Expression of PARs in DRG neurons: in situ hybridisation
In situ hybridization (ISH) was implemented to determine the cellular distribution of PAR subtype mRNAs in DRGs from adult mice as previously described [40
]. In Fig. , bright-field photomicrographs of representative examples of autoradiographs show the localization of oligonucleotide probes complementary to mouse PAR1, 2, 3, 4 mRNA (arrowheads). Silver grains are visualised as small black dots overlying tissue sections. Probes with comparable activity were used for each receptor. PAR3 mRNA was intensely expressed in many DRG neurons and the mRNAs for PAR1, PAR2 and PAR4 were more weakly expressed. A comparison of expression intensity between receptor subtypes is subject to several variables, such as efficiency of the labelling reaction to incorporate 35S-dATP tails into the oligonucleotide probes, and hybridisation strength, though these effects were minimised as much as possible. However, within these limitations it seems clear that the expression of PAR3 is much more intense than that of PAR1, 2 and 4. In the case of PAR1, 2 and 4 a quantitative method was used to distinguish neurons with signal above background, see Methods and [40
Fig. shows histograms of cells positive for PAR1-4 against mean cross-sectional area of neuronal profiles (between 912 and 1072 cells obtained from five sections for each PAR and from three adult animals). Grey histograms indicate all neuronal profiles with nuclei present that were measured, and black bars show profiles with a positive in situ hybridization signal/noise ratio. PAR1 mRNA was found to be expressed in 15.0 ± 1.5% of DRG neurones across all size classes. PAR2 mRNA was present in 21.5 ± 3.4% of total neuronal profiles, almost exclusively in neurones with a small cross-sectional area; there was only a low level of mRNA expression in medium sized neurones and no detectable expression in neurones with a large cross-sectional area. PAR3 was present in 49.5 ± 4.5% of neurons and was expressed mainly in neurones with a small cross-sectional area, but unlike PAR2, PAR3 mRNA was also expressed in medium-sized neurones. PAR4 mRNA expression was found in a similar proportion of neurones to PAR1 (14.5% ± 4.3) and with a distribution of expression across the neuronal size range similar to that found for PAR1.
Expression of PARs in glia was difficult to detect unequivocally using ISH because of the small size of these cells and the scatter of silver grains. We show below that there is clear functional expression of PAR1 and PAR2 in glial cells.
Expression of PARs in DRG neurons: immunohistochemistry
PAR1, 3 and 4 immunoreactivity (IR) was detected in DRG neurons (Fig. ). The PAR2 antibodies available to us showed clear evidence of non-specific bands on Western blot and results are therefore not shown. Preabsorption controls with a 20-fold excess of immunising peptide completely ablated the signals for PAR1, 3 and 4 in adjacent sections, as did incubation in the absence of primary antibody (data not shown). As with ISH, the distribution of expression was determined by measuring neuronal cross-sectional area, and by only including profiles in which there was a visible nucleus (Fig. ).
PAR1-IR was restricted to a small percentage of neurones (10.28 ± 2.54%) and was expressed in cells across the neuronal size range. The results for PAR1-IR were similar to those obtained with ISH (compare Fig. and ). Expression was punctate and appeared particularly intense in vesicular structures surrounding the nucleus, suggesting the presence of large intracellular stores of protein. PAR3-IR was detected in 42.03 ± 4.95% of neuronal profiles (Fig. ), similar to results obtained using ISH (Fig. ). PAR4-IR was also detected in DRG neurons but PAR4-IR was particularly strongly expressed in glial cells, and it was not always possible to distinguish positive neurones stained at the plasma membrane from surrounding ensheathing glial cells (see Fig. ). For this reason PAR4-IR was not quantified.
Calcium signals activated by PAR agonists in DRG neurons
We next examined functional activation of PAR receptors. A sub-population of small DRG neurons responded to the specific PAR2 activator peptide SLIGRL (PAR2-AP), which is derived from the activator domain of PAR2 (Fig. ). The proportion of neurons responding to the PAR2-AP with an increase in [Ca]i was 15.6% in neonatal rats and 12.1% in neurons from adult mice. A large majority of the PAR2-AP responsive neuronal population also expressed TRPV1 and TRPA1, as shown from the increase in [Ca]i in response to the specific TRPV1 agonist capsaicin and to the specific TRPA1 agonist mustard oil, and bound the plant isolectin B4 (IB4), which identifies a non-peptidergic subpopulation of nociceptors (see Fig. and ). These PAR2+ neurons therefore have the characteristics of IB4-positive nociceptors. None of these neurons, however, responded to thrombin and so are unlikely to express PAR1 or 4 (PAR3 appears unresponsive in DRG neurons, see below).
