Neuroscience Letter.s, 110 (1990) 52 59 Elsevier ScientilicPublishers lreland Ltd.
52
NSL 06703
Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurones as well as the activation of peripheral nociceptors in vitro A. Dray, C.A. Forbes and G.M. Burgess Sandoz Institute for Medical Research. London (U.K.)
(Received 3 October 1989; Revised version received 19 October 1989;Accepted 19 October 1989) Key words." Capsaicin: Ruthenium red; Calcium blocker; Membrane current; Single channel; Fura 2;
Nociceptor In a number of sensory neuron preparations, Ruthenium red (RR) selectivelyattenuated the response to capsaicin. First, RR (100 nM) reversibly abolished capsaicin but not bradykinin induced increases in [Ca2+]1 measured in single DRG neurons from neonatal rats, using the calcium sensitive dye Fura-2. Second, RR completely but reversiblyabolished capsaicin-activated single ion channel currents measured in membrane patches from rat DRG neurons. This effect of RR differed from that produced by lanthanum. Finally, in a neonatal rat spinal cord-tail preparation maintained in vitro, RR selectivelyattenuated the activation of peripheral nociceptors produced by capsaicin but not by bradykinin or noxious heat. These data indicate that RR inhibits capsaicin mediated effects on sensory neurons by an action on the plasma membrane to prevent opening of capsaicin activated ion channels.
Capsaicin is an algogenic vanillyamide which acts selectively on a subset of sensory n e u r o n s including p o l y m o d a l nociceptors a n d w a r m thermoceptors [7, 8]. The activation of sensory n e u r o n s a n d axons is a c c o m p a n i e d by m e m b r a n e depolarization a n d a n increased permeability to cations including sodium, p o t a s s i u m a n d calcium [3, 6, 12, 17, 24]. In addition, capsaicin produces a calcium d e p e n d e n t release of sensory neuropeptides from p r i m a r y afferent nerve terminals which m a y in t u r n activate s m o o t h muscles a n d other tissues [8, 13, 14]. Recently the polyvalent cationic dye, R u t h e n i u m red (RR) has been shown to prevent the capsaicin-induced increase in 45Ca c o n t e n t of sensory n e u r o n e s [24]. Other indirect measures of sensory n e u r o n a l activation by capsaicin such as s m o o t h muscle c o n t r a c t i o n a n d n e u r o p e p t i d e release are also a t t e n u a t e d by R R [15, 16]. Since R R
Corre,spondence: A. Dray, Sandoz Institute of Medical Research, 5 Gower Place, London WCIE 6BN,
U.K. 0304-3940/90/$ 03.50 i('~J1990 ElsevierScientificPublishers Ireland Ltd.
53 blocks transmembrane calcium movements [10, 22] and mitochondrial calcium sequestration [4, 18, 20, 21] it was concluded [15, 16, 24] that this was the basis for its effects on capsaicin-mediated events. Here we show that RR prevents a number of capsaicin effects on sensory neurons in culture including the opening of capsaicincoupled ion channels, the capsaicin-induced rise in intracellular calcium and selectively attenuates the capsaicin induced activation of peripheral nociceptors. DRG neurons were prepared from all spinal ganglia of neonatal rats as described previously [24]. Neurons were plated onto polylysine and laminin coated No 0 glass coverslips and then maintained in culture in HAMS F-14 medium, supplemented with nerve growth factor (5/zg/ml), horse serum (10%), penicillin (100/zg/ml) and streptomycin (100 U/ml) for between 4 and 10 days before use. For measurements of intracellular calcium, cells were incubated for 20 min at 37°C with 10 pM of the acetoxymethyl ester of the fluorescent calcium indicator Fura-2. They were then washed and the coverslip placed in a perfusion chamber on the stage of a Zeiss inverted microscope. The neurons were continuously superfused with HEPES-buffered Dulbecco's modified Eagle medium with Earles salts, pH 7.4, at room temperature. Agonists were applied by means of a U-tube while RR or other blockers were applied both in the perfusion fluid and in the U-tube together with the agonist. Fluorescent signals were measured using a Deltascan D104B system from Photon Technology Inc. The Fura-2 inside the cell was alternately excited at 340 and 380 nM at a frequency of 50 Hz. The fluorescence signal at 510 nm, from a single cell body, was measured using a photomultiplier-tube and intracellular calcium concentration [Ca2+]i was calculated from the ratio of the fluorescence at the two excitation wavelengths [11]. The maximum and minimum signal values for this calculation were obtained using ionomycin (2 pM) in the presence of either 2 mM extracellular calcium or 4 mM EGTA. Cellular autofluorescence was minimal compared to the signal obtained from Fura-2-1oaded cells and did not change in response to capsaicin. Membrane ion channel recordings were made using outside-out patches obtained from cultured adult DRG neurons. Recordings were made in a flow chamber with continuous perfusion using conventional recording methods. Capsaicin was applied by means of a U-tube at 500 nM in