The effects of membrane lipid raft disruption on glucocorticoid, progesterone, and antagonist Mifepristone-induced rapid responses in human T cells

• Main Authors: Pin-Yi Lin, 林品儀
• Other Authors: Eileen Jea Chien
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/50135006040944505974

碩士 === 國立陽明大學 === 生理學研究所 === 101 === Background: In our previous study, hydrocortisone (HC), dexamethasone (DEX), and progesterone (P4) trigger T cell rapid-responses on inhibition of Na+/H+ exchanger 1 (NHE1) activity, the increases of intracellular acidification and C-terminal resides phosphorylation of ezrin/radixin/moesin, ERM, (cp-ERM) level. In addition, P4 induces intracellular calcium elevation. HC, DEX, and P4 can immunosuppress T cell proliferation. Glucocorticoid receptor antagonist RU486 has antagonistic effects on immunosuppression caused by HC and DEX, but has no effect on immunosuppression by P4. Moreover, RU486 does not have antagonistic effect on cp-ERM level in T cells within 5 minutes by HC. Therefore, this study will further investigate (1) the roles of lipid rafts on inhibition of NHE1 activity by HC, DEX, and P4; (2) whether RU486 antagonizes the effects on cp-ERM caused by DEX and P4; (3) whether RU486 antagonizes the effects on the inhibition of NHE1 activity caused by HC, DEX, and P4; (4) the roles of lipid rafts on RU486 antagonistic effects on inhibition of NHE1 activity caused by HC, DEX, and P4. Methods: Methyl-β-cyclodextrin (MβCD) was used to disrupt lipid rafts. Protein kinase C (PKC) activator phorbol myristate acetate (PMA) and phospholipase C (PLC) inhibitor U73122 were used to investigate the mechanisms on NHE1 activation. Phytohemagglutinin (PHA) was used as control stimulant to activate T cells. BCECF was used to detect the change of intracellular pH and NHE1 activity. Fura-2 and Western blotting analysis were used to detect the level of intracellular calcium elevation and cp-ERM, separately. Results: (1) Both the activities of NHE1 and the level of cp-ERM were increased significantly after the disruption of lipid rafts in resting T cells. (2) The enhancement on NHE1 activities by PMA was not affected in T cells by lipid raft disruption. The inhibition on NHE1 activities by U73122 was also not affected by disruption. (3) The inhibition on NHE1 activities by HC, DEX, and P4 were vanished, whereas, the increases on NHE1 activities were instead significantly after the disruption of lipid rafts in T cells. Under the same situations, the increases on NHE1 activities by PHA were also enhanced. (4) The intracellular acidification by HC, DEX, and P4 was significantly inhibited and the intracellular alkalinization by PHA was enhanced in T cells after lipid raft disruption. In contrast, the intracellular calcium elevation by P4 or PHA was not affected after the disruption. (5) The cp-ERM level was enhanced significantly by HC, DEX and P4 in cells after lipid raft disruption. (6) RU486 did not exert antagonistic effects on cp-ERM by DEX within 5 min. But RU486 did have antagonistic effect on the inhibition of NHE1 activities by HC, DEX, and P4 in stimulated cells. (7) After lipid raft disruption, RU486 had no antagonistic effect on the inhibition of NHE1 activity by HC. Conclusions: (1) Both membrane NHE1 activity and cp-ERM level were increased in human T cells after lipid raft disruption. (2) The inhibition on NHE1 activities by HC, DEX, and P4 required the presences of lipid rafts in cells. After the disruption of rafts, NHE1 activities were increased by all stimulants. (3) RU486 alone could increase cp-ERM level but not affect the activities of NHE1 in T cells. Thus, RU486 did not antagonize the increase of cp-ERM level by HC and DEX. But, with intact lipid rafts in cells, RU486 did antagonize the inhibition of NHE1 activities caused by HC and DEX. RU486 lost its antagonistic effect on NHE1 activities after disruption of lipid rafts in T cells.