Eosinophils are known to be the important effector cells in asthmatic airway inflammation.The purpose of this study was to investigate the effects of nobiletin,a polymethoxyflavonoid,on eosinophilic airway inflammation of asthmatic rats,and explore its possible mechanisms.Animals were actively sensitized by subcutaneous injection of ovalbumin(OVA).The inflammation in lung tissues of asthmatic rats was observed by hematoxylin and eosin(HE)staining.The eosinophils in blood and BALF were separated by Percoll density gradient centrifugation and counted under microscope.The level of Eotaxin was detected by enzyme-linked immunosorbent assay(ELISA).In addition,the apoptosis of eosinophils was labeled by TdT-mediated dUTP nick end labeling(TUNEL)technique,the semi-quantitative detection for Fas mRNA expression of eosinophils was performed by reverse transcription-polymerase chain reaction(RT-PCR).The airway inflammation of asthmatic rats pretreated with nobiletin was obviously alleviated.Nobiletin(1.5 and 5.0 mg/kg given intraperitoneally)significantly reduced OVA-induced increases in eosinophils,remarkably lowered the level of Eotaxin in blood and broncho-alveolar lavage fluid(BALF)of asthmatic rats.On the other hand,semi-quantitative RT-PCR analysis for Fas of eosinophils from OVA aerosol-challenged sensitized rats showed that Fas mRNA expression of eosinophils was obviously enhanced by nobiletin.Meanwhile,the apoptosis index of cultured eosinophils was significantly elevated after treatment with different doses of nobiletin.These results indicated that nobiletin could inhibit the eosinophilic airway inflammation.Lowering the levels of Eotaxin,relieving airway infiltration of eosinophils and promoting apoptosis of eosinophils by enhancing expression of Fas mRNA may be important mechanisms for nobiletin to antagonize eosinophilic airway inflammation of asthmatic rats.D 2005 Elsevier Inc.All rights reserved.

Urocortin(UCN),a newly isolated peptide,has been found to play an important role mainly in female reproductive system.In order to investigate the effect of UCN on T-type calcium currents(ICa,T), exploring the mechanisms of UCN s role in male reproductive system,especially in acrosome reaction,we directly measured the ICa,T in mouse spermatogenic cells exposed to UCN using standard whole-cell patch clamp recording technique.Our results showed that UCN reversibly inhibited the T-type Ca2+ currents in the cells in a concentration-dependent manner.The current density was inhibited by about 19% after exposure of the cells to UCN(0.1 lM)for 5 min,from the control value of 6.75±1.17 to 5.26±0.82 pA/pF.UCN up-shifted the current-voltage(I-V)curve.Frequency-dependence of UCN’s effects on ICa,T was also observed.Moreover,UCN at 0.1 lM did not markedly affect the activation of ICa,T but shifted the inactivation curve of ICa,T to the left.The inhibitory effect of UCN on the T-type Ca2+ current was not affected by Astressin,the CRF receptor blocker.Since T-type calcium channels are a key component in acrosome reaction,our data suggest that UCN might be a significant factor in male reproductive action and a potential contraceptive agent.

The newly isolated peptide,urocortin(UCN)has been found to have potent cardioprotective effects.In order to investigate the effectof UCN on L-type calcium currents(ICa,L),exploring the mechanisms of UCN’s cardioprotective effects,we directly measured the ICa,Lin the adult rat cardiac myocytes exposed to UCN using standard whole-cell patch-clamp recording technique.Our results showed that UCN exerted decreasing effects on the ICa,L of the single adult rat cardiac myocytes.The current density was inhibited by about 35% after exposure of the cells to UCN(0.1 mol L−1)for 10 min,from the control value of 7.19±1.44 pA/pF to 4.74±0.75 pA/pF(n=5,P<0.05).This ICa,L-inhibiting action of UCN was concentration dependent.Moreover,no frequency dependence of UCN effects on ICa,L was observed.In combination with previous reports,our results suggest that there might be a close relationship between the cardioprotective effects of UCN and L-type calcium channels.

Urocortin(UCN),a newly isolated peptide related to hypothalamic corticotrophin releasing factor(CRF)family,had been reported to play biologically diverse roles in several systems such as cardiovascular,reproductive,appetite,stress,and inflammatory responses,etc.It was thought previously to be an endogenous agonist,producing the several actions previously attributed to CRF. But, recently, it was shown to directly reduce L-type calcium currents of acute isolated cardiac myocytes and T-type calcium currents in mouse spermatogenic cells via inhibiting calcium channel instead of binding first to its CRF-R2 receptors.UCN could also reduce the intracellular calcium in vascular smooth muscle cells via inhibiting calcium channel directly.Furthermore,UCN could increase the gene expression of ATP-sensitive potassium channels(KATP)and activate sarcolemmal ATP-sensitive potassium current during normal or hypoxia,which could be inhibited by glibenclamide,a specific KATP blocker.This review will highlight the current novel findings on the ionic mechanisms by which UCN may exert its several actions.

The newly isolated peptide,urocortin(UCN),is a member of the corticotropin-releasing factor(CRF)-related peptides that has been found to have potent cardiovascular protective effects.In order to investigate the effect of UCN on the viability of adult rat vascular smooth muscle cells(VSMC)and the relevant mechanisms,we exposed the VSMC to UCN to observe the change in cell viability using MTT assay and intracellular calcium concentration using confocal laser scanning microscope methods.Our results showed that UCN(10−7M)inhibited the viability of VSMC by about 26% (P<0.05, compared to control).The effect was concentration-dependent, but it was not dependent on the affecting time.Glybenclamide (Gly, 10−5M), the ATP-sensitive potassium channel(KATP channel)blocker,and astressin (10−6 M), a competitive antagonist of CRF receptors,had no influence on this inhibition.Bay K8644 (10−6 M), a special L-type calcium channel activator,increased the viability of VSMC. Pre-treatment of the cells with UCN diminished the effect of Bay K8644 (n=6, P<0.05). UCN was also observed to reduce the intracellular Ca2+ increase induced by KCl and Bay K8644. There was no significant difference in nitrite accumulation between UCN groups and the control.In conclusion,UCN reduced the viability of VSMC through L-type calcium channels.These interesting results might suggest that UCN may be a new vasoactive agent involved in hindering vascular remodeling in combination with previous reports about UCN's hypotensive effects.