Role of [Ca2+]i in the ATP-Induced Heat Sensitization Process of Rat Nociceptive Neurons

M. Kress and S. Guenther

Institut für Physiologie und Experimentelle Pathophysiologie, Friedrich Alexander University, D91054 Erlangen-Nuremberg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kress, M. and S. Guenther. Role of [Ca2+]i in the ATP-induced heat sensitization process of rat nociceptive neurons. In inflamed tissue, nociceptors show increased sensitivity to noxious heat, which may account for heat hyperalgesia. In unmyelinated nociceptive afferents in rat skin in vitro, a drop of heat threshold and an increase in heat responses were induced by experimental elevation of intracellular calcium ([Ca2+]i) levels with the calcium ionophore ionomycin (10 µM). Similar results were obtained in experiments employing [Ca2+]i release from preloaded "caged calcium" (NITR-5/AM) via UV photolysis. In both cases, sensitization was prevented by preventing rises in [Ca2+]i with the membrane-permeant calcium chelator BAPTA-AM (1 mM). No pronounced change of mechanical sensitivity was observed. Heat-induced membrane currents (Iheat) were investigated with patch-clamp recordings, and simultaneous calcium measurements were performed in small sensory neurons isolated from adult rat dorsal root ganglia (DRG). Ionomycin-induced rises in [Ca2+]i resulted in reversible sensitization of Iheat. In the same subset of DRG neurons, the endogenous algogen ATP (100 µM) was used to elevate [Ca2+]i, which again resulted in significant sensitization of Iheat. In correlative recordings from the skin-nerve preparation, ATP induced heat sensitization of nociceptors, which again could be blocked by preincubation with BAPTA-AM. Rises in [Ca2+]i in response to inflammatory mediators, e.g., ATP, thus appear to play a central role in plastic changes of nociceptors, which may account for hypersensitivity of inflamed tissue.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pain is normally evoked by stimuli sufficiently intense to induce excitation of unmyelinated or thinly myelinated nociceptors. Most of these are polymodal transducers of physical (e.g., pressure or heat) as well as chemical stimuli such as capsaicin, the pungent ingredient of hot chili peppers. In inflammation, the sensitivity of nociceptors toward heating can dramatically increase so that even nonnoxious warming becomes painful. Inflammatory mediators, e.g., bradykinin, and exogenous algogens such as capsaicin have been found to induce such heat sensitization that outlasts the presence of the algogen (Keele and Armstrong 1964; Kress and Reeh 1996; Reeh and Kress 1995). Acting at specific membrane receptors, capsaicin activates a nonselective cation current that is partially carried by calcium ions and that itself can be activated by noxious heat (Caterina et al. 1997; Kirschstein et al. 1997; Reichling and Levine 1997; Zeilhofer et al. 1997). This results in an increase of intracellular calcium concentration ([Ca2+]i) by calcium influx (Wood et al. 1988; Zeilhofer et al. 1997). Similar rises in [Ca2+]i have also been reported for endogenous compounds. ATP, which is physiologically released in synaptic transmission (Bardoni et al. 1997; Gu and MacDermott 1997) and which is liberated in high concentration on destruction of cells, e.g., in inflammation, activates membrane conductances via P2X receptor binding (Chen et al. 1995; Cook et al. 1997). These currents are also partially carried by calcium ions and yield rises in [Ca2+]i (Bouvier et al. 1990; Lewis et al. 1995). In addition, rises in [Ca2+]i may also occur by release from intracellular stores, e.g., after bradykinin exposure after the activation of the PLC/IP3 pathway, and this could also mediate nociceptor sensitization to heat. However data are not yet available to support this hypothesis. We hypothesize that rises in [Ca2+]i could be a central mechanism mediating plasticity of nociceptor responsiveness similar, e.g., to calcium-dependent long-term potentiation in the CNS.

Therefore we used three different approaches to elevate [Ca2+]i in nociceptive terminals in a skin-nerve in vitro preparation and recorded single-fiber responses to experimental heat stimuli before and after the treatment. In addition, the membrane permeant calcium chelator BAPTA-AM was used to prevent calcium-dependent sensitization. However, in this relatively complex model, nonspecific signal cascades secondary to rises in [Ca2+]i,, e.g., neuropeptide release, cannot be excluded. In addition, the small size of nociceptive nerve terminals of <1 µm diam does not allow for direct investigation of membrane mechanisms and ionic currents subthreshold to action potential firing. Therefore in a second series of experiments correlative patch-clamp recordings with simultaneous calcium measurements were performed in sensory neurons isolated from DRG. We investigated whether experimental increases in [Ca2+]i with ionomycin and ATP as an endogenous mediator, respectively, can induce sensitization of Iheat in nociceptors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Skin nerve preparation

Single-fiber recordings were performed in a rat skin-saphenous nerve in vitro preparation (Reeh 1986). Briefly, 17 adult male Wistar rats (200-310 g body weight) from an inbred colony were killed by breathing 100% CO2. The hairy skin of the dorsal hind paw together with the saphenous nerve were subcutaneously dissected and transferred, epidermal side down, to an organ bath. Small filaments were dissected with sharpened watchmaker forceps from the nerve in a second chamber filled with liquid paraffin. Single-unit activity was recorded monopolarly via gold wire electrodes with the reference electrode positioned nearby. The skin was superfused (16 ml/min) with "synthetic interstitial fluid" (SIF) (Bretag 1969) consisting of (in mM) 108 NaCl, 26.2 NaHCO3, 9.64 sodium gluconate, 5.55 glucose, 7.6 sucrose, 3.48 KCl, 1.67 NaH2PO4, 1.53 CaCl2, and 0.69 MgSO4, saturated with carbogen (95% O2-5% CO2) at pH 7.38 and 32°C. Receptive fields of single units were searched by probing the skin with a blunt glass rod. Electrical stimuli were applied to receptive fields via a Teflon-insulated steel semimicroelectrode with bare tip (impedance approx 1 MOmega ) to determine conduction velocities (cv) and identify unmyelinated afferents (cv <1.4 m/s). Mechanical (von Frey) thresholds were determined with a set of nylon filaments with uniform tips (0.9 mm diam) calibrated in a geometric series [from 0.4 to 256 mN (<= 7.2 bar)]. For heat stimulation, the receptive field was isolated from the surrounding organ bath with a metal ring (8 mm ID), and SIF was evacuated to prevent feedback errors caused by convection. Radiant heat stimuli were applied to the epidermal surface of the skin from a halogen bulb that was feedback controlled with a thermocouple placed on the corium side. Standard heat stimuli consisted of a linear temperature rise from 32 to 46°C within 21 s (0.67°C/s; corresponding epidermal temperature 32-52°C) (Reeh 1986) and were applied in 5-min intervals. Under these conditions, heat-response magnitudes and threshold temperatures neither sensitized nor desensitized with repetition if not otherwise manipulated (n = 9, Fig. 1A). Only mechano-heat-receptive C-fibers (CMH units) were included. After digitization (sampling rate 3 kHz), unitary action potentials were recorded with a PC-type laboratory computer and the Spike/Spidi software package, which was also used for off-line spike discrimination (Forster and Handwerker 1990).



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Fig. 1. Repeating heat stimuli at 5-min intervals in control experiments did not alter heat responses and heat thresholds throughout the recording period. A: mean heat responses and thresholds were recorded for a control population of 9 mechano-heat sensitive C-fibers. Large error bars (SE) result from pronounced interindividual differences between individual mechano-heat-receptive C- (CMH)-fiber responses to heat in this and all following samples. B: control experiments with exposure to DMSO (1%) () and UV light (black-square) neither changed heat responses nor heat thresholds. C: example of heat-activated currents of a single dorsal root ganglion (DRG) neuron in response to repeated stimulation (top trace). On the average, current amplitudes neither sensitized nor desensitized under the conditions used (bottom panel).

Chemical stimulation and UV photolysis in rat skin

Nerve terminals were loaded with the membrane-permeant "caged calcium" NITR-5/AM for 9 min (Calbiochem; 100 µM in SIF, 1% DMSO) with the nondisruptive acetoxymethylester-loading technique (Tsien 1981). Spontaneous decomposition of the compound was minimized by darkening the preparation during the experiment. To liberate Ca2+, the receptive field was exposed to continuous UV light with an Elipar II UV-lamp (600-700 mW/cm2, 400-500 nm; ESPE GmbH, Seefeld) for 2 min. Treatment with the pure vehicle and UV illumination did not introduce a change in heat responses (n = 4, Fig. 1B).

ATP-Na (Sigma, from a 1 mM stock solution in SIF) or ionomycin (Sigma, from a 1 mM stock solution in DMSO, final content 1%) diluted in SIF were applied between heat stimuli and removed 30 s before the next heat stimulus was started. To buffer [Ca2+]i increases, neurons were loaded with the membrane-permeant Ca2+ chelator BAPTA-AM (Molecular Probes; Leiden, The Netherlands) again with acetoxymethylester loading (Tsien 1981). BAPTA-AM was initially dissolved in DMSO and diluted to a final concentration of 1 mM in SIF (final DMSO content 1%).

Cell culture

Details of dissociation procedures have been published elsewhere (Wood et al. 1988). Briefly, lumbar DRG were harvested from adult female Wistar rats (100-160 g) from an inbred colony that had been killed by breathing 100% CO2. Ganglia were harvested from levels T13-L5 and transferred into DMEM supplemented with 0.5 ml/100 ml gentamycin (Sigma). The connective tissue was removed, and ganglia were treated with collagenase (0.28 units/ml in DMEM, Boehringer Mannheim, Germany, 75 min) and, after two washes in DMEM, in trypsin (25,000 units/ml, Sigma, 12 min). After washing, cells were dissociated with a fire-polished Pasteur pipette, centrifuged at 3,500 rpm, and finally resuspended in supplemented culture medium. After plating on glass coverslips coated with poly-L-lysine (200 µg/ml, Sigma) cultures were kept in serum-free TNB 100 medium (Biochrom; Berlin) supplemented with penicillin-streptomycin (each 20.000 IU/100 ml), 2 mM L-glutamin (both from GIBCO), and 100 ng/ml NGF (Calbiochem) at 37°C in a humid atmosphere containing 5% CO2.

Electrophysiology in isolated neurons from DRG

Whole cell current measurements in the voltage-clamp configuration of the patch-clamp technique were performed between 4 and 36 h after dissociation at -80-mV holding potential and 3-kHz sampling rate with the Axopatch 200A amplifier and pClamp6.0 software running on a PC-type computer (Axon Instruments; Foster City, CA). The same software package was used for evaluation of the currents and for calculating single-exponential inactivation time constants with the Chebychev algorithm. Only experiments in which leak currents did not exceed 90 pA were included, and therefore no compensation procedures for leak currents were performed. From the cultures, small-size neurons (<25 µm) were selected because they are generally assumed to represent nociceptors, and only those neurons were included that exhibited heat-activated currents of >150-pA current amplitude in response to the standard heat stimuli used. These were considered to correspond best to the population of polymodal nociceptors investigated in the single-fiber recordings. Electrodes from borosilicate glass (Science Products; Hofheim, Germany) were filled with (in mM) 148 KCl, 4 MgCl2, 2 Na-ATP, 10 HEPES, and 0.2 Li-GTP, pH adjusted to 7.3 with KOH, and had typical resistances of 2-4 MOmega . External solution consisted of (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3 adjusted with NaOH. For calcium measurements, 100 µM fura-2 (pentapotassium salt, Molecular Probes; Leiden, The Netherlands) was added to the intracellular solution (Grynkiewicz et al. 1985). Background-corrected fluorescent images were taken with a slow-scan charge-coupled device camera system with fast monochromator (PTI; New Jersey) coupled to an Axiovert with ×40 fluotar oil objective (Zeiss; Oberkochen, Germany). Fura-2 was excited at 340- and 380-nm wavelengths (lambda ), and fluorescence was collected at lambda  > 420 nm at a frequency of 1 Hz with equal exposure time of 200 ms. [Ca2+]i was calculated as previously published (Grynkiewicz et al. 1985; Zeilhofer et al. 1996), and calibration constants obtained in vitro were Rmin = 0.44, Rmax = 8.0, and Keff = 1.2 µM.

Heat stimulation

A fast, 10-channel system with common outlet was used for drug application, which allowed for independent heating of all 10 solutions. Solenoids were controlled manually from a switchboard, or a sequence was programmed and controlled from the computer. Step-voltage commands for heat stimuli were obtained from the pClamp6.0 clampex part and yielded time constants for temperature rises of ~150 ms. When the formation of air bubbles was avoided during filling of the system, solutions were flowing at constant speed, which resulted in good reproducibility of heat stimuli (Dittert et al. 1998). To exclude, however, differences in temperature reached with different lines heat stimuli were only applied during application of control solution running always through the same tube in the course of the experiment. Temperature was constantly measured inside the tip of the common outlet via a thermocouple that feedback controlled the heating-voltage command. The distance between the recorded cell and the site of temperature measurement was estimated to be 100 µm. At low temperatures (~25°C) a good correlation was obtained of the temperature in the tip of the application system and the temperature that was measured with a second thermocouple at the site where the cell normally was located. Differences of <= 5°C were observed at high temperatures of ~50°C (Dittert et al. 1998). Standard heat stimuli of 2-s duration and ~49°C amplitude (measured inside the tip of the applicator) were applied at 1-min intervals. In the time course of a single experiment heat stimuli were well reproducible with very few exceptions. In these cases the whole experiment was excluded from the study. Under these conditions and with the solutions used, heat currents neither sensitized nor desensitized if no other manipulations were performed (see Fig. 1C).

Data analyses

For detailed statistical analysis of the data, the CSS software package was used (StatSoft; Tulsa, OK). All summarizing data in figures are given as means ± SE. For intraindividual comparisons, MANOVA and a post hoc Wilcoxon matched-pairs tests were calculated if not otherwise stated and refer to median and upper and lower quartiles given in figure legends. Differences were considered significant at P < 0.05.


    RESULTS
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INTRODUCTION
METHODS
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A total of 61 CMH afferents were isolated with standard teased fiber techniques, of which two exhibited a very low level of ongoing activity (<1 action potential/min) for which no correction procedure was performed.

To explore the effects of increased [Ca2+]i, membrane-permeant "caged calcium" (NITR-5/AM 100 µM) was loaded nondisruptively into the nociceptive nerve terminal. The loading of the nerve terminals itself did not introduce discharge activity or changes in sensitivity to heat stimulation (Fig. 2). After three consecutive heat stimuli, calcium ions were released from the cage by UV photolysis. After the UV illumination, a facilitation of the response as well as a drop of heat thresholds were observed (n = 7, Figs. 2 and 3). This facilitation was fully abolished after coadministration of the membrane-permeant calcium chelator BAPTA-AM (1 mM), which itself did not induce any change of heat sensitivity (n = 7, Fig. 3).



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Fig. 2. Intracellular UV photolysis of calcium ions from NITR-5/AM-induced heat sensitization. A: heat responses of 1 unit before and after UV photolysis of calcium ions. After calcium ions were released intracellularly, heat responses became larger and started at a lower temperature than before. B: time course of the experiment; 7 uniform heat stimuli were applied to the receptive field. After the first stimulation (H1), NITR-5/AM was applied to the receptive field indicated by the shaded bars. UV illumination started 3 min before H4 and is indicated by arrow. Numbers correspond to those in A.



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Fig. 3. Averaged heat responses are significantly increased and heat thresholds significantly lowered by UV photolysis of "caged calcium" (NITR-5/AM; left panels). Changes of medians and lower and upper quartiles in heat responses (15 spikes/heat stimulus as median and 8 and 57 as quartiles before vs. 38 spikes/heat stimulus as median and 13 and 75 as quartiles after release) and heat thresholds (42.7°C median and 38 and 46°C as quartiles before vs. 40.7°C as median and 36.7 and 43°C as quartiles) are significant (* P < 0.05). Both effects are abolished by coadministration of BAPTA-AM (right panels).

