Li-hua Yu

17β-Estradiol Rapidly Attenuates P2X3 Receptor-Mediated Peripheral Pain Signal Transduction via ERα and GPR30

Estrogen has been reported to affect pain perception, although the underlying mechanisms remain unclear. In this investigation, pain behavior testing, patch clamp recording, and immunohistochemistry were used on rats and transgenic mice to determine which estrogen receptors (ERs) and the related signaling pathway are involved in the rapid modulation of estrogen on P2X3 receptor-mediated events. The results showed that 17β-estradiol (E2) rapidly inhibited pain induced by α,β-methylene ATP (α,β-me-ATP), a P2X1 and P2X3 receptor agonist in ovariectomized rats and normal rats in diestrus. The ERα agonist 4,49,499-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) and G protein-coupled receptor 30 (GPR30) agonist G-1 mimicked the estrogen effect, whereas the ERβ agonist diarylpropionitrile (DPN) had no effect. In cultured rat dorsal root ganglion (DRG) neurons, PPT and G-1 but not DPN significantly attenuated α,β-me-ATP-mediated currents, with the dose-response curve of these currents shifted to the right. The inhibitory effect of E2 on P2X3 currents was blocked by G-15, a selective antagonist to the GPR30 estrogen receptor. E2 lacked this effect in DRG neurons from ERα-knockout mice but partly remained in those from ERβ-knockout mice. The P2X3 and GPR30 receptors were coexpressed in the rat DRG neurons. Furthermore, the ERK1/2 inhibitor U0126 reversed the inhibitory effect of E2 on α,β-me-ATP-induced pain and of PPT or G-1 on P2X3 receptor-mediated currents. The cAMP-protein kinase A (PKA) agonist forskolin, but not the PKC agonist phorbol-12-myristate-13-acetate (PMA), mimicked the estrogen-inhibitory effect on P2X3 receptor currents, which was blocked by another ERK1/2 inhibitor, PD98059. These results suggest that estrogen regulates P2X3-mediated peripheral pain by acting on ERα and GPR30 receptors expressed in primary afferent neurons, which probably involves the intracellular cAMP-PKA-ERK1/2 pathway.

Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

BackgroudsATP and P2X receptors play important roles in the modulation of trigeminal neuropathic pain, while the role of G protein-coupled P2Y2 receptors and the underlying mechanisms are less clear. The threshold and frequency of action potentials, fast inactivating transient K+ channels (IA) are important regulators of membrane excitability in sensory neurons because of its vital role in the control of the spike onset. In this study, pain behavior tests, QT-RT-PCR, immunohistochemical staining, and patch-clamp recording, were used to investigate the role of P2Y2 receptors in pain behaviour.ResultsIn control rats: 1) UTP, an agonist of P2Y2/P2Y4 receptors, caused a significant decrease in the mean threshold intensities for evoking action potentials and a striking increase in the mean number of spikes evoked by TG neurons. 2) UTP significantly inhibited IA and the expression of Kv1.4, Kv3.4 and Kv4.2 subunits in TG neurons, which could be reversed by the P2 receptor antagonist suramin and the ERK antagonist U0126. In ION-CCI (chronic constriction injury of infraorbital nerve) rats: 1) mRNA levels of Kv1.4, Kv3.4 and Kv4.2 subunits were significantly decreased, while the protein level of phosphorylated ERK was significantly increased. 2) When blocking P2Y2 receptors by suramin or injection of P2Y2R antisense oligodeoxynucleotides both led to a time- and dose-dependent reverse of allodynia in ION-CCI rats. 3) Injection of P2Y2 receptor antisense oligodeoxynucleotides induced a pronounced decrease in phosphorylated ERK expression and a significant increase in Kv1.4, Kv3.4 and Kv4.2 subunit expression in trigeminal ganglia.ConclusionsOur data suggest that inhibition of P2Y2 receptors leads to down-regulation of ERK-mediated phosphorylation and increase of the expression of IA–related Kv channels in trigeminal ganglion neurons, which might contribute to the clinical treatment of trigeminal neuropathic pain.

P2Y2 receptor-mediated modulation of estrogen-induced proliferation of breast cancer cells

It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y(2) receptors promote or suppress proliferation in different cancers. In the present study, the methods of methylthiazoltetrazolium (MTT) assay, real-time RT-PCR, Western blot and fluorescent calcium imaging analysis were used to investigate whether P2Y(2) receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y(2) receptors were expressed in both the estrogen receptor alpha (ER(α))-positive breast cancer cell line MCF-7 and the ER(α)-negative breast cancer cell line MDA-MB-231. 17β-Estradiol (17β-E(2)) (1 pM to 1000 nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1 μM) and the ER(α) antagonist methyl-piperidino-pyrazole (MPP, 50 μM), but not by the ER(β) antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50 μM) or ER(β) small interfering RNA. The P2Y(2) and P2Y(4) receptor agonist UTP (10-100 μM) suppressed the viability of breast cancer cells in both MCF-7 and MDA-MB-231 cells. The effect was blocked by suramin (10-100 μM), known to be an effective antagonist against P2Y(2), but not P2Y(4), receptor-mediated responses. 17β-E(2) played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y(2) receptors. 17β-E(2) (0.1-1000 nM) downregulated the expression of P2Y(2) receptors in terms of both mRNA and protein levels in MCF-7 cells. The effect was blocked by ICI 182,780 and MPP, but not PHTPP or ER(β) small interfering RNA. 17β-E(2) did not affect the expression of P2Y(2) receptors in MDA-MB-231. UTP (10-100 μM) led to a sharp increase in intracellular Ca(2+) in MCF-7 cells. Pre-incubation with 17β-E(2) (0.1 μM) attenuated UTP-induced [Ca(2+)](i), which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER(α) receptors, promotes proliferation of breast cancer cells by down-regulating P2Y(2) receptor expression and attenuating P2Y(2)-induced increase of [Ca(2+)](i).

Estrogen altered visceromotor reflex and P2X3 mRNA expression in a rat model of colitis

P2X(3) and P2X(2/3) receptors are expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in peripheral pain. However, the mechanisms underlying P2X receptor-mediated nociception at different ovarial hormone levels has not been examined. In this study, 24 female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), estrogen-treated, and estrogen-progesterone-treated groups with colitis. In each group, the visceromotor reflex (VMR) to colorectal distension was tested and the DRG were harvested for a real-time PCR analysis of P2X(3) and P2X(2) receptor mRNA. In OVX rats with colitis we found that the VMR to colorectal distension and P2X(3) receptor mRNA in DRG were both significantly decreased. Estrogen replacement reversed the decrease. However, neither the VMR nor the P2X(3) mRNA level in DRG from OVX colitis rats was reversed by the complex of estrogen and progesterone. Patch-clamp recording showed that in colitis rats, estradiol rapidly potentiated the sustained and transient currents evoked by ATP to 336+/-49% and 122+/-12% of controls, respectively, in a subpopulation of DRG neurons, which were blocked by ICI 182, 780, an antagonist of the estrogen receptor. Whereas progesterone rapidly inhibited the transient currents induced by ATP to 67+/-10% of control and had no effect on the sustained currents evoked by the same agonist. These results indicate that P2X(3) receptors are likely to be an important contributor to the altered colonic functions in colitis rats, where the underlying mechanisms are closely related to endogenous estrogen modulation.

The role of large-conductance, calcium-activated potassium channels in a rat model of trigeminal neuropathic pain

Background Trigeminal neuralgia is a disorder of paroxysmal and severely disabling facial pain and continues to be a real therapeutic challenge. At present there are few effective drugs. Here the aim of this study was to investigate the role of BKCa channels in trigeminal neuropathic pain. Methods Rats were divided into two groups: a sham and a chronic constriction injury of infraorbital branch of trigeminal nerve (ION-CCI) group. Nociceptive behavior testing, immunohistochemistry, RT-PCR, Western blotting and whole-cell patch clamp recording were used. Results Relative to the sham group, rats with ION-CCI consistently displayed lower mechanical pain thresholds in the vibrissal pad region from day 6 to 42 after ION-CCI operation. ION-CCI induced a significant down-regulation of BKCa channels both in mRNA and protein levels in the ipsilateral trigeminal ganglion (TG), a lower threshold intensity of action potential, and decreased total BKCa currents in cultured TG neurons. TG target injection of NS1619 (20–100 µg), an opener of BKCa channels, dose-dependently increased the mechanical pain threshold, which was blocked by the BKCa channel inhibitor iberiotoxin (IbTX, 20 µg). NS1619 (10 µM) significantly increased the mean threshold intensities of action potentials in ION-CCI rats, while failing to affect those in the sham rats. The levels of phosphorylated extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinases (JNK) in TG were significantly increased after ION-CCI operation. The ERK1/2 antagonist U0126, p38 antagonist SB203580 and JNK antagonist SP600125 significantly reversed the facial mechanical allodynia in ION-CCI rats. However, the ERK1/2 antagonist U0126, p38 antagonist SB203580 but not JNK antagonist SP600125 significantly increased BKCa currents in ION-CCI TG neurons. Conclusions Our results indicate the important involvement of mainly ERK and p38 MAPK pathways in modulating BKCa channels in ION-CCI TG neurons. BKCa channels represent a new therapeutic target for the clinical treatment of trigeminal neuropathic pain.

