Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
(2020) In Bio-protocol 10(16).- 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... (More)
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 (τ, 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: Pgly = 1/[(S/V)τ] where τ (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.
(Less)
- author
- Gena, Patrizia ; Portincasa, Piero ; Matera, Sabino LU ; Sonntag, Yonathan LU ; Rützler, Michael LU and Calamita, Giuseppe
- organization
- publishing date
- 2020
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Aquaglyceroporins, Aquaporin inhibitors, Erythrocytes, Glycerol membrane permeability, Stopped-flow light scattering
- in
- Bio-protocol
- volume
- 10
- issue
- 16
- article number
- e3723
- publisher
- Bio-protocol LLC
- external identifiers
-
- scopus:85150612710
- ISSN
- 2331-8325
- DOI
- 10.21769/BioProtoc.3723
- language
- English
- LU publication?
- yes
- id
- b22f0256-499d-45e8-a5ff-18dfc0cb5890
- date added to LUP
- 2023-05-30 10:20:22
- date last changed
- 2023-05-30 10:20:22
@article{b22f0256-499d-45e8-a5ff-18dfc0cb5890,
abstract = {{<p>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 (τ, 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: Pgly = 1/[(S/V)τ] where τ (s) is the fitted exponential time constant and S/V is the surface-to-volume ratio (cm<sup>-1</sup>) 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.</p>}},
author = {{Gena, Patrizia and Portincasa, Piero and Matera, Sabino and Sonntag, Yonathan and Rützler, Michael and Calamita, Giuseppe}},
issn = {{2331-8325}},
keywords = {{Aquaglyceroporins; Aquaporin inhibitors; Erythrocytes; Glycerol membrane permeability; Stopped-flow light scattering}},
language = {{eng}},
number = {{16}},
publisher = {{Bio-protocol LLC}},
series = {{Bio-protocol}},
title = {{Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability}},
url = {{http://dx.doi.org/10.21769/BioProtoc.3723}},
doi = {{10.21769/BioProtoc.3723}},
volume = {{10}},
year = {{2020}},
}