Description:

Volume regulation in response to a hypotonic environment requires the movement of anions out of the cell. Chloride is the most commonly thought to move across the plasma membrane although the movement of a sufficient amount of any anion would contribute to returning cell volume to normal. Glutamate is an anion at physiological pH and may move through the volume regulated anion channel. To determine the composition of the volume regulated anion flux, self-referencing anion-selective electrodes were positioned 1-3 µm from human embryonic kidney cells (tsA201a) and used to record flux during a sustained hyposmotic challenge. The ionophore used to make the anion selective electrode was ~3-fold more selective for Cl^- over glutamate or gluconate but less than 2-fold selective over bicarbonate, acetate, citrate or thiosulfate. An outward anion efflux was recorded from cells challenged with hypotonic (250 ± 5 mOsm) solution. The increase in efflux peaked at 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 µM tamoxifen. These results establish the feasibility of using modulation of electrochemical, anion-selective electrodes to monitor anion movement during volume regulatory events.

Progress:

The role of glutamate, a bio-active amino acid and neurotransmitter, in hypotonic-induced volume recovery was further investigate using a glutamate probe developed at the BRC This probe is small enough to record from groups of 10-20 mammalian cells. Preliminary results show that a signal was detected from tsA201a cells 20-30 mins after exposure to hypotonicity, indicating the release of glutamate into the bath solution. Addition of 10 µM tamoxifen blocked this signal. Control experiments are currently being carried out to determine the veracity of this signal. These preliminary results suggest that glutamate is released in response to volume regulation in a hypotonic environment. Glutamate release during volume regulation may lead to localized cell toxicity and/or epileptic activity.