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Characterisation of the calcium responses to histamine in capsaicin-sensitive and capsaicin-insensitive sensory neurones
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 5 6 1 - 9
Neuroscience Vol. 110, No. 2, pp. 329^338, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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CHARACTERISATION OF THE CALCIUM RESPONSES TO HISTAMINE IN CAPSAICIN-SENSITIVE AND CAPSAICIN-INSENSITIVE SENSORY NEURONES T. A. NICOLSON,a S. BEVANb and C. D. RICHARDSa * a
Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK b
Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BN, UK
AbstractöAdult rat sensory neurones were maintained in short-term tissue culture and their response to histamine was studied by monitoring changes in intracellular [Ca2 ] with Fura-2. The proportion of histamine-sensitive neurones increased as the concentration increased from 10 WM to 10 mM. The fraction of responding cells did not change signi¢cantly over the ¢rst week in culture. About 60% of histamine-sensitive cells were insensitive to capsaicin and these cells tended to be of small diameter. The integrated calcium response to histamine was greatest at 100 WM when the response consisted of two phases: an initial short-lasting transient followed by a sustained plateau that was dependent on extracellular calcium. This response was blocked by the histamine H1 receptor antagonist mepyramine but not by cimetidine or thioperamide which block H2 and H3 receptors, respectively. Moreover, application of histamine increased the intracellular concentration of inositol 1,4,5-trisphosphate ^ an e¡ect blocked by mepyramine. These data show that the response is mediated by H1 receptors. The phospholipase C inhibitor U73122 blocked the response to 100 WM histamine and signi¢cantly reduced the fraction of cells responding to 1 mM and 10 mM histamine as did removal of extracellular calcium. A combination of U73122 and calcium-free medium abolished all responses to histamine. These data suggest that in addition to activating phospholipase C, high concentrations of histamine gate an in£ux of calcium that is independent of store depletion. The implications of these results for the transduction of pruritic stimuli is discussed. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: itch, H1 receptor, histamine, capsaicin, calcium, sensory neurone.
burning pain following block of cutaneous C-¢bres by local anaesthetics but persists when a nerve trunk is blocked by pressure at a time when only C-¢bres remain active. For these reasons it is likely that the sensation is mediated by unmyelinated cutaneous a¡erents. In support of this view, recent microneurography studies on human volunteers have found that a small subset of unmyelinated nerve ¢bres responded with sustained discharge after ionophoretic injection of histamine into the skin (Handwerker et al., 1991; Schmelz et al., 1997). Moreover, the time course of the discharge corresponded with the time course of the itch sensations. Very recently Andrew and Craig (2001) have described a class of lamina I spinothalamic tract neurones in the cat that have properties that parallel the pure itching sensation in man and match the responses of peripheral C-¢bres that respond selectively to histamine. Despite these insights, the mechanisms by which histamine excites cutaneous nerve ¢bres remain obscure as does the relationship between nociceptive and pruritic a¡erents (see McMahon and Koltzenburg, 1992). In an attempt to address these issues, we have used changes in intracellular calcium to characterise the response of adult sensory neurones maintained in short-term tissue culture to application of histamine. In particular, we have examined the relationship between the sensitivity of sensory neurones to histamine and that to capsaicin which is diagnostic of polymodal nociceptive neurones. Preliminary accounts
Histamine is released into the dermis following mast cell degranulation and this event appears to be the primary trigger for the sensation of itch. Ionophoretic application of histamine into the super¢cial regions of the skin elicits a pure sensation of itch (Magerl et al., 1990; Ward et al., 1996) and concentrations of histamine which produce the sensation have been shown to excite unmyelinated a¡erent nerve ¢bres (Fjallbrant and Iggo, 1961; Tuckett and Wei, 1987). Furthermore, the skin possesses speci¢c itch points with a punctate distribution like other skin sensations (Shelley and Arthur, 1957). Excision of these points followed by histological examination reveals no speci¢c sensory structures but an increased density of bare nerve endings. The sensation of itch disappears along with
*Corresponding author. Tel.: +44-020-7679-6089; fax: +44-0207387-6368. E-mail address: [email protected] (C. D. Richards). Abbreviations : 4Br-A23187, 4-bromo-A23187 (4-bromo-calcimycin) ; EGTA, ethylene glycol-bis(L-aminoethyl-ether)-N,N,NP,NPtetra acetic acid; Fura-2 AM, Fura-2 acetoxy methyl ester ; HEPES, N-(2-hydroxyethylpiperazine)-NP-(2-ethane sulphonic acid); 5-HT, 5-hydroxytryptamine; IP3 , inositol 1,4,5-trisphosphate; NGF, nerve growth factor; U73122, 1-[6-((17L-3-methoxyestra-1,3,5(10)-triene 17-yl)amino)hexyl]1H-pyrrole-2,5-dione ; Y25130, N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-methyl-3-oxo3,4,dihydro-2H-1,4-benzoxazine-8-carboxamide. 329
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of our ¢ndings have been published elsewhere (Nicolson et al., 1998; Nicolson et al., 1999).
EXPERIMENTAL PROCEDURES
Cell culture Primary sensory neurones were isolated and maintained in culture as described by Bevan and Winter, (1995). In brief, adult Sprague^Dawley rats from the Royal Free Hospital School of Medicine colony were killed by asphyxiation with a rising concentration of carbon dioxide followed by cervical dislocation. The vertebral column was removed and a 3^4-mm strip of bone was cut along the length of the vertebral column to expose the spinal cord and the recessed bony cavities which contain the spinal ganglia. The ganglia were carefully dissected out under aseptic conditions and placed in Ham's F14 medium containing 4% Ultroser-G. The dorsal and ventral roots were trimmed and the individual ganglia transferred to medium containing 1.25 mg ml31 collagenase type IV for 3^4 h. After the period of incubation, the ganglia were washed free of collagenase and dispersed to form a cell suspension by trituration with £ame-polished Pasteur pipettes. The cell suspension was centrifuged in Ham's F 14 medium containing 15% bovine serum albumin. The resulting pellet was resuspended in F14 medium containing 4% Ultroser-G supplemented with nerve growth factor (NGF) 0.2 Wg ml31 . The isolated sensory neurones were then plated onto glass coverslips that had previously been coated with poly-ornithine and laminin. The cells were maintained in identical medium for up to 8 days. Intracellular Ca2 measurements Ca2 measurements were made with the dual excitation calcium indicator Fura-2 acetoxymethyl ester (Fura-2, Molecular Probes, Eugene, OR, USA) which was loaded into the cells as its AM-ester as follows: glass coverslips with the adherent cells were incubated in standard bicarbonate-bu¡ered Locke's solution containing 10 WM Fura-2 acetoxy methyl ester (Fura-2 AM) for 30 min at 37³C. Following loading, the cells were then washed for 15 min in bicarbonate-bu¡ered Locke at 37³C prior to their transfer to HEPES-bu¡ered Locke's solution. All experiments were conducted at room temperature (approximately 22³C) and carried out within approximately 1 h of loading the cells with the indicator. The dye was excited alternately with light of 340 and 380 nm and the light emitted above 420 nm was collected via a cooled CCD camera (supplied by Digital Pixel, Brighton, UK). To improve the signal-to-noise ratio, the collection times were adjusted to increase the total number of photons accumulated at the lowest intensity of emission. Thus the collection period for 340-nm excitation was three times that for 380-nm excitation (the collection period was generally 600 ms compared to 200 ms). The resulting images were analysed using software supplied by Kinetic Imaging (Liverpool, UK). In each experiment, ratio values were calculated after subtraction of the background signal which was determined at the end of every experiment by treating the cells with 0.1% Triton X-100. To monitor the local Ca2 from the ratio images, speci¢c areas of interest were chosen and the average ratio value for the designated areas was then calculated and plotted as a function of time. The ratio values in the ¢gures are not corrected for the di¡erent collection times. Apart from one short series of experiments to investigate tachyphylaxis to histamine, cells were only exposed to a single application of histamine. Furthermore, to check the viability of the cultured cells, the experimental protocol invariably included the following steps after stimulation with histamine. They were ¢rst exposed to medium containing 500 nM capsaicin and then to medium containing 50 mM potassium. Those cells that did not respond to either capsaicin or to high potassium were con-
sidered unviable and were excluded from the study. This protocol also allowed us to classify the sensory neurones into those which were sensitive to capsaicin and those which were not. Calibration of the ratios in terms of [Ca2 ] was carried out in situ in several experiments using the ionophore 4-bromo-A23187 (4-bromo-calcimycin, 4Br-A23187). Cells were exposed to a `zero' calcium solution containing 20 mM EGTA and 0 mM Ca2 to obtain a minimum ratio and a `high' calcium solution containing 2 mM Ca2 to obtain a maximum ratio, both in the presence of 5 WM 4Br-A23187. Cells were exposed to these zero and high-calcium solutions alternately until consistent ratio values were obtained. The average values from these calibrations have been used to quantify the changes in intracellular Ca2 where this was considered appropriate using the following equation (Grynkiewicz et al., 1985): Ca2 K d R3Rmin = Rmax 3R Sf2= S b2 where R is the measured ratio of interest, Rmin and Rmax the ratios recorded with zero and high extracellular calcium respectively, Sf2 and Sb2 the signals at 380 nM in zero and high calcium and Kd is the apparent dissociation constant for Fura2 which has been taken as 224 nM (Grynkiewicz et al., 1985). For the sensory neurones Rmin and Rmax were 0.88 þ 0.05 and 4.2 þ 0.28 and Sf2 /Sba was 3.12 þ 0.39 (n = 12 þ S.E.M.). Inositol 1,4,5-trisphosphate (IP3 ) assays Intracellular [3 H]IP3 formation was measured in unstimulated cells and in cells stimulated by histamine. These assays were based on those of Irvine et al., (1985). Neurones on coverslips were incubated with [3 H]inositol overnight. Following stimulation, the inositol phosphates were separated by anion-exchange chromatography on AG-1 columns before radiochemical assay by scintillation counting. As the density of neurones varied between coverslips [3 H]IP3 formation was expressed as a percentage of total lipid radioactivity. All experiments were performed in triplicate and data plotted as mean þ S.E.M. Solutions Cells were bathed in Locke's solution bu¡ered with 16 mM NaHCO3 equilibrated with 95% O2 : 5% CO2 or HEPES or 20 mM HEPES. The unbu¡ered Locke's solution had the following composition (mM) NaCl 140, KCl 5, MgCl2 1, CaCl2 2, and glucose 5.5. The HEPES-bu¡ered solutions were adjusted to pH 7.3 using NaOH. The agonists were diluted from concentrated stocks into the appropriate bathing solution before use and the pH adjusted to 7.3 if necessary. Agonists and inhibitors were added to the bath as indicated by the bars in the ¢gures. Suppliers Except where otherwise indicated, all chemicals and tissue culture materials were supplied by Sigma-Aldrich (Poole, Dorset, UK). Ultroser G was supplied by Gibco-BRL (Life Technologies, Paisley, UK). NGF was supplied by Alemone Laboratories, Jerusalem, Israel. Forskolin, H89 and 1-[6-((17L3-methoxyestra-1,3,5(10)-triene 17-yl)amino)hexyl]1H-pyrrole2,5-dione (U73122) were supplied by Calbiochem Novabiochem (Beeston, Nottingham UK). Toris Cookson (Avonmouth, Bristol, UK) supplied cimetidene, mepyramine, methiothepin, thioperamide and the 5-hydroxytryptamine (5-HT) antagonist Y25310. Statistical analysis This was carried out on the pooled data from several experiments, comparisons between groups being made either with Fisher's Exact Test or, in the case of the IP3 assays by means of a two-tailed t-test. Di¡erences between data sets were regarded as statistically signi¢cant when P 6 0.05.
