Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3

Neuroscience Letters 241 (1998) 159–162

Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3 Caroline Brechenmacher a, Yves Larmet b, Paul Feltz b, Jean-Luc Rodeau a ,* a

Laboratoire de Neurobiologie Cellulaire, CNRS UPR 9009, 5 rue Blaise Pascal, F-67084 Strasbourg, France Laboratoire de Physiologie Ge´ne´rale, Universite´ Louis-Pasteur, URA 1446, 21 rue Descartes, F-67084 Strasbourg, France

b

Received 29 September 1997; received in revised form 15 December 1997; accepted 19 December 1997

Abstract The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (,20 mm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.  1998 Elsevier Science Ireland Ltd.

Keywords: Substance P; Tachykinin receptors; Sensory neurones; Calcium; Fura-2; Capsaicin

Processing of nociceptive information begins with the release of neurotransmitter by primary afferent sensory neurones that act on specific subpopulations of dorsal horn neurones. Substance P (SP), co-liberated with glutamate, is involved in the transmission and modulation of noxious stimuli conveyed by unmyelinated primary afferent C-fibres [17,18]. Besides these postsynaptic effects, an additional presynaptic action has been suggested [16] and evidence has been provided for the existence of autoreceptors for SP [8]. If such effects of SP on dorsal horn neurones have been extensively studied, little is known on its effects on primary sensory neurones (i.e. dorsal root ganglion (DRG) neurones) or on the pharmacology of the SP receptors involved. Three distinct classes of tachykinin receptors (NK1, NK2 and NK3) have been described in mammals [13,14], preferentially activated by SP, neurokinin A and neurokinin B, respectively. All three types have been found in the central nervous system, especially in the dorsal horn of the spinal cord, where NK1 and NK2 receptors are involved in the transmission of nociceptive messages [5,20], whereas the * Corresponding author. Tel.: +33 3 88456691; fax: +33 3 88601664; e-mail: [email protected]

NK3 receptor has been proposed to possibly play an antinociceptive role [9]. Tachykinin receptors act through a G protein, causing production of inositol trisphosphate and elevation of the intracellular free calcium concentration ([Ca2+]i) [13]. SP is thus able to induce a [Ca2+]i rise in dorsal horn neurones, which is thought to contribute to the long term changes in the excitable properties of these cells following primary afferent fibre stimulation [21]. In DRG cells SP was also shown to elicit such a transient rise in [Ca2+]i [2]. In the present work, we attempted to identify the pharmacological properties of the tachykinin receptors present on DRG sensory neurones. Lumbar and thoracic dorsal root ganglia were removed from Wistar rat pups (0 to 1-day-old). Cells were dissociated as previously described [2], collected in culture medium (MEM-alpha; Gibco) supplemented with heat inactivated horse and foetal calf serum (5% v/v each; Gibco), and finally plated on glass coverslips coated with collagen (500 mg/ml in water) and poly-L-ornithine (500 mg/ ml in water). Cultures were incubated in 95% air/5% CO2 at 37°C for 24 h prior to experimentation. Neurones were identified by their reactivity to an antibody directed against neuronal specific enolase (NSE, C.B.R). Stained cells had a characteristic morphology and refractiveness.

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00045- 7

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[Ca2+]i was measured at room temperature (20–25°C) using the calcium sensitive dye, Fura-2. DRG neurones were incubated for 30 min in recording medium (in mM: 145 NaCl, 2.5 CaCl2, 3 KCl, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.2) containing 3 mM Fura-2-acetoxymethyl ester (Molecular Probes; USA). Cells were illuminated alternatively at 350–380 nm on an inverted microscope (Axiovert 35M; Zeiss, Germany) with an oil immersion objective (40×, DApo 40 UV; Olympus, Japan). Emitted light was detected at 520 nm by an intensified CCD camera (Extended ISIS; Photonic Science). A pair of images was recorded every second using an imaging system (Fluostar; Imstar, France). [Ca2+]i was calculated as [Ca2+]i = K(R − R min)/ (R max − R), where R is the ratio of the fluorescence intensities excited at 350 and 380 nm [7]. In vitro calibration was performed to obtain R min and R max (in the absence of calcium and at saturating calcium, respectively), and K, the apparent dissociation constant of Fura-2 for calcium [19]. For morphometric analysis, bright field images were acquired and cell diameter measurements were performed off-line using a morphometric software (Morphostar; Imstar, France). Substance P, the NK1 agonist [Sar9, Met(O2)11]SP, the NK2 agonist [Ala5, a-Ala8]-a-neurokinin fragment 4–10 and capsaicin were obtained from Sigma (USA). The NK3-receptor agonist senktide was from Cambridge Research Biochemicals (UK). The antagonists SR 140333A (NK1), SR 48968A (NK2) and SR 142801A (NK3), gifts from SANOFI (France), were kept in dimethylsulfoxide (DMSO) stock solutions (10−5, 10−2, 10−2 M, respectively). Capsaicin was dissolved in ethanol (10−3 M). Drugs were diluted to the appropriate concentration in the recording medium (≤0.1% DMSO or ethanol). Recorded cells were continuously superfused with recording medium (0.5 ml/min; total bath volume: 2 ml). The NK1, NK2 and NK3 antagonists (10 nM, 1 mM and 1 mM, respectively), and capsaicin (1 mM), were added to bath solution. SP and NK1, NK2, and NK3 agonists (1 mM in saline) were pressure-applied from a glass micropipette

