Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcel...Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.展开更多
BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism o...BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Two pathways are involved in the purinoreceptor-mediated [Ca^2＋]i rise observed in nociceptive trigeminal ganglion neurons. One pathway involves the metabotropic P2Y receptors, which are associated with the IP3 sensitive Ca^2＋store, and the second pathway is coupled to ionotropic P2X receptors that induce the Ca^2＋ influx.展开更多
Alzheimer’s disease（AD）is the most common form of dementia representing a major problem for public health.In 2017 there were an estimated 50 million patients worldwide and this number is expected to almost double e...Alzheimer’s disease（AD）is the most common form of dementia representing a major problem for public health.In 2017 there were an estimated 50 million patients worldwide and this number is expected to almost double every 20years,reaching 75 million in 2030 and 131.5 million in 2050（https：//www.alz.co.uk/research/statistics）.展开更多
In single rat pancreatic βcells, using fura-2 microfluorometry to measure [Ca2+] response upon different stimuli, the ways of calcium regulation have been studied. When the extracellular calcium concentration was 2.5...In single rat pancreatic βcells, using fura-2 microfluorometry to measure [Ca2+] response upon different stimuli, the ways of calcium regulation have been studied. When the extracellular calcium concentration was 2.5 mmol/L, either 60 mmol/L KCI, 20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]. Such increase in [Ca2+] was absent when the same stimuli were applied under zero extracellular calcium. These results indicate that the increase of [Ca2+] is induced by the activation of voltage-dependent calcium channels in p celis. The manifold forms of [Ca2+] change induced by glucose imply that the effects of glucose are complex. 5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+], which is independent of the external calcium, suggesting that [Ca2+] can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in p celis. The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s), it is展开更多
AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwe...AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.展开更多
Calcium （Ca） plays an important role in the metabolism of higher plants. Recently, research on Ca^2＋ in plants has been focused especially at the cellular and molecular levels. Uptake, transport, and distribution a...Calcium （Ca） plays an important role in the metabolism of higher plants. Recently, research on Ca^2＋ in plants has been focused especially at the cellular and molecular levels. Uptake, transport, and distribution are also very important for Ca to accomplish its function at the whole-plant level. In this experiment, one-year-old apple seedlings （M. hupehensis Rehd.） were investigated to determine the distribution of stored Ca, the different forms of Ca, and Ca^2＋-ATPase activity after treatment with indole butyric acid （IBA）. The results showed that the total Ca measured in mature leaves and Ca^2＋- ATPase activity in tender leaves were higher compared with those in the control （CK）. Calcium nitrate and calcium chloride （ALe-Ca） and calcium phosphate and calcium carbonate （HAC-Ca） decreased in both mature leaves and shoots, whereas water-soluble calcium （H2O-Ca）, calcium pectate （NaCl-Ca）, and calcium oxalate （HCl-Ca） increased. The percentage of active calcium, calcium pectate, and water-soluble calcium increased, whereas the percentage of calcium phosphate and calcium carbonate decreased. When treated with IBA, calcium fractions and percentage of the different forms of Ca was enhanced in 40 part per million （ppm） IBA compared with 20 ppm IBA and water. The results indicated that IBA increased the percentage of both active calcium （NaCl-Ca and H2O-Ca） in tender shoots and boosted the transportation of stored Ca in plants. IBA promoted Ca^2＋-ATPase activity and Ca^2＋ uptake in tender shoots of M. hupehensis. It can improve the total Ca contents and the relative percentage of Ca.展开更多
The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subce...The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.展开更多
The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (...The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC 50 value of 64.63±10.56 μmol/L (n=8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.展开更多
文摘Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.
基金the National Natural Science Foundation of China, No.30670694 the Natural Science Foundation of Anhui Province Department of Education in China, No.2006KJ361B+2 种基金 the National Science Fund for Distinguished Young Scholars of Anhui Medical University, No.GJJQ-0801 the Scientific Research Foundation for Doctor of Anhui Medical University, No. XJ2005006the Special Foundation for Young Scientists in Higher Education Institutions of Anhui Province, No.2010SQRL078
文摘BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Two pathways are involved in the purinoreceptor-mediated [Ca^2＋]i rise observed in nociceptive trigeminal ganglion neurons. One pathway involves the metabotropic P2Y receptors, which are associated with the IP3 sensitive Ca^2＋store, and the second pathway is coupled to ionotropic P2X receptors that induce the Ca^2＋ influx.
基金supported by the Volkswagen Stiftung(grant No.90233)to OG
文摘Alzheimer’s disease（AD）is the most common form of dementia representing a major problem for public health.In 2017 there were an estimated 50 million patients worldwide and this number is expected to almost double every 20years,reaching 75 million in 2030 and 131.5 million in 2050（https：//www.alz.co.uk/research/statistics）.
文摘In single rat pancreatic βcells, using fura-2 microfluorometry to measure [Ca2+] response upon different stimuli, the ways of calcium regulation have been studied. When the extracellular calcium concentration was 2.5 mmol/L, either 60 mmol/L KCI, 20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]. Such increase in [Ca2+] was absent when the same stimuli were applied under zero extracellular calcium. These results indicate that the increase of [Ca2+] is induced by the activation of voltage-dependent calcium channels in p celis. The manifold forms of [Ca2+] change induced by glucose imply that the effects of glucose are complex. 5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+], which is independent of the external calcium, suggesting that [Ca2+] can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in p celis. The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s), it is
基金Supported by The Natural Science Foundation of China,No.81271950,to Ji QMProjects of International/HMT(Hong Kong,Macao,and Taiwan)Cooperation and Innovation Platform in Science and Technology of Guangdong Higher Education Institutions,No.2012gjhz0009,to Liu ZG+2 种基金Key Laboratory Construction Program of Shenzhen,No.SW201110010,to Liu ZGBasic Research Program of Shenzhen University,No.201101,to Liu ZGBasic Research Foundation of Shenzhen,No.JC201005250059A,JCYJ20120613115535998
文摘AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.
基金The study was supported financially by the National Natural Science Foundation of China(30170655&30270923).
文摘Calcium （Ca） plays an important role in the metabolism of higher plants. Recently, research on Ca^2＋ in plants has been focused especially at the cellular and molecular levels. Uptake, transport, and distribution are also very important for Ca to accomplish its function at the whole-plant level. In this experiment, one-year-old apple seedlings （M. hupehensis Rehd.） were investigated to determine the distribution of stored Ca, the different forms of Ca, and Ca^2＋-ATPase activity after treatment with indole butyric acid （IBA）. The results showed that the total Ca measured in mature leaves and Ca^2＋- ATPase activity in tender leaves were higher compared with those in the control （CK）. Calcium nitrate and calcium chloride （ALe-Ca） and calcium phosphate and calcium carbonate （HAC-Ca） decreased in both mature leaves and shoots, whereas water-soluble calcium （H2O-Ca）, calcium pectate （NaCl-Ca）, and calcium oxalate （HCl-Ca） increased. The percentage of active calcium, calcium pectate, and water-soluble calcium increased, whereas the percentage of calcium phosphate and calcium carbonate decreased. When treated with IBA, calcium fractions and percentage of the different forms of Ca was enhanced in 40 part per million （ppm） IBA compared with 20 ppm IBA and water. The results indicated that IBA increased the percentage of both active calcium （NaCl-Ca and H2O-Ca） in tender shoots and boosted the transportation of stored Ca in plants. IBA promoted Ca^2＋-ATPase activity and Ca^2＋ uptake in tender shoots of M. hupehensis. It can improve the total Ca contents and the relative percentage of Ca.
文摘The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.
基金This project was supported by the National Natural Sci ence Foundation of China (No. 30270532), the Trans Cen tury Excellent Talent Development Plan Fund of Ministry ofEducation of China (Official Letter No. 2002 48) and Shu guang Program Project of Shanghai Educational Committee(No. 02SG20).
文摘The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC 50 value of 64.63±10.56 μmol/L (n=8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.
