Fig. 6.
Effect of bumetanide (BUM) on basal cell water volume (CWV) in choroid plexus epithelial cells (CPECs) from Na+-K+-2Cl− cotransporter 1 (NKCC1) wild-type (NKCC1+/+) and knockout (NKCC1−/−) mice. A and B: relative CWV changes with respect to time [(Vt/V0) where V0 is the cell water volume in basal control condition (V0 = 1), and Vt is the percentage change in cell water reached at time t, divided by 100] were measured in single CPECs loaded with calcein, as described in materials and methods. Cells were equilibrated in isosmotic control solution (ISO). Osmotic calibration pulses (±10% anisosmotic) preceded the exposure to BUM (10 µm). Bars at the bottom of each trace indicate the duration of exposure to each solution. All of the solutions had the same osmolality (290 ± 1 mosmol/kgH2O) except for those used for the calibration pulses, which were nominally −10% (hypoosmotic) and +10% (hyperosmotic) with respect to the ISO control solution. A: BUM (10 µm) produced shrinkage in CPECs from NKCC1+/+. In the example shown [NKCC1+/+, postnatal day 18 (P18), male], BUM produced a maximum decrease in CWV of −13.1% at an initial rate of −3.8%/min. Cell recovered its initial volume on returning to the isosmotic (ISO) control solution. BUM exposure was preceded by 2 pulses of osmotic calibration solutions nominally −10 and +10% hypoosmotic and hyperosmotic, respectively. Double-headed gray arrows indicate the points in which the maximum of each response is measured for statistical analysis. B: BUM (10 µm) had no effect on basal CWV of CPECs lacking NKCC1. Example shown is from an NKCC1−/−, P11, female. Thus BUM-induced shrinkage is due to a specific pharmacological inhibition of NKCC1. C: maximum percentage change in CWV (% volume response) produced by 10 µM BUM in NKCC1+/+ and NKCC1−/− CPECs. In NKCC1+/+ cells, BUM produced an average CWV decrease of −16.0% (SD 6.4%; SEM 1.7%, n = 14 cells, 3 mice). BUM had no effect in CWV of NKCC1−/− (mean 0.4%, SD 1.4%, SEM 0.4, n = 10 cells, 3 mice). Difference between means is significant, P < 0.0001. D: percentage change in CWV produced by exposure to ±10% anisosmotic calibration pulses preceding BUM exposure. Amplitudes of the responses were measured as indicated by the double-headed gray arrows in A. Differences between hypoosmotic and hyperosmotic pulses recorded in NKCC1+/+ and NKCC1−/− cells were not statistically significant (NS). Bars in C and D represent the mean % change in CWV response. Error bars represent standard error of the mean (SEM). Black bars in C and D represent data from NKCC1+/+, and white bars correspond to data from NKCC1−/−.