Estrogens Exacerbate Nociceptive Pain via Up-Regulation of TRPV1 and ANO1 in Trigeminal Primary Neurons of Female Rats

ORIGINAL RESEARCH Estrogens Exacerbate Nociceptive Pain via Up-Regulation of TRPV1 and ANO1 in Trigeminal Primary Neurons of Female Rats Kazuaki Yamagata, Mitsutaka Sugimura, Miki Yoshida, Shinichi Sekine, Akiyo Kawano, Aiko Oyamaguchi, Hiroharu Maegawa, and Hitoshi Niwa Department of Dental Anesthesiology (K.Y., M.Y., A.K., A.O., H.M., H.N.), Osaka University Graduate School of Dentistry, Suita City, Osaka, 565-0871 Japan; Department of Dental Anesthesiology (M.S.), Field of Oral Maxillofacial Rehabilitation, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima City, Kagoshima, Japan; and Division of Special Care Dentistry (S.S.), Osaka University Graduate School of Dentistry, Suita City, Osaka, Japan Several trigeminal pain disorders show sex differences, and high levels of estrogens may underlie these differences. The interaction between transient receptor potential vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) plays an important role in peripheral nociception. However, whether TRPV1 and ANO1 are involved in estrogen-modulated trigeminal pain sensitivity is unclear. In this study, we examined estradiol (E2) modulation of nociception through behavioral and immunohistological experiments after application of capsaicin (Cap), a selective TRPV1 agonist, onto the ocular surface in ovariectomized rats treated with high-dose E2 (HE) or low-dose E2 (LE) for 2 days. In addition, we used real-time PCR to study the effects of E2 on the expression levels of TRPV1 and ANO1 mRNA in trigeminal ganglia. In the behavioral experiment, the HE group showed significant potentiation of Cap-evoked nocifensive behavior compared with the LE group. Immunohistochemistry showed that Cap evoked a significantly greater number of cells that were immunoreactive for c-Fos, a marker of nociceptive activation, in the trigeminal subnucleus caudalis/upper cervical cord in the HE group than in the LE group. The number of c-Fos-immunoreactive cells in the ventral trigeminal interpolaris/caudalis were similar in the 2 groups. Real-time PCR showed that the levels of TRPV1 and ANO1 mRNA in the HE group were significantly higher than levels in the LE group. Thus, high levels of estrogens may be a risk factor for Cap-evoked nociceptive pain, and estrogen-dependent increases in TRPV1 and ANO1 are likely involved in modulating the nociceptive response in the trigeminal area. (Endocrinology 157: 4309 – 4317, 2016) n certain pain disorders, women experience more severe levels of pain, more frequent pain, and a longer duration of pain than men (1–3). Several trigeminal pain disorders, including temporomandibular disorders, trigeminal neuralgia, and burning tongue, show similar sex differences (4 – 6). Furthermore, several of these disorders are exacerbated in the presence of high levels of the typical female hormone estrogens (7). Therefore, high levels of estrogens may be a risk factor for trigeminal pain disorders in women. I Estrogens affect cells through genomic and nongenomic pathways (8). In the former pathway, estrogens bind to estrogen receptors (ERs), which are located in the cytoplasm and nucleus, and form an estrogen-ER complex. This complex binds to estrogen-response element sequences in the promoter region of some genes and modulates the levels of associated mRNAs and proteins. ERs are expressed in the rat trigeminal ganglia (TG), and their activation alters the expression of certain mRNAs and ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in USA Copyright © 2016 by the Endocrine Society This article is published under the terms of the Creative Commons Attribution-Non Commercial License (CC-BY-NC; https://creativecommons.org/licenses/by-nc/4.0/). Received April 5, 2016. Accepted September 27, 2016. First Published Online September 30, 2016 Abbreviations: ANO1, anoctamin 1; Cap, capsaicin; E2, 17␤-estradiol; ER, estrogen receptor; HE, high-dose E2; IR, immunoreactive; LE, low-dose E2; OVX, ovariectomized; TG, trigeminal ganglia; TRPV1, transient receptor potential vanilloid 1. doi: 10.1210/en.2016-1218 Endocrinology, November 2016, 157(11):4309 – 4317 press.endocrine.org/journal/endo 4309 4310 Yamagata et al Estrogens Up-Regulate Expression of TRPV1 and ANO1 proteins in sensory neurons (8, 9). These observations suggest that estrogens may modulate pain sensitivity by affecting signaling pathways in the TG that are involved in peripheral nociception. Recent work has shown that the interaction between transient receptor potential vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) is a significant pain-enhancing mechanism in peripheral nociception (10). TRPV1 is a polymodal sensor that is activated by heat (43°C), acid, or capsaicin (Cap) (11–13). TRPV1 is up-regulated and activated in the mouse TG after peripheral nerve injury, resulting in thermal hyperalgesia (14). The expression of TRPV1 is abnormally high in patients with burning mouth syndrome (15). Thus, TRPV1 plays an important role in the signaling pathways involved in TG peripheral nociception. ANO1, which is also known as the transmembrane protein 16A component of Ca2⫹-activated chloride channels, possesses 8 transmembrane domains and conducts chloride current that is activated by intracellular Ca2⫹. Cho and Oh (16) reported that ANO1 is highly colocalized with TRPV1 in small-diameter dorsal root ganglion neurons and is activated by temperatures over 44°C. Takayama et al (10) reported that Cap-evoked depolarization is composed of 2 components: calcium ion influxmediated depolarization caused by TRPV1 activation and subsequent chloride ion efflux, which is mediated by ANO1 activation caused by the entry of calcium ions through TRPV1. In addition, 76.2% of ANO1-positive small- or medium-diameter TG neurons coexpress TRPV1 (17). Thus, similar to TRPV1, ANO1 plays an important role in the signaling pathways involved in TG peripheral nociception. However, whether TRPV1 and ANO1 are involved in estrogen-modulated trigeminal pain sensitivity is unclear. In the present study, we examined estradiol (E2) modulation of orofacial nociception after Cap application onto the ocular surface. The ocular surface is suitable for nociception studies in the trigeminal system, because the nociceptive receptors on the ocular surface have a large representation in the TG via the ophthalmic branch of the trigeminal nerve, and the ocular surface receives the densest nociceptive innervation of the body (18). Thus, to determine whether the estrogen status modulates orofacial nociception, we performed behavioral and immunohistological experiments after Cap application onto the ocular surface in ovariectomized (OVX) rats treated with E2 for a short period. In addition, we used real-time PCR (RTPCR) to study the effects of estrogens on the expression levels of TRPV1 and ANO1 mRNA in TG. Endocrinology, November 2016, 157(11):4309 – 4317 Materials and Methods Experimental procedures The study protocols for animal experiments were appro (...truncated)