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Intracellular free calcium responses to protons and capsaicin in cultured trigeminal neurons
Neuroscience Vol. 67, No. I, pp. 235-243, 1995
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Pergamon
0306-4522(95)00055-0
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
INTRACELLULAR FREE CALCIUM RESPONSES TO PROTONS A N D CAPSAICIN IN CULTURED TRIGEMINAL NEURONS J. G A R C I A - H I R S C H F E L D , L. G. L O P E Z - B R I O N E S , C. B E L M O N T E and M. V A L D E O L M I L L O S * Departamento de Fisiologia and Instituto de Neurociencias, Facultad de Medicina, Universidad de Alicante, Aptdo. 374. 03080 Alicante, Spain Abstract--Acidic solutions and capsaicin are selective chemical stimuli for nociceptive neurons. The effect of these stimuli on intracellular calcium concentration was analysed in cultured trigeminal neurons of newborn rabbits. Rapid reductions in pH (from 7.4 to 5) evoked a transient rise in intracellular calcium concentration of 270% on average over the basal level (162.5 + 3.5 nM; n = 174) in 86% of the neurons. Maximal responses were found at pH 5.5. Proton-induced transients were diminished or abolished by 20 mM CaC12, by zero CaC12 and by 1/~M Ruthenium Red. In response to 1/tM capsaicin, 40% of the cells that were sensitive to protons also increased their intracellular calcium concentration to 218% of control. Capsaicin-induced intracellular calcium concentration rises were composed of an initial peak followed by a second, slower intracellular calcium concentration elevation. The capsaicin response was completely blocked by 1 pM Ruthenium Red, and disappeared in zero calcium, but was augmented in high extracellular calcium. Intracellular calcium concentration responses to capsaicin were still observed in neurons whose response to protons was desensitized by sustained exposure to low pH (pH 6.5). Cells surviving a 10-24 h capsaicin (10#M) treatment, still displayed responses to pH reductions. These results suggest that intracellular calcium concentration rises induced by moderate reductions in pH 0 and capsaicin occur through different mechanisms.
Transduction of injurious stimuli into a discharge of nerve impulses occurs in nociceptors and is often the first step in the genesis of pain sensations. Polymodal nociceptors are free nerve terminals of a subpopulation of primary neurons located in sensory ganglia, that respond to noxious mechanical forces, irritant chemicals and heat applied to peripheral tissues. 5 The discharge characteristics of nociceptive fibers in response to tissue damage have been extensively studied. 32 However, the mechanisms involved in stimulus transduction in nociceptive terminals are largely unknown. Acidic solutions and capsaicin are selective chemical stimuli for nociceptive neurons. 3'~5'22'23 Decreases of pHo are known to evoke nerve discharges in pain fibers; 4'29 moreover, proton accumulation in inflammatory exudates were proposed as one of the causes of nociceptive stimulation in injured tissues. 24 Capsaicin, a neurotoxin with well known pungent effects, selectively excites and eventually inactivates peripheral nociceptors? °'3~ Due to this specific effect,
capsaicin sensitivity of primary sensory neurons has been often employed to identify nociceptive cells.3,6,7,33 The soma of cultured mammalian trigeminal or dorsal root ganglion neurons has served as a model to study transduction of chemical stimuli by nociceptors. 3'22 When applied to the cell body, low pH 0 solutions activate two different inward currents? the first is a fast inactivating current that is evoked by small, rapid changes in p H 0 values and is present in a large proportion of primary sensory neurons, as well as in other excitable cells. 13'2° The second is a sustained, slowly inactivating cationic current evoked by moderate p H 0 (pH 6.2) reductions. This second current is restricted to the subpopulation of capsaicin-sensitive sensory neurons. 8 Although the bulk of p H 0 evoked currents is carried by monovalent cations, there are recent suggestions that Ca 2+ ions may also contribute to the fast inactivating current. 21 A long-lasting current, that resembles the sustained current produced by protons, is also evoked by capsaicin in a proportion of dorsal root ganglion primary sensory n e u r o n s ) This effect appears to be mediated by a specific membrane receptor for capsaicin that activates a cationic channel. 28 The channel is relatively nonselective, being permeable to both monovalent and divalent cations. 7'~2'~5'~s'-'5 Based on the analogy of currents evoked in nociceptive neurons by acid and capsaicin, it has been proposed that both
*To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin;
DMEM, Dulbecco's modified essential medium; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid; FCS, fetal calf serum; HBS, HEPES-buffered saline solution; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; INDO-1/AM, indo-1 acetoxymethylester; NGF, nerve growth factor. 235
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substances activate the same ionic channel. 9 The purpose o f this work was to analyse the changes of intracellular calcium concentration ([Ca 2÷ ]i) induced by acidic solutions and by capsaicin in the soma of cultured trigeminal neurons and to explore the relationship between Ca 2÷ Signals activated by both stimuli.
EXPERIMENTAL PROCEDURES
Culture of trigeminal neurons Newborn rabbit (pseudocalifornia from our local breeding house) trigeminal ganglia were used. Animals were killed with an overdose of nembutal (1 ml of a 15 mg/ml solution). For trigeminal neuron culture, standard procedures were employedY Briefly, animals were washed with 70% ethanol and trigeminal ganglia were isolated under a dissecting microscope and settled in 1 ml of Leibovitz medium (L-15, Flow). Ganglia were washed with culture medium and incubated in collagenase 0.025mg/ml (Boehringer) for 45 rain in a humidified atmosphere of 5% CO2, 95% air. Enzymatic treatment was stopped by adding fresh L15 medium, t9 Chunks of tissue were dissociated by several passages through a stainless-steel hypodermic needle (22 or 23 G) using a 1 ml syringe. Cell suspensions were passed through a nylon filter (pore size: 15/~m) and then centrifuged at 1000 r.p.m, for 3 min. The pellet was resuspended in Dulbecco's modified essential medium (DMEM, Flow) supplemented with 1.2 mg of NaHCO 3, 2 mM glutamine, 0.1/~g/ml 7S nerve growth factor (NGF), 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (100 iu penicillin, 100#g/ml streptomycin); to prevent non-neuronal cells growth, 5 # M cytosine arabinoside was added to the culture medium at the second day of culture and removed one day later (all drugs from Sigma). Neurons were cultured on a thin coverglass coated with polylysine, surrounded by a grease ring (vacuum grease, Beekmann) inside of a 35-petri dish, at a density of I ganglion/2 petri dishes. Neurons were grown for three to seven days at 37°C and 5% CO 2, before being used. Measurement of Cytosolic Ca 2+ The day before the experiment, trigeminal neurons were incubated in a N2 serum-free medium," containing: 1:1 DMEM and F-12 with 3.7 g/l NaHCO3, supplemented with 2mM glutamine, 1% penicillin-streptomycin (100 iu penicillin,. 100#g/ml streptomycin), 20/~M ascorbic acid, 1.2 mg/ml NaHCO3, 5/~g/ml insulin, 100/~g/ml transferrin, 20 nM progesterone, 100/~M putrescine, 30 nM Na2SeO3, and 0.1/~g/ml 7S nerve growth factor. Trigeminal neurons, that had been maintained for three to seven days in culture were loaded with the Ca2+-sensitive fluorescent probe Indo1,16 by incubation in DMEM with 5% bovine serum albumin (BSA), containing the acetoxymethylester form of Indo-1 (Indo-1/AM, 2#M, Molecular Probes), for 45 min at 37°C and 5% CO2 atmosphere. Subsequently, the coverglass with trigeminal neurons was attached to the base of a 300-/~l-ehamber mounted on the stage of an epifluorescence inverted microscope (Nikon Diaphot). Cultured neurons were superfused at I ml/min with a control medium composed by: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HBS) solution (20ram HEPES, l l 5 m M NaC1, 5.4mM KCI, 2.2mM CaCI2, 0.SmM MgCI2 and 13.SmM glucose, pH 0 7.4). Different pH 0 solutions were obtained by adding HC1 to the stock HEPES solution. The medium was kept at 37°C. The Indo-1 fluorescence (excitation, 350 nm) was split into two beams. These were passed through band-pass filters (centered at 410nm and 480nm) and detected by two photomultipliers. The fluorescence ratio (F410/F4s0) was