the external bathing solution. When indicated RR (100 nM) and lanthanum chloride (100 pM) were applied together with capsaicin. For studies of peripheral nociceptors the intact spinal cord with the functionally connected tail were removed from 1 to 2-day-old rats following decapitation. The superficial surface of the skin was carefully removed from the tail and the cord and tail were separately superfused (2-4 ml/min) with a physiological salt solution (composition mM: NaC1 138.6, KCI 3.35, CaCI2 1.26, MgC12 1.16, NaHCO3 21.0, NaHPO4 0.58, glucose 10) at 24°C and gassed with 95% 02/5% CO2. Peripheral nociceptive fibres were activated by superfusion of the tail with capsaicin, bradykinin, 5-hydroxytryptamine (serotonin, 5-HT) or by superfusate heated to 48°C. Innocuous stimulation was produced by lightly brushing the tail with a fine sable-hair paint brush. Each stimulus was applied for 20 s with an intervening period of 15 min between stimuli. Bradykinin and 5-HT administrations were separated by at least 40-60 rain to avoid tachyphylaxis. The activation of peripheral fibres was assessed
54
by measuring the depolarization produced in a spinal ventral root (L3-Ls). The ventral root potential was recorded dc with respect to the spinal cord which was earthed. Signals were amplified and displayed using conventional methods. Drugs were obtained from the following sources: Ruthenium red (Sigma); bradykinin (synthesized at Sandoz, Basel), cadmium chloride (Sigma), lanthanum chloride (BDH), Fura-2 (Sigma). A brief 10 s application of submaximal concentration of capsaicin (300 nM) to a single D R G neuron resulted in a transient rise in [Ca2+]i from a basal level of 150 to about 600 nM (Fig. IA). In this figure, switching from normal medium to one containing 100 nM RR is indicated by the bar. A second application of capsaicin, in the presence of RR, evoked only a very small response. Following the removal of RR from the perfusion fluid there was complete recovery of responsiveness to capsaicin (Fig. I B). Similar effects were seen in 8 other D R G neurons. Bradykinin also increased [Ca2+]i in these cells but this effect was unchanged by RR (100 nM, n = 3). Fig. 1C and 1D show a similar experiment in another D R G neuron. In this case, following a control response to 100 nM capsaicin (Fig. IC), 30/~M cadmium was A
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Fig. 1. Action of R R and cadmiun on the increase in [Ca2+]i (/tM) produced by capsaicin in single D R G neurons from neonatal rat. A and B are successive recordings from the cell body of the same single neuron. Capsaicin (300 nM) was applied for 10 s (indicted by a dot) and produced an increase in [Ca2~],. In A, RR (100 nM) applied continuously (bar above trace) blocked the response to a second application of capsaicin. In B, the response to capsaicin was restored following the removal of RR. C and D are also successive recordings from another single neuron. C shows the response to 100 nM capsaicin and in D a second capsaicin application, in the presence of 30 #M cadmium (applied for the period indicated by the bar above the trace), induced a larger, maintained increase in [Ca2+],.
55
added to the bathing medium (Fig. 1D) causing a gradual rise in [Ca2+]i. Reapplication of 100 nM capsaicin, in the presence of cadmium, resulted in a prolonged elevation of [Ca2+]i. This increase was much greater than seen in the control responses and was never observed to return to control levels. Membrane ion channel activity evoked by applications of 500 nM capsaicin was measured in inside-out patches which were voltage clamped at - 80mV. In the patch recording illustrated in Fig. 2A, two capsaicin-activated channels can be seen. The
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10pAl__ 2s Fig. 2. Action of Ruthenium red (RR) and lanthanum chloride on capsaicin-activated single channels in an outside-out patch (clamped at - 8 0 mV) recording from a cultured sensory neuron. The bathing solution at the extracellular face was of the following composition (raM): NaCl 130, KC1 3.5, CaCl2 2.0, MgCI2 1.0, HEPES 5.0, glucose 5.0, sucrose 40) and at the intracellular face was (mM): NaC1 5.0, KC1 140, CaC12 1.0, EGTA 1l, HEPES 10, glucose 5, Na ATP 1,0. Drug application periods are denoted by solid bars. A, shows the current evoked by 500 nM capsaicin in control conditions. The channel openings are indicated by a downward deflection of the trace. In B, capsaicin (500 nM) and RR (100 nM) were applied together, but no channel activity was seen. In C, application of capsaicin alone ten minutes after washout of RR again evoked single-channel activity which was very similar to the control currents in amplitude and kinetics. In contrast, the application of 500 nM capsaicin and 100/~M lanthanum chloride showed a different pattern, with single channel currents showing much greater flicker than the control conditions. E, recorded I min after D, shows that this effect was reversed much more quickly than the effect of RR.
