Abstract
We have used H2O2 as a pharmacologic agent to examine the effects of oxidizing conditions on lymphocyte signal pathways. Treatment of Ramos cells with 5-10 mM H2O2 gave rapid and strong tyrosine phosphorylation of multiple cellular proteins and activation of p72syk to levels equal to or greater than that observed upon surface Ig cross-linking. Strong Ca2+ signals that could be blocked by the tyrosine kinase inhibitor herbimycin A were also observed under these conditions. However, there was no increase in activity for the Src family kinases p56lck, p59fyn, or p56/p53lyn. Our findings that the p72syk tyrosine kinase responds to H2O2 treatment of cells suggest that this kinase is likely to contribute to cellular tyrosine phosphorylation and calcium signaling induced by oxidizing conditions. Furthermore, H2O2 may be useful as a pharmacologic agent to distinguish the effects of p72syk-related kinases from those of Src family kinases.
Highlights
We haveused H202 as apharmacologicagentto this study, we have employed H2OZalone as a pharmacologic examine theeffects of oxidizingconditionson lympho- agent to examine effects ofoxidationonproteintyrosine cyte signal pathways
H202and reactive oxygenintermediates such as superoxide change was observedforlower concentrations
In the current study we used kinase assays of p561ek, p59"",and p56/p53IY" were performed H202as a pharmacologic agent over a range of doses to to determine whether these kinases increased in activity following H202 treatment of Ramos B cells(Fig. 4C).The autophosphorylated kinases are observed above the enolase band used as an exogenous substrate
Summary
Lymphocyte activation responses have been shown to be sensitive to the effects of oxidants suchas HzO2.Exposure to. Lymphocytes may be exposed to oxidizing conditions at sites of inflammation due to superoxide anion and HzOzproducedby neutrophilsand monocytes These gene, La Jolla, CA) with 254-nm lamps. Increased activity upon stimulation via the TCR' in T cells Immunoblots and Immune Complex Kinase Assays-Cellswere or surfaceimmunoglobulin (sIg) in B cells. Immune complexes were collected on Protein A-Sepharose beads (Repligen, Cambridge, MA) and washed four times with Nonidet P-40 lysis buffer. Immune complex kinase assays of samples from Ramos cells were performed as previously described That enolase was not employed as a substrate for assays of ~72'~'F. or kinase assaysof samples from Bal 17 cells, cellswere lysed in a buffer. MM Hepes pH 7.5, 150 mM NaCl, 1 mM vanadate, 10 pg/ml aprotinin and vanadate, 0.1% Brij 96, and two times in the same buffer but withoutdetergent.Kinasereactions were performed for 1 min a t "c 30 in 25 mM Hepes pH 7.5, 10 mM MnCI2,5 mM p-nitrophenyl. Phosphoamino acid analysis was performed as previously described [16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