A drop of heat thresholds and a facilitation of the heat response similar to the one induced by intracellular photorelease of calcium ions were obtained when exposing the receptive field of cutaneous nociceptors to the calcium ionophore ionomycin to allow calcium ions to enter the neuron (10 µM, n = 7, Fig. 4). Both effects were significant (P < 0.05). Again BAPTA-AM preincubation itself did not change heat sensitivity but showed a protective effect against heat sensitization (n = 7, Fig. 4). Neither treatment induced a conspicuous change of mechanical thresholds. These results hint to calcium ions as a trigger for increasing heat responsiveness of nociceptors.



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Fig. 4. The calcium ionophore ionomycin induced calcium-dependent heat sensitization in cutaneous nociceptors. A: response of a single neuron to 5 consecutive heat stimuli at 5-min intervals. Ionomycin induced action potential firing at profoundly lower temperature and an increase of the total heat response. B, left panels: averaged heat responses are increased and mean heat thresholds significantly lowered after ionomycin (*:significant differences, P < 0.05 and refer to 22 spikes/heat stimulus as median and 19 and 41 as quartiles before vs. 57 spikes/heat stimulus as median and 21 and 83 as quartiles after ionomycin and 40.7°C median and 38.7 and 41.8°C as quartiles before vs. 35.8°C as median and 32.7 and 40.0°C as quartiles after ionomycin). Preincubation with BAPTA-AM (right panels, light bar) fully prevented heat sensitization by ionomycin (solid bars).

To explain this calcium-dependent heat hypersensitivity we hypothesized that the recently reported heat-activated current (Cesare and McNaughton 1996) could present as a possible target of second-messenger cascades initiated by calcium. To investigate this hypothesis, current measurements in the whole cell voltage-clamp configuration of the patch-clamp technique were performed. With the short application time of 5 s, no inward or outward currents were observed in response to ionomycin application. In some experiments, [Ca2+]i concentration was monitored microfluorimetrically with the calcium indicator dye fura-2, which was loaded into the neuron via the patch pipette. Ionomycin induced a profound increase in [Ca2+]i levels, and current amplitudes were greatly facilitated (Fig. 5). On the average, a significant facilitation of Iheat occurred, which only partially recovered after several minutes (425 ± 127 pA vs. 1,071 ± 334 pA; n = 6, P < 0.05).



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Fig. 5. Specimen of a capsaicin-sensitive DRG neuron that initially at low calcium concentration responds to heat with an inward current of ~400 pA. Ionomycin induces a rise in [Ca2+]i that is followed by an increase in current amplitude to >1,500 pA. Only partial recovery is obtained in this neuron.



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Fig. 6. Heat-induced currents before and after ATP application, which itself evokes a fast and transient inward current and recovery. Inset: intracellular calcium ([Ca2+]i) concentration () and heat-induced current () before and after ATP. *: signficant changes (P < 0.05; n = 9)

The endogenous mediator ATP has been considered an algogen, and in a subset of DRG neurons it has been reported to activate a cation current that is partially carried by calcium ions (Bouvier et al. 1990; Burnstock and Wood 1996; Chen et al. 1995). In small-size DRG neurons, ATP-activated transient inward currents had a peak amplitude of 680 ± 320 pA and an inactivation time constant of ~140 ms. At the same time, a rise in calcium concentration was observed (53.7 ± 8.8 nM vs. 151.8 ± 66.1 nM, n = 8, P < 0.05). Simultaneously, a significant facilitation of Iheat (476 ± 111 pA vs. 1,961 ± 353 pA, n = 8, P < 0.05) at high calcium concentration was obtained (Fig. 6, n = 6). These changes only occurred in neurons that exhibited ATP-activated ionic currents. The threshold of rises in [Ca2+]i effective to induce sensitization of heat-induced currents was not determined systematically, but with ATP a rise from ~50 nM to only 150 nM was sufficient to yield the effect. Together there was a significant correlation between [Ca2+]i and current amplitudes of Iheat with a correlation coefficient of 0.59 (n = 15, P < 0.05).

