Abstract
Hyperalgesic priming, an estrogen dependent model of the transition to chronic pain, produced by agonists at receptors that activate protein kinase C epsilon (PKCε), occurs in male but not in female rats. However, activation of second messengers downstream of PKCε, such as the ryanodine receptor, induces priming in both sexes. Since estrogen regulates intracellular calcium, we investigated the interaction between estrogen and ryanodine in the susceptibility to develop priming in females. The lowest dose of ryanodine able to induce priming in females (1 pg) is 1/100,000th that needed in males (100 ng), an effect dependent on the activation of ryanodine receptors. Treatment of female rats with antisense to estrogen receptor alpha (ERα), but not beta (ERβ), mRNA, prevented the induction of priming by low dose ryanodine, and the ERα agonist, PPT, induced ryanodine receptor-dependent priming. In vitro application of ryanodine in low concentration (2 nM) to small DRG neurons cultured from females, significantly potentiated calcium release via ryanodine receptors induced by caffeine. This effect was only observed in IB4+ neurons, cultured in the presence of β-estradiol or PPT. Our results demonstrate a profound regulatory role of ERα in ryanodine receptor-dependent transition to chronic pain.
Highlights
Activation of mechanisms downstream of PKCε in the induction of priming, e.g. by ryanodine – modulator of the ryanodine receptor, which can release calcium from the endoplasmic reticulum at low concentration18,19 – and calcium/calmodulin-dependent protein kinase II alpha, induces priming in both sexes[20]
Since estrogen-dependent sexual dimorphism in mechanisms for calcium homeostasis has been demonstrated in diverse cell types[21,22], including in neurons[25,26], we tested the hypothesis that the observed resistance to priming induced by activation of PKCε in female rats[13], was due to the requirement for more intense stimuli to activate second messenger signaling pathways downstream of PKCε (e.q., a higher concentration of ryanodine could induce priming in the female rat)
In marked contrast to our initial hypothesis, we found that ryanodine induces priming in the female rat, expressed as prolongation of the hyperalgesia induced by prostaglandin E2 (PGE2), observed 4 h after its injection[12,20], at a markedly lower dose than needed to induce priming in the male
Summary
Activation of mechanisms downstream of PKCε in the induction of priming, e.g. by ryanodine – modulator of the ryanodine receptor, which can release calcium from the endoplasmic reticulum at low concentration18,19 – and calcium/calmodulin-dependent protein kinase II alpha (αCaMKII), induces priming in both sexes[20]. In the groups of female rats that had received doses of ryanodine 1 pg and higher, but not 0.1 pg, and in the group of male rats previously treated with 100 ng of ryanodine, but not with 100 pg, 10 ng or 30 ng, the hyperalgesia induced by PGE2 was still present at the 4th h (female rats: 0.1 pg, t5 = 1.170, p = 0.1479 (NS); 1 pg, t5 = 5.509, p = 0.0027 (**); 10 pg, t5 = 11.59, p < 0.0001 (****); 30 pg, t5 = 20.07, p < 0.0001 (****); 100 pg, t5 = 12.25, p < 0.0001 (****); 10 ng, t5 = 15.41, p < 0.0001 (****); male rats: 100 pg, t5 = 1.151, p = 0.3019 (NS); 10 ng, t5 = 2.534, p = 0.0522 (NS); 30 ng, t5 = 0.0, p > 0.9999 (NS); 100 ng, t5 = 28.76, p < 0.0001 (****), when the mechanical nociceptive thresholds before and 4 h after the injection of PGE2, for each group, are compared, paired Student’s t-test). Since estrogen regulates intracellular calcium homeostasis[21,22], as well as priming, we determined if any dependence of sex differences in ryanodine-induced priming was dependent on the action of estrogen at either of its classic receptors, estrogen receptor alpha (ERα) and/or estrogen receptor beta (ERβ), both present in nociceptors[23,24]
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