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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Oct 15, 2004; 560(Pt 2): 391–401.
Published online Aug 5, 2004. doi:  10.1113/jphysiol.2004.067462
PMCID: PMC1665249

Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF

Abstract

Bradykinin (BK) has long been recognized as an important mediator of pain and inflammation. In normal tissue bradykinin causes an acute sensation of pain by an action at B2 receptors, but in inflamed tissue the pharmacology of the response changes to that of B1 receptors. Attempts to demonstrate the presence of functional B1 receptors in sensory neurones have failed, however, and the actions of B1 agonists have therefore been presumed to be indirect. Here we show that specific B1 receptor activation causes translocation of the epsilon isoform of protein kinase C (PKC-epsilon) to the membrane of a small fraction of freshly isolated sensory neurones from rats and mice. The proportion of neurones in which PKC-epsilon translocation was observed increased to around 20% of neurones after 3 days in culture with the neurotrophins glial cell line derived neurotrophic factor (GDNF) and neurturin, but not with nerve growth factor (NGF). Using in situ hybridization we found that the proportion of neurones expressing B1 mRNA increased from close to zero to 20.4% after 8 h culture in GDNF. Neurones expressing functional B1 receptors were negative for the neuropeptides CGRP and substance P, but most expressed functional TRPV1 receptors for capsaicin (60%) and bound the lectin IB4 (68%), both markers characteristic of nociceptors. B1 activation enhanced the heat-activated membrane current ~3-fold, and the enhancement was much more prolonged than was the case with B2 activation, consistent with a role for B1 receptors in sustained pain. We conclude that GDNF and neurturin potently upregulate functional B1 receptor expression in small non-peptidergic nociceptive neurones.

Bradykinin (BK) is a small proinflammatory peptide released during tissue damage and inflammation by proteolytic cleavage of precursor kininogens (Regoli & Barabe, 1980). The C terminal arginine of BK is removed in vivo by carboxypeptidases to generate des-Arg9-BK, which is also physiologically active. Two receptors for bradykinin have been characterized: a B2 receptor, for which BK is the normal physiological agonist, although des-Arg9-BK can also activate B2 receptors with much lower affinity; and a B1 receptor for which the reverse is true (Regoli & Barabe, 1980; Marceau et al. 1998). B2 receptors are expressed in many tissues, while B1 receptors are in general not constitutively expressed, but their expression can be rapidly upregulated following tissue damage (Marceau et al. 1998; Regoli et al. 2001).

Bradykinin causes an acute sensation of pain in vivo by an action at B2 receptors expressed in the surface membranes of nociceptive neurones (Steranka et al. 1988; Burgess et al. 1989; Dray et al. 1992; McGuirk & Dolphin, 1992; Dray & Perkins, 1993; Lee et al. 2002). In normal tissue injection of B1 agonists is without effect, but following injury or inflammation B1 agonists become effective in producing pain (Perkins & Kelly, 1993; Davis & Perkins, 1994; Davis et al. 1994; Rupniak et al. 1997; Belichard et al. 2000), consistent with an upregulation of B1 receptor expression. B1 receptor mRNA and protein appear to be expressed in nociceptive neurones (Seabrook et al. 1997; Petersen et al. 1998; Levy & Zochodne, 2000; Wotherspoon & Winter, 2000), and expression is upregulated following inflammation (Fox et al. 2003), but attempts to demonstrate the presence of functional B1 receptors in sensory neurones have failed (Davis et al. 1996; Seabrook et al. 1997). The actions of B1 agonists have therefore been presumed to be mediated by an indirect action on B1 receptors on non-neuronal cells, followed by release of an unidentified mediator responsible for activating nociceptors (Davis et al. 1996; Seabrook et al. 1997).

