Carbonic acid buffer species measured in real time with an intracellular microelectrode array.
Carbonic acid buffer anions, HCO3- and CO3(2-), play an instrumental role in a host of vital processes in animal cells and tissues. Yet study of carbonic acid buffer species is hampered because no means are available to simultaneously monitor them at a cellular level in a rapid and dynamic fashion. An ion-selective cocktail, previously reported to measure changes in bicarbonate activity (alpha HCO3-), was instead shown to be principally selective for alpha CO3(2-). Ion-selective micropipettes (ISMs) based on this exchanger and consisting of a 3:1:6 (volume) mixture of tri-n-octylpropylammonium chloride, 1-octanol, and trifluoroacetyl-p-butylbenzene showed no