Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 22;37(8):2032-2044.
doi: 10.1523/JNEUROSCI.2911-16.2017. Epub 2017 Jan 23.

Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming

Eugen V Khomula  1 , Luiz F Ferrari  1 , Dionéia Araldi  1 , Jon D Levine  2
Affiliations

Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming

Eugen V Khomula et al. J Neurosci. .

Abstract

Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 μm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with β-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3 IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of β-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming.SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic reticulum receptors, IP3 and ryanodine, in the induction of priming, regulated by estrogen receptor α in the nociceptor of female rats. The presence of this signaling pathway modulating the susceptibility of nociceptors to develop plasticity may contribute to our understanding of sex differences observed clinically in chronic pain syndromes.

Keywords: IP3 receptor; endoplasmic reticulum; hyperalgesia; hyperalgesic priming; nociceptor; ryanodine receptor.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Ryanodine-induced priming is IP3 receptor dependent. The IP3 receptor antagonist xestospongin C (0.2 μg, black bars) was injected intradermally on the dorsum of the hindpaw of male (left) and female (right) rats; control groups (white bars) received vehicle. After 10 min, the doses of ryanodine previously shown to induce priming (100 ng in males and 1 pg in females) were injected at the same site. One week later, evaluation for the presence of priming was performed by intradermal injection of PGE2 (100 ng) at the same site as ryanodine. Two-way repeated-measures ANOVA, followed by Bonferroni post hoc test, showed a significant attenuation of the hyperalgesia induced by PGE2 at the fourth hour, in groups that had been pretreated with xestospongin C, compared with the control groups, pretreated with vehicle (males: F(1,5) = 130.6; ***p < 0.0001; females: F(1,5) = 89.10; ****p = 0.0002, when the hyperalgesia in the vehicle- and xestospongin C-treated groups is compared at the fourth hour), demonstrating that the induction of priming by ryanodine is dependent on the activation of IP3 receptors (N = 6 paws per group).
Figure 2.
Figure 2.
Induction of hyperalgesic priming by IP3 in male and female rats. a, Different doses of IP3 were injected on the dorsum of the hindpaw in different groups of female (open circles represent 10 pg; 100 pg; 1 ng; 10 ng; 100 ng; 1 and 10 μg) and male (filled circles represent 1 μg; 3 μg; 10 μg) rats. No change in the mechanical nociceptive threshold was observed after the injection of IP3 (data not shown). PGE2 (100 ng) was injected at the same site, 1 week later, and the mechanical hyperalgesia evaluated by the Randall-Sellitto paw-withdrawal test. This figure shows the mechanical hyperalgesia at the fourth hour after the injection of PGE2; the presence of hyperalgesia at that time point was used to confirm the induction of priming by the previous injection of IP3. In the groups of female rats that had received a dose of 100 pg and higher, and in the group of male rats previously treated with 3 or 10 μg, but not with 1 μg, the hyperalgesia induced by PGE2 was still present at the fourth hour (female rats: 10 pg, t(5) = 1.627, p = 0.1647 (not significant, NS); 100 pg, t(5) = 8.788, p = 0.0003 (***); 1 ng, t(5) = 27.81; 10 ng, t(5) = 12.59; 100 pg, t(5) = 21.24; 1 μg, t(5) = 15.53; 10 μg, t(5) = 13.94, all p < 0.0001 (****); male rats: 1 μg, t(5) = 0.4385, p = 0.6793 (NS); 3 μg, t(5) = 16.54; 10 μg, t(5) = 23.27, both 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). These results support the suggestion that nociceptors in females are significantly more sensitive to induction of priming by IP3 because a dose much lower was required. b, Groups of male (left) and female (right) rats received an injection of vehicle (white bars), the IP3 receptor inhibitor xestospongin C (0.2 μg, light gray bars), the SERCA inhibitor thapsigargin (1 μg, dark gray bars), or the ryanodine receptor inhibitor dantrolene (1 μg, black bars) on the dorsum of the hindpaw. Ten minutes later, the smallest doses of IP3 that induced priming (a; 3 μg in male; 100 pg in female) were injected at the same site. No significant change in mechanical nociceptive threshold was observed after injection of IP3 (data not shown). One week later, the presence of priming was determined by the evaluation of the prolonged mechanical hyperalgesia induced by intradermal injection of PGE2 (100 ng), at the same site as IP3. Two-way repeated-measures ANOVA, followed by Bonferroni post hoc test, showed that, while the