Figure 2 Calcium signals elicited by PAR agonists. A. Adult mouse neuron in which an increase in [Ca]i was elicited by a specific PAR2 activator peptide (PAR2-AP, SLIGRL, 100 μM), but not by thrombin (100 nM) which activates PAR1, 3 and 4. The neuron also (more ...)
Thrombin, which activates PAR1, 3 and 4, elicited robust increases in [Ca]i
in a distinct sub-population of sensory neurons (Fig. ). The proportion of neurons responding to thrombin with an increase in [Ca]i
was 17.5% in neonatal rats and 15.2% in neurons from adult mice. No neuron responsive to thrombin also responded to PAR2-AP (Fig. ). Among these thrombin-responsive neurons, around 25-33% responded to capsaicin, mustard oil, and to the peptides Bv8 and bradykinin, both of which act on G-protein coupled receptors expressed in nociceptors [34
], but none bound IB4 (Fig. ). About a third of thrombin-responsive neurons are therefore IB4-negative nociceptors, while the remainder are non-nociceptive. As PAR2 is predominantly expressed in IB4+
nociceptors (see above) this shows that functional PAR2 and PAR1/3/4 receptors are located in separate subpopulations of nociceptors. PAR4 was found to be colocalised with PAR1 expression in neonatal rat neurons, because calcium responses to a PAR4-AP (AYPGKFR) were elicited in a subset of PAR1 expressing neurons (see below) and all cells responding to PAR4-AP also exhibited a calcium signal in response to PAR1-AP (not shown).
Glial cells are clearly distinguishable from neurons both on morphological grounds and because they do not exhibit a calcium increase in response to elevated [K+] (Fig. ). Most glial cells responded to thrombin (Fig. ). A few glial cells responding to thrombin also responded to PAR2-AP (3.6% -see Fig. ), showing that in contrast to neurons, PAR2 and PAR1/3/4 are co-expressed in a small subset of glial cells. The calcium response to thrombin and PAR1-AP in glial cells was ablated by genetic deletion of PAR1 but was unaffected by deletion of PAR2 (Fig. ).
PAR3 is highly expressed in DRG neurons (Fig. ). PAR3 mRNA is seen in about 50% of neurons and PAR3 protein is seen in about 42% of neurons. Functional responses to thrombin, which should activate PAR3 (along with PAR1 and PAR4), are seen in a significantly lower number of neurons, however, suggesting that PAR3 is non-functional, at least when expressed in the absence of other PARs. To test this more conclusively it would be desirable to activate PAR3 alone, but specific activation of PAR3 in neurons coexpressing PAR1 and PAR4 is not possible because PAR3 peptides also activate PAR1 and PAR4 [45
]. We therefore tested for PAR3 responses by desensitizing PAR1 and 4 with their specific activator peptides, and then retesting with thrombin (Fig. ). Following desensitization of PAR1 and PAR4, calcium signals in response to thrombin are seen in only a very small number of neurons, far smaller than the proportion in which histological studies had shown expression of PAR3. These results support the idea the PAR3 is largely non functional by itself in DRG neurons, but they do not rule out the possibility that PAR3 may heteromerise with other PARs to form functional receptors, as has been found in other studies [47
Figure 3 Desensitization of PAR1 and PAR4 ablates calcium signals in response to thrombin. A. Increase in [Ca]i recorded as in Fig. 2. Calcium increase elicited by application of PAR1-AP completely desensitizes response to a subsequent application of PAR1-AP but (more ...)
Sensitization of TRPV1 by PAR activation
Activation of the heat and capsaicin gated ion channel TRPV1 is potentiated by PAR2 activation [22
]. Fig. shows a similar potentiation of heat-activated inward currents by specific PAR1 and PAR4 activator peptides. Both PAR agonists caused substantial sensitization of TRPV1 in a subset of neurons (c. 10% of total neurons, consistent with studies of expression of PAR1 or PAR4, see above). Sensitization was long-lasting and subsequent PAR-AP applications were ineffective (Fig. ). Thrombin and trypsin also caused a substantial enhancement in the inward current activated by heat (Fig. ).
Figure 4 Sensitization of TRPV1 by PAR activation. A - D. Heat-activated currents were significantly enhanced in c. 10% of neurons by application of PAR1-AP and PAR4-AP. Single traces in panels to left are taken from time courses shown in right hand panels. Both (more ...)
Many pro-inflammatory mediators sensitize TRPV1 via downstream activation of PKCε, reviewed in [49
]. Consistent with this also being the principal signaling pathway activated by PAR1/3/4, Fig. shows that the sensitization caused by thrombin was reduced at least 5-fold by the specific PKC inhibitor Ro-318220 or by the broad-spectrum kinase inhibitor staurosporine.
We next tested sensitization of TRPV1 by thrombin in wild-type and PAR1-/-
mice. In order to improve cell yield we employed a calcium imaging protocol similar to that used by Bonnington & McNaughton [43
]. We activated TRPV1 by applying brief pulses of the specific agonist capsaicin, and tested the effect of thrombin in enhancing TRPV1 activation (Fig. ). Ratios of responses to capsaicin before and after application of thrombin were calculated, and sensitized cells were identified when the ratio exceeded the 99.7% confidence limits of a distribution obtained from control experiments (see Additional file 1
). In PAR1-/-
animals the percentage of sensitized neurons using thrombin as a PAR activator was 8.3%, significantly lower than in WT neurons (Fig. ). Thus removal of PAR1 reduces but does not completely abolish the response to thrombin, consistent with the idea (see above) that DRG neurons also express functional PAR4 receptors.
Note that the percentage of neurons from PAR1-/- mice responding to thrombin is similar to the proportion of neurons expressing PAR4 by ISH (14.5%, see Fig. above) but is very much smaller than the proportion expressing PAR3 by both ISH and immunohistochemistry (49.5% and 42.03% respectively, see Fig. above). Combined with the results in Fig. (above) these results suggest that PAR4 receptors in DRG neurons are functionally activated by thrombin but that PAR3 receptors are not.
PAR1/4 agonists cause translocation of PKC-ε in sensory neurons
The activation of PKCε can be visualized as a translocation from the cytoplasm to the cell surface membrane, and provides a sensitive indicator of those neurons activated by bradykinin [21
] or by other pro-inflammatory mediators [34
]. We found that thrombin and PAR1-AP caused a pronounced translocation of PKC-ε to the neuronal cell membrane in a subset of neurons from adult and neonatal rats and from adult mice (Fig. ). PKCε translocation, expressed as the percentage of neurons in which clear translocation was observed, peaked at 30 s after application of a maximal concentration of 100 nM thrombin (Fig. ). At longer application times PKCε was internalized into peri-nuclear vesicles (Fig. , right hand panel), as is seen after longer exposures to bradykinin [21
] and to the prokineticin receptor agonist Bv8 [34
]. Translocation of PKCε was half-activated by a concentration of 2.0 ± 0.4 nM thrombin and was fully saturated at 100 nM thrombin (Fig. ). In adult mouse neurons cultured without NGF translocation was observed in 15.6 ± 0.5% of the population (Fig. ), a proportion which increased to 19.3 ± 1.0% with NGF (see below). Responsive neurons were distributed across all neuronal size classes, in agreement with histological data for expression of PAR1 and 4 (Fig. ).
Figure 5 Translocation of PKCε to neuronal surface membrane caused by thrombin. A. Translocation of PKCε to neuronal surface membrane in control conditions (left) and following exposure to thrombin (100 nM, 30 and 60 sec). PKCε translocated (more ...)
Other proteases known to activate PAR1 and PAR4 were also effective in causing translocation of PKCε (Fig. ). Trypsin, a broad-spectrum PAR activator, produced translocation in 17.1 ± 1.6% of neurons. Cathepsin G, which preferentially activates PAR4 over PAR1, caused translocation in 11.8 ± 2.3% of neurons. Type IV collagenase was ineffective. The PAR1-AP TFLLR caused translocation in a similar proportion of neurons to thrombin, while the specific PAR4-AP AYPGKF caused translocation in a significantly lower percentage of neurons than thrombin (9.1 ± 2.1%, n = 5 p < 0.05). Application of the PAR1-AP TFLLR in combination with PAR4-AP gave only a slightly higher percentage than PAR1-AP applied alone (17.3 ± 0.6%, n = 6). These data agree with those above (Fig. ) in showing that PAR4 receptors are expressed in a subset of the PAR1-expressing sensory neurons. PAR2-AP was ineffective in causing translocation of PKCε in any neuron.
