The central nervous system catecholaminergic cell line CAD initially swells then undergoes a regulatory volume decrease (RVD) when exposed to a hypotonic environment (Harvey et al. 2002). RVD is usually accompanied by the loss of K⁺ and Cl^– from the cell. A possible candidate for the anion exit pathway is the volume-regulated anion channel (VRAC) reported to be present in many cell types (Lang et al. 1998).

Using the whole-cell configuration of the patch-clamp technique, CAD cells exhibited reversible activation of an outwardly rectifying current in response to hypotonic stress. Data reported below were all obtained by 100 ms step depolarisations from -80 to +100 mV from a holding potential of -60 mV, and are expressed as mean ± S.E.M. current densities (pA pF^-1). Significant differences were determined using Student’s paired t test or ANOVA followed by Tukey’s HSD test.

Cells were bathed in isotonic bath solution containing (mM) NaCl 110, CaCl₂ 1, MgCl₂ 1, Hepes 10, adjusted with NaOH (pH7.3) and mannitol (300 mosmol (kg H₂O)^-1), and dialysed with a pipette solution containing (mM) NaCl 50, EGTA 1, MgCl₂ 1, Hepes 10, adjusted with NaOH (pH7.3) and mannitol (290 mosmol (kg H₂O)^-1). Under these conditions CAD cells displayed an outward current of 7.8 ± 1.9 pA pF^-1 and inward current of -0.5 ± 0.5 pA pF^-1 (n = 13). After a 5 min exposure to an iso-ionic hypotonic solution (230 mosmol (kg H₂O)^-1) the outward current increased to 40.6 ± 7.1 pA pF^-1 and the inward current increased to -11.8 ± 2.7 pA pF^-1 (n = 13, P < 0.001, Student’s paired t test). The reversal potential (E_rev), -15.6 ± 1.6 mV (n = 13) was similar to the theoretical E_rev for a Cl^–-selective conductance under these conditions.

The swelling-activated current was inhibited by the Cl^– channel antagonist 4,4â-diisothiocyanatostilbene-2,2â-disulphonic acid (DIDS). Bath perfusion with 100 µM DIDS reduced the outward current from 50.3 ± 11.8 to 21.9 ± 4.4 pA pF^-1 (n = 4, P < 0.05, ANOVA), but had no significant effects on the inward current. At positive potentials the channel block mediated by DIDS was dependent on the applied transmembrane potential. This inhibition was reversed to 43.2 ± 14.5 pA pF^-1 (n = 4) following washout with hypotonic solution.

In conclusion, CAD cells possess swelling-activated currents that display properties consistent with those of the VRAC reported in other cell types. Since 100 µM DIDS also inhibits RVD in CAD cells (Harvey et al. 2002), these currents may represent the pathway for the extrusion of Cl^– during RVD.