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We have previously demonstrated that parathyroid hormone 2 (PTH2) receptors are expressed in dorsal root ganglion (DRG) neurons and that its endogenous agonist tuberoinfundibular peptide of 39 residues (TIP39) causes nociceptive paw flexor responses after intraplantar administration. Here we found that the PTH2 receptor is selectively localized on myelinated A-, but not unmyelinated C-fibers using immunohistochemical labeling, based on PTH2 receptor expression on antibody N52-positive medium/large-sized DRG neurons, but not on TRPV1, substance P, P2X3 receptor or isolectin B4-binding protein-positive small-sized DRG neurons. Pharmacological studies showed that TIP39-induced nociceptive responses were mediated by activation of Gs and cAMP-dependent protein kinase. We also found that nociceptive responses induced by TIP39- or the cAMP analog 8-bromo-cAMP were significantly greater following partial sciatic nerve injury induced neuropathic pain, without changes in PTH2 receptor expression. Together these data suggest that activation of PTH2 receptors stimulates nociceptive A-fiber through Gs-cAMP-dependent protein kinase signaling, and this pathway has elevated sensitization following nerve injury.
The parathyroid hormone 2 (PTH2) receptor, which has ~50% amino acid sequence identity with PTH1 receptor, was identified in 1995 (Usdin et al. 1995). The PTH2 receptor is expressed at relatively high levels in several nervous system areas, including parts of the limbic system, several hypothalamic nuclei and the median eminence (Usdin et al. 1999a, 2003; Wang et al. 2000). It is expressed at very low levels in kidney and bone, where the PTH1 receptor is highly expressed (Urena et al. 1993). Interestingly, PTH2 receptors are also expressed in dorsal root ganglion (DRG) neurons as well as the spinal cord dorsal horn, suggesting that PTH2 receptors may play a role in pain regulation (Usdin et al. 1999a; Dobolyi et al. 2002). Indeed, we found that intraplantar injection of its endogenous agonist tuberoinfundibular peptide of 39 (TIP39) elicited nociceptive flexor responses in mice (Dobolyi et al. 2002). In addition, the intrathecal administration of TIP39 potentiated thermal and mechanical responses, as well inducing a nocifensive response (Dobolyi et al. 2002). These findings suggest that TIP39 may have pharmacological and physiological effects on nociceptive fibers, but these effects remain to be defined.
Through a series of studies, we have developed several strategies to characterize the in vivo signal transduction of specific key molecules involved in pain regulation (Ueda 2006, 2008). One of these strategies is the use of the algogenic-induced paw flexion (APF) test, in which the amplitude of nociceptive flexor responses induced by intraplantar injection (i.pl.) of algogenics is quantitatively evaluated (Inoue et al. 2003b; Ueda 2006). From its careful characterization, the APF test was found to be less stressful and more sensitive than other nociceptive tests. Using this test, the i.pl. administration of test substances can be combined with intrathecal delivery of antisense oligodeoxynucleotides (AS-ODNs) targeting potential effector molecules, such as G proteins (specifically α-subunits) to characterize the in vivo signal transduction of pain-producing stimuli. We previously showed study that large amounts of intrathecally administered FITC-labeled AS-ODN accumulate within DRG, while little if any, is detectable in the superficial region of spinal dorsal horn (Ueda 1999). This approach has been used a number of times to inhibit gene expression in DRG (Inoue et al. 2003a, 2004; Rashid et al. 2004; Matsumoto et al. 2007). Neonatal capsaicin-treatment allows responses to be classified as mediated by myelinated A- or unmyelinated C-fibers, as this treatment destroys unmyelinated C-fibers (Hiura and Ishizuka 1989; Rashid et al. 2003). We also used pharmacological blockade of nociceptive responses by intrathecal administration of specific antagonists for substance P (NK1 receptor) or glutamate (NMDA or non-NMDA receptors), which are representative spinal pain transmitters. Using these strategies, we have successfully characterized signal transduction and sensory fibers that are involved in nociceptive responses caused by pain-producing stimuli, such as bradykinin, substance P, ATP, prostaglandin I2 agonist, nociceptin and nocistatin (Inoue et al. 1998a, 2003a,c; Matsumoto et al. 2006; Ueda 2006).