hyperalgesia induced by PGE2 in control groups (vehicle-treated) was still present 4 h after injection, and not different from the 30 min time point, in the groups pretreated with xestospongin C, thapsigargin, or dantrolene, its magnitude was significantly smaller at the 4 h time point (males, xestospongin C group: F(1,5) = 81.30, ***p = 0.0003; thapsigargin group: F(1,5) = 31.11, **p = 0.0026; dantrolene group: F(1,5) = 44.44, *p = 0.0011; females: xestospongin C group: F(1,5) = 230.5, ****p < 0.0001; thapsigargin group: F(1,5) = 58.63, ##p = 0.0006; dantrolene group: F(1,5) = 16.48, ****p < 0.0001, when the hyperalgesia in the vehicle- and inhibitor-treated groups is compared at the fourth hour), indicating that the induction of priming by IP3 is dependent on the activation of IP3 and ryanodine receptors (N = 6 paws all groups).
Figure 3.
Figure 3.
EsRα regulates the induction of hyperalgesic priming by IP3 in females. a, Female rats were treated with ODN AS (black bars) or MM (white bars) for EsRα mRNA, for 6 consecutive days. IP3 (100 pg, left; 3 μg, right) was injected on the dorsum of the left hindpaws on the fourth day of ODN treatment. On the seventh day, PGE2 (100 ng) was injected at the same site as IP3, and the mechanical nociceptive threshold evaluated, 30 min and 4 h later. No significant difference was observed in the mechanical nociceptive thresholds before the injections of IP3 and immediately before injection of PGE2 (data not shown). PGE2-induced hyperalgesia was still present 4 h after injection in all groups, except in the group that received the low dose of IP3 (100 pg) treated with ODN AS (F(1,10) = 31.89; **p = 0.0002, when the low-dose groups treated with ODNs are compared; F(1,10) = 0.1479; p = 0.7086, not significant, when the high-dose groups treated with ODNs are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These results support the suggestion that EsRα regulates the ability of a low dose of IP3 to induce priming in the female rat. b, Female rats received an intradermal injection of vehicle (white bars) or the IP3 receptor inhibitor xestospongin C (0.2 μg, black bars) on the dorsum of the hindpaw. Ten minutes later, the specific EsRα agonist PPT (1 μg) was injected at the same site. After 1 week, testing for the presence of hyperalgesic priming was performed by injecting PGE2 (100 ng). Mechanical hyperalgesia was observed in both groups when evaluated 30 min after PGE2 injection. However, at the fourth hour, the magnitude of the PGE2-induced hyperalgesia was significantly smaller in the group previously treated with xestospongin C (F(1,5) = 69.81, ***p = 0.0004; when both groups are compared at the fourth hour; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the inhibition of IP3 receptors prevented the induction of priming by PPT (N = 6 paws all groups).
Figure 4.
Figure 4.
IP3 in the presence of β-estradiol potentiates depolarization-induced calcium transients. a, Representative recording of calcium transients in an IB4+ small DRG neuron incubated without β-estradiol. Calcium transients were induced by two identical depolarizing stimuli (arrows indicate short applications of K20, 20 mm), before and after exposure to IP3 (gray horizontal bar represents 400 μm), which was applied for 10 min when [Ca2+]i returned to baseline after the first application of K20. No changes in the response to K20, even after the application of IP3, and no changes in baseline in response to application of IP3 by itself, are observed. b, Pooled relative changes in the amplitudes of the second response to K20, as percentage of the first response in the following: neurons incubated without β-estradiol submitted to application of vehicle between the applications of K20 (as control, white bar, N = 32), neurons incubated without β-estradiol submitted to the application of IP3 alone (as described in a, gray bar, N = 34), and neurons incubated with β-estradiol (100 nm) submitted to application of vehicle between applications of K20 (black bar, N = 28). Negative values indicate lack of potentiation of the response to K20 application in all cases. The absence of a significant difference among the groups (F(2,91) = 2.22, p = 0.12; one-way ANOVA) suggests that neither the exposure to IP3 alone nor the incubation with β-estradiol by itself significantly affects the responses to K20 compared with control conditions (no IP3/no β-estradiol). c, Recordings of calcium transients in IB4+ small DRG neurons, incubated in the presence of β-estradiol (100 nm). IP3 was applied as described in a. Although no response was produced by IP3 itself, a significant potentiation of the response to K20 occurred after exposure to IP3 (second transient). d, Pooled relative changes in the amplitudes of the second response to K20, as percentage of the first response, in IB4+ small DRG neurons incubated in the presence of β-estradiol (100 nm), after exposure to different concentrations of IP3: 0 (N = 28, the same as the black bar in b); 0.1 μm (N = 4); 1 μm (N = 23); 10 μm (N = 13); 100 μm (N = 13, same recording shown in c); and 400 μm (N = 21). Maximum potentiation was observed for 100 μm of IP3, with no significant difference between the magnitudes of potentiation produced by 100 and 400 μm (t(25) = 0.34, p = 0.73; two-tailed unpaired Student's t test with Welch's correction). Therefore, all further experiments were performed with 100 μm of IP3. a, c, Horizontal scale bars: 200 s; vertical bars, 0.2 arbitrary units (a.u.) of the fluorescence ratio F340/F380. b, + and −, located under the bars, indicate presence and absence of the corresponding agent, respectively.
Figure 5.
Figure 5.
Potentiation of depolarization-induced calcium transients by IP3 is dependent on both IP3 and ryanodine receptors. Pooled relative changes in the amplitudes of the second response to K20, as percentage of the first response, after exposure to IP3 (100 μm) in IB4+ small DRG neurons, incubated in the presence of β-estradiol (100 nm), shows lack of potentiation in the neurons preincubated with the selective IP3 receptor antagonist xestospongin C (xestC, 1 μm, light gray bar; N = 25), or the selective ryanodine receptor antagonist dantrolene (dantrl, 1 μm, dark gray bar; N = 13). The “vehicle alone” group (white bar) and the “vehicle + IP3” group (black bar) are the “0 μm” and “100 μm” groups, respectively, from Figure 4d and are shown here for comparison. Both antagonists significantly attenuated the potentiation of the response to the second application of K20 compared with the control group (black bar) (F(3,75) = 39, ***p < 0.0001; one-way ANOVA followed by Tukey's multiple comparison test). No significant difference was observed compared with the “vehicle alone” group (white bar).
Figure 6.
Figure 6.
Potentiation of depolarization-induced calcium transients by ryanodine is dependent on IP3 receptors and enhanced in the presence of β-estradiol. a, Pooled relative changes in the amplitude of the second response to K20 after exposure to ryanodine (2 nm) in IB4+ small DRG neurons, incubated without (control, white bar, N = 36) or in the presence of β-estradiol (100 nm, black bar, N = 28), reveal significant potentiation in both groups (positive values significantly >0: control group, t(35) = 4.1, p = 0.0002; β-estradiol group, t(27) = 6.5, p < 0.0001). Importantly, potentiation was significantly higher in the group incubated with β-estradiol (t(58) = 2.1, *p = 0.04) when both groups are compared (two-tailed unpaired Student's t test with Welch's correction). b, Pooled relative changes in the amplitude of the second response to K20 after exposure to ryanodine (2 nm) in IB4+ small DRG neurons, incubated with β-estradiol (100 nm), show lack of potentiation in neurons preincubated with the selective IP3 receptor antagonist xestospongin C (1 μm, gray bar; N = 8). Black bar is the same as the “β-estradiol” bar in a. White bar (no ryanodine) is the same as “β-estradiol alone” from Figure 4b, shown here for comparison. Xestospongin C significantly inhibited the ryanodine-induced potentiation of calcium transients compared with the control group (black bar) (F(2,61) = 35, ***p = 0.0004; one-way ANOVA followed by Tukey's multiple comparison test).
Figure 7.
Figure 7.
Schematic diagram of proposed mechanism of reciprocal interaction between IP3 and ryanodine receptors in the induction of hyperalgesic priming. Activation of PKCε (in males, only) by the priming stimulus in the peripheral terminal of an IB4+ nociceptor (right side of figure) triggers a cascade of events that ultimately produces hyperalgesic priming (Aley et al., 2000; Joseph et al., 2003; Joseph and Levine, 2010; Ferrari et al., 2013b), which includes calcium release from ER and requires both ryanodine (RyR) and IP3 (IP3R) receptors, activation of αCaMKII, and cytoplasmic polyadenylation element binding protein (CPEB) (Ferrari et al., 2013b). Direct activation of RyR and IP3R also produces priming, in males and females. We hypothesize that release of calcium ions (Ca2+) from ER stores, causing a local increase in [Ca2+]i (dashed and magnified boxes, middle) (Ehrlich et al., 1994; MacKrill, 1999, 2012) is a key factor required for the induction of priming. The suggested interaction between RyR and IP3R, by Ca2+ released through these ionotropic receptors, is shown in the magnified box. Once outside the ER, Ca2+ can activate nearby RyR and IP3R, increasing [Ca2+]i even more, via CICR, to reach a level high enough to trigger mechanisms, such as calmodulin and αCaMKII (Shakiryanova et al., 2007, 2011; Wong et al., 2009), leading