56 channel openings are indicated by downward deflections of the trace. Channel activity could be evoked repeatedly by application of capsaicin without any marked signs of desensitization. During the simultaneous application of 500 nM capsaicin and 100 nM RR (Fig. 2B) no activity was evoked by the dose of capsaicin which had been effective previously. After the application of RR, capsaicin was applied periodically to the cells at the same concentration, and over a period of 5--10 min channel activity in response to capsaicin gradually recovered (Fig. 2C). This complete abolition of capsaicin-evoked activity by RR was in contrast to the effect of 100/~M lanthanum chloride (Fig. 2D). The effect of lanthanum, to increase the flickering of the activated channel, was more consistent with that expected of a blocker of the ion channel, such as the action of local anaesthetics on the acetylcholine-activated channel [18]. Stimulation of peripheral nociceptors by submaximal doses of capsaicin (0.3-0.6 /iM), administered in the tail superfusate, produced a consistent response that was reproducible over many hours (Fig. 3). In most experiments noxious heat was also used to evoke consistent responses while in others, noxious chemicals such as bradykinin (0.1 0.5 llM) or 5-HT (10/iM) were also used. Continuous superfusion of the tail with RR (100 nM), 15 min before and during each test stimulus, consistently attenuated the responses to capsaicin without affecting those to noxious heat (8 out of 8 experiments) (Fig. 3A), bradykinin (3 out of 3 experiments; Fig. 3B) or 5-HT (2 experiments). In other studies (n = 3) continuous superfusion of the tail with cadmium (200/~M; 20 min), did not reduce the effects of capsaicin or noxious heat when these were tested in the presence of cadmium. In the present study we have shown that ruthenium red antagonises the effect of capsaicin in a number of sensory neuron preparations. The effect of RR was selective, producing an attenuation of the responses to capsaicin but not of the responses to other stimuli (bradykinin, 5-HT, noxious heat, light brush). The selectivity of RR against capsaicin induced nociceptor stimulation confirms previous observations on nociceptors in an isolated rabit ear preparation [2]. Capsaicin-induced activation of susceptible sensory neurons is accompanied by membrane depolarization, caused by an increase in membrane permeability, resulting from the opening of ion channels selective for cations, particularly Na +, K + and Ca 2+ [6, 7, 12, 24]. The capsaicin-induced increase in membrane permeability is insensitive to sodium channel blockers and organic calcium channel blockers [24], suggesting that these voltage-activated channels contribute minimally to the capsaicinevoked membrane current. It is significant in the present context, that excitation of peripheral nociceptors by capsaicin occurs in the absence of extracellular calcium [5]~ consistent with the non selective nature of the ion channels activated by capsaicin. We have demonstrated here that capsaicin causes a transient increase in [Ca2+]i in single D R G neurons measured by the calcium-sensitive dye Fura-2. We have also shown that this capsaicin-induced increase in [Ca 2 +]i can be reversibly abolished by RR. Previous studies have shown that RR prevents capsaicin-induced accumulation of 45calcium, an effect attributed to inhibition of calcium buffering by mitochondria [24]. If this were its mechanism of action, RR would not be expected to prevent the capsaicin-induced rise in [Ca 2+]i. The action of RR contrasts therefore with the effect
57
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Fig. 3. The selectiveinhibition of capsaicin evoked responses by Ruthenium red on peripheral nociceptors. The top traces in A and B show control responses to capsaicin noxious heat (48°C) or bradykinin applied for 10 s. There was a 15 min period between successiveapplications of capsaicin and heat and 60 min between applications of bradykinin. The tail was then superfused with ruthenium red (100 nM) for 15 min before peripheral stimuli were retested (middle traces). Ruthenium red (applied during the continuous bar below the traces) produced a selective reduction of capsaicin-evoked responses but did not change the responses to (A) noxious heat, or (B) bradykinin. Followinga 20450 min wash of the tissue, partial recovery of the capsaicin response was observed.
of cadmium ions, which have also been shown to block 45Ca accumulation [24], but which did not reduce the capsaicin induced elevation of [Ca2+]i. Indeed, application of cadmium ions induced a slow rise in [Ca2+]i and both potentiated and prolonged the response to capsaicin. It is unlikely that cadmium acts by blocking the activation of capsaicin-coupled membrane ion channels as had been suggested by Wood et al. (1988). We believe its effects are best explained by an inhibition of Ca2+-ATPase mediated Ca 2+ pumps (see e.g. ref. 23) with the consequent failure of both intracellular calcium sequestration and cellular calcium extrusion. Consistent with this interpretation are our findings that cadmium ions did not prevent the capsaicin-induced activation of nociceptive neurons in vitro. The characteristics of the capsaicin-activated single channels shown here are similar to those previously described [9]. Capsaicin-activated ion channel activity was completely abolished by application of RR, but this action did not have the characteristics typical of ion channel blockade. The action of lanthanum, however, was more like that expected of an ion channel blocker, increasing the flickering of channels between the open and closed states. This effect is similar to the block of acetylcholine-activated ion channels by local anaesthetics [19], and has been interpreted as being due to the rapid blocking and unblocking of the open ion channels. Hence it
58 a p p e a r s t h a t R R m a y act in a m a n n e r m o r e like a p h a r m a c o l o g i c a l a n t a g o n i s t , e i t h e r i n t e r f e r i n g w i t h the b i n d i n g o f c a p s a i c i n to the m e m b r a n e site o r p r e v e n t i n g c h a n n e l o p e n i n g a f t e r c a p s a i c i n has b o u n d . In p r e v i o u s studies o f R R i n h i b i t i o n o f c a p s a i c i n - m e d i a t e d effects, i n d i r e c t m e a s ures i n v o l v i n g c a l c i u m - d e p e n d e n t p r o c e s s e s o r c a l c i u m a c c u m u l a t i o n , w e r e used to i n d i c a t e the a c t i v a t i o n o f s e n s o r y n e u r o n s by c a p s a i c i n [15, 16, 24]. As R R has been s h o w n to b l o c k c a l c i u m ion t r a n s p o r t a n d c a l c i u m s t o r a g e its m o d e o f a c t i o n a g a i n s t c a p s a i c i n was t h o u g h t to relate to these effects. T h e p r e s e n t o b s e r v a t i o n s , u s i n g m o r e d i r e c t m e a s u r e s , offer a n a l t e r n a t i v e e x p l a n a t i o n . W e suggest t h a t R R acts at the level o f the p l a s m a m e m b r a n e by p r e v e n t i n g the o p e n i n g o f c a t i o n - s e l e c t i v e i o n c h a n n e l s by c a p s a i c i n .
