ml/paw of saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M.
ID50 values were 14.8 (8–27.4) and 63.9 (42–97) pmol/paw for the acid and non-pH-adjusted glutamate solutions, respectively. Capsazepine (100 pmol/paw) had a maximal inhibition of 64 6 5 and 73 6 6% for the acid and nonpH-adjusted glutamate solutions, respectively. To investigate whether the inflammatory mediator prostaglandins are produced in the nociception caused by non-pH-adjusted glutamate, pH-adjusted glutamate and acid solutions, the mice were pretreated with the cyclooxygenase inhibitor indomethacin. Indomethacin from 1 ng/paw caused a significant inhibition of the licking behavior induced by acid solution (2% acetic acid, pH 2). In contrast, only 100 ng/paw and 200 mg/paw indomethacin inhibited significantly the nociception caused by the non-pH-adjusted glutamate (3 mmol/paw, pH 2) and pH-adjusted glutamate solutions (20 mmol/paw, pH 7), respectively (Fig 5). The ID50 values were 7.8
(3.3–18.4) and 636.3 (203.1–1993.2) ng/paw for acid and non-pH-adjusted glutamate solutions, respectively. The maximal inhibitions were 75 6 10 % (1 mg/paw), 71 6 11% (100 mg/paw) and 48.8 6 11% (200 mg/paw) for the nociception induced by acid, non-pH-adjusted glutamate and pH-adjusted glutamate solution, respectively. The disruption of the sensorial C fibers, through the neonatal treatment with capsacin, significantly decreased the nociception mediated by acid (38 6 2%), pH-adjusted glutamate (55 6 7%) and acid glutamate (55 6 5%) solutions (Fig 6). The confirmation of the disruption of the C fibers in animals treated with capsaicin was obtained through the topical application of capsaicin on the right eye. Only mice that had a number of wiping motions less than 6 were considered for the experiment; this proved that capsaicin treatment was effective in disrupting the sensorial C-fibers.
Figure 2. Effect of different concentrations of amiloride on the nociceptive response in non-pH-adjusted glutamate (white bars), pHadjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw of amiloride was injected immediately before the injection of non-pH-adjusted or pH-adjusted glutamate or acid solution (20 ml/paw) and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw of saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05 and **P < .01) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
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Figure 3. Effect of different concentrations of MK-801 on the nociceptive response in non-pH-adjusted glutamate (white bars), pHadjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw MK-801 was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw of saline solution (NaCl .9%, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05, **P < .01 and ***P < .001) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
Discussion The stimulation of glutamate receptors, after an intraplantar or intrathecal injection of glutamate, has been extensively used to investigate the mechanisms of nociception and in the search for new analgesic drugs.2,10,33,43 Previous studies have demonstrated that the nociception induced by glutamate occurs through the release of neurokinins, kinins, CGRP, and nitric oxide at the site of the injection3,11 and by opening of the cation channels sensitive to amiloride.10 Our concern is that these mechanisms may not be due to the action of the glutamate on its receptors, but rather to the activation of cation channels that are sensitive to the protons dissociated from L-glutamic acid in the solution.
In this study, we found that the protons released from L-glutamic acid induced nociception in mice. As seen by the ED50 values, the protons present in the solution potentiated 6-fold the nociceptive effect of glutamate. In fact, a concentration of 10 mmol/paw of neutral glutamate solution was necessary to induce a consistent licking behavior, while 1 mmol/paw of non-pH-adjusted glutamate solution was sufficient to cause this effect. To compare the contribution of the protons in the nociception induced by glutamate, we used an acid solution at pH 2, which corresponded to the pH of the glutamate in a non-pH-adjusted solution. The nociception induced by protons acts as a sensor when the pH drops in conditions like ischemia (eg,
Figure 4. Effect of different concentrations of capsazepine on the nociceptive response in non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw capsazepine was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/ paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw saline solution (NaCl .9%, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference ***P < .001 from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
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Figure 5. Effect of different concentrations of indomethacin on the nociceptive response in non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/ paw indomethacin was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05, **P < .01 and *** P< .001) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
angina) or intense muscle work (anaerobic metabolism). In these situations a very small variation in the pH (.2) is able to sensitize specific receptors, such as ASIC receptors.21,22 In this study, the mice showed a nociceptive behavior only when the pH decreased by at least 2 units, pH 4. Interestingly, we did not observe any significant licking behavior at pH 6. This was probably because the intensity of the stimulus at pH 6 is not sufficient to induce a spontaneous response in our model of nociception. Therefore, although the pH is low and ASICs were likely sensitized to cause a phenomenon of hypernoci-
Figure 6. Effect of the neonatal treatment with capsaicin on the nociceptive behavior induced by non-pH-adjusted glutamate (3 mmol/paw, pH 2), pH-adjusted glutamate (20 mmol/ paw, pH 7) or acetic acid (2%, pH 2) solutions injected into the mouse hindpaw. The mice were treated with capsaicin (50 mg/ kg, sc) or sterile saline (.9% NaCl, sc) on the second day of life. The licking behavior was evaluated in the 6th week of life. The columns represent the amount of time the mice spent licking the injected paw within 15 minutes. Each column represents the mean of 4 mice 6S.E.M. The symbols denote a significant difference (*P < .05 and **P < .01) from the group treated with saline by t-test.
ception, this effect did not induce an overt nociceptive behavior. The role of the ASICs in nociception has been studied using gene deletion for this receptor. However, it is hard to identify the exact role of these receptors using knockout mice because these receptors form heteromultimers leading to conflicting results regarding their role in sensitivity.8,34,46 A good tool to study the function of this receptor is the pharmacological antagonism with amiloride. Amiloride is a K1-sparing diuretic that blocks the members of the ASICs family. ASICs are responsible for controlling pH homeostasis, transduction of mechanical stimuli, and nociception.10,13,15,37 In our study, the blocking of ASICs by amiloride prevented, with a similar potency, the nociception induced by the acid solution and by the non-pH-adjusted glutamate solution. However, amiloride did not prevent the nociception induced by the pH-adjusted glutamate solution. This demonstrates that the nociception induced by an injection of a non-pH-adjusted glutamate solution is mostly caused by the activation of the ASICs. The effect of protons in potentiating the nociception induced by glutamate was further confirmed by pretreatment with the NMDA receptor antagonist MK-801. The concentration of MK-801 that decreased 50 % of the response induced by the non-pH-adjusted glutamate solution (ID50) was 3-fold higher than the ID50 value for decreasing the nociception induced by the pH-adjusted glutamate solution. In addition, the antagonism of the NMDA receptor reduced the nociception induced by acid solution. Taking this into account, it is likely that protons and glutamate that are present in the non-pH-adjusted solution are acting synergistically to depolarize the cell. These results are in agreement with previous findings that showed a regulatory effect of ASICs on the activity of NMDA receptors. In fact, a rapid acidic transient accompanying synaptic transmission provides a membrane depolarization by ASICs. This depolarization
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facilitates the release of the Mg from NMDA receptors (voltage-dependent activation).25,26 As a result, the NMDA receptor is susceptible to activation by glutamate that is released from synaptic vesicles or supplied by an exogenous source. The lower potency of MK-801 in decreasing the nociception induced by the non-pH-adjusted glutamate solution compared to acid and pH-adjusted glutamate solution suggests that, beside the modulatory effect of ASICs on NMDA receptors, the acid solution might activate others mediators or intracellular pathways involved in neuron sensitization.1,30 It is worthwhile to mention that the nociception caused by glutamate is also dependent on the activation of non-NMDA receptors in neurons from the periphery. However, the participation of the non-NMDA receptor is clearly less significant than the NMDA receptor.2 In this concern, we have chosen the most representative receptor that is being activated during intraplantar injection of glutamate to compare the effects between glutamate, acid, and acid plus glutamate solution. The activation of TRPV1 was crucial for the nociceptive transmission induced by acid and by acid glutamate. This was not surprising since TRPV1 works as a sensor for the alteration of pH.5 On the other hand, the TRPV1 antagonist capsazepine did not inhibit the nociception induced by glutamate in a pH-adjusted solution. The TRPV1 receptor contains phosphorylation sites in the intracellular loop responsible for modulating the activity of the receptor. The phosphorylation of TRPV1 by protein kinases is a secondary mechanism triggered, for example, by activation of calcium/calmodulin.36 This phosphorylation promotes the insertion of the channel into the plasma membrane and decreases the threshold for the activation of TRPV1 by the agonists, but does not activate the receptor directly.35,38,49 In accordance with this, our results demonstrated that the activation of glutamate receptors does not affect TRPV1 activity in the absence of protons in the solution. ASICs are overexpressed during inflammatory processes and their expression is increased by mediators like neural growth factor (NGF), serotonin, interleukin-1b, and bradykinin.32 In agreement with this, nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the expression and
the membrane potential triggered by ASICs.50 Similarly to the ASICs, the vanilloid receptor TRPV1 is also sensitive to activation by products of cyclooxygenase.9,19 Herein, we demonstrated, for the first time, that the inhibition of the synthesis of prostanoids by indomethacin greatly decreased the nociception induced by protons in vivo. The inhibitory effect of indomethacin was greater in mice treated with acid solution than in those treated with acid glutamate solution. Thus, the inhibition of COX activity could, in part, hinder the direct increase in sensitivity of the ASICs and TRPV1 caused by arachidonic acid metabolites. However, the eicosanoids derived from COX may also contribute to the nociceptive response induced by acid solution by other mechanisms beside the activation of ASICs and TRPV1.30,45,52 The nociception caused by the 3 algogens tested were essentially dependent on the integrity of sensory C fibers. The disruption of C fibers by the neonatal treatment with capsaicin reduced in a similar manner the nociceptive transmission induced by acid, pH-adjusted and non-pHadjusted glutamate. A previous study had already demonstrated that the nociception induced by glutamate solution was decreased after the depletion of C fibers.3 However, no data had demonstrated that both components, glutamate and protons, have their nociceptive effect dependent on activation of sensory C fibers. In conclusion, our results demonstrated that the protons released from L-glutamic acid in aqueous solution can themselves cause nociception and can potentiate the nociception caused by glutamate. These effects were mainly dependent on the activation of ASICs and TRPV1, but were also antagonized by the blocking of the NMDA receptor, inhibition of the prostanoids synthesis, and disruption of C fibers. In this regard, when the nociceptive effect is desired only on the glutamatergic system, it is important to exclude the protons from the solution.
References
5. Caterina MJ, Julius D: The vanilloid receptor: A molecular gateway to the pain pathway. Annu Rev Neurosci 24: 487-517, 2001
1. Allen NJ, Attwell D: Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals. J Physiol 543:521-529, 2002 2. Beirith A, Santos ARS, Calixto JB: Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Res 924:219-228, 2002 3. Beirith A, Santos ARS, Calixto JB: The role of neuropeptides and capsaicin-sensitive fibres in glutamate-induced nociception and paw oedema in mice. Brain Res 969:110-116, 2003 4. Carlton SM: Peripheral excitatory amino acids. Curr Opin Pharmacol 1:52-56, 2001
Acknowledgments The authors are grateful to Siobhan Wiese by the critical revision of the manuscript.
6. Coggeshall RE, Carlton SM: Receptor localization in the mammalian dorsal horn and primary afferent neurons. Brain Res Brain Res Rev 24:28-66, 1997 7. Dickenson AH: Central acute pain mechanisms. Ann Med 27:223-227, 1995 8. Drew LJ, Rohrer DK, Price MP, Blaver KE, Cockayne DA, Cesare P, Wood JN: Acid-sensing ion channels ASIC2 and ASIC3 do not contribute to mechanically activated currents in mammalian sensory neurones. J Physiol 556:691-710, 2004 9. Ferreira J, da Silva GL, Calixto JB: Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol 141:787-794, 2004
