Analysis Of Membrane PIP₂ In Xenopus Oocytes By RP-HPLC

Phospholipids are characteristic components of cell membrane, mainly including phosphatidylcholine, phosphatidylserine and phosphatidylinositol. Phosphatidylinositol-4,5-bisphosphate (PIP2) is one type of phosphatidylinositol and plays an important role in cell signaling. PIP2, although comprises less than 1% of cell membrane phospholipids, plays multiple important roles in a wide variety of cellular processes, such as generation of second messengers, regulation of both endocytosis and exocytosis, membrane attachment to the cytoskeleton, etc. In addition, PIP2 can also modulate the function of many ion transporters and channels, such as Na/Ca exchanger, Na/H exchanger, voltage-dependent Ca channels, TRP channels, Kir channels and KCNQ channels, etc. The metabolism of PIP2 is involved in modulation of ion channel currents. In recent years, it has been found that neurotransmitters, protein kinases, pH , charged ions, drugs and metabolites of lipids can be involved in the regulation of ion channels by affecting the interaction of membrane PIP2 and ion channels. PIP2 may be the ultimate and crucial mediator in the current modulation of ion channels. Therefore, to develop the method of quantification of membrane PIP2, which will provide direct evidence for the involvement of PIP2, is greatly important for the study of mechanism for the modulation of ion channels.There are generally two ways of measuring PIP2. The classical way is to use radioisotope, which requires high specific activities and long labeling times, and the other way is to measure derivatives of PIP2 with suppressed conductivity detection or UV detection, which requires modification of the PIP2 structure. In our work, we set up a nonradioactive, fast and convenient method by reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection, which separated and quantified the membrane PIP2 in Xenopus oocytes effectively and directly. We also used the pharmacological agent wortmannin, high performance liquid chromatography/mass spectrometry (HPLC/MS) technique and laser scanning confocal microscopy (LSCM) to verify the accuracy and reliability of the established RP-HPLC method.Objective: To establish and test the reliability of a method of reversed-phase high performance liquid chromatography (RP-HPLC) to measure membrane PIP2 in Xenopus oocytes.Methods: (1) The establishment of RP-HPLC method: Chromatography was carried out on a Diamonsil C18 column (250mm×4.6mm,5μm) with a Diamonsil C18 guard column(4mm×4.6mm,5μm). The mobile phase contained methanol-acetonitrile-water (40:40:20, v/v/v) and the flow rate was 1.0ml/min using isocratic elution. The detection wavelength was at 205nm and column temperature at 25℃. (2) The analysis of membrane PIP2 in Xenopus oocytes by RP-HPLC: For wortmannin-treated group, oocytes were first incubated with wortmannin for 2 h, and then were manually lysed in 1ml cold lysis buffer and homogenized in the crushed ice. The homogenate was transferred to a 2-mL microfugetube and centrifuged for 5min at 5500rpm at 4℃. The supernatant was then centrifuged for 2h at 17500rpm at 4℃. The pellets were resuspended with 50μl lysis buffer (1μl per oocyte). 900μl methanol-chloroform (1:9, v/v) solution was then added to the suspension and phospholipids were extracted. Membrane PIP2 from wortmannin-treated group and control group (without wortmannin) were determined by RP-HPLC. Both the standard PIP2 and sample membrane PIP2 were dissolved with mobile phase solution containing methanol-acetonitrile-water (40:40:20, v/v/v), 20μl of each solution was injected for analysis. (3) The qualitative verification of standard PIP2 and sample membrane PIP2 by HPLC/MS: The preparation of standard PIP2 and sample membrane PIP2 and chromatographic conditions were the same as described above. The main MS parameters were as follows: source type: turbo spray (ESI); polarity: negative; scan type: Q1 MS. (4) Further verification of the established chromatographic method employed laser scanning confocal microscopic (LSCM) technique: PLCδ1PH-GFP cDNA subcloned into the pOMU plasmid vector was linearized with XbaⅠrestriction endonuclease. PLCδ1PH-GFP cRNA was transcribed using RibomaxTM Large Scale RNA Production Systems T7 Kit in vitro. Each oocyte was injected with 50nl of water containing the desired cRNA. The injected oocytes were cultivated in ND96 solution containing 2.5mM sodium pyruvate at 19-20℃. After expressing for 24h, oocytes were incubated with wortmannin for 2h and PIP2 hydrolysis from wortmannin-treated group and control group were monitored using LSCM.Results: (1) Membrane PIP2 in Xenopus oocytes was separated and quantified effectively and directly by RP-HPLC with the retention time of 2.1min. The calibration curve of PIP2 was linear in the range of 0.025~0.3μg/μl. The regression equation was y=100.09x+0.7483 (r=0.9979). The lowest detectable limit was 6.25×10-3μg/μl. The precision was determined by analyzing the same standard PIP2 solution for five times, the relative standard deviation (RSD) value of peak height was calculated as 6.0%. The repeatability was determined by analyzing five sample solutions, the RSD value of peak height was calculated as 5.7%. (2) Phospholipids were extracted from 50 Xenopus oocytes for each group and dissolved with 100μl mobile phase solution. The peak height of PIP2 for control group and wortmannin-treated group was 11.16±0.28 (n=5) and 5.30±0.13 (n=5) respectively. The difference was significant between these two groups (P<0.01). The peak height of PIP2 from wortmannin-treated group decreased by 52.5% compared with control group. (3) The mass spectrum was obtained at the same retention time and there was good agreement between standard PIP2 and sample membrane PIP2. The values of m/z181, m/z233, m/z249, m/z317, m/z369, m/z385, m/z453, m/z589, m/z657, m/z673, m/z725, m/z809 were all the same in both sample groups. The characteristic MS peak of PIP2 including m/z1133 ([M-5H+4Na]-),m/z 929 ([M-1-H3PO4-H2O]-),m/z 521 ([M-2H]2-),m/z 437 and m/z 369 ([M-2H-R2COOH]2-) were found in the spectrum of standard PIP2 and m/z 741 ([M-2H-R2COO-]-), m/z 401,m/z 369 ([M-2H-R2COOH]2-),m/z 348 ([M-3H]3-) in the spectrum of oocyte membrane PIP2. (4) Membrane PIP2 hydrolysis in oocytes monitored by LSCM: The fluorescence intensity of membrane for control group and wortmannin-treated group was 127.90±3.6 (n=7) and 63.74±4.3 (n=6) respectively. There was significant difference between these two groups (P<0.01). The fluorescence intensity of oocyte membrane from wortmannin-treated group decreased by 50.2% compared with control group.Conclusion: The established RP-HPLC method can be applied in detecting the content of membrane PIP2 in Xenopus oocytes with high efficiency, accuracy and reliability.