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May 2019

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Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
红细胞甘油通透性的Stopped-flow 光散射分析   

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Abstract

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes.

Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins allowing transport of glycerol across membranes. Suspensions of RBCs (1% hematocrit) are exposed to an inwardly directed gradient of 100 mM glycerol in a SFLS apparatus at 20 °C and the resulting changes in scattered light intensity are recorded at a monochromatic wavelength of 530 nm for 120 s. The SFLS apparatus is set up to have a dead time of 1.6-ms and 99% mixing efficiency in less than 1 ms. Data are fitted to a single exponential function and the related time constant (, seconds) of the cell-swelling phase of light scattering corresponding to the osmotic movement of water that accompanies the entry of glycerol into erythrocytes is measured. The coefficient of glycerol permeability (Pgly, cm/s) of RBCs is calculated with the following equation:





where (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm-1) of the analyzed RBC specimen. Pharmacokinetics of the isoform-specific inhibitors of AQP3, AQP7 and AQP9 are assessed by evaluating the extent of RBC Pgly values resulting after the exposure to serial concentrations of the blockers.

Keywords: Stopped-flow light scattering (Stopped-flow 光散射), Erythrocytes (红细胞), Glycerol membrane permeability (甘油膜通透性), Aquaglyceroporins (水甘油通道蛋白), Aquaporin inhibitors (水通道抑制剂)

Background

The movement of water and solutes across biological and artificial membranes can be analyzed using different approaches. Assessing the rate with which the volume of any sealed particle lined by a membrane changes following the osmotically-driven movement of water represents an indirect approach widely used to measure biophysically the water and solute permeability of the particle specimen.

The Stopped-Flow Light Scattering (SFLS) method was devised to analyze the kinetics of rapid enzymatic reactions accompanied by significant changes of the molecular weight and/or shape of the reacting substrates (Riesner and Buenemann, 1973). SFLS soon proved to be an implementable, reliable and reproducible spectroscopic technique with a number of advantages and only few limitations. While the original method remains extensively used to study the chemical kinetics of fast reactions in solution the conventional SFLS apparatus has been modified to allow the measurement of the coefficient of solute and osmotic water permeability (Ps and Pf, respectively; cm/s) of both biological and artificial membranes (Terwilliger and Solomon, 1981; Mlekoday et al., 1983; van Heeswijk and van Os, 1986). SFLS is largely employed to assess the Ps and Pf of whole cells (Yang and Verkman, 2002; Maggio et al., 2018), organelles, extracellular vesicles (Calamita et al., 2005 and 2006; Ivanova et al., 2008; Soria et al., 2010), and vesicles of any kind of biological membrane (Shi et al., 1990; Calamita et al., 2012; Gena et al., 2017; Miyazawa et al., 2018). The method is also used to measure the permeability of artificial membranes such as liposomes (Zeidel et al., 1992 and 1994; Harris et al., 1994; Agre et al., 1999; Müller-Lucks et al., 2013) and polymersomes (Erbakan et al., 2014; Loo et al., 2017). SFLS is also employed to evaluate the Arrhenius activation energy (Ea; kcal/mol) of water and solute transport based on the temperature dependence with which molecules cross the membrane (Agre et al., 1999; Yang and Verkman, 2002; Mathai et al., 2001; Mouro-Chanteloup et al., 2010).

Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human RBCs in presence or absence of three isoform-specific blockers of AQP3, AQP7 and AQP9 (Jelen et al., 2011; Sonntag et al., 2019), three aquaglyceroporin channels conducting glycerol, water and some other molecules. AQP3, AQP7 and AQP9 are variously expressed in the body playing roles both in health and disease (Yang et al., 2001; Hara-Chikuma and Verkman, 2006) and triggering strong pharmacological interest (Calamita et al., 2018). RBC glycerol permeability is measured after their exposure to different concentrations of the three blockers (vs. control RBCs treated with the vehicle alone). Useful information is acquired regarding the efficiency, selectivity and potency of the compounds in blocking the Aquaglyceroporin-facilitated glycerol transport as well as their preclinical sustainability (solubility, toxicity) for drug developments.