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Histamine responses of sensory neurons RESULTS
Basic characteristics of the histamine response In this study we have used changes in intracellular calcium to determine the proportion of cells responding to histamine and to follow the time course of the response. A total of 421 cells were used to obtain the concentration^response relationship. To avoid any desensitisation, each cell was tested with a single concentration of histamine. Before application of histamine the 340/380 nm ratio typically had a value between 1.0 and 1.3. Application of histamine elicited a rise in intracellular calcium in a sub-population of sensory neurones (Fig. 1). The percentage of responsive cells depended on the concentration of histamine (Fig. 1B). At the lowest concentration tested (1 WM), no cells responded to histamine (0/54). Increasing the concentration to 10 WM elicited a response in only 1/77 cells and increasing the concentration to 100 WM resulted in 16/103 (15%) of cells responding. The proportion of responding cells increased further to 19/80 (24%) when cells were challenged with 1 mM histamine and rose to 37/107 (35%) at 10 mM histamine which was the highest concentration tested.
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To facilitate our studies on histamine receptor sensitisation (Nicolson et al., 1999) we chose 100 WM as our standard stimulus and, at this concentration, the percentage of cells responding remained essentially constant over a week in culture (the data from 500 neurones are summarised in Fig. 1C). Fig. 1A shows the time course of the change in intracellular calcium concentration for those cells that responded to application of histamine. When 100 WM histamine was applied, the 340/380 ratio rose within 10 s from a resting ratio between 1.0 and 1.2 to a peak value around 1.7 before relaxing back to a steady-state value of about 1.45 (the plateau phase). These ratio values correspond to a rise in the free calcium level from a resting level of approximately 60^90 nM to a peak value in the range 230^260 nM before relaxing to a plateau value in the range 130^160 nM. Washing o¡ the agonist resulted in a slow decline in intracellular free calcium towards baseline. When the histamine concentration was increased to 1 mM, the ratio increased from 1 to 1.8 and then declined to basal values with a half time of approximately 60s. There was no obvious plateau phase. With 10 mM histamine, the initial rise in the calcium response was slightly smaller (the peak value was
Fig. 1. The response of adult sensory neurones in short-term tissue culture to histamine. (A) Shows the time course of the calcium signal when the cells were challenged with 100 WM, 1 mM and 10 mM histamine. (B) Shows the percentage of sensory neurones responding to concentrations of histamine between 1 WM and 10 mM (logarithmic scale). (C) Shows the percentage of cells responding to 100 WM histamine at various times in culture (each point gives the number of responding cells from samples of 51^139 neurones).
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Fig. 2. The relationship between histamine sensitivity and capsaicin sensitivity for cultured sensory neurones. The time course of the response to histamine for those cells that were not sensitive to capsaicin (A) and for those that were (B). (C) Shows the time course of the responses to 500 nM capsaicin. Note that a subsequent application of 55 mM K had very little additional e¡ect. (D) Shows the size distribution of those cells sensitive to both capsaicin and histamine (n = 29, bottom) and to histamine only (n = 43, top).
approximately 1.6) and then rapidly declined to the baseline level with a half time of approximately 25 s. This decline appeared to be biphasic with an initial rapid phase followed by a slow phase. After an initial challenge with 100 WM histamine, a subsequent application failed to elicit a response in the majority of cells tested (in this series of experiments 9/84 cells responded to 100 WM histamine but only one of these cells responded to a second application given between 5 and 60 min later; this response was elicited after washing the preparation for 1 h and was much smaller and less long-lasting than the ¢rst). In order to aid the characterisation of the histaminesensitive neurones, we examined the responses to 100 WM histamine followed by 500 nM capsaicin as described in Section 2. Of one group of 72 sensory neurones that responded to histamine, 29 (40.2%) also responded to capsaicin while the remainder were capsaicin-insensitive but did respond to a depolarising challenge with high K . Although the percentage of cells responding to histamine increased with increasing concentration, the proportion of histamine-sensitive cells that also responded to capsaicin did not change signi¢cantly: 42% of cells responding to 1 mM histamine and 37.5% of cells responding to 10 mM histamine were also sensitive to capsaicin. Typical responses to capsaicin are shown in Fig. 2C. For capsaicin-insensitive cells, a challenge with 50 mM potassium increased the ratio from its resting value of approximately 1.0 to a value close to 2.5 within 10 s. Histamine responses were found predominantly in
small-diameter neurones with the sizes skewed towards those with smallest diameters (see Fig. 2D). Ninety percent of the histamine-responsive neurones had diameters in the range 11^25 Wm with a mean diameter of 17.8 Wm. The diameters of the neurones sensitive to both capsaicin and histamine ranged from 15 to 25 Wm (mean 19.7 Wm) while those that were histamine-sensitive but capsaicininsensitive had diameters of 11^20 Wm (mean 16.4 Wm). Overall, the histamine-sensitive small-diameter neurones ( 6 20 Wm) were less likely to be capsaicin-sensitive than the larger diameter neurones ( s 20 Wm, P = 0.005). In addition to the di¡erence in the size distribution, the response to 100 WM histamine also di¡ered between the capsaicin-sensitive and capsaicin-insensitive populations. In response to stimulation with 100 WM histamine, the capsaicin-insensitive population showed a large rise in intracellular calcium (by V1 ratio units) followed by a clear plateau phase in the response (Fig. 2A) whereas the capsaicin-sensitive neurones showed a smaller rise of about 0.3 ratio units which was slower in onset and was not sustained (Fig. 2B). This di¡erence in response was not seen when higher concentrations of histamine were used. H1 receptor activation underlies the response to histamine To determine the receptor sub-type that was responsible for the rise in intracellular calcium, histamine was applied in the presence of maximal inhibitory concentrations of the selective histamine receptor antagonists
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Fig. 3. The e¡ects of histamine receptor antagonists on calcium response to 100 WM, 1 mM and 10 mM histamine. Only mepyramine signi¢cantly reduced the number of cells responding to the agonist.
mepyramine (1 WM), cimetidine (50 WM) and thioperamide (1 WM). The H1 receptor antagonist mepyramine abolished the response to both 100 WM (Fig. 3A) and 1 mM histamine (Fig. 3B) and signi¢cantly reduced the number of neurones responding to 10 mM histamine (Fig. 3C). In contrast, 50 WM cimetidine and 1 WM thioperamide, which inhibit H2 and H3 receptors respectively, had no signi¢cant e¡ect on the percentage of neurones responding to these concentrations of histamine (Fig. 3). In agreement with the pharmacological characterisation and the expected coupling of H1 receptors to activation of phospholipase C, application of histamine increased IP3 production by the isolated sensory neurones in a concentration-dependent manner over the range 100 WM to 10 mM histamine (Fig. 4A). The increase in IP3 concentration was inhibited by 1 WM mepyramine (Fig. 4B). Consistent with this was the ¢nding that pre-treatment with the phospholipase C inhibitor U73122 (10 WM) abolished the calcium response to 100 WM histamine (n = 0/100; P 6 0.0001 ^ see Fig. 4C) and signi¢cantly reduced the proportion responding to
10 mM histamine from 35% (37/107) to 9% (5/55; P 6 0.0003) ^ see Fig. 4D. Stimulation with 10 mM histamine activates a calcium in£ux pathway Removal of extracellular calcium had no e¡ect on the proportion of cells responding to 100 WM histamine but did alter the time course of the response (Figs. 4C and 5A). In the absence of extracellular Ca2 , the response consisted of a rise in calcium that reached its peak within about 10 s and decayed with a half time of approximately 60 s (see Fig. 5A). In the presence of 2 mM Ca2 , this initial response was followed by a sustained elevation in intracellular Ca2 as described above (see Fig. 1A). The percentage of responding cells was not signi¢cantly di¡erent in Ca2 containing medium and Ca2 -free medium containing 100 WM EGTA (15% or 16/103 compared to 13% or 10/79; P = 0.67). In contrast, the percentage of neurones responding to 10 mM histamine fell from 35% in medium containing 2 mM Ca2 to 15% in Ca2 -free medium (n = 12/79; P = 0.004 ^ see
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Fig. 4. Histamine increases the formation of IP3 in sensory neurones. (A) Application of histamine increases IP3 production in sensory neurones (*P 6 0.05 compared to control). (B) This e¡ect is blocked by mepyramine. (*P = 0.003 compared to control ; 2 P = 0.004 compared to 10 mM histamine). (C) The phospholipase C inhibitor U71322 (10 WM) but not removal of extracellular calcium abolished the response to 100 WM histamine. Cell numbers are (responding cells to number tested): control 10/79, U73122 0/100 and 0 mM Ca2 16/103. (D) The percentage of cells responding to 10 mM histamine was signi¢cantly reduced by both 10 WM U73122 and removal of extracellular calcium but a combination of 10WM U73122 and Ca2 free medium was required to completely block the Ca2 response to histamine. Cell numbers are: control 37/107, U73122 5/55, 0 mM Ca2 12/79 and 0 mM Ca2 plus U73122 0/47.
Fig. 4D). This suggests that 10 mM histamine raises intracellular calcium via two pathways: one dependent on the generation of IP3 via phospholipase C and the other by directly gating a calcium in£ux. In agreement with this idea, pre-treatment of neurones with the speci¢c phospholipase C inhibitor U73122 (10 WM) reduced the percentage of neurones responding to 10 mM histamine when the medium contained 2 mM Ca2 (Fig. 4D) but did not block all responses. Complete abolition of the response to 10 mM histamine was only achieved when 10 WM U73122 was administered in Ca2 -free bathing medium (n = 0/30, P 6 0.0001 ^ see Fig. 4D). The time course of the calcium responses to 10 mM histamine was not signi¢cantly a¡ected by the removal of extracellular calcium or by pre-treatment with U73122 (Fig. 5B,C). The apparent increase in the amplitude of the response to 10 mM histamine in Ca2 -free medium presumably re£ects the large mobilisation of Ca2 from the internal stores. Pre-treatment of neurones with 50 WM ryanodine for 5 min prior to stimulating the cells was carried out to establish whether calcium-induced calcium release from ryanodine-sensitive stores played a role in the response to histamine. Ryanodine failed to evoke a calcium response in any of the 98 neurones tested and the num-
ber of cells responding to histamine after pre-treatment with ryanodine was not signi¢cantly reduced. Without ryanodine pre-treatment 37/107 cells responded to application of 10 mM histamine compared to 27/98 after pretreatment. This di¡erence was not statistically signi¢cant (P = 0.29). There have been reports that histamine is able to activate 5-HT receptors in smooth muscle (Todorov and Petkov, 1980) and 5-HT1c receptors expressed in Xenopus oocytes (Shichijo et al., 1991). To determine whether the e¡ects of histamine were due to non-speci¢c stimulation of 5-HT receptors we examined the e¡ects of 1 WM methiothepin (which is a speci¢c inhibitor of 5-HT1 and 5-HT2 receptors) and 2 WM N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4,dihydro2H-1,4-benzoxazine-8-carboxamide (Y25130) which is speci¢c for 5-HT3 receptors. The 5-HT receptor antagonists applied in combination had no signi¢cant e¡ect on the percentage of cells responding to 100 WM or 10 mM histamine. The percentage of cells responding to 100 WM histamine in the presence of the 5-HT receptor antagonists was 19% (16/103, P = 0.55) compared to control (15%) and 24% (19/79, P = 0.15) responded to 10 mM histamine compared to control (35%). In contrast, the percentage of cells responding to 100 WM 5-HT fell
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Fig. 5. The time course of the response to 100 WM and 10 mM histamine in medium containing 2 mM Ca2 and Ca2 -free medium (A, B). (C) Shows the time course of those cells that responded to 10 mM histamine after treatment with the phospholipase C inhibitor U71322.
from 21% (15/71) in the absence of antagonists to 4% (3/67, P = 0.005) in the presence of 1 WM methiothepin plus 2 WM Y25130. When 5-HT was applied in the presence of a mixture of mepyramine (1 WM), cimetidine (50 WM) and thioperamide (1 WM) 21% of cells responded which was the same as the proportion responding in the absence of antagonists (data not shown).
DISCUSSION
We have used changes in intracellular calcium to char-
acterise the sensitivity of cultured adult sensory neurones to histamine and have shown that application of this agonist elicits an increase in intracellular calcium in a sub-population of small-diameter cells. The proportion of cells responding increased as the concentration of histamine increased from 10 WM to 10 mM. A second application of histamine failed to elicit a response in all but one of the histamine-sensitive neurones. Sensitivity to histamine was evident from the ¢rst day in culture and the proportion of sensitive cells remained essentially constant over 7 days. The response to histamine was blocked by mepyramine, an antagonist of H1 receptors, but not
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by cimetidine or thioperamide which are antagonists of H2 and H3 receptors respectively. Nor did 5-HT receptor antagonists in combination reduce the proportion of cells responding to histamine. They did, however, block the response to 5-HT as expected. From this we conclude that the response to histamine results from activation of H1 histamine receptors. This view is supported by the ¢nding that application of histamine resulted in an increased formation of IP3 which could also be blocked by mepyramine. Our ¢nding that histamine increases the formation of IP3 in sensory neurones contrasts with an earlier study by Malhotra et al., (1990) who found that IP3 formation by sensory neurones was increased by acetylcholine, 5-HT and vasoactive polypeptide but not by histamine, dopamine or noradrenaline. These authors used only one concentration of histamine (30 WM) which is not su¤cient to activate all the histamine-sensitive neurones. In other neural tissues histamine has been shown to increase IP3 formation via H1 receptor activity (Sarri et al., 1995; Xu and Chuang, 1987). The relatively high proportion of histamine-sensitive neurones in our cultures (up to 35%) re£ects our method of cell isolation which results in a sample that is biased towards smalldiameter cells. The observation that the population of sensory neurones that respond to histamine is biased towards smalldiameter cells is in agreement with a recent study by Kashiba et al., (1999) in which around 15% of guineapig sensory neurones in the trigeminal and lumbar regions were found to contain H1 receptor mRNA. These neurones were exclusively of small diameter. Schmelz et al., (1997) found that a¡erents excited by histamine were slowly conducting (approximately 0.5 m s31 ) and their data are also in accord the notion that histamine-sensitive neurones are of small diameter. In those cells that did respond to histamine, the magnitude of the calcium response did not increase with increasing concentration over the range 100 WM to 10 mM. Indeed, the most sustained calcium response to histamine was seen at a concentration of 100 WM. At this concentration the response consisted of two phases in the continuing presence of the agonist: an initial short-lasting phase which decayed over approximately 20 s to a steady-state value which was signi¢cantly higher than the resting level. Higher concentrations of histamine did not elicit signi¢cantly larger responses but did elicit responses that showed a marked decline towards the resting level. The initial component of the calcium response is the result of calcium release from internal stores as it was not abolished by removal of extracellular calcium, unlike the sustained component of the signal. Moreover, this phase of the response could be prevented by pre-incubation of the cells with the speci¢c phospholipase C inhibitor U71322 strongly suggesting that calcium was released from the endoplasmic reticulum stores following the formation of IP3 whereas application of ryanodine had no e¡ect on resting calcium levels nor did it a¡ect the calcium response to histamine. The sustained phase of the response is clearly due to a calcium in£ux as it was abolished when the cells were challenged with histamine in calcium-free medium. This phase of the calcium
response most likely re£ects in£ux via a store-operated entry which has been described in other mammalian CNS neurones stimulated by agonists that activate phospholipase C (Prothero et al., 1998, 2000). Pre-incubation of cells with U73122 signi¢cantly reduced the proportion of cells responding to 1 and 10 mM histamine. A somewhat smaller reduction in the proportion of responding cells was found when they were challenged in Ca2 -free medium in the absence of U73122. When histamine was applied in Ca2 -free medium after prior incubation with U71322, no responses to histamine were seen. This suggests that in some sensory neurones, high concentrations of histamine are able to gate an in£ux of Ca2 that is independent of store depletion. The nature of this pathway remains unclear but it may correspond to that reported by Tani et al. (1990) for rat trigeminal ganglion cells in which histamine increased the [Ca2 ]i of the neurones via H1 receptors. Unlike the responses to low concentrations of histamine reported here for dorsal root ganglion cells, removal of extracellular calcium abolished the response of the trigeminal cells. The majority of histamine-sensitive cells were not sensitive to capsaicin and tended to be smaller in diameter than those cells that responded to both capsaicin and histamine. Moreover, the two populations exhibited different patterns of response to 100 WM histamine. In cells that did not respond to capsaicin, 100 WM histamine evoked a large and sustained rise in intracellular calcium. In those cells that responded to capsaicin the rise in calcium in response to histamine was slower in onset, smaller in amplitude and tended to decline towards baseline, even in the continuing presence of the agonist. This di¡erence was not evident, however, when cells were challenged with higher concentrations of histamine. Nevertheless, the proportion of histamine-sensitive neurones that also responded to capsaicin did not change signi¢cantly as the histamine concentration increased. From this ¢nding we may also conclude that exposure to histamine did not induced a cross-desensitisation of the capsaicin response (otherwise we should expect the proportion of capsaicin responsive cells to decrease as the concentration of histamine increased). As the proportion of cells responding to histamine increases with concentration, it is important to have an estimate of the range of concentrations of histamine likely to be present in the environs of a degranulating mast cell. It is di¤cult to ascribe a precise value for the histamine content of mast cells in the skin as no reliable method exists for isolating such cells in their native state. It is, however, well known that rat peritoneal mast cells contain 5HT in addition to histamine with a histamine/ 5HT ratio in the range 30^100 (Enerba«ck and Wingren, 1980). As the histamine/5HT ratio for skin is only slightly less than that reported for puri¢ed peritoneal mast cells (Beaven et al., 1983; Enerba«ck and Wingren, 1980), it is reasonable to conclude that rat skin mast cells will have a histamine/5HT ratio similar to that of peritoneal mast cells which contain around 10^30 pg of histamine/cell (Soll et al., 1981; Beaven et al., 1983). Since each cell has 500^1000 granules with a mean diameter of
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Histamine responses of sensory neurons
0.6 Wm (Helander and Bloom, 1974), the concentration of histamine per granule will range from approximately 0.8 M to 1.6 M. As it is packaged with heparin in a matrix, this histamine is not present in free solution. Nevertheless, as histamine is a small molecule with a high aqueous solubility, it will rapidly di¡use from the matrix following the secretion of a granule. From this it would appear that immediately following degranulation, the histamine concentration in the extracellular space is likely to be close to a saturating concentration for histamine ^ probably in the region of 15^30 mM although this will progressively fall with time. Given that some nerve endings are closely apposed to mast cells [ 6 100 nm separation (Dimitriadou et al., 1997)], they are likely to experience millimolar concentrations of histamine as the mast cells degranulate. In this respect, it is of some interest that Jinks and Carstens (2000) used injections of 3% ( = 163 mM) histamine to activate chemosensitive a¡erents in rat skin. Neural mechanisms of itch As we have not labelled skin a¡erents, the sensory neurones we have studied are of mixed origin. Moreover, the transduction processes we have described may di¡er from those of the histamine-sensitive nerve endings in the skin. Nevertheless, our ¢ndings have some bearing on the peripheral mechanisms of the sensation of itch. Four distinct theories have been proposed (see McMahon and Koltzenburg, (1992). They are: (i) the speci¢city theory which proposes that a speci¢c set of a¡erents respond to pruritic stimuli; (ii) the intensity theory which proposes that low levels of activity in nociceptive a¡erents signal itch while higher levels of activity signal pain; (iii) the selectivity theory which proposes that there are no speci¢c populations of a¡erents that signal itch, rather the sensation is mediated via a subset
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of nociceptive a¡erents that activate separate central connections responsible for the sensation of itch; (iv) the pattern theory which proposes that itch is encoded by temporal or spatial discharge patterns in cutaneous a¡erents that also signal other modalities. These theories are not mutually exclusive as more than one mechanism may be involved. However, since application of histamine to the super¢cial regions of the skin is known to elicit a pure sensation of itch (Magerl et al., 1990; Ward et al., 1996) and since a small population of C-¢bre a¡erents responds to histamine but not to other forms of stimulation (Schmelz et al., 1997), the pattern and selectivity theories are unlikely explanations for the sensation of itch. Nevertheless, there is some recent evidence from H1 knockout mice that histamine may play a role in nociception (Mobarakeh et al., 2000). This view is supported by the well-established ¢ndings that injection of histamine into the deeper layers of the skin can elicit pain (Keele and Armstrong, 1964). Moreover, many of the histamine-sensitive a¡erents are also sensitive to mechanical stimulation, capsaicin or mustard oil, suggesting that some histamine-sensitive a¡erents are polymodal nociceptors (Handwerker et al., 1991; Schmelz et al., 1997). Since, however, we have found that most of the histamine-sensitive sensory neurones are not sensitive to capsaicin, it is clear that most are not polymodal nociceptors. This implies that the intensity theory cannot fully account for the sensation of itch. Taken together, the available data are consistent with the notion that most itch sensations are mediated via a speci¢c subset of a¡erent ¢bres, although it is possible that other mechanisms may be involved.
AcknowledgementsöWe thank the MRC for an Industrial Collaborative Studentship awarded to T.A.N.
REFERENCES
Andrew, D., Craig, A.D., 2001. Spinothalamic lamina I neurons selectively sensitive to histamine: a central neural pathway for itch. Nat. Neurosci. 4, 72^77. Beaven, M.A., Aiken, D.L., Woldemussie, E., Soll, A.H., 1983. Changes in histamine synthetic activity, histamine contant and rersponsiveness to compound 48/80 with maturation of rat peritoneal mast cells. J. Pharmacol. Exp. Ther. 224, 620^626. Bevan, S., Winter, J., 1995. Nerve growth factor (NGF) di¡erentially regulates the chemosensitivity of adult rat cultured sensory neurons. J. Neurosci. 15, 4918^4926. Dimitriadou, V., Rouleau, A., Trung Tuong, M.D., Newlands, G.J., Miller, H.R., Lu¡au, G., Schwartz, J.C., Garbarg, M., 1997. Functional relationships between sensory nerve ¢bers and mast cells of dura mater in normal and in£ammatory conditions. Neuroscience 77, 829^839. Enerba«ck, L., Wingren, U., 1980. Histamine content of peritoneal and tissue mast cells of growing rats. Histochemistry 66, 113^124. Fjallbrant, N., Iggo, A., 1961. The e¡ect of histamine, 5-hydroxytryptamine and acetylcholine on cutaneous a¡erent ¢bres. J. Physiol. 156, 578^ 590. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2 indicators with greatly improved £uorescence properties. J. Biol. Chem. 260, 3440^3450. Handwerker, H.O., Forster, C., Kirchho¡, C., 1991. Discharge patterns of human C-¢bers induced by itchning and burning stimuli. J. Neurophysiol. 66, 307^315. Helander, H.F., Bloom, G.D., 1974. Quantitative analysis of mast cell structure. J. Microsc. 100, 315^321. Irvine, R.F., Anggard, E.E., Letcher, A.J., Downes, C.P., 1985. Metabolism of inositol 1, 4, 5-trisphosphate and inositol 1, 3, 4- trisphosphate in rat parotid glands. Biochem. J. 229, 505^511. Jinks, S.L., Carstens, E., 2000. Super¢cial dorsal horn neurons identi¢ed by intracutaneous histamine: chemonociceptive responses and modulation by morphine. J. Neurophysiol. 84, 616^627. Kashiba, H., Fukui, H., Morikawa, Y., Senba, E., 1999. Gene expression of histamine H1 receptor in guinea pig primary sensory neurons: a relationship between H1 receptor mRNA-expressing neurons and peptidergic neurons. Mol. Brain Res. 66, 24^34. Keele, C. and Armstrong, D. (1964) Substances producing pain and itch, Edward Arnold, London. Magerl, W., Westerman, R.A., Mohner, B., Handwerker, H.O., 1990. Properties of transdermal histamine iontophoresis: di¡erential e¡ects of season, gender, and body region. J. Invest. Dermatol. 94, 347^352.
