Carbonic acid buffer species measured in real time with an intracellular microelectrode array

Area of Science:

• Physiology
• Biochemistry
• Cell Biology

Background:

• Carbonic acid buffer anions, bicarbonate (HCO3-) and carbonate (CO3(2-)), are vital for animal cell and tissue processes.
• Current methods lack the ability to simultaneously monitor these species dynamically at the cellular level.

Purpose of the Study:

• To develop a novel method for the rapid, simultaneous, and dynamic measurement of intracellular carbonate (CO3(2-)) and pH.
• To enable calculation of intracellular bicarbonate (HCO3-) and CO2 tension.

Main Methods:

• Fabrication of triple-barrel ion-selective micropipettes (ISMs) with a CO3(2-)-sensitive barrel.
• Testing the selectivity of the ion-exchanger cocktail against various physiological ions.
• In vivo penetration of skeletal muscle cells to measure membrane potential, intracellular pH (pHi), and intracellular alpha CO3(2-).

Main Results:

• The developed ISMs demonstrated principal selectivity for alpha CO3(2-) with minimal interference from other ions.
• Measurements in skeletal muscle cells yielded pHi of 6.94 +/- 0.09 and intracellular alpha CO3(2-) of 11 +/- 5 microM.
• Calculated intracellular alpha HCO3- was 25 +/- 10 mM and CO2 tension was 120 +/- 55 Torr.
• Ion measurements reached a steady state within 9 +/- 2 seconds.

Conclusions:

• This study presents a novel technique for real-time intracellular measurement of CO3(2-), pH, HCO3-, and CO2 tension.
• The method offers unprecedented spatial and temporal resolution for studying carbonic acid buffering in biological systems.
• This advancement is critical for understanding cellular physiology and disease states.