Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells

• Published in: The Journal of physiology 1999-04, Vol.516 (1), p.75-84
• Main Authors: Weikersthal, Sophia F., Barrand, Margery A., Hladky, Stephen B.
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Adenosine Triphosphate - pharmacology; Adenosine Triphosphate - physiology; Animals; Brain Chemistry - drug effects; Brain Chemistry - genetics; Brain Chemistry - physiology; Calcium - physiology; Calcium Channel Blockers - pharmacology; Chloride Channels - antagonists & inhibitors; Chloride Channels - chemistry; Chloride Channels - genetics; Electrophysiology; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme Activation - physiology; Hypotonic Solutions; In Vitro Techniques; Male; Nitrobenzoates - pharmacology; Original; Patch-Clamp Techniques; Protein Kinase C - metabolism; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Verapamil - pharmacology
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.1999.075aa.x

Volume-activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole-cell voltage-clamp technique and on a molecular level using the reverse transcriptase-polymerase chain reaction (RT-PCR). Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time-dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I − (1.7) > Br − (1.2) > Cl − (1.0) > F − (0.7) > gluconate (0.18). The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 μM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 100 μM) also blocked the hypotonicity-induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume-activated current was also inhibited by the antioestrogen tamoxifen (10 μM). The current was dependent on intracellular ATP and independent of intracellular Ca 2+ . Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. Verapamil (100 μM) decreased both the inward and the outward hypotonicity-activated chloride current. RT-PCR analysis was used to determine possible molecular candidates for the volume-sensitive current. Expression of the ClC-2, ClC-3 and ClC-5 chloride channels, as well as pI Cln , could be shown at the mRNA level. We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC-3 channel currents as described in other cell types.