These strategies led to the finding that the neuropathic pain model with partial injury of the sciatic nerve shows a decrease in C-fiber-mediated responses, whereas significant enhancement in A-fiber-mediated responses (Rashid et al. 2003; Inoue et al. 2006; Ueda 2006). These findings are supported by a study using biochemical markers, in which C-fiber stimulation-induced spinal neuronal activation was reduced while A-fiber stimulation-induced neuronal activation was up-regulated (Matsumoto et al. 2008). Thus, we have proposed a dominant role of A-fibers in neuropathic pain.
In the present study, we first performed an immunohistochemical characterization of the cell population that expresses PTH2 receptors in DRG, and second performed a pharmacological characterization of this receptor’s role in acute and chronic pain.
Male ddY mice and male C57BL/6J mice weighing 20–24 g were used after adaptation to the laboratory conditions: 22 ± 2°C, 55 ± 5% relative humidity and a 12 h light/dark cycle with food and water ad libitum. Neonatal capsaicin treatment, which causes degeneration of unmyelinated C-fibers, was performed as described previously (Rashid et al. 2003) by subcutaneous (s.c.) injection of 50 mg/kg capsaicin (Nacalai Tesque, Kyoto, Japan) into newborn (P4) pups. Animal procedures were approved by the Nagasaki University Animal Care Committee, and complied with the fundamental guidelines for proper conduct of animal experiments and related activities in academic research institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Partial ligation of sciatic nerve of mice was performed under pentobarbital (50 mg/kg, i.p.) anesthesia, as described previously (Rashid et al. 2003; Inoue et al. 2004; Matsumoto et al. 2008). Briefly, the common sciatic nerve of the right hind limb was exposed at high thigh level through a small incision and half of the nerve thickness was tightly ligated with a silk suture. Sham surgery was performed similarly, except without touching the sciatic nerve. Most of the experiments in this study were carried out on the 7th day after the sham or nerve injury surgery. At this time point, significant thermal hyperalgesia and mechanical allodynia were observed (Inoue et al. 2004).
The following drugs were used: Capsaicin (Nacalai Tesque), (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Research Biochemicals International, Natick, MA, USA), 8-bromo-cAMP (8-Br-cAMP), H-89 dihydrochloride hydrate (Sigma, St. Louis, MO, USA), pertussis toxin and U-73122 (Funakoshi, Tokyo, Japan), and KT-5720 (Wako, Tokyo, Japan). Mouse TIP39 was synthesized by Midwest Biomolecules (Waterloo, IL, USA). CP-99994 was generously provided by Pfizer Pharmaceuticals (Sandwich, Kent, UK). All drugs except KT-5720 and capsaicin were dissolved in physiological saline. KT-5720 was dissolved in 30% dimethyl sulfoxide, while capsaicin was dissolved in 10% ethanol and 10% Tween 80 in physiological saline.
The AS-ODN against the PTH2 receptor (5′-ATCCACAAATGTAGGTGAAA-3′) and its mismatch scrambled oligodeoxynucleotide (MS-ODN; 5′-TACCAACAATTGAGGGTAAA-3′), and the AS-ODN against Gαs (5′-AGTCACCCATTAGTGACGCC-3′), and its MS-ODN (5′-AGCTACCACTATGTGCAGCC-3′) were synthesized by Operon Biotechnologies (Tokyo, Japan). The oligodeoxynucleotides were freshly dissolved in artificial CSF comprised of 125 mM NaCl, 3.8 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 1.2 mM KH2PO4, 26 mM NaHCO3 and 10 mM D-glucose (pH 7.4) and injected intrathecally (i.t.) between the L5 and L6 lumbar space in unanesthetized mice using a 30-gauge needle. The treatment was performed (10 μg/5 μL) on the 1st, 3rd and 5th days, as stated in our previous study (Ueda and Inoue 2000; Matsumoto et al. 2007). On the 6th day, the treated mice were assessed in behavioral tests, and tissues were isolated for western blot and immunohistochemical experiments.
To confirm the effect of AS-ODN for PTH2 receptor and Gαs, sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 12% polyacrylamide gel and immunoblot analysis were performed as described (Ueda and Inoue 2000). Twenty μg of protein extracted from the L4–6 dorsal root ganglion (DRG) was used. To get equal transfer efficiency, we applied all samples to the same gel and carried out the immunoblot transfer using the same membrane. Visualization of immunoreactive bands was performed by use of an enhanced chemiluminescent substrate for detection of horseradish peroxidase, Super Signaling Substrate (PIERCE, Rockford, IL, USA). The intensities of the immunoreactive bands were analyzed by NIH Image for Macintosh after scanning exposed films.