In the present study we re-investigated the possible presence of functional B1 receptors in sensory neurones. One convenient index of B2 receptor activation is translocation of the epsilon isoform of PKC to the cell membrane (Cesare et al. 1999), and we established in initial experiments that specific B1 activation also causes translocation of PKC-epsilon in sensory neurones. Using this index we found that functional B1 receptors are expressed in only a small percentage of freshly isolated dorsal root ganglion (DRG) neurones, but that expression is upregulated by exposure to the related neurotrophins GDNF and neurturin, while NGF is inactive. In situ hybridization confirmed that B1 mRNA is rapidly upregulated by GDNF but not by NGF. Consistent with this, the B1 receptor expression following GDNF treatment is found exclusively in the non-peptidergic population of DRG neurones which express the Ret receptor for GDNF (Snider & McMahon, 1998). A functional consequence of B1 activation is that the heat-gated ion current is sensitized, and the effect is much more prolonged than for B2 activation, consistent with a more important role for B1 receptors in long-term inflammatory pain.

Methods

Cell culture, electrophysiology, Ca2+ imaging

Experiments were performed on isolated DRG neurones taken from neonatal mice (B2−/−, postnatal day 1–5) or rats (Wistar, postnatal day 2–7), killed by cervical dislocation followed by decapitation. All animal procedures were carried out in accordance with UK Home Office guidelines. DRG neurones were isolated and cultured on polylysine- and laminin-coated glass coverslips as previously described (Cesare & McNaughton, 1996; Vellani et al. 2001), in the presence of NGF (100 ng ml−1, Promega), and where appropriate also GDNF or neurturin (50 ng ml−1, PeproTech). For calcium imaging cells were loaded with the acetoxymethyl (AM) ester of the calcium-sensitive dye Fluo-4 (Molecular Probes; 10 μm for 10 min at room temperature) and imaged using a ×20 high-NA lens and a Micro-Radiance inverted confocal microscope (Bio-Rad). Images were typically acquired at 3 s intervals, and the calcium signal from up to 150 individual neurones could be tracked simultaneously. For whole-cell patch clamp experiments, recordings were made from small-diameter neurones (15–20 μm) using an Axopatch amplifier and pCLAMP software (Axon Instruments Inc.). Heat stimuli were applied using a multibarrel rapid-change system in which the temperature in the heated barrel was maintained constant using a Peltier device under feedback control; final monitoring of the temperature was done with a miniature thermocouple located in the outlet tube within 8 mm of the preparation. Temperature changes were complete within 20–50 ms. See Vellani et al. (2001) for further details.

Immunocytochemistry

DRG neurones on coverslips were exposed to agonists and antagonists, usually for 30 s (indicated in the text), then rapidly fixed by immersion in 4% formaldehyde (w/v) plus 4% sucrose (w/v) in 50% phosphate-buffered saline (PBS; Gibco) for 5 min at room temperature, and permeabilized with 0.1% Triton in PBS (5 min). Fish skin gelatin (Sigma, 0.5% w/v) was used throughout to reduce non-specific antibody binding. Fixed neurones were exposed to primary antibodies specific for PKC isoforms (as described in Cesare et al. 1999) for 2 h, washed and stained with Alexa 488-labelled secondary antibodies (goat anti-rabbit or donkey anti-rabbit, from Molecular Probes). Double labelling was performed with goat anti-substance P or anti-CGRP polyclonal (Santa Cruz) or anti-parvalbumin mouse monoclonal antibodies (Sigma) and visualized with appropriate secondary antibodies labelled with Alexa 594 (Molecular Probes). Isolectin B4 (IB4) conjugated to Alexa 594 (Molecular Probes; 1 μg ml−1 for 10 min in PBS) was used to visualize IB4 binding. In the results of immunocytochemistry experiments we give the number n of separate experiments, in each of which neurones were obtained from a different animal on a different experimental day. At least two coverslips were counted per experiment, with at least 350 neurones per coverslip for all results shown in this paper.

Cell sorting

IB4-positive (IB4+) neurones were isolated using a fluoresence-activated cell sorter (FACS; MoFlo cytometer, Dako Cytomation). DRG suspensions prepared as for cultures were centrifuged and resuspended at a density of about 1 million per millilitre in Dulbecco's modified Eagle's medium (DMEM) containing 10% serum, passed through a cell strainer (40 μm), stained with isolectin IB4 coupled to Alexa 488 (100 ng ml−1 for 10 min, Molecular Probes) and sorted with the sorting window set to select IB4+ neurones. A plot of pulse width versus fluorescence was used to exclude doublets.

In situ hybridization

To detect bradykinin B1 mRNA, cRNA anti-sense and sense probes were transcribed from a plasmid containing full length mouse B1 cDNA (Hess et al. 1996), concurrently 35S-UTP-labelled and cleaved with alkaline hydrolysis to an average length of 200 bp (see Lee et al. 2002). Rat DRG neurones were isolated using the methods described above and were allowed to attach to coverslips for 2 h. Once attached (Time zero in Fig. 2B) the medium was exchanged for one containing either NGF (100 ng ml−1) or NGF plus GDNF (50 ng ml−1). After incubation at 37°C for 4 h or 8 h cells were fixed in 4% formaldehyde, 5% acetic acid and 0.9% NaCl for 30 min, washed with PBS, incubated in 70% ethanol at 4°C for 24 h and dehydrated in 90 and 100% ethanol before removal of lipids in xylene for 5 min. After rehydration the cells were deproteinated for 15 min in 0.1 m HCl and acetylated in 0.25% acetic anhydride in a 0.1 m triethanolamine solution for 20 min. The coverslips were then incubated at 55°C for 16 h with 100 μl hybridization buffer per coverslip containing 52% formamide w/v, 10% dextran sulphate w/v, 210 mm NaCl, 10 mm dithiothreitol (DTT), 10 mm Tris-HCl pH 7.6, 1 × Denhardt's solution, 0.5 mg yeast tRNA and 50 × 106 c.p.m. probe. After hybridization, coverslips were treated with RNAse A (10 μg ml−1) at 37°C for 30 min and washed in 2 × saline sodium citrate (SSC; 0.15 m sodium chloride, 0.015 m trisodium citrate pH 7.0) and 1 mm DTT for 10 min at 48°C, 0.5 × SSC and 1 mm DTT for 10 min, 0.1 × SSC and 1 mm DTT for 30 min at 70°C and 0.1 × SSC and 1 mm DTT for 2 min at room temperature. Cells were dehydrated in a graded series of ethanol and the coverslips glued cell-side uppermost onto microscope slides with cyanoacrylate glue. The slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1 : 1, exposed for 10 weeks, developed and fixed at 12°C and counterstained with cresyl violet. Silver grains were visualized and images acquired using dark-field illumination at ×40 magnification (Nikon Eclipse E400 equipped with a Nikon Coolpix camera). Cell type and dimensions could be clearly identified from epi-illumination images acquired using a DAPI/FITC/Texas red triple wavelength block (see Fig. 2A). Digitized dark-field and epi-illumination images were transferred to a PC, neurones were identified in the epi-illumination image, outlined using Bio-Rad LaserPix software and mean neuronal diameter calculated. These outlines were then transferred to the dark-field image (see Fig. 2A) and a numerical value of mean scattered light intensity per unit area was calculated. The dark-field illumination was kept low to ensure that the camera output remained within the linear range, so the measured value of scattered light intensity was proportional to silver grain density. No hybridization signal was obtained using sense probes (data not shown).

Figure 2
Effect of NGF and GDNF on expression of B1 mRNA in DRG neurones

Results

B1 activation causes specific translocation of PKC-epsilon

We first sought to establish a means by which the expression of functional B1 receptors could be quantified. Several possibilities have been suggested by previous work on the homologous B2 receptors. Following B2 activation with bradykinin (BK), the epsilon isoform of protein kinase C (PKC-epsilon) is translocated from a cytoplasmic location to the neuronal plasma membrane (Cesare et al. 1999); an inward membrane current is induced and calcium released from intracellular stores (Baccaglini & Hogan, 1983; Burgess et al. 1989; Bleakman et al. 1990); and the membrane current activated by a heat stimulus is enhanced (Cesare & McNaughton, 1996; Vellani et al. 2001). We examined whether any of these indices correlated with B1 receptor activation in nociceptive neurones.