determined on-line and filtered at 10 Hz. Calibration of the fluorescence signals was achieved using an in vitro calibration procedure. Indo-K was added to saline solution containing Ca2÷-EGTA buffers giving minimum and saturating levels of Ca 2+, which allowed determination of the minimum and maximum fluorescence ratios, required for the equation: [Ca2+ ]i = Kafl(R - Rmin)/(Rmax-- R) where fl is the ratio of the maximum/minimum fluorescence emission at 410 zero Ca2+/410 sat Ca 2+, [Ca2+ ]i was calculated using Kd = 210 nmol/1. Drugs were applied or solutions changed by switching between reservoirs. The small bath (300 #1) and the high flow rates (250/~l/second) allowed rapid exchange of solutions: in some experiments (not shown) the pH 0 was changed slowly by switching the lines flowing at a rate of 1 ml/min. In such case the time necessary to attain the new pH0 is around 15 s. When serial stimuli were applied, basal Ca 2+ levels were allowed to recover before the next stimulus was applied. Neurons exhibiting an increase in [Ca2÷]~of over 200% when challenged with low pH or capsaicin were selected for experiments where attempts were made to block the response. Statistical comparisons were made using either a paired samples t-test or a Wilcoxon test for non parametric data. Data are presented as mean of percentage of basal [Ca2+]i q- SE of t h e m e a n . Capsaicin was dissolved in 1.5% ethanol, 8.5% Tween 80, and 90% HBS, to provide a 33 mM stock solution. Ruthenium Red (I mM) was dissolved in distilled water. Stock solutions of the drugs were diluted in the perfusing HBS to a final concentration of l/aM. Solutions containing 20 mM Ca 2+ were made by adding CaC12. In zero Ca 2+ solutions, CaC12 was substituted by an equimolar amount of MgC12; in some experiments, I mM EGTA was added to this solution. RESULTS
The mean resting level of [Ca2+]i in trigeminal neurons maintained in primary cultures for periods o f three to seven days was 158.7 _ 3.23 n M (n = 204). The functional status of the cell was assessed at the beginning of the experiment, by measuring the increase in [Ca2+]i elicited by high K ÷ ( 2 5 m M ) solutions (not shown). Intracellular calcium concentration changes induced by acidic solutions and capsaicin Figure 1 shows the effect of rapid changes of pH0 using different buffered solutions of pH0 ranging from 7.4 down to 6.0. Cells were superfused with each test solution for 3 s. O f a total of 204 tested cells, 86% responded to pH0 changes with a fast Ca 2+ rise, that reached a peak in about 1 s (Fig.l, inset) and then declined gradually, even when perfusion with the low pH solution was maintained (Fig. 1, inset; see also Fig. 8). Although we used a p H 6.5 solution to test for responses to acid, we also investigated in some cells the effect of other p H changes. As shown in Figure 2, Ca 2+ peak amplitude was proportional to the magnitude of the pH0 drop from the starting value o f 7.4, being maximal for decreases down to p H 0 5.5 and minimal when pH0 was lowered to 7.0.
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Fig. I. Changes in [Ca2+]i induced by application of acid to cultured newborn rabbit trigeminal neurons, measured with Indo-1 fluorescence. [Ca2+]i increases evoked by solutions of different pH (6.0, 6.5, 7.0), applied for 3 s (arrows). Inset: response to a pH 6.5 solution shown in an expanded time scale.
The mean peak [Ca2+]i value after application of pH 6.5, was 435.2 _ 21.2 nM which is an increase of 270.5 ___12.64% over the mean basal level 162.46 _ 3.51 nM (n = 174, P < 0.0001, Wilcoxon test). Successive challenges with the same low PH0 solution led to progressively smaller Ca 2÷ peaks. Desensitization of the response varied from cell to cell but was evident when the effect of successive challenges applied with short ( < 1 min) time intervals were compared, as shown in Fig. 3A. Desensitization was not observed when a resting period of 4 min or more was left between test. When pH 0 changes were applied at a slow rate (see Experimental Procedures), Ca 2÷ transients were not observed in most of the cases (data not shown). The amplitude of the [Ca2+]~ peak was also dependent on the initial pH0 at which the cells were continuously bathed. As shown in Figure 3B, keeping the cells at pH 0 7.9 and changing it to pH0 6.5 elicited
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a larger [Ca 2+]i increase than when the initial pH0 was more acidic. The effect was particularly evident if the cell was kept at pH 0 7.1 (second challenge in Fig. 3B); from this value, a further decrease to pH06.5 produced a very small elevation of [Ca 2+]~. Capsaicin (1-2/~M) also elicited a rise of neuronal [Ca2+]~ in 39% of cells (53 out of 135) (Fig. 4). The time course of the response to capsaicin was quite variable, ranging from a sharp peak to a smooth rise in [Ca ~+]i or a mixture of both (compare the effects of capsaicin shown in Figs 4 and 8). On the average, capsaicin, induced a significant elevation of [Ca:+]~ to a peak mean value that was 381.9+ 33.2nM which is an increment of 218.4 + 17.6% above resting level (n = 53, P < 0.001, Wilcoxon test).
Comparison o f the responses to acid and capsaicin Among 135 cells challenged with both pH and capsaicin, applied in either order, 39% responded to both stimuli, 46% were activated exclusively by low pH 0 and the remaining neurons did not respond to any of the stimuli. The response of the cells could not be correlated with cell diameter or any other apparent morphological characteristic of the explored neurons. Figure 4 depicts an example of a cell responding both to pH0 decreases and to capsaicin. The capsaicin-induced [Ca2+]~ elevation was longer-lasting than the response to low pH0. Desensitization appeared after repeated challenges with capsaicin (data not shown) as previously reported in the literature) 2,~4
pH Fig. 2. Peak changes in [Caz+ ]i of cultured trigeminal neurons, induced by different pHo values. Responses for individual cells are expressed as per cent of basal [Ca2+]~at pH 7.4. The mean data are represented by black dots. Individual values for pH 6.5 (n = 174) are not represented and only the average value has been included. NSC
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Effects o f zero extracellular calcium The source of Ca 2+ for the [Ca2+]i response to pH and capsaicin was explored in a separate set of experiments. When cells were superfused at zero [Ca2+]0, (no EGTA added), the response to both
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Fig. 3. Desensitization of pH-induced increases in [Ca2+]~. (A) Attenuation of the response to repeated stimulation with a pH 6.5 solution (arrows). Inset: peak [Ca2+ ]~ represented as per cent of the response to the first stimulus. (B). Response of a cultured trigeminal neuron to stimulation with pH 6.5 (arrows) performed at different initial pH 0 values 7.9, 7.1 (applied during the time indicated by the corresponding horizontal bar) or control pH 0 value (7.4), representative of eight experiments.
agents was absent or minimal (Fig. 5), being attributable in the latter cases to residual Ca 2+ in the medium. This residual response was never present when 1 m M E G T A was added to the zero Ca 2+ solution. When Ca 2÷ was reintroduced in the superfusion medium, cells were again able to respond to pH and capsaicin, as shown in Fig. 5. N o differences in basal [Ca 2÷ ]~ were found between cells superfused with zero and with 2.2 m M Ca 2+ solutions.
Effect o f high extracellular calcium The inward current activated by protons in sensory neurons is blocked by high external calciumJ 3'z°
Therefore we have tested whether a similar effect occurs with the calcium transients. This manoeuvre did not modify significantly basal [Ca 2÷ ]i. When cells were subsequently challenged with protons, the evoked rise in [Ca 2÷ ]i was smaller than seen with the regular (2.2mM) [Ca2+]0 concentration (Fig. 6, inset). Figure 6 shows an example of the reduced [Ca2+]i elevation obtained by stimulation with pH 0 6.5, in the presence of a high [Ca 2÷ ]0. Such small response was not due to desensitization, as the [Ca2+ ]i peak recovered when [Ca 2÷ ]0 was returned to 2.2raM (not shown). The effects of capsaicin in 20 m M [Ca2+]0 are also depicted in Fig. 6. In the presence of high [Ca2+]0, the [CaZ+]i response to
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Fig. 4. Modification of [Ca 2+ ]~ in a trigeminal neuron, elicited by application of a pH 6.5 stimulus and a 1/~M capsaicin solution.
capsaicin was always present, although the shape of the response differed from that of the control response. The mean amplitude of the [Ca2+]i peak elicited by capsaicin was not statistically different in normal and in 2 0 m M [Ca2+]0 (Fig. 6, inset). However, the areas under the [Ca 2+ ]i curves evoked by capsaicin in high [Ca2+]0 were 740.02 ___276.5% of the corresponding capsaicin responses obtained in control medium (n = 7, P < 0.01, paired t-test), indicating a larger influx of calcium under these experimental conditions. In these experiments, basal
Ca 2+ levels did not usually recover after capsaicin treatment.
Effects of Ruthenium Red It has been previously shown that the inward current elicited by capsaicin in primary sensory neurons can be blocked with Ruthenium Red. 2,m'14 Therefore, the possibility that this drug antagonized [Ca 2+ ]~ rises induced by acid and capsaicin was explored in another set of experiments. Basal [Ca2+]i decreased
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Fig. 5. Influence of zero [Ca2+ ]0 on the [Ca 2+ ]i response to acid and capsaicin. During the period indicated by the horizontal bar, the cell was superfused with a calcium-free medium (no EGTA added). Thin arrow: stimulation with pH 6.5 solution. Thick arrow: application of 1 p M capsaicin. Inset: changes in [Ca 2÷ ]~, expressed as per cent of basal [Ca 2+ ]i, elicited by pH 6.5 and capsaicin in normal (open bars) and zero (hatched bars) [Ca 2÷ ]0. n = 11 for control and acid-stimulated neurons, n = 9 for capsaicin-stimulated neurons. The responses to pH 6.5 and capsaicin in zero and 2.2 mM [Ca 2÷ ]0 were statistically different, *P < 0.001, paired t-test.
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time (min) Fig. 6. Influence of 20 mM [Ca2+]0 on the [Ca2+]i response to acid and capsaicin. During the period indicated by the horizontal bar, the cell was superfused with a 20 mM calcium solution. Thin arrow: stimulation with pH 6.5 solution. Thick arrow: application of 1/~M capsaicin. Inset: changes in [Ca 2+ ]i, expressed as percent of basal [Ca2÷ ]i, elicited by pH 6.5 and capsaicin in normal (open bars) and 20 mM [Ca2÷ ]0 (hatched bars), n = 9 for control and acid-stimulated neurons; n = 6 for capsaicin-stimulated neurons. The responses to pH 6.5 in 20 mM and 2.2 mM [Ca2+]0 were statistically different, *P < 0.05, paired t-test.
slightly (not significant) u n d e r the effect o f R u t h e n i u m Red ( 1 / t M ) . T h e drug blocked fully the effect o f capsaicin a n d reduced the response to low p H 0 (Fig. 7). Cells did n o t recover usually after washing the R u t h e n i u m Red.
Influence o f sustained p H 0 decreases In a n effort to dissociate fast from slow comp o n e n t s in the [Ca 2+ ]i responses to acid a n d capsaicin, a low p H ( 6 . 5 ) solution was applied for a
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Fig. 7. Effect of 1 # M Ruthenium Red (RR, horizontal bar) on the changes of [Ca2+]i induced by application of acid (thin arrow) and capsaicin (thick arrow). Inset: changes in [Ca 2+ ]i, expressed as per cent of basal [Ca2+ ]~, elicited by pH 6.5 and capsaicin during superfusion with the control solution (open bars) and the solution containing RR (hatched bars), n = 11 for control and acid-stimulated neurons, n = 8 for capsaicin-stimulated neurons. *P < 0.002; **P < 0.005.
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Fig. 8. Desensitization of the response to acid by continuous exposure of the cell to low pH 0. During the time indicated by the horizontal bar, the cell was superfused with a pH 6.5 solution. The thick arrow signals the applications of 1 #M capsaicin. Representative of six experiments. prolonged period of time. This produced a transient, initial [Ca2+]~ peak that decayed spontaneously in about 1 min to resting [Ca2+]~ levels. Under these conditions, capsaicin still produced an increase in [Ca2+]i, as shown in Fig. 8.
Influence of long-term capsaicin pretreatment In the next group of experiments, we analysed the effects of long-term capsaicin treatment. Cells were cultured for periods of 10-24 h in a medium containing 10/~M capsaicin. It is known that the subpopulation of capsaicin-sensitive cells disappears when primary sensory neurons are grown for a prolonged period of time in the presence of capsaicin. 8 Accordingly, incubation of the cells in the presence of 10 # M capsaicin greatly diminished the total number of sensory neurons. The response to acidic solutions (pH0 = 6.5) was tested in a total of nine cells from five different culture dishes and was not different from the control ones (not shown). However, none of these neurons responded to capsaicin. DISCUSSION The present experiments demonstrate that sudden decreases of pH 0 down to 6.5 elicit a transient elevation of [Ca 2+ ]i. In agreement with the results of previous authors, a rise in [Ca 2+ ]i was also evoked by application of capsaicin, a substance that has been claimed to selectively activate polymodal nociceptive neurons. 3'6'2s~33All neurons showing a [Ca 2+ ]i increase when challenged with capsaicin also exhibited a sensitivity to protons; however, 47% of the cells responded to acid but not to capsaicin.