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Malhotra, R.K., Bhave, S.V., Wakade, T.D., Bhave, A.S., Wakade, A.R., 1990. E¡ects of neurotransmitters and peptides on phospholipid hydrolysis in sympathetic and sensory neurons. Faseb J. 4, 2492^2498. McMahon, S.B., Koltzenburg, M., 1992. Itching for an explanation. Trends Neurosci. 15, 497^501. Mobarakeh, J.I., Sakurada, S., Katsuyama, S., Kutsuwa, M., Kuramasu, A., Lin, Z.Y., Watanabe, T., Hashimoto, Y., Yanai, K., 2000. Role of histamine H(1) receptor in pain perception: a study of the receptor gene knockout mice. Eur. J. Pharmacol. 391, 81^89. Nicolson, T., Bevan, S. and Richards, C.D. (1999). The prostaglandin-induced sensitization of cultured dorsal root ganglion neurones to histamine is mediated via a cyclic AMP-dependent mechanism. J. Physiol., 518, 152P. Nicolson, T., Bevan, S. and Richards, C.D. (1998). The sensitivity of rat cultured dorsal root ganglion neurones to histamine is augmented by prostaglandin E-2. J. Physiol., 511, 122P. Prothero, L.S., Mathie, A., Richards, C.D., 2000. Purinergic and muscarinic receptor activation activates a common calcium entry pathway in rat neocortical neurons and glial cells. Neuropharmacol. 39, 1768^1778. Prothero, L.S., Richards, C.D., Mathie, A., 1998. Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia. Br. J. Pharmacol. 125, 1551^1561. Sarri, E., Picatoste, F., Claro, E., 1995. Neurotransmitter-speci¢c pro¢les of inositol phosphates in rat brain cortex: relation to the mode of receptor activation of phosphoinositide phospholipase C. J. Pharmacol. Exp. Ther. 272, 77^84. Schmelz, M., Schmidt, R., Bickel, A., Handwerker, H.O., Torebjork, H.E., 1997. Speci¢c C-receptors for itch in human skin. J. Neurosci. 17, 8003^8008. Shelley, W.E., Arthur, R.P., 1957. The neurohistology and neurophysiology of the itch sensation in man. Arch. Dermatol. 76, 296^323. Shichijo, S., Payan, D.G., Harrowe, G., Mitsuhashi, M., 1991. Histamine e¡ects on the 5-HT1c receptor expressed in Xenopus oocytes. J. Neurosci. Res. 30, 316^320. Soll, A.H., Lewin, K.J., Beaven, M.A., 1981. Isolation of histamine containing cells from rat gastric mucosa : biochemical and morphological di¡erences from mast cells. Gastroenterology 80, 717^727. Tani, E., Shiosaka, S., Sato, M., Ishikawa, T., Tohyama, M., 1990. Histamine acts directly on calcitonin gene-related peptide- and substance P-containing trigeminal ganglion neurons as assessed by calcium in£ux and immunocytochemistry. Neurosci. Lett. 115, 171^176. Todorov, S., Petkov, V., 1980. On the interaction of histamine with the tryptaminergic smooth-muscle receptors. Acta Physiol. Pharmacol. Bulg. 6, 47^54. Tuckett, R.P., Wei, J.Y., 1987. Response to an itch-producing substance in cat II. Cutaneous receptor populations with unmyelinated axons. Brain Res. 413, 95^103. Ward, L., Wright, E., McMahon, S.B., 1996. A comparison of the e¡ects of noxious and innocuous counterstimuli on experimentally induced itch and pain. Pain 64, 129^138. Xu, J., Chuang, D.M., 1987. Serotonergic, adrenergic and histaminergic receptors coupled to phospholipase C in cultured cerebellar granule cells of rats. Biochem. Pharmacol. 36, 2353^2358. (Accepted 26 October 2001)
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Neuroscience Vol. 110, No. 2, pp. 329^338, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
CHARACTERISATION OF THE CALCIUM RESPONSES TO HISTAMINE IN CAPSAICIN-SENSITIVE AND CAPSAICIN-INSENSITIVE SENSORY NEURONES T. A. NICOLSON,a S. BEVANb and C. D. RICHARDSa * a
Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK b
Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BN, UK
AbstractöAdult rat sensory neurones were maintained in short-term tissue culture and their response to histamine was studied by monitoring changes in intracellular [Ca2 ] with Fura-2. The proportion of histamine-sensitive neurones increased as the concentration increased from 10 WM to 10 mM. The fraction of responding cells did not change signi¢cantly over the ¢rst week in culture. About 60% of histamine-sensitive cells were insensitive to capsaicin and these cells tended to be of small diameter. The integrated calcium response to histamine was greatest at 100 WM when the response consisted of two phases: an initial short-lasting transient followed by a sustained plateau that was dependent on extracellular calcium. This response was blocked by the histamine H1 receptor antagonist mepyramine but not by cimetidine or thioperamide which block H2 and H3 receptors, respectively. Moreover, application of histamine increased the intracellular concentration of inositol 1,4,5-trisphosphate ^ an e¡ect blocked by mepyramine. These data show that the response is mediated by H1 receptors. The phospholipase C inhibitor U73122 blocked the response to 100 WM histamine and signi¢cantly reduced the fraction of cells responding to 1 mM and 10 mM histamine as did removal of extracellular calcium. A combination of U73122 and calcium-free medium abolished all responses to histamine. These data suggest that in addition to activating phospholipase C, high concentrations of histamine gate an in£ux of calcium that is independent of store depletion. The implications of these results for the transduction of pruritic stimuli is discussed. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: itch, H1 receptor, histamine, capsaicin, calcium, sensory neurone.
burning pain following block of cutaneous C-¢bres by local anaesthetics but persists when a nerve trunk is blocked by pressure at a time when only C-¢bres remain active. For these reasons it is likely that the sensation is mediated by unmyelinated cutaneous a¡erents. In support of this view, recent microneurography studies on human volunteers have found that a small subset of unmyelinated nerve ¢bres responded with sustained discharge after ionophoretic injection of histamine into the skin (Handwerker et al., 1991; Schmelz et al., 1997). Moreover, the time course of the discharge corresponded with the time course of the itch sensations. Very recently Andrew and Craig (2001) have described a class of lamina I spinothalamic tract neurones in the cat that have properties that parallel the pure itching sensation in man and match the responses of peripheral C-¢bres that respond selectively to histamine. Despite these insights, the mechanisms by which histamine excites cutaneous nerve ¢bres remain obscure as does the relationship between nociceptive and pruritic a¡erents (see McMahon and Koltzenburg, 1992). In an attempt to address these issues, we have used changes in intracellular calcium to characterise the response of adult sensory neurones maintained in short-term tissue culture to application of histamine. In particular, we have examined the relationship between the sensitivity of sensory neurones to histamine and that to capsaicin which is diagnostic of polymodal nociceptive neurones. Preliminary accounts
Histamine is released into the dermis following mast cell degranulation and this event appears to be the primary trigger for the sensation of itch. Ionophoretic application of histamine into the super¢cial regions of the skin elicits a pure sensation of itch (Magerl et al., 1990; Ward et al., 1996) and concentrations of histamine which produce the sensation have been shown to excite unmyelinated a¡erent nerve ¢bres (Fjallbrant and Iggo, 1961; Tuckett and Wei, 1987). Furthermore, the skin possesses speci¢c itch points with a punctate distribution like other skin sensations (Shelley and Arthur, 1957). Excision of these points followed by histological examination reveals no speci¢c sensory structures but an increased density of bare nerve endings. The sensation of itch disappears along with
*Corresponding author. Tel.: +44-020-7679-6089; fax: +44-0207387-6368. E-mail address: [email protected] (C. D. Richards). Abbreviations : 4Br-A23187, 4-bromo-A23187 (4-bromo-calcimycin) ; EGTA, ethylene glycol-bis(L-aminoethyl-ether)-N,N,NP,NPtetra acetic acid; Fura-2 AM, Fura-2 acetoxy methyl ester ; HEPES, N-(2-hydroxyethylpiperazine)-NP-(2-ethane sulphonic acid); 5-HT, 5-hydroxytryptamine; IP3 , inositol 1,4,5-trisphosphate; NGF, nerve growth factor; U73122, 1-[6-((17L-3-methoxyestra-1,3,5(10)-triene 17-yl)amino)hexyl]1H-pyrrole-2,5-dione ; Y25130, N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-methyl-3-oxo3,4,dihydro-2H-1,4-benzoxazine-8-carboxamide. 329
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of our ¢ndings have been published elsewhere (Nicolson et al., 1998; Nicolson et al., 1999).