Experiments were performed as described previously (Dobolyi et al. 2002; Inoue et al. 2003b; Ueda 2006). Briefly, mice were held in a cloth sling with their four limbs hanging free through holes. The sling was suspended from a metal bar. All limbs were tied with strings, and three were fixed to the floor, while the 4th was connected to an isotonic transducer and recorder. A polyethylene cannula (0.61 mm in outer diameter) filled with drug solution was connected to microsyringe and then carefully inserted into the undersurface of the right hind paw. In the dose-response experiments and the experiment with AS-ODN, we used the biggest response among spontaneous and non-specific flexor responses occurring immediately after cannulation as the maximal reflex, as the intensity of flexor responses differs from mouse to mouse. In these experiments, TIP39 was administered i.pl. through the cannula at 5-min intervals. Averages of two responses to each TIP39 dose were evaluated. In the experiment with inhibitors, TIP39 was administered 10 and 5 min (control TIP39-response) prior to and 5, 10, 20 and 30 min after the inhibitor (or vehicle)-injection. The results were expressed as percent of control response, using the following equation: [TIP39-response (mm) after the inhibitor administration/the average of two control TIP39-responses (mm)] × 100 (%).
Experiments were performed as described previously (Inoue et al. 2003b). Mice were placed individually in plastic cages for 1 h to adapt to the environment. Algogenics in a volume of 20 μL were injected into the right hind paw of mice (i.pl.) using a 30-gauge needle fitted to 50 μL volume Hamilton microsyringe. In the experiments using antagonists, 2 μL antagonist was injected (i.pl) 20 min prior to the algogen in a 20 μL volume. Each mouse was immediately put back in its cage and the time spent biting and licking the injected paw was measured for a period of 10 min after injection. Saline was injected as a control.
Anesthetized mice were transcardially perfused with phosphate-buffered saline, followed by cold 4% paraformaldehyde solution. The L4–6 DRG were isolated, post-fixed for 3 h, and cryoprotected overnight in 25% sucrose solution. The tissues were fast-frozen in cryo-embedding compound on a mixture of ethanol and dry ice and stored at −80°C until use. DRG were cut on a cryostat at a thickness of 10 μm, thaw-mounted on silane-coated glass slides, and air-dried overnight at 25°C. Sections were incubated with blocking buffer containing 2% bovine serum albumin in PBST (0.1% Triton X-100 in phosphate-buffered saline) and subsequently reacted overnight at 4°C with rabbit antibody against PTH2 receptor (1: 3000) in the blocking buffer (Usdin et al. 1999a,b). After thorough washing, the sections were incubated with secondary antibody, Alexa Fluor 488-conjugated anti-rabbit IgG (1: 500; Invitrogen, Carlsbad, CA, USA), for 120 min at 25°C, and coverslipped with PermaFluor (Thermo Shandon, Pittsburgh, PA, USA). For double immunolabeling, we used the following antibodies: a mouse monoclonal antibody against N52 clone of Neurofilament 200, a marker of myelinated fibers (anti-N52; 1: 30 000; Sigma); a goat polyclonal antibody against transient receptor potential vanilloid 1 (TRPV1); a rabbit polyclonal antibody against Substance P; a guinea pig polyclonal antibody against P2X3 (Chemicon, Temecula, CA, USA); FITC-conjugated isolectin B4 (IB4; 10 μg/mL; Sigma); Alexa Fluor 488-conjugated anti-rabbit IgG; Alexa Fluor 594-conjugated anti-rabbit IgG; Alexa Fluor 488-conjugated anti-mouse IgG; Alexa Fluor 488-conjugated anti-goat IgG; Alexa Fluor 488-conjugated anti-guinia pig IgG (1: 300; Molecular probes). Sections were examined under a BX-50 fluorescence microscopy (Olympus, Tokyo, Japan) or a BZ-8000 fluorescence microscopy (Keyence, Tokyo, Japan). Digital images were acquired with either ×10 objective or ×20 objective, and neurons with visible nuclei were evaluated. The diameters of DRG neurons were measured and calculated from a line drawn by the operator using AxioVision 3.1 software (Carl Zeiss, Oberkochen, Germany) and BZ-analyzer software (Keyence), respectively.
