Analysis of dynamic changes in HeLa cell metabolism with an on-line microperifusion system.

Abstract

The pattern of input signals received by cells can be the major component in determining their response. Additionally, cells frequently employ end-product inhibition to help regulate metabolism. Therefore, a system addressing these issues by allowing the dynamic introduction and removal of metabolites to cells should be valuable for examining cellular behavior. For this purpose, a perifusion system, a device used for culturing living cells in the presence of flowing medium, has been developed. The system described in this thesis is unique because it contains on-line hydrogen ion monitoring capabilities, and exceptionally small dead volumes. A dual electrode system consisting of pre-cell control and post-cell response detectors was employed to distinguish cellular responses from test compound introduction. Since these electrodes are sensitive to an exceptionally small pH change (0.0005 pH units), it is virtually impossible to introduce test solutions that are identical in pH to the baseline medium. Because some dispersion occurs as chemicals pass through perifusion systems, the shape of the pH change induced by switching medium will be different at the two electrodes. Therefore, the analytical technique of convolution was used to correct for these differences. The analyzed data demonstrate for the first time that the cellular response to the loss of oxidative phosphorylation induced by the uncoupler CCCP contains different components. The system was also used to compare the dynamics of HeLa cell glycolytic modification induced by starvation and the glycolytic inhibitor iodoacetate. Both methods resulted in a rapid, continuous decrease in the quantity of acid released by the cells. Iodoacetate took longer to act; however, once begun, inhibition was greater with a slower recovery time. Additionally, the dynamics of cellular conversion from a glucose-dependent to a glutamine-dependent metabolism were examined. The response was fast, without measurable delay. The cells preferred glutamine as their energy source; however, some glucose was still used. By combining the mathematical technique of convolution with a perifusion system capable of measuring acidification changes on the order of seconds, new insights into the dynamics of cellular metabolism have been obtained.