Calcium concentration changes induced by acidic ~olutions Rapid changes in pH 0 produced an increase in [Ca2+]~ in a large percentage of cultured trigeminal
cells. The effect was transitory and exhibited desensitization. [Ca 2+ ]i rises appear to be related to protoninduced changes in membrane currents. It has been described that acidic solutions activate two types of currents in the soma of dorsal root ganglion neurons "in vitro.". Sudden pH 0 drops from 7.4 to 6.0 elicited a fast, rapidly inactivating inward current carried mainly by sodium. 13'2° In about half of the neurons, strong pH reductions (<6.2) also evoked a second, inward current with a much slower time course) This sustained inward current was attributed to activation of an non-specific cationic conductance for K +, Cs + and Na +. In most of our experiments, test pH0 was 6.5; therefore, the effects of protons on [Ca 2+]~ described here are presumably associated to the proton-induced fast inactivating current. It has been proposed that this current is driven by monovalent cations through the transient transformation by protons of a voltage dependent Ca 2+ channel into a non-selective cationic channelJ '2° Many of the described characteristics of such current are compatible with the changes in [Ca2+]i observed in the present experiments: Current activation depend on fast changes of pH0 and was graded, being maximum for changes from pH07.4 to 5.5; furthermore, small increases of external concentration of H +, which by themselves failed to activate this current, inactivated the proton-induced responseJ s'2° Likewise, changes in [Ca2+]i in our experiments were maximal for pH0 reductions to 5.5 but were still apparent at pH 0=7.0. As reported for the fast proton-induced currents, slow acidification decreased the effect of rapid changes of pH 0 on [Ca 2+ ]i- In contrast, acidification from an alkaline solution (PH07.9) elicited larger [Ca2+]~ transients. A further parallel between fast inactivating current and [Ca2+ ]i rises elicited by protons was the blockade by high [Ca2+]0 of both processes. Moreover, the fast proton-induced current is blocked by inorganic Ca 2+
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channel blockers. 2° Likewise, Ni 2+ produce a reduction of [Ca2+]i peaks in cultured trigeminal neurons (Garcia-Hirschfeld and L6pez-Briones, unpublished observations). There is experimental evidence suggesting that when the proton-induced Na + current is activated, voltage-gated Ca 2+ currents are temporarily suppressed. 2° Our results with zero [Ca2+]0 prove that Ca 2+ entering the cell after acidic stimuli was of extracellular origin. Therefore, we can conclude either that under normal [Ca2+]o "transformed" Ca 2+ channels retain some permeability to Ca 2+ or that this ion permeates through other type of Ca 2+ channel, that would be gated by membrane depolarization resulting from proton-induced current. 8'27 The contribution of the sustained inward current described in presumed nociceptive cells, to the [Ca2+]~ rises elicited by protons is difficult to assess; ~'2° none the less, the time course and duration of the proton-induced [Ca2+]i responses, as well as the relative independence of pH and capsaicin effects (see below) speak against a significant contribution of this current to the pH-induced Ca 2÷ peaks reported here. Calcium concentration changes induced by capsaicin. Comparison with the response to acidic solutions
Our experiments confirmed that capsaicin increases
[Ca2+ ]i in primary sensory neurons. 12'1°'33The source of Ca 2+ was extracellular as the response disappeared when the cells were bathed in a Ca2+-free medium. Capsaicin was still able to induce a rise in [Ca 2+ ]i even after the fast pH0-induced [Ca 2+ ]i transient had inactivated during maintained low pH application. In fact, in some of the experiments, the [Ca2+]~ change elicited by capsaicin was considerably larger when the initial pH was under 7.4. Such sensitization most likely correspond to pH effects on capsaicin-evoked currents, which are markedly augmented in the presence of protons. 27 Thus, the response to capsaicin is maintained or augmented when the calcium entry evoked by low pH has inactivated, suggesting that the Ca 2+ transients produced by low pH and capsaicin occur via independent mechanisms. This is supported by the following additional evidence: (i) the capsaicin effect was enhanced at high extracellular Ca 2+ levels while this manoeuvre blocked the response to low pH0; (ii) the effect of capsaicin was evoked even when the fast pH-induced [Ca2+]i elevation had returned to resting values in the continuous presence of an acidic pH0; (iii) Ruthenium Red
blocked the response to capsaicin but only attenuated the effect of protons; (iv) normal responses to pH 0 reductions were observed in surviving neurons, after elimination of capsaicin-sensitive cells by prolonged incubation with this agent. Bevan and Yeats 8 proposed that the ionic channel involved in the response to capsaicin was also activated by pH values below 6.2. We did not test systematically such low pH's; thus, it remains to be determined whether pH drops below 6.2 also induced a sustained [Ca2+]i elevation as does capsaicin. Responses of peripheral nociceptors to acid include a phasic burst of impulses followed by a sustained discharge. 4'29A parallel between these firing responses and the fast and slow currents has been suggested. 29 Our results further show that initial activation of sensory neurons by protons is accompanied by an entrance of calcium. This phenomenon is presumably present also in nociceptive neurons and may contribute to activation processes of peripheral nociceptive terminals. CONCLUSIONS Our results demonstrate that rapid reductions in pH evoke a transient rise in [Ca2+]i in cultured trigeminal neurons; we also report a [Ca 2÷ ]~ increase in response to capsaicin. The characteristics of these responses suggest that [Ca 2÷ ]~ rises evoked by rapid reductions in pH and by capsaicin occur through different mechanisms because: (i) the effect of capsaicin was enhanced in high extracellular Ca 2÷ while this manoeuvre blocked the response to low pH0; (ii) the effect of capsaicin was still evoked even when the [Ca2+]i elevation induced by acid stimulation had returned to resting values in the continuous presence of a low pH0; (iii) Ruthenium Red blocked the response to capsaicin but only attenuated the effect of protons; (iv) Normal responses to pH0 reductions were present in surviving neurons, after elimination of capsaicin-sensitive cells by prolonged incubation with this agent. Acknowledgements--The collaboration of Ms Rosa Garcia Velasco in preparing the cultures and the technical assistance of Mr Sim6n Moya and Alfonso Prrez-Vegara are gratefully acknowledged. We thank S. Bevan, R. Gallego and J. Gallar for critical reading of the manuscript. Supported by grants PM90-0113 and SAF93-0267of the Comisi6n Nacional de Ciencia y Tecnologia, Direcci6n General de Investigaci6n Cientifica y T6cnica, and F.I.S. 93/134 of the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
REFERENCES
1. Akaike N., Krishtal O. A. and Maruyama T. (1990) Proton-induced sodium current in frog isolated dorsal root ganglion cells. J. Neurophysiol. 63, 805-813. 2. Amann R., Donnerer J. and Lembeck F. (1989) Capsaicin-induced stimulation of polymodal nociceptors is antagonized by Ruthenium Red independently of extracellular calcium. Neuroscience 32, 255 259. 3. Baccaglini P. I. and Hogan P. G. (1983) Some rat sensory neurones express characteristics of different pain sensory cells. Proc. natn. Acad. Sci. U.S.A. 80, 594 598.
Intracellular calcium in cultured trigeminal neurons