The L4–6 DRGs were isolated and RNA was purified using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) was used for cDNA synthesis with Superscript II reverse transcriptase and random hexamer primers (Invitrogen). Real-time quantitative PCR (RT-PCR) was performed using an ABI Prism 7000 Sequence Detection system (Applied Biosystems, Tokyo, Japan), using qPCR MasterMix Plus for SYBR® Green I (Eurogentec, Seraing, Belgium) containing dNTPs (+dUTP), Hot Goldstar DNA polymerase, and Urasil-N Glycosilase, according to the manufactures instructions. The cycling conditions for all primers were the following: 10 min at 95°C to activate the Hot Goldstar DNA polymerase, followed by 50 cycles consisting of two steps, 15 s at 95°C (denaturation) and 1 min at 60°C (annealing-extension). The PTH2 receptor mRNA levels were evaluated by comparison to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences were as follows; PTH2 receptor, (F)5′-GATGGCTGATTCTCAGTAGCTGTCT-3′ and (R)5′-CACACTGCATTTTAGCCTTCATAAC-3′ corresponding to the mouse PTH2 receptor gene (NCBI accession number; AK045576), GAPDH, (F)5′-TATGACTCCACTCACGGCAAAT-3′ and (R)5′-GGGTCTCGCTCCTGGAAGAT-3′ corresponding to the mouse GAPDH (NCBI accession number; NM_001001303). In all cases, the validity of amplification was confirmed by the presence of a single peak in the melting temperature analysis and linear amplification against the PCR cycle number.
Data were analyzed using an unpaired Student’s t-test. Experiments with multiple groups were analyzed using one-way ANOVA with Tukey-Kramer multiple comparison post-hoc analysis. The criterion of significance was set at p < 0.05. All results are expressed as mean ± SEM from four to six separate mice.
Parathyroid hormone 2 receptor immunoreactive neurons in dorsal root ganglia (DRG) appeared larger than neurons expressing several C-fiber marker proteins, including TRPV1, substance P, the P2X3 receptor and IB4-binding protein (Fig. 1a). There appeared to be no neurons that co-expressed the PTH2 receptor and any of the C-fiber marker proteins (Fig. 1b). Instead, approximately 70% of the neurons expressing the PTH2 receptor were also positive for N52, a monoclonal antibody against neurofilament 200, which is a representative molecular marker for myelinated A-fibers (Fig. 1a and b). A size distribution study demonstrated that PTH2 receptor labeling was mostly present on medium/large-sized DRG neurons (> 24 μm) that largely overlapped the size distribution of N52-immunoreactive cells. This size distribution was clearly distinguishable from the distribution of TRPV1-immunoreactivitity, which was present on the small-sized DRG neurons (< 24 μm) (Fig. 1c). However, it should be noted that the N52-positive population included larger diameter neurons than the PTH2 receptor-positive population, and there were a few PTH2 receptor-positive cells that were smaller than N52-positive cells.
When neonatal mice (postnatal day 4) were pre-treated with 50 mg/kg (s.c.) capsaicin, TRPV1-positive neurons were substantially lost in DRG, while PTH2 receptor-positive ones were not affected (Fig. 2a). In a quantitative analysis, the population of TRPV1-positive neurons was significantly reduced, from 24% to 8%, by the neonatal capsaicin-treatment, while that of PTH2 receptor-positive neurons was slightly but not significantly increased by the capsaicin-treatment (Fig. 2b). This increase in the ratio of PTH2 receptor-positive neurons to total number of neurons could be attributed to the specific loss of the C-fibers induced by the neonatal capsaicin-treatment (Hiura 2000; Nakagawa and Hiura 2006).
As previously reported (Dobolyi et al. 2002), intraplantar injection of TIP39, the endogenous agonist for the PTH2 receptor (Usdin et al. 1999a), dose-dependently caused nociceptive flexor responses in the range between 10 fmol and 100 pmol in the APF test in ddY mice (data not shown). The flexor responses elicited by TIP39 (100 pmol, i.pl.) were significantly reduced by i.t. pre-treatment with an AS-ODN against the PTH2 receptor, but not with a scrambled MS-ODN, as shown in Fig. 3(a). Western blot analysis of DRG preparations showed that AS-ODN markedly reduced the level of PTH2 receptor immunoreactivity, while MS-ODN did not (Fig. 3a–i). TIP39 (100 pmol, i.pl.)-induced nociception in the APF test was not affected by neonatal capsaicin-pretreatment, which abolished the acute capsaicin (i.pl.)-induced nocisponsive biting and licking behaviors (Fig. 3b and c).
