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The Journal of Neurophysiology Vol. 79 No. 2 February 1998, pp. 670-676
Copyright ©1998 by the American Physiological Society
ka Vitásková1,
1 Institute of Physiology, AS CR, 14220 Prague 4, Czech Republic; 2 Institut für Physiologie und Experimentelle Pathophysiologie, D-91054 Erlangen, Germany
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Vyklický, Ladislav, Helena Knotková-Urbancová, Zdeka Vitásková, Viktorie Vlachová, Michaela Kress, and Peter W. Reeh. Inflammatory mediators at acidic pH activate capsaicin receptors in cultured sensory neurons from newborn rats. J. Neurophysiol. 79: 670-676, 1998. Whole cell membrane currentsinduced by the inflammatory mediators, bradykinin, 5-hydroxytryptamine (5-HT) and prostaglandin E2, were investigated in capsaicin-sensitive dorsal root ganglion (DRG) neurons from newborn rats grown on a monolayer of hippocampal glia without nerve growth factor (NGF). When firmly attached to an underlying cell, the neurons survived >14 days without growing extensive processes. A majority of the small diameter neurons (~80%) exhibited sensitivity to capsaicin (3-6 µM) and this was enhanced in solution of low pH. In acidic extracellular solution (pH 6.1), the combination of bradykinin (10 µM), 5-HT (10 µM) and prostaglandin E2 (1 µM) induced an inward membrane current in all capsaicin-sensitive DRG neurons (n = 43). The current exceeded the sustained, low pH-induced membrane current by 205 ± 53 (SE) pA. The combination of acidic inflammatory mediators was ineffective in cells that were insensitive to capsaicin. In capsaicin-sensitive neurons, the inflammatory mediators when applied singly or in any combination of two, induced no membrane currents or small current at pH 7.3 and 6.1. Capsazepine (10 µM), the capsaicin antagonist, completely inhibited the facilitatory action of inflammatory mediator combination but not the sustained inward current induced by acidic extracellular solution (pH 6.1 or 5.5). It is suggested that the inflammatory mediators, bradykinin,5-HT, and prostaglandin E2 together act as endogenous mediators at capsaicin receptors to generate an inward current when the ion channel is protonized.
A distinct subpopulation of thinly myelinated and unmyelinated primary afferents serves to convey signals from nociceptive "free" nerve endings in peripheral tissue. It is reasonable to assume that a variety of membrane receptors and ion channels exist to transduce chemical or physical stimuli of potentially damaging intensity. Owing to their small size (<1 µM), these nerve endings are not suitable for measuring membrane currents. However the notion that the receptors and ion channels are proteins that may be incorporated in the plasma membrane of the cell body, suggests that a distinct subpopulation of sensory neurons in culture may serve for the study of transduction mechanisms involved in nociception (Baccaglini and Hogan 1983 Cell cultures
Primary cultures of DRG neurons were prepared in two steps as described previously (Guthrie et al. 1987 Recording and perfusion techniques
The single electrode patch clamp technique was used to record whole cell membrane currents employing an Axopatch 1D preamplifier, and pClamp 6 programs (Axon Instruments) with a laboratory PC for storing and evaluating the data. Electrodes were pulled from borosilicate glass and after fire polishing and filling they had resistances of 2-4 M DRG neurons cultured on a monolayer of hippocampal glia
DRG neurons cultured on the monolayer of hippocampal glia without added NGF survived Membrane currents induced by inflammatory mediators and acidic pH
At negative holding membrane potential (usually
Inhibition of responses to acidic inflammatory mediators by the capsaicin antagonist capsazepine
The effects of inflammatory mediators were observed exclusively in DRG neurons that were sensitive to capsaicin and a 100% overlap of capsaicin-sensitivity and sensitivity to IM at low pH was observed. In addition, the IM seemed to share one principle of action with capsaicin, that is to facilitate the pH-gated sustained inward current. Alternatively, membrane currents induced by the IM subthreshold at neutral pH, may have been facilitated by lowering extracellular pH. The alternatives are not mutually exclusive. If this action of the combined IM were the result of an effect on the putative capsaicin receptor, capsazepine, the capsaicin receptor antagonist, should block it (Bevan et al. 1992
Our results demonstrate that the combined inflammatory mediators, bradykinin, serotonin, and prostaglandin E2 together in acidic extracellular solution produce a marked inward membrane current in capsaicin-sensitive DRG neurons from newborn rats. The response is smaller or absent if any one of the four constituents is lacking. The data further suggest that the combination operates on the capsaicin receptor, because the responses to the acidic mixture of IM can be blocked by capsazepine, a competitive capsaicin receptor antagonist.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
).
) and discharge activity in C polymodal nociceptors (Szolcsanyi et al. 1988). In ~80% of small dorsal root ganglion (DRG) neurons in culture, capsaicin induces an inward current that is carried by cations, including Ca2+, through channels of ~30 pS conductance (Bevan and Forbes 1987; Bevan and Szolcsanyi 1990; Vlachová and Vyklický 1993). This membrane current can be blocked by capsazepine, a competitive capsaicin antagonist that might also act on endogenous ligands acting at putative capsaicin receptors (Bevan et al. 1992). Because capsaicin is an agent foreign to the mammalian body, acting on a distinct class of DRG neurons, it seems likely that such endogenous mediator(s) exist.
; Steen and Reeh 1993). The underlying sustained membrane current induced by protons in DRG neurons (Bevan and Yeats 1991) exhibits many similarities to the capsaicin induced membrane current. This led to the suggestion that protons may act as endogenous operators of the putative capsaicin receptor-channel complex (Bevan and Geppetti 1994).
).
). This study is in agreement with previous findings in a skin-nerve preparation and with psychophysical studies demonstrating that the combination of protons together with inflammatory mediators (including BK, 5-HT, HIS, and PGE2) had synergistic excitatory and algogenic effects (Steen et al. 1995, 1996).
). The similarity between inflammatory mediators and capsaicin in facilitating proton-induced currents (Kress et al. 1997) suggested that IM may act as endogenous ligands or operators at the capsaicin receptor that seems to be unique to nociceptive neurons.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Hippocampi from newborn rats were removed, dissociated with trypsin and plated on collagen-coated glass coverslips in a nutrient medium composed of 90% Eagle's minimum essential medium (MEM) and 10% fetal bovine serum. When the glial cultures became confluent, usually after 6-9 days, the medium was switched to nutrient-supplemented MEM with 5-fluoro-2-deoxyuridine and uridine and 10% horse serum to minimize cell division. In the second step, dorsal root ganglia were dissected from 2 to 3 day old Sprague-Dawley rats and incubated at 37°C in phosphate-buffered saline (PBS) containing 2% collagenase for 45 min and then in PBS with 0.3% trypsin for 10 min. The ganglia were then rinsed with calcium- and magnesium-free PBS and dissociated by trituration by using a fire-polished Pasteur pipette. DRG cells were plated on coverslips with the glial feeder layer cultures and grown in a medium composed of 90% MEM and 10% fetal calf serum and maintained at 37°C in a water-saturated atmosphere with 5% CO2. This medium was replaced by a nutrient supplemented MEM containing 10% horse serum and 5-fluoro-2-deoxyuridine (15 mg/ml) and uridine (35 mg/ml) the next day (Guthrie et al.1987). The nutrient supplement contained the following components added to 30 ml PBS: 2 ml bovine albumin (10 mg/ml PBS), 4 ml human transferrin (400 mg/4 ml PBS), 0.8 ml putrescin dihydrochloride (80 mg/ml PBS), 2 ml sodium selenite (1.04 mg/100 ml PBS), 0.2 ml triiodo-thyronine Na (1 mg/5 ml 0.01 N NaOH), 0.8 ml insulin (21.8 mg/ml 20 mM HCl), 0.2 ml progesterone [1 mg/8 ml absolute (abs) ethanol], and 0.04 ml corticosterone grade A (2 mg/ml abs ethanol]. One milliliter of this supplement was added to 50 ml MEM. Neither antibiotics nor nerve growth factor (NGF) were added at any step of culturing because the glia monolayer supported the survival of DRG neurons. Under these conditions, the neurons had previously been observed to develop only short processes if at all (Vlachová and Vyklický 1993).
