Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane fluidity of mitochondria were investigated between the wolf spider(Pirata subpiraticus(Boes.et Str.))and the rice stem bo...Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane fluidity of mitochondria were investigated between the wolf spider(Pirata subpiraticus(Boes.et Str.))and the rice stem borer(Chilo suppressalis(Walker)).Based on a comparison of LD_(50) values,the toxicities of the tested insecticides were higher to the wolf spider than to the rice stem borer.Cyhalothrin at 1×10^(–4) mmol L^(–1) caused inhibition of the mitochondrial Na~+-K~+-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities,and it’s inhibitions on Na+-K+-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities were significantly higher in the wolf spider(44 and 28%)than in the rice stem borer(19 and 11%).Methamidophos at 1×10^(–4) mmol L^(–1) decreased Ca^(2+)-Mg^(2+)-ATPase activity by 16 and 27%in the wolf spider and the rice stem borer,respectively,but no significant effect on the specific activity of Na+-K+-ATPase was observed.The DPH(1,6-diphenyl-1,3,5-hexatriene)fluorescence polarization values of mitochondrial membranes were not significantly affected by methamidophos in either species.However,cyhalothrin and alpha-cypermethrin induced the values of DPH polarization of mitochondrial membrane increasing with the concentration of cyhalothrin and alpha-cypermethrin from 20 to 100μmol L^(–1) in the rice stem borer and the wolf spider.Effect of ethofenprox on fluidity of the wolf spider and the rice stem borer was contrary.These results suggest that both inhibition of membrane ATPase and changes of membrane fluidity could be appended to the action mechanisms of pyrethroid insecticides.展开更多
Plasma membrane calcium ATPase (PMCA) plays a critical role in transporting Ca^2+ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2+ homeostasis and in biominerali...Plasma membrane calcium ATPase (PMCA) plays a critical role in transporting Ca^2+ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2+ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2+ homeostasis and the mechanism of mineralization in pearl oyster.展开更多
The activation and deactivation of Ca^(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Con...The activation and deactivation of Ca^(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca^(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca 2+ concentrations ([Ca^(2+)]_(i)), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca^(2+)]_(i) caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.展开更多
Background Chenodeoxycholic acid (CDC) is an apolar bile salt and damages hepatocytes, whereas ursodeoxycholic acid (UDC) is a more polar bile salt and protects liver cell against toxic bile salts. We therefore inves...Background Chenodeoxycholic acid (CDC) is an apolar bile salt and damages hepatocytes, whereas ursodeoxycholic acid (UDC) is a more polar bile salt and protects liver cell against toxic bile salts. We therefore investigated the activity of membrane associated Na + K + ATPase, a coenzyme of the sodium dependent bile salt carrier. Methods Liver plasma membranes (LPM) were isolated from rat livers according to the method of Song et al (J Cell Biol 1969; 41:124). The LPM were incubated with bile salts (TUDC, UDC, TCDC, CDC) in concentrations of 0.1 2 mmol/L for 0 30 minutes at room temperature. To study reversibility of the effect of CDC, LPM were diluted with buffer 50 times of volume after incubation. The activity of membrane associated Na + K+ ATPase was determined enzymatically at 37℃ and the phospholipid (PL) release into the supernatant was measured. Results CDC and TCDC both showed a dose dependent inhibition of the enzyme activity (P<0.01 vs control). Gastroenterology, Center of Internal Medicine, University Hospital, D 60590 Frankfurt Main, Germany (You T, Guldutuna S, Bhatti S and Leuschner U)Initially TCDC induced an increase in enzyme activity at concentrations of 0.1 1 mmol/L, however, after 3 minutes activity repidly decreased to less than 30% of controls. Up to a concentration of 1 mmol/L CDC the inhibition of enzyme activity could be reversed by diluting the bile salt in the incubation medium. At a concentration of 2 mmol/L CDC activity was only partially restored and at this concentration a marked PL release into the supernatant was observed, indicating solubilization of the membranes. UDC did not decrease the enzyme activity at concentrations of 0.1 2 mmol/L. At a concentration of 2 mmol/L TUDC inhibited the Na + K + ATPase by about 20%. Solubilization of membrane PL was not observed. Conclusion CDC and TCDC inhibited Na + K + ATPase. Dilution of the bile salt with buffer reversed the inhibitory effect up to concentrations of 1 mmol/L CDC. The inhibitory effect is probably due to alteration of the plasma membrane. 2 mM CDC caused irreparable membrane damage. Physiological concentrations of UDC and TUDC did not affect membrane ATPase.展开更多
