Use of 51Cr cell labelling to distinguish between release of radiolabelled amino acids from primary astrocyte cultures being due to efflux or cell damage.
Continuous perfusion methods are widely used to monitor release of substances, particularly transmitters, from brain cell cultures growing as monolayers. However, if stimuli used to produce release also cause loss or lysis of cells, the appearance of label in the perfusate due to such effects will be indistinguishable from release. Using a perfusion method we have studied release of preloaded, radiolabelled amino acids from primary astrocyte cultures due to a variety of stimuli; hypotonic or high K+ media, activation of beta-receptors or swelling-induced release due to isosmotic ethanol. In this study primary astrocyte cultures were simultaneously labelled with Na2(51)CrO4 and allowed to take up radiolabelled D-aspartate or taurine. It was found that while all of the above methods caused release of radiolabelled amino acids none caused release of 51Cr into the perfusion fluid. In contrast, perfusion with 0.05% (v/v) Triton X-100 did lead to release of 51Cr. Thus a variety of means of inducing swelling or shape changes in astrocytes causes true release of radiolabelled amino acids and simultaneously monitoring 51Cr release seems a convenient means of distinguishing such release from cell loss or lysis.
Kimelberg
H K
HK
Division of Neurosurgery, Albany Medical College, NY 12208.
Bonville
D J
DJ
Goderie
S K
SK
D000596:Amino Acids / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D003902:Detergents; D000431:Ethanol / Q000494:pharmacology; D007038:Hypotonic Solutions; D009638:Norepinephrine / Q000494:pharmacology; D017830:Octoxynol; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism
D000596:Amino Acids; D002860:Chromium Radioisotopes; D003902:Detergents; D007038:Hypotonic Solutions; D013654:Taurine; D001224:Aspartic Acid; D000431:Ethanol; D017830:Octoxynol; D011188:Potassium; D009638:Norepinephrine