Washed human platelets were loaded with the Ca2+-sensitive photoprotein, aequorin. using hypoosmotic shock treatment-technique. Then aggregation and cytoplasmic ionized calcium concentration ( [Ca2+] i) changes in res...Washed human platelets were loaded with the Ca2+-sensitive photoprotein, aequorin. using hypoosmotic shock treatment-technique. Then aggregation and cytoplasmic ionized calcium concentration ( [Ca2+] i) changes in response to collagen or thrombin were measured simultaneously in the aequorin-loaded human platelets with a Platelet Ionized Calcium Aggregometer. 764-3. an active component isolated from the Chinese medicinal herb Salvia Miltiorrhiza Bge, inhibited platelet [Ca2+]i rise as well as aggregation evoked by collagen or thrombin in the presence of extracellular Ca2+. After the extracellular Ca2+. was removed by addition of EGTA, collagen or thrombin. causing no aggregation. still elicited platelet [Ca2+] i rise which reflected Ca2+ mobilization from intraplatelet stores. Under this condition, 764-3 could also suppress platelet [Ca2+] i rise. Analysis shows that 764-3 inhibrts platelet Ca2+ influx and Ca2+ mobilization with similar potency. which accounts for its suppression of platelet [Ca2+] i rise, and must contribute to its inhibition of platelet aggregation.展开更多
AIM:To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI).METHODS:Using freshly...AIM:To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI).METHODS:Using freshly isolated hepatocytes from a rat model of HIRI (and controls),we measured cyto-solic free Ca 2+ concentration (by calcium imaging),net Ca 2+ fluxes (by a non-invasive micro-test technique),the SOC current (I SOC ;by whole-cell patch-clamp record-ing),and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays].RESULTS:Ca 2+ oscillations and net Ca 2+ fluxes medi-ated by Ca 2+ entry via SOCs were observed in rat he-patocytes.I SOC was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA,P <0.05) and was inhibited by La 3+.Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls,an effect reversed by SOC blockers.CONCLUSION:SOCs are pivotal in HIRI.SOC blockers protected against HIRI and assisted the recovery of se-cretory function in hepatocytes.Thus,they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.展开更多
AIM: To investigate the effects of anti-fibrosis I herbal compound on intracellular Ca2+ in activated hepatic stellate cell (HSC) and to try to survey its molecular mechanism in treatment and prevention of hepatic fib...AIM: To investigate the effects of anti-fibrosis I herbal compound on intracellular Ca2+ in activated hepatic stellate cell (HSC) and to try to survey its molecular mechanism in treatment and prevention of hepatic fibrosis and portal hypertension. METHODS: The activated HSC line was plated on small glass cover slips in 24 wells culture dishes at a density of 5×106 /mL, and incubated in RPMI-1640 media for 24 h. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured with laser scanning confocal microscopy (LSCM). The dynamic changes of intracellular Ca2+, stimulated by carbon tetrachloride, TGF-β1 antibody and the drug serum of anti-fibrosis I herbal compound and under orthogonal design were determined by LSCM. The effect of anti-fibrosis I herbal compound on intracellular Ca2+ was observed before and after the addition of TGF-β1 antibody. RESULTS: The intracellular Ca2+ were significantly different in different dosage of carbon tetrachloride anti-fibrosis I formula drug serum, TGF-β1 antibody and different turn of these substance, but their interval time between CCl4 and TGF-β1 antibody, CCl4 and anti-fibrosis I drug serum had no influence on intracellular Ca2+. The result showed intracellular Ca2+ wasn't significantly different between rat serum without anti-fibrosis I and untreated group. After carbon tetrachloride stimulation, intracellular Ca2+ of activated HSC increased significantly when the dosage of CCl4 from 5 to 15 mmol/L, however, decreased significantly after stimulation by 5-20 μg/mL TGF-β1 antibody or 5-20 mL/L drug serum. Moreover, before and after the addition of TGF-β1 antibody, intracellular Ca2+ was significantly different. These results suggested that the molecular mechanism was independent of blocking TGF-β1 effects. CONCLUSION: Anti-fibrosis I herbal compound may treat hepatic fibrosis and decrease portal hypertension by inhibiting activated HSC contractility through decrease of intracellular Ca2+.展开更多
This study focused on the influences of opioids on the generation of antibody against sheep erythrocyte in vitro, It was found that morphine. a-CAO, DADLE, MENK were able to inhibit the capacity of murine spleen cell...This study focused on the influences of opioids on the generation of antibody against sheep erythrocyte in vitro, It was found that morphine. a-CAO, DADLE, MENK were able to inhibit the capacity of murine spleen cells to generate antibody and leukotriene C4 and conversely. dynorphin was able to stimulate the capacity of murine spleen cells to generate antibody and leukotriene C4. Morphine, a-CAO, MENK, DADLE, dynorphin decreased intracellular cAMP level, increased [Ca(2+)]i and calmodulin activity. The effects were completely blocked by naloxone, the specific opioid antagonist. Our results showed that opioids regulate the production of antibody in murine spleen cells, and alter intracellular cAMP, [Ca(2+)]i calmodulin activity. and leukotriene C4 production by way of binding to different receptor types.展开更多
Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation. Prior data from synchronized cell population could not actually stand for various CaM levels in different ph...Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation. Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle. Here, based upon quantitative measurement of fluorescence in individual cells, a method was developed to investigate intracellular total CaM and Ca2+-activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level, and Ca2+ -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP). In mouse erythroleuke-mia (MEL) cells, total CaM level increased from G1 through S to G2 M, reaching a maximum of 2-fold increase, then reduced to half amount after cell division. Meanwhile, Ca2+-activated CaM also in creased through the cell cycle (G1 , S, G2M). Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation, and, equally or even more important, Ca2+-dependent activation of CaM. Ca2+- activated CaM decreased after cell divi-sion. The results suggested that CaM gene expression and Ca2+-modulated CaM activation act synergistically to accomplish the cell cycle progression.展开更多
Background Recently, it has been proposed that the autoantibodies against various cardiovascular receptors play a role in the pathogenesis of primary hypertension. In this study, we aimed to identify whether or not th...Background Recently, it has been proposed that the autoantibodies against various cardiovascular receptors play a role in the pathogenesis of primary hypertension. In this study, we aimed to identify whether or not there are autoantibodies against cardiovascular L-type Ca^2＋ channels in patients with primary hypertension. Methods A peptide corresponding to the sequence 2-16 of the alc-subunit of L-type Ca^2＋ channel was used as an antigen to screen the autoantibodies from 90 patients with primary hypertension and 45 healthy controls by an enzyme-linked immunosorbent assay （ELISA）. The clinical data of 90 hypertensive patients were compared between patients with and without these autoantibodies. Results Serum from 3 （6.7%） of the 45 healthy controls, 33 （36.7%） of 90 hypertensives showed positive responses in ELISA （P 〈0.01）. The prevalence of such autoantibodies in two subgroups of hypertensives with coronary heart disease （9/21, 57.14%, P 〈0.05） and left ventricular diastolic dysfunction （28/63, 44.4%, P 〈0.05） was higher than in those without the corresponding complications. And the patients with such autoantibodies had lower E/A than patients without such autoantibodies （0.803±0.191 vs 1.004±0.322, P=0.002）. Conclusion There are autoantibodies against vascular L-tyPe Ca^2＋ channels in patients with primary hvnertension.展开更多
文摘Washed human platelets were loaded with the Ca2+-sensitive photoprotein, aequorin. using hypoosmotic shock treatment-technique. Then aggregation and cytoplasmic ionized calcium concentration ( [Ca2+] i) changes in response to collagen or thrombin were measured simultaneously in the aequorin-loaded human platelets with a Platelet Ionized Calcium Aggregometer. 764-3. an active component isolated from the Chinese medicinal herb Salvia Miltiorrhiza Bge, inhibited platelet [Ca2+]i rise as well as aggregation evoked by collagen or thrombin in the presence of extracellular Ca2+. After the extracellular Ca2+. was removed by addition of EGTA, collagen or thrombin. causing no aggregation. still elicited platelet [Ca2+] i rise which reflected Ca2+ mobilization from intraplatelet stores. Under this condition, 764-3 could also suppress platelet [Ca2+] i rise. Analysis shows that 764-3 inhibrts platelet Ca2+ influx and Ca2+ mobilization with similar potency. which accounts for its suppression of platelet [Ca2+] i rise, and must contribute to its inhibition of platelet aggregation.
基金Supported by The National Natural Science Foundation ofChina,No.30670744and81071996Tsinghua-Yue-Yuen Medical Science Foundation,No.20240000531and20240000547
文摘AIM:To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI).METHODS:Using freshly isolated hepatocytes from a rat model of HIRI (and controls),we measured cyto-solic free Ca 2+ concentration (by calcium imaging),net Ca 2+ fluxes (by a non-invasive micro-test technique),the SOC current (I SOC ;by whole-cell patch-clamp record-ing),and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays].RESULTS:Ca 2+ oscillations and net Ca 2+ fluxes medi-ated by Ca 2+ entry via SOCs were observed in rat he-patocytes.I SOC was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA,P <0.05) and was inhibited by La 3+.Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls,an effect reversed by SOC blockers.CONCLUSION:SOCs are pivotal in HIRI.SOC blockers protected against HIRI and assisted the recovery of se-cretory function in hepatocytes.Thus,they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.
