Activation of ATP-sensitive potassium channels antagonize nociceptive behavior and hyperexcitability of DRG neurons from rats
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Nociceptive responses to noxious stimuli are initiated at peripheral nociceptor terminals. Ion channels play a vital role in pain signal initiation and conduction. Activation of KATP channels has been implicated in mediating the analgesic effects of agents such as morphine. However, systematic studies regarding the effects of KATP activators on nociception and neuronal excitability are scarce.
In this study, we describe the antagonistic effects of KATP activators pinacidil and diazoxide on nocifensive behavior induced by bradykinin (BK), thermo and mechanical stimuli, and the bradykinin-induced hyperexcitability of DRG neurons. We also found that KATP activators can moderately activate KATP in DRG neurons. Because the effects of KATP activators can be reversed by the KATP blocker glyburide, direct activation of KATP is most likely the underlying mechanism.
This systematic study clearly demonstrates that activation of KATP could have significant modulatory effects on the excitability of sensory neurons and thus on sensory behaviors, such as nociception. KATP activators can be evaluated clinically for the treatment of pain symptoms.
KeywordsKATP potassium channels DRG excitability nociception pinacidil diazoxide glyburide
Ion channels play a vital role in pain signal initiation and conduction . For example, activation of TRPV1 following a heat stimulus generates inward currents in the nociceptor peripheral terminal and results in action potentials in the nociceptor axon, leading to pain sensation [2, 3, 4, 5]. The TRP channel family and voltage gated sodium channels are among the most intensively studied ion channels in pain signaling. Until recently, less attention has been paid to the role of potassium (K+) channels in pain [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. K+ channels play an essential role in setting the resting membrane potential and in controlling the excitability of neurons. Thus, K+ channels represent potentially attractive peripheral targets for the treatment of pain. One K+ channel that is known to regulate excitability in a variety of central and peripheral neurons is the M channel [18, 19]. Activation of the M channel by retigabine inhibits responses to the intrapaw application of carrageenan  and bradykinin  in rat nociceptive behavioral studies. The M channel blocker XE991 evokes spontaneous pain in rats [12, 20].
Another family of K+ channels that has recently been indicated in pain responses is the ATP-sensitive potassium channel (KATP) family. These channels are widely expressed in central neurons, wherein they regulate membrane excitability and neurotransmitter release, and they provide neuroprotection [21, 22]. It has been suggested that KATP may mediate the analgesic effects of morphine , clonidine  and 5-HT1 agonists  because the antinociceptive effects of these agents could be reversed by pretreatment with selective KATP antagonists but not other potassium channels blockers . Studies indicate that the nitric oxide (NO) pathway mediates the morphine activation of KATP [16, 27]. Indeed, NO donors produce peripheral antinociceptive effects in inflammatory pain [27, 28] and directly activate KATP in rat DRG neurons .
The existence of KATP in peripheral sensory neurons has only recently been confirmed [8, 13, 14, 15, 17]. However, in vitro studies on DRG neurons demonstrate whole-cell KATP currents that either show unusual rectification properties or are expressed in only a subpopulation of the neurons [8, 11]. Activation or inhibition of KATP has only minor effects on the resting membrane potential of the subpopulation of neurons [11, 15]. It has been suggested [13, 15] that KATP may play a major role in large diameter DRG neuron-mediated neuropathic pain. There is also evidence suggesting that the anti-nociceptive effects of KATP activators may stimulate mechanisms that produce anti-nociception through opioid receptor activation [29, 30]. However, studies on the effects of direct KATP activation on peripheral antinociception are scarce [31, 32, 33].
The primary purpose of the present study is to establish the effects of direct activation of KATP on pain sensation and the excitability of sensory neurons. We performed experiments testing the effects of two KATP activators, diazoxide and pinacidil, on pain behaviors induced by bradykinin and thermal and mechanical stimuli. We also studied the effects of KATP activators on the excitability and whole-cell currents of DRG neurons. The results show that activation of KATP antagonizes the rat nociceptive behavior induced by all tested stimuli and dampens the hyperexcitability of DRG neurons induced by bradykinin.
