The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino ...The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (△611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72 3 and the last seven amino acids of C-terminal are essential to the transport activity of NET.展开更多
The Na+/Ca2+ exchanger plays an important role in regulation of airway smooth muscle contraction by regulating intracellular calcium, and is a potential target for treatment of asthma. To test modulation of exchanger ...The Na+/Ca2+ exchanger plays an important role in regulation of airway smooth muscle contraction by regulating intracellular calcium, and is a potential target for treatment of asthma. To test modulation of exchanger activity, we used Xenopus oocytes as model system. Na+/Ca2+ exchanger was expressed in the cells by microinjection of cRNA of the exchanger isoform NCX1. The activity of NCX1 was determined as Ni2+-sensitive current under voltage clamp in low Cl– medium and in the presence of the Cl–-channel inhibitor niflumic acid. Only this composition of solution allowed determining NCX1-mediated current with sufficient accuracy. Among a few tested Chinese herbal drugs, glycyrrhizic acid turned out to be a potent inhibitor of NCX1 with an apparent IC50 value of 40 μM. Previous work had revealed elevated cyclophylin A concentration in serum of asthmatic rats after receiving acupuncture treatment. Extracellular incubation of the oocytes in cyclophylin A for one day led to significant inhibition with an apparent IC50 value of about 1 μM. We suggest that effects of acupuncture and application of glycyrrhizic acid as an active constituent of Chinese medicine for treatment of asthma symptoms may partially be attributed to inhibition of the reversed mode of NCX1 and that these compounds may stimulate the search for new anti-asthmatic drugs.展开更多
OBJECTIVE:To investigate the role of adenosine triphosphate(ATP) purinergic signaling in mast cells(MCs) modulated by heat to further understand the molecular mechanisms of moxibustion.METHODS:Skin temperatures induce...OBJECTIVE:To investigate the role of adenosine triphosphate(ATP) purinergic signaling in mast cells(MCs) modulated by heat to further understand the molecular mechanisms of moxibustion.METHODS:Skin temperatures induced by monkshood cake moxibustion were evaluated by measuring the Neiguan acupoint(PC 6) from 31 participants with a digital thermocouple thermometer.Temperatures of 43 ℃ and 52 ℃ were applied to cultured human leukemia mast cell line HMC-1 in vitro.Calcium fluorescence was applied to detect intracellular Ca^(2+)([Ca^(2+)]_i).Extracellular ATP contents were measured by luciferin-luciferase assay.RESULTS:Maximum skin temperatures mostly ranged from 40-45 ℃,but some reached up to50 ℃.Both 43 ℃,and 52 ℃ induced MC degranulation,which was accompanied by an increase in[Ca^(2+)]_i and ATP release.Complexing extracellular Ca^(2+) with 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA) inhibited the noxious heat-induced elevation of[Ca^(2+)]_i and prevented the enhanced ATP secretion by those cells at 52℃ but not 43 ℃.CONCLUSION:Monkshood cake moxibustion can generate heat sufficient to trigger cellular events of MCs,including degranulation,[Ca^(2+)]_i elevation,and ATP release,suggesting that purinergic signals originating from MCs are possibly the initiating response of acupoints to moxibustion.展开更多
Objective:To better understand the function of mast cells in acupuncture points(acupoints)inacupuncture-induced analgesia.The author tested their sensitivity to mechanical,thermo and lightstimulation.Methods:The tail ...Objective:To better understand the function of mast cells in acupuncture points(acupoints)inacupuncture-induced analgesia.The author tested their sensitivity to mechanical,thermo and lightstimulation.Methods:The tail flick model was applied to measure analgesia in rats,and the authordetermined the density of mast cells in tissue slices and their degranulation ratio before/after acupuncture.The author also applied the patch-clamp technique to investigate activation of human mast cells(HMC1cell line)by mechanical stress or noxious heat,and the author optically observed degranulation展开更多
Objective:Cyclophilin A (Cyp A) is a ubiquitously distributed protein belonging to the immunophilin family and is recognized as the host cell receptor for the potent immunosuppressive
Objective:To establish a new and better model to investigate the properties of mast cells that couldbe involved in acupuncture process mechanisms.Methods:Connective tissue under the corium at thearea of acupuncture po...Objective:To establish a new and better model to investigate the properties of mast cells that couldbe involved in acupuncture process mechanisms.Methods:Connective tissue under the corium at thearea of acupuncture point Zusanli(ST 36)from rat was acutely bluntly separated with forceps andscissors,and incubated in bath solution up to several hours.Mast cells in slices of that tissue wereirradiated with laser light of 650 nm,and changes in the appearance were observed under microscope.In addition,patch-clamp technique in whole-cell configuration was employed to induce mechano-展开更多