In initial experiments we activated B1 receptors without activating B2 receptors by applying the B1 agonist des-Arg9-BK to sensory neurones isolated from B2−/− mice (Borkowski et al. 1995). We found PKC-epsilon translocation in a small but significant proportion of DRG neurones from B2−/− mice (see Fig. 1A), while no translocation was observed in any neurone in the absence of the B1 agonist. PKC-epsilon translocation was observed in 2.4 ± 0.5% of neurones from B2−/− mice after 1 day of culture in NGF (mean ± s.e.m., n = 3 separate experiments using neurones from different animals; at least 2 coverslips were counted per experiment and at least 350 neurones counted per coverslip for all results shown in this paper). Similar results were obtained in experiments on rat neurones (Fig. 1C), in which specific B1 activation was achieved by applying des-Arg9-BK in the presence of the potent and specific B2 blocker HOE140 (Hock et al. 1991). We observed PKC-epsilon translocation in 2.8 ± 0.6% of freshly isolated rat neurones (n = 4), a proportion similar to that observed in experiments on B2−/− neurones from mouse. When neurones were cultured in the presence of NGF the proportion of neurones in which PKC-epsilon translocation was seen declined steadily, suggesting that some factor necessary to maintain B1 expression was missing from the NGF-containing culture medium (see Fig. 1B and D), even though substantial upregulation of B2 receptor expression was observed in the same medium (Lee et al. 2002).

Figure 1
B1 receptor activation causes PKC-epsilon translocation in dorsal root ganglion (DRG) neurones

We used the absence of PKC-epsilon translocation following B1 activation after 6 days in culture to check the effectiveness of HOE140 in blocking responses to B2 activation. When B2 receptors in rat neurones were activated with BK in the absence of HOE140, PKC-epsilon translocation was observed in 32 ± 2% (n = 13) of neurones after 6 days of culture in NGF, so the observation that no translocation was seen in the presence of HOE140 shows that block of B2 receptors was complete.

We examined whether other PKC isoforms are also translocated by B1 activation. In agreement with Cesare et al. (1999) we found PKC-βI and PKC-βII were already membrane-localized in small DRG neurones, and no change was seen following B1 activation. The locations of PKC-δ and PKC-ζ, which are cytoplasmic, were unaffected by B1 activation (see Fig. 1A for PKC-δ), results which are similar to those obtained with B2 activation (Cesare et al. 1999).

We next investigated the possibility that calcium release or induction of an inward current might correlate with B1 activation, as they do with B2 activation. We observed no detectable calcium release in response to B1 activation in any freshly isolated neurone, however, although PKC-epsilon translocation was observed in a small but significant fraction of these neurones (see above). We therefore attempted to enhance B1 expression using methods which have been successful in other preparations. Exposure to interleukin-1β (IL1β) enhances the responses to B1 agonists in non-neuronal preparations (Marceau et al. 1998; Ni et al. 1998; Zhou et al. 1998; Phagoo et al. 2000), but no detectable calcium signal was elicited in B2−/− mouse neurones by B1 activation following 3 days of exposure to IL1β (50 ng ml−1, calcium signal ΔFFmax < 0.05, 240 neurones, where F is fluorescence). NGF increases the expression of B2 receptors in DRG neurones (Von Banchet et al. 1996; Lee et al. 2002), and might have a similar effect on B1 receptor expression, but no calcium signal in response to B1 activation was seen after 3 days of culture of B2−/− mouse neurones in NGF (100 ng ml−1, 290 neurones). We show below that GDNF enhances the expression of B1 receptors, but even after culture for 3 days in GDNF (50 ng ml−1), when other measures showed robust B1 expression, we found no detectable calcium release in response to B1 activation in either B2−/− mouse neurones (40 neurones) or in rat neurones (120 neurones). Many of the same neurones in which B1 activation failed to generate a calcium signal were subsequently found to generate large calcium increases in response to B2 activation with bradykinin or in response to activation of protease-activated receptor 1 (PAR1) receptors by the use of a PAR1 agonist peptide (Schmidlin & Bunnett, 2001). A calcium-dependent fluorescence increase greater than 5% of the maximum (where the maximum was determined at the end of the experiment by exposure to ionomycin) was not seen in any neurone (312 neurones) in response to B1 activation, but was subsequently observed in 25% of the same neurones in response to B2 activation, and in 20.5% in response to PAR1 activation. There was therefore no defect in the release of calcium from internal stores in these neurones. We also examined the possibility that B1 activation, like B2 activation, might elicit an inward membrane current, but in rat neurones no inward current was seen following culture for 3 days in GDNF (< 2 pA, 18 neurones), while obvious inward currents were produced by B2 agonists in neurones from the same cultures (see also Cesare & McNaughton, 1996; Vellani et al. 2001). These experiments confirm earlier results in which no effect of B1 activation on [Ca2+]i was seen (Davis et al. 1996) and suggest that an increase in [Ca2+]i or direct activation of inward membrane current are not useful indicators of functional B1 expression in DRG neurones. A possible explanation for the failure of B1 activation to release calcium in significant quantities from internal stores, while still being able to cause PKC-epsilon translocation, is that the rate of IP3 production may be low, and because IP3 is rapidly degraded, its concentration never reaches the threshold for activation of IP3 receptors. DAG is not rapidly degraded, and as B1 activation is sustained compared to B2 activity (see Fig. 5) the concentration of DAG therefore builds to levels sufficient to activate PKC-epsilon.