1 Amann, R., Donnerer, J. and Lembeck, F., Ruthenium red selectively inhibits capsaicin-induced release of calcitonin gene-retated peptide from the isolated perfused guinea pig lung, Neurosci. Lett., 101(1989) 311 315. 2 Amann, R. and Lembeck, F., Ruthenium red selectively prevents capsaicin-induced nociceptor stimulation, Eur. J. Pharmacol., 161 (1989) 227 229. 3 Baccaglini, P.I. and Hogan, P.G., Some rat sensory neurons in culture express characteristics of differentiated pain sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 594~598. 4 Baker, P.F. and Umbach, J.A., Calcium buffering in axons and axoplasm of Loligo, J. Physiol. (Lond.), 383 (1987) 369 394. 5 Bettaney, J., Dray, A. and Forster, P., Calcium and the effects of capsaicin on afferent fibres in a neonatal rat isolated spinal cord-tail preparation, J. Physiol. (Lond.) 406 (1988) 37P. 6 Bevan, S.J. and Forbes, C.A., Membrane effects of capsaicin on dorsal root ganglion neurons in cell culture, J. Physiol. (Lond.), 398 (1988) 28P. 7 Bevan, S.J., James, I.F., Rang, H.P., Winter, J. and Wood, J.N., The mechanism of action of capsaicin a sensory neurotoxin. In P. Jenner (Ed.), Neurotoxins and their Pharmacological Implications, Raven, New York, 1987, pp. 261 277. 8 Buck, S.H. and Burks, T.F., The neuropharmacology ofcapsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179 226. 9 Forbes, C.A. and Bevan, S.J., Properties of single capsaicin-activated channels, Soc. Neurosci. Abstr., 14 (1988) 642. 10 Goddard, G.A. and Robinson, J.D., Uptake and release of calcium by rat brain synaptosomes, Brain Res., 110(1976) 331 350. 11 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2~ indicators with greatly improved fluorescent properties, J. Biol. Chem., 260 (1985) 3440-3450. 12 Heyman, I. and Rang, H.P., Depolarizing responses to capsaicin in a subpopulation of rat dorsal root ganglion cells, Neurosci. Lett. 56 (1985) 69-75. 13 Holzer, P., Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience, 24 (1988) 738 768. 14 Maggi, C.A. and Meli, A., The sensory-efferent function of capsaicin-sensitive sensory neurons, Gen. Pharmacol. 19 (1988) 1~43. 15 Maggi, C.A., Patacchini, R., Santicioli, P., Guiliani, S. Geppeni, P. and Meli, A., Protective action of ruthenium red toward capsaicin desensitization of sensory fibres, Neurosci. Lett., 88 (1988) 201 205. 16 Maggi, C.A., Santiciolli, P., (3eppetti, P., Parlani, M., Astolfi, M., Pradelles, P., Patacchini, R. and Meli, A., The antagonism induced by ruthenium red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies, Eur. J. Pharmacol., 154 (1988) 1 10. 17 Marsh, S.J., Stansfeld, C.E., Brown, D.A., Davey, R. and McCarthy, D., The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience, 23 (1987) 275-286.
59 18 Moore, C.L., Specific inhibition ofmitochondrial Ca 2+ transport by ruthenium red, Biochem. Biophys. Res. Commun., 42 (1971) 298-305. 19 Neher, E. and Steinbach, J.H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1976) 153-176. 20 Nicholls, D.G. and Crompton, M., Mitochondrial calcium transport, FEBS Lett., 111, (1980) 261 268. 21 Reed, K.C. and Bygrave, F.L., The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red, Biochem. J., 140 (1974) 143-155. 22 Swanson, P.D., Anderson, L. and Stahl, W.L., Uptake of calcium by synaptosomes from rat brain, Biochim. Biophys. Acta, 356 (1974) 174-183. 23 Verbost, P.M., Flik, G., Pang, P.K.T., Lock, R.A.C. and Wedelaar Bonga, S.E., Cadmium inhibition of the erythrocyte Ca 2÷ pump, J. Biol. Chem., 264 (1989) 5613-5615. 24 Wood, J.N., Winter, J., James, I.F., Rang, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988) 3208 3220.
