This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labele...This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.展开更多
OBJECTIVE Two-pore domain potassium channel subtype TREK-1 was widely proved to be activated by inhalational anesthet⁃ics such as chloroform,diethyl ether,halothane,and isoflurane.But little is known about whether TRE...OBJECTIVE Two-pore domain potassium channel subtype TREK-1 was widely proved to be activated by inhalational anesthet⁃ics such as chloroform,diethyl ether,halothane,and isoflurane.But little is known about whether TREK-1 was also a potentially important target of intravenous anesthetics.Etomidate is a popularly used intravenous anesthetic with good safety in clinic.The action of etomidate on TREK-1 was seldom reported.METHODS AND RESULTS By using patch-clamp whole-cell recording tech⁃niques,we found for the first time that etomidate could bidirectionally regulate the TREK-1 potassi⁃um channel in CHO/TREK-1 cells.TREK-1 current amplitudes were observed after the administra⁃tion of etomidate at concentrations ranging from 3 to 100μmol·L-1.Etomidate activated TREK-1 current at concentrations of 3,10,and 15μmol·L-1 with maximum activation at 10μmol·L-1.Interest⁃ingly,at higher concentrations from 20 to 100μmol·L-1,etomidate inhibited TREK-1 current in a concentration-dependent way.According to the concentration-response curve,the fitted criti⁃cal concentration of etomidate between TREK-1 activation and inhibition was 20.7μmol·L-1,which close to the result that etomidate had no obvious effect on TREK-1 at 20μmol·L-1.In addition,etomidate 10μmol·L-1 induced a significant mem⁃brane potential hyperpolarization while etomidate 30μmol·L-1 showed obvious membrane potential depolarization.Furthermore,the bidirectional regulation still existed when the extracellular pH of CHO/TREK-1 cells was decreased.CONCLUSION TREK-1 is activated by etomi⁃date at clinically relevant concentrations but inhib⁃ited by supraclinical concentrations of etomidate,which is different to other volatile anesthetics.TREK-1 might be a potential target for anesthetic such as etomidate and the complicated bidirec⁃tional regulation mechanism of etomidate needed to be fully studied in the future.展开更多
基金supported by grants from the National Natural Science Foundation of China (No.30971007)the Natural Science Foundation of Hubei Province for Outstanding Young Scholars (No.2010CDA103)the National Basic Research Program(No.2011CB504403)
文摘This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.
文摘OBJECTIVE Two-pore domain potassium channel subtype TREK-1 was widely proved to be activated by inhalational anesthet⁃ics such as chloroform,diethyl ether,halothane,and isoflurane.But little is known about whether TREK-1 was also a potentially important target of intravenous anesthetics.Etomidate is a popularly used intravenous anesthetic with good safety in clinic.The action of etomidate on TREK-1 was seldom reported.METHODS AND RESULTS By using patch-clamp whole-cell recording tech⁃niques,we found for the first time that etomidate could bidirectionally regulate the TREK-1 potassi⁃um channel in CHO/TREK-1 cells.TREK-1 current amplitudes were observed after the administra⁃tion of etomidate at concentrations ranging from 3 to 100μmol·L-1.Etomidate activated TREK-1 current at concentrations of 3,10,and 15μmol·L-1 with maximum activation at 10μmol·L-1.Interest⁃ingly,at higher concentrations from 20 to 100μmol·L-1,etomidate inhibited TREK-1 current in a concentration-dependent way.According to the concentration-response curve,the fitted criti⁃cal concentration of etomidate between TREK-1 activation and inhibition was 20.7μmol·L-1,which close to the result that etomidate had no obvious effect on TREK-1 at 20μmol·L-1.In addition,etomidate 10μmol·L-1 induced a significant mem⁃brane potential hyperpolarization while etomidate 30μmol·L-1 showed obvious membrane potential depolarization.Furthermore,the bidirectional regulation still existed when the extracellular pH of CHO/TREK-1 cells was decreased.CONCLUSION TREK-1 is activated by etomi⁃date at clinically relevant concentrations but inhib⁃ited by supraclinical concentrations of etomidate,which is different to other volatile anesthetics.TREK-1 might be a potential target for anesthetic such as etomidate and the complicated bidirec⁃tional regulation mechanism of etomidate needed to be fully studied in the future.
