Beeson Group Research

Quantitative Analysis of Cell Metabolism

Microphysiometry is a relatively new technique in which a silicon-based sensor is used to monitor cell metabolism. The conventional mode of operation for the Cytosensor® microphysiometer is to measure the rate of acid release for a small number of cells entrapped within a perfusion chamber that is in contact with the sensor. The rate of acid release is a general measure of cell metabolism. We, and others, have used this instrument to monitor cell activation for the purposes of drug screening and basic physiology studies. We've also used the instrument to study the cell surface reactions involved in T-cell activation.

A mixture of T cells and antigen presenting cells were exposed to MBP(1-14)A4 antigen peptide for 30 min starting at t= 0 min. Shown in blue is the decrease in acidification rate as this short-lived peptide dissociates from MHC protein. Addition of anti-MHC antibody at different times (arrows) confirms that the signaling at longer times involves active TCR ligation. An exponential fit to the decay shown in blue was used to calculate the peptide dissociation rate. Comparison of this rate with that for cells not in contact with TCR was used to estimate the fraction of peptide-MHC complexes bound by TCR.

We have recently used a modified microphysiometer to measure redox reactions in live cells. The pH sensitive sensor was modified with a thin layer of gold which functions as an inert electrode. The buffer contains a mixture of potassium ferri- and ferrocyanide. A redox cycling agent, such as menadione, is used to couple the iron redox pair with an intracellular enzymatic reaction. We've shown that in many cells menadione is reduced primarily by the cytosolic DT-diaphorase and that the rate of reduction is a measure of the enzymatic rate. We've also shown that the primary reductant for this reaction is NADPH which is produced in the hexose monophosphate (HMP) shunt. Thus, the rate of menadione reduction by cells is an indirect measure of the flux through the HMP shunt. Additional redox cycling agents are being used to measure the flux through different pathways. When combined with measurem,ents of acid release, we are able to provide a quantitative meaure of the metabolic state of a cell with high kinetic resolution. We are using these techniques to study the role of energy metabolism in cell signaling pathways.

Potentiometric measurements of cell redox reactions Shown in the top panel is the rate of menadione reduction by CHO cells. Stimulation of the cells with insulin (shaded region) causes a decrease in the rate of menadione reduction and an increase in the rate of acid release (not shown). Both respiration and glycolysis are upregulated by the insulin. The decrease in menadione reduction is apparently due to a transient increase in the concentration of reactive oxygen species. The bottom panel illustrates how energy metabolism is measured by microphysiometry. Acid release rates (yellow) are measured in the pH mode. The flux of reducing equivalents (red) is measured in the redox mode and oxygen consumption is measured independently with a micro electrode.

With recent modifications of the microphysiometer we can now measure the rates of total acid extrusion, lactic acid extrusion, and oxygen consumption. From these measurements we can calculate the fluxes through aerobic glycolysis (TCA flux), anaerobic glycolysis and the HMP shunt. Shown in the panel on the left is a comparison of fluxes measured for cardiac myoblasts using microphysiometry or radioisotope-labeled glucose. Shown on the right is a comparison of the changes in these fluxes as measured by the two techniques. The advantage of the microphysiometer is its superior kinetic resolution. With this technique we can study the regulation of energy metabolism during cell stimulation.

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