Materials and Reagents

  1. Fifteen ml conical tubes (FalconTM) (Fisher Scientific, catalog number: 14-959-53A )
  2. Sterile individually packaged 5 ml pipettes (SARSTEDT, catalog number: 86.1253.001 )
  3. BD Vacutainer spray coated K2EDTA tubes (BD, catalog number: 366643 )
  4. Fresh human whole blood from healthy donors
    Collected following standard clinical procedures. The blood is drawn by direct puncture to the human vein located in the antecubital area of the arm and collected in in K2EDTA tubes. After washings in DPBS, keep the blood at room temperature and proceed directly to stopped-flow light scattering measurements.
  5. HTS13286 [AQP9 inhibitor (Jelen et al., 2011; Sonntag et al., 2019)], 2-[(4-Methyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]-N-(5-thiophen-2-yl-1,3,4-thiadiazol-2-yl) acetamide, MolPort, MolPort-002-839-693, stable, storage 2.5 mM stock solutions dissolved in DMSO at -20 °C. After freezing, warm well (15 min at 37 °C and vortex) before use
  6. DFP00173 [(AQP3 inhibitor (Sonntag et al., 2019)], 1-(2,6-dichlorophenyl)-3-(5-nitrothiophen-3-yl) urea, MolPort, MolPort-002-897-426, stable, storage 2.5 mM stock solutions dissolved in DMSO at -20 °C. After freezing, warm well (15 min at 37 °C and vortex) before use
  7. Z433927330 [(AQP7 inhibitor (Sonntag et al., 2019)], ethyl 4-(3-(4-(1H-pyrazol-1-yl)benzyl)ureido) benzoate, ENAMINE, MolPort-002-897-426, stable, storage 2.5 mM stock solutions dissolved in DMSO at -20 °C. After freezing, warm well (15 min at 37 °C and vortex) before use
  8. Dulbecco’s Phosphate Buffer Saline (DPBS) (EuroClone SpA, catalog number: ECB4004L ), storage at 4 °C; shelf-life: 24 months from date of manufacture
  9. Glycerol (Sigma-Aldrich, catalog number: G7757 ), very stable, storage at room temperature, shelf-life: 3 to 5 years from date of manufacture
  10. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 ), very stable, storage at room temperature in a dry place, since DMSO is known to be hygroscopic, shelf-life: 3 to 5 years from date of manufacture
  11. Hypertonic solution consisting of DPBS added of glycerol (final osmolarity: 500 mOsM; final volume: 50 ml) (see Recipes)

Equipment

  1. Refrigerate benchtop centrifuge (Thermo Fisher Scientific, Heraeus, model: Multifuge 1L-R , catalog number: 75004330 )
  2. Osmometer (EliTech, model: VAPRO Vapor Pressure Osmometer , catalog number: 5600 )
  3. Stopped-Flow Module (BioLogic Science Instruments, model: SFM20 )
  4. Spectrophotometer (Jasco EUROPE Srl, model: FP 6200 )
  5. P20 pipetman (Gilson, Fisher Scientific, catalog number: F123600 )
  6. P200 pipetman (Gilson, Fisher Scientific, catalog number: F123601 )
  7. P1000 pipetman (Gilson, Fisher Scientific, catalog number: F123601 2)
  8. Bibbyjet pipette controller (Bibby Sterilin Ltd, catalog number: PC1002 )
  9. Block heater (Stuart Scientific, catalog number: SBH130D )
  10. Maxi Mixer Vortex 714 (Hosmotic Srl, catalog number: 29970003 )
  11. 10 ml Gastight Hamilton Syringes for SFM-20 (Jasco EUROPE Srl, catalog number: 950-12/3 )

Software

  1. Bio-Kine 32 software (BioLogic Science Instruments, available here)
  2. GraphPad Prism 5.0 software (GraphPad Software, Inc., www.graphpad.com)

Procedure

  1. Isolation of human RBCs
    1. Collect a sample of human venous blood in a test-tube with anticoagulant (BD Vacutainer spray coated K2EDTA tube).
    2. Dilute the human venous blood sample 1:10 with Dulbecco’s phosphate buffered saline (DPBS).
    3. Wash the human venous blood sample for three times in DPBS (800 x g for 10 min at 4 °C) to remove serum and cellular buffy coat.
    4. Resuspend the resulting pellet of RBCs in DPBS to attain 1% hematocrit.

