Identification Of Mini P-glycoprotein In Rat Islets-A Preliminary Study

Objective In our preliminary study,we detected a 65-kDa membrane protein named mini P-glycoprotein(encoded by mdrl,abcb1a/b)by its specific monoclonal antibodies,C219,in rat islets.The mini P-glycoprotein was further evaluated to posses some biological functions similar to sulfonylureas receptor.Hence we speculated that the mini P-glycoprotein had regulatory effects mediating biphasic insulin secretion,but not acted as the classical P-glycoprotein to serve as an efflux pump.Real-time Fluorescence Quantitative Technology(FQ-PCR)and Northern Blot techniques were applied in this study to identify the existence of the mini P-glycoprotein.Methods Indentifying bio-information of the rat P-glycoprotein:(1)Physical and chemical properties(2)Conservative sequence domains analysis.(3)The comparison of two conservative sequence domains by three-dimensional structure.(4)Hydrophilic prediction.Detecting the amino-terminal and carboxy-terminal expression of abcblb and abcbla gene by real-time fluorescence quantitative PCR:(1)Rat islets were isolated from pancreas by collagenase V digestion,insulinoma cells(INS-1)were cultured.Then total RNAs of rat islets and INS-1 cells,liver,kidney and brain were extracted,respectively.The cDNAs were run with specific primers selected from the mRNA of abcblb and abcb1a.(2)The PCR products were purified,transformed,cloned and extracted.(3)Three standard curves were made for the N-terminal,C-terminal expressions of abcb1b and abcb1a respectively(4)INS-1 cells,kidney,liver and brain were used to analyze expression the two genes severed as controls to rat islets.(5)The data were analyzed statistically.identifying alternative splicing form of abcblb gene in rat islets by northern blot:(1)Ttotal RNA of the INS-1 cells were extracted.The cDNAs were run with specific primers selected from the abcblb mRNA.(2)The PCR products were labeled by DIG as the probe for northern blot.(3)Total RNAs of isolated rat islets were extracted.The RNAs were separated by formaldehyde modified electrophoresis,and then transferred to nylon membrane by siphon imprinting method.(4)Hybridization of the probe was carried out on the nylon membrane.Results Indentifying bio-information of the rat P-glycoprotein:P-glycoprotein composes of 1275 amino acids with a relative molecular weight around 140,655.P-glycoprotein has two similar conservative sequence domains with 46%homology.N-terminal has 10 or 15-kDa areas with glycosylation.P-glycoprotein is a hydrophobic protein.Detecting the amino-terminal and carboxy-terminal expression of abcblb and abcb1a gene by real-time fluorescence quantitative PCR:(1)The quantity of the N-terminal of abcb1b was(26.29 + 26.29)× 104;C-terminal was(43.42 + 10.72)× 104,and abcb1a was(9.23 + 0.09)× 104.(2)The compassion of abcb1b expression quantities of different tissues and cells were INS-1 cell>islets>liver>kidney>brain.(3)In different tissues or cells,the compassion of abcbla expression quantities of different tissues and cells were liver>islets>kidney>brain>INS-1 cells.Identifying alternative splicing form of abcblb gene in rat islets by northern blot:(1)The 673 base pair fragments were labeled by DIG,then efficiency determination showed success of probe labeling.(2)The hybridization of northern blot was failed,but the dot hybridization confirmed that the probe could hybrid with RNA of rat islets.Conclusion(1)The expression quantity of abcb1b in rat islets is about 5 times greater than abcb1a.Since our previous studies showed that alpha cells almost had no expression of P-glycoprotein,P-glycoprotein existed rat islets is encoded by abcb1b.(2)The expressions of P-glycoprotein are tissue-specific.abcb1b expression is major in INS-1 cells and liver tissues,but abcbla is the one in brain tissues.A similar expression level of abcbla and abcblb was found in the kidney tissues.(3)The expression of the C-terminal of abcb1b is 1.7 times greater than its N-terminal.Hence an alternative splicing form of the abcblb gene can be presumed,which might be encoding the mini P-glycoprotein.(4)Northern bolt experiments were designed to identify lower molecular mRNA expressions in rat islets,and confirm the existence of an alternative splicing.Although probe-labeling was succeeded,the probe hybridization with RNAs was not successful.