52
NSL 06703
Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurones as well as the activation of peripheral nociceptors in vitro A. Dray, C.A. Forbes and G.M. Burgess Sandoz Institute for Medical Research. London (U.K.)
(Received 3 October 1989; Revised version received 19 October 1989;Accepted 19 October 1989) Key words." Capsaicin: Ruthenium red; Calcium blocker; Membrane current; Single channel; Fura 2;
Nociceptor In a number of sensory neuron preparations, Ruthenium red (RR) selectivelyattenuated the response to capsaicin. First, RR (100 nM) reversibly abolished capsaicin but not bradykinin induced increases in [Ca2+]1 measured in single DRG neurons from neonatal rats, using the calcium sensitive dye Fura-2. Second, RR completely but reversiblyabolished capsaicin-activated single ion channel currents measured in membrane patches from rat DRG neurons. This effect of RR differed from that produced by lanthanum. Finally, in a neonatal rat spinal cord-tail preparation maintained in vitro, RR selectivelyattenuated the activation of peripheral nociceptors produced by capsaicin but not by bradykinin or noxious heat. These data indicate that RR inhibits capsaicin mediated effects on sensory neurons by an action on the plasma membrane to prevent opening of capsaicin activated ion channels.
Capsaicin is an algogenic vanillyamide which acts selectively on a subset of sensory n e u r o n s including p o l y m o d a l nociceptors a n d w a r m thermoceptors [7, 8]. The activation of sensory n e u r o n s a n d axons is a c c o m p a n i e d by m e m b r a n e depolarization a n d a n increased permeability to cations including sodium, p o t a s s i u m a n d calcium [3, 6, 12, 17, 24]. In addition, capsaicin produces a calcium d e p e n d e n t release of sensory neuropeptides from p r i m a r y afferent nerve terminals which m a y in t u r n activate s m o o t h muscles a n d other tissues [8, 13, 14]. Recently the polyvalent cationic dye, R u t h e n i u m red (RR) has been shown to prevent the capsaicin-induced increase in 45Ca c o n t e n t of sensory n e u r o n e s [24]. Other indirect measures of sensory n e u r o n a l activation by capsaicin such as s m o o t h muscle c o n t r a c t i o n a n d n e u r o p e p t i d e release are also a t t e n u a t e d by R R [15, 16]. Since R R
Corre,spondence: A. Dray, Sandoz Institute of Medical Research, 5 Gower Place, London WCIE 6BN,
U.K. 0304-3940/90/$ 03.50 i('~J1990 ElsevierScientificPublishers Ireland Ltd.
53 blocks transmembrane calcium movements [10, 22] and mitochondrial calcium sequestration [4, 18, 20, 21] it was concluded [15, 16, 24] that this was the basis for its effects on capsaicin-mediated events. Here we show that RR prevents a number of capsaicin effects on sensory neurons in culture including the opening of capsaicincoupled ion channels, the capsaicin-induced rise in intracellular calcium and selectively attenuates the capsaicin induced activation of peripheral nociceptors. DRG neurons were prepared from all spinal ganglia of neonatal rats as described previously [24]. Neurons were plated onto polylysine and laminin coated No 0 glass coverslips and then maintained in culture in HAMS F-14 medium, supplemented with nerve growth factor (5/zg/ml), horse serum (10%), penicillin (100/zg/ml) and streptomycin (100 U/ml) for between 4 and 10 days before use. For measurements of intracellular calcium, cells were incubated for 20 min at 37°C with 10 pM of the acetoxymethyl ester of the fluorescent calcium indicator Fura-2. They were then washed and the coverslip placed in a perfusion chamber on the stage of a Zeiss inverted microscope. The neurons were continuously superfused with HEPES-buffered Dulbecco's modified Eagle medium with Earles salts, pH 7.4, at room temperature. Agonists were applied by means of a U-tube while RR or other blockers were applied both in the perfusion fluid and in the U-tube together with the agonist. Fluorescent signals were measured using a Deltascan D104B system from Photon Technology Inc. The Fura-2 inside the cell was alternately excited at 340 and 380 nM at a frequency of 50 Hz. The fluorescence signal at 510 nm, from a single cell body, was measured using a photomultiplier-tube and intracellular calcium concentration [Ca2+]i was calculated from the ratio of the fluorescence at the two excitation wavelengths [11]. The maximum and minimum signal values for this calculation were obtained using ionomycin (2 pM) in the presence of either 2 mM extracellular calcium or 4 mM EGTA. Cellular autofluorescence was minimal compared to the signal obtained from Fura-2-1oaded cells and did not change in response to capsaicin. Membrane ion channel recordings were made using outside-out patches obtained from cultured adult DRG neurons. Recordings were made in a flow chamber with continuous perfusion using conventional recording methods. Capsaicin was applied by means of a U-tube at 500 nM in the external bathing solution. When indicated RR (100 nM) and lanthanum chloride (100 pM) were applied