When MK-801 (3 mol), a non-competitive NMDA receptor antagonist, was given intrathecally 20 min prior to TIP39 (100 pmol, i.pl.), TIP39-induced flexor responses were completely abolished (Fig. 3d). However, as shown in Fig. 3(d), the TIP39-induced responses were not affected by the NK1 receptor antagonist CP-99994 (3 mol, i.t.), which significantly blocks bradykinin (i.pl.)- or substance P (i.pl.)-induced flexor responses (Inoue et al. 2003c).
Similarly, MK-801 (3 nmol, i.t.) but not CP-99994 (3 nmol, i.t.) attenuated the TIP39 (100 fmol, i.t.)-induced nociceptive behaviors, such as reciprocal hindlimb scratching, and caudally directed scratching, biting, and licking during 20 min after TIP39 administration (Fig. S1). On the other hand, CNQX (3 nmol, i.t.), an α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist, failed to inhibit the TIP39-induced scratching, biting, and licking responses (Fig. S1).
The pre-treatment with AS-ODN against G protein subunit, Gαs, markedly reduced the Gαs levels in the DRG (Fig. 4a–i). TIP39 (100 pmol, i.pl.)-induced flexor responses were significantly reduced by the pre-treatment with AS-ODN against Gαs, but not by its MS-ODN, as shown in Fig. 4(a). Repeated challenges with TIP39 (100 pmol, i.pl.) at intervals of 5–10 min caused constant responses for at least 30 min (Fig. 4b), suggesting that these repeated TIP39 challenges did not cause desensitization of the PTH2 receptor. When KT-5720, a specific cAMP-dependent protein kinase (PKA) inhibitor, was given (3 nmol, i.pl.) (Inoue et al. 2003a) after the confirmation of control responses to TIP39, subsequent TIP39 responses were inhibited as early as 10 min after the inhibitor infusion (Fig. 4b), suggesting that the activation of the PTH2 receptor, Gs and PKA is required for TIP39-mediated nociception. In contrast, TIP39 responses were not affected by pertussis toxin (10 ng, i.pl.) or by U-73122 (3 pmol, i.pl.), which block receptor-Gi/o coupling or a phospholipase C signaling, respectively (Inoue et al. 1998b) (Fig. 4c).
Tuberoinfundibular peptide of 39-induced nociceptive responses were analyzed in the nerve injury-induced neuropathic pain model using C57BL/6J mice. In this experiment, mice were injected with vehicle or TIP39, returned to their cage, and algogenic biting and licking (ABL) behaviors quantitated. In the absence of TIP39 (see dose ‘0’ in Fig. 5b), no significant ABL response was observed in sham-operated or nerve-injured mice. As shown in Fig. 5(a), characteristic ABL responses to TIP39 (1 pmol, i.pl.) were observed between 2 and 5 min in injured mice, and not in sham-operated ones, although transient responses within 1 min were observed in both groups. The TIP39-induced nociceptive responses continued for 10 min after the injection in injured mice and were dose-dependent in the range between 0.1 and 10 pmol (Fig. 5b). However, no substantial responses were observed in sham-operated mice. When the ABL test was carried out 28 days after the injury, the injury-specific TIP39 (10 nmol)-responses were still observed, while the level (35.2 s) was slightly lower than that (60.7 s) at 7 days (Fig. 5c). Similar enhanced TIP39-induced responses were also observed in ddY mice (Fig. S2).
Quantitative real-time PCR indicated no change in the level of PTH2 receptor mRNA in DRG for up to 7 days following nerve injury, as compared to GAPDH (Fig. 6a). The lack of change of PTH2 receptor level in the DRG following nerve injury was also observed when the PTH2 receptor level was evaluated by immunohistochemistry (Fig. 6b). In addition, the nerve injury did not cause substantial change in the size distribution of PTH2-positive neurons (data not shown).
