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. 2010 Apr;2(4):632-48.
doi: 10.3390/toxins2040632. Epub 2010 Apr 8.

The effects of cholera toxin on cellular energy metabolism

Rachel M Snider  1 , Jennifer R McKenzie, Lewis Kraft, Eugene Kozlov, John P Wikswo, David E Cliffel
Affiliations

The effects of cholera toxin on cellular energy metabolism

Rachel M Snider et al. Toxins (Basel). 2010 Apr.

Abstract

Multianalyte microphysiometry, a real-time instrument for simultaneous measurement of metabolic analytes in a microfluidic environment, was used to explore the effects of cholera toxin (CTx). Upon exposure of CTx to PC-12 cells, anaerobic respiration was triggered, measured as increases in acid and lactate production and a decrease in the oxygen uptake. We believe the responses observed are due to a CTx-induced activation of adenylate cyclase, increasing cAMP production and resulting in a switch to anaerobic respiration. Inhibitors (H-89, brefeldin A) and stimulators (forskolin) of cAMP were employed to modulate the CTx-induced cAMP responses. The results of this study show the utility of multianalyte microphysiometry to quantitatively determine the dynamic metabolic effects of toxins and affected pathways.

Keywords: PC-12; cholera toxin; cyclic AMP; forskolin; metabolism; microphysiometry; multianalyte.

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Figures

Figure 1
Figure 1
Average metabolic response of 5 × 105 HepG2 cells to thirty minutes exposure to 1,000 nM CTx. The black bar indicates the 30 minutes 1,000 nM CTx was being perfused through the cell chamber. Cell necrosis is triggered at 200 minutes.
Figure 2
Figure 2
Electrochemical measurements of the metabolic response of PC-12 cells to 1000 nM Cholera Toxin. (a) Electrochemical currents corresponding to MAMP signals for 5 × 105 PC-12 cells in response to 1000 nM CTx. The black bars indicate where CTx was flowed through the cell chamber. Cell death was triggered at 200 minutes, and CTx was re-introduced into the chamber to determine the effect on the sensors prior to calibration at 275 minutes. A baseline shift can be seen in the glucose signal. (b) Close-up of current at glucose electrode when CTx is present for live cells and dead cells. A shift in current during dead-cell exposure indicates the CTx affect the glucose oxidase-based glucose sensor. The shift proved to be correctable; however, no measurable change was seen in glucose consumption due to CTx exposure.
Figure 3
Figure 3
The average metabolic rates and acidification of PC-12 cells exposed to thirty minutes 1,000 nM (a) and 500 nM CTx (b). The black bar indicates the 30 minutes CTx was being flown through the cell chamber. Cell necrosis is triggered at 200 minutes.
Figure 4
Figure 4
The average metabolic rates and acidification of PC-12 cells exposed to three 2-minute pulses of (a) 1,000 nM CTx (●) and (b) 100 nM CTx (●). Cell necrosis is triggered at 200 minutes.
Figure 5
Figure 5
Average metabolic responses of two chambers of PC-12 cells exposed to (a) one 2 minute dose of 100 nM CTx (●) or (b) a ten minute dose of 5 nM CTx (●). The spike in oxygen at 58 minutes is the result of noise spike in one of the chambers. Cell necrosis is triggered near 300 minutes.
Figure 6
Figure 6
Average metabolic responses of PC-12 cells exposed to either 100 nM CTx or 500 nM CTxB subunit. Cell necrosis is triggered at 300 minutes. (a) Lactate production. The average of four chambers () treated with three 2-minute pulse of 500 nM CTxB (◊). The other trace is the average of four chambers () treated with three 2-minute pulse of 100 nM CTx (♦). (b) Oxygen Consumption. The average of four chambers () treated with three 2-minute pulse of 500 nM CTxB (◊). The other trace is the average of three chambers () treated with three 2-minute pulse of 100 nM CTx (■). (c) Acid production. The average of four chambers () treated with three 2-minute pulse of 500 nM CTxB (◊). The other trace is the average of four chambers () treated with three 2-minute pulse of 100 nM CTx (■).
Figure 7
Figure 7
The metabolic response of PC-12 cells pre-treated with 1 µg/mL BrA. Necrosis is triggered at 330 minutes. (a) Lactate production. One chamber () treated with brefeldin A (black bar), and then exposed to ten minutes of 10 nM CTx (♦). The lactate sensor in the replicate chamber failed to calibrate, so N = 1. The other chamber () received only CTx. (b) Acid production. The average of two chambers () treated with brefeldin A (black bar), and then exposed to ten minutes of 10 nM CTx (♦). The other chamber () received only CTx.
Figure 8
Figure 8
The average metabolic rates and acidification of PC-12 cells exposed to thirty minutes 30 µM H-89 (black bar) and a single two minute dose of 100 nM CTx (♦). Cell necrosis is triggered at 220 minutes.
Figure 9
Figure 9
The average metabolic response of PC-12 cells exposed to thirty minutes 10 µM forskolin. The black bar indicates the 10 µM forskolin was being flown through the cell chamber. Cell necrosis is triggered at 230 minutes.
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