Fig. 1. Pressure applications (200 ms) of 1 mM substance P or NK1-, NK2-, NK3-specific agonist typically induced a transient rise of [Ca2+]i in DRG neurones. The responses illustrated were obtained from four different neurones.

Fig. 2. (A) Proportion of DRG neurones responding to substance P or specific tachykinin agonists (NK), in the absence or presence of the corresponding antagonist (ANK), as indicated on the bottom scale. (B) Average rise in [Ca2+]i measured at peak in responding cells. Error bars correspond to SEM. Responses to SP, the NK1 and the NK3 agonists were not significantly different from each other, but differed from that to the NK2 agonist (P , 0.05). The NK2 antagonist SR 48968A had no significant effect on the calcium response induced by the agonist.

about 50 mm from the cells. These applications (200 ms) allowed rapid changes around the cells, but the final concentrations were unknown. The three antagonists tested were applied 10 to 15 min before the agonist. A second application of the agonist was performed after 10 to 15 min of washout. Since not all neurones responded to tachykinins, 50 mM KCl was applied for 5 s at the beginning of each record to check their viability. Data are the mean ± SEM, n corresponds to the number of observations. Multiple comparisons between responses to agonists were performed using the Student-Newman–Keuls test after one-way ANOVA (significance level: 0.05). After 1 day in culture, DRG neurones had a rounded aspect and showed some processes. The average basal level of [Ca2+]i was 37.2 ± 0.6 nM (n = 213). Application of substance P elicited a transient rise in [Ca2+]i in 45% (17/ 38) of neurones tested. A typical SP response presented a rapid rise from basal level to a mean peak value of 146.8 ± 10.0 nM (n = 17) followed by a slow (20–25 s) decrease back to the resting free calcium concentration (Fig. 1). The average [Ca2+]i variation from basal level to peak value was 106.6 ± 10.0 nM. On a second application of the same agonist, after a delay ranging from 5 to 30 min, only about 27% (6/22) of the initially responsive neurones showed a calcium rise. The effects of specific agonists and antagonists for the three

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Fig. 3. (A) Histogram of somatic diameters of DRG neurones after 24 h in culture (n = 626). Cells were tentatively grouped in two categories according to their size: small (,20 mm) and large (.20 mm). Solid lines show the corresponding sum of two gaussian curves. (B) Stacked bar chart of cell body size for neurones responding to tachykinins. All tachykinin-responsive neurones had a cell diameter smaller than 16 mm.

neurokinin receptor subtypes are summarised in Fig. 2. None of the antagonists used had any effect by itself on [Ca2+]i. Desensitisation prevented us from comparing the effect of each agonist in the presence or absence of an antagonist successively on the same neurone. We checked that the response to an agonist of one receptor type was not blocked by an antagonist of the other types. The NK1 receptor agonist [Sar9, Met(O2)11]SP induced an increase in [Ca2+]i (123.3 ± 10.0 nM, n = 21) in 40% (21/ 52) of neurones. In the presence of 10 nM SR 140333A, a selective NK1 receptor antagonist [15], we never observed any [Ca2+]i response to the selective agonist (0/22). No response to a second application of the agonist could ever be observed after removal of the antagonist. About 27% (12/45) of cells were responsive to the NK2 receptor agonist, [Ala5, b-Ala8]-a-neurokinin fragment 4– 10. The induced change in calcium was 60.6 ± 8.0 nM. SR 48968A, a specific NK2 receptor antagonist [4], had no blocking effect at 10 or 100 nM. In the presence of 1 mM SR 48968A, we still observed a calcium variation of 47.6 ± 6.6 nM in 33% (8/24) of cells tested. This response was about 21% smaller than without the antagonist, but the decrease was not statistically significant. Application of the selective NK3-receptor agonist senktide increased intracellular calcium by 102.0 ± 8.7 nM in 37% (20/54) of cells tested. In the presence of 1 mM of the NK3 antagonist SR 142801A [3], no calcium variation was observed (0/18). After 10 min wash, two cells showed a small calcium response to the NK3 agonist (24.5 ± 3.3 nM). No significant difference was observed in the percentage of cells responsive to each agonist (Fig. 2A; Chi square test, P = 0.67). The overall percentage of responsive cells was about 37%. Similarly, the amplitude of the response was not significantly different when evoked by either SP, the NK1 or the NK3 agonist (Fig. 2B). At the concentrations used, the calcium rise induced by the NK2 agonist was significantly smaller by about 50%, whether in the absence or in the presence of the antagonist. Thus the NK2 antagonist used did not seem to be efficient on our preparation: the proportion of neurones responding to the NK2 agonist was the same in the presence or absence of the NK2 antagonist; it