together with capsaicin. For studies of peripheral nociceptors the intact spinal cord with the functionally connected tail were removed from 1 to 2-day-old rats following decapitation. The superficial surface of the skin was carefully removed from the tail and the cord and tail were separately superfused (2-4 ml/min) with a physiological salt solution (composition mM: NaC1 138.6, KCI 3.35, CaCI2 1.26, MgC12 1.16, NaHCO3 21.0, NaHPO4 0.58, glucose 10) at 24°C and gassed with 95% 02/5% CO2. Peripheral nociceptive fibres were activated by superfusion of the tail with capsaicin, bradykinin, 5-hydroxytryptamine (serotonin, 5-HT) or by superfusate heated to 48°C. Innocuous stimulation was produced by lightly brushing the tail with a fine sable-hair paint brush. Each stimulus was applied for 20 s with an intervening period of 15 min between stimuli. Bradykinin and 5-HT administrations were separated by at least 40-60 rain to avoid tachyphylaxis. The activation of peripheral fibres was assessed
54
by measuring the depolarization produced in a spinal ventral root (L3-Ls). The ventral root potential was recorded dc with respect to the spinal cord which was earthed. Signals were amplified and displayed using conventional methods. Drugs were obtained from the following sources: Ruthenium red (Sigma); bradykinin (synthesized at Sandoz, Basel), cadmium chloride (Sigma), lanthanum chloride (BDH), Fura-2 (Sigma). A brief 10 s application of submaximal concentration of capsaicin (300 nM) to a single D R G neuron resulted in a transient rise in [Ca2+]i from a basal level of 150 to about 600 nM (Fig. IA). In this figure, switching from normal medium to one containing 100 nM RR is indicated by the bar. A second application of capsaicin, in the presence of RR, evoked only a very small response. Following the removal of RR from the perfusion fluid there was complete recovery of responsiveness to capsaicin (Fig. I B). Similar effects were seen in 8 other D R G neurons. Bradykinin also increased [Ca2+]i in these cells but this effect was unchanged by RR (100 nM, n = 3). Fig. 1C and 1D show a similar experiment in another D R G neuron. In this case, following a control response to 100 nM capsaicin (Fig. IC), 30/~M cadmium was A
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Time (s)
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Fig. 1. Action of R R and cadmiun on the increase in [Ca2+]i (/tM) produced by capsaicin in single D R G neurons from neonatal rat. A and B are successive recordings from the cell body of the same single neuron. Capsaicin (300 nM) was applied for 10 s (indicted by a dot) and produced an increase in [Ca2~],. In A, RR (100 nM) applied continuously (bar above trace) blocked the response to a second application of capsaicin. In B, the response to capsaicin was restored following the removal of RR. C and D are also successive recordings from another single neuron. C shows the response to 100 nM capsaicin and in D a second capsaicin application, in the presence of 30 #M cadmium (applied for the period indicated by the bar above the trace), induced a larger, maintained increase in [Ca2+],.
55
added to the bathing medium (Fig. 1D) causing a gradual rise in [Ca2+]i. Reapplication of 100 nM capsaicin, in the presence of cadmium, resulted in a prolonged elevation of [Ca2+]i. This increase was much greater than seen in the control responses and was never observed to return to control levels. Membrane ion channel activity evoked by applications of 500 nM capsaicin was measured in inside-out patches which were voltage clamped at - 80mV. In the patch recording illustrated in Fig. 2A, two capsaicin-activated channels can be seen. The
A
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i
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10pAl__ 2s Fig. 2. Action of Ruthenium red (RR) and lanthanum chloride on capsaicin-activated single channels in an outside-out patch (clamped at - 8 0 mV) recording from a cultured sensory neuron. The bathing solution at the extracellular face was of the following composition (raM): NaCl 130, KC1 3.5, CaCl2 2.0, MgCI2 1.0, HEPES 5.0, glucose 5.0, sucrose 40) and at the intracellular face was (mM): NaC1 5.0, KC1 140, CaC12 1.0, EGTA 1l, HEPES 10, glucose 5, Na ATP 1,0. Drug application periods are denoted by solid bars. A, shows the current evoked by 500 nM capsaicin in control conditions. The channel openings are indicated by a downward deflection of the trace. In B, capsaicin (500 nM) and RR (100 nM) were applied together, but no channel activity was seen. In C, application of capsaicin alone ten minutes after washout of RR again evoked single-channel activity which was very similar to the control currents in amplitude and kinetics. In contrast, the application of 500 nM capsaicin and 100/~M lanthanum chloride showed a different pattern, with single channel currents showing much greater flicker than the control conditions. E, recorded I min after D, shows that this effect was reversed much more quickly than the effect of RR.
