Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge. This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels. Using whole-cell recordings, we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge. These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions: I ->NO 3 ->Cl ->Gluc -≥MeS ->Ise -. Interestingly, extracellular I -, albeit showing the greatest permeability, blocked the currents with an IC 50 of ≈50 mM. Currents were almost completely inhibited by 123 μM NPPB and partially inhibited by 200 μM niflumic acid or 200 μM DIDS. Additionally, the total number of Cl -ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation. We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes. To examine potential candidate chloride channel genes expressed by astrocytes, we employed RTPCR to demonstrate the presence of transcripts for ClC-2, 3, 4, 5, and 7, as well as for VDAC and CFTR in cultured astrocytes. Moreover, we performed immunostaining with antibodies against each of these channels and showed the strongest expression of ClC-2 and ClC-3, strong expression of ClC-5 and VDAC, weak expression of ClC-7 and very weak expression of ClC-4 and CFTR. Intriguingly, although we found at least seven Cl -channel proteins from three different gene families in astrocytes, none appeared to be active in resting cells.
The objective of this study was to determine the relative contribution of Cl− channels to volume regulation of cultured rat cortical astrocytes after hypotonic cell swelling. Using a Coulter counter, we showed that cortical astrocytes regulate their cell volume by ∼60% within 45 min after hypotonic challenge. This volume regulation was supported when Cl− was replaced with Br−, NO[Formula: see text], methanesulfonate−, or acetate− but was inhibited when Cl− was replaced with isethionate− or gluconate−. Additionally, substitution of Cl− with I−completely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl− channel blockers, including 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and niflumic acid. Surprisingly, the combination of Cd2+ with NPPB, DIDS, or niflumic acid inhibited regulation to a greater extent than any of these drugs alone. Volume regulation did not differ among astrocytes cultured from different brain regions, as cerebellar and hippocampal astrocytes exhibited behavior identical to that of cortical astrocytes. These data suggest that Cl− flux through ion channels rather than transporters is essential for volume regulation of cultured astrocytes in response to hypotonic challenge.
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