Characteristics of neurons expressing functional thrombin receptors
We next examined the histological characteristics of thrombin-responsive neurons, using translocation of PKCε as a marker. Around half of the thrombin-responsive neuronal population, predominantly medium-sized and large neurons, stained for neurofilament H (NFH+), a marker for myelinated neurons (second bar in Fig. ). Only a small fraction (around 6%) of these NFH+ neurons also expressed functional TRPV1 receptors, as demonstrated by a calcium increase in response to application of capsaicin (see white bar at bottom of second bar in Fig. ), showing that this class of thrombin-responsive large neurons is predominantly non-nociceptive.
Figure 6 Co-localisation of thrombin-induced translocation of PKCε with other neuronal markers. A. PKCε translocation (green) following exposure to thrombin (100 nM, 30 s) colocalises with other neuronal markers as shown. PKCε translocation (more ...)
A distinct population of thrombin-responsive neurons, mainly small neurons, co-expressed the neuropeptides CGRP and/or substance P (bars 3 and 4 in Fig. ). Neurons in this population gave a calcium increase in response to capsaicin and therefore express TRPV1 (final bar in Fig. ). Very few thrombin-responsive small neurons were IB4-positive (5% of the thrombin-responsive population), in agreement with Fig. above where neurons responding to thrombin with a calcium increase were found to be IB4-negative. The presence of neuropeptides and the lack of binding of IB4 identifies a TrkA positive C-fibre nociceptor sub-population [50
] as the location of nociceptor PAR1/4 expression. In addition, thrombin-responsive neurons were negative for cyclooxygenase 1 (COX-1), an enzyme expressed in a subpopulation of small-sized nociceptive neurons [51
], and for parvalbumin, expressed in non-nociceptive sensory neurons innervating muscle spindles [52
]. In summary, our data show that functional receptors for thrombin are expressed broadly across all neuronal size classes, in neurons subtending both myelinated and unmyelinated fibres. In the unmyelinated neuronal population thrombin-responsive neurons are found in the peptidergic/IB4-
class of nociceptors.
Release of CGRP by heat is potentiated by PAR1
The results outlined above show that PAR1/4 receptors in small neurons co-express with TRPV1 and the neuropeptide CGRP, suggesting that neuropeptide release caused by TRPV1 activation should be potentiated by PAR1. Fig. shows an experiment in which this hypothesis was tested using a rat skin preparation, which contains nerve terminals from which CGRP can be released by noxious heat stimulation [53
]. In mouse skin, this heat response is markedly reduced, though not abolished, if the TRPV1 gene is deleted, and it is sensitized by pre-treatment with the weak TRPV1/2/3 agonist 2-APB which is ineffective in TRPV1 knockouts [54
]. In the present experiments, the heat stimulation caused about a tenfold increase in CGRP release from cutaneous nerves (p < 0.001, t-test). The basal CGRP release was unaffected by the presence of PAR1-AP. The release in response to heat was approximately doubled by exposure to the PAR1-AP, consistent with expression of PAR1 in peptidergic neurons.
Figure 7 Heat-induced CGRP release from isolated rat skin is facilitated by PAR1 activation. A. CGRP release elicited by heat (closed circles, n = 16) was increased approximately two-fold by the rat PAR1-AP SFLLRN-OH (100 μM, open circles, applied during (more ...)
Upregulation of PAR expression by neurotrophins
Fig. examines the effect of exposure to neurotrophic factors on expression of functional PAR1/4 receptors, measured from PKCε translocation following exposure to thrombin. NGF and neurturin (NTN) applied individually significantly increased the number of thrombin-responsive small neurons, while the effects of NGF and NTN applied together were additive, consistent with the known expression of TrkA and Ret receptors in separate neuronal populations (Fig. ). In the absence of neurotrophins few thrombin-responsive neurons bind IB4 (first bar in Fig. ). NGF increased the proportion of thrombin-responsive neurons but IB4 binding was not significantly increased. NTN, on the other hand, significantly upregulated the proportion of the thrombin-responsive population stained by IB4.
Figure 8 Upregulation of proportion of thrombin-responsive cells by neurotrophic factors. A. Percentage of neurons in which PKCε translocation was observed following exposure to thrombin (100 nM, 30 s) (white bars) was increased significantly by NGF (100 (more ...)
In neurons from PAR1-/- animals the proportion showing PKCε translocation was significantly reduced but was not zero (Fig. ), consistent with an action of thrombin on PAR4 as discussed above. The fraction of responsive neurons was upregulated by NGF and NTN. In neurons from PAR2-/- animals the proportion of neurons activated by thrombin, and the effects of NGF and NTN in upregulating the proportion of thrombin-responsive neurons, were similar to wild-type neurons (Fig. ), confirming that PAR2 receptors are not involved in responses to thrombin.