56 channel openings are indicated by downward deflections of the trace. Channel activity could be evoked repeatedly by application of capsaicin without any marked signs of desensitization. During the simultaneous application of 500 nM capsaicin and 100 nM RR (Fig. 2B) no activity was evoked by the dose of capsaicin which had been effective previously. After the application of RR, capsaicin was applied periodically to the cells at the same concentration, and over a period of 5--10 min channel activity in response to capsaicin gradually recovered (Fig. 2C). This complete abolition of capsaicin-evoked activity by RR was in contrast to the effect of 100/~M lanthanum chloride (Fig. 2D). The effect of lanthanum, to increase the flickering of the activated channel, was more consistent with that expected of a blocker of the ion channel, such as the action of local anaesthetics on the acetylcholine-activated channel [18]. Stimulation of peripheral nociceptors by submaximal doses of capsaicin (0.3-0.6 /iM), administered in the tail superfusate, produced a consistent response that was reproducible over many hours (Fig. 3). In most experiments noxious heat was also used to evoke consistent responses while in others, noxious chemicals such as bradykinin (0.1 0.5 llM) or 5-HT (10/iM) were also used. Continuous superfusion of the tail with RR (100 nM), 15 min before and during each test stimulus, consistently attenuated the responses to capsaicin without affecting those to noxious heat (8 out of 8 experiments) (Fig. 3A), bradykinin (3 out of 3 experiments; Fig. 3B) or 5-HT (2 experiments). In other studies (n = 3) continuous superfusion of the tail with cadmium (200/~M; 20 min), did not reduce the effects of capsaicin or noxious heat when these were tested in the presence of cadmium. In the present study we have shown that ruthenium red antagonises the effect of capsaicin in a number of sensory neuron preparations. The effect of RR was selective, producing an attenuation of the responses to capsaicin but not of the responses to other stimuli (bradykinin, 5-HT, noxious heat, light brush). The selectivity of RR against capsaicin induced nociceptor stimulation confirms previous observations on nociceptors in an isolated rabit ear preparation [2]. Capsaicin-induced activation of susceptible sensory neurons is accompanied by membrane depolarization, caused by an increase in membrane permeability, resulting from the opening of ion channels selective for cations, particularly Na +, K + and Ca 2+ [6, 7, 12, 24]. The capsaicin-induced increase in membrane permeability is insensitive to sodium channel blockers and organic calcium channel blockers [24], suggesting that these voltage-activated channels contribute minimally to the capsaicinevoked membrane current. It is significant in the present context, that excitation of peripheral nociceptors by capsaicin occurs in the absence of extracellular calcium [5]~ consistent with the non selective nature of the ion channels activated by capsaicin. We have demonstrated here that capsaicin causes a transient increase in [Ca2+]i in single D R G neurons measured by the calcium-sensitive dye Fura-2. We have also shown that this capsaicin-induced increase in [Ca 2 +]i can be reversibly abolished by RR. Previous studies have shown that RR prevents capsaicin-induced accumulation of 45calcium, an effect attributed to inhibition of calcium buffering by mitochondria [24]. If this were its mechanism of action, RR would not be expected to prevent the capsaicin-induced rise in [Ca 2+]i. The action of RR contrasts therefore with the effect
57
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Fig. 3. The selectiveinhibition of capsaicin evoked responses by Ruthenium red on peripheral nociceptors. The top traces in A and B show control responses to capsaicin noxious heat (48°C) or bradykinin applied for 10 s. There was a 15 min period between successiveapplications of capsaicin and heat and 60 min between applications of bradykinin. The tail was then superfused with ruthenium red (100 nM) for 15 min before peripheral stimuli were retested (middle traces). Ruthenium red (applied during the continuous bar below the traces) produced a selective reduction of capsaicin-evoked responses but did not change the responses to (A) noxious heat, or (B) bradykinin. Followinga 20450 min wash of the tissue, partial recovery of the capsaicin response was observed.
of cadmium ions, which have also been shown to block 45Ca accumulation [24], but which did not reduce the capsaicin induced elevation of [Ca2+]i. Indeed, application of cadmium ions induced a slow rise in [Ca2+]i and both potentiated and prolonged the response to capsaicin. It is unlikely that cadmium acts by blocking the activation of capsaicin-coupled membrane ion channels as had been suggested by Wood et al. (1988). We believe its effects are best explained by an inhibition of Ca2+-ATPase mediated Ca 2+ pumps (see e.g. ref. 23) with the consequent failure of both intracellular calcium sequestration and cellular calcium extrusion. Consistent with this interpretation are our findings that cadmium ions did not prevent the capsaicin-induced activation of nociceptive neurons in vitro. The characteristics of the capsaicin-activated single channels shown here are similar to those previously described [9]. Capsaicin-activated ion channel activity was completely abolished by application of RR, but this action did not have the characteristics typical of ion channel blockade. The action of lanthanum, however, was more like that expected of an ion channel blocker, increasing the flickering of channels between the open and closed states. This effect is similar to the block of acetylcholine-activated ion channels by local anaesthetics [19], and has been interpreted as being due to the rapid blocking and unblocking of the open ion channels. Hence it
58 a p p e a r s t h a t R R m a y act in a m a n n e r m o r e like a p h a r m a c o l o g i c a l a n t a g o n i s t , e i t h e r i n t e r f e r i n g w i t h the b i n d i n g o f c a p s a i c i n to the m e m b r a n e site o r p r e v e n t i n g c h a n n e l o p e n i n g a f t e r c a p s a i c i n has b o u n d . In p r e v i o u s studies o f R R i n h i b i t i o n o f c a p s a i c i n - m e d i a t e d effects, i n d i r e c t m e a s ures i n v o l v i n g c a l c i u m - d e p e n d e n t p r o c e s s e s o r c a l c i u m a c c u m u l a t i o n , w e r e used to i n d i c a t e the a c t i v a t i o n o f s e n s o r y n e u r o n s by c a p s a i c i n [15, 16, 24]. As R R has been s h o w n to b l o c k c a l c i u m ion t r a n s p o r t a n d c a l c i u m s t o r a g e its m o d e o f a c t i o n a g a i n s t c a p s a i c i n was t h o u g h t to relate to these effects. T h e p r e s e n t o b s e r v a t i o n s , u s i n g m o r e d i r e c t m e a s u r e s , offer a n a l t e r n a t i v e e x p l a n a t i o n . W e suggest t h a t R R acts at the level o f the p l a s m a m e m b r a n e by p r e v e n t i n g the o p e n i n g o f c a t i o n - s e l e c t i v e i o n c h a n n e l s by c a p s a i c i n .