When 8-bromo-cAMP (8-Br-cAMP), a stable and cell permeable analog of cAMP was given intraplantarly, a characteristic enhancement of ABL behaviors was observed following nerve injury (Fig. 6c). Both TIP39 (10 pmol) and 8-Br-cAMP (10 nmol)-induced hypersensitized ABL responses in nerve-injured mice were significantly attenuated by the PKA inhibitor H-89 (10 nmol), which was given intraplantarly 20 min prior to the test drug administration (Fig. 6d).
Finally, we examined the co-expression of the PTH2 receptor and TRPV1 in the DRG following nerve injury, as TRPV1 is a representative target for PKA (Lopshire and Nicol 1998; Bhave et al. 2002), and it is newly expressed in myelinated A-fibers after the nerve injury (Rashid et al. 2003). TRPV1 is dramatically up-regulated 7 days after nerve injury and partially co-expressed with PTH2 receptor (Fig. 6e). On the other hand, substantially no co-expression was observed in the DRG of sham-operated mice.
In the present study, PTH2 receptor-immunoreactivity was observed in medium/large-sized DRG neurons, and found to be substantially co-localized with the myelinated A-fiber marker N52, but not with C-fiber markers. Consistently, the intraplantar injection of TIP39, a potent endogenous agonist of the PTH2 receptor induced nociceptive flexor response through capsaicin-insensitive glutamatergic A-fibers. These results suggest that the PTH2 receptor could be a novel neurochemical marker for nociceptive A-fibers. Many neurochemical markers have been identified for C-fibers, including TRPV1, substance P, calcitonin gene-related peptide, P2X3 receptors, IB4-binding protein, voltage-gated sodium channel Nav1.8, and Nav1.9 (Dib-Hajj et al. 1998; Snider and McMahon 1998; Tate et al. 1998; Ueda 2006). These molecules are useful not only as pharmacological targets to evaluate or modify C-fiber function (Ueda 2006), but also as genetic targets to specifically label or ablate C-fibers (Braz et al. 2005; Abrahamsen et al. 2008). On the other hand, fewer neurochemical markers have been identified in A-fibers. The best known marker is neurofilament 200 (N52 or RT97), which is expressed in both Aδ-fibers and Aα/β-fibers (Lawson and Waddell 1991). Using this marker, we found that the size distribution of PTH2-positive DRG neurons overlapped significantly with the distribution of N52-positive cells, but was somewhat smaller on average. This suggests that PTH2 receptor might be expressed in smaller sized A-fibers or Aδ-like-fibers, in view of the fact that there is a positive co-relationship between the conduction velocity and the soma size of DRG neurons (Lawson and Waddell 1991). In addition, TIP39-induced nociceptive responses share the similar pharmacological characteristics in terms of spinal neurotransmission with those by Aδ-fiber stimulation using the Neurometer® method (Matsumoto et al. 2008). Both responses were sensitive to MK-801, but not to CP-99994 or CNQX (Matsumoto et al. 2006, 2008). The lack of CNQX blockade of the nociceptive responses induced by peripherally applied chemicals was consistent with our previous reports (Inoue et al. 2003c; Matsumoto et al. 2006, 2008), though α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-mediated action is known to precede the NMDA receptor-mediated one. Although details remain to be determined, the lack of CNQX blockade may be explained by recent studies in which the ephrinB-EphB signaling closely related to the NMDA receptor-mediated spinal neurotransmission (Liu et al. 2009), facilitates the glutamate-induced NMDA receptor activation in the presence of CNQX (Takasu et al. 2002). Although further characterization of A-fibers (Aδ vs. Aβ) requires electrophysiological studies, it is interesting to speculate that the PTH2 receptor might be a functional marker for nociceptive A(δ) fibers. There are reports that other functional molecules, such as Nav1.1, Nav1.6 (Fukuoka et al. 2008), Kv1.1, Kv1.2 (Rasband et al. 2001) and TRPV2 (Caterina et al. 1999; Lewinter et al. 2004) are also expressed by medium/large-sized DRG neurons, though little pharmacological information is available how these molecules are functionally assigned to Aδ-fibers.
It is interesting to discuss the potential source of endogenous TIP39. We have reported that fine scattered TIP39-containing fibers were observed in the lateral funiculus adjacent to the gray matter, an area that contains both descending and primary afferent fibers, and TIP39 mRNA was found in the DRG, but not in the hindpaw or spinal cord (Dobolyi et al. 2002). These findings suggest that the source of TIP39 for primary afferent PTH2 receptors seems to be from DRG primary afferent neurons. However, as TIP39-knock out mice showed normal nociception in the mechanical and the thermal test (Fegley et al. 2008), endogenous TIP39 is unlikely involved in pain sensation. Thus, primary afferent PTH2 receptors may be the pharmacological target for TIP39, but unlikely have significant physiological roles in pain regulation.