EXPERIMENTAL PROCEDURES
Cell culture Primary sensory neurones were isolated and maintained in culture as described by Bevan and Winter, (1995). In brief, adult Sprague^Dawley rats from the Royal Free Hospital School of Medicine colony were killed by asphyxiation with a rising concentration of carbon dioxide followed by cervical dislocation. The vertebral column was removed and a 3^4-mm strip of bone was cut along the length of the vertebral column to expose the spinal cord and the recessed bony cavities which contain the spinal ganglia. The ganglia were carefully dissected out under aseptic conditions and placed in Ham's F14 medium containing 4% Ultroser-G. The dorsal and ventral roots were trimmed and the individual ganglia transferred to medium containing 1.25 mg ml31 collagenase type IV for 3^4 h. After the period of incubation, the ganglia were washed free of collagenase and dispersed to form a cell suspension by trituration with £ame-polished Pasteur pipettes. The cell suspension was centrifuged in Ham's F 14 medium containing 15% bovine serum albumin. The resulting pellet was resuspended in F14 medium containing 4% Ultroser-G supplemented with nerve growth factor (NGF) 0.2 Wg ml31 . The isolated sensory neurones were then plated onto glass coverslips that had previously been coated with poly-ornithine and laminin. The cells were maintained in identical medium for up to 8 days. Intracellular Ca2 measurements Ca2 measurements were made with the dual excitation calcium indicator Fura-2 acetoxymethyl ester (Fura-2, Molecular Probes, Eugene, OR, USA) which was loaded into the cells as its AM-ester as follows: glass coverslips with the adherent cells were incubated in standard bicarbonate-bu¡ered Locke's solution containing 10 WM Fura-2 acetoxy methyl ester (Fura-2 AM) for 30 min at 37³C. Following loading, the cells were then washed for 15 min in bicarbonate-bu¡ered Locke at 37³C prior to their transfer to HEPES-bu¡ered Locke's solution. All experiments were conducted at room temperature (approximately 22³C) and carried out within approximately 1 h of loading the cells with the indicator. The dye was excited alternately with light of 340 and 380 nm and the light emitted above 420 nm was collected via a cooled CCD camera (supplied by Digital Pixel, Brighton, UK). To improve the signal-to-noise ratio, the collection times were adjusted to increase the total number of photons accumulated at the lowest intensity of emission. Thus the collection period for 340-nm excitation was three times that for 380-nm excitation (the collection period was generally 600 ms compared to 200 ms). The resulting images were analysed using software supplied by Kinetic Imaging (Liverpool, UK). In each experiment, ratio values were calculated after subtraction of the background signal which was determined at the end of every experiment by treating the cells with 0.1% Triton X-100. To monitor the local Ca2 from the ratio images, speci¢c areas of interest were chosen and the average ratio value for the designated areas was then calculated and plotted as a function of time. The ratio values in the ¢gures are not corrected for the di¡erent collection times. Apart from one short series of experiments to investigate tachyphylaxis to histamine, cells were only exposed to a single application of histamine. Furthermore, to check the viability of the cultured cells, the experimental protocol invariably included the following steps after stimulation with histamine. They were ¢rst exposed to medium containing 500 nM capsaicin and then to medium containing 50 mM potassium. Those cells that did not respond to either capsaicin or to high potassium were con-
sidered unviable and were excluded from the study. This protocol also allowed us to classify the sensory neurones into those which were sensitive to capsaicin and those which were not. Calibration of the ratios in terms of [Ca2 ] was carried out in situ in several experiments using the ionophore 4-bromo-A23187 (4-bromo-calcimycin, 4Br-A23187). Cells were exposed to a `zero' calcium solution containing 20 mM EGTA and 0 mM Ca2 to obtain a minimum ratio and a `high' calcium solution containing 2 mM Ca2 to obtain a maximum ratio, both in the presence of 5 WM 4Br-A23187. Cells were exposed to these zero and high-calcium solutions alternately until consistent ratio values were obtained. The average values from these calibrations have been used to quantify the changes in intracellular Ca2 where this was considered appropriate using the following equation (Grynkiewicz et al., 1985): Ca2 K d R3Rmin = Rmax 3R Sf2= S b2 where R is the measured ratio of interest, Rmin and Rmax the ratios recorded with zero and high extracellular calcium respectively, Sf2 and Sb2 the signals at 380 nM in zero and high calcium and Kd is the apparent dissociation constant for Fura2 which has been taken as 224 nM (Grynkiewicz et al., 1985). For the sensory neurones Rmin and Rmax were 0.88 þ 0.05 and 4.2 þ 0.28 and Sf2 /Sba was 3.12 þ 0.39 (n = 12 þ S.E.M.). Inositol 1,4,5-trisphosphate (IP3 ) assays Intracellular [3 H]IP3 formation was measured in unstimulated cells and in cells stimulated by histamine. These assays were based on those of Irvine et al., (1985). Neurones on coverslips were incubated with [3 H]inositol overnight. Following stimulation, the inositol phosphates were separated by anion-exchange chromatography on AG-1 columns before radiochemical assay by scintillation counting. As the density of neurones varied between coverslips [3 H]IP3 formation was expressed as a percentage of total lipid radioactivity. All experiments were performed in triplicate and data plotted as mean þ S.E.M. Solutions Cells were bathed in Locke's solution bu¡ered with 16 mM NaHCO3 equilibrated with 95% O2 : 5% CO2 or HEPES or 20 mM HEPES. The unbu¡ered Locke's solution had the following composition (mM) NaCl 140, KCl 5, MgCl2 1, CaCl2 2, and glucose 5.5. The HEPES-bu¡ered solutions were adjusted to pH 7.3 using NaOH. The agonists were diluted from concentrated stocks into the appropriate bathing solution before use and the pH adjusted to 7.3 if necessary. Agonists and inhibitors were added to the bath as indicated by the bars in the ¢gures. Suppliers Except where otherwise indicated, all chemicals and tissue culture materials were supplied by Sigma-Aldrich (Poole, Dorset, UK). Ultroser G was supplied by Gibco-BRL (Life Technologies, Paisley, UK). NGF was supplied by Alemone Laboratories, Jerusalem, Israel. Forskolin, H89 and 1-[6-((17L3-methoxyestra-1,3,5(10)-triene 17-yl)amino)hexyl]1H-pyrrole2,5-dione (U73122) were supplied by Calbiochem Novabiochem (Beeston, Nottingham UK). Toris Cookson (Avonmouth, Bristol, UK) supplied cimetidene, mepyramine, methiothepin, thioperamide and the 5-hydroxytryptamine (5-HT) antagonist Y25310. Statistical analysis This was carried out on the pooled data from several experiments, comparisons between groups being made either with Fisher's Exact Test or, in the case of the IP3 assays by means of a two-tailed t-test. Di¡erences between data sets were regarded as statistically signi¢cant when P 6 0.05.
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Histamine responses of sensory neurons RESULTS
Basic characteristics of the histamine response In this study we have used changes in intracellular calcium to determine the proportion of cells responding to histamine and to follow the time course of the response. A total of 421 cells were used to obtain the concentration^response relationship. To avoid any desensitisation, each cell was tested with a single concentration of histamine. Before application of histamine the 340/380 nm ratio typically had a value between 1.0 and 1.3. Application of histamine elicited a rise in intracellular calcium in a sub-population of sensory neurones (Fig. 1). The percentage of responsive cells depended on the concentration of histamine (Fig. 1B). At the lowest concentration tested (1 WM), no cells responded to histamine (0/54). Increasing the concentration to 10 WM elicited a response in only 1/77 cells and increasing the concentration to 100 WM resulted in 16/103 (15%) of cells responding. The proportion of responding cells increased further to 19/80 (24%) when cells were challenged with 1 mM histamine and rose to 37/107 (35%) at 10 mM histamine which was the highest concentration tested.
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To facilitate our studies on histamine receptor sensitisation (Nicolson et al., 1999) we chose 100 WM as our standard stimulus and, at this concentration, the percentage of cells responding remained essentially constant over a week in culture (the data from 500 neurones are summarised in Fig. 1C). Fig. 1A shows the time course of the change in intracellular calcium concentration for those cells that responded to application of histamine. When 100 WM histamine was applied, the 340/380 ratio rose within 10 s from a resting ratio between 1.0 and 1.2 to a peak value around 1.7 before relaxing back to a steady-state value of about 1.45 (the plateau phase). These ratio values correspond to a rise in the free calcium level from a resting level of approximately 60^90 nM to a peak value in the range 230^260 nM before relaxing to a plateau value in the range 130^160 nM. Washing o¡ the agonist resulted in a slow decline in intracellular free calcium towards baseline. When the histamine concentration was increased to 1 mM, the ratio increased from 1 to 1.8 and then declined to basal values with a half time of approximately 60s. There was no obvious plateau phase. With 10 mM histamine, the initial rise in the calcium response was slightly smaller (the peak value was
Fig. 1. The response of adult sensory neurones in short-term tissue culture to histamine. (A) Shows the time course of the calcium signal when the cells were challenged with 100 WM, 1 mM and 10 mM histamine. (B) Shows the percentage of sensory neurones responding to concentrations of histamine between 1 WM and 10 mM (logarithmic scale). (C) Shows the percentage of cells responding to 100 WM histamine at various times in culture (each point gives the number of responding cells from samples of 51^139 neurones).