Objective:a-asarone is a major effective component that can be isolated from Acorus tatarinowii Schott,a Chinese herbal medicine.Clinical investigations have shown that a-asarone has strong sedative andanti-convulsive...Objective:a-asarone is a major effective component that can be isolated from Acorus tatarinowii Schott,a Chinese herbal medicine.Clinical investigations have shown that a-asarone has strong sedative andanti-convulsive action in the central nervous system.In recent years,several medicines containinga-asarone were applied in treatment of asthma,bronchitis,expectorant,or epilepsy.However,theunderlying cellular mechanism of a-asarone is still unknown.Here the authors considered EAAC1。展开更多
Conserved Ser 354, Ser 357 in the seventh transmembrane domain of NET were mutated and the function of mutants was studied by 3H-NE influx measurement. Double mutation at these two serine sites reduced the activity of...Conserved Ser 354, Ser 357 in the seventh transmembrane domain of NET were mutated and the function of mutants was studied by 3H-NE influx measurement. Double mutation at these two serine sites reduced the activity of NET to base level. It is inferred that these two sites may be involved in the recognition and transport activity of the NET protein.展开更多
文摘The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (△611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72 3 and the last seven amino acids of C-terminal are essential to the transport activity of NET.
文摘The Na+/Ca2+ exchanger plays an important role in regulation of airway smooth muscle contraction by regulating intracellular calcium, and is a potential target for treatment of asthma. To test modulation of exchanger activity, we used Xenopus oocytes as model system. Na+/Ca2+ exchanger was expressed in the cells by microinjection of cRNA of the exchanger isoform NCX1. The activity of NCX1 was determined as Ni2+-sensitive current under voltage clamp in low Cl– medium and in the presence of the Cl–-channel inhibitor niflumic acid. Only this composition of solution allowed determining NCX1-mediated current with sufficient accuracy. Among a few tested Chinese herbal drugs, glycyrrhizic acid turned out to be a potent inhibitor of NCX1 with an apparent IC50 value of 40 μM. Previous work had revealed elevated cyclophylin A concentration in serum of asthmatic rats after receiving acupuncture treatment. Extracellular incubation of the oocytes in cyclophylin A for one day led to significant inhibition with an apparent IC50 value of about 1 μM. We suggest that effects of acupuncture and application of glycyrrhizic acid as an active constituent of Chinese medicine for treatment of asthma symptoms may partially be attributed to inhibition of the reversed mode of NCX1 and that these compounds may stimulate the search for new anti-asthmatic drugs.
基金National Natural Science Foundation of China(P2Y Receptors in Acupoints Involved in the Analgesic Effects of Laser Acupuncture(No.81102635)Efficacy and Neurobiological Mechanisms of Traditional MoxibustionBased Laser Moxibustion with Specific Wavelength on Inflammatory Pain(No.81320108028))+2 种基金The National Basic Research Program of China(973 programs,Basic Research on Trigger Signals in Acupoints and Specific Modulation of Correlated Target Viscera(No.2012CB518502)Biologic Research on Characteristics of Infrared Physics and Mechanisms in Moxibustion,(No.2015CB554505))The Project of State Administration of Traditional Chinese Medicine of China(Acupuncture,No.ZYSNXD-CC-ZDXK-07)
文摘OBJECTIVE:To investigate the role of adenosine triphosphate(ATP) purinergic signaling in mast cells(MCs) modulated by heat to further understand the molecular mechanisms of moxibustion.METHODS:Skin temperatures induced by monkshood cake moxibustion were evaluated by measuring the Neiguan acupoint(PC 6) from 31 participants with a digital thermocouple thermometer.Temperatures of 43 ℃ and 52 ℃ were applied to cultured human leukemia mast cell line HMC-1 in vitro.Calcium fluorescence was applied to detect intracellular Ca^(2+)([Ca^(2+)]_i).Extracellular ATP contents were measured by luciferin-luciferase assay.RESULTS:Maximum skin temperatures mostly ranged from 40-45 ℃,but some reached up to50 ℃.Both 43 ℃,and 52 ℃ induced MC degranulation,which was accompanied by an increase in[Ca^(2+)]_i and ATP release.Complexing extracellular Ca^(2+) with 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA) inhibited the noxious heat-induced elevation of[Ca^(2+)]_i and prevented the enhanced ATP secretion by those cells at 52℃ but not 43 ℃.CONCLUSION:Monkshood cake moxibustion can generate heat sufficient to trigger cellular events of MCs,including degranulation,[Ca^(2+)]_i elevation,and ATP release,suggesting that purinergic signals originating from MCs are possibly the initiating response of acupoints to moxibustion.