Figure 5
B1 receptor activation enhances the heat-gated membrane current

B1 expression is upregulated by GDNF

When neurones were cultured in the presence of GDNF the proportion in which specific B1 activation caused PKC-epsilon translocation increased markedly, to 23.2 ± 1.2% after 3 days in culture in B2−/− mouse neurones (n = 4) and to 18.9 ± 1.3% (n = 9) in rat neurones (see Fig. 1B and D). The neurotrophin neurturin, which is a member of the same family as GDNF, had similar effects (23.3 ± 1.2% showing PKC-epsilon translocation after 3 days in rat, n = 3, results not shown). Translocation was an all-or-none effect, with only background levels of fluorescence observed in the cytoplasm in most responding neurones after 30 s application of agonist (see Fig. 1A and C). The only difference in PKC-epsilon translocation between neurones cultured in the presence and absence of GDNF was in the number of responding neurones, with the degree of translocation in responding neurones unaffected by addition of the neurotrophin. Partial translocation, with some fluorescence above background remaining in the cytoplasm, was observed with short exposures to the B1 agonist (5 s or less) but not for exposures of 30 s as used in the majority of experiments.

An increase in the proportion of B1-responsive neurones could be caused by selective cell death in the non-responsive population (see Molliver et al. 1997), but quantitative arguments show that this cannot explain the magnitude of the increase. By counting cells in defined areas of neuronal cultures we found that 69% of rat neurones survived to 3 days and 38% to 6 days. In the extreme case that all B1-expressing neurones had survived in the presence of GDNF, and that cell death had occurred only amongst the non-B1-expressing population, then the 2% of B1-positive neurones seen in freshly isolated cells would increase to 3% after 3 days and 5% after 6 days – an order of magnitude less than the observed effect. A second argument originates from the finding that B1-responsive neurones were found in the population of neurones that bound the lectin IB4 (see below). Figure 1F shows that the proportion of IB4+ neurones present in cultures with NGF alone did not change by more than 10% over 3 days (open symbols), showing that these neurones did not die more rapidly than IB4-negative neurones. There was a small but significant effect of GDNF on the survival of the IB4+ population (compare open and filled symbols in Fig. 1F) but the effect was too small by an order of magnitude to explain the increase in B1-expressing neurones.

We next used 35S-labelled riboprobes and in situ hybridization to measure changes in B1 mRNA expression in cultured neurones from rat DRG isolated under identical conditions to those used for experiments on functional B1 expression. Neurones are readily distinguished from non-neuronal cells in epi-illumination images (right-hand panels in Fig. 2A). Identified neuronal cell bodies were outlined in the epi-illumination image, the outlines were transferred to the dark-field images of silver grains (left-hand panels in Fig. 2A) and the intensity of scattered light per unit area (proportional to grain density per unit area, see Methods) was calculated. Histograms of intensity are shown in Fig. 2B. A low intensity, but still significantly above background, was seen over neurones fixed immediately after isolation (‘time zero’ in Fig. 2B) and was unchanged after culture for 4 and 8 h in NGF alone (Fig. 2B and upper left image in Fig. 2A). Because this label was relatively uniformly spread across the whole neuronal population, while only a minority of neurones in the same conditions express functional B1 receptors (see Fig. 1), we believe this label to be non-specific. We do not believe this non-specific signal to be simply background, however, because no signal was observed over any non-neuronal cell under any conditions (see Fig. 2A), and the most likely origin is a partial match between the B1 probe and an unidentified non-B1 mRNA sequence present in all DRG neurones. The absence of signal over non-neuronal cells shows that B1 mRNA is not present in non-neuronal cells under the any of the conditions of these experiments.

