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- Intracellular Hypertonicity Is Responsible for Water Flux As...Intracellular Hypertonicity Is Responsible for Water Flux Associated with Na/Glucose Cotransport
Biophysical Journal, Volume 90, Issue 10, 15 May 2006, Pages 3546-3554
François M. Charron, Maxime G. Blanchard and Jean-Yves Lapointe
AbstractDetection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na/glucose cotransporter (SGLT1) of two Na, one glucose, and 264 water molecules. Volumetric measurements of oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2mM of the substrate -methyl-glucose (MG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na injection and Na-selective electrode, the intracellular diffusion coefficient for Na was estimated at 0.29±0.03×10cm s. Using the effect of intracellular MG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of MG was estimated at 0.15±0.01×10cms. Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of convection, or the diffusion in cytosol of an oocyte is more complex than depicted by a simple model.
Abstract | Full Text | PDF (183 kb) - Response to Zeuthen and Zeuthen's Comment to the Editor: Eno...Response to Zeuthen and Zeuthen's Comment to the Editor: Enough Local Hypertonicity Is Enough
Biophysical Journal, Volume 93, Issue 4, 15 August 2007, Pages 1417-1419
Jean-Yves Lapointe
AbstractIn a Comment to the Editor, Zeuthen and Zeuthen criticize our treatment of the water cotransport hypothesis. In this response, we argue that we calculated water cotransport as if there were no significant local osmotic gradient generated in the first minute of Na/glucose cotransport. It is surprising to receive this type of criticism from Zeuthen and Zeuthen, as the same treatment was used in at least six studies from his laboratory where it is systematically assumed that “intracellular unstirred layers effects” are negligible. Zeuthen and Zeuthen also state that “the cotransport hypothesis predicts the measurements better than the osmotic hypothesis”. We present a quantitative comparison that challenges this contention. We would like to conclude by stating that our article was not about comparing different numerical models but about an experimental measurement of the local osmotic gradient generated after 20, 40, or 60s of cotransport. Osmotic gradients were indeed detected, and were of appropriate amplitude to explain virtually all water transport observed.
Abstract | Full Text | PDF (96 kb) - Hypoosmotic Cell Swelling as a Novel Mechanism for Modulatio...Hypoosmotic Cell Swelling as a Novel Mechanism for Modulation of Cloned HCN2 Channels
Biophysical Journal, Volume 89, Issue 3, 1 September 2005, Pages 2159-2169
Kirstine Calloe, Pernille Elmedyb, Soren-Peter Olesen, Nanna K. Jorgensen and Morten Grunnet
AbstractThis work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to −100mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.
Abstract | Full Text | PDF (294 kb) - …more
Copyright © 1998 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 74, Issue 4, 2121-2128, 1 April 1998
doi:10.1016/S0006-3495(98)77919-6
High Microvascular Endothelial Water Permeability in Mouse Lung Measured by a Pleural Surface Fluorescence Method
Ethan P. Carter, Bence P. Ölveczky, Michael A. Matthay and A.S. Verkman
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Address reprint requests to Dr. Alan S. Verkman, Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847.Abstract
Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23°C and was reduced 54% by 0.5mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12–37°C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23°C). Immunoblot analysis showed ∼1.4×1012 aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium.
