Extracellular glutamine is a critical modulator for regulatory volume increase in human glioma cells

Abstract

Mammalian cells regulate their volume to prevent unintentional changes in intracellular signaling, cell metabolism, and DNA integrity. Intentional cell volume changes occur as cells undergo proliferation, apoptosis, or cell migration. To regulate cell volume, cells use channels and transport systems to flux osmolytes across the plasma membrane followed by the obligatory movement of water. While essentially all cells are capable of regulatory volume decrease (RVD), regulatory volume increase (RVI) mechanisms have only been reported in some cell types. In this investigation, we used human glioma cells as a model system to determine conditions necessary for RVI. When exposed to hyperosmotic conditions through the addition of 30 mosM NaCl or sucrose, D54-MG and U251 glioma cell lines and glioma cells from acute patient biopsies shrunk transiently but were able to fully recover their original cell volume within 40-70 min. This ability was highly temperature sensitive and absolutely required the presence of low millimolar concentrations of l-glutamine in the extracellular solution. Other known substrates of glutamine transporters such as methyl-amino isobutyric acid (MeAIB), alanine, and threonine were unable to support RVI. The ability of cells to undergo RVI also required the presence of Na+, K+, and Cl- and was inhibited by the NKCC inhibitor, bumetanide, consistent with the involvement of a Na+/K+/2Cl- cotransporter (NKCC). Moreover, the expression of NKCC1 was demonstrated by Western blot. We concluded that regulatory volume increase in human glioma cells occurs through the uptake of Na+, K+, and Cl- by NKCC1 and is modulated by the presence of glutamine.

Figure 1. Glioma cells exposed to a hyperosmotic challenge undergo a regulatory volume increase in the presence of supplemented cell culture medium

Normalized mean cell volumes recorded during a hyperosmotic challenge induced by the addition of 15mM NaCl. D54-MG cells (A), U251 cells (B), and cells cultured acutely from a patient biopsy, labeled GBM50 (C), were suspended in normal NaCl bath solution or cell culture medium supplemented with 2mM glutamine and 7% bovine growth serum. (p<0.05 at 20, 40, or 60 minutes is indicated by *.)

Figure 2. The ability of D54-MG cells to undergo RVI is independent of the extent of the hyperosmotic challenge

A: Normalized mean cell volumes recorded during a hyperosmotic challenge induced by the addition of various concentrations of NaCl. B: Normalized mean cell volumes recorded at the initiation of the hyperosmotic challenge (V_0’, both actual observed and those expected for a perfect osmometer) compared to the volumes attained after 60 minutes of volume regulation (V_60’).

Figure 3. Extracellular glutamine is necessary for RVI in D54-MG cells

A: Normalized mean cell volumes of glioma cells were recorded during a hyperosmotic challenge induced by an addition of 15mM NaCl. Cells were suspended in unsupplemented cell culture medium, cell culture medium supplemented with glutamine and bovine growth serum, normal NaCl bath solution supplemented with glutamine alone, or normal NaCl bath solution supplemented with bovine growth serum alone. B: A dose-response curve was generated by plotting the maximum RVI response observed versus the concentration of extracellular glutamine at which the observed response occurred.C: Known glutamine transport systems are not involved in RVI of D54-MG cells Normalized mean cell volumes at the initiation of the hyperosmotic challenge (0′) compared to those recorded after 60′ of volume regulation in the presence or absence of 5mM amino acids or amino acid derivatives. When compared to bath alone, p<0.05 is indicated by *.

Figure 4. Ion uptake through a Na⁺/K⁺/Cl⁻ cotransporter (NKCC) is necessary for RVI in D54-MG cells

Normalized mean cell volumes recorded during a hyperosmotic challenge induced by the addition of 15mM NaCl. A: A comparison of the response of cells maintained at 37ºC versus room temperature (R.T.). B: A comparison of the response of cells suspended in normal NaCl bath versus those suspended in bath solutions lacking extracellular Na⁺, K⁺, or Cl⁻. C: Volumes recorded at the initiation of the hyperosmotic challenge (0′) compared to those recorded after 40′ of volume regulation in the presence or absence of glutamine and the NKCC inhibitor, bumetanide. (p<0.05 indicated by *.)

Figure 5. NKCC1 but not NKCC2 is expressed in human glioma cell lines and cells from a patient biopsy

Whole cell lysates from D54-MG and U251 cells as well as the patient sample GBM50 were separated by SDS-PAGE and probed with commercial antibodies generated against NKCC1 and NKCC2.