[Protective effects of ouabain conjugated peptide from Ph.D.-7 Library on vascular endothelial cell].
OBJECTIVE
To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension.


METHODS
In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph.D.-7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na(+)-K(+)-ATPase alpha1-subunit and beta1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na(+) in EAhy926 cells were measured by laser confocal microscope.


RESULTS
The ouabain conjugated peptide was found out, and it was occupied in 0.64 (9/14). The analysis of protein showed that the obtained peptides had no homology with Na(+)-K(+)-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and (3)H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na(+)-K(+)-ATPase alpha1-subunit and down-regulated expression of Na(+)-K(+)-ATPase beta1-subunit induced by ouabain in EAhy926 cells.


CONCLUSIONS
The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na(+)-K(+)-ATPase and explore novel anti-ouabain agents.
Xu
Zhong-Wei
ZW
Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Tianjin 300162, China. yunaixu@163.com
Xu
Rui-Cheng
RC
Chen
Xiao-Yi
XY
Liu
Ying-Fu
YF
D000595:Amino Acid Sequence; D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006973:Hypertension / Q000188:drug therapy; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology* / Q000627:therapeutic use; D010042:Ouabain / Q000737:chemistry* / Q000494:pharmacology; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
D009842:Oligopeptides; D019151:Peptide Library; D012333:RNA, Messenger; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase