Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Abstract

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl(-) channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V(Post-RVD) < V(baseline)). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl(-) channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd(2+) (V(Post-RVD) > 1.4*V(baseline)). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K(+)-Cl(-) cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15 degrees C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15 degrees C, the channel blockers NPPB + Cd(2+) completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl(-) channels vs. transporters to this process is approximately 60-70% and approximately 30-40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl(-) channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a.

Fig. 1

D54-MG cells exhibit regulatory volume decrease (RVD) in response to a hyposmotic challenge. A and B: normalized mean cell volumes (MCVs) during a hyposmotic challenge induced by the addition of double-distilled H₂O (ddH₂O) in D54-MG cells (A) and cultured astrocytes (B). Vt, volume at time t; V_baseline, original volume. C: normalized mean peak volumes (both observed and those expected for a perfect osmometer) compared with the final volumes attained after volume regulation in D54-MG cells.

Fig. 2

Cl^- channel inhibitors limit RVD in human glioma cells. A: comparison of normalized mean volumes of D54-MG cells exposed to a 50% addition of ddH₂O and treated with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a combination of NPPB + Cd²⁺, or no drug (control). B: normalized peak mean and post-RVD volumes. *P < 0.05 compared with control; #P < 0.05 compared with NPPB alone. C: current-voltage (V) relationship of whole cell recordings obtained by eliciting voltage ramps from -120 to +40 mV in cells exposed to a 50% hypotonic challenge in the presence of no drug (1), NPPB (2), or NPPB + Cd²⁺ (3).

Fig. 3

An intact Cl^- gradient is necessary for volume recovery. A comparison of normalized mean volumes of D54-MG cells exposed to a 50% addition of ddH₂O and treated with the Cl^- channel inhibitors NPPB + Cd²⁺ or the Cl^- ionophore tributyltin chloride (TBT).

Fig. 4

Cl^--cation transport inhibitors also limit RVD in human glioma cells. A: comparison of normalized mean volumes of D54-MG cells exposed to a 50% addition of ddH₂O and treated with bumetanide, R-(+)- [(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3- dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), furosemide, or no drug (control). B: normalized peak mean and post-RVD volumes. C: current-voltage relationship of whole cell recordings obtained by eliciting voltage ramps from -120 to +40 mV in cells exposed to a 50% hypotonic challenge in the presence of no drug (1), DIOA (2), or furosemide (3). D: current density values normalized to the control average at 0 mV. *P < 0.05 compared with control.

Fig. 5

Inhibition of Cl^- channels and transporters synergistically inhibits RVD. A and B: comparison of normalized mean cell volumes (A) and mean peak and post-RVD volumes (B) attained from D54-MG cells exposed to a 50% addition of ddH₂O and treated with NPPB + Cd²⁺, NPPB + Cd²⁺ + DIOA or no drug (control). C: current-voltage relationship of whole cell recordings obtained by eliciting voltage ramps from -120 to +40 mV in cells exposed to a 50% hypotonic challenge in the presence of no drug (1), NPPB + Cd²⁺ (2), or NPPB + Cd²⁺ + DIOA (3). D: current density values normalized to the control average at 0 mV. *P < 0.05 compared with control (unless otherwise indicated); #P < 0.05 compared with NPPB + Cd²⁺.

Fig. 6

The transport-mediated component of volume regulation can be inhibited at higher temperatures than the channel-mediated component. A: normalized mean cell volumes of D54-MG cells exposed to a 50% addition of ddH₂O at 4, 10, 15, 25, and 37°C. B: normalized mean cell volumes of D54-MG cells exposed to a 50% addition of ddH₂O while maintaining the temperature of the experiment at 15°C. Cells were treated with DIOA, NPPB + Cd²⁺, or no drug (control).

Fig. 7

Estimated number of osmolytes effluxed from cells during 10-min intervals after the addition of ddH₂O. Experiments were performed at 22°C (A) or 15°C (B) in the presence of DIOA, NPPB + Cd²⁺, DIOA + NPPB + Cd²⁺, or no drug. *P < 0.05 compared with control; #P < 0.05 compared with 22°C control.

Fig. 8

RVD in glioma cells from patient biopsies is also inhibited by both Cl^- channel and transport inhibitors. A and B: comparison of normalized mean volumes of cells from patient samples [identified as GBM-1 (A) and GBM-2 (B)] exposed to a 50% addition of ddH₂O and treated with DIOA, NPPB + Cd²⁺, or no drug (control).

Fig. 9

Glioma cells show expression of Cl^- channels and transporters. Whole cell lysates of D54-MG cells, patient samples GBM-1 and GBM-2, and cortical astrocytes were separated by SDS-PAGE and probed with antibodies to ClC-1–7 (A) and K+-Cl^- cotransporter (KCC)1–4 (B).