to induction of priming (Ferrari et al., 2013b). The ER calcium pump, SERCA, transports the Ca2+ released into the cytosol, back to stores, to refill them, restricting Ca2+ signals spatially and temporally, a “loop” mechanism that has been associated with the development of neuroplasticity (Pal et al., 2001; Scullin and Partridge, 2010; Kato et al., 2012; Komin et al., 2015; Yasuda and Mukai, 2015). Additional Ca2+ buffering in the cytosol limits the fast diffusion of Ca2+, supporting its local accumulation. Thus, the whole system of RyR and IP3R can be triggered by administration of IP3 or ryanodine. When the concentration of agonists reaches a certain level, the effective doses able to induce priming (Ferrari et al., 2016) (Fig. 2a), the local cascade of propagating CICR becomes self-supported. We believe this “small” (in the scale of the whole cell), but long-lasting, change in Ca2+ homeostasis is a crucial factor for the development of hyperalgesic priming. In contrast, when the Ca2+ signal produced is strong but of short duration, for example, that produced by calcium ionophore, due to the bell-shaped sensitivity of RyR and IP3R for [Ca2+]i, the production of the self-supported long-lasting local [Ca2+]i increase is prevented, and hyperalgesic priming does not develop. Although the direct interaction between the receptors is important to generate amplification, we consider Ca2+ a key mediator of the reported reciprocal modulation of RyR and IP3R, and the amount of Ca2+ initially released is the most important factor in the mechanism, regardless of which receptor is stimulated. This constitutes our paradigm of “the common (shared) amplifier,” postulating the amplification of Ca2+ signals produced by either input (RyR or IP3R), which explains the similarity in the difference between males and females in the sensitivity to be primed by either IP3 or ryanodine. Although this amplification system is present in both males and females, it is less potent in males (a 100,000-fold difference). In regard of the higher sensitivity of females to be primed by ryanodine or IP3, the action of estrogen on EsRα (left) potentiates the amplification system, likely by sensitizing the ER receptors, as suggested by previous reports: modulation of RyR by estrogen-PKA signaling (Lin et al., 2007) or potentiation of IP3R by estrogen through metabotropic glutamate receptor 1, phospholipase C, and generation of IP3 (Tabatadze et al., 2015) (left, large arrow). This latter pathway could also result in tonic sensitization of IP3R in females, due to an increased basal IP3 levels. As shown in Figures 4 and 6, tonic activation of EsR in cultured male nociceptors, by incubation in the presence of β-estradiol, also potentiates their sensitivity to both ryanodine and IP3. This finding suggests that the sensitizing machinery of EsR is also present in males, but probably not active enough due to a lower level (or lack) of estrogen. Nevertheless, there is still a possibility that the EsRα-dependent sensitization in females (as well as in males, when activated) is indirect, due to the suppression of some blocking pathway active in males and females when EsRα is not stimulated. Although little is known about endogenous competitive antagonists shared by RyR and IP3R, according to our “shared amplification system paradigm,” suppression of either RyR and IP3R by their antagonists, including endogenous (Lenzen and Rustenbeck, 1991; Uehara et al., 1996; Vervliet et al., 2015), could indeed reduce the sensitivity of the whole system (as shown in Figs. 1, 2b). Whether this mechanism is involved in the observed difference between males and females in our study remains to be a determined.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adasme T, Haeger P, Paula-Lima AC, Espinoza I, Casas-Alarcón MM, Carrasco MA, Hidalgo C (2011) Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation. Proc Natl Acad Sci U S A 108:3029–3034. 10.1073/pnas.1013580108 - DOI - PMC - PubMed
    1. Adasme T, Paula-Lima A, Hidalgo C (2015) Inhibitory ryanodine prevents ryanodine receptor-mediated Ca2+ release without affecting endoplasmic reticulum Ca2+ content in primary hippocampal neurons. Biochem Biophys Res Commun 458:57–62. 10.1016/j.bbrc.2015.01.065 - DOI - PubMed
    1. Alessandri-Haber N, Dina OA, Chen X, Levine JD (2009) TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization. J Neurosci 29:6217–6228. 10.1523/JNEUROSCI.0893-09.2009 - DOI - PMC - PubMed
    1. Aley KO, Levine JD (1999) Role of protein kinase A in the maintenance of inflammatory pain. J Neurosci 19:2181–2186. - PMC - PubMed
    1. Aley KO, Messing RO, Mochly-Rosen D, Levine JD (2000) Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci 20:4680–4685. - PMC - PubMed

Publication types

MeSH terms