Although the expression of ATP-activated inward currents is well accepted in isolated sensory neurons (Bouvier et al. 1990; Burnstock and Wood 1996; Chen et al. 1995), the excitatory action of ATP on nociceptors has not been well established. In the skin-nerve preparation, ATP only exceptionally (in 2/19 neurons) induced excitation of nociceptors in <= 1 mM concentration. However, in the doses used, ATP induced a dose-dependent heat sensitization as shown in Fig. 7B. The sensitivity increase was characterized by more-pronounced heat responses and by a simultaneous drop of heat thresholds similar to the sensitization that has been found before for ionomycin and caged calcium. The ATP-induced heat sensitization was again abolished after preincubation of the receptive field with the [Ca2+]i chelator BAPTA-AM (Fig. 8). This again corroborates our hypothesis of an effect mediated by calcium ions.



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Fig. 7. ATP-induced calcium-dependent heat sensitization in cutaneous nociceptors. A: responses of a single neuron to heat before and after exposure to ATP. B: dose-response curve of ATP action on heat responses and heat thresholds of CMH-fibers. Relative magnitude of the responses refers to the previous baseline stimulus to which the response was set to equal 1 and only relative temperature changes are depicted (star  P < 0.05 and star star P < 0.01, for details see Figs. 8 and 9).



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Fig. 8. ATP causes calcium-dependent heat sensitization in polymodal nociceptors of rat skin. Left panel: mean numbers of action potentials per heat response; average heat thresholds were significantly altered after ATP application during black-square (P < 0.05 indicated by * refers to 12 spikes/heat stimulus as median and 5 and 51 spikes/heat stimulus as lower and upper quartiles before ATP vs. 33 spikes/heat stimulus as median and 12 and 65 spikes/heat stimulus as quartiles after ATP; 41.3°C as median and 38.7 and 43.3 as quartiles before vs. 39.0°C as median and 37.3 and 42.7 as quartiles after ATP). Preincubation with 1 mM BAPTA-AM () fully prevented heat sensitization (right panel).

Both signs of sensitization were also abolished by coadministration of the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; Calbiochem; 50 µM, n = 9, Fig. 9). In pilot experiments, PPADS at 10 µM was ineffective and at higher concentrations (100 µM) induced ongoing activity that may be caused by a P2X-independent membrane depolarization of unknown mechanism that has been found previously (McLaren et al. 1994).



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Fig. 9. Mean numbers of action potentials per heat response before and after ATP application. Significant heat sensitization induced by 100 µM ATP (left panels, indicated by *, P < 0.05, refers to 17.5 spikes/heat stimulus as median and 3.5 and 22 spikes/heat stimulus as quartiles before vs. 20.0 spikes/heat stimulus as median and 4.5 and 33.5 as quartiles after ATP; 42.7°C as median and 38.7 and 43.7 as quartiles vs. 39.67°C as median and 38.2 and 41.2 as quartiles) was fully abolished by coadministration of 50 µM pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results provide first evidence that sensitization of nociceptors to heat can be induced by agents that elevate [Ca2+]i in the skin-nerve in vitro model. Furthermore, evidence is presented that the molecular target of the sensitization is a heat-activated membrane current that is specifically facilitated by a calcium-dependent mechanism in small DRG neurons. Three different approaches have been successfully used to increase [Ca2+]i levels in nociceptors in vitro; intracellular release of caged calcium as well as calcium influx either through artificially formed calcium permeable pores, or constitutive cation channels were effective to facilitate heat responses. Buffering [Ca2+]i prevented this heat sensitization in all three approaches.

ATP was used as an endogenous mediator to induce calcium influx. The substance is known to activate P2X receptors that are coupled to nonselective cation channels permeable for calcium ions, and these receptors are expressed in the soma membrane of DRG neurons (Bouvier et al. 1990; Chen et al. 1995; Cook et al. 1997). Although the effects of ATP in the CNS are well established (Driessen et al. 1994; Gu and MacDermott 1997; Salter and Henry 1985) its action on peripheral nociceptors is still controversially discussed. Although it has been found to cause short-lasting excitation in a subpopulation of nociceptive afferents in the rat knee joint (Dowd et al. 1998), in another study ATP facilitated the second phase of the formalin response but had little excitatory effect (Sawynok and Reid 1997). However, algesic effects have long been proposed (Burnstock and Wood 1996), and only recently increased ATP release from epithelia has been found during inflammation, suggesting a role in inflammatory pain or hyperalgesia (Bodin and Burnstock 1998).