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4. Belmonte C., Gallar J., Pozo M. A. and Rebollo I. (1991) Excitation by irritant chemical substances of sensory afferent units in the cat's cornea. J. Physiol. 437, 709-725. 5. Bessou P. and Perl E. R. (1969) Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J. Neurophysiol. 32, 1025-1043. 6. Bevan S. and Forbes C. A. (1988) Membrane effects of capsaicin on rat dorsal root ganglion neurones in cell culture. J. Physiol. 398, 28P. 7. Bevan S. and Szolcs~inyi J. (1990) Sensory neuron-specific actions of capsaicin: mechanisms and applications. Trends Pharmae. Sci. 11, 330-333. 8. Bevan S. and Yeats J. (1991) Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurones. J. Physiol., Lond. 433, 145-161. 9. Bevan S. J., Forbes C. A. and Winter J. (1993) Protons and capsaicin activate the same ion channels in rat isolated dorsal root ganglion neurones. J. Physiol. 549, 401P. 10. Bleackman D., Brorson J. R. and Miller R. J. (1990) The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells. Br. J. Pharmac. 101, 423-431. l l. Bottenstein J. E. and Sato G. H. (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. natn. Acad. Sci. U.S.A. 76, 514-517. 12. Cholewinski A., Burgess G. M. and Bevan S. (1993) The role of calcium in capsaicin-induced desensitization in rat cultured dorsal root ganglion neurons. Neuroscienee 55, 1015 1023. 13. Davies N. W., Lux H. D. and Morad M. (1988) Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones. J. Physiol., Lond. 400, 159-187. 14. Dray A., Forbes C. A. and Burgess G. M. (1990) Ruthenium Red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurons as well as the activation of peripheral nociceptor in vitro. Neurosci. Lett. 110, 52-59. 15. Fitzgerald M. (1983) Capsaicin and sensory neurons, a review. Pain 15, 109 130. 16. Grynkiewicz G., Poenie M. and Tsien R. Y. (1985) A new generation of Ca z+ indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440-3450. 17. Hawrot E. and Patterson P. H. (1979) Long-term culture of dissociated sympathetic neurons. Meth. Enzymol. 58, 574-584. 18. Heyman I. and Rang H. P. (1985) Depolarizing responses to capsaicin in a subpopulation of dorsal root ganglion cells. Neurosci. Lett. 56, 69-75. 19. Higgins D., Lien P. J., Osterhout D. J. and Johnson M. I. (1991) Tissue culture of mammalian autonomic neurons. Culturing nerve cells. In: Cellular and Molecular Neuroseience Series. (eds Banker G. and Goslin K.) pp. 177-205. Massachusetts Institute of Technology Press, Cambridge, MA. 20. Konnerth A., Lux H. D. and Morad M. (1987) Proton-induced transformation of calcium channel in chick dorsal ganglion cells. J. Physiol., Lond. 386, 603-633. 21. Kovalchuk Y. N., Krishtal O. A. and Nowycky M. C. (1990) The proton-activated inward current of rat sensory neurons includes a calcium component. Neurosci. Lett. 115, 237-242. 22. Krishtal O. A. and Pidoplichko V. I. (1981) Receptor for protons in the membrane of sensory neurons. Brain Res. 214, 150-154. 23. Krishtal O. A. and Pidoplichko V. I. (1981) A receptor for protons in the membrane of sensory neurons may participate in nociception. Neuroscience 6, 2599 2601. 24. Lindahl O. (1962) Pain: a chemical explanation. Acta Rheum. Scand. 8, 161-169. 25. Lynn B. (1990) Capsaicin: actions on nociceptive C-fibres and therapeutic potential. Pain 41, 61~59. 26. Marsh S. J., Stanfeld C. E., Brown D. A., Davey R. and McCarthy D. (1987) The mechanism of action of capsaicin on sensory c-type neurons and their axons in vitro. Neuroseience 23, 275-289. 27. Petersen M. and LaMotte R. H. (1993) Effect of protons on the inward current evoked by capsaicin in isolated dorsal root ganglion cells. Pain 54, 37-42. 28. Rang H. P. (1991) The nociceptive afferent neurone as a target for new types of analgesic drug. Proceeding of the Vllth Worm Congress on Pain, 119-127. Elsevier Science Publishers BV. 29. Steen K. H., Reeh P. W., Anton F. and Handwerker H. O. (1992) Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin in vitro. J. Neurosci. 12, 86-95. 30. Szolcsfinyi J. (1977) A pharmacological approach to elucidation of the role of different nerve fibres and receptor endings in mediation of pain. J. Physiol., Paris 73, 251 259. 31. Szolcs~inyi J. (1982) In Handbook of Experimental Pharmacology (ed. Milton A. S.), Vol. 60, pp. 437-478. Springe~Verlag, Berlin. 32. Willis W. D. Jr (1985) In The Pain System. The Neural Basis of Nociceptive Transmission in the Mammalian Nervous System. Karger, New York. 33. Wood J. N., Winter J., James I. F., Rang H. P., Yeats J. and Bevan S. (1988) Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture. J. Neurosci. 8, 3208-3230. (Accepted 20 December 1994)
~
Pergamon
0306-4522(95)00055-0
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
INTRACELLULAR FREE CALCIUM RESPONSES TO PROTONS A N D CAPSAICIN IN CULTURED TRIGEMINAL NEURONS J. G A R C I A - H I R S C H F E L D , L. G. L O P E Z - B R I O N E S , C. B E L M O N T E and M. V A L D E O L M I L L O S * Departamento de Fisiologia and Instituto de Neurociencias, Facultad de Medicina, Universidad de Alicante, Aptdo. 374. 03080 Alicante, Spain Abstract--Acidic solutions and capsaicin are selective chemical stimuli for nociceptive neurons. The effect of these stimuli on intracellular calcium concentration was analysed in cultured trigeminal neurons of newborn rabbits. Rapid reductions in pH (from 7.4 to 5) evoked a transient rise in intracellular calcium concentration of 270% on average over the basal level (162.5 + 3.5 nM; n = 174) in 86% of the neurons. Maximal responses were found at pH 5.5. Proton-induced transients were diminished or abolished by 20 mM CaC12, by zero CaC12 and by 1/~M Ruthenium Red. In response to 1/tM capsaicin, 40% of the cells that were sensitive to protons also increased their intracellular calcium concentration to 218% of control. Capsaicin-induced intracellular calcium concentration rises were composed of an initial peak followed by a second, slower intracellular calcium concentration elevation. The capsaicin response was completely blocked by 1 pM Ruthenium Red, and disappeared in zero calcium, but was augmented in high extracellular calcium. Intracellular calcium concentration responses to capsaicin were still observed in neurons whose response to protons was desensitized by sustained exposure to low pH (pH 6.5). Cells surviving a 10-24 h capsaicin (10#M) treatment, still displayed responses to pH reductions. These results suggest that intracellular calcium concentration rises induced by moderate reductions in pH 0 and capsaicin occur through different mechanisms.
Transduction of injurious stimuli into a discharge of nerve impulses occurs in nociceptors and is often the first step in the genesis of pain sensations. Polymodal nociceptors are free nerve terminals of a subpopulation of primary neurons located in sensory ganglia, that respond to noxious mechanical forces, irritant chemicals and heat applied to peripheral tissues. 5 The discharge characteristics of nociceptive fibers in response to tissue damage have been extensively studied. 32 However, the mechanisms involved in stimulus transduction in nociceptive terminals are largely unknown. Acidic solutions and capsaicin are selective chemical stimuli for nociceptive neurons. 3'~5'22'23 Decreases of pHo are known to evoke nerve discharges in pain fibers; 4'29 moreover, proton accumulation in inflammatory exudates were proposed as one of the causes of nociceptive stimulation in injured tissues. 24 Capsaicin, a neurotoxin with well known pungent effects, selectively excites and eventually inactivates peripheral nociceptors? °'3~ Due to this specific effect,
capsaicin sensitivity of primary sensory neurons has been often employed to identify nociceptive cells.3,6,7,33 The soma of cultured mammalian trigeminal or dorsal root ganglion neurons has served as a model to study transduction of chemical stimuli by nociceptors. 3'22 When applied to the cell body, low pH 0 solutions activate two different inward currents? the first is a fast inactivating current that is evoked by small, rapid changes in p H 0 values and is present in a large proportion of primary sensory neurons, as well as in other excitable cells. 13'2° The second is a sustained, slowly inactivating cationic current evoked by moderate p H 0 (pH 6.2) reductions. This second current is restricted to the subpopulation of capsaicin-sensitive sensory neurons. 8 Although the bulk of p H 0 evoked currents is carried by monovalent cations, there are recent suggestions that Ca 2+ ions may also contribute to the fast inactivating current. 21 A long-lasting current, that resembles the sustained current produced by protons, is also evoked by capsaicin in a proportion of dorsal root ganglion primary sensory n e u r o n s ) This effect appears to be mediated by a specific membrane receptor for capsaicin that activates a cationic channel. 28 The channel is relatively nonselective, being permeable to both monovalent and divalent cations. 7'~2'~5'~s'-'5 Based on the analogy of currents evoked in nociceptive neurons by acid and capsaicin, it has been proposed that both
*To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin;
DMEM, Dulbecco's modified essential medium; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid; FCS, fetal calf serum; HBS, HEPES-buffered saline solution; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; INDO-1/AM, indo-1 acetoxymethylester; NGF, nerve growth factor. 235
236
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substances activate the same ionic channel. 9 The purpose o f this work was to analyse the changes of intracellular calcium concentration ([Ca 2÷ ]i) induced by acidic solutions and by capsaicin in the soma of cultured trigeminal neurons and to explore the relationship between Ca 2÷ Signals activated by both stimuli.
EXPERIMENTAL PROCEDURES
Culture of trigeminal neurons Newborn rabbit (pseudocalifornia from our local breeding house) trigeminal ganglia were used. Animals were killed with an overdose of nembutal (1 ml of a 15 mg/ml solution). For trigeminal neuron culture, standard procedures were employedY Briefly, animals were washed with 70% ethanol and trigeminal ganglia were isolated under a dissecting microscope and settled in 1 ml of Leibovitz medium (L-15, Flow). Ganglia were washed with culture medium and incubated in collagenase 0.025mg/ml (Boehringer) for 45 rain in a humidified atmosphere of 5% CO2, 95% air. Enzymatic treatment was stopped by adding fresh L15 medium, t9 Chunks of tissue were dissociated by several passages through a stainless-steel hypodermic needle (22 or 23 G) using a 1 ml syringe. Cell suspensions were passed through a nylon filter (pore size: 15/~m) and then centrifuged at 1000 r.p.m, for 3 min. The pellet was resuspended in Dulbecco's modified essential medium (DMEM, Flow) supplemented with 1.2 mg of NaHCO 3, 2 mM glutamine, 0.1/~g/ml 7S nerve growth factor (NGF), 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (100 iu penicillin, 100#g/ml streptomycin); to prevent non-neuronal cells growth, 5 # M cytosine arabinoside was added to the culture medium at the second day of culture and removed one day later (all drugs from Sigma). Neurons were cultured on a thin coverglass coated with polylysine, surrounded by a grease ring (vacuum grease, Beekmann) inside of a 35-petri dish, at a density of I ganglion/2 petri dishes. Neurons were grown for three to seven days at 37°C and 5% CO 2, before being used. Measurement of Cytosolic Ca 2+ The day before the experiment, trigeminal neurons were incubated in a N2 serum-free medium," containing: 1:1 DMEM and F-12 with 3.7 g/l NaHCO3, supplemented with 2mM glutamine, 1% penicillin-streptomycin (100 iu penicillin,. 100#g/ml streptomycin), 20/~M ascorbic acid, 1.2 mg/ml NaHCO3, 5/~g/ml insulin, 100/~g/ml transferrin, 20 nM progesterone, 100/~M putrescine, 30 nM Na2SeO3, and 0.1/~g/ml 7S nerve growth factor. Trigeminal neurons, that had been maintained for three to seven days in culture were loaded with the Ca2+-sensitive fluorescent probe Indo1,16 by incubation in DMEM with 5% bovine serum albumin (BSA), containing the acetoxymethylester form of Indo-1 (Indo-1/AM, 2#M, Molecular Probes), for 45 min at 37°C and 5% CO2 atmosphere. Subsequently, the coverglass with trigeminal neurons was attached to the base of a 300-/~l-ehamber mounted on the stage of an epifluorescence inverted microscope (Nikon Diaphot). Cultured neurons were superfused at I ml/min with a control medium composed by: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HBS) solution (20ram HEPES, l l 5 m M NaC1, 5.4mM KCI, 2.2mM CaCI2, 0.SmM MgCI2 and 13.SmM glucose, pH 0 7.4). Different pH 0 solutions were obtained by adding HC1 to the stock HEPES solution. The medium was kept at 37°C. The Indo-1 fluorescence (excitation, 350 nm) was split into two beams. These were passed through band-pass filters (centered at 410nm and 480nm) and detected by two photomultipliers. The fluorescence ratio (F410/F4s0) was
determined on-line and filtered at 10 Hz. Calibration of the fluorescence signals was achieved using an in vitro calibration procedure. Indo-K was added to saline solution containing Ca2÷-EGTA buffers giving minimum and saturating levels of Ca 2+, which allowed determination of the minimum and maximum fluorescence ratios, required for the equation: [Ca2+ ]i = Kafl(R - Rmin)/(Rmax-- R) where fl is the ratio of the maximum/minimum fluorescence emission at 410 zero Ca2+/410 sat Ca 2+, [Ca2+ ]i was calculated using Kd = 210 nmol/1. Drugs were applied or solutions changed by switching between reservoirs. The small bath (300 #1) and the high flow rates (250/~l/second) allowed rapid exchange of solutions: in some experiments (not shown) the pH 0 was changed slowly by switching the lines flowing at a rate of 1 ml/min. In such case the time necessary to attain the new pH0 is around 15 s. When serial stimuli were applied, basal Ca 2+ levels were allowed to recover before the next stimulus was applied. Neurons exhibiting an increase in [Ca2÷]~of over 200% when challenged with low pH or capsaicin were selected for experiments where attempts were made to block the response. Statistical comparisons were made using either a paired samples t-test or a Wilcoxon test for non parametric data. Data are presented as mean of percentage of basal [Ca2+]i q- SE of t h e m e a n . Capsaicin was dissolved in 1.5% ethanol, 8.5% Tween 80, and 90% HBS, to provide a 33 mM stock solution. Ruthenium Red (I mM) was dissolved in distilled water. Stock solutions of the drugs were diluted in the perfusing HBS to a final concentration of l/aM. Solutions containing 20 mM Ca 2+ were made by adding CaC12. In zero Ca 2+ solutions, CaC12 was substituted by an equimolar amount of MgC12; in some experiments, I mM EGTA was added to this solution. RESULTS
The mean resting level of [Ca2+]i in trigeminal neurons maintained in primary cultures for periods o f three to seven days was 158.7 _ 3.23 n M (n = 204). The functional status of the cell was assessed at the beginning of the experiment, by measuring the increase in [Ca2+]i elicited by high K ÷ ( 2 5 m M ) solutions (not shown). Intracellular calcium concentration changes induced by acidic solutions and capsaicin Figure 1 shows the effect of rapid changes of pH0 using different buffered solutions of pH0 ranging from 7.4 down to 6.0. Cells were superfused with each test solution for 3 s. O f a total of 204 tested cells, 86% responded to pH0 changes with a fast Ca 2+ rise, that reached a peak in about 1 s (Fig.l, inset) and then declined gradually, even when perfusion with the low pH solution was maintained (Fig. 1, inset; see also Fig. 8). Although we used a p H 6.5 solution to test for responses to acid, we also investigated in some cells the effect of other p H changes. As shown in Figure 2, Ca 2+ peak amplitude was proportional to the magnitude of the pH0 drop from the starting value o f 7.4, being maximal for decreases down to p H 0 5.5 and minimal when pH0 was lowered to 7.0.
Intracellular calcium in cultured trigeminal neurons
_pH6.5
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Fig. I. Changes in [Ca2+]i induced by application of acid to cultured newborn rabbit trigeminal neurons, measured with Indo-1 fluorescence. [Ca2+]i increases evoked by solutions of different pH (6.0, 6.5, 7.0), applied for 3 s (arrows). Inset: response to a pH 6.5 solution shown in an expanded time scale.
The mean peak [Ca2+]i value after application of pH 6.5, was 435.2 _ 21.2 nM which is an increase of 270.5 ___12.64% over the mean basal level 162.46 _ 3.51 nM (n = 174, P < 0.0001, Wilcoxon test). Successive challenges with the same low PH0 solution led to progressively smaller Ca 2÷ peaks. Desensitization of the response varied from cell to cell but was evident when the effect of successive challenges applied with short ( < 1 min) time intervals were compared, as shown in Fig. 3A. Desensitization was not observed when a resting period of 4 min or more was left between test. When pH 0 changes were applied at a slow rate (see Experimental Procedures), Ca 2÷ transients were not observed in most of the cases (data not shown). The amplitude of the [Ca2+]~ peak was also dependent on the initial pH0 at which the cells were continuously bathed. As shown in Figure 3B, keeping the cells at pH 0 7.9 and changing it to pH0 6.5 elicited
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a larger [Ca 2+]i increase than when the initial pH0 was more acidic. The effect was particularly evident if the cell was kept at pH 0 7.1 (second challenge in Fig. 3B); from this value, a further decrease to pH06.5 produced a very small elevation of [Ca 2+]~. Capsaicin (1-2/~M) also elicited a rise of neuronal [Ca2+]~ in 39% of cells (53 out of 135) (Fig. 4). The time course of the response to capsaicin was quite variable, ranging from a sharp peak to a smooth rise in [Ca ~+]i or a mixture of both (compare the effects of capsaicin shown in Figs 4 and 8). On the average, capsaicin, induced a significant elevation of [Ca:+]~ to a peak mean value that was 381.9+ 33.2nM which is an increment of 218.4 + 17.6% above resting level (n = 53, P < 0.001, Wilcoxon test).
Comparison o f the responses to acid and capsaicin Among 135 cells challenged with both pH and capsaicin, applied in either order, 39% responded to both stimuli, 46% were activated exclusively by low pH 0 and the remaining neurons did not respond to any of the stimuli. The response of the cells could not be correlated with cell diameter or any other apparent morphological characteristic of the explored neurons. Figure 4 depicts an example of a cell responding both to pH0 decreases and to capsaicin. The capsaicin-induced [Ca2+]~ elevation was longer-lasting than the response to low pH0. Desensitization appeared after repeated challenges with capsaicin (data not shown) as previously reported in the literature) 2,~4
pH Fig. 2. Peak changes in [Caz+ ]i of cultured trigeminal neurons, induced by different pHo values. Responses for individual cells are expressed as per cent of basal [Ca2+]~at pH 7.4. The mean data are represented by black dots. Individual values for pH 6.5 (n = 174) are not represented and only the average value has been included. NSC
67/1..
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Effects o f zero extracellular calcium The source of Ca 2+ for the [Ca2+]i response to pH and capsaicin was explored in a separate set of experiments. When cells were superfused at zero [Ca2+]0, (no EGTA added), the response to both
J. Garcia-Hirschfeld et al.
238
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Fig. 3. Desensitization of pH-induced increases in [Ca2+]~. (A) Attenuation of the response to repeated stimulation with a pH 6.5 solution (arrows). Inset: peak [Ca2+ ]~ represented as per cent of the response to the first stimulus. (B). Response of a cultured trigeminal neuron to stimulation with pH 6.5 (arrows) performed at different initial pH 0 values 7.9, 7.1 (applied during the time indicated by the corresponding horizontal bar) or control pH 0 value (7.4), representative of eight experiments.
agents was absent or minimal (Fig. 5), being attributable in the latter cases to residual Ca 2+ in the medium. This residual response was never present when 1 m M E G T A was added to the zero Ca 2+ solution. When Ca 2÷ was reintroduced in the superfusion medium, cells were again able to respond to pH and capsaicin, as shown in Fig. 5. N o differences in basal [Ca 2÷ ]~ were found between cells superfused with zero and with 2.2 m M Ca 2+ solutions.
Effect o f high extracellular calcium The inward current activated by protons in sensory neurons is blocked by high external calciumJ 3'z°
Therefore we have tested whether a similar effect occurs with the calcium transients. This manoeuvre did not modify significantly basal [Ca 2÷ ]i. When cells were subsequently challenged with protons, the evoked rise in [Ca 2÷ ]i was smaller than seen with the regular (2.2mM) [Ca2+]0 concentration (Fig. 6, inset). Figure 6 shows an example of the reduced [Ca2+]i elevation obtained by stimulation with pH 0 6.5, in the presence of a high [Ca 2÷ ]0. Such small response was not due to desensitization, as the [Ca2+ ]i peak recovered when [Ca 2÷ ]0 was returned to 2.2raM (not shown). The effects of capsaicin in 20 m M [Ca2+]0 are also depicted in Fig. 6. In the presence of high [Ca2+]0, the [CaZ+]i response to
239
Intracellular calcium in cultured trigeminal neurons
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Fig. 4. Modification of [Ca 2+ ]~ in a trigeminal neuron, elicited by application of a pH 6.5 stimulus and a 1/~M capsaicin solution.
capsaicin was always present, although the shape of the response differed from that of the control response. The mean amplitude of the [Ca2+]i peak elicited by capsaicin was not statistically different in normal and in 2 0 m M [Ca2+]0 (Fig. 6, inset). However, the areas under the [Ca 2+ ]i curves evoked by capsaicin in high [Ca2+]0 were 740.02 ___276.5% of the corresponding capsaicin responses obtained in control medium (n = 7, P < 0.01, paired t-test), indicating a larger influx of calcium under these experimental conditions. In these experiments, basal
Ca 2+ levels did not usually recover after capsaicin treatment.
Effects of Ruthenium Red It has been previously shown that the inward current elicited by capsaicin in primary sensory neurons can be blocked with Ruthenium Red. 2,m'14 Therefore, the possibility that this drug antagonized [Ca 2+ ]~ rises induced by acid and capsaicin was explored in another set of experiments. Basal [Ca2+]i decreased
I
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Fig. 5. Influence of zero [Ca2+ ]0 on the [Ca 2+ ]i response to acid and capsaicin. During the period indicated by the horizontal bar, the cell was superfused with a calcium-free medium (no EGTA added). Thin arrow: stimulation with pH 6.5 solution. Thick arrow: application of 1 p M capsaicin. Inset: changes in [Ca 2÷ ]~, expressed as per cent of basal [Ca 2+ ]i, elicited by pH 6.5 and capsaicin in normal (open bars) and zero (hatched bars) [Ca 2÷ ]0. n = 11 for control and acid-stimulated neurons, n = 9 for capsaicin-stimulated neurons. The responses to pH 6.5 and capsaicin in zero and 2.2 mM [Ca 2÷ ]0 were statistically different, *P < 0.001, paired t-test.
J. Garcia-Hirschfeld et al.
240
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30
time (min) Fig. 6. Influence of 20 mM [Ca2+]0 on the [Ca2+]i response to acid and capsaicin. During the period indicated by the horizontal bar, the cell was superfused with a 20 mM calcium solution. Thin arrow: stimulation with pH 6.5 solution. Thick arrow: application of 1/~M capsaicin. Inset: changes in [Ca 2+ ]i, expressed as percent of basal [Ca2÷ ]i, elicited by pH 6.5 and capsaicin in normal (open bars) and 20 mM [Ca2÷ ]0 (hatched bars), n = 9 for control and acid-stimulated neurons; n = 6 for capsaicin-stimulated neurons. The responses to pH 6.5 in 20 mM and 2.2 mM [Ca2+]0 were statistically different, *P < 0.05, paired t-test.
slightly (not significant) u n d e r the effect o f R u t h e n i u m Red ( 1 / t M ) . T h e drug blocked fully the effect o f capsaicin a n d reduced the response to low p H 0 (Fig. 7). Cells did n o t recover usually after washing the R u t h e n i u m Red.
Influence o f sustained p H 0 decreases In a n effort to dissociate fast from slow comp o n e n t s in the [Ca 2+ ]i responses to acid a n d capsaicin, a low p H ( 6 . 5 ) solution was applied for a
[""7
~.~
pH6.5
800
~~
2.2
m~/
Ca
4oo 200
,m
0
600
capsaicin
control
pH-6.5
caps.
1 /zM RR
+"q~400 u 200
I
0
I
I
4
I
time (min)
I
8
I
I
12
Fig. 7. Effect of 1 # M Ruthenium Red (RR, horizontal bar) on the changes of [Ca2+]i induced by application of acid (thin arrow) and capsaicin (thick arrow). Inset: changes in [Ca 2+ ]i, expressed as per cent of basal [Ca2+ ]~, elicited by pH 6.5 and capsaicin during superfusion with the control solution (open bars) and the solution containing RR (hatched bars), n = 11 for control and acid-stimulated neurons, n = 8 for capsaicin-stimulated neurons. *P < 0.002; **P < 0.005.
Intracellular calcium in cultured trigeminal neurons 400
241
pH 6.5 pH 6.5 capsaicin capsaicin
+
200
I
I
0
2
I
time
4 (rain)
i
I
6
8
Fig. 8. Desensitization of the response to acid by continuous exposure of the cell to low pH 0. During the time indicated by the horizontal bar, the cell was superfused with a pH 6.5 solution. The thick arrow signals the applications of 1 #M capsaicin. Representative of six experiments. prolonged period of time. This produced a transient, initial [Ca2+]~ peak that decayed spontaneously in about 1 min to resting [Ca2+]~ levels. Under these conditions, capsaicin still produced an increase in [Ca2+]i, as shown in Fig. 8.