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Fig. 2. The relationship between histamine sensitivity and capsaicin sensitivity for cultured sensory neurones. The time course of the response to histamine for those cells that were not sensitive to capsaicin (A) and for those that were (B). (C) Shows the time course of the responses to 500 nM capsaicin. Note that a subsequent application of 55 mM K had very little additional e¡ect. (D) Shows the size distribution of those cells sensitive to both capsaicin and histamine (n = 29, bottom) and to histamine only (n = 43, top).
approximately 1.6) and then rapidly declined to the baseline level with a half time of approximately 25 s. This decline appeared to be biphasic with an initial rapid phase followed by a slow phase. After an initial challenge with 100 WM histamine, a subsequent application failed to elicit a response in the majority of cells tested (in this series of experiments 9/84 cells responded to 100 WM histamine but only one of these cells responded to a second application given between 5 and 60 min later; this response was elicited after washing the preparation for 1 h and was much smaller and less long-lasting than the ¢rst). In order to aid the characterisation of the histaminesensitive neurones, we examined the responses to 100 WM histamine followed by 500 nM capsaicin as described in Section 2. Of one group of 72 sensory neurones that responded to histamine, 29 (40.2%) also responded to capsaicin while the remainder were capsaicin-insensitive but did respond to a depolarising challenge with high K . Although the percentage of cells responding to histamine increased with increasing concentration, the proportion of histamine-sensitive cells that also responded to capsaicin did not change signi¢cantly: 42% of cells responding to 1 mM histamine and 37.5% of cells responding to 10 mM histamine were also sensitive to capsaicin. Typical responses to capsaicin are shown in Fig. 2C. For capsaicin-insensitive cells, a challenge with 50 mM potassium increased the ratio from its resting value of approximately 1.0 to a value close to 2.5 within 10 s. Histamine responses were found predominantly in
small-diameter neurones with the sizes skewed towards those with smallest diameters (see Fig. 2D). Ninety percent of the histamine-responsive neurones had diameters in the range 11^25 Wm with a mean diameter of 17.8 Wm. The diameters of the neurones sensitive to both capsaicin and histamine ranged from 15 to 25 Wm (mean 19.7 Wm) while those that were histamine-sensitive but capsaicininsensitive had diameters of 11^20 Wm (mean 16.4 Wm). Overall, the histamine-sensitive small-diameter neurones ( 6 20 Wm) were less likely to be capsaicin-sensitive than the larger diameter neurones ( s 20 Wm, P = 0.005). In addition to the di¡erence in the size distribution, the response to 100 WM histamine also di¡ered between the capsaicin-sensitive and capsaicin-insensitive populations. In response to stimulation with 100 WM histamine, the capsaicin-insensitive population showed a large rise in intracellular calcium (by V1 ratio units) followed by a clear plateau phase in the response (Fig. 2A) whereas the capsaicin-sensitive neurones showed a smaller rise of about 0.3 ratio units which was slower in onset and was not sustained (Fig. 2B). This di¡erence in response was not seen when higher concentrations of histamine were used. H1 receptor activation underlies the response to histamine To determine the receptor sub-type that was responsible for the rise in intracellular calcium, histamine was applied in the presence of maximal inhibitory concentrations of the selective histamine receptor antagonists
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Fig. 3. The e¡ects of histamine receptor antagonists on calcium response to 100 WM, 1 mM and 10 mM histamine. Only mepyramine signi¢cantly reduced the number of cells responding to the agonist.
mepyramine (1 WM), cimetidine (50 WM) and thioperamide (1 WM). The H1 receptor antagonist mepyramine abolished the response to both 100 WM (Fig. 3A) and 1 mM histamine (Fig. 3B) and signi¢cantly reduced the number of neurones responding to 10 mM histamine (Fig. 3C). In contrast, 50 WM cimetidine and 1 WM thioperamide, which inhibit H2 and H3 receptors respectively, had no signi¢cant e¡ect on the percentage of neurones responding to these concentrations of histamine (Fig. 3). In agreement with the pharmacological characterisation and the expected coupling of H1 receptors to activation of phospholipase C, application of histamine increased IP3 production by the isolated sensory neurones in a concentration-dependent manner over the range 100 WM to 10 mM histamine (Fig. 4A). The increase in IP3 concentration was inhibited by 1 WM mepyramine (Fig. 4B). Consistent with this was the ¢nding that pre-treatment with the phospholipase C inhibitor U73122 (10 WM) abolished the calcium response to 100 WM histamine (n = 0/100; P 6 0.0001 ^ see Fig. 4C) and signi¢cantly reduced the proportion responding to
10 mM histamine from 35% (37/107) to 9% (5/55; P 6 0.0003) ^ see Fig. 4D. Stimulation with 10 mM histamine activates a calcium in£ux pathway Removal of extracellular calcium had no e¡ect on the proportion of cells responding to 100 WM histamine but did alter the time course of the response (Figs. 4C and 5A). In the absence of extracellular Ca2 , the response consisted of a rise in calcium that reached its peak within about 10 s and decayed with a half time of approximately 60 s (see Fig. 5A). In the presence of 2 mM Ca2 , this initial response was followed by a sustained elevation in intracellular Ca2 as described above (see Fig. 1A). The percentage of responding cells was not signi¢cantly di¡erent in Ca2 containing medium and Ca2 -free medium containing 100 WM EGTA (15% or 16/103 compared to 13% or 10/79; P = 0.67). In contrast, the percentage of neurones responding to 10 mM histamine fell from 35% in medium containing 2 mM Ca2 to 15% in Ca2 -free medium (n = 12/79; P = 0.004 ^ see
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Fig. 4. Histamine increases the formation of IP3 in sensory neurones. (A) Application of histamine increases IP3 production in sensory neurones (*P 6 0.05 compared to control). (B) This e¡ect is blocked by mepyramine. (*P = 0.003 compared to control ; 2 P = 0.004 compared to 10 mM histamine). (C) The phospholipase C inhibitor U71322 (10 WM) but not removal of extracellular calcium abolished the response to 100 WM histamine. Cell numbers are (responding cells to number tested): control 10/79, U73122 0/100 and 0 mM Ca2 16/103. (D) The percentage of cells responding to 10 mM histamine was signi¢cantly reduced by both 10 WM U73122 and removal of extracellular calcium but a combination of 10WM U73122 and Ca2 free medium was required to completely block the Ca2 response to histamine. Cell numbers are: control 37/107, U73122 5/55, 0 mM Ca2 12/79 and 0 mM Ca2 plus U73122 0/47.
Fig. 4D). This suggests that 10 mM histamine raises intracellular calcium via two pathways: one dependent on the generation of IP3 via phospholipase C and the other by directly gating a calcium in£ux. In agreement with this idea, pre-treatment of neurones with the speci¢c phospholipase C inhibitor U73122 (10 WM) reduced the percentage of neurones responding to 10 mM histamine when the medium contained 2 mM Ca2 (Fig. 4D) but did not block all responses. Complete abolition of the response to 10 mM histamine was only achieved when 10 WM U73122 was administered in Ca2 -free bathing medium (n = 0/30, P 6 0.0001 ^ see Fig. 4D). The time course of the calcium responses to 10 mM histamine was not signi¢cantly a¡ected by the removal of extracellular calcium or by pre-treatment with U73122 (Fig. 5B,C). The apparent increase in the amplitude of the response to 10 mM histamine in Ca2 -free medium presumably re£ects the large mobilisation of Ca2 from the internal stores. Pre-treatment of neurones with 50 WM ryanodine for 5 min prior to stimulating the cells was carried out to establish whether calcium-induced calcium release from ryanodine-sensitive stores played a role in the response to histamine. Ryanodine failed to evoke a calcium response in any of the 98 neurones tested and the num-
ber of cells responding to histamine after pre-treatment with ryanodine was not signi¢cantly reduced. Without ryanodine pre-treatment 37/107 cells responded to application of 10 mM histamine compared to 27/98 after pretreatment. This di¡erence was not statistically signi¢cant (P = 0.29). There have been reports that histamine is able to activate 5-HT receptors in smooth muscle (Todorov and Petkov, 1980) and 5-HT1c receptors expressed in Xenopus oocytes (Shichijo et al., 1991). To determine whether the e¡ects of histamine were due to non-speci¢c stimulation of 5-HT receptors we examined the e¡ects of 1 WM methiothepin (which is a speci¢c inhibitor of 5-HT1 and 5-HT2 receptors) and 2 WM N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4,dihydro2H-1,4-benzoxazine-8-carboxamide (Y25130) which is speci¢c for 5-HT3 receptors. The 5-HT receptor antagonists applied in combination had no signi¢cant e¡ect on the percentage of cells responding to 100 WM or 10 mM histamine. The percentage of cells responding to 100 WM histamine in the presence of the 5-HT receptor antagonists was 19% (16/103, P = 0.55) compared to control (15%) and 24% (19/79, P = 0.15) responded to 10 mM histamine compared to control (35%). In contrast, the percentage of cells responding to 100 WM 5-HT fell
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Fig. 5. The time course of the response to 100 WM and 10 mM histamine in medium containing 2 mM Ca2 and Ca2 -free medium (A, B). (C) Shows the time course of those cells that responded to 10 mM histamine after treatment with the phospholipase C inhibitor U71322.
from 21% (15/71) in the absence of antagonists to 4% (3/67, P = 0.005) in the presence of 1 WM methiothepin plus 2 WM Y25130. When 5-HT was applied in the presence of a mixture of mepyramine (1 WM), cimetidine (50 WM) and thioperamide (1 WM) 21% of cells responded which was the same as the proportion responding in the absence of antagonists (data not shown).