Meotti, Coelho, and Santos 10. Ferreira J, Santos ARS, Calixto JB: Antinociception produced by systemic, spinal and supraspinal administration of amiloride in mice. Life Sci 65:1059-1066, 1999 11. Ferreira J, Santos ARS, Calixto JB: The role of systemic, spinal and supraspinal L-arginine-nitric oxide-cGMP pathway in thermal hyperalgesia caused by intrathecal injection of glutamate in mice. Neuropharmacology 38:835-842, 1999 12. Funez MI, Ferrari LF, Duarte DB, Sachs D, Cunha FQ, Lorenzetti BB, Parada CA, Ferreira SH: Teleantagonism: A pharmacodynamic property of the primary nociceptive neuron. Proc Natl Acad Sci U S A 105:19038-19043, 2008 13. Fyfe GK, Quinn A, Canessa CM: Structure and function of the Mec-ENaC family of ion channels. Semin Nephrol 18: 138-151, 1998 14. Gadotti VM, Tibola D, Paszcuk AF, Rodrigues AL, Calixto JB, Santos ARS: Contribution of spinal glutamatergic receptors to the antinociception caused by agmatine in mice. Brain Res 1093:116-122, 2006 15. Garty H, Benos DJ: Characteristics and regulatory mechanisms of the amiloride-blockable Na1 channel. Physiol Rev 68:309-373, 1988 16. Hesselager M, Timmermann DB, Ahring PK: pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits. J Biol Chem 279:11006-11015, 2004 17. Holzer P: Capsaicin as a tool for studying sensory neuron functions. Adv Exp Med Biol 298:3-16, 1991 18. Hudspith MJ: Glutamate: A role in normal brain function, anaesthesia, analgesia and CNS injury. Br J Anaesth 78:731-747, 1997 19. Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D, Oh U: Direct activation of capsaicin receptors by products of lipoxygenases: Endogenous capsaicin-like substances. Proc Natl Acad Sci U S A 97: 6155-6160, 2000
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of acid sensing ion channels in brain and dorsal root ganglion cells. J Biol Chem 272:29778-29783, 1997 29. Liu XJ, White TD, Sawynok J: Intraplantar injection of glutamate evokes peripheral adenosine release in the rat hind paw: Involvement of peripheral ionotropic glutamate receptors and capsaicin-sensitive sensory afferents. J Neurochem 80:562-570, 2002 30. Lopez JC: ASIC-nal integrator. Nature Review Neuroscience 3:763, 2002 31. Malmberg AB, Yaksh TL: Cyclooxygenase inhibition and the spinal release of prostaglandin E2 and amino acids evoked by paw formalin injection: A microdialysis study in unanesthetized rats. J Neurosci 15:2768-2776, 1995 32. Mamet J, Baron A, Lazdunski M, Voilley N: Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci 22: 10662-10670, 2002 33. Meotti FC, Luiz AP, Pizzolatti MG, Kassuya CA, Calixto JB, Santos ARS: Analysis of the antinociceptive effect of the flavonoid myricitrin: Evidence for a role of the L-arginine-nitric oxide and protein kinase C pathways. J Pharmacol Exp Ther 316:789-796, 2006 34. Mogil JS, Breese NM, Witty MF, Ritchie J, Rainville ML, Ase A, Abbadi N, Stucky CL, Seguela P: Transgenic expression of a dominant-negative ASIC3 subunit leads to increased sensitivity to mechanical and inflammatory stimuli. J Neurosci 25:9893-9901, 2005 35. Montell C: The TRP superfamily of cation channels. Sci STKE 2005:re3, 2005 36. Numazaki M, Tominaga T, Takeuchi K, Murayama N, Toyooka H, Tominaga M: Structural determinant of TRPV1 desensitization interacts with calmodulin. Proc Natl Acad Sci U S A 100:8002-8006, 2003 37. Palmer LG: Epithelial Na channels: Function and diversity. Annu Rev Physiol 54:51-66, 1992
20. Ikeda Y, Ueno A, Naraba H, Oh-ishi S: Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 69:2911-2919, 2001
38. Premkumar LS, Ahern GP: Induction of vanilloid receptor channel activity by protein kinase C. Nature 408: 985-990, 2000
21. Immke DC, McCleskey EW: ASIC3: A lactic acid sensor for cardiac pain. ScientificWorldJournal 1:510-512, 2001
39. Reeh PW, Kress M: Molecular physiology of proton transduction in nociceptors. Curr Opin Pharmacol 1:45-51, 2001
22. Immke DC, McCleskey EW: Protons open acid-sensing ion channels by catalyzing relief of Ca21 blockade. Neuron 37:75-84, 2003 23. Julius D, Basbaum AI: Molecular mechanisms of nociception. Nature 413:203-210, 2001
40. Sakurada T, Sugiyama A, Sakurada C, Tanno K, Sakurada S, Kisara K, Hara A, Abiko Y: Involvement of nitric oxide in spinally mediated capsaicin- and glutamateinduced behavioural responses in the mouse. Neurochem Int 29:271-278, 1996
24. Juranek I, Lembeck F: Afferent C-fibres release substance P and glutamate. Can J Physiol Pharmacol 75: 661-664, 1997
41. Santos ARS, Calixto JB: Ruthenium red and capsazepine antinociceptive effect in formalin and capsaicin models of pain in mice. Neurosci Lett 235:73-76, 1997
25. Krishtal O: The ASICs: Signaling molecules? Modulators? Trends Neurosci 26:477-483, 2003
42. Savegnago L, Pinto LG, Jesse CR, Alves D, Rocha JB, Nogueira CW, Zeni G: Antinociceptive properties of diphenyl diselenide: Evidences for the mechanism of action. Eur J Pharmacol 555:129-138, 2007
26. Krishtal OA, Osipchuk YV, Shelest TN, Smirnoff SV: Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Res 436:352-356, 1987 27. Lawand NB, McNearney T, Westlund KN: Amino acid release into the knee joint: Key role in nociception and inflammation. Pain 86:69-74, 2000
43. Scheidt C, Santos ARS, Ferreira J, Malheiros A, CechinelFilho V, Yunes RA, Calixto JB: Evidence for the involvement of glutamatergic receptors in the antinociception caused in mice by the sesquiterpene drimanial. Neuropharmacology 43:340-347, 2002
28. Lingueglia E, de Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M: A modulatory subunit
44. Sluka KA, Westlund KN: An experimental arthritis model in rats: The effects of NMDA and non-NMDA antagonists on
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aspartate and glutamate release in the dorsal horn. Neurosci Lett 149:99-102, 1993 45. Smith HS: Arachidonic acid pathways in nociception. J Support Oncol 4:277-287, 2006 46. Staniland AA, McMahon SB: Mice lacking acidsensing ion channels (ASIC) 1 or 2, but not ASIC3, show increased pain behaviour in the formalin test. Eur J Pain 13:554-563, 2009 47. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple painproducing stimuli. Neuron 21:531-543, 1998 48. Ugawa S, Ueda T, Ishida Y, Nishigaki M, Shibata Y, Shimada S: Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Invest 110:1185-1190, 2002
Protons and Glutamate Induce Nociception 49. Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA: Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol 534:813-825, 2001 50. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 21:8026-8033, 2001 51. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 386:173-177, 1997 52. Zeilhofer HU: Prostanoids in nociception and pain. Biochem Pharmacol 73:165-174, 2007 53. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110, 1983
The Nociception Induced by Glutamate in Mice Is Potentiated by Protons Released into the Solution Flavia Carla Meotti, Igor dos Santos Coelho, and Adair Roberto Soares Santos Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil.
Abstract: In this study we compare the effect of a glutamate solution with pH adjusted to 7 (3–30 mmol/paw), a non-pH-adjusted glutamate solution (.3–30 mmol/paw, pH range 2.24–1.14), and an acid solution (2% acetic acid, pH 1.4–7) in terms of causing licking behavior in mice. The sum of licking seconds was recorded in the first 15minutes following the intraplantar (i.pl.) injection of the solutions. Protons potentiated the nociception induced by glutamate. The ED50 values were 2.5 (1.5–4.2) and 15.1 (11.5–19.9) mmol/paw for the non-pH-adjusted and pH-adjusted glutamate solutions, respectively. The acid solutions at pH 1.4, 2 and 4 induced a similar nociception. The blocking of the acid-sensitive ion channels (ASICs) by amiloride and the antagonism of the transient receptor potential vanilloid subtype-1 (TRPV1) by capsazepine, injected via i.pl., significantly decreased the nociception mediated by acid and by non-pH-adjusted glutamate solutions, but did not affect the nociception caused by the pH-adjusted glutamate solution. The pretreatment with the NMDA-receptor antagonist (MK-801, i.pl.), with the cyclooxygenase inhibitor (indomethacin, i.pl.) or the disruption of the sensorial C fibers by capsaicin, decreased the nociceptive effect of the 3 algogen tested. In summary, the protons present in aqueous solution of glutamate can cause nociception per se or can potentiate the nociception caused by glutamate. These effects are related to the activation of ASICs, TRPV1 and NMDA receptors, inhibition of the synthesis of prostanoids, and disruption of the C fibers. Perspective: The nociception induced by glutamate is a useful method for investigation of the mechanisms of nociception and the effects of new analgesic drugs. Our findings showed that the protons released from glutamic acid have to be removed from the solution to avoid misinterpretation of results in the search for new analgesic drugs. ª 2010 by the American Pain Society Key words: Glutamate, protons, nociception, receptors.