Following culture in GDNF a clear increase in silver grain density was seen over a subset of small neurones (lower left image in Fig. 2A). This increase in B1-specific labelling was quantified by measuring the signal intensity per unit area over a large number of neurones cultured with and without GDNF (open and filled columns, respectively, in Fig. 2B). Culture in NGF alone for 4 h or 8 h had no significant effect on the distribution of signal intensity when compared to that in freshly isolated neurones (filled columns in Fig. 2B). In the presence of GDNF, however, a progressive and significant increase in B1-specific label was observed in a subpopulation of neurones (open columns; the difference between the two distributions shown in Fig. 2B at both 4 h and 8 h was highly significant, t test, P < 10−9). A criterion level for distinguishing between B1-positive and B1-negative neurones was set so as to exclude 99% of freshly isolated neurones (arrows in Fig. 2B), on the assumption (see above) that the signal in these neurones did not reflect the presence of B1 mRNA. Using this significance level 19.5% of neurones were found to be B1-positive after 4 h culture in GDNF, and 20.4% positive after 8 h. Following culture in NGF alone the corresponding figures were 2.5% and 3.4%, neither of which was significantly different from the 1% level of ‘false positives’ expected on the basis of the criterion level adopted. The B1-positive neurones had a significantly smaller diameter than the B1-negative population (17.1 ± 0.9 μm versus 19.7 ± 0.4 μm, significantly different, P = 0.002). Similar results were obtained in four separate experiments.

Other possible mediators causing PKC-epsilon translocation

Davis et al. (1996) found no evidence for functional B1 receptor expression in DRG neurones, and suggested that the effects of B1 agonists in vivo must be due to an action of B1 agonists on B1 receptors located on non-neuronal cells, followed by release of an unidentified extracellular messenger which then acts on sensory neurones. A similar explanation could account for the translocation of PKC-epsilon resulting from B1 activation, because non-neuronal cells present in the cultures used in the present study could express B1 receptors, although there was in fact no evidence for this in the in situ hybridization experiments described above. We investigated the possibility of cross-talk from non-neuronal cells in two ways: by removing contaminating cells with a cell sorter, and by investigating the effects of possible extracellular messengers.

Sensory neurones were labelled with fluorescently tagged IB4, which binds to the subset of sensory neurones in which upregulation of B1 receptors is observed (see below). These neurones were isolated in a FACS and cultured in the presence of GDNF (see Methods). Glial cells were almost completely eliminated by this procedure, and neurones were selected for analysis that were clearly not in contact with the few remaining glial cells. PKC-epsilon translocation was still observed in response to B1 activation in a subset of these neurones (Fig. 1C).

Arachidonic acid (AA) is known to be liberated by B2 receptor activation (Gammon et al. 1989), and is therefore a possible candidate for an extracellular messenger liberated in response to B1 activation. We found, however, that AA (1–50 μm applied for 0.5–5 min) did not induce PKC-epsilon translocation in any neurone (n = 8, not shown), showing that AA cannot be an extracellular messenger mediating the B1 actions on PKC-epsilon. These experiments also argue against the possibility that PGE2 is the extracellular messenger, with AA liberated by B1 activation providing the substrate for the release of PGE2. PGE2 itself does in fact cause PKC-epsilon translocation (see below), consistent with a possible role as extracellular messenger, but the absence of any translocation following the application of even high concentrations of AA argues that any possible AA liberation by B1 activation cannot result in the release of PGE2 in quantities sufficient to cause PKC-epsilon translocation.

