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Monitoring Cl- movement in single cells exposed to hypotonic solution

Monitoring Cl- movement in single cells exposed to hypotonic solution
Monitoring Cl- movement in single cells exposed to hypotonic solution
Self-referencing ion--selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 microm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (VN) measured in 100 and 10 mM NaCl (deltaVN = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (deltaVN = 70 +/- 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 microM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was approximately 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 microM 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 microM tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level
0022-2631
101-110
Garber, S.S.
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Messerli, M.A.
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Hubert, M.
dfba2230-87e7-472d-a533-5d619ecd161e
Lewis, R.
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Hammar, K.
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Indyk, E.
2c034b57-0a44-4cc2-8160-ac07ed157c28
Smith, P.J.S
003de469-9420-4f12-8f0e-8e8d76d28d6c
Garber, S.S.
8e7580a6-a20c-43a1-a628-71738fefd411
Messerli, M.A.
0d35456b-47c1-4590-81cd-12b483a422ca
Hubert, M.
dfba2230-87e7-472d-a533-5d619ecd161e
Lewis, R.
6f1ba3ae-babd-4e0e-9fc4-7dee2e4d57af
Hammar, K.
a39cddad-b4b1-4019-854f-3eb196bb9d5c
Indyk, E.
2c034b57-0a44-4cc2-8160-ac07ed157c28
Smith, P.J.S
003de469-9420-4f12-8f0e-8e8d76d28d6c

Garber, S.S., Messerli, M.A., Hubert, M., Lewis, R., Hammar, K., Indyk, E. and Smith, P.J.S (2005) Monitoring Cl- movement in single cells exposed to hypotonic solution. The Journal of Membrane Biology, 203 (2), 101-110. (doi:10.1007/s00232-005-0735-x). (PMID:15981714)

Record type: Article

Abstract

Self-referencing ion--selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 microm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (VN) measured in 100 and 10 mM NaCl (deltaVN = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (deltaVN = 70 +/- 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 microM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was approximately 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 microM 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 microM tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level

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Published date: January 2005
Organisations: University of Southampton

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Local EPrints ID: 188805
URI: http://eprints.soton.ac.uk/id/eprint/188805
ISSN: 0022-2631
PURE UUID: 19b7698e-6f72-415a-b84e-6fe82da3b4ba
ORCID for P.J.S Smith: ORCID iD orcid.org/0000-0003-4400-6853

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Date deposited: 03 Jun 2011 10:41
Last modified: 20 Jul 2019 00:44

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Contributors

Author: S.S. Garber
Author: M.A. Messerli
Author: M. Hubert
Author: R. Lewis
Author: K. Hammar
Author: E. Indyk
Author: P.J.S Smith ORCID iD

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