The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechani...The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.展开更多
AIM:To explore the possibility of using the Non-invasive Micro-test Technique(NMT)to investigate the role of Transient Receptor Potential Canonical 1(TRPC1) in regulating Ca 2+ influxes in HL-7702 cells,a normal human...AIM:To explore the possibility of using the Non-invasive Micro-test Technique(NMT)to investigate the role of Transient Receptor Potential Canonical 1(TRPC1) in regulating Ca 2+ influxes in HL-7702 cells,a normal human liver cell line.METHODS:Net Ca 2+ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces,non-invasively.The expression levels of TRPC1 were increased by liposomal transfection,whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction. RESULTS:Ca 2+ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells.They were enhanced by addition of 20μmol/L noradrenalin and inhibited by 100μmol/L LaCl3(a non-selective Ca 2+ channel blocker);5μmol/L nifedipine did not induce any change.Overexpression of TRPC1 caused increased Ca 2+ influx.Five micromoles per liter nifedipine did not inhibit this elevation,whereas 100μmol/L LaCl3 did.CONCLUSION:In HL-7702 cells,there is a type of TRPC1-dependent Ca 2+ channel,which could be detected via NMT and inhibited by La3+.展开更多
AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concen...AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.展开更多
目的观察离体活膀胱肌条铺片中卡哈尔间质细胞(interstitial cells of cajal,ICCs)自发性钙瞬变,并检测胞外钙及胞内钙库钙对ICCs细胞自发性钙瞬变的影响,探讨ICCs细胞自发性钙瞬变产生的机制。方法制作离体膀胱肌条铺片,生理液灌流保...目的观察离体活膀胱肌条铺片中卡哈尔间质细胞(interstitial cells of cajal,ICCs)自发性钙瞬变,并检测胞外钙及胞内钙库钙对ICCs细胞自发性钙瞬变的影响,探讨ICCs细胞自发性钙瞬变产生的机制。方法制作离体膀胱肌条铺片,生理液灌流保持肌条铺片活性。待肌条自发性收缩活动出现后,Fluo-4负载肌条铺片,激光共聚焦显微镜观察其中ICCs细胞自发性钙瞬变,随后观察尼莫地平(Nimodipine)、2-氨基乙基二苯硼酸盐(2-aminoethoxydip henylborate,2-APB)、雷诺丁(Ryanodine)、毒胡萝卜素(Thapsigargin)及无钙、高钙生理液对ICCs细胞自发性钙瞬变的影响,探讨ICCs细胞自发性钙瞬变产生的机制。结果可见离体活膀胱肌条铺片中平滑肌细胞和ICCs细胞均产生节律性自发性钙瞬变,平滑肌细胞钙瞬变频率较快,而ICCs细胞钙瞬变频率相对较慢。尼莫地平能完全抑制平滑肌细胞的自发性钙瞬变,但对ICCs细胞的自发性钙瞬变无显著影响。2-APB、雷诺丁、毒胡萝卜素及无钙生理液均可完全抑制ICCs细胞的自发性钙瞬变,高钙生理液可显著增加ICCs细胞自发性钙瞬变的频率。结论细胞外钙离子通过非L型钙离子通道入胞及细胞内钙库放钙均参与ICCs细胞自发性钙瞬变的产生。展开更多
基金supported by the National Natural Science Foundation of China,Nos.81870732(to DZ),82171161(to DZ),81900933(to YS),and 82000978(to ZL).
文摘The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.
基金Supported by The National Natural Science Foundation of China,No.30270532 and No.30670774Tsinghua-Yue-Yuen Medical Science Foundation,No.20240000531 and No.20240000547
文摘AIM:To explore the possibility of using the Non-invasive Micro-test Technique(NMT)to investigate the role of Transient Receptor Potential Canonical 1(TRPC1) in regulating Ca 2+ influxes in HL-7702 cells,a normal human liver cell line.METHODS:Net Ca 2+ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces,non-invasively.The expression levels of TRPC1 were increased by liposomal transfection,whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction. RESULTS:Ca 2+ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells.They were enhanced by addition of 20μmol/L noradrenalin and inhibited by 100μmol/L LaCl3(a non-selective Ca 2+ channel blocker);5μmol/L nifedipine did not induce any change.Overexpression of TRPC1 caused increased Ca 2+ influx.Five micromoles per liter nifedipine did not inhibit this elevation,whereas 100μmol/L LaCl3 did.CONCLUSION:In HL-7702 cells,there is a type of TRPC1-dependent Ca 2+ channel,which could be detected via NMT and inhibited by La3+.
基金Supported by Grants from the Korean National Research Foun-dation(2010-0014617)the Myung Sun Kim Memorial Founda-tion(2009)the Yonsei University Faculty Research Grant(2004)
文摘AIM:To investigate serotonergic Ca 2+ signaling and the expression of 5-hydroxytryptamine(5-HT) receptors,as well as Ca 2+ transporting proteins,in hepatic stellate cells(HSCs) . METHODS:The intracellular Ca 2+ concentration([Ca 2+ ]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system.Quantitative PCR was per-formed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum(ER) proteins involved in Ca 2+ storage and release in cultured rat HSCs. RESULTS:Distinct from quiescent cells,activated HSCs exhibited[Ca 2+ ]i transients following treatment with 5-HT,which was abolished by U-73122,a phospholipase C inhibitor.Upregulation of 5-HT2A and 5-HT2B receptors,but not 5-HT3,was prominent during trans-differentiation of HSCs.Pretreatment with ritanserin,a 5-HT2 antagonist,inhibited[Ca 2+ ]i changes upon application of 5-HT.Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca 2+ storage and release in activated HSCs.Ca 2+ binding chaperone proteins of the ER,including calreticulin,calnexin and calsequestrin,were up-regulated following activation of HSCs. CONCLUSION:The appearance of 5-HT-induced[Ca 2+ ]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca 2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.
文摘目的观察离体活膀胱肌条铺片中卡哈尔间质细胞(interstitial cells of cajal,ICCs)自发性钙瞬变,并检测胞外钙及胞内钙库钙对ICCs细胞自发性钙瞬变的影响,探讨ICCs细胞自发性钙瞬变产生的机制。方法制作离体膀胱肌条铺片,生理液灌流保持肌条铺片活性。待肌条自发性收缩活动出现后,Fluo-4负载肌条铺片,激光共聚焦显微镜观察其中ICCs细胞自发性钙瞬变,随后观察尼莫地平(Nimodipine)、2-氨基乙基二苯硼酸盐(2-aminoethoxydip henylborate,2-APB)、雷诺丁(Ryanodine)、毒胡萝卜素(Thapsigargin)及无钙、高钙生理液对ICCs细胞自发性钙瞬变的影响,探讨ICCs细胞自发性钙瞬变产生的机制。结果可见离体活膀胱肌条铺片中平滑肌细胞和ICCs细胞均产生节律性自发性钙瞬变,平滑肌细胞钙瞬变频率较快,而ICCs细胞钙瞬变频率相对较慢。尼莫地平能完全抑制平滑肌细胞的自发性钙瞬变,但对ICCs细胞的自发性钙瞬变无显著影响。2-APB、雷诺丁、毒胡萝卜素及无钙生理液均可完全抑制ICCs细胞的自发性钙瞬变,高钙生理液可显著增加ICCs细胞自发性钙瞬变的频率。结论细胞外钙离子通过非L型钙离子通道入胞及细胞内钙库放钙均参与ICCs细胞自发性钙瞬变的产生。