In the skin-nerve preparation in vitro, ATP induced a calcium-dependent sensitization of nociceptors to heat that was inhibited by the P2 receptor antagonist PPADS. Although the majority of fibers exposed to ATP were sensitized without previously being excited, the results are in agreement with a sensitization of heat-activated currents in DRG neurons exhibiting ATP-activated inward currents. Approximately 40% of DRG cells show ATP-activated currents, which however inactivate very rapidly (Bouvier et al. 1990). This may explain the small proportion of fibers excited by ATP even at high concentrations. The current results also confirm previous work suggesting ionotropic P2X receptors to play an important role in nociception (Gu and MacDermott 1997; McLaren et al. 1994).

Relatively small rises reaching peak calcium concentrations of 150 nM were effective to induce sensitivity changes, and in these experiments heat sensitization recovered within few minutes; this recovery followed the recovery of [Ca2+]i. However, after application of the calcium ionophore ionomycin the facilitation of heat-activated currents partially persisted, and this may be due to the high calcium concentrations of <= 500 nM obtained in these recordings. Large rises in [Ca2+]i might induce prolonged effects compared with moderate or small elevations, but mechanisms or signaling pathways of such differential effects in nociceptors are yet unknown.

Although the results are supporting our initial hypothesis, the acetoxymethylester-loading technique in the skin-nerve preparation could also possibly exert unspecific effects on neurons. Because, however, heat responses during loading were unchanged in all series of experiments and because specific and opposing effects were observed for BAPTA-AM and NITR-5/AM, respectively, unspecific side effects of the loading itself seem unlikely. Similarly, the release of calcium ions from NITR-5 by photolysis appeared to be specific because ionomycin produced the same effect, and in both cases BAPTA-AM buffering completely prevented sensitization.

An obvious restriction of the in vitro organ preparation, however, is the complexity of the tissue. Neurons are closely surrounded by other cell types, and the possibility of secondary release of substances from various cells as a consequence of rises in [Ca2+]i needs to be taken into account. Well-known candidates that are however released from the nociceptive nerve terminal itself are the neuropeptides substance P and calcitonin gene-related peptide (Kress and Reeh 1996). However, neither direct application of substance P nor the induction of neurogenic inflammation by antidromic nerve stimulation was effective to induce heat sensitization in nociceptors, suggesting no major contribution of these neuropeptides in the heat sensitization process (Cohen and Perl 1990; Meyer et al. 1988; Reeh et al. 1986). Still other pathways may exist that cannot fully be excluded in this type of preparation. Therefore correlative recordings were performed in isolated sensory neurons that allow for better control of the composition of the extracellular environment. In these experiments similar effects were observed as in the skin-nerve preparation. Thus a specific role of [Ca2+]i in neurons rather than the contribution of released mediators seems to mediate the heat sensitization process.

Calcium ions as second messengers can activate various enzymatic signal cascades, yielding phosphorylation or dephosphorylation of proteins, including ion channels in the cell membrane. The phosphorylation status of ion channels, respectively, may exhibit different kinetic states and may thus account for changes in sensitivity toward adequate stimuli in nociceptors (for review see Cesare and McNaughton 1997; Ghosh and Greenberg 1995; Kress and Reeh 1996). First evidence for calcium-dependent sensitivity changes in nociceptors came from recordings in DRG neurons of capsaicin-activated membrane currents that exhibited strong desensitization and tachyphylaxis (Caterina et al. 1997; Cholewinski et al. 1993; Koplas et al. 1997). These could be prevented when either high intracellular concentrations of calcium buffers or blockers of calcium-dependent phosphatases, e.g., calcineurin, were used (Cholewinski et al. 1993; Koplas et al. 1997). Assuming that both heat- and capsaicin-activated currents are mediated by the same ion channel (Caterina et al. 1997), the desensitizing effect of increasing [Ca2+]i on native capsaicin-activated ion channels seems to contradict our current findings. However, depending on the distribution and local concentration of calcium, a variety of changes in neurons may be initiated, and either phosphatases or protein kinases may become active.