基金Supported by the National Science Foundation of Hebei Province,No. 302489
文摘AIM: To investigate the effects of anti-fibrosis I herbal compound on intracellular Ca2+ in activated hepatic stellate cell (HSC) and to try to survey its molecular mechanism in treatment and prevention of hepatic fibrosis and portal hypertension. METHODS: The activated HSC line was plated on small glass cover slips in 24 wells culture dishes at a density of 5×106 /mL, and incubated in RPMI-1640 media for 24 h. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured with laser scanning confocal microscopy (LSCM). The dynamic changes of intracellular Ca2+, stimulated by carbon tetrachloride, TGF-β1 antibody and the drug serum of anti-fibrosis I herbal compound and under orthogonal design were determined by LSCM. The effect of anti-fibrosis I herbal compound on intracellular Ca2+ was observed before and after the addition of TGF-β1 antibody. RESULTS: The intracellular Ca2+ were significantly different in different dosage of carbon tetrachloride anti-fibrosis I formula drug serum, TGF-β1 antibody and different turn of these substance, but their interval time between CCl4 and TGF-β1 antibody, CCl4 and anti-fibrosis I drug serum had no influence on intracellular Ca2+. The result showed intracellular Ca2+ wasn't significantly different between rat serum without anti-fibrosis I and untreated group. After carbon tetrachloride stimulation, intracellular Ca2+ of activated HSC increased significantly when the dosage of CCl4 from 5 to 15 mmol/L, however, decreased significantly after stimulation by 5-20 μg/mL TGF-β1 antibody or 5-20 mL/L drug serum. Moreover, before and after the addition of TGF-β1 antibody, intracellular Ca2+ was significantly different. These results suggested that the molecular mechanism was independent of blocking TGF-β1 effects. CONCLUSION: Anti-fibrosis I herbal compound may treat hepatic fibrosis and decrease portal hypertension by inhibiting activated HSC contractility through decrease of intracellular Ca2+.
文摘This study focused on the influences of opioids on the generation of antibody against sheep erythrocyte in vitro, It was found that morphine. a-CAO, DADLE, MENK were able to inhibit the capacity of murine spleen cells to generate antibody and leukotriene C4 and conversely. dynorphin was able to stimulate the capacity of murine spleen cells to generate antibody and leukotriene C4. Morphine, a-CAO, MENK, DADLE, dynorphin decreased intracellular cAMP level, increased [Ca(2+)]i and calmodulin activity. The effects were completely blocked by naloxone, the specific opioid antagonist. Our results showed that opioids regulate the production of antibody in murine spleen cells, and alter intracellular cAMP, [Ca(2+)]i calmodulin activity. and leukotriene C4 production by way of binding to different receptor types.
文摘Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation. Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle. Here, based upon quantitative measurement of fluorescence in individual cells, a method was developed to investigate intracellular total CaM and Ca2+-activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level, and Ca2+ -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP). In mouse erythroleuke-mia (MEL) cells, total CaM level increased from G1 through S to G2 M, reaching a maximum of 2-fold increase, then reduced to half amount after cell division. Meanwhile, Ca2+-activated CaM also in creased through the cell cycle (G1 , S, G2M). Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation, and, equally or even more important, Ca2+-dependent activation of CaM. Ca2+- activated CaM decreased after cell divi-sion. The results suggested that CaM gene expression and Ca2+-modulated CaM activation act synergistically to accomplish the cell cycle progression.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. C03030201）.
文摘Background Recently, it has been proposed that the autoantibodies against various cardiovascular receptors play a role in the pathogenesis of primary hypertension. In this study, we aimed to identify whether or not there are autoantibodies against cardiovascular L-type Ca^2＋ channels in patients with primary hypertension. Methods A peptide corresponding to the sequence 2-16 of the alc-subunit of L-type Ca^2＋ channel was used as an antigen to screen the autoantibodies from 90 patients with primary hypertension and 45 healthy controls by an enzyme-linked immunosorbent assay （ELISA）. The clinical data of 90 hypertensive patients were compared between patients with and without these autoantibodies. Results Serum from 3 （6.7%） of the 45 healthy controls, 33 （36.7%） of 90 hypertensives showed positive responses in ELISA （P 〈0.01）. The prevalence of such autoantibodies in two subgroups of hypertensives with coronary heart disease （9/21, 57.14%, P 〈0.05） and left ventricular diastolic dysfunction （28/63, 44.4%, P 〈0.05） was higher than in those without the corresponding complications. And the patients with such autoantibodies had lower E/A than patients without such autoantibodies （0.803±0.191 vs 1.004±0.322, P=0.002）. Conclusion There are autoantibodies against vascular L-tyPe Ca^2＋ channels in patients with primary hvnertension.