E
xcitatory amino acids have received considerable attention in the study of the mechanisms that underlie nociception because these neurotransmitters lead to excitatory synapses in both afferent sensorial neurons and the central nervous system.6,7,18 The excitatory amino acids glutamate and aspartate are released in the spinal cord or in the hindpaw in response to noxious stimuli24,31,40 and inflammation.4,27,44 A direct injec-
Received May 12, 2009; Revised August 19, 2009; Accepted September 23, 2009. Supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Programa de Apoio aos Nu´cleos de Exceleˆncia (PRONEX), Fundac¸a˜o de Apoio a` Pesquisa Cientı´fica Tecnolo´gica do Estado de Santa Catarina (FAPESC) and Financiadora de Estudos e Projetos [FINEP, Rede Instituto Brasileiro de Neurocieˆncia (IBN-Net)], Brazil. Address reprint requests to Dr. Adair R. S. Santos, Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal de Santa Catarina, Campus Universita´rio – Trindade, 88040-900, Floriano´polis, SC, Brazil. E-mail: arssantos@ ccb.ufsc.br 1526-5900/$36.00 ª 2010 by the American Pain Society doi:10.1016/j.jpain.2009.09.012
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tion of glutamate in the mouse footpad or lumbar area causes an immediate nociceptive response identified by licking and biting behavior.2,14,43 These models of nociception have been broadly used to understand the mechanisms of nociceptive transmission and the mechanism of action for analgesic drugs.2,14,29,33,42,43 The nociception triggered by the intraplantar injection of glutamate has been mainly attributed to the activation of both ionotropic and metabotropic glutamate receptors.2,29 However, the chemical profile of glutamate confers to this molecule an acidic property by releasing its protons from the carboxyl group in aqueous solution. Thus, the nociception caused by glutamate could result from the release of protons in solution and not only from the action of glutamate on its receptors. Protons that are released in the interstitial fluid can directly activate the receptors TRPV1 (transient receptor potential vanilloid subtype-1) and ASICs (acid-sensing ion channels).23,39 The ASIC functional channel results from an association of the ASIC subunits (ASIC1a, ASIC1b,
Meotti, Coelho, and Santos ASIC1b2, ASIC2a, ASIC2b, ASIC3 and ASIC4) forming trimers with homomeric or heteromeric structures.16,28 ASICs are predominantly, but not exclusively, expressed in neurons at both central and peripheral levels.25 Together with TRPV1, they rapidly respond to a fall in pH, leading to cation influx. The consequences are changes in the membrane potential and nociceptive transmission by the sensory neurons.25,47,51 The main difference in the activation of ASICs and of TRPV1 is that ASICs are sensitive to small changes in pH, whereas TRPV1 responds to a more significant change (pH less than 6).5,48 TRPV1 can also respond to endogenous products of inflammation, phospholipase C, protein kinase A and C and both, TRPV1 and ASICs, are sensitive to products of arachidonic acid metabolism.9,19,50 Since the intraplantar injection of glutamate is a broadly applied and useful method to evaluate the mechanisms of nociception and analgesic drugs,2,14,29,33,42,43 and considering that the acidification of the solution could lead to misinterpretation of the results, this study was designed to investigate the mechanisms of nociception induced by a glutamate solution at neutral pH (solution adjusted to pH 7) by a glutamate solution with no pH adjustment and by an acid solution. For this purpose, we compared the nociceptive effect of these solutions and evaluated their effects using an antagonist of the ionotropic glutamate receptor N-methyl-Daspartate (NMDA), a blocker of ASICs, and a TRPV1 antagonist.
Methods Animals Experiments were conducted using adult female Swiss mice (25–35 g) housed at 22 6 2 C under a 12-hour light/ 12-hour dark cycle (lights on at 6:00) with access to food and water ad libitum. The animals were from our own breeding colony. They were acclimatized to the laboratory for at least 1 hour before testing and were used only once during the experiments. The experiments were performed after approval of the protocol by the Institutional Ethics Committee of the Universidade Federal de Santa Catarina (UFSC) (protocol number 23080.0011700/2005-03/UFSC). All experiments were carried out in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations on experimental pain in conscious animals as previously specified.53 The number of animals and intensity of noxious stimuli used were the minimum necessary to demonstrate consistent effects of the drug treatments.
Reagents The following substances were used: glutamic acid, was purchased from Sigma Aldrich (St. Louis, MO); amiloride, capsazepine, capsaicin, indomethacin and (5S,10R)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] (MK-801) were purchased from Tocris Cookson Inc (Ellisville, MO); acetic acid, sodium chloride and sodium hydroxide were purchased from Merck (Darmstadt,
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Germany). All other chemicals were of analytical grade and obtained from standard commercial suppliers.
Algogen-Induced Overt Nociception in Mice The nociceptive behavior was induced by an i.pl. injection of 20 mL non-pH-adjusted glutamate solution (.3, 1, 3, 10, 20 or 30 mmol/paw), a pH 7 glutamate solution (3, 10, 20 or 30 mmol/paw) or 2% acetic acid at different pH values (1.4, 2, 4, 6 or 7). The glutamate was prepared in a sterile isotonic saline solution (.9% NaCl). The corresponding pH values for the non-pH-adjusted glutamate solution were 2.24, 2.1, 2, 1.56, 1.34 and 1.14 for the .3, 1, 3, 10, 20 or 30 mmol/paw solutions, respectively. The neutral glutamate solution was prepared by adding 1 M sodium hydroxide to obtain a solution at pH 7. The acetic acid was diluted to 2% and the different values of pH were obtained by adding 1 M chloridric acid or 1 M sodium hydroxide. The solutions were injected into the ventral surface of the right hind paw. After the injection, mice were observed individually for 60 minutes and the amount of time that they spent licking or biting the injected paw was timed in intervals of 5 minutes. The sum of licking seconds accumulated in the first 15 minutes was considered representative of the nociception induced by all solutions. The glutamate concentration was chosen based in previous experiments.2
Analysis of the Mechanisms Involved in the Nociception Induced by pH-Adjusted, Non-pH-Adjusted Glutamate and Acetic Acid Solutions To evaluate the mechanisms involved in the nociceptive effect of pH-adjusted glutamate, non-pH-adjusted glutamate or acetic acid solutions, the mice were treated intraplantary (i.pl.) with 10 mL/paw amiloride10 (an antagonist of the acid-sensitive ion channel); MK-8012 (an antagonist of the glutamate receptor subtype NMDA, N-methyl-D-aspartate); capsazepine41 (an antagonist of the vanilloid receptor) or indomethacin12 (an inhibitor of cyclooxigenase) immediately before an injection of 20 mL/paw of pH-adjusted glutamate (20 mmol/paw, pH 7), non-pH-adjusted glutamate (3 mmol/paw, pH 2) or acetic acid (2%, pH 2). The concentrations of 3 mmol/ paw non-pH-adjusted glutamate and 20 mmol/paw pHadjusted glutamate were chosen for the subsequent tests because these concentrations had a similar response (seconds of licking) when injected into the hindpaw. A stock solution of indomethacin, capsazepine or amiloride was made up in DMSO plus ethanol. The final work solution was prepared immediately before use and contained 5% DMSO and 5% ethanol in saline. MK-801 was dissolved in saline solution. After the injection, the mice were observed individually for 60 minutes and the amount of time that they spent licking or biting the injected paw was timed in intervals of 5 minutes. The sum of licking seconds accumulated in the first 15 minutes was chosen as representative of nociception induced by all solutions.
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Involvement of Capsaicin-Sensitive Fibers
Table 1.
To investigate the role of capsaicin-sensitive fibers in the nociceptive response induced by pH-adjusted, nonpH-adjusted glutamate, and acid solutions, newborn mice were treated by the subcutaneous (sc) route with 50 mg/kg capsaicin on the second day of life. This treatment induces an irreversible degeneration of unmyelinated afferent neurons, especially C fibers.17 Control animals received a subcutaneous injection of the vehicle used to dissolve the capsaicin (10% ethanol, 10% Tween 80, and 80% saline). The nociceptive response caused by pH-adjusted glutamate, non-pH-adjusted glutamate or acid solutions was analyzed 6 to 7 weeks after the neonatal capsaicin or vehicle treatment. The efficiency of the treatment of mice with capsaicin was confirmed by a topical application of 50 mL capsaicin (.01%) on the right eye. The number of wiping motions occurred during the subsequent 1-minute period was counted as described previously.20 In the group treated with capsaicin neonatal, only mice that had a number of wiping motions less than 6 were considered for the experiment.
GLUTAMATE SOLUTION
Statistical Analyses The results are presented as mean 1 S.E.M. The ED50 or ID50 values (ie, the dose that induced or decreased the nociceptive response by 50% relative to the control value) were reported as geometric mean accompanied by their respective 95% confidence limits. The ED50 or ID50 values were determined by linear regression from individual experiments using linear regression GraphPad software (GraphPad software, San Diego, CA). The percentage of inhibition was calculated taking each individual response and compared to the average of the respective control group, providing a standard error for each calculated percentage. Comparisons between experimental and control groups were performed by ANOVA followed by the Newman-Keuls or Student t test when appropriate. P-values less than .05 (P < .05) were considered as indicative of significance.
Results The nociceptive effect of glutamate was measured using a non-pH-adjusted and a pH-adjusted glutamate solution. The pH of the non-pH-adjusted glutamate solution decreased with increasing glutamate concentration (Table 1). Besides, the nociceptive action of glutamate was directly increased raising the glutamate concentration (Table 1). A comparative study with the non-pH-adjusted glutamate, pH-adjusted glutamate and acid solution showed that the nociceptive effect of glutamate is more potent in an acid condition. The ED50 values were 2.5 (1.5–4.2) mmol/paw and 15.1 (11.5–19.9) mmol/paw for the non-pH-adjusted and pHadjusted glutamate, respectively. The licking behavior was initiated from 1 mmol/paw in mice injected with a non-pH-adjusted glutamate solution. However, a solution of 10 mmol/paw was necessary to initiate the licking behavior in mice injected with a pH-adjusted glutamate
Licking Behavior and Correspondent pH in Different Concentrations of Glutamate
.3 mmol/paw 1 mmol/paw 3 mmol/paw 10 mmol/paw 20 mmol/paw 30 mmol/paw
PH
LICKING (S)
2.24 2.1 2 1.56 1.34 1.14
762 63 6 14 136 6 26 200 6 17.2 212 6 30.3 240 6 30.1
NOTE. The nociception was evaluated within 15 minutes after glutamate injection. The data are mean 6 S.E.M. of 6 mice.
solution (Fig 1). This demonstrated that the protons present in the glutamate solution potentiate the algesic effect of this neurotransmitter. Taking this into account, the mice were injected with an acetic acid solution at different pH values to evaluate the sensitization caused by the protons. The mice presented a nociceptive behavior from the lowest pH used (pH 1.4). The same nociceptive behavior was observed at pH 2 and 4, but it was absent at pH 6 and 7 (Fig 1). To investigate the activation of the acid-sensitive ion channel in the nociception caused by the acid glutamate, neutral glutamate, and acid solutions, the mice received an i.pl. injection of amiloride prior to the algogens. The nociception caused by the non-pH-adjusted glutamate (3 mmol/paw, pH 2) and by the acid solution (2% acetic acid, pH 2) was inhibited by amiloride at the very low doses of 10 and 30 nmol/paw, respectively. As expected, amiloride was unable to inhibit the nociception induced by the pHadjusted glutamate solution (Fig 2). The calculated doses of amiloride that inhibited 50% (ID50) of the response to the non-pH-adjusted glutamate and acid solution were 18.5 (13.9–24.5) and 25.64 (23.6–28.5) nmol/paw, respectively. The maximal inhibition caused by amiloride (100 nmol/paw) was 66 6 6% for the non-pH-adjusted glutamate and 66 6 8% for the acid solution. The treatment with the NMDA-receptor antagonist, MK-801, blocked the nociceptive effect of the 3 algogens tested (Fig 3). MK-801 had a similar potency to inhibit the nociception induced by the pH-adjusted glutamate solution (20 mmol/paw, pH 7) and acid solution (2% acetic acid, pH 2) with ID50 values of 6.6 (3.3–13.3) and 7.7 (4.4–13.6) nmol/paw, respectively. In contrast, a dose of 18.7 (15–23.2) nmol/paw was necessary to inhibit 50% of the response induced by the non-pH-adjusted glutamate (3 mmol/paw, pH 2). MK-801 (50 nmol/paw) was more efficacious in inhibiting the nociception caused by the acid solution, achieving an inhibition of 89 6 8%. When tested against pH-adjusted and non-pH-adjusted glutamate solution, MK-801 (50 nmol/paw) inhibited 62 6 6 and 68 6 11% of the nociception, respectively. The TRPV1 antagonist, capsazepine, greatly inhibited the nociception induced by the acid solution (2% acetic acid, pH 2). A higher dose of capsazepine was necessary to inhibit significantly the nociception induced by nonpH-adjusted glutamate (3 mmol/paw, pH 2). Capsazepine was ineffective against the nociception induced by pHadjusted glutamate (20 mmol/paw, pH 7) (Fig 4). The
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Figure 1. Nociceptive effect of different concentrations of non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 20 ml/paw was injected into the right hindpaw and the licking behavior was monitored for 15 minutes. The control group (C) received 20 ml/paw of saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M.
