Xin Ni

Functional up-regulation of P2X3 receptors in the chronically compressed dorsal root ganglion

Abstract P2X receptors on dorsal root ganglion (DRG) neurons have been strongly implicated in pathological nociception after peripheral nerve injuries or inflammation. However, nothing is known of a role for purinergic receptors in neuropathic pain produced by a chronic compression of DRG (CCD) – an injury that may accompany an intraforaminal stenosis, a laterally herniated disc or other disorders of the spine leading to radicular pain. In a rat model of DRG compression, hyperexcitable neurons retain functioning axonal connections with their peripheral targets. It is unknown whether such hyperexcitability might enhance chemically mediated nociceptive stimulation of the skin. In this study, CCD facilitated the nocifensive behavior and mechanical hyperalgesia‐induced by the P2X3 agonist, α,β‐methylene ATP (α,β‐meATP). An injection of α,β‐meATP into the hind paw of CCD rats resulted in a significantly greater decrease in the mean threshold to von Frey stimuli and a greater duration of paw lifts than in sham‐operated control rats. CCD also increased the levels of P2X3 receptor protein and the number of P2X3 immunoreactive, small diameter DRG neurons in the compressed ganglion. P2X3 receptors were co‐labeled with the isolectin IB4, consistent with a role in nociception. In addition, a α,β‐meATP induced significantly larger fast‐inactivating currents in CCD‐ than in sham‐operated acutely dissociated DRG neurons. These currents were accompanied by the generation of action potentials – but only in the CCD neurons. U0126, a specific inhibitor of the MEK1/2, greatly down‐regulated the enhanced current. Taken together, these observations suggest that enhanced purinergic responses after CCD are mediated by P2X3 receptors.

Differential Regulation of Prostaglandin Production Mediated by Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts

Prostaglandin (PG) production by intrauterine tissues plays a key part in the control of pregnancy and parturition. The present study was to investigate the role of placenta-derived CRH and CRH-related peptides in the regulation of PG synthesis and metabolism. We found that placental trophoblasts expressed both CRH-R1 and CRH-R2. Treatment of cultured placental cells with either a CRH or urocortin I (UCNI) antibody resulted in a significant decrease in PGE2 release. Both CRH and UCNI antibodies significantly decreased mRNA and protein expression of synthetic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2 and increased mRNA and protein expression of 15-hydroxyprostaglandin dehydrogenase (PGDH), the key enzyme of PG metabolism. CRH-R1/-R2 antagonist astressin and CRH-R1 antagonist antalarmin significantly inhibited PGE2 release, whereas CRH-R2 antagonist astressin-2b had no effect on PGE(2) release. Administration of astressin decreased expression of cPLA2 but had no effect on COX-2 expression. Antalarmin reduced cPLA2 and COX-2 expression, whereas astressin-2b did not alter cPLA2 expression but increased COX-2 expression. PGDH expression was enhanced by these three antagonists. Cells treated with exogenous CRH and UCNI showed an increase in PGE(2) release and expression of cPLA2 and COX-2 but a decrease in PGDH expression. UCNII and UCNIII had no effect on PGE2 release but decreased COX-2 and PGDH expression. Our results suggested CRH and CRH-related peptides act on CRH-R1 and CRH-R2 to exert different effects on PG biosynthetic enzymes cPLA2 and COX-2 and thereby modulate output of PGs from placenta, which would be important for controlling pregnancy and parturition.

Protective effects of the free radical scavenger edaravone on acute pancreatitis-associated lung injury.

Impaired lung function is the primary contributor to most deaths associated with severe acute pancreatitis. It is widely accepted that oxidative stress plays a central role in the pathogenesis of pancreatitis and associated complications. Therefore, in the present study, we investigated whether therapeutic treatment with the free radical scavenger edaravone could protect rats against acute pancreatitis and the associated lung injury. Acute pancreatitis was induced by infusion of 1ml/kg of sodium taurocholate (3% solution) into the biliopancreatic duct. Edaravone (8mg/kg) was administered 1h and 13h after inducing pancreatitis, the severity of pancreatic and pulmonary injuries was evaluated 24h after inducing pancreatitis. Edaravone treatment significantly reduced the elevated malondialdehyde levels in rat lungs after acute pancreatitis, suggesting an important role for free radicals in acute pancreatitis-associated lung injury. In addition, edaravone showed significant protective effects against neutrophil infiltration and tissue injury in both pancreas and lung, as demonstrated by serum amylase levels, myeloperoxidase activity and histopathological analysis. Edaravone treatment also attenuated the elevated mRNA levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) in rat lungs after acute pancreatitis. In conclusion, edaravone protects rats against acute pancreatitis-associated lung injury, probably through its antioxidant and anti-inflammatory effects. Thus, edaravone shows promise as a treatment for lung injury in patients with acute pancreatitis.

Expression of Cystathionine β-synthase and Cystathionine γ-lyase in Human Pregnant Myometrium and Their Roles in the Control of Uterine Contractility

Background Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H2S produced locally in the control of uterine contractility during labour is unknown. Methodology/Principal Findings Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H2S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H2S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10−7–10−2 mol/L), a precursor of H2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10−3 mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips. Conclusion/Significance H2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H2S synthetic enzymes is down-regulated during labour, suggesting that H2S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.

Swimming exercise ameliorates depression-like behaviors induced by prenatal exposure to glucocorticoids in rats.

Prenatal exposure to glucocorticoids (GCs) leads to affective dysfunction in adulthood, which may be associated with the alterations in hypothalamic-pituitary-adrenal (HPA) axis. Physical exercise has been shown to ameliorate depressive symptoms. The objectives of present study were to investigate whether prenatal exposure to GCs induces depression-like behaviors in adult offspring rats, and determine whether swimming exercise alleviates the depression-like behaviors induced by this paradigm. Pregnant rats received dexamethasone (DEX) (0.1mg/kg/day) in the last third of pregnancy or vehicle. DEX treatment reduced body weight in 1, 3, 6, 9-week old male offspring, and 3, 6, 9-week old female offspring. DEX treatment resulted in an elevated level of serum corticosterone in adult offspring (9weeks). Female and male adult offspring rats exhibited decreased number of poking into holes and rearing and decreased central distance traveled in open field test (OFT), and reduced sucrose consumption, suggesting prenatal DEX exposure increase depression-like behaviors in the adult offspring rats. Four-week swimming exercise reduced serum corticosterone levels, and alleviated the depressive behavior by reversing the decreased number of poking into holes and rearing as well as decreased central distance traveled, and reversing the reduced sucrose consumption in male and female adult offspring. These findings suggested prenatal exposure to GCs increase the activity of HPA axis and depression-like behaviors of adult offsprings. Swimming exercise decreases HPA activity and ameliorates depression in rats exposed to DEX prenatally.

Estrogen modulation of peripheral pain signal transduction: involvement of P2X3 receptors

There is evidence that gonadal hormones may affect the perception of painful stimulation, although the underlying mechanisms remain unclear. This investigation was undertaken to determine whether the adenosine 5′-triphosphate (ATP) receptor subunit, P2X3, is involved in the modulatory action of estrogen in peripheral pain signal transduction in dorsal root ganglion (DRG). The mechanical pain behavior test, real-time quantitative reverse transcription–polymerase chain reaction analysis, and Western blot methods were used to determine the mean relative concentrations and functions of P2X3 receptors in DRG in sham, ovariectomized (OVX), and estradiol replacement (OVX+E2) female rats and in sham and orchiectomized male rats. The mechanical hyperalgesia appeared after ovariectomy, which was subsequently reversed after estradiol replacement, whereas it was not observed after orchiectomy in male rats. Plantar injection of 2′(3′)-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a P2X3 and P2X2/3 receptor antagonist, resulted in an increase of the pain threshold force in OVX rats while had no effect on sham rats. Furthermore, A-317491, a selective P2X3/P2X2/3 receptor antagonist, significantly reversed the hyperalgesia of OVX rats. Injection of ATP into the plantars also caused a significant increase of the paw withdrawal duration in OVX rats compared with that seen in the sham group, which became substantially attenuated by TNP-ATP. P2X3 receptors expressed in DRG were significantly increased in both mRNA and protein levels after ovariectomy and then reversed after estrogen replacement, while a similar increase was not observed after orchiectomy in male rats. Furthermore, P2X3 mRNA was significantly decreased 24xa0h after the application of 17β-estradiol in a concentration-dependent manner in cultured DRG neurons. ICI 182,780, an estrogen receptor antagonist, blocked the reduction in the protein level. These results suggest that the female gonadal hormone, 17β-estradiol, might participate in the control of peripheral pain signal transduction by modulating P2X3 receptor-mediated events in primary sensory neurons, probably through genomic mechanisms.