基金supported by the National Basic Research Program of China(2012CB114103)
文摘Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane fluidity of mitochondria were investigated between the wolf spider(Pirata subpiraticus(Boes.et Str.))and the rice stem borer(Chilo suppressalis(Walker)).Based on a comparison of LD_(50) values,the toxicities of the tested insecticides were higher to the wolf spider than to the rice stem borer.Cyhalothrin at 1×10^(–4) mmol L^(–1) caused inhibition of the mitochondrial Na~+-K~+-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities,and it’s inhibitions on Na+-K+-ATPase and Ca^(2+)-Mg^(2+)-ATPase activities were significantly higher in the wolf spider(44 and 28%)than in the rice stem borer(19 and 11%).Methamidophos at 1×10^(–4) mmol L^(–1) decreased Ca^(2+)-Mg^(2+)-ATPase activity by 16 and 27%in the wolf spider and the rice stem borer,respectively,but no significant effect on the specific activity of Na+-K+-ATPase was observed.The DPH(1,6-diphenyl-1,3,5-hexatriene)fluorescence polarization values of mitochondrial membranes were not significantly affected by methamidophos in either species.However,cyhalothrin and alpha-cypermethrin induced the values of DPH polarization of mitochondrial membrane increasing with the concentration of cyhalothrin and alpha-cypermethrin from 20 to 100μmol L^(–1) in the rice stem borer and the wolf spider.Effect of ethofenprox on fluidity of the wolf spider and the rice stem borer was contrary.These results suggest that both inhibition of membrane ATPase and changes of membrane fluidity could be appended to the action mechanisms of pyrethroid insecticides.
基金the National Natural Science Foundation of China (Nos. 30530600, 30371092, and 30221003)
文摘Plasma membrane calcium ATPase (PMCA) plays a critical role in transporting Ca^2+ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2+ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2+ homeostasis and the mechanism of mineralization in pearl oyster.
基金supported by grants from the National Basic Research Program of China(Grant Nos.2010CB833701 and 2012CB934003)the National Natural Science Foundation of China(Grant No.31070736).
文摘The activation and deactivation of Ca^(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca^(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca 2+ concentrations ([Ca^(2+)]_(i)), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca^(2+)]_(i) caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.
文摘Background Chenodeoxycholic acid (CDC) is an apolar bile salt and damages hepatocytes, whereas ursodeoxycholic acid (UDC) is a more polar bile salt and protects liver cell against toxic bile salts. We therefore investigated the activity of membrane associated Na + K + ATPase, a coenzyme of the sodium dependent bile salt carrier. Methods Liver plasma membranes (LPM) were isolated from rat livers according to the method of Song et al (J Cell Biol 1969; 41:124). The LPM were incubated with bile salts (TUDC, UDC, TCDC, CDC) in concentrations of 0.1 2 mmol/L for 0 30 minutes at room temperature. To study reversibility of the effect of CDC, LPM were diluted with buffer 50 times of volume after incubation. The activity of membrane associated Na + K+ ATPase was determined enzymatically at 37℃ and the phospholipid (PL) release into the supernatant was measured. Results CDC and TCDC both showed a dose dependent inhibition of the enzyme activity (P<0.01 vs control). Gastroenterology, Center of Internal Medicine, University Hospital, D 60590 Frankfurt Main, Germany (You T, Guldutuna S, Bhatti S and Leuschner U)Initially TCDC induced an increase in enzyme activity at concentrations of 0.1 1 mmol/L, however, after 3 minutes activity repidly decreased to less than 30% of controls. Up to a concentration of 1 mmol/L CDC the inhibition of enzyme activity could be reversed by diluting the bile salt in the incubation medium. At a concentration of 2 mmol/L CDC activity was only partially restored and at this concentration a marked PL release into the supernatant was observed, indicating solubilization of the membranes. UDC did not decrease the enzyme activity at concentrations of 0.1 2 mmol/L. At a concentration of 2 mmol/L TUDC inhibited the Na + K + ATPase by about 20%. Solubilization of membrane PL was not observed. Conclusion CDC and TCDC inhibited Na + K + ATPase. Dilution of the bile salt with buffer reversed the inhibitory effect up to concentrations of 1 mmol/L CDC. The inhibitory effect is probably due to alteration of the plasma membrane. 2 mM CDC caused irreparable membrane damage. Physiological concentrations of UDC and TUDC did not affect membrane ATPase.