  2. Assessment of RBCs glycerol permeability by SFLS
    1. Prepare two solutions: A) isotonic solution of 1× DPBS (300 mOsM) and B) hypertonic solution consisting of 1× DPBS added with glycerol (final osmolarity: 500 mOsM).
    2. Verify the osmolarity of the above reported solutions by using an osmometer. Mixing of solutions A and B will result in a 100 mM inwardly direct gradient of glycerol.
    3. Set up the stopped-flow light scattering device (Figure 1) as follows:


      Figure 1. Stopped-flow light scattering apparatus. The SFM-20 injection system coupled to a spectrophotometer is an ideal system for 90° light scattering assays. In this protocol, the SFLS apparatus is employed for the assessment of erythrocyte glycerol permeability. The SFM-20 injection driving motor triggers the syringe pistons allowing the solutions to pass simultaneously into the mixing chamber directly connected to an observation cell irradiated with a xenon source lamp. With the SFM-20’s driving control of the total flow rate shear forces are minimized. A synchronized electro-valve stops the sample flow and prevents the occurrence of overpressure artifacts. All the data collected by a detector are then analyzed by the software Bio-Kine 32.

      1. Set the cryostat temperature at 20 °C;
      2. Initialize the injection system and the spectrophotometer by the MPS-20 and Bio-Kine 32 software, respectively;
      3. Set the SFLS apparatus using the following MPS-20 parameters (Figure 2):
        1. Dead time (time required for the solutions to flow from the mixing chamber to the observation cell): 1.6 ms;
        2. 99% mixing efficiency in < 1 ms;
        3. Mixing time: 20 ms;
        4. Volume injected by syringe 1: 100 μl;
        5. Volume injected by syringe 2: 100 μl;
        6. Set the excitation and emission wavelengths on the spectrophotometer to 530 nm. The bandwidth of excitation and emission must be set to 20 nm.


      Figure 2. SFM-20 software for single mixing applications. SFM-20 is set up for rapid and easy SFLS experimental design. The user sets the mixing ratio, the size of syringes and cuvette, and the volume samples. Color-coded windows display calculated values and alert the user to out of range experimental conditions. The estimated dead time is automatically displayed, and a pretrigger is also available to be sure the stationary state is reached accurately.

    4. Equilibrate 15 μl of RBCs suspension in 2.5 ml of isotonic DPBS for 5 min and then fill one of the two syringes with the cell suspension.
    5. Fill the other syringe with the hypertonic solution (DPBS added of glycerol, final osmolarity: 500 mOsM).
    6. Inject the erythrocyte suspension together with the hypertonic solution (osmotic gradient, 100 mOsM) into a mixing chamber. As a result of a glycerol gradient, at first, a rapid water outflow occurs leading to cell shrinkage and causing an increase in scattered light intensity (I) over time (s) (Figure 3), subsequently, as a consequence of the glycerol entry, water flows into the cells causing cell swelling whose kinetics is displayed as decrease in scattered light intensity (I) over time (s) (Figure 3).


      Figure 3. Stopped-flow light scattering measurements of erythrocyte glycerol permeability. The addition of extracellular glycerol causes a rapid increase in the intensity of scattered light due to the water efflux induced by the osmotic gradient (shrinkage) followed by a slower scattered light decrease (swelling) induced by the osmotic influx of water the accompanies the entry of glycerol into the cells. Glycerol permeability is calculated by measuring the exponential time constant (, s) fitted to the cell swelling phase of the 90° light scattering time course associated to the influx of glycerol and water.

    7. By using the Bio-Kine 32 software, fit the data to a single exponential function and measure the time rate constant (, s) of the cell swelling phase during the entry phase of glycerol and water into the erythrocytes (Figure 4).


      Figure 4. Bio-Kine 32 for data acquisition and analysis. Acquisition and analysis of signals produced by SFLS device are carried out using the Bio-Kine 32 software. Bio-Kine 32 has been designed to complement the SFM-20 software and to connect the spectrometer to the mixing device.

    8. For the inhibition studies (Figure 5), dissolve the compounds in DMSO as previously reported, add the right volume of inhibitor solution to the erythrocyte suspension and incubate for 10 min at 20 °C prior to the measurements of light scattering. The final concentration of HTS13286, DFP00173 and Z433927330 is 25 μM in 1% DMSO. Carry out all the inhibition assays while keeping the final concentration of DMSO constant at 1%. To keep constant the concentration of DMSO needed for inhibition studies, prepare a series of DMSO solutions of the drug with different concentrations, and do the same dilution with the buffer solution, in this way is possible to get buffer solutions of different drug concentrations while maintaining the same percentage of DMSO.


      Figure 5. Inhibition studies of glycerol transport across erythrocyte plasma membrane. Stopped-flow light scattering technique is a valuable tool for assessing water and solute movement across biological membranes and the characterization of pharmacokinetic properties (efficiency, specificity, potency) of protein channels and carrier inhibitors, as well. Representative tracings of glycerol permeation into human red blood control cells (no inhibitor) or incubated for 10 min with two different concentrations of each inhibitor, one blocking mostly the glycerol-facilitated diffusion and the other being ineffective. A significant inhibition of glycerol permeability is seen adding 25 μM of all three inhibitors, HTS 13286 (A), DFP00173 (B) and Z433927220 (C) compared to the control cells (1% DMSO).

    9. RBCs suspensions incubated with 1% DMSO (solvent of all the three inhibitors) (10 min at 20 °C as well as the experimental conditions) represent the control condition.

  3. Calculation of the glycerol permeability coefficient (Pgly, cm/s).
    The osmotic water influx following glycerol entry into the erythrocytes and therefore the resulting swelling leads to a decrease of the scattered light intensity over time (Figure 2). Calculate the glycerol permeability coefficient (Pgly, cm/s) by measuring the  value (using the Bio-Kine 32 software), exponential time constant fitted to the data related to the cell swelling phase of the curve, and using the following equation:


         where S/V is the surface-to-volume ratio of the analyzed RBCs.

    A practical example for the calculation of the Pgly of the RBC plasma membrane is described below:


    where S/V is the initial surface/volume ratio of RBCs. Assuming that the cell shape is roughly spherical and the cell outer surface is regular and completely smooth, S/V may be obtained as follows:
    where r is the cell radius.
    The above Pgly equation can now be also written as follows:


    The RBC mean radius is of 4 x 10-5 cm and the  value corresponds to the reciprocal of a time constant, Ki (s-1), calculated from the exponential fitting of the SFLS traces (RBC = 1/Ki (RBC) = 1/0.110 s-1 = 9.0 s). The Pgly value is then calculated as follows:

Data analysis

Stopped-flow light scattering experiments should be performed at least in triplicate. All data resulted from three to five different human red blood cell suspensions are expressed as mean ± SEM. Data are analyzed statistically by two-way ANOVA in GraphPad Prism 5.0 and the results are considered statistically significant when P < 0.05.

Recipes

  1. Hypertonic solution consisting of PBS added of glycerol (final osmolarity: 500 mOsM; final volume: 50 ml)
    Add 0.730 ml of 100% glycerol to 1× DPBS and mix gently until complete mixing.

Acknowledgments

Financial support to GC from Italian “Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale 2017” (PRIN2017; grant # 2017J92TM5) and to YS from Stiftelsen Olle Engkvist Byggmästare is gratefully acknowledged.
  The protocol object of the present article is derived from a previous research paper from our laboratories (Sonntag et al., 2019).

Competing interests

The authors declare no conflict of interest.

Ethics

All human subjects provided written informed consent for participation in the study, in accordance with ICH/GCP guidelines. The protocol was approved by the local ethics committee (authorization # FMF01-5870-27-02-2019).

References

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简介

[摘要] 停流光散射(SFLS)是一种用于分析快速化学反应动力学的方法,该化学反应导致平均分子量和/或反应底物形状发生显着变化。已对原始停流系统进行了几处修改,从而大大扩展了其技术应用范围。这些修饰之一允许水的生物物理表征以及生物膜和人造膜的溶质渗透性。

在这里,我们描述了一种SFLS协议,用于测量分离的人红细胞(RBC)的甘油渗透性,并评估AQP3,AQP7和AQP9的同种型特异性抑制剂的药代动力学特性(选择性和效价),这三种哺乳动物的水甘油糖蛋白允许转运甘油跨膜。在20°C的SFLS设备中,将RBC的悬浮液(1%的血细胞比容)暴露于100 mM甘油的向内定向,并在530 nm的单色波长下记录120 s的散射光强度变化。该设备的死区时间为1.6毫秒,混合效率在不到1毫秒的时间内达到99%。数据拟合到单指数函数和有关的时间constan 吨( ,对应于伴随甘油的进入红细胞的水渗透运动的光散射的细胞膨胀相的秒)进行测定。RBC 的甘油渗透系数(P gly ,cm / s)通过以下公式计算:



P gly = 1 / [[((S / V) ]



其中 (s)是拟合的指数时间常数,S / V是分析的RBC 样本的表面积与体积之比(cm -1 )。AQP3,AQP7和AQP9异构体特异性抑制剂的药代动力学通过评估暴露于一系列浓度的阻滞剂后产生的RBC P gly 值的程度来评估。

关键词:停流光散射红细胞甘油膜通透性 Aquaglyceroporins,Aquaporin抑制剂



[背景] 可以使用不同的方法来分析水和溶质在生物膜和人工膜上的运动。评估随水的渗透驱动运动而被膜衬里的任何密封颗粒的体积变化的速率代表了一种间接方法,该方法广泛用于生物物理地测量颗粒样品的水和溶质渗透率。

设计了“ 停流光散射(SFLS)”方法来分析快速酶促反应的动力学,并伴随着反应底物的分子量和/或形状的显着变化(Riesner和Buenemann,1973)。SFLS很快被实现为一种可实施,可靠且可重现的光谱技术,它具有许多优点,但也有一些局限性。尽管原始方法仍广泛用于研究溶液中快速反应的化学动力学,但对常规SFLS装置进行了改进,可以测量溶质系数和渗透水渗透率(分别为P s 和P f ; cm / s) (Terwilliger和Solomon,1981; Mlekoday 等,1983; van Heeswijk和van Os,1986)。SFLS主要用于评估全细胞的P s 和P f (Yang和Verkman,2002; Maggio 等,2018),细胞器,细胞外囊泡(Calamita 等,2005 和2006; Ivanova 等,2008)。 ; Soria 等人,2010),以及任何种类的生物膜的囊泡(Shi 等人,1990; Calamita 等人,2012; Gena 等人,2017; Miyazawa 等人,2018)。该方法也被用于测量人工膜如脂质体的渗透性(•塞得尔等人,1992 和1994;哈里斯等人,1994;阿格雷等人,1999; M ü 米勒-LUCKS 。等人,20 13 )和聚合物囊泡(Erbakan 等人,2014; Loo 等人,2017)。SFLS还用于根据分子穿过膜的温度依赖性来评估水和溶质的Arrhenius活化能(E a ; kcal / mol)(Agre 等,1999; Yang和Verkman,2002; Mathai 等。等人,2001; Mouro-Chanteloup 等人,2010)。

在此,我们描述了一种SFLS协议,用于在存在或不存在三种AQP3,AQP7和AQP9异构体特异性阻滞剂的情况下测量分离的人RBC的甘油通透性(Jelen 等,2011; Sonntag 等,2019),三甘油水通道可以传导甘油,水和其他一些分子。AQP3,AQP7和AQP9在人体中以多种形式表达,在健康和疾病中发挥作用(Yang 等人,2001; Hara-Chikuma和Verkman,2006)并引发强烈的药理学兴趣(Calamita 等人,2018)。RBC甘油的渗透性是在它们暴露于三种浓度的不同浓度的三种阻滞剂(相对于仅用赋形剂处理的对照RBC)之后测量的。获得了有关化合物在阻断水甘油三磷酸酯促进的甘油运输中的效率,选择性和效力以及它们在药物开发中的临床前可持续性(溶解性,毒性)的有用信息。

关键字:Stopped-flow 光散射, 红细胞, 甘油膜通透性, 水甘油通道蛋白, 水通道抑制剂

材料和试剂


 


15 ml锥形管(Falcon TM )(Fisher Scientific,目录号:14-959-53A)
无菌独立包装的5 ml移液器(SARSTEDT,目录号:86.1253.001)
BD Vacutainer喷涂K 2 EDTA管(BD,目录号:366643)
来自健康捐献者的新鲜人类全血
ç ollected以下标准的临床程序。通过直接穿刺将血液抽到位于手臂肘前区域的人体静脉,并收集在K 2 EDTA管中。在DPBS中洗涤后,将血液保持在室温下,直接进行停止流光散射测量。


HTS13286 [AQP9抑制剂(Jelen et al。,2011; Sonntag et al 。,2019)],2-[(4-甲基-5-吡啶-4-基-1,2,4-三唑-3-基)硫烷基] -N-(5-噻吩-2-基) -1,3,4-噻二唑-2-基)乙酰胺,MolPort ,MolPort-002-839-693,稳定,存储的2.5mM储备溶液溶于DMSO在-20℃下 冷冻后,在使用前充分加热(在37°C和涡旋下15分钟)
DFP00173 [(AQP3抑制剂(桑塔格等人,2019))],1-(2,6-二氯苯基)-3-(5-硝基噻吩-3-基)脲,MolPort ,MolPort-002-897-426,稳定,储存在-20°C的DMSO中溶解的2.5 mM储备溶液。冷冻后,在使用前充分温热(在37°C并涡旋15分钟)
Z433927330 [(AQP7抑制剂(Sonntag et al。,2019))],4-(3-(4-(4-(1H-吡唑-1-基)苄基)脲基)脲基)乙基苯甲酸乙酯,ENAMINE,MolPort-002-897-426,稳定,在-20°C下储存2.5 mM溶于DMSO的储备溶液。冷冻后,在使用前充分温热(在37°C和涡旋下15分钟)
Dulbecco磷酸盐缓冲盐溶液(DPBS)(EuroClone SpA ,目录号:ECB4004L),在4°C下储存;保质期:自生产之日起24个月
甘油(西格玛-奥德里奇(Sigma-Aldrich),目录号:G7757),非常稳定,可在室温下保存,保质期:自生产之日起3-5年
二甲基亚砜(DMSO)(西格玛奥德里奇(Sigma-Aldrich),目录号:D8418),非常稳定,可在室温下干燥的地方储存,因为已知DMSO具有吸湿性,保质期:自生产之日起3-5年
高渗溶液,由添加了甘油的DPBS组成(最终渗透压:500 mOsM ;最终体积:50 ml)(请参见食谱)
 


设备


 


冷藏台式离心机(热Fisher Scientific公司,贺利氏,米Odel等:Multifuga 1L-R,目录号:75004330)
渗压计(ELITECH ,米Odel等:蒸气压渗透压VAPRO,目录号:5600)
停流模块(的BioLogic 科学仪器,米穿上:SFM20)
分光光度计(Jasco EUROPE Srl ,米通:FP 6200)
P20 移液器(Gilson,费舍尔科学公司,目录号:F123600)
P200 移液器(Gilson,Fisher Scientific,目录号:F123601)
P1000 移液器(Gilson,Fisher Scientific,目录号:F1236012)
Bibbyjet 移液器控制器(Bibby Sterilin Ltd ,目录号:PC1002)
块状加热器(Stuart Scientific,目录号:SBH130D)
Maxi Mixer Vortex 714(Hosmo tic Srl ,目录号:29970003)
10毫升用于SFM-20的Gastight Hamilton注射器(Jasco EUROPE Srl ,目录号:950-12 / 3)
 


软件


 


生物中国32软件(的BioLogic 科学仪器,可在这里)
GraphPad Prism 5.0软件(GraphPad Software,Inc.,www.graphpad.com)
 


程序


 


人类红细胞的分离
用抗凝剂(BD Vacutainer喷涂K 2 EDTA管)在试管中收集人静脉血样品。
用Dulbecco磷酸盐缓冲盐水(DPBS)稀释人类静脉血样品1:10。
在DPBS中洗涤人体静脉血样3次(在4°C下800 x g 持续10分钟),以去除血清和细胞血沉棕黄层。
将所得的RBC沉淀重悬于DPBS中,以达到1%的血细胞比容。
 


通过SFLS评估红细胞的甘油渗透性
准备两种溶液:A)1 × DPBS(300 mOsM )的等渗溶液,以及B)由1 × DPBS和甘油组成的高渗溶液(最终渗透压:500 mOsM )。
使用渗透压计验证上述报告的溶液的渗透压。溶液A和B的混合将产生100 mM的甘油向内直接梯度。
如下设置停止流动的光散射装置(图1):
 


              D:\重新格式化\ 2020-6-1 \ 1902858--1457朱塞佩·卡拉米塔781994 \图jpg \图1.jpg


图1.停止流动的光散射设备。与分光光度计耦合的SFM-20注射系统是90°光散射测定的理想系统。在该协议中,SFLS设备用于评估红细胞甘油的渗透性。SFM-20注射驱动马达触发注射器活塞,使溶液同时进入直接与氙气源灯照射的观察室相连的混合室。利用SFM-20的总流量驱动控制,剪切力得以最小化。同步电动阀可停止样品流动并防止超压伪影的发生。然后通过Bio- China 32 软件分析检测器收集的所有数据。


 


小号等人20的低温恒温器温度℃;
我分别通过MPS-20和BioKine 32 软件彻底消除了进样系统和分光光度计。
小号等使用以下MPS-20参数(图2)的装置SFLS:
死区时间(溶液从混合室流到观察池所需的时间):1.6 ms ;
<1 毫秒内达到99%的混合效率;
混合时间:20 毫秒;
注射器注射量1:100μl ;
注射器2注入的体积:100μl ;
将分光光度计上的激发和发射波长设置为530 nm。激发和发射的带宽必须设置为20 nm。
 


D:\重新格式化\ 2020-6-1 \ 1902858--1457朱塞佩·卡拉米塔781994 \图jpg \图2.jpg


图2.用于单一混合应用的SFM-20软件。SFM-20的设置是为了快速,轻松地进行SFLS实验设计。用户设置混合比例,注射器和比色皿的尺寸以及样品体积。颜色编码的窗口显示计算出的值并警告用户超出实验条件的范围。估计的停滞时间将自动显示,并且还可以使用预触发来确保准确到达静止状态。


 


平衡15 微升的RBC悬浮液在2.5ml 5分钟等渗DPBS中,然后填充该两个注射器与细胞悬浮液中的一个。
用高渗溶液(添加了DPBS的甘油,最终渗透压:500 mOsM )填充另一个注射器。
将红细胞悬液与高渗溶液(渗透梯度,100 mOsM )一起注入混合室。作为甘油梯度的结果,首先,由于甘油的进入,首先会出现快速的水流出,导致细胞收缩,并导致散射光强度(I)随时间的推移而增加(图3)(图3)。 ,水流入细胞引起细胞肿胀,其动力学表现为散射光强度(I)随时间(s)的降低(图3)。
 


D:\重新格式化\ 2020-6-1 \ 1902858--1457朱塞佩·卡拉米塔781994 \图jpg \图3.jpg


图3.红细胞甘油渗透率的停止流光散射测量。加入的胞外甘油Ç auses 迅速增加的强度由于散射光的水通过渗透梯度流出诱导(收缩),随后是较慢的散射光降低(肿胀通过水的渗透潮诱导的)的accompan IES 的的条目甘油到细胞中。甘油的渗透性是通过测量与甘油和水的流入有关的90°光散射时间过程的细胞溶胀阶段的指数时间常数( ,s)来计算的。


 


通过使用Bio- Kine 32软件,将数据拟合为一个指数函数,并测量甘油和水进入红细胞阶段的细胞溶胀阶段的时间速率常数( ,s)(图4)。
 


D:\重新格式化\ 2020-6-1 \ 1902858--1457朱塞佩·卡拉米塔781994 \无花果jpg \图4.jpg


图4. Bio-China 32用于数据采集和分析。SFLS设备产生的信号的采集和分析使用Bio-China 32软件进行。Bio-China 32设计用于补充SFM-20软件,并将光谱仪连接到混合设备。


 


对于抑制研究(图5),按照先前报道的方法将化合物溶解在DMSO中,在红细胞悬液中添加适当体积的抑制剂溶液,并在测量光散射之前在20°C下孵育10分钟。HTS13286,DFP00173和Z433927330的最终浓度在1%DMSO中为25μM 。进行所有抑制试验,同时将DMSO的最终浓度保持恒定在1%。为了使抑制研究所需的DMSO浓度保持恒定,准备一系列不同浓度的DMSO溶液,并用缓冲液进行相同的稀释,这样就可以得到不同药物浓度的缓冲液,同时保持相同百分比的DMSO。
 


D:\重新格式化\ 2020-6-1 \ 1902858--1457朱塞佩·卡拉米塔781994 \图jpg \图5.jpg


图5.甘油跨红细胞质膜运输的抑制研究。停流光散射技术是评估水和溶质在生物膜上运动以及蛋白质通道和载体抑制剂的药代动力学特性(功效,特异性,效力)表征的有价值的工具。甘油渗透到人红细胞控制细胞(无抑制剂)或与两种不同浓度的每种抑制剂孵育10分钟的代表性示踪图,一种主要阻断甘油促进的扩散,另一种无效。阿甘油渗透性显著抑制被认为是添加25 μ所有三种抑制剂,HTS 13286的M(A),DFP00173(B)和Z433927220(C)与对照相比细胞(1%DMSO)。


 


用1%DMSO(所有三种抑制剂的溶剂)孵育的RBC悬浮液(在20°C以及实验条件下10分钟)代表对照条件。
 


甘油渗透系数的计算(P gly ,cm / s)。
甘油进入红血球后,渗透水大量涌入,因此导致的肿胀导致散射光强度随时间下降(图2)。通过测量 值(使用Bio - Kine 32软件),拟合与该曲线的细胞溶胀阶段有关的数据的指数时间常数,来计算甘油的渗透系数(P gly ,cm / s):


 


P gly = 1 / [[((S / V) ]


              其中S / V是分析的RBC的表面积与体积之比。


 


RBC质膜的P gly 的计算的一个实际例子如下:


 


P gly = 1 / [[((S / V) ]


 


其中,S / V为初始的表面/体积比的红细胞。假设单元格形状大致为球形,并且单元格外表面规则且完全光滑,则可以按以下方式获得S / V:


 


S / V =4πR 2 /(4/3)πR 3 = 3 / R


 


                                                                                                  WH ERE - [R 是第Ë小区半径。


上面的P gly 方程现在也可以写成:


 


 =  


 


红细胞平均半径为4×10 -5 厘米和 值对应于一个时间常数,的倒数ķ 我(S -1 ),从SFLS迹线的指数拟合计算( RBC = 1 / ķ 我(RBC)= 1 / 0.110 s -1 = 9.0 s)。然后,P gly 值计算如下:


 


 = 1.48 × 10 -6 厘米/秒


 


数据分析


 


停止流光散射实验应至少重复三次。由三至五个不同的人类红细胞悬液得到的所有数据均表示为平均值±SEM。在GraphPad Prism 5.0中通过双向ANOVA对数据进行统计分析,当P <0.05 时,结果被认为具有统计学意义。


 


菜谱


 


高渗溶液,由加入甘油的PBS组成(最终渗透压:500 mOsM ;最终体积:50 ml)
将0.730 ml的100%甘油添加到1 × DPBS中,轻轻混合直至完全混合。






已确认发言:


 


非常感谢意大利为“意大利国立科学基金会计划”(2017年,PRIN2017;拨款号为2017J92TM5)向GC提供的财政支持,以及Stiftelsen Olle EngkvistByggmästare对YS的财政支持。


  本文的协议对象源自我们实验室的先前研究论文(Sonntag 等,2019)。


 


利益争夺


 


在一个uthors宣称没有利益冲突。


 


伦理


 


根据ICH / GCP指南,所有人类受试者均提供了书面知情同意书以参与研究。该协议已由当地道德委员会批准(授权号为FMF01-5870-27-02-2019 )。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gena, P., Portincasa, P., Matera, S., Sonntag, Y., Rutzler, M. and Calamita, G. (2020). Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability. Bio-protocol 10(16): e3723. DOI: 10.21769/BioProtoc.3723.
  2. Sonntag, Y., Gena, P., Maggio, A., Singh, T., Artner, I., Oklinski, M. K., Johanson, U., Kjellbom, P., Nieland, J. D., Nielsen, S., Calamita, G. and Rutzler, M. (2019). Identification and characterization of potent and selective aquaporin-3 and aquaporin-7 inhibitors. J Biol Chem 294(18): 7377-7387.
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