The KATP channel activators antagonize bradykinin-induced spontaneous pain behavior
To confirm that the observed effects of the KATP activators were the results of direct actions on the nociceptors rather than some indirect systemic effects of the KATP activators, two extra control experiments were performed. First, to exclude the possibility that the decreased behavioral responses to BK was the result of a systemic effect of the KATP activators (i.e. lowered blood pressure), pinacidil was injected into the contralateral paw (left) of the paw (right) injected with BK, and the BK-induced behavioral responses were compared with those when pinacidil was co-injected with BK in the same paw (right). As shown in Figure S1A (Additional file 1, Figure S1A), contralateral paw injection of pinacidil did not affect BK-induced behavioral responses. The second control experiments were performed to exclude the possibility that the observed anti-nociceptive effects of KATP activators were the results of the vasodilatation, since the primary effect of pinacidil is a vasodilator. The increase in arteriolar blood flow may potentially affect the behavioral outcome. Although apparent vasodilatation was not observed upon injection of KATP activators visually, a control vasodilator, phentolamine, was used as a control vasodilator to be tested for its effect on nociceptive behavior. Clearly, as shown in Figure S1B (Additional file 1, Figure S1B), phentolamine did not affect the BK-induced behavioral responses.
The KATP channel activators antagonize thermal pain behavior
We used the Hargreaves test , in which the paw is heated by a radiant heat source, to study the effects of the KATP activators on thermal nociception. In this case, the latency to paw licking or withdrawal was measured in the absence or presence of KATP activators and blockers. Thermal nociceptive behavior was studied 8 min after the intraplantar injection of 50 μl of one of the following solutions: solvent alone, the KATP channel activators, and the activators plus blockers. As demonstrated in Figure 1B, compared with the solvent control, both diazoxide (100 μM) and pinacidil (10 μM) significantly increased the time latencies for thermal nociceptive behavior (Solvent, 23 ± 2.8 s, n = 12; Pin, 36 ± 2.9 s, n = 12; Dia 35 ± 3.0 s, n = 13). Pre-injection of glyburide together with the KATP activators reversed the anti-thermal nociceptive effects of the KATP channel activators, although the effects of glyburide on diazoxide did not reach statistical significance (Figure 1B, Gly + Pin, 26 ± 1.7 s, n = 12; Gly + Dia, 31 ± 4.1 s, n = 10). Glyburide alone did not affect thermal nociceptive behavior (26 ± 2.5 s, n = 13). These results show that activation of KATP can reduce the sensitivity to noxious heat stimuli.
The KATP channel activators antagonize the nociceptive response to mechanical stimuli
We used von Frey filaments to test withdrawal thresholds for mechanical stimuli applied to the hind paw of rats. The solvent control or KATP activators (50 μl) were injected into the plantar of the rat hind paw, and the response to the mechanical stimuli was measured 8 min later. The KATP activators significantly increased the threshold for nocifensive withdrawal of the hind paw in response to mechanical stimuli (Figure 1C, Solvent, 23 ± 3.8 s, n = 12; Pin, 41 ± 6.5 s, n = 10; Dia, 44 ± 5.8 s, n = 12). Pre-injection of glyburide together with the KATP activators reversed the effects of the KATP channel activators (Figure 1C, Gly + Pin, 26 ± 4.9 s, n = 12; Dia + Glib, 23 ± 4.2 s, n = 11). Glyburide alone did not affect mechanical nociceptive behavior (25 ± 5.9 s, n = 11). These results show that activation of KATP can reduce the sensitivity to noxious mechanical stimuli.
Effects of the KATP activators on the increased excitability of DRG neurons induced by BK
In our earlier study, we showed that BK can depolarize the membrane potential and increase the firing frequency of DRG neurons by inhibiting M-type K+ currents and activating Ca2+ activated Cl- currents . We examined whether activation of KATP could affect the increased excitability of DRG neurons induced by BK.