The Inhibitory Effect of Doxycycline on Cisplatin-Sensitive and -Resistant Epithelial Ovarian Cancer

Background Detecting a new effective and hypotoxic anticancer drug is an emerging new strategy for cancer chemotherapy. Doxycycline (DC) is a kind of antibiotics but also inhibits tumorigenesis. Methods MTT and cell invasion assay, flow cytometry, western-blot analysis and nude mice were used to investigate the effects and underlying mechanisms of doxycycline on epithelial ovarian cancer cells. Results Doxycycline inhibited the proliferation and invasion of SKOV3 and SKOV3/DDP; induced moderate apoptosis of SKOV3/DDP. CXCR4 expression at both mRNA and protein levels was downregulated in both cell lines when treated with doxycycline. Akt and ERK1/2 were involved in doxycycline effect on cell proliferation of SKOV3 but not of SKOV3/DDP. Akt and EKR1/2 phosphorylation were activated by SDF-1α, which was then inhibited by doxycycline in SKOV3. Pro-caspase-3 expression was significantly higher in SKOV3 than that in SKOV3/DDP which was upregulated when treated with doxycycline. In vivo, doxycycline inhibited peritoneal tumor xenograft and decreased malignant ascites. Conclusion Doxycycline not only has an inhibitory effect on ovarian cancer, but also can increase sensitivity to cisplatin. SDF-1α/CXCR4-regulated Akt and ERK 1/2 activations are probably involved in the antitumor effect of doxycycline on SKOV3 cells, while upregulation of pro-caspase-3 may be the main mechanism involved in SKOV3/DDP cells.

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats.

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freunds adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.

Progesterone Rapidly Attenuates ATP-Evoked Transient Currents in Cultured Rat Dorsal Root Ganglion Neurons

Background: Progesterone has been shown to play a role in pain perception. However, the effects of progesterone on P2X3 receptors, the nociception-related receptors in primary sensory neurons, remain unclear. Methods: We investigated the effects of progesterone on P2X3-receptor-mediated responses of rat dorsal root ganglion neurons by using whole-cell patch clamp techniques. Results: Progesterone (10 pmol/l to 1 µmol/l) inhibited ATP (100 µmol/l)-induced transient currents and the transient component of biphasic currents in a dose-dependent manner, but had no effect on the sustained currents. The effect of progesterone could be blocked by its receptor inhibitor, RU38486. Actidione, an inhibitor of protein synthesis, did not change the rapid effect. Investigations of the signaling pathways of progesterone showed that a nonselective antagonist of protein kinase, H-9, totally blocked the depressive effect of progesterone on ATP currents in a dose-dependent manner. Blocking protein kinase A with H-89 or KT5720 also affected progesterone-induced depression. The protein kinase A activator, forskolin, mimicked the effect of progesterone on ATP currents. Conclusion: These results suggest that progesterone may modulate pain signal transmission on dorsal root ganglia via regulating P2X3 receptor function. The cAMP-PKA signaling pathway is involved in the downregulating effect of progesterone on P2X3 receptors.

Inhibitory effect of estrogen receptor beta on P2X3 receptors during inflammation in rats

Estrogen receptor beta (ERβ) has been shown to play a therapeutic role in inflammatory bowel disease (IBD). However, the mechanism underlying how ERβ exerts therapeutic effects and its relationship with P2X3 receptors (P2X3R) in rats with inflammation is not known. In our study, animal behavior tests, visceromotor reflex recording, and Western blotting were used to determine whether the therapeutic effect of ERβ in rats with inflammation was related with P2X3R. In complete Freund adjuvant (CFA)-induced chronic inflammation in rats, paw withdrawal threshold was significantly decreased which were then reversed by systemic injection of ERβ agonists, DPN or ERB-041. In 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, weight loss, higher DAI scores, increased visceromotor responses, and inflammatory responses were reversed by application of DPN or ERB-041. The higher expressions of P2X3R in dorsal root ganglia (DRG) of CFA-treated rats and those in rectocolon and DRG of TNBS-treated rats were all decreased by injection of DPN or ERB-041. DPN application also inhibited P2X3R-evoked inward currents in DRG neurons from TNBS rats. Mechanical hyperalgesia and increased P2X3 expression in ovariectomized (OVX) CFA-treated rats were reversed by estrogen replacements. Furthermore, the expressions of extracellular signal-regulated kinase (ERK) in DRG and spinal cord dorsal horn (SCDH) and c-fos in SCDH were significantly decreased after estrogen replacement compared with those of OVX rats. The ERK antagonist U0126 significantly reversed mechanical hyperalgesia in the OVX rats. These results suggest that estrogen may play an important therapeutic role in inflammation through down-regulation of P2X3R in peripheral tissues and the nervous system, probably via ERβ, suggesting a novel therapeutic strategy for clinical treatment of inflammation.