Currently, at least three different families of protein kinases are known that can potentially be activated by calcium ions, e.g., protein kinase C (PKC) and A (PKA) families and calcium-calmodulin (CaM) kinases. PKC has been previously reported to facilitate heat activated currents (Cesare and McNaughton 1996), and, e.g., PKC gamma  or delta  are activated by calcium. A recent study has reported reduced neuropathic pain and heat allodynia in a neuropathic pain model in mutant mice lacking the PKC gamma  isoform (Malmberg et al. 1997b). However, this PKC isoform could not be found in DRG with an immunocytochemical assay, and a major contribution to the calcium-dependent heat sensitization of nociceptors seems therefore improbable. The changes in pain processing accompanied by impaired synaptic plasticity rather suggest a role in the CNS of this PKC isoform (Abeliovich et al. 1993).

Second, calcium ions can activate the type I-like adenylyl cyclase isoenzymes (Mons and Cooper 1995), and the resulting increase in cAMP levels activates PKA (Brandon et al. 1997). Evidence for a role of PKA in heat sensitization comes from mice that carry a null mutation for the neuron-specific isoform of the type I regulatory subunit of PKA (RIbeta ). In these animals, acute pain processing was not noticeably altered, but PGE2-induced heat hyperalgesia was clearly reduced compared with wild type (Malmberg et al. 1997a).

From the molecular structure of the cloned capsaicin receptor VR-1, which probably serves as a heat transducer in nociceptors, three conserved PKA phosphorylation sites were predicted, and further support comes from a study that demonstrates that the prostaglandin E2 enhancement of capsaicin-induced currents was mediated by the cAMP/PKA transduction cascade (Caterina et al. 1997; Lopshire and Nicol 1998; Pitchford and Levine 1991). Single-channel recordings revealed that the open probability of capsaicin-activated channels was increased after PKA activation (Lopshire and Nicol 1998). This is in line with previous studies in which heat sensitization was observed in cutaneous nociceptors after exposure to membrane-permeant and stable analogs of cAMP (Ferreira and Nakamura 1979; Kress et al. 1996). Although VR-1 has conserved sites for PKA, it remains to be demonstrated that these sites are indeed phosphorylated and involved in the heat sensitization process.

Other mechanisms at present cannot be excluded. Thus PKA activation has been reported to induce a facilitation of TTX-resistant sodium channels, and this might yield a general excitability increase of the neuron nonspecific for heat sensitization (England et al. 1996; Gold et al. 1996). Furthermore, other kinases such as CaM-kinase may well be expressed in nociceptive neurons; however, nothing is known about this pathway in the peripheral nociceptive system.

In the CNS, similar rises in [Ca2+]i initiate plasticity in many types of neurons. Our results present first hints that increased [Ca2+]i may mediate the sensitizing effect of algogens that cause increases of [Ca2+]i and heat sensitization in nociceptors. Another connection of calcium and heat sensitivity comes from opioid effects on peripheral nociceptors. Opioid µ or delta  receptors are expressed in cutaneous unmyelinated afferents (Coggeshall et al. 1997) and recently have been reported to play a certain role in the prevention of heat hyperalgesia under certain conditions (Koppert et al. 1998). This study may help to explain this effect because opioids reduce the calcium influx through voltage-activated calcium channels and thus may reduce the amount of calcium influx and prevent calcium-dependent heat sensitization of nociceptors (Taddese et al. 1995).


    ACKNOWLEDGMENTS

The authors thank H. O. Handwerker for carefully reading the manuscript and I. Mueller-Roth, A. Wirth-Huecking, and I. Izydorzyk for expert technical assistance.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 353, A10) and the Wilhelm-Sander-Stiftung (96.058.1).


    FOOTNOTES

Address for reprint requests: M. Kress, Institute of Physiology and Experimental Pathophysiology, Universitätsstr. 17, D-91054 Erlangen, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 September 1998; accepted in final form 26 February 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society