Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat

Abstract

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N⁶-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming.

Perspective: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Keywords: Nociceptor; chronic pain; ecto-5′nucleotidase; estrogen receptor; hyperalgesic priming.

Fig. 1. AMP induces mechanical hyperalgesia in primed male and female rats

Male and female rats were divided in two groups, primed (filled symbols) and non-primed (open symbols). The priming inducer, ryanodine (100 ng in males and 1 pg in females), or its vehicle (saline) were injected on the dorsum of the right or left hind paw, respectively. One week later, AMP (1 μg) was injected at the same site. Mechanical nociceptive thresholds were then evaluated 10, 20 and 30 min after AMP injection. We observed that, in non-primed paws, AMP did not induce significant change in the mechanical nociceptive threshold, whereas in primed paws, hyperalgesia was observed at all time points in both male and female rats. Two-way repeated measures ANOVA followed by the Bonferroni post-hoc test showed significant difference between the effect of AMP in primed and non-primed paws in both male and female groups (males: F_3,30 = 18.18, ****p < 0.0001; females: F_3,30 = 8.157, ***p = 0.0004, when the primed male or female groups are compared to their respective non-primed groups). (N = 6 paws per group)

Fig. 2. AMP-induced mechanical hyperalgesia in primed male and female rats is dependent on its conversion to adenosine and activation of A1 adenosine receptors

Male (left panel) and female (right panel) rats received an intradermal injection of ryanodine (100 ng in males and 1 pg in females) on the dorsum of both hind paws. One week later, the ecto-5′nucleotidase inhibitor AMPCP (1 μg, gray symbols) was injected in the left paws, and the A1 adenosine receptor antagonist DPCPX (1 μg, black symbols) was injected in the right paws, at the same site as ryanodine. The control groups, represented by the white symbols, are the same primed groups shown in Fig. 1. After 10 min, AMP (1 μg) was injected in all paws and the mechanical nociceptive threshold evaluated, 10, 20 and 30 min later. While in primed paws AMP induced significant hyperalgesia (control groups), in the groups pretreated with AMPCP or DPCPX the AMP-induced hyperalgesia was markedly attenuated (males: AMPCP, F_1,10 = 32.22, *p = 0.0002; DPCPX, F_1,10 = 65.62, ****p < 0.0001; females: AMPCP, F_1,10 = 14.96, **p = 0.0031; DPCPX, F_1,10 = 27.07, ***p = 0.0004, when the inhibitors groups are compared to the control groups, two-way repeated measures ANOVA followed by the Bonferroni post-hoc test) demonstrating that the mechanical hyperalgesia induced by AMP in primed rats is dependent on its conversion to adenosine. (N = 6 paws per group)

Fig. 3. AMP-, but not CPA-, induced mechanical hyperalgesia in female primed rats is dependent on estrogen receptor alpha (EsRα)

A: Male (left panel) and female (right panel) rats received an intradermal injection of ryanodine (100 ng in males and 1 pg in females) on the dorsum of both hind paws. One week later, intrathecal treatment with ODN antisense or mismatch against EsRα mRNA was performed for 3 consecutive days. On the 4^th day, AMP (1 μg) was injected at the same site as ryanodine, and mechanical nociceptive threshold evaluated 10, 20 and 30 min later. While AMP induced significant hyperalgesia in both male and female rats treated with mismatch and in the male antisense group, in the group of females that had been treated with EsRα antisense, AMP-induced hyperalgesia was prevented [males: F_1,10 = 1.179, p = 0.3030 (non-significant); females: F_1,10 = 52.35, ****p < 0.0001, when the antisense and mismatch groups are compared, two-way repeated measures ANOVA followed by the Bonferroni post-hoc test] indicating a dependence on EsRα for the hyperalgesic effect of AMP in female, but not in male, primed rats; B: Female rats that had been primed with an intradermal injection of ryanodine (1 pg) on the dorsum of both hind paws, were treated, one week later, with intrathecal injections of ODN antisense or mismatch against EsRα mRNA for 3 consecutive days. On the 4^th day, CPA (1 μg) was injected at the same site as ryanodine, and mechanical nociceptive thresholds were then evaluated 10, 20 and 30 min later. No difference in the hyperalgesia induced by CPA was observed between the antisense and mismatch groups [F_1,10 = 1.700, p = 0.2215 (non-significant), when the antisense and mismatch groups are compared, two-way repeated measures ANOVA followed by the Bonferroni post-hoc test]. (N = 6 paws per group)

Fig. 4. Autocrine mechanism of hyperalgesic priming expression in male and female nociceptors

In the primed nociceptor, neuroplasticity manifests as a PKCε-dependent mechanical hyperalgesia ^{, , ,} . While in the normal nociceptor PGE₂ induces hyperalgesia by activating PKA, which lasts ~2h ², in the primed nociceptor an autocrine signaling pathway at the plasma membrane is also triggered, culminating in the activation of PKCε, prolonging the PGE₂-induced hyperalgesia to more than 4 h ^, . The activation of this additional pathway involves the transport of cAMP, produced by adenyl cyclase (AC) stimulation, across the plasma membrane into the extracellular compartment. In sequence, cAMP is metabolized to AMP and then adenosine, by ecto-phosphodiesterase (PDE) and ecto-5′nucleotidase (E5NT), respectively, ultimately activating Gi-coupled A1 adenosine receptor (A1). This stimulates PKCε, responsible for the late component of PGE₂-induced hyperalgesia in the hyperalgesic priming. While this autocrine mechanism is present in both males and females, in females (bigger circle) it is regulated by estrogen, which acts at the estrogen receptor alpha (EsRα), regulating either the expression or the activity, or both, of the E5NT, thus modulating the conversion of AMP to adenosine, and the downstream signaling that will produce PKCε-dependent hyperalgesia. The regulation of E5NT by EsRα has been suggested to involve either a genomic mechanism, regulating the expression of this enzyme, or the interaction between EsRα and E5NT, directly or through second messengers. In the first case, transcription factors such as AP-1 and Sp1, which are active at the E5NT gene promoter ^, , are regulated by activation of EsRα ^{, ,} . Of note, this interaction has been observed in hippocampal neurons, and associated to differences in neuroplasticity between the sexes . The second possibility would be the direct interaction of the receptor with the enzyme, supported by studies demonstrating co-immunoprecipitation of estrogen receptors and E5NT, suggesting that these elements can interact through physical association . In addition, activation of messengers/kinases that increase either the expression or the activity of the E5NT, such as PKC, has also been reported ^{, ,} . Hence, even though these signaling pathways have not been demonstrated in nociceptors, it is plausible that they are involved in the regulation of processes in which E5NT plays a role, therefore explaining why the knock down of EsRα in the nociceptor impairs the expression of priming in females.