The present study demonstrated that TIP39-induced nociceptive responses in naïve mice are mediated by activation of Gs and PKA, being consistent to the findings with COS-7 cells expressing the PTH2 receptor (Wang et al. 2000; Hoare and Usdin 2001). As it is known that Gs-PKA activation has an important role in nociceptor sensitization (Hucho and Levine 2007), the administration of TIP39 may also cause robust sensitization toward physical stimuli, as seen in the report that i.pl. injection of 8-Br-cAMP or forskolin (an activater of adenylate cyclase) in naïve rat produced significant mechanical hyperalgesia (Dina et al. 2005).
It should be noted that the nociceptive responses mediated through PTH2 receptors were significantly enhanced in nerve injury-induced neuropathic pain. In the present study, we adopted the ABL test, in which mice are allowed to bite and lick their paw freely to alleviate increased hyperalgesia following injury. The hyperalgesia is unlikely to result from quantitative change in PTH2 receptor expression, as there were no significant changes in the PTH2 receptor protein and gene expression in DRG. Instead the cause seems to be related to post-PTH2 receptor mechanisms, which may include the hypersensitization at the level of primary afferents (Ueda 2006; Hucho and Levine 2007) and spinal neurons (Ji et al. 2003). The involvement of post-PTH2 receptor mechanisms was confirmed by the finding that nociceptive behaviors induced by 8-Br-cAMP (i.pl.) was significantly enhanced in nerve-injured mice, possibly through mimicking post-PTH2 receptor signaling. Indeed, the pre-treatment with H-89, a PKA inhibitor, specifically blocked the hyperalgesia to TIP39 or to 8-Br-cAMP in nerve-injured mice. Thus, unidentified mechanisms at the level of PKA activation, or further downstream, seem to underlie the hypersensitivity of PTH2 receptor-expressing A-fibers in neuropathic pain.
One of the targets for PKA would be TRPV1, whose expression occurs in A-fibers following the partial ligation of sciatic nerve of mice, though it is predominantly expressed in C-fibers of naïve mice (Rashid et al. 2003). In the present study, we found that co-expression of TRPV1 and PTH2 was significantly increased in the DRG neurons following nerve injury, which strongly suggests functional interaction in terms of nociceptor sensitization. There are a number of reports that PKA activation causes neuronal hyper-excitability by increasing TRPV1 currents (Lopshire and Nicol 1998; Bhave et al. 2002). Other possible targets involved in PKA-mediated A-fiber activation would be voltage-dependent Nav1.3 and calcium channel α2δ-1 subunit in A-fibers, which are up-regulated by nerve injury (Kim et al. 2001; Hains et al. 2004). Thus, these mechanisms seem to facilitate generation of action potentials in nociceptive A-fibers, causing hyperalgesic nociceptive response in neuropathic pain. This speculation may be supported by a previous study in which local delivery of a PKA inhibitor to DRG suppressed neuropathic thermal hyperalgesia (Song et al. 2006).
In conclusion, we provide evidence that PTH2 receptor-expressing sensory neurons are nociceptive glutamatergic A(δ)-fibers, and that TIP39 as the endogenous ligand of PTH2 receptor is a novel tool to activate this type of nociceptive fiber, and to examine its role in neuropathic hyperalgesia followed by nerve injury. Thus, the PTH2 receptor may be a valuable neurochemical marker that will facilitate investigation of nociceptive A-fibers involved in neuropathic pain.
TIP39 stimulates glutamate-NMDA receptor pain transmission.
Hypersensitization of responses to TIP39 in nerve injured ddY mice.
We thank Makoto Inoue for his kind discussion on experimental design. The research described in this article was supported by MEXT KAKENHI-S to H.U. (17109015) and MEXT KAKENHI-C to M.M. (21600007), and Health Labour Sciences Research Grant “Third Term Comprehensive Control Research for Cancer” (398-49) from the Ministry of Health, Labor and Welfare of Japan to H.U. T. U. was supported by the Intramural Program of the National Institute of Mental Health, NIH, USA.