Populations of neurones expressing B1 and B2 receptors

BK, which activates B2 receptors relatively specifically, caused PKC-epsilon translocation in 32 ± 2% of neurones cultured for 3 days in the presence of NGF (Fig. 3). GDNF upregulated B1 expression, as can be seen from the substantial proportion of neurones activated by des-Arg9-BK in the presence of HOE140 following addition of GDNF to the culture medium, but did not increase the proportion of neurones activated by BK (Fig. 3). When both B1 and B2 receptors were activated together the combined effect was no greater than with B2 activation alone. This experiment shows that B1 expression is upregulated by GDNF in a subset of those neurones which already express B2 receptors.

Figure 3
Subpopulations of neurones activated by B1 and B2 agonists and by PGE2

We also examined the action of PGE2 in translocating PKC-epsilon. The hyperalgesic action of PGE2 is usually attributed to PKA activation (England et al. 1996) but an action via PKC has also been suggested (Gold et al. 1998). We found that PGE2 potently translocates PKC-epsilon in 19 ± 3% of neurones (Fig. 3), in support of the proposal that the hyperalgesic actions of PGE2 are at least partly mediated by PKC. The population of neurones in which PKC-epsilon was translocated by PGE2 was, however, entirely distinct from that expressing B2 receptors (and from the arguments above, also B1 receptors) because the combined effect of BK plus PGE2 was not significantly different from the sum of each individually (56 ± 2% of neurones activated, see Fig. 3).

In combined functional and immunocytochemical experiments we examined the populations of neurones expressing B1 receptors, TRPV1 receptors for capsaicin (formerly named VR1, see Caterina et al. 1997) and binding sites for the lectin IB4 (Fig. 4A). Calcium imaging was first used to identify neurones expressing functional TRPV1 receptors, from the calcium increase observed following capsaicin application. Neurones were then exposed to des-Arg9-BK to activate B1 receptors, fixed, and then PKC-epsilon translocation was determined as above, with IB4 binding also determined in the same cells. In these experiments 60.2 ± 5.3% of neurones expressing functional B1 receptors were also found to express functional TRPV1, and 68 ± 6% were IB4 positive (n = 4).

Figure 4
Populations of neurones expressing B1 receptors

Colocalization of functional B1 receptors with the neuropeptides CGRP and substance P was also examined. Expression of functional B1 receptors was low in neurones expressing CGRP (1.8%, Fig. 4B, n = 2) and absent in neurones expressing substance P (SP, Fig. 4B, n = 4). We also examined colocalization with parvalbumin, which is expressed in neurones projecting to muscle spindles (Copray et al. 1994). No colocalization of functional B1 receptors with parvalbumin was observed in any neurone (not shown).

B1 activation enhances the heat-gated membrane current

We used rat neurones cultured in the presence of GDNF to investigate the effect of B1 receptor activation on the membrane current activated by heat. An inward membrane current was generated in around half of these neurones in response to a suprathreshold pulse of heat (Fig. 5A). B1 activation caused an increase in the amplitude of the heat-gated current, in around half of the heat-sensitive neurones. The mean increase was 295 ± 57% (mean ± s.e.m., n = 9; see Fig. 5B), an increase which is similar to, but somewhat larger than, the 209 ± 19% increase observed in response to B2 receptor activation (Cesare & McNaughton, 1996). A major difference between the two, though, is that B1 activation caused a sustained enhancement of the heat-gated current, unlike B2 activation, which causes only a transient enhancement (Cesare & McNaughton, 1996). B1 receptor activation is therefore more likely to be able to mediate the prolonged sensitization to heat stimuli which is observed in inflammatory conditions (Perkins & Kelly, 1993).

Discussion

In previous studies we showed that the activation of B2 receptors by BK specifically translocates PKC-epsilon, and that this action underlies the sensitization by BK of the nociceptor heat response (Cesare & McNaughton, 1996; Cesare et al. 1999). In the present study we show that B1 activation has similar effects in causing PKC-epsilon translocation (Fig. 1) and increasing the heat-gated ion current (Fig. 5). Translocation of PKC-epsilon is a particularly useful index of functional B1 receptor expression, as it can be measured in a large number of cells, and can readily be combined with a number of other histological indices of neuronal type. Using this index we find that functional B1 receptors are expressed in only ~2% of freshly isolated sensory neurones, a proportion which declines to zero in the presence of NGF alone. In the presence of GDNF or the related neurotrophin neurturin, however, functional B1 expression is rapidly upregulated in approximately 20% of the total neuronal population. It seems likely that the low level of B1 expression in freshly isolated neurones results from a low level of exposure to GDNF in vivo, and that expression declines to zero because of GDNF withdrawal when neurones are cultured in NGF alone. It is perhaps surprising that the trauma of dissection and dissociation of these neurones alone was not sufficient to cause upregulation of the B1 receptor. However, we would propose that B1 upregulation is caused not by processes intrinsic to the neurone but by the release of GDNF from surrounding tissue following injury or inflammation. During dissociation the surrounding tissue is removed and pro-inflammatory factors are rapidly washed away.