ID50 values were 14.8 (8–27.4) and 63.9 (42–97) pmol/paw for the acid and non-pH-adjusted glutamate solutions, respectively. Capsazepine (100 pmol/paw) had a maximal inhibition of 64 6 5 and 73 6 6% for the acid and nonpH-adjusted glutamate solutions, respectively. To investigate whether the inflammatory mediator prostaglandins are produced in the nociception caused by non-pH-adjusted glutamate, pH-adjusted glutamate and acid solutions, the mice were pretreated with the cyclooxygenase inhibitor indomethacin. Indomethacin from 1 ng/paw caused a significant inhibition of the licking behavior induced by acid solution (2% acetic acid, pH 2). In contrast, only 100 ng/paw and 200 mg/paw indomethacin inhibited significantly the nociception caused by the non-pH-adjusted glutamate (3 mmol/paw, pH 2) and pH-adjusted glutamate solutions (20 mmol/paw, pH 7), respectively (Fig 5). The ID50 values were 7.8
(3.3–18.4) and 636.3 (203.1–1993.2) ng/paw for acid and non-pH-adjusted glutamate solutions, respectively. The maximal inhibitions were 75 6 10 % (1 mg/paw), 71 6 11% (100 mg/paw) and 48.8 6 11% (200 mg/paw) for the nociception induced by acid, non-pH-adjusted glutamate and pH-adjusted glutamate solution, respectively. The disruption of the sensorial C fibers, through the neonatal treatment with capsacin, significantly decreased the nociception mediated by acid (38 6 2%), pH-adjusted glutamate (55 6 7%) and acid glutamate (55 6 5%) solutions (Fig 6). The confirmation of the disruption of the C fibers in animals treated with capsaicin was obtained through the topical application of capsaicin on the right eye. Only mice that had a number of wiping motions less than 6 were considered for the experiment; this proved that capsaicin treatment was effective in disrupting the sensorial C-fibers.
Figure 2. Effect of different concentrations of amiloride on the nociceptive response in non-pH-adjusted glutamate (white bars), pHadjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw of amiloride was injected immediately before the injection of non-pH-adjusted or pH-adjusted glutamate or acid solution (20 ml/paw) and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw of saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05 and **P < .01) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
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Figure 3. Effect of different concentrations of MK-801 on the nociceptive response in non-pH-adjusted glutamate (white bars), pHadjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw MK-801 was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw of saline solution (NaCl .9%, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05, **P < .01 and ***P < .001) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
Discussion The stimulation of glutamate receptors, after an intraplantar or intrathecal injection of glutamate, has been extensively used to investigate the mechanisms of nociception and in the search for new analgesic drugs.2,10,33,43 Previous studies have demonstrated that the nociception induced by glutamate occurs through the release of neurokinins, kinins, CGRP, and nitric oxide at the site of the injection3,11 and by opening of the cation channels sensitive to amiloride.10 Our concern is that these mechanisms may not be due to the action of the glutamate on its receptors, but rather to the activation of cation channels that are sensitive to the protons dissociated from L-glutamic acid in the solution.
In this study, we found that the protons released from L-glutamic acid induced nociception in mice. As seen by the ED50 values, the protons present in the solution potentiated 6-fold the nociceptive effect of glutamate. In fact, a concentration of 10 mmol/paw of neutral glutamate solution was necessary to induce a consistent licking behavior, while 1 mmol/paw of non-pH-adjusted glutamate solution was sufficient to cause this effect. To compare the contribution of the protons in the nociception induced by glutamate, we used an acid solution at pH 2, which corresponded to the pH of the glutamate in a non-pH-adjusted solution. The nociception induced by protons acts as a sensor when the pH drops in conditions like ischemia (eg,
Figure 4. Effect of different concentrations of capsazepine on the nociceptive response in non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/paw capsazepine was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/ paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw saline solution (NaCl .9%, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference ***P < .001 from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
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Figure 5. Effect of different concentrations of indomethacin on the nociceptive response in non-pH-adjusted glutamate (white bars), pH-adjusted glutamate (crossed bars), and acid (black bars) solutions injected into the mouse hindpaw. A volume of 10 ml/ paw indomethacin was injected immediately before the injection of non-pH-adjusted glutamate, pH-adjusted glutamate or acid solution (20 ml/paw), and the licking behavior was evaluated for 15 minutes. The control group (C) received 10 ml/paw saline solution (.9% NaCl, pH 7). Each column represents the mean of 6 mice 6S.E.M. The symbols denote a significant difference (*P < .05, **P < .01 and *** P< .001) from the control group. The statistical analyses were performed by 1-way ANOVA, followed by the Newman-Keuls test.
angina) or intense muscle work (anaerobic metabolism). In these situations a very small variation in the pH (.2) is able to sensitize specific receptors, such as ASIC receptors.21,22 In this study, the mice showed a nociceptive behavior only when the pH decreased by at least 2 units, pH 4. Interestingly, we did not observe any significant licking behavior at pH 6. This was probably because the intensity of the stimulus at pH 6 is not sufficient to induce a spontaneous response in our model of nociception. Therefore, although the pH is low and ASICs were likely sensitized to cause a phenomenon of hypernoci-
Figure 6. Effect of the neonatal treatment with capsaicin on the nociceptive behavior induced by non-pH-adjusted glutamate (3 mmol/paw, pH 2), pH-adjusted glutamate (20 mmol/ paw, pH 7) or acetic acid (2%, pH 2) solutions injected into the mouse hindpaw. The mice were treated with capsaicin (50 mg/ kg, sc) or sterile saline (.9% NaCl, sc) on the second day of life. The licking behavior was evaluated in the 6th week of life. The columns represent the amount of time the mice spent licking the injected paw within 15 minutes. Each column represents the mean of 4 mice 6S.E.M. The symbols denote a significant difference (*P < .05 and **P < .01) from the group treated with saline by t-test.