Expression of the calcium-activated potassium channel in upper and lower segment human myometrium during pregnancy and parturition

BackgroundLarge conductance calcium-activated potassium channel (BKCa) plays an important role in the control of uterine contractility during pregnancy. The change from uterine quiescence to enhanced contractile activity may be associated with the spatial and temporal expression of BKCa within myometrium. The objectives of this study were to examine the expression of BKCa alpha- and beta-subunit in upper segment (US) and lower segment (LS) regions of uterus, and to investigate for the possibly differential expression of these proteins in US and LS myometrium obtained from three functional states: (1) non-pregnant (NP); (2) term pregnant not in labour (TNL) and (3) term pregnant in labour (TL).MethodsMyometrial biopsies were collected from non-pregnant women at hysterectomy and pregnant women at either elective caesarean section or emergency caesarean section. Protein expression level and cellular localization of BKCa alpha- and beta-subunit in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively.ResultsBKCa alpha- and beta-subunit were predominantly localized to myometrial smooth muscle in both US and LS myometrium obtained from non-pregnant and pregnant patients. The level of BKCa alpha-subunit in US but not in LS was significantly higher in NP myometrium than those measured in myometrium obtained during pregnancy. Lower expression of BKCa alpha-subunit in both US and LS was found in TL than in TNL biopsies. Expression of beta-subunit in both US and LS myometrium was significantly reduced in TL group compared with those measured in TNL group. There was no significant difference in BKCa beta-subunit expression in either US or LS between NP and TNL group.ConclusionOur results suggest that expression of BKCa alpha- and beta-subunit in pregnant myometrium is reduced during labour, which is consistent with the myometrial activity at the onset of parturition.

GABAergic mechanism in the rostral ventrolateral medulla contributes to the hypotension of moxonidine

AIMSnThe depressor action of the centrally antihypertensive drug moxonidine has been attributed to activation of I(1)-imidazoline receptor in the rostral ventrolateral medulla (RVLM). The objective of this study was to determine the role of the γ-aminobutyric acid (GABA) mechanisms in the RVLM in mediating the effect of moxonidine in anaesthetized normotensive rats.nnnMETHODS AND RESULTSnThe relationship between the effects of microinjection or picoinjection of moxonidine and the functional state of GABA receptors at the level of the RVLM or pre-sympathetic neuron was determined. Microdialysis was performed to detect the effect of moxonidine on the release of GABA in the RVLM. Western blot analysis was carried out to test the effect of chronic intracerebroventricular injection of moxonidine on the protein expression of GABA receptors in the RVLM. Pre-treatment with the GABA(A) or GABA(B) receptor antagonist bicuculline (5 pmol) or CGP35348 (200 pmol), respectively, microinjected into the RVLM significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity induced by moxonidine. In 22 moxonidine-sensitive pre-sympathetic neurons in the RVLM, picoinjection of bicuculline (100 fmol/5 nL) significantly attenuated the neuronal inhibition evoked by moxonidine (100 pmol/5 nL). The release of GABA in the RVLM was increased after intravenous moxonidine (50 μg/kg). Central infusion of moxonidine upregulated the protein expression of both GABA(A) and GABA(B) receptors in the RVLM.nnnCONCLUSIONnThe current data demonstrate that GABAergic mechanisms in the RVLM are responsible for the hypotension and sympathoinhibition of moxonidine.

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).

Reduced expression of CRH receptor type 1 in upper segment human myometrium during labour

BackgroundCorticotropin-releasing hormone (CRH) and CRH-related peptide are shown to modulate uterine contractility through two CRH receptor subtype, CRH-R1 and CRH-R2 during pregnancy. Through different signaling pathways, CRH-R1 maintains myometrial quiescence whereas CRH-R2 promotes smooth muscle contractility. We hypothesized that the expression of CRH receptors in myometrium might be changed during pregnancy and labour.MethodImmunohistochemistry, Western blot and RT-PCR were used to quantify the cellular localization, the protein levels and the mRNA variants of both CRH-R1 and CRH-R2 in upper segment (US) and lower segment (LS) myometrium from nonpregnant and pregnant women at term before or after labour.ResultsCRH-R1 and CRH-R2 were predominately localized to myometrial smooth muscle cells in US and LS. The protein level of CRH-R1 in US was significantly down-regulated in pregnancy, with a further decrease at the onset of labour. However, the expression of CRH-R1 in LS remained unchanged during pregnancy and labour. No significant changes in CRH-R2 expression were observed in US or LS. Six variants of CRH-R1, CRH-R1alpha,-R1beta,-R1c, -R1e,-R1f and -R1g, were identified in nonpregnant and pregnant myometrium. CRH-R2alpha was identified in pregnant myometrium, whereas CRH-R2beta was identified in nonpregnant myometriumConclusionCRH-R1 and CRH-R2 are expressed in nonpregnant and pregnant US and LS myometrium. Changed expression of CRH receptors during labour may underlie the initiation of uterine contractility during parturition.