DISCUSSION
We have used changes in intracellular calcium to char-
acterise the sensitivity of cultured adult sensory neurones to histamine and have shown that application of this agonist elicits an increase in intracellular calcium in a sub-population of small-diameter cells. The proportion of cells responding increased as the concentration of histamine increased from 10 WM to 10 mM. A second application of histamine failed to elicit a response in all but one of the histamine-sensitive neurones. Sensitivity to histamine was evident from the ¢rst day in culture and the proportion of sensitive cells remained essentially constant over 7 days. The response to histamine was blocked by mepyramine, an antagonist of H1 receptors, but not
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by cimetidine or thioperamide which are antagonists of H2 and H3 receptors respectively. Nor did 5-HT receptor antagonists in combination reduce the proportion of cells responding to histamine. They did, however, block the response to 5-HT as expected. From this we conclude that the response to histamine results from activation of H1 histamine receptors. This view is supported by the ¢nding that application of histamine resulted in an increased formation of IP3 which could also be blocked by mepyramine. Our ¢nding that histamine increases the formation of IP3 in sensory neurones contrasts with an earlier study by Malhotra et al., (1990) who found that IP3 formation by sensory neurones was increased by acetylcholine, 5-HT and vasoactive polypeptide but not by histamine, dopamine or noradrenaline. These authors used only one concentration of histamine (30 WM) which is not su¤cient to activate all the histamine-sensitive neurones. In other neural tissues histamine has been shown to increase IP3 formation via H1 receptor activity (Sarri et al., 1995; Xu and Chuang, 1987). The relatively high proportion of histamine-sensitive neurones in our cultures (up to 35%) re£ects our method of cell isolation which results in a sample that is biased towards smalldiameter cells. The observation that the population of sensory neurones that respond to histamine is biased towards smalldiameter cells is in agreement with a recent study by Kashiba et al., (1999) in which around 15% of guineapig sensory neurones in the trigeminal and lumbar regions were found to contain H1 receptor mRNA. These neurones were exclusively of small diameter. Schmelz et al., (1997) found that a¡erents excited by histamine were slowly conducting (approximately 0.5 m s31 ) and their data are also in accord the notion that histamine-sensitive neurones are of small diameter. In those cells that did respond to histamine, the magnitude of the calcium response did not increase with increasing concentration over the range 100 WM to 10 mM. Indeed, the most sustained calcium response to histamine was seen at a concentration of 100 WM. At this concentration the response consisted of two phases in the continuing presence of the agonist: an initial short-lasting phase which decayed over approximately 20 s to a steady-state value which was signi¢cantly higher than the resting level. Higher concentrations of histamine did not elicit signi¢cantly larger responses but did elicit responses that showed a marked decline towards the resting level. The initial component of the calcium response is the result of calcium release from internal stores as it was not abolished by removal of extracellular calcium, unlike the sustained component of the signal. Moreover, this phase of the response could be prevented by pre-incubation of the cells with the speci¢c phospholipase C inhibitor U71322 strongly suggesting that calcium was released from the endoplasmic reticulum stores following the formation of IP3 whereas application of ryanodine had no e¡ect on resting calcium levels nor did it a¡ect the calcium response to histamine. The sustained phase of the response is clearly due to a calcium in£ux as it was abolished when the cells were challenged with histamine in calcium-free medium. This phase of the calcium
response most likely re£ects in£ux via a store-operated entry which has been described in other mammalian CNS neurones stimulated by agonists that activate phospholipase C (Prothero et al., 1998, 2000). Pre-incubation of cells with U73122 signi¢cantly reduced the proportion of cells responding to 1 and 10 mM histamine. A somewhat smaller reduction in the proportion of responding cells was found when they were challenged in Ca2 -free medium in the absence of U73122. When histamine was applied in Ca2 -free medium after prior incubation with U71322, no responses to histamine were seen. This suggests that in some sensory neurones, high concentrations of histamine are able to gate an in£ux of Ca2 that is independent of store depletion. The nature of this pathway remains unclear but it may correspond to that reported by Tani et al. (1990) for rat trigeminal ganglion cells in which histamine increased the [Ca2 ]i of the neurones via H1 receptors. Unlike the responses to low concentrations of histamine reported here for dorsal root ganglion cells, removal of extracellular calcium abolished the response of the trigeminal cells. The majority of histamine-sensitive cells were not sensitive to capsaicin and tended to be smaller in diameter than those cells that responded to both capsaicin and histamine. Moreover, the two populations exhibited different patterns of response to 100 WM histamine. In cells that did not respond to capsaicin, 100 WM histamine evoked a large and sustained rise in intracellular calcium. In those cells that responded to capsaicin the rise in calcium in response to histamine was slower in onset, smaller in amplitude and tended to decline towards baseline, even in the continuing presence of the agonist. This di¡erence was not evident, however, when cells were challenged with higher concentrations of histamine. Nevertheless, the proportion of histamine-sensitive neurones that also responded to capsaicin did not change signi¢cantly as the histamine concentration increased. From this ¢nding we may also conclude that exposure to histamine did not induced a cross-desensitisation of the capsaicin response (otherwise we should expect the proportion of capsaicin responsive cells to decrease as the concentration of histamine increased). As the proportion of cells responding to histamine increases with concentration, it is important to have an estimate of the range of concentrations of histamine likely to be present in the environs of a degranulating mast cell. It is di¤cult to ascribe a precise value for the histamine content of mast cells in the skin as no reliable method exists for isolating such cells in their native state. It is, however, well known that rat peritoneal mast cells contain 5HT in addition to histamine with a histamine/ 5HT ratio in the range 30^100 (Enerba«ck and Wingren, 1980). As the histamine/5HT ratio for skin is only slightly less than that reported for puri¢ed peritoneal mast cells (Beaven et al., 1983; Enerba«ck and Wingren, 1980), it is reasonable to conclude that rat skin mast cells will have a histamine/5HT ratio similar to that of peritoneal mast cells which contain around 10^30 pg of histamine/cell (Soll et al., 1981; Beaven et al., 1983). Since each cell has 500^1000 granules with a mean diameter of
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0.6 Wm (Helander and Bloom, 1974), the concentration of histamine per granule will range from approximately 0.8 M to 1.6 M. As it is packaged with heparin in a matrix, this histamine is not present in free solution. Nevertheless, as histamine is a small molecule with a high aqueous solubility, it will rapidly di¡use from the matrix following the secretion of a granule. From this it would appear that immediately following degranulation, the histamine concentration in the extracellular space is likely to be close to a saturating concentration for histamine ^ probably in the region of 15^30 mM although this will progressively fall with time. Given that some nerve endings are closely apposed to mast cells [ 6 100 nm separation (Dimitriadou et al., 1997)], they are likely to experience millimolar concentrations of histamine as the mast cells degranulate. In this respect, it is of some interest that Jinks and Carstens (2000) used injections of 3% ( = 163 mM) histamine to activate chemosensitive a¡erents in rat skin. Neural mechanisms of itch As we have not labelled skin a¡erents, the sensory neurones we have studied are of mixed origin. Moreover, the transduction processes we have described may di¡er from those of the histamine-sensitive nerve endings in the skin. Nevertheless, our ¢ndings have some bearing on the peripheral mechanisms of the sensation of itch. Four distinct theories have been proposed (see McMahon and Koltzenburg, (1992). They are: (i) the speci¢city theory which proposes that a speci¢c set of a¡erents respond to pruritic stimuli; (ii) the intensity theory which proposes that low levels of activity in nociceptive a¡erents signal itch while higher levels of activity signal pain; (iii) the selectivity theory which proposes that there are no speci¢c populations of a¡erents that signal itch, rather the sensation is mediated via a subset
337
of nociceptive a¡erents that activate separate central connections responsible for the sensation of itch; (iv) the pattern theory which proposes that itch is encoded by temporal or spatial discharge patterns in cutaneous a¡erents that also signal other modalities. These theories are not mutually exclusive as more than one mechanism may be involved. However, since application of histamine to the super¢cial regions of the skin is known to elicit a pure sensation of itch (Magerl et al., 1990; Ward et al., 1996) and since a small population of C-¢bre a¡erents responds to histamine but not to other forms of stimulation (Schmelz et al., 1997), the pattern and selectivity theories are unlikely explanations for the sensation of itch. Nevertheless, there is some recent evidence from H1 knockout mice that histamine may play a role in nociception (Mobarakeh et al., 2000). This view is supported by the well-established ¢ndings that injection of histamine into the deeper layers of the skin can elicit pain (Keele and Armstrong, 1964). Moreover, many of the histamine-sensitive a¡erents are also sensitive to mechanical stimulation, capsaicin or mustard oil, suggesting that some histamine-sensitive a¡erents are polymodal nociceptors (Handwerker et al., 1991; Schmelz et al., 1997). Since, however, we have found that most of the histamine-sensitive sensory neurones are not sensitive to capsaicin, it is clear that most are not polymodal nociceptors. This implies that the intensity theory cannot fully account for the sensation of itch. Taken together, the available data are consistent with the notion that most itch sensations are mediated via a speci¢c subset of a¡erent ¢bres, although it is possible that other mechanisms may be involved.
AcknowledgementsöWe thank the MRC for an Industrial Collaborative Studentship awarded to T.A.N.
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