. The series resistance was usually <10 M and was not compensated.
-2-ethanesulfonic acid (HEPES), and 10 glucose 10; pH was adjusted to 7.3 with NaOH. In acidic ECS, 2-[N-morpholino]ethane-sulfonic acid was used instead of HEPES and the pH was adjusted as indicated. Only one neuron was tested per coverslip and capsaicin was always applied at the end of the sequence to minimize desensitization. All experiments were performed at room temperature (20-22°C). The intracellular solution (ICS) contained the following (in mM): 140 KCl 140, 0.5 CaCl2, 2 MgCl2, 5 ethylene glycol-bis(
-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 10 HEPES, pH was adjusted to 7.3 with KOH. Stock solutions of BK, 5-HT, and PGE2 were prepared in the absence of oxygen and were stored in aliquots at 
20°C. Capsaicin (CAPS) and capsazepine (CZP) were dissolved in 100 µl 96% ethanol and dimethylsulfoxide, respectively, and diluted with distilled water to a concentration of 1 mM, which was used to prepare the test solutions by adding ECS. All other drugs were purchased from Sigma. Data are given as means ± SE. For statistical comparisons the Student t-test and Pearson product moment correlation were calculated with SigmaPlot and SigmaStat (Jandel Corporation). Differences were considered significant at a P value < 0.05.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
14 days provided that they were firmly attached to an underlying cell, probably an astrocyte. Signs of healthy appearance were a smooth plasma membrane, sharp boundary of the nucleus and one or two spot-like nucleoli. After several days some of the neurons exhibited one or several thin processes. Only small neurons (<30 µM diam) without visible processes were selected for electrophysiological recordings. These neurons had high probability of being capsaicin sensitive and their membrane potential was well controlled by voltage clamp. Most of the experiments were performed between 4 and 10 days after plating.
60 mV), ~80% of the small DRG neurons exhibited a slowly activating inward current in response to application of 3 or 6 µM capsaicin at pH 7.3 for 10 s, which slowly deactivated during washout. In this series of experiments (n = 30), illustrated in Fig. 1, the inflammatory mediators (IM), BK (10 µM), 5-HT (10 µM), and PGE2 (1 µM), were tested singly, and in combination of all three at extracellular pH 7.3 and pH 6.1, in a sequence of 10-s applications separated by 20 s intervals of washing with control ECS. Fast application of acidic extracellular solution at pH 6.1 alone induced, in this set of neurons, a fast inactivating inward membrane current shown previously to be carried by sodium ions (Konnerth et al. 1987; Krishtal and Pidoplichko 1981). This fast inactivating current was regularly followed by a sustainedinward current (Fig. 1A) usually not exceeding 50 pA (48 ±13 pA). The effects of exposing neurons to ECS of pH 6.1 on input resistance was tested in 10 of these cells. It was found that acidification to pH 6.1 was accompanied by increased input resistance, reflecting closure of ion channels.
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FIG. 1.
Responses to inflammatory mediators and to capsaicin in a dorsal root ganglion (DRG) neuron (soma diam 22 µM) cultured on a monolayer of central glia for 10 days. A: whole cell membrane currents. Sequence of responses from left to right: extracellular solution (ECS) at pH 6.1; 1 µM prostaglandin E2 (PGE2) at pH 7.3; PGE2 at pH 6.1; IM [inflammatory mediators: bradykinin 10 µM, 5-hydroxytryptamine (5-HT) 10 µM, and PGE2 1 µM] at pH 7.3; IM at pH 6.1; control ECS at pH 6.1, CAPS (capsaicin; 6 µM) at pH 7.3; CAPS at pH 6.1. B: changes of membrane potential recorded in current-clamp mode induced by same sequence of drug applications in same neuron as in A. Resting membrane potential was 62 mV. Symbols are same as in A. Bars indicate duration of drug application; open bars indicate pH 6.1.
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FIG. 2.
Sustained membrane currents in capsaicin-sensitive DRG neurons induced by extracellular pH 6.1 and increase induced in presence of combination of IM (BK 10 µM, 5-HT 10 µM, PGE2 1 µM) or capsaicin. Mean currents induced by combination of inflammatory mediators at pH 6.1 are significantly larger than currents induced by pH 6.1 alone (n = 30). Inflammatory mediators at extracellular pH 7.3 did not produce any membrane current. In 18 of these 30 cells effects of 3 µM CAPS at pH 7.3 and 6.1 were tested.