1 Amann, R., Donnerer, J. and Lembeck, F., Ruthenium red selectively inhibits capsaicin-induced release of calcitonin gene-retated peptide from the isolated perfused guinea pig lung, Neurosci. Lett., 101(1989) 311 315. 2 Amann, R. and Lembeck, F., Ruthenium red selectively prevents capsaicin-induced nociceptor stimulation, Eur. J. Pharmacol., 161 (1989) 227 229. 3 Baccaglini, P.I. and Hogan, P.G., Some rat sensory neurons in culture express characteristics of differentiated pain sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 594~598. 4 Baker, P.F. and Umbach, J.A., Calcium buffering in axons and axoplasm of Loligo, J. Physiol. (Lond.), 383 (1987) 369 394. 5 Bettaney, J., Dray, A. and Forster, P., Calcium and the effects of capsaicin on afferent fibres in a neonatal rat isolated spinal cord-tail preparation, J. Physiol. (Lond.) 406 (1988) 37P. 6 Bevan, S.J. and Forbes, C.A., Membrane effects of capsaicin on dorsal root ganglion neurons in cell culture, J. Physiol. (Lond.), 398 (1988) 28P. 7 Bevan, S.J., James, I.F., Rang, H.P., Winter, J. and Wood, J.N., The mechanism of action of capsaicin a sensory neurotoxin. In P. Jenner (Ed.), Neurotoxins and their Pharmacological Implications, Raven, New York, 1987, pp. 261 277. 8 Buck, S.H. and Burks, T.F., The neuropharmacology ofcapsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179 226. 9 Forbes, C.A. and Bevan, S.J., Properties of single capsaicin-activated channels, Soc. Neurosci. Abstr., 14 (1988) 642. 10 Goddard, G.A. and Robinson, J.D., Uptake and release of calcium by rat brain synaptosomes, Brain Res., 110(1976) 331 350. 11 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2~ indicators with greatly improved fluorescent properties, J. Biol. Chem., 260 (1985) 3440-3450. 12 Heyman, I. and Rang, H.P., Depolarizing responses to capsaicin in a subpopulation of rat dorsal root ganglion cells, Neurosci. Lett. 56 (1985) 69-75. 13 Holzer, P., Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience, 24 (1988) 738 768. 14 Maggi, C.A. and Meli, A., The sensory-efferent function of capsaicin-sensitive sensory neurons, Gen. Pharmacol. 19 (1988) 1~43. 15 Maggi, C.A., Patacchini, R., Santicioli, P., Guiliani, S. Geppeni, P. and Meli, A., Protective action of ruthenium red toward capsaicin desensitization of sensory fibres, Neurosci. Lett., 88 (1988) 201 205. 16 Maggi, C.A., Santiciolli, P., (3eppetti, P., Parlani, M., Astolfi, M., Pradelles, P., Patacchini, R. and Meli, A., The antagonism induced by ruthenium red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies, Eur. J. Pharmacol., 154 (1988) 1 10. 17 Marsh, S.J., Stansfeld, C.E., Brown, D.A., Davey, R. and McCarthy, D., The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience, 23 (1987) 275-286.
59 18 Moore, C.L., Specific inhibition ofmitochondrial Ca 2+ transport by ruthenium red, Biochem. Biophys. Res. Commun., 42 (1971) 298-305. 19 Neher, E. and Steinbach, J.H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1976) 153-176. 20 Nicholls, D.G. and Crompton, M., Mitochondrial calcium transport, FEBS Lett., 111, (1980) 261 268. 21 Reed, K.C. and Bygrave, F.L., The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red, Biochem. J., 140 (1974) 143-155. 22 Swanson, P.D., Anderson, L. and Stahl, W.L., Uptake of calcium by synaptosomes from rat brain, Biochim. Biophys. Acta, 356 (1974) 174-183. 23 Verbost, P.M., Flik, G., Pang, P.K.T., Lock, R.A.C. and Wedelaar Bonga, S.E., Cadmium inhibition of the erythrocyte Ca 2÷ pump, J. Biol. Chem., 264 (1989) 5613-5615. 24 Wood, J.N., Winter, J., James, I.F., Rang, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988) 3208 3220.
