Continuous on-line hydrogen ion monitoring to study flow dynamics of perifusion systems and cellular metabolism.
Time-dependent concentration profiles of input signals and feedback of metabolic products can strongly influence cellular responsiveness. To study these parameters, we developed a perifusion system that can deliver biological signals to cells with minimal dispersion, monitor real time responses, and remove waste products continuously. By monitoring pH with miniature hydrogen ion-selective electrodes at intervals of 1 s, effects of dispersion, flow rate, pumping system, and changes in cellular metabolism were demonstrated. Dynamic responses of a human cell line to a series of 10-min pulses of the metabolic uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) were monitored. A rapid 1-min increase in acid release occurred on exposure to CCCP, followed by a decrease in acidification and then a gradual return to a baseline slightly more acidic than before administration of CCCP. These observations demonstrate that this perifusion system can reveal small changes in pH (+/- 0.0005 units) induced by metabolic perturbations and has the potential to reveal the dynamics of cellular responsiveness to a wide range of hormonal, metabolic, and other chemical signals.
Brand
R M
RM
Reproductive Sciences Program, University of Michigan, Ann Arbor 48109-0404.
Ghazzi
M N
MN
Rolfes-Curl
A
A
Cantor
H C
HC
Midgley
A R
AR
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002477:Cells / Q000378:metabolism*; D004867:Equipment Design; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008839:Microelectrodes; D010477:Perfusion / Q000295:instrumentation / Q000379:methods*; D013594:Syringes
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone