V L Harvey1, M W Saul, C Garner, R L McDonald. 1. Neuroscience, Pharmacology & Physiology, University College London, Gower Street, London WC1E 6BT, UK. viclharvey@googlemail.com
Abstract
AIM: The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. METHODS: Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl(-) channel antagonists tamoxifen (10 microm) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microm) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize ICl(swell), the current underlying the VRAC. RESULTS: Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg(-1) H(2)O to 210 mOsmol kg(-1) H(2)O. In the presence of tamoxifen (10 microm) or DIDS (100 microm) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg(-1) H(2)O) evoked ICl(swell), an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (E(rev)) for ICl(swell) was -14.7 + or - 1.4 mV, similar to the theoretical E(rev) for a selective Cl(-) conductance. ICl(swell) was inhibited in the presence of DIDS (100 microm) and tamoxifen (10 microm), the DIDS inhibition being voltage dependent. CONCLUSIONS: Osmotic swelling elicits an outwardly rectifying Cl(-) conductance in CAD cells. The ICl(swell) observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl(-) which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation.
AIM: The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. METHODS: Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl(-) channel antagonists tamoxifen (10 microm) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microm) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize ICl(swell), the current underlying the VRAC. RESULTS: Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg(-1) H(2)O to 210 mOsmol kg(-1) H(2)O. In the presence of tamoxifen (10 microm) or DIDS (100 microm) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg(-1) H(2)O) evoked ICl(swell), an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (E(rev)) for ICl(swell) was -14.7 + or - 1.4 mV, similar to the theoretical E(rev) for a selective Cl(-) conductance. ICl(swell) was inhibited in the presence of DIDS (100 microm) and tamoxifen (10 microm), the DIDS inhibition being voltage dependent. CONCLUSIONS: Osmotic swelling elicits an outwardly rectifying Cl(-) conductance in CAD cells. The ICl(swell) observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl(-) which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation.
Authors: Edward G Hawkins; William L Dewey; Mallappa Anitha; Shanthi Srinivasan; John R Grider; Hamid I Akbarali Journal: Dig Dis Sci Date: 2013-01-31 Impact factor: 3.199