ception, this effect did not induce an overt nociceptive behavior. The role of the ASICs in nociception has been studied using gene deletion for this receptor. However, it is hard to identify the exact role of these receptors using knockout mice because these receptors form heteromultimers leading to conflicting results regarding their role in sensitivity.8,34,46 A good tool to study the function of this receptor is the pharmacological antagonism with amiloride. Amiloride is a K1-sparing diuretic that blocks the members of the ASICs family. ASICs are responsible for controlling pH homeostasis, transduction of mechanical stimuli, and nociception.10,13,15,37 In our study, the blocking of ASICs by amiloride prevented, with a similar potency, the nociception induced by the acid solution and by the non-pH-adjusted glutamate solution. However, amiloride did not prevent the nociception induced by the pH-adjusted glutamate solution. This demonstrates that the nociception induced by an injection of a non-pH-adjusted glutamate solution is mostly caused by the activation of the ASICs. The effect of protons in potentiating the nociception induced by glutamate was further confirmed by pretreatment with the NMDA receptor antagonist MK-801. The concentration of MK-801 that decreased 50 % of the response induced by the non-pH-adjusted glutamate solution (ID50) was 3-fold higher than the ID50 value for decreasing the nociception induced by the pH-adjusted glutamate solution. In addition, the antagonism of the NMDA receptor reduced the nociception induced by acid solution. Taking this into account, it is likely that protons and glutamate that are present in the non-pH-adjusted solution are acting synergistically to depolarize the cell. These results are in agreement with previous findings that showed a regulatory effect of ASICs on the activity of NMDA receptors. In fact, a rapid acidic transient accompanying synaptic transmission provides a membrane depolarization by ASICs. This depolarization
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facilitates the release of the Mg from NMDA receptors (voltage-dependent activation).25,26 As a result, the NMDA receptor is susceptible to activation by glutamate that is released from synaptic vesicles or supplied by an exogenous source. The lower potency of MK-801 in decreasing the nociception induced by the non-pH-adjusted glutamate solution compared to acid and pH-adjusted glutamate solution suggests that, beside the modulatory effect of ASICs on NMDA receptors, the acid solution might activate others mediators or intracellular pathways involved in neuron sensitization.1,30 It is worthwhile to mention that the nociception caused by glutamate is also dependent on the activation of non-NMDA receptors in neurons from the periphery. However, the participation of the non-NMDA receptor is clearly less significant than the NMDA receptor.2 In this concern, we have chosen the most representative receptor that is being activated during intraplantar injection of glutamate to compare the effects between glutamate, acid, and acid plus glutamate solution. The activation of TRPV1 was crucial for the nociceptive transmission induced by acid and by acid glutamate. This was not surprising since TRPV1 works as a sensor for the alteration of pH.5 On the other hand, the TRPV1 antagonist capsazepine did not inhibit the nociception induced by glutamate in a pH-adjusted solution. The TRPV1 receptor contains phosphorylation sites in the intracellular loop responsible for modulating the activity of the receptor. The phosphorylation of TRPV1 by protein kinases is a secondary mechanism triggered, for example, by activation of calcium/calmodulin.36 This phosphorylation promotes the insertion of the channel into the plasma membrane and decreases the threshold for the activation of TRPV1 by the agonists, but does not activate the receptor directly.35,38,49 In accordance with this, our results demonstrated that the activation of glutamate receptors does not affect TRPV1 activity in the absence of protons in the solution. ASICs are overexpressed during inflammatory processes and their expression is increased by mediators like neural growth factor (NGF), serotonin, interleukin-1b, and bradykinin.32 In agreement with this, nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the expression and
the membrane potential triggered by ASICs.50 Similarly to the ASICs, the vanilloid receptor TRPV1 is also sensitive to activation by products of cyclooxygenase.9,19 Herein, we demonstrated, for the first time, that the inhibition of the synthesis of prostanoids by indomethacin greatly decreased the nociception induced by protons in vivo. The inhibitory effect of indomethacin was greater in mice treated with acid solution than in those treated with acid glutamate solution. Thus, the inhibition of COX activity could, in part, hinder the direct increase in sensitivity of the ASICs and TRPV1 caused by arachidonic acid metabolites. However, the eicosanoids derived from COX may also contribute to the nociceptive response induced by acid solution by other mechanisms beside the activation of ASICs and TRPV1.30,45,52 The nociception caused by the 3 algogens tested were essentially dependent on the integrity of sensory C fibers. The disruption of C fibers by the neonatal treatment with capsaicin reduced in a similar manner the nociceptive transmission induced by acid, pH-adjusted and non-pHadjusted glutamate. A previous study had already demonstrated that the nociception induced by glutamate solution was decreased after the depletion of C fibers.3 However, no data had demonstrated that both components, glutamate and protons, have their nociceptive effect dependent on activation of sensory C fibers. In conclusion, our results demonstrated that the protons released from L-glutamic acid in aqueous solution can themselves cause nociception and can potentiate the nociception caused by glutamate. These effects were mainly dependent on the activation of ASICs and TRPV1, but were also antagonized by the blocking of the NMDA receptor, inhibition of the prostanoids synthesis, and disruption of C fibers. In this regard, when the nociceptive effect is desired only on the glutamatergic system, it is important to exclude the protons from the solution.
References
5. Caterina MJ, Julius D: The vanilloid receptor: A molecular gateway to the pain pathway. Annu Rev Neurosci 24: 487-517, 2001
1. Allen NJ, Attwell D: Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals. J Physiol 543:521-529, 2002 2. Beirith A, Santos ARS, Calixto JB: Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Res 924:219-228, 2002 3. Beirith A, Santos ARS, Calixto JB: The role of neuropeptides and capsaicin-sensitive fibres in glutamate-induced nociception and paw oedema in mice. Brain Res 969:110-116, 2003 4. Carlton SM: Peripheral excitatory amino acids. Curr Opin Pharmacol 1:52-56, 2001
Acknowledgments The authors are grateful to Siobhan Wiese by the critical revision of the manuscript.
6. Coggeshall RE, Carlton SM: Receptor localization in the mammalian dorsal horn and primary afferent neurons. Brain Res Brain Res Rev 24:28-66, 1997 7. Dickenson AH: Central acute pain mechanisms. Ann Med 27:223-227, 1995 8. Drew LJ, Rohrer DK, Price MP, Blaver KE, Cockayne DA, Cesare P, Wood JN: Acid-sensing ion channels ASIC2 and ASIC3 do not contribute to mechanically activated currents in mammalian sensory neurones. J Physiol 556:691-710, 2004 9. Ferreira J, da Silva GL, Calixto JB: Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol 141:787-794, 2004
Meotti, Coelho, and Santos 10. Ferreira J, Santos ARS, Calixto JB: Antinociception produced by systemic, spinal and supraspinal administration of amiloride in mice. Life Sci 65:1059-1066, 1999 11. Ferreira J, Santos ARS, Calixto JB: The role of systemic, spinal and supraspinal L-arginine-nitric oxide-cGMP pathway in thermal hyperalgesia caused by intrathecal injection of glutamate in mice. Neuropharmacology 38:835-842, 1999 12. Funez MI, Ferrari LF, Duarte DB, Sachs D, Cunha FQ, Lorenzetti BB, Parada CA, Ferreira SH: Teleantagonism: A pharmacodynamic property of the primary nociceptive neuron. Proc Natl Acad Sci U S A 105:19038-19043, 2008 13. Fyfe GK, Quinn A, Canessa CM: Structure and function of the Mec-ENaC family of ion channels. Semin Nephrol 18: 138-151, 1998 14. Gadotti VM, Tibola D, Paszcuk AF, Rodrigues AL, Calixto JB, Santos ARS: Contribution of spinal glutamatergic receptors to the antinociception caused by agmatine in mice. Brain Res 1093:116-122, 2006 15. Garty H, Benos DJ: Characteristics and regulatory mechanisms of the amiloride-blockable Na1 channel. Physiol Rev 68:309-373, 1988 16. Hesselager M, Timmermann DB, Ahring PK: pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits. J Biol Chem 279:11006-11015, 2004 17. Holzer P: Capsaicin as a tool for studying sensory neuron functions. Adv Exp Med Biol 298:3-16, 1991 18. Hudspith MJ: Glutamate: A role in normal brain function, anaesthesia, analgesia and CNS injury. Br J Anaesth 78:731-747, 1997 19. Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D, Oh U: Direct activation of capsaicin receptors by products of lipoxygenases: Endogenous capsaicin-like substances. Proc Natl Acad Sci U S A 97: 6155-6160, 2000
The Journal of Pain
577
of acid sensing ion channels in brain and dorsal root ganglion cells. J Biol Chem 272:29778-29783, 1997 29. Liu XJ, White TD, Sawynok J: Intraplantar injection of glutamate evokes peripheral adenosine release in the rat hind paw: Involvement of peripheral ionotropic glutamate receptors and capsaicin-sensitive sensory afferents. J Neurochem 80:562-570, 2002 30. Lopez JC: ASIC-nal integrator. Nature Review Neuroscience 3:763, 2002 31. Malmberg AB, Yaksh TL: Cyclooxygenase inhibition and the spinal release of prostaglandin E2 and amino acids evoked by paw formalin injection: A microdialysis study in unanesthetized rats. J Neurosci 15:2768-2776, 1995 32. Mamet J, Baron A, Lazdunski M, Voilley N: Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci 22: 10662-10670, 2002 33. Meotti FC, Luiz AP, Pizzolatti MG, Kassuya CA, Calixto JB, Santos ARS: Analysis of the antinociceptive effect of the flavonoid myricitrin: Evidence for a role of the L-arginine-nitric oxide and protein kinase C pathways. J Pharmacol Exp Ther 316:789-796, 2006 34. Mogil JS, Breese NM, Witty MF, Ritchie J, Rainville ML, Ase A, Abbadi N, Stucky CL, Seguela P: Transgenic expression of a dominant-negative ASIC3 subunit leads to increased sensitivity to mechanical and inflammatory stimuli. J Neurosci 25:9893-9901, 2005 35. Montell C: The TRP superfamily of cation channels. Sci STKE 2005:re3, 2005 36. Numazaki M, Tominaga T, Takeuchi K, Murayama N, Toyooka H, Tominaga M: Structural determinant of TRPV1 desensitization interacts with calmodulin. Proc Natl Acad Sci U S A 100:8002-8006, 2003 37. Palmer LG: Epithelial Na channels: Function and diversity. Annu Rev Physiol 54:51-66, 1992
20. Ikeda Y, Ueno A, Naraba H, Oh-ishi S: Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 69:2911-2919, 2001
38. Premkumar LS, Ahern GP: Induction of vanilloid receptor channel activity by protein kinase C. Nature 408: 985-990, 2000
21. Immke DC, McCleskey EW: ASIC3: A lactic acid sensor for cardiac pain. ScientificWorldJournal 1:510-512, 2001
39. Reeh PW, Kress M: Molecular physiology of proton transduction in nociceptors. Curr Opin Pharmacol 1:45-51, 2001
22. Immke DC, McCleskey EW: Protons open acid-sensing ion channels by catalyzing relief of Ca21 blockade. Neuron 37:75-84, 2003 23. Julius D, Basbaum AI: Molecular mechanisms of nociception. Nature 413:203-210, 2001
40. Sakurada T, Sugiyama A, Sakurada C, Tanno K, Sakurada S, Kisara K, Hara A, Abiko Y: Involvement of nitric oxide in spinally mediated capsaicin- and glutamateinduced behavioural responses in the mouse. Neurochem Int 29:271-278, 1996
24. Juranek I, Lembeck F: Afferent C-fibres release substance P and glutamate. Can J Physiol Pharmacol 75: 661-664, 1997
41. Santos ARS, Calixto JB: Ruthenium red and capsazepine antinociceptive effect in formalin and capsaicin models of pain in mice. Neurosci Lett 235:73-76, 1997
25. Krishtal O: The ASICs: Signaling molecules? Modulators? Trends Neurosci 26:477-483, 2003
42. Savegnago L, Pinto LG, Jesse CR, Alves D, Rocha JB, Nogueira CW, Zeni G: Antinociceptive properties of diphenyl diselenide: Evidences for the mechanism of action. Eur J Pharmacol 555:129-138, 2007
26. Krishtal OA, Osipchuk YV, Shelest TN, Smirnoff SV: Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Res 436:352-356, 1987 27. Lawand NB, McNearney T, Westlund KN: Amino acid release into the knee joint: Key role in nociception and inflammation. Pain 86:69-74, 2000
43. Scheidt C, Santos ARS, Ferreira J, Malheiros A, CechinelFilho V, Yunes RA, Calixto JB: Evidence for the involvement of glutamatergic receptors in the antinociception caused in mice by the sesquiterpene drimanial. Neuropharmacology 43:340-347, 2002
28. Lingueglia E, de Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M: A modulatory subunit
44. Sluka KA, Westlund KN: An experimental arthritis model in rats: The effects of NMDA and non-NMDA antagonists on
578
The Journal of Pain
aspartate and glutamate release in the dorsal horn. Neurosci Lett 149:99-102, 1993 45. Smith HS: Arachidonic acid pathways in nociception. J Support Oncol 4:277-287, 2006 46. Staniland AA, McMahon SB: Mice lacking acidsensing ion channels (ASIC) 1 or 2, but not ASIC3, show increased pain behaviour in the formalin test. Eur J Pain 13:554-563, 2009 47. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple painproducing stimuli. Neuron 21:531-543, 1998 48. Ugawa S, Ueda T, Ishida Y, Nishigaki M, Shibata Y, Shimada S: Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Invest 110:1185-1190, 2002
Protons and Glutamate Induce Nociception 49. Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA: Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol 534:813-825, 2001 50. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 21:8026-8033, 2001 51. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 386:173-177, 1997 52. Zeilhofer HU: Prostanoids in nociception and pain. Biochem Pharmacol 73:165-174, 2007 53. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110, 1983