A chloride current with mild outward rectification was induced in the native bovine non‐pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. ATP blocked 88 and 61 % of the outward and inward components of the volume‐activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. The volume‐activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC‐3 expression using a ClC‐3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC‐3 and was inhibited by ATP. ClC‐3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC‐3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC‐3 protein was diminished by the ClC‐3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. The size of the volume‐activated chloride current was positively correlated with the ClC‐3 immunofluorescence level. Regulatory volume decrease of the NPCE cells was reduced by ClC‐3 antisense oligonucleotide. We conclude that endogenous ClC‐3 is associated with the volume‐activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. The nuclear predominance of ClC‐3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC‐3 and ICl,vol argue against ClC‐3 being the only, or even the main, volume‐activated chloride channel in NPCE cells.

Of those people who are anosmic to androstenone, a proportion can acquire sensitivity to it by repeated exposure and even those who are able to smell it can lower their threshold with this treatment. Using olfactory threshold testing, intranasal electrophysiology and EEG we show for the first time that: (1) the subjects' detection threshold is proportional to the amplitude of the olfactory evoked potential (EOG) recorded inside the nose; (2) the EOG amplitude is correlated with the amplitude of the olfactory event-related potential (OERP) recorded on the scalp; and (3) with repetitive exposure, human subjects acquire a reduced threshold for androstenone and, as they do so, their EOG and OERP increase. These observations support the existence of odourant-specific plasticity in the peripheral olfactory system.

Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl− current, I Cl(vol), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl− current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl− equilibrium potential. The sequence of anion permeability was I− > Br− > Cl− > gluconate. The Cl− channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I Cl(vol) was cell cycle dependent, being high in G1 phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl− channel blockers NPPB, tamoxifen, and ATP. The expression of I Cl(vol) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I Cl(vol) by NPPB (100 μM) arrested cells in G0/G1. The data also suggest that expression of I Cl(vol) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl− current associated with RVD may therefore play an important role during the cell cycle progress.

1. The whole‐cell patch clamp technique was used to investigate the swelling‐activated currents in bovine non‐pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB). The I‐V relationship was shifted in the direction expected for a Cl‐ current when the external Cl‐ was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS) and 4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (DIDS), also inhibited this current. 5. The volume‐activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time‐dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl‐ current. 8. An antibody (C219) to the MDR1 gene product, P‐glycoprotein, caused a functional block of the swelling‐activated Cl‐ current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB‐1 demonstrated the presence of P‐glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non‐pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)‐independent, ATP‐dependent Cl‐ current and that the activity of this current is associated with P‐glycoprotein. 11. It is suggested that this Cl‐ current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are highaffinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens. (J. Clin. Invest. 1994. 94:1690-1697