Effects of KATP activators and blockers on currents recorded from rat DRG neurons
The currents recorded using the protocol described above are shown in Figure 4. The dotted line in Figure 4A indicates the zero current level. Substantial basal outward currents were observed at -20 mV, whereas small inward currents were seen at -140 mV. Small slow deactivating tail currents were observed when the membrane potentials were step changed from -20 mV to -50 mV or -60 mV, and these currents eventually reached the zero current level when the membrane potential was at -60 mV (Figure 4A, control). These characteristics of the basal currents are consistent with the presence of outwardly rectifying K+ currents (K+ reversal potential is calculated to be around -100 mV). The resting membrane potential for this cell should be around -60 mV. Pinacidil (10 μM) evoked a small increase of both the outward currents at -20 mV and the inward currents at -140 mV, and these effects were blocked by glyburide (10 μM). Neither pinacidil nor glyburide significantly affected the currents at -50 mV or -60 mV (Figure 4A). Figure 4B shows the time course of the currents recorded at -20 mV and - 140 mV. Pinacidil and diazoxide slightly increased the inward current at -140 mV and the outward currents at -20 mV, and these effects were also reversed by glyburide, consisted with the increased currents being KATP currents. To demonstrate the effect of pinacidil and diazoxide more clearly, the current traces from -140 mV were rescaled (Figure 4B, squared box). Capsaicin, an agonist of the TRPV channel, was applied at the end of the experiments and usually evoked a large inward current at both -140 mV and -20 mV (Figure 4B).
The basal currents were large compared with the increased currents by KATP activators (Figure 4). Because we do not know the source of these currents, including these currents in the analysis of the effects of the KATP activators and blocker (Figure 5A) could underestimate the effects of the KATP activators. In addition, not all neurons responded to the KATP activators, but these neurons were included in the analysis shown in Figure 5A. To better assess the actions of the KATP activators and blocker, we re-analyzed the data, and the results are shown in Figure 5B. In this case, only neurons that had a visible response to the KATP activators (31 out of 60) and blocker (6 out of 60) were included for analysis. Furthermore, the basal current levels were taken as a zero current level, and only the currents increased or reduced by the KATP activators and blocker were used for the analysis. Thus, the basal currents were excluded from the analysis. Figure 5B shows that both pinacidil (n = 25) and diazoxide (n = 16) significantly activated currents recorded at -140 mV, and these effects were abolished by glyburide. Of the 60 neurons tested, 23 did not respond to the KATP activators or blocker and were not included in the analysis shown in Figure 5B. These results suggest that the KATP activators activate the KATP currents in a subpopulation of DRG neurons.
In this study, we described the antagonistic effects of the KATP activators, pinacidil and diazoxide, on nocifensive behavior induced by bradykinin, thermal and mechanical stimuli and the bradykinin-induced hyperexcitability of DRG neurons. We also found that the KATP activators can moderately activate KATP in DRG neurons. Because the effects of the KATP activators could be reversed by the KATP blocker glyburide, the direct activation of KATP is the most likely the underlying mechanism. This systematic study clearly demonstrates that activation of KATP could have significant modulatory effects on excitability of the sensory neurons and thus on sensory behavior, such as nociception.
Many studies have provided evidence suggesting that analgesic agents, such as opioid and non-opioid receptor agonists, exert their antinociceptive effects, at least partly, through activation of KATP . In these studies, the involvement of KATP was suggested from the fact that the antinociceptive effects of these analgesic agents could be inhibited by a KATP blocker. Thus, KATP activators are expected to show antinociceptive effects. However, conflicting results were obtained when KATP activators were tested against different pain models. Furthermore, even in the studies that did observe antinociceptive effects, the effects were attributed to the KATP-mediated activation of opioid receptors . In these studies, KATP activators were given via intracerebroventricular (i.c.v.), intrathecal (i.t.) or epidural injection. Thus, the direct effects of KATP on the excitability of sensory neurons could not be determined from these studies. A few studies have also observed the effects of KATP activators on peripheral nociception. In these studies, KATP activators were tested against the hyperalgesia caused by carrageenan or prostaglandin E(2) or in a formalin pain model. The activators reduced pain behavior in these studies [31, 32, 33]. In the first part of our present study, we studied the effects of two KATP activators, pinacidil and diazoxide, on three different pain models, and the results unequivocally demonstrate that activation of KATP antagonizes the nocifensive behaviors induced by bradykinin and thermal and mechanical noxious stimuli. Bradykinin is a well-known inflammatory mediator and one of the most potent endogenous pain-inducing substances [35, 36]. In our earlier study, we attributed BK-induced spontaneous pain to the inhibition of M channel K+ currents and the activation of Ca2+-activated Cl- currents in DRG neurons . BK can also induce hyperalgesia and allodynia [36, 37], possibly mediated by sensitization of TRPV1  and TRPA1  through the activation of PKCε . Activation of M channels by the specific channel activator retigabine antagonizes BK-induced pain behavior, indicating an important role for M-type K+ channels in the control of sensory neuronal excitability . On the other hand, thermal and mechanical nociception are mediated by TRPV channels [2, 41, 42, 43, 44]. Clearly these different nociceptive stimuli employ different mechanisms for inducing nociception, but they should all initiate the nociceptive signals by increasing the excitability of nociceptors. The non-discriminating effects of KATP activators in antagonizing the nocifensive behaviors induced by different stimuli indicate the functional presence of KATP in all the nociceptors mediating bradykinin-induced and thermal and mechanical nociception. On the other hand, the KATP blocker glyburide did not affect any of the three nocifensive behaviors in the absence of KATP activators. This suggests that KATP does not necessarily play a role in the nociception induced by these stimuli, merely that the existence of these channels provides a means of reducing hyperexcitability when KATP activity is enhanced.
Effects of KATP activators on excitability of DRG neurons are in agreement with the results from our nocifensive behavioral study. Thus, pinacidil reversed the hyperexcitability induced by BK in DRG neurons. In addition, pinacidil also reversed the membrane depolarization induced by BK. These effects of pinacidil are very efficient and striking, and in all cases, the effects of BK were completely reversed by pinacidil (Figure 2, 3 and 4). On the other hand, neither the KATP activators nor the blocker per se affected the firing frequency or resting membrane potentials of DRG neurons in the absence of BK. Thus, as we observed in the nocifensive behavior study, the effects of KATP can only be manifested when nociception/DRG hyperexcitability/depolarization were initiated by nociceptive stimuli. An increase in the inward currents and/or a decrease in the outward currents is the first step in the activation of nociceptors, which subsequently depolarizes the membrane and increases the firing of APs. This scenario may provide a setting for the manifestation of KATP function. As demonstrated in a previous report, BK increases the excitability of DRG neurons through the inhibition of M-type currents and the activation of Ca2+-activated Cl- currents . M-type K+ channels might be the dominate contributor to the resting K+ conductance  in neurons, thus masking the contribution of other K+ channels, such as KATP. The reduction of M-type K+ currents would allow the function of other K+ channels to be unmasked. In addition, membrane depolarization (Ca2+ influx and inward currents through Cl- currents) would increase the driving force for outward KATP currents. The combination of these two changes could explain the observed effects of KATP activators under nociceptor activation. This possibility may not be limited to the explanation of KATP activators antagonizing BK effects. In fact, with regard to the modulation of M-type currents and membrane depolarization, other nociceptive stimuli may result in same molecular environment as BK. The core of the BK mechanism is an increase in intracellular Ca2+ , which should be shared by many other stimuli which activate TRPV channels.
