Stereoselectivity Analysis Of Propranolol Enantiomer And Study Function Of GBLP

ObjectiveTo further elucidate the chiral biological performances of Pro and offer new experimental basis of stereoselectivity mechanism ofβ1 andβ2 adenoreceptor blocker enantiomers through study of the transepithelial transport of Propranolol(Pro) enantiomers (R-Pro and S-Pro) in the human colon carcinoma cell (Caco-2) monolayer model system , changes in the Protein spectrum of whole cell extracts isolated from R/S-Propranolol-treated Caco-2,hepatoma cell(HepG-2) and human umbilical vein endothelial cell(HUVEC) with comparative Proteome research ,and function of GBLP.Methods1.Set up the Caco-2 cell monolayer model: Planted and cultured the Caco-2 cells in the Transsell polycarbonate for certain days and evaluated the integrity and stability of the cell monolayer with several methods including the transepithelial electrical resistance (TEER),the apparent permeability coefficients(Papp) and morphology ( scanning electron microscopy(SEM) and transmission electron microscope(TEM)). 2.Investigation of the transepithelial transport of R-Pro and S-Pro: The Caco-2 cell monolayer was treated with R-Pro and S-Pro and detected the drug concentration of two sides of the cell monolayer by HPLC to elucidate the absorption and transport modality of the two enantiomers.3.Analysis of differently expressed Proteins in R/S-Propranolol treated Caco-2.HepG-2 and HUVEC cells with comparative proteome: First,suitable conditions for two-dimensional electrophoresis(2-DE) were ascertained, including suitable loading quantity of sample and staining method. The whole cell extracts of Caco-2,HepG-2,HUVEC treated with R/S-Propranolol were separated by two dimensional electrophoresis respectively. Image analysis was performed using PDQuest(7.4.0) image analysis software. Matrix assisted laser desorption/isonization time-of-flying mass spectrometry(MALDI-TOF-MS) was adopted to identity differential Protein spots and the mascot software was used to search for Proteins in SwissProt database of Mascot scour engine.4.To examine whether the expression of GBLP had difference in R-Pro and S-Pro -treated HUVEC: After cells treated with R-Pro and S-Pro respectively,Western blot analysis was used to detect the expression of GBLP;GBLP location was assessed by confocal microscopy.5.Analysis of Proteins which may interact with GBLP: The whole cell extracts were isolated from R-Pro and S-Pro treated HUVEC and control cells.Immunity precipitation(IP) and Liquid Chronatography-Electrospray Ionization-Mass Spectrometer/ Mass Spectrometer (LC-ESI-MS/MS) were used to isolate and identify Proteins which can interact with GBLP respectively.Results1.The Caco-2 cells were confluenced to a successive and integrity cell monolayer in morphology. The junction of cells was tight , cells were covered by brush border microvilli vertically ,and these morphology were similar with small intestine epithelial cells; The apparent permeability coefficients(Papp) was under 1.0×10-6 cm/s,meanwhile the transepithelial electrical resistance(TEER) was up 200(?*cm2)after the cell monolayer was cultured in DMEM medium for 21～25 days which hinted that the cell monolayer had good integrity; After the cell monolayer was cultured in Hank'balanced salt solution (HBSS) for 8h, the Papp was under 1.0×10-6 cm/s,meanwhile the TEER exceeded 200(?*cm2)which hinted that the cell monolayer had good stability.2.The Papp of R- and S-Propranolol in absorprive direction(apical to basolateral) and secretory direction(basolateral to apical) on Caco-2 cell monolayer were similar and had no significant difference.3.The loading quantity of sample and staining of SDS-PAGE gel were selected as 200μg and silver staining respectively via the optimizing conditions.Compared with the 2-DE gels of whole cell extracts isolated from R-Pro treated Caco-2,HepG-2 and HUVEC respectively,tweenty-five,thirty and thirty-six Protein spots respectively showed differential expression in S-Pro treated cells.Among them,25 ,15 and 18 Protein spots in Caco-2,HepG-2 and HUVEC respectively were selected to MALDI-TOF-MS analysis,of 8,4 and 10 spots were identified respectively.4.The Western blot and LSCM analysis both showed that the expression of GBLP in S-Pro treated HUVEC was obviously lower than that in R-Protreated cell.In addition,LSCM displayed the Protein GBLP locationed in Protoplasmic membrane and kytoplasm.5.The same eight Protein bands were observed both in control group and R-Pro /S-Pro treated group via immunity precipitation and nine Proteins in these bands were identified,of sevev Proteins were identified as casein alpha-S1,casein alpha-S1,beta casein,desmoglein 1 precursor,alpha enolase, peroxiredoxin 2 and fructose-bisphosphate aldolase C.Conclusions1.The Caco-2 cell monolayer model was set up successfully and it had good integrity and stablity.The model system can be uese to study of drug transepithelial transport.2. R-Pro and S-Pro belong to high-flux drug mainly through passive absorption and the two enantiomers have no stereoselectivity on transepithelial transport.3.Those differentialy expressed proteins identified in the study might play important roles in the mechanism of chiral biological performance of R-Pro and S-Pro.4.Expression of GBLP in R-Pro and S-Pro treated HUVEC showed manifest difference.According to the result of bioinformatics searching,we presume that GBLP might involved in the mechanism of chiral biological performance of R-Pro and S-Pro ,it might an important adaptor protein in protein G signal transduction,and it might a new potential drug target.5.Seven Proteins might have interactions with GBLP.The bioinformatics searching of these proteins displayed that GBLP might involved in adapter of energy metabolism and antioxidation mechanism which are closely related to biological effect of Propranolol.