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. 2016 Mar;157(3):698-709.
doi: 10.1097/j.pain.0000000000000421.

Adenosine-A1 receptor agonist induced hyperalgesic priming type II

Dioneia Araldi  1 , Luiz F Ferrari, Jon D Levine
Affiliations

Adenosine-A1 receptor agonist induced hyperalgesic priming type II

Dioneia Araldi et al. Pain. 2016 Mar.

Abstract

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala, N-Me-Phe, Gly-ol]-enkephalin acetate salt) induces a model of transition to chronic pain that we have termed type II hyperalgesic priming. Similar to type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, type II hyperalgesic priming differs from type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that, as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N-cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced type II hyperalgesic priming. In this study, we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms, as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor.

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Figures

Fig. 1
Fig. 1. Repeated exposure to CPA induces acute mechanical hyperalgesia and prolongation of PGE2 hyperalgesia in male rats
A. Male rats received repeated (hourly x4) intradermal injections of vehicle (control, black bars) or CPA (1 µg, white bars) on the dorsum of the hind paw, and the mechanical nociceptive threshold was evaluated at the injection site 30 min after the 1st, 3rd and 4th administration, by the Randall-Sellitto paw withdrawal test. After 3 injections of CPA, significant mechanical hyperalgesia was observed, which increased in magnitude after a 4th injection (F1,20 = 77.41; ***p < 0.001, when both groups are compared; two-way repeated measures ANOVA followed by Bonferroni post hoc test), demonstrating that repeated administration of CPA produces changes in the function of the nociceptor; B (Left panel). Rats that were treated with 4 injections of vehicle (black bars) or CPA (white bars) one week before, received PGE2 (100 ng), injected at the same site. Mechanical nociceptive threshold was evaluated 30 min and 4 h later. Of note, at the time PGE2 was injected, the mechanical nociceptive thresholds were not different from pre CPA-injection control baseline (t5 = 0.3384; p = 0.7488, for the vehicle group; t5 = 1.112; p = 0.3165, for the CPA group; paired Student’s t-test). In both groups PGE2 induced significant hyperalgesia. However, while in the repeated vehicle-treated group the effect of PGE2 was no longer present at the 4th h, in the group previously treated with repeated injection of CPA (hourly x4) the hyperalgesia induced by PGE2 was still present, indicating the presence of priming (***p < 0.0001, when both groups are compared at the 4th h, two-way repeated measures ANOVA followed by Bonferroni post hoc test); B (Right panel). At 38 days after injection of the vehicle (x4) or CPA (x4) treatments [no difference in the average mechanical nociceptive thresholds from pre-treatments levels was observed: p = 0.0706 for the vehicle (t5 = 2.291) and p = 0.5717 for the CPA group (t5 = 0.6049), paired Student’s t-test], PGE2 was injected at the same site. We observed that it produced prolonged hyperalgesia in the group previously treated with CPA (x4), but not in the vehicle-treated control group (***p < 0.0001, when both groups are compared at the 4th h, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the repeated injection of CPA produced long-term plastic changes in nociceptors; C. Mechanical nociceptive threshold was evaluated 1, 3, 5, 10, 15, 20 and 30 min after a 3rd injection of CPA (1 µg) on the dorsum of the hind paw in male rats. Significant hyperalgesia was already observed 5 min after the 3rd injection (*p < 0.05, **p <0.005 and ***p < 0.0005, when compared with the baseline (BL), paired Student’s t-test). (n = 6 paws per group)
Fig. 2
Fig. 2. Type II priming induced by repeated exposure to CPA is not dependent on IB4-positive neurons or local protein translation
A. (Left panel). Male rats were treated with vehicle (control, black bars) or IB4-saporin (3.2 µg/20 µL; white bars), by intrathecal injection. 2 weeks later, CPA (1 µg) was injected (hourly, x4) on the dorsum of the hind paw. Significant mechanical hyperalgesia was observed in both groups after the 3rd injection of CPA. However, in the IB4-saporin-treated group the magnitude of the CPA-induced hyperalgesia was significantly higher than in the vehicle-treated group (F1,20 = 6.94; ***p < 0.001, when both groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); A. (Right panel). Six days later, when the mechanical thresholds were not different from pre-CPA baseline, (t5 = 1.419; p = 0.2150, for the control group; t5 = 0.4732; p = 0.6560, for the IB4-saporin group, paired Student’s t-test), PGE2 (100 ng) was injected intradermally at the same site on the dorsum of the hind paw, and the mechanical hyperalgesia evaluated 30 min and 4 h later. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test showed PGE2-induced hyperalgesia at 30 min, that was still present at the 4th h after injection, in both groups, with no significant difference between the groups (p > 0.05, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that IB4-positive nociceptors do not contribute to the prolonged hyperalgesia induced by PGE2 observed in the type II priming; B. Male rats that were treated with repeated intradermal administration of CPA (1 µg, hourly, x4) on the dorsum of the hind paw received, one week later, an injection of PGE2 (100 ng) at the same site, in the presence of vehicle (control, black bars) or the inhibitor of protein translation, cordycepin (1 µg, white bars), administered 15 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after injection of PGE2. We observed no difference between the groups in the prolongation of the PGE2-induced hyperalgesia (F1,20 = 0.19; p = 0.6737, when both groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). (n = 6 paws per group)
Fig. 3
Fig. 3. Repeated exposure to CPA induces acute mechanical hyperalgesia and prolongation of PGE2 hyperalgesia in female rats
Left panel. Female rats received repeated hourly (x4) intradermal injections of CPA (1 µg, white bars) or vehicle (black bars) on the dorsum of the hind paw. The mechanical nociceptive threshold was evaluated before and 30 min after the 1st, 3rd and 4th administration. Significant hyperalgesia was observed after the 3rd injection of CPA (F1,30 = 203.98; ***p < 0.0001, when both groups are compared, two-way repeated measures ANOVA followed by Bonferroni post hoc test); Right panel. One week later, when the mechanical thresholds were not different from pre-vehicle or pre-CPA baseline levels (t5 = 0.8155; p = 0.4519, for the vehicle group; t5 = 1.112; p = 0.3165, for the CPA group, paired Student’s t-test), PGE2 (100 ng) was injected at the same site, and the mechanical hyperalgesia evaluated 30 min and 4 h later. One way ANOVA followed by Bonferroni post hoc test showed that PGE2 induced significant hyperalgesia that was still present 4 h after its injection (F1,20 = 24.99; ***p < 0.001, when compared to the vehicle group). Taken together, these data demonstrate that, as in males, repeated injection of CPA also produces neuroplasticity in female rats. (n = 6 paws per group)
Fig. 4
Fig. 4. PKA but not PKCε plays a role in the expression of Type II priming induced by repeated exposure to CPA
A. Male rats that were treated with repeated intradermal administration of CPA (1 µg, hourly, x4) on the dorsum of the hind paw received, 4 days later, an injection of PGE2 (100 ng) at the same site, in the presence of vehicle (control, black bars) or the PKCε inhibitor PKCεV1-2 (1 µg, white bars), administered 10 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after PGE2. We observed no difference between the groups in the prolongation of the PGE2-induced hyperalgesia (F1,10 = 0.27; p = 0.6167, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the prolongation of PGE2-induced hyperalgesia in Type II priming produced by CPA is not dependent on PKCε; B (Left panel). Rats were treated with intradermal injection of vehicle (control, black bars) or H-89 (1 µg, white bars) on the dorsum of the hind paw. 10 min later, 4 injections of CPA (1 µg, hourly) were performed in both groups of rats. We observed, in the group pretreated with H-89, significant attenuation of the mechanical hyperalgesia induced by the 4th injection of CPA, when compared with the control group (F1,10 = 22.76; ***p < 0.001, two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Four days later, a time point when the mechanical nociceptive threshold was not different from the pre-treatment levels (t5 = 0.3384; p = 0.7488, for the control group; t5 = 0.5547; p = 0.6030, for the H-89 group; paired Student’s t-test), PGE2 (100 ng) was injected at the same site, and the mechanical hyperalgesia evaluated 30 min and 4 h later. Although the hyperalgesia induced by PGE2 was present 30 min after injection in the group previously treated with H-89, it was significantly inhibited at the 4th h (F1,20 = 35.11, ***p < 0.001, when both groups are compared at the 4th h; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); B (Right panel). To determine if Type II priming was permanently prevented by H-89, PGE2 was injected again, at the same site, 10 days later. At this time we observed that the hyperalgesia induced by PGE2 was prolonged in both groups; C. Rats received repeated (hourly, x4) intradermal injection of CPA (1 µg) on the dorsum of the hind paw. Four days later, when the mechanical thresholds were not different from pre-CPA levels (t5 = 0.3953; p = 0.7089, for the control group; t5 = 0.4060; p = 0.7015, for the H-89 group; paired Student’s t-test), vehicle (control, black bars) or H-89 (1 µg, white bars) was injected at the same site, followed, 10 min later, by PGE2 (100 ng). While PGE2-induced hyperalgesia was still present 4 h later in the group that received vehicle, in the group treated with H-89 it was attenuated at both time points (F1,20 = 119.89 ***p < 0.0001, two-way repeated-measures ANOVA followed by Bonferroni post hoc test). (n = 6 paws per group)
Fig. 5
Fig. 5. Role of inhibitory G-protein αi subunit in CPA-induced Type II priming
A (Left panel). Rats received an intradermal injection of vehicle (control, black bars) or pertussis toxin (PTX, 1 µg, white bars) on the dorsum of the hind paw. 30 min later, CPA (1 µg) was injected (hourly x4) in the same site. We observed significant inhibition of the mechanical hyperalgesia induced by the repeated injection of CPA in the group treated with PTX, compared with the vehicle group (F1,20 = 135.49; ***p < 0.0001; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating a role of the αi subunit in the hyperalgesia induced by repeated injection of CPA; A (Right panel). Four days later, when the mechanical thresholds were not different from the pre-CPA injections levels, rats received intradermal injection of PGE2 (100 ng) in the same site, and the mechanical hyperalgesia evaluated 30 min and 4 h later. A significant difference was observed in the mechanical hyperalgesia induced by PGE2 at 4th h after injection (F1,20 = 14.43; ***p < 0.001, when comparing both groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the αi subunit plays a role in the prolongation of PGE2 hyperalgesia in the type II priming; B. In order to evaluate if the reversal of Type II priming by treatment with PTX was permanent, PGE2 was again injected, in the same site, 10 days after the PTX injection. We observed that PGE2-induced mechanical hyperalgesia was still present at the 4th h, indicating the presence of Type II priming. (n = 6 paws per group)
Fig. 6
Fig. 6. Type II priming induced by repeated exposure to CPA is not dependent on the G-protein β/γ subunit
Rats were treated with intradermal injection of vehicle (control, black bars) or the G-protein β/γ inhibitor, gallein (1 µg, white bars), on the dorsum of the hind paw. 30 min later, repeated injections of CPA (1 µg, hourly, x4) were performed at the same site, and the mechanical nociceptive threshold evaluated 30 min after the 3rd and 4th administration. We observed significant mechanical hyperalgesia, with no significant difference, in both groups (F1,20 = 0.69, p = 0.4261, when both groups are compared, two-way repeated-measures ANOVA followed by Bonferroni post hoc test). One week later, a time point when the mechanical thresholds were not different from pre-injection levels (t5 = 1.661; p = 0.1576, for the vehicle group, and t5 = 0.7559; p = 0.4838, for the gallein group; paired Student’s t-test), rats received an intradermal injection of PGE2 (100 ng), at the same site, and the mechanical nociceptive threshold was evaluated 30 min and 4 h later. In both groups, PGE2 induced mechanical hyperalgesia was still present 4 h after injection (F1,20 = 3.78, p = 0.0808, when control and gallein groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the inhibition of the β/γ subunit did not affect the induction of Type II priming by repeated injection of CPA. (n = 6 paws per group)
Fig. 7
Fig. 7. Second messengers involved in Type II priming: PLC-β3 and Src
A (Left panel). Rats were treated with daily spinal intrathecal injections of ODN MM (black bars) or AS (white bars) for PLC-β3 mRNA, for 3 consecutive days. On the 4th day, repeated (hourly x4) intradermal injections of CPA (1 µg) on the dorsum of the hind paw were performed, and the mechanical nociceptive threshold evaluated 30 min after the 3rd and 4th CPA injection. We observed that in the AS-treated group, but not in the MM-treated group, after the 3rd and 4th injection of CPA the mechanical hyperalgesia was significantly inhibited (F1,20 = 164.20; ***p < 0.0001, when both groups are compared, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), suggesting a role for PLC-β3 in Type II hyperalgesic priming. The ODN treatment continued for 2 more days, and, at a time point when the mechanical thresholds were not different from the pre-CPA levels (t5 = 0.6956; p = 0.5177, for the ODN MM group; t5 = 0.5423; p = 0.6109, for the ODN AS group; paired Student’s t-test), PGE2 (100 ng) was administered in both groups. Evaluation of the mechanical thresholds 30 min and 4 h after injection showed that, while PGE2 induced hyperalgesia that was still present 4 h later in the group treated with MM, in the group treated with AS it was significantly attenuated at the 4th h (F1,20 = 33.23; ***p < 0.001, two-way repeated-measures ANOVA followed by Bonferroni post hoc test); A (Right panel). In order to evaluate if the induction of Type II priming was prevented by the treatment with the PLC-β3 ODN AS, PGE2 was injected, in the same site, 10 days after the last ODN injection. We observed that, in both groups, PGE2 induced mechanical hyperalgesia was still present 4 h after its injection, indicating the presence of priming; B (Left panel). Rats were treated with intradermal injection of vehicle (black bars) or SU6656 (1 µg, white bars). 10 min later, repeated injections of CPA (1 µg) were performed on the dorsum of the hind paw. We observed inhibition of the mechanical hyperalgesia induced by repeated injection of CPA in the group previously treated with SU6656, when compared with vehicle group (F1,20 = 107.42; ***p < 0.0001, two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Four days later, when the mechanical thresholds were not different from pre-CPA levels (t5 = 0.7559; p = 0.4838, for the control group; t5 = 0.3152; p = 0.7654, for the SU6656 group; paired Student’s t-test), PGE2 (100 ng) was injected at the same site and the mechanical hyperalgesia evaluated 30 min and 4 h later. In the group previously treated with SU6656, PGE2-induced hyperalgesia was significantly attenuated at the 4th h (F1,20 = 16.92; ***p < 0.001, when both groups are compared at the 30 min and 4th h, respectively; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); B (Right panel). To determine if CPA-induced Type II priming was permanently prevented by SU6656, PGE2 was injected again, at the same site, 10 days later. At this time we observed that the hyperalgesia induced by PGE2 was prolonged in both groups; C. Male rats received repeated (hourly, x4) intradermal injections of CPA (1 µg) on the dorsum of the hind paw. Four days later, when the mechanical thresholds were not different from the pre-CPA levels (t5 = 0.1152; p = 0.9128, for the control group; t5 = 0.8305; p = 0.4441, for the SU6656 group; paired Student’s t-test), vehicle (black bars) or the Src inhibitor SU6656 (1 µg, white bars) was injected at the same site, followed by, 10 min later, PGE2 (100 ng). In the group treated with SU6656, PGE2-induced hyperalgesia was almost completely inhibited at the 4th h (F1,20 = 30.71; ***p < 0.001, two-way repeated-measures ANOVA followed by Bonferroni post hoc), indicating a role of Src in the prolongation of PGE2 hyperalgesia in CPA-induced Type II priming. (n = 6 paws per group)
Fig. 8
Fig. 8. Schematic representation of the differences in the signaling pathways in Type I and Type II hyperalgesic priming
As shown in “A” (left side), inflammatory stimuli, such as carrageenan, IL-6 or TNFα, applied at the terminal of the IB4-positive nociceptor, triggers the events that will lead to the development of Type I hyperalgesic priming. Activation of PKCε stimulates CPEB, release of calcium, activation of αCaMKII and local protein translation [5; 8; 20; 22; 52], ultimately producing neuroplastic changes that are expressed as prolongation of the PGE2-induced hyperalgesia (A, right side). PGE2 hyperalgesia, which is dependent only on PKA in the normal state [26], in the primed state is prolonged due to activation of an additional pathway involving Gαi-protein, PKCε and ERK/MEK [23]. In “B” (left side) the induction of Type II hyperalgesic priming by repeated activation of Gαi-protein coupled receptor in IB4-negative nociceptors is illustrated. The repeated administration of CPA (an A1-adenosine receptor agonist) or DAMGO (a mu-opioid receptor agonist)(*) [7], stimulates a yet-to-be determined pathway that produces neuroplastic changes in the nociceptor, also expressed as prolongation of PGE2-induced hyperalgesia (B, right side). However, in contrast to Type I, in Type II priming the prolongation of PGE2-induced hyperalgesia involves the G-protein subunits αi or β/γ, depending on the inducer (**), PKA and Src. Abbreviations: IL-6, Interleukin 6; TNFα, tumor necrosis factor alpha; PKCε, protein kinase C epsilon; CPEB, cytoplasmic polyadenylation element binding protein; αCaMKII, α calmodulin-dependent protein kinase II; ERK/MEK, extracellular signal-related kinase (ERK)⁄mitogen-activated protein kinase (MEK); GPCR, G-protein coupled receptor; PGE2, prostaglandin-E2; PKA, protein kinase A; Src, Src tyrosine kinase.
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