In the present study, KATP only induced small glyburide-sensitive currents in a subpopulation of rat DRG neurons. This modest effect of KATP activators indicates that KATP function is not subject to modulation under resting conditions. Furthermore, lack of significant effects of glyburide on the basal currents of DRG neurons further implies that KATP does not contribute substantially to the resting membrane conductance of DRG neurons. This is in line with the observations from our nocifensive and excitability experiments. A systematic analysis of KATP distribution in subgroup DRG neurons is lacking. Population of the small size DRG neurons are believed to be the nociceptors. Clearly, these small DRG neurons have functional KATP . However, the activity of the KATP in the small DRG neurons seems lower than that of the KATP in the large DRG neurons, and furthermore, these KATP in two sizes of DRG neurons may contributed differently to the development of the neuronpathic pain . The small nociceptive DRG neurons are polymodal and are categorized into two major classes based on peripheral and central target fields, trophic factor dependence, and biochemical properties . These two classes of nociceptors, nonoverlapping populations of trkA positive (tyrosine kinase A) peptidgeric and isolectin-B4 (IB4) positive neurons may have different mechanisms in controlling the excitability and have different responsiveness to different stimuli. The expression of KATP channels in peptidergic nociceptors (CGRP positive) has been shown . However, a systematic functional and expression study of KATP in defined subpopulation of DRG neurons will help to understand the role of the KATP in nociception further.
It seems that KATP-like currents can be readily recorded at a single channel level from cell-attached or isolated inside-out patches [13, 14, 15, 17, 20]. Although all KATP channel subunits (Kir6.1 and Kir6.2) and SUR receptors (SUR1 and SUR2) can be found in DRG neurons at mRNA level, pharmacological, western blots and immunohistochemistry studies suggest that the single channel activity of KATP in DRG neuron may be the results of heteromeric Kir6.2/SUR1 complex . The reports for whole-cell KATP-like currents in DRG neurons are scarce; only two published papers have reported KATP-like whole cell currents from DRG neurons. In one of these works, KATP activator- and blocker-sensitive outwardly rectifying currents were recorded . This study also reported that the currents conveyed by KATP account for only 10-25% of the total potassium currents recorded . In the present study, we recorded a 10% increase over the basal currents in response to KATP activators (Figure 5A). In another report, the KATP activator diazoxide activated currents in only a subpopulation of DRG neurons . However, the diazoxide-activated currents were not subjected to testing for inhibition by a KATP blocker . Further experiments are needed to define the pharmacological and biophysical properties of the whole-cell KATP-like currents in DRG neurons. Taken together, our work and that of other groups suggest that KATP is not a dominant contributor to conductance under resting conditions in DRG neurons.
In summary, our behavioral and electrophysiological results support the notion that functional KATP channels are present in nociceptors. KATP opening could antagonize the nociceptive responses evoked by noxious stimuli by dampening the hyperexcitability of the nociceptors. On the other hand, KATP may not contribute significantly to the resting membrane conductance of the DRG neurons. Nevertheless, this should not dampen enthusiasm for further investigation of KATP activators as potential candidates for antinociceptive measures. In light of the findings that analgesic compounds, such as morphine, may exert antinociceptive effects through activation of KATP [16, 27], direct activators of KATP, such as pinacidil and diazoxide, are certainly worthy of further investigation for their potential analgesic actions. In this regard, our behavioral results concerning these compounds provide a solid foundation for future studies. The characteristics of molecular compositions, pharmacological modulation and biophysical properties of the KATP in DRG neurons need to be further investigated in order to find whether the KATP in DRG neurons can be specifically modulated by the channel activator to relieve the pain induced by different stimuli.
Male Sprague-Dawley rats (180-220 g) were randomly grouped and allowed to acclimatize for at least 20 min to the environment prior to the experiment. Behavioral studies were conducted in three separate experimental settings: (1) Bradykinin-induced acute spontaneous pain. The right hind paw of the animal received an intraplantar injection (50 μl) of BK (200 μM, 10 nmol/site) or solvent (saline) and the nocifensive responses (licking, biting, lifting and flinching) were recorded using a video camera for 30 min. The videos were analyzed by an observer unaware of the treatment allocations. To study the effects of activation of KATP channels on bradykinin-induced nociceptive behavior, one group of animals was pre-injected with the KATP activators. After 5 min, bradykinin(BK) was co-injected with the activators into the same site of the hind paw. Control animals were injected with solvent (0.5% DMSO in saline) instead of the KATP activators. In a second experimental group, the KATP blocker was co-applied with the KATP activators. Drugs were diluted in saline (pH 7.4) from stock solutions (dissolved in DMSO) and applied at a volume of 50 μl at the following concentrations: diazoxide, 100 μM; pinacidil, 10 μM; glyburide, 10 μM. (2) Mechanical withdrawal thresholds were measured using calibrated von Frey filaments (a set of monofilaments made from nylon filaments of varying diameter) (North Coast Medical, Inc. Morgan Hill, CA) applied to the plantar surface of the paw. Testing was initiated with an Evaluator Size 5.07 (10 g). If the animal withdrew the paw, the next weaker hair was applied. In the case of no withdrawal, the next stronger hair was applied . The cut-off was Evaluator Size 6.10 (100 g). (3) To test for thermal hyperalgesia, radiant heat was applied to the plantar surface of a hind paw from underneath a glass floor using a ray of light from a high-intensity lamp bulb. The paw withdrawal latency was recorded automatically when the paw was withdrawn from the light (TaiMeng Technology Co., Ltd. Chengdu).
Rat DRG cell culture
The dorsal root ganglia were extracted from all spinal levels of 32 adult male Sprague Dawley rats, and the neurons were dissociated as previously described . Briefly, the rats were anesthetized with an intraperitoneal (i.p.) injection of pelltobarbitalum natricum (10-20 mg/kg) and then sacrificed. The ganglia were cut into pieces, transferred into collagenase solution (1 mg/ml) and incubated for 50 min at 37°C. The ganglia were then placed into a trypsin solution (2.5 mg/ml) for 20 min at 37°C. The digested fragments were then rinsed with 2 ml DMEM plus 10% fetal bovine serum three times, centrifuged and dissociated by trituration. The ganglia were plated onto glass coverslips pre-coated with poly-D-lysine and incubated at 37°C. After the neurons had attached to the coverslips, the cell culture medium was changed to Neurobasal plus B27 supplement (Invitrogen). Neurons were used within 24 h of isolation. The diameter of the DRG neuron was measured using a calibrated micrometer mounted in the eyepiece of the microscope.
Patch electrodes were pulled from borosilicate glass and fire-polished to a final resistance of 1-2 MΩ when filled with internal solution. An Axon 700B (Axon Instruments) patch clamp amplifier was used for voltage and current clamp experiments. All recordings were performed using the amphotericin B (250 μg/ml, Sigma) perforated patch technique. The internal pipette solution contained (in mM) 150 KCl, 5 MgCl2, 10 HEPES, pH 7.4. The external solution contained (in mM) 160 NaCl, 2.5 KCl, 5 CaCl2, 1 MgCl2, 10 HEPES, and 8 glucose, pH 7.4. A low-profile perfusion chamber fed by a gravity perfusion system was used for solution exchange (2 ml/min, bath exchange time of ~15 s).
All data are given as mean ± SEM. Differences between groups were assessed by Student's t-test or one-way ANOVA followed by Bonferroni's post-hoc test. The differences were considered significant at p ≤ 0.05.
Diazoxide; pinacidil; glyburide and capsaicin were obtained from Sigma.
The use of animals in this study was approved by the Animal Care and Ethical Committee of Hebei Medical University, (Shijiazhuang, China) under the IASP guidelines for animal use.
This work was supported by National Natural Science Foundation of China (30500112) to XD and (30730031) to HZ, and by the National Basic Research Program (2007CB512100) to HZ.
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