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was apparently not affected either by the NK1 (4/13 cells) or the NK3 antagonist (3/8 cells). Our results suggest that SP-responsive DRG neurones express the three different classes of tachykinin receptor subtypes. Each subtype may be expressed by a distinct subpopulation of neurones, or the different subtypes may coexist on each DRG neurone. The latter hypothesis would account for the fact that roughly the same proportion of cells responded to either agonist. We tested this by applying two different selective agonists consecutively on the same neurones. Since distinct receptors are involved, desensitisation of the first receptor stimulated should not interfere with the second one. We performed such experiments on a few neurones only, but colocalised receptors were indeed observed. In a population of NK1-responsive neurones 1/4 responded to NK2 and 0/4 to NK3. For NK2-responsive neurones, 1/3 responded to NK1 and 1/7 to NK3. For NK3-responsive neurones, 0/3 responded to NK1 and 1/5 to NK2. The same neurone can thus co-express the different tachykinin receptor subtypes. However this colocalisation seems to be sparse and cannot be easily quantified with the present experimental approach. An immunocytochemical study using antibodies directed against the various receptor subtypes would be better suited to confirm this point. DRG neurones can belong to various subpopulations. An in vivo study has shown that several populations of sensory neurones could be distinguished according to their soma size [10], and it has been demonstrated that the smaller neurones are those involved in the nociceptive pathway [11,12]. We measured neuronal diameters in our cultures in order to identify which subpopulation possesses tachykinin receptors. The size distribution of DRG cells after 1 day in culture (Fig. 3A) suggested the presence of a small group (18%) of large cells (diameter .20 mm) distinct from a large population (82%) of small cells (diameter ,20 mm). However the histogram of cell sizes was not significantly better fitted with a sum of two gaussian curves than with a single one. We therefore could not demonstrate the existence of two subpopulations of cells based on their somatic diameter

Fig. 4. DRG neurones responding to tachykinins (here the NK2 agonist [Ala5, b-Ala8]-a-neurokinin fragment 4–10) also responded to a bath application (1–2 s) of 1 mM capsaicin by a large long-lasting increase in [Ca2+]i.

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in our in vitro model. We also determined the somatic diameter of the neurones found to respond to tachykinins (Fig. 3B). All SP-, NK1-, NK2- and NK3-responsive cells had a small somatic diameter, none was larger than 15 mm. Since neurone subpopulations could not be reliably identified according to body size, we characterised nociceptive neurones by their response to capsaicin. Capsaicin, the pungent ingredient in peppers of the Capsicum family, has been shown to act specifically on a subpopulation of nociceptive primary afferent sensory neurones, where it opens cationselective ion channels and induces a large calcium rise [1]. We thus assayed capsaicin on neurones responding to tachykinins. A brief application (1–2 s) of 1 mM capsaicin induced a transient rise in [Ca2+]i in almost all the tachykinin-responsive neurones tested (23/24 cells). This [Ca2+]i elevation was large (≥500 nM) and lasted for several minutes (20–25 min; Fig. 4). According to this last criterion, i.e. a capsaicin-induced [Ca2+]i rise, our results suggest that neonatal DRG neurones expressing functional tachykinin receptors belong to the nociceptive sensory neurones. It was recently shown that capsaicin sensitivity is mostly found in the smaller cells [6], which correlates with our observations on tachykinin sensitivity. Our pharmacological analysis thus showed that all three known tachykinin receptor subtypes (NK1, NK2 and NK3) are present in rat DRG and can be found on the same neurones. Tachykinin-responsive neurones in our cultures were also capsaicin-responsive, which strongly suggests that they are involved in the nociceptive pathway. In our in vitro model the putative receptors are expressed on the soma, and we have no indication of the possible expression pattern in vivo. However other studies provided evidence for the existence of functional tachykinin receptors in vivo. The recent work of Hu et al. [8] suggests that these receptors may be autoreceptors, since a large fraction of the capsaicinsensitive cells synthesise SP. Stimulation of such SP autoreceptors on primary nociceptive sensory neurones could lower their activation threshold and thus be involved in hyperalgesia.

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This paper is dedicated to the memory of Paul Feltz, deceased on January 24th, 1996, who initiated this study. We wish to thank SANOFI (France) for the gracious gift of antagonists, and Drs. R. Schlichter and J. Trouslard for their helpful advises and critical reading of the manuscript.

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