An upregulation of B1 mRNA expression by GDNF was confirmed using in situ hybridization, which showed that the level of B1 mRNA was enhanced in ca 20% of the neuronal population following 4–8 h exposure to GDNF, a proportion which agrees well with the proportion of neurones expressing functional B1 receptors after 3 days in GDNF. While an upregulation of B1 mRNA expression does not of itself prove that receptor expression is increased by GDNF, when taken together with the functional data demonstrating an increase in the functional response to a selective B1 agonist, it represents an important supporting argument for the upregulation of functional B1 protein by GDNF.

The neuronal population which expresses B1 receptors following exposure to GDNF has some interesting characteristics. The B1-expressing population is a subset of the B2-expressing population, as combined activation of B1 and B2 receptors activates no more neurones than B2 activation alone. The majority express TRPV1 (60%) and bind the lectin IB4 (68%), as would be expected for nociceptors. Strikingly, almost all are non-peptidergic, and as the non-peptidergic population comprises approximately half of the total, B1 receptors are expressed in ~40% of non-peptidergic neurones following exposure to GDNF. The observation that B1 expression is confined to the non-peptidergic population is consistent with our finding that B1 expression is upregulated by GDNF and neurturin but not by NGF, because TrkA receptors for NGF are expressed only in the peptidergic population, while Ret receptors for GDNF and neurturin are expressed in the non-peptidergic population (Snider & McMahon, 1998).

This study has demonstrated a possible basis for the known shift in responsiveness of nociceptive neurones from a B2 pharmacology in the normal state to a B1 pharmacology in chronic inflammation. We show here that exposure to GDNF upregulates B1 expression, in much the same way as NGF upregulates the expression of B2 receptors (Lee et al. 2002), neuropeptides (Lindsay & Harmar, 1989; Verge et al. 1995), TRPV1 (Michael & Priestley, 1999) and NaV1.8 (Waxman et al. 1999). Direct evidence for a release of GDNF by non-neuronal tissues following damage has not to the best of our knowledge been obtained, but both GDNF and NGF have been shown to be released following nerve damage, in which activated macrophages invading the degenerating nerve secrete neurotrophins (Trupp et al. 1995; Naveilhan et al. 1997). GDNF or related neurotrophins are therefore likely candidates to drive upregulation of B1 expression following injury or inflammation, leading to a switch in agonist responsiveness from a B2 to a B1 profile. We propose that the small number of B1-expressing neurones in freshly isolated preparations (see Fig. 1B and D) reflects a low level of supply of GDNF available to nociceptive axons in normal animals, but that an increase in the supply of GDNF following injury or inflammation drives upregulation of B1 expression and consequently contributes to long-term maintenance of hyperalgesia. The B1 receptor may play a more important role in chronic inflammatory pain than the B2 receptor, because the enhancement of the membrane current activated by a heat stimulus caused by B2 activation is only transient (Cesare & McNaughton, 1996), while B1 activation induces a sustained enhancement of the heat response (see Fig. 5), which is more in keeping with the sustained hyperalgesia observed during chronic inflammation.

Acknowledgments

We thank Drs G. Seabrook and T. O'Neill for arranging the supply of B2−/− mice, Dr Nigel Miller for assistance with cell sorting, and Drs H. Cadiou, S. Honan and X. Zhang for helpful comments. This work was supported by the Wellcome Trust and by Fondazione Cassa di Risparmio di Modena and Fondazione Cassa di Risparmio di Carpi (Italy).

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