Influence of long-term capsaicin pretreatment In the next group of experiments, we analysed the effects of long-term capsaicin treatment. Cells were cultured for periods of 10-24 h in a medium containing 10/~M capsaicin. It is known that the subpopulation of capsaicin-sensitive cells disappears when primary sensory neurons are grown for a prolonged period of time in the presence of capsaicin. 8 Accordingly, incubation of the cells in the presence of 10 # M capsaicin greatly diminished the total number of sensory neurons. The response to acidic solutions (pH0 = 6.5) was tested in a total of nine cells from five different culture dishes and was not different from the control ones (not shown). However, none of these neurons responded to capsaicin. DISCUSSION The present experiments demonstrate that sudden decreases of pH 0 down to 6.5 elicit a transient elevation of [Ca 2+ ]i. In agreement with the results of previous authors, a rise in [Ca 2+ ]i was also evoked by application of capsaicin, a substance that has been claimed to selectively activate polymodal nociceptive neurons. 3'6'2s~33All neurons showing a [Ca 2+ ]i increase when challenged with capsaicin also exhibited a sensitivity to protons; however, 47% of the cells responded to acid but not to capsaicin.
Calcium concentration changes induced by acidic ~olutions Rapid changes in pH 0 produced an increase in [Ca2+]~ in a large percentage of cultured trigeminal
cells. The effect was transitory and exhibited desensitization. [Ca 2+ ]i rises appear to be related to protoninduced changes in membrane currents. It has been described that acidic solutions activate two types of currents in the soma of dorsal root ganglion neurons "in vitro.". Sudden pH 0 drops from 7.4 to 6.0 elicited a fast, rapidly inactivating inward current carried mainly by sodium. 13'2° In about half of the neurons, strong pH reductions (<6.2) also evoked a second, inward current with a much slower time course) This sustained inward current was attributed to activation of an non-specific cationic conductance for K +, Cs + and Na +. In most of our experiments, test pH0 was 6.5; therefore, the effects of protons on [Ca 2+]~ described here are presumably associated to the proton-induced fast inactivating current. It has been proposed that this current is driven by monovalent cations through the transient transformation by protons of a voltage dependent Ca 2+ channel into a non-selective cationic channelJ '2° Many of the described characteristics of such current are compatible with the changes in [Ca2+]i observed in the present experiments: Current activation depend on fast changes of pH0 and was graded, being maximum for changes from pH07.4 to 5.5; furthermore, small increases of external concentration of H +, which by themselves failed to activate this current, inactivated the proton-induced responseJ s'2° Likewise, changes in [Ca2+]i in our experiments were maximal for pH0 reductions to 5.5 but were still apparent at pH 0=7.0. As reported for the fast proton-induced currents, slow acidification decreased the effect of rapid changes of pH 0 on [Ca 2+ ]i- In contrast, acidification from an alkaline solution (PH07.9) elicited larger [Ca2+]~ transients. A further parallel between fast inactivating current and [Ca2+ ]i rises elicited by protons was the blockade by high [Ca2+]0 of both processes. Moreover, the fast proton-induced current is blocked by inorganic Ca 2+
242
J. Garcia-Hirschfeld et al.
channel blockers. 2° Likewise, Ni 2+ produce a reduction of [Ca2+]i peaks in cultured trigeminal neurons (Garcia-Hirschfeld and L6pez-Briones, unpublished observations). There is experimental evidence suggesting that when the proton-induced Na + current is activated, voltage-gated Ca 2+ currents are temporarily suppressed. 2° Our results with zero [Ca2+]0 prove that Ca 2+ entering the cell after acidic stimuli was of extracellular origin. Therefore, we can conclude either that under normal [Ca2+]o "transformed" Ca 2+ channels retain some permeability to Ca 2+ or that this ion permeates through other type of Ca 2+ channel, that would be gated by membrane depolarization resulting from proton-induced current. 8'27 The contribution of the sustained inward current described in presumed nociceptive cells, to the [Ca2+]~ rises elicited by protons is difficult to assess; ~'2° none the less, the time course and duration of the proton-induced [Ca2+]i responses, as well as the relative independence of pH and capsaicin effects (see below) speak against a significant contribution of this current to the pH-induced Ca 2÷ peaks reported here. Calcium concentration changes induced by capsaicin. Comparison with the response to acidic solutions
Our experiments confirmed that capsaicin increases
[Ca2+ ]i in primary sensory neurons. 12'1°'33The source of Ca 2+ was extracellular as the response disappeared when the cells were bathed in a Ca2+-free medium. Capsaicin was still able to induce a rise in [Ca 2+ ]i even after the fast pH0-induced [Ca 2+ ]i transient had inactivated during maintained low pH application. In fact, in some of the experiments, the [Ca2+]~ change elicited by capsaicin was considerably larger when the initial pH was under 7.4. Such sensitization most likely correspond to pH effects on capsaicin-evoked currents, which are markedly augmented in the presence of protons. 27 Thus, the response to capsaicin is maintained or augmented when the calcium entry evoked by low pH has inactivated, suggesting that the Ca 2+ transients produced by low pH and capsaicin occur via independent mechanisms. This is supported by the following additional evidence: (i) the capsaicin effect was enhanced at high extracellular Ca 2+ levels while this manoeuvre blocked the response to low pH0; (ii) the effect of capsaicin was evoked even when the fast pH-induced [Ca2+]i elevation had returned to resting values in the continuous presence of an acidic pH0; (iii) Ruthenium Red
blocked the response to capsaicin but only attenuated the effect of protons; (iv) normal responses to pH 0 reductions were observed in surviving neurons, after elimination of capsaicin-sensitive cells by prolonged incubation with this agent. Bevan and Yeats 8 proposed that the ionic channel involved in the response to capsaicin was also activated by pH values below 6.2. We did not test systematically such low pH's; thus, it remains to be determined whether pH drops below 6.2 also induced a sustained [Ca2+]i elevation as does capsaicin. Responses of peripheral nociceptors to acid include a phasic burst of impulses followed by a sustained discharge. 4'29A parallel between these firing responses and the fast and slow currents has been suggested. 29 Our results further show that initial activation of sensory neurons by protons is accompanied by an entrance of calcium. This phenomenon is presumably present also in nociceptive neurons and may contribute to activation processes of peripheral nociceptive terminals. CONCLUSIONS Our results demonstrate that rapid reductions in pH evoke a transient rise in [Ca2+]i in cultured trigeminal neurons; we also report a [Ca 2÷ ]~ increase in response to capsaicin. The characteristics of these responses suggest that [Ca 2÷ ]~ rises evoked by rapid reductions in pH and by capsaicin occur through different mechanisms because: (i) the effect of capsaicin was enhanced in high extracellular Ca 2÷ while this manoeuvre blocked the response to low pH0; (ii) the effect of capsaicin was still evoked even when the [Ca2+]i elevation induced by acid stimulation had returned to resting values in the continuous presence of a low pH0; (iii) Ruthenium Red blocked the response to capsaicin but only attenuated the effect of protons; (iv) Normal responses to pH0 reductions were present in surviving neurons, after elimination of capsaicin-sensitive cells by prolonged incubation with this agent. Acknowledgements--The collaboration of Ms Rosa Garcia Velasco in preparing the cultures and the technical assistance of Mr Sim6n Moya and Alfonso Prrez-Vegara are gratefully acknowledged. We thank S. Bevan, R. Gallego and J. Gallar for critical reading of the manuscript. Supported by grants PM90-0113 and SAF93-0267of the Comisi6n Nacional de Ciencia y Tecnologia, Direcci6n General de Investigaci6n Cientifica y T6cnica, and F.I.S. 93/134 of the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
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J. Neurosci. 8, 3208-3230. (Accepted 20 December 1994)