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China(973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
文摘目的:研究阿片受体与神经递质转运蛋白和钠钾泵在针刺镇痛中的可能机制。方法:利用爪蟾卵母细胞所建立的异源性蛋白表达模型,通过基因显微注射技术,应用双电极电压钳方法检测所表达靶蛋白的跨膜稳态电流。结果:δ-阿片受体(DOR)与γ-氨基丁酸转运蛋白(GAT1)、谷氨酸转运蛋白(EAAC1)或钠钾泵共表达均可降低神经递质转运蛋白的活性,阿片受体的激活以不同方式影响转运蛋白的活性:1)GAT1活性被进一步抑制;2)EAAC1活性增强;3)钠钾泵被抑制会导致 DOR 对激动剂(DPDPE)的敏感性增加。结论:DOR 的激活可使突触间隙的 GABA 水平增加,而谷氨酸浓度减少,钠钾泵的抑制导致阿片受体激动剂的敏感性增加,我们认为内源性哇巴因可能放大了这些效应。这些协同性效应可能是痛觉抑制和/或针刺镇痛的分子机制。
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China(973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
文摘Objective:To better understand the function of mast cells in acupuncture points(acupoints)inacupuncture-induced analgesia.The author tested their sensitivity to mechanical,thermo and lightstimulation.Methods:The tail flick model was applied to measure analgesia in rats,and the authordetermined the density of mast cells in tissue slices and their degranulation ratio before/after acupuncture.The author also applied the patch-clamp technique to investigate activation of human mast cells(HMC1cell line)by mechanical stress or noxious heat,and the author optically observed degranulation
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China (973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China(973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
文摘Objective:Cyclophilin A (Cyp A) is a ubiquitously distributed protein belonging to the immunophilin family and is recognized as the host cell receptor for the potent immunosuppressive
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China(973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
文摘Objective:To establish a new and better model to investigate the properties of mast cells that couldbe involved in acupuncture process mechanisms.Methods:Connective tissue under the corium at thearea of acupuncture point Zusanli(ST 36)from rat was acutely bluntly separated with forceps andscissors,and incubated in bath solution up to several hours.Mast cells in slices of that tissue wereirradiated with laser light of 650 nm,and changes in the appearance were observed under microscope.In addition,patch-clamp technique in whole-cell configuration was employed to induce mechano-
基金the Science Foundation of Shanghai Municipal Commission of Science and Technology(05DZ19745,06DZ19732,064319053,07DZ19722,07DZ19733)the National Basic Research Program of China(973 Program,2005CB523306)Shanghai Leading Academic Discipline Project(B112 and T0302)
文摘Objective:a-asarone is a major effective component that can be isolated from Acorus tatarinowii Schott,a Chinese herbal medicine.Clinical investigations have shown that a-asarone has strong sedative andanti-convulsive action in the central nervous system.In recent years,several medicines containinga-asarone were applied in treatment of asthma,bronchitis,expectorant,or epilepsy.However,theunderlying cellular mechanism of a-asarone is still unknown.Here the authors considered EAAC1。
文摘Conserved Ser 354, Ser 357 in the seventh transmembrane domain of NET were mutated and the function of mutants was studied by 3H-NE influx measurement. Double mutation at these two serine sites reduced the activity of NET to base level. It is inferred that these two sites may be involved in the recognition and transport activity of the NET protein.