. Whether these two groups of capsaicin sensitive neurons differ in further respects representing distinct subpopulations of DRG neurons is not known.
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FIG. 3.
Membrane currents induced by pairs of inflammatory mediators: BK (10 µM) and 5-HT (10 µM), BK (10 µM) and PGE2 (1 µM), PGE2 (1 µM) and 5-HT (10 µM) in a set of capsaicin-sensitive DRG neurons (n = 8) that exhibited large sustained membrane currents induced by pH 6.1. In these neurons, pairs of inflammatory mediators induced at pH 7.3 small, sustained membrane currents. At acidic pH they increased response induced by pH 6.1 alone by 81 ± 18%, 69 ± 21%, and 86 ± 34% (Student's paired t-test P < 0.05).
).
). The column diagram in Fig. 4C summarizes the effects of 10 µM capsazepine on the responses induced by acidic IM in seven capsaicin-sensitive neurons. Capsazepine (10 µM) had no significant effect on the sustained inward membrane current induced by acidic ECS at pH 6.1. The suppressive effect of capsazepine on responses to acidic IM was observed in every single cell tested. In some experiments, the responses to acidic IM were reduced even more than was the control magnitude of sustained membrane current induced by pH 6.1 alone. This reduction may result from desensitization that was observed after repeated applications of capsaicin or acidic IM (see Fig. 4A).
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FIG. 4.
Inhibition of membrane currents induced by combination of inflammatory mediators (BK, 5-HT, and PGE2) at pH 6.1 by capsazepine. A: capsazepine blocks increase of membrane currents produced by combination of inflammatory mediators (BK, 5-HT, and PGE2) at pH 6.1. Capsazepine (CZP), at concentration 10 and 3 µM, as indicated. B: capsazepine blocks membrane current induced by inflammatory mediators or by capsaicin but not that induced by pH 5.5. To diminish disuse, effects of capsazepine were tested before control. Smaller magnitude of membrane current to pH 5.5 after response to pH 5.5 with capsazepine is apparently the result of inactivation. CZP, capsazepine (6 µM); CAPS, capsaicin (5 µM). C: mean sustained membrane current induced by ECS at pH 6.1, capsazepine 10 µM (CZP) at pH 6.1, combination of inflammatory mediators at pH 6.1, combination of IMs with capsazepine 10 µM (CZP) at pH 6.1 (n = 7).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
).
). The receptor-channel complex can be effectively modified by protons, which dramatically increase the capsaicin-induced, whole cell membrane current even at extracellular H+ concentrations that are subthreshold to induce any membrane current by themselves (Petersen and LaMotte 1993). The facilitation is apparently exerted by protonation of the capsaicin gated ion channel and not by increasing the affinity of the receptor for capsaicin (Kress et al. 1996). This finding is in striking contrast to the effects of increased H+ on N-methyl-D-aspartate (NMDA) receptors that are allosterically blocked by lowering extracellular pH (Traynelis et al. 1990; Vyklický et al. 1990). It is also in contrast to the pH-dependent inactivation of various voltage-sensitive ion channels (see Hille 1992), which, in our results, may explain the increase in membrane resistance as a net-effect in all those neurons that exhibited only small sustained responses to low pH itself.
; Kress and Reeh 1996; Szolcsanyi 1993) the search for endogenous ligands has primarily been focused on agents that can play a role in signaling pain in inflammation. It has been suggested that H+ may play this role by activating capsaicin receptors (Bevan and Gepetti 1994), although not necessarily at the same recognition site (Rang et al. 1991). Indeed, pH in the exudates of inflammatory processes has been found to be below 5.5, which led to the idea that H+ might be the common principle of producing pain in inflammation (Lindahl 1962). Acidic extracellular solutions were found to induce a sustained inward current exclusively in small capsaicin-sensitive DRG neurons cultured with NGF added to the medium (Bevan and Yeats 1991). This current is likely to underlie pain induced by experimental acidosis in humans and excitation of nociceptors in skin nerve preparation or in the cornea (Belmonte et al. 1991; Steen et al. 1992; Steen and Reeh 1993).
; Vlachová and Vyklický 1993). This can be explained by differences in the strength of binding to the receptor, which is apparently very weak for protons, allowing their release at a speed exceeding the limits of solution exchange of the drug application system (~100 ms). In contrast, the long relaxation time of capsaicin-induced responses suggests tight binding to the receptor or slow diffusion of this lipophilic stimulant out from the lipid bilayer of the neuronal membrane.
). A difference, however, is that protons open the ion channels by themselves whereas glycine only supports the action of glutamate. If capsaicin receptors opened spontaneously, similar to nicotinic acetylcholine receptors (Brehm et al.1984; Jackson 1984) or NMDA receptors (Ture
ek et al. 1997), it could be speculated that a high concentration of H+ may increase the probability of spontaneous openings and thus induce significant membrane current. The idea of identity of capsaicin and proton gated channels is further supported by the recent finding that the sustained proton induced membrane current is carried not only by monovalent cations but also by Ca2+, as is the case for the capsaicin-gated channel (Zeilhofer et al. 1996).
). However loss of their excitatory potency during prolonged application cannot fully explain sustained pain (Kanaka et al. 1985; Kumazawa et al. 1987; Lang et al. 1990). Nevertheless, they are essential mediators of nociceptor sensitization, and combining BK and 5-HT with experimental acidosis, (in the presence of prostaglandin E2 and histamine) produces more than additive excitatory and algogenic effects (Kessler et al.1992; Lang et al. 1990; Steen et al. 1995, 1996). The psychophysiological study, which demonstrated that IM induce a sustained sensitizaton to low pH stimulation (Steen et al. 1996), was recently corroborated by the finding that the combination of BK, 5-HT, PGE2, and HIS markedly increased the sustained membrane current induced by pH 6.1 (Kress et al. 1997). This analogy to the pH-capsaicin interaction leads to the idea that the IM combination may represent endogenous mediators acting directly or indirectly on the capsaicin receptor-channel complex.
; Liu et al. 1996; Petersen et al. 1996). However in all capsaicin-sensitive neurons, the combination of three inflammatory mediators, BK, 5-HT, and PGE2 at acidic extracellular pH, induced depolarizing membrane currents of magnitudes that are likely to induce spike activity in primary afferent nerve endings.
). In contrast to capsaicin-induced responses, capsazepine did not block the sustained pH induced membrane current. However, as already pointed out by Rang et al. (1991), this finding does not exclude the possibility that capsaicin and protons act at the same receptor protein, because the ion channel could be gated from two recognition sites distinct for capsaicin and protons. Our results (Fig. 4) demonstrate that capsazepine, at concentrations that effectively inhibited responses to capsaicin, completely blocked the sensitizing effects of the inflammatory mediators on the pH responses but was ineffective in producing significant changes of the control responses induced by low extracellular pH alone. Although the specificity of capsazepine as an antagonist of capsaicin has been questioned because it was found in some preparations to antagonize the neuropeptide release by protons (see Bevan and Geppetti 1994), this does not contradict the original finding that capsazepine is a competitive antagonist at the putative capsaicin receptor, which is functionally expressed in small mammalian DRG neurons. These results together with the findings that acidic inflammatory mediators induce membrane current exclusively in capsaicin sensitive DRG neurons with capsaicin-like kinetics of activation, strongly support our view that the inflammatory mediators play a role as endogenous mediators at capsaicin receptor channels and produce excitatory membrane currents provided that the ion channels are protonized.
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ACKNOWLEDGEMENTS |
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The authors thank R. K. Orkand for critical reading of the manuscript.
The study was supported by the grants from EU CIPA, National Institutes of Health FIRCA, and Grant Agency of the Czech Republic Grant 305/97/0519. Capsazepine was generously supplied by Novartis, London.
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FOOTNOTES |
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Address for reprint requests: L. Vyklický, Institute of Physiology, 142 20 Prague 4, Vídeská 1083, Czech Republic.
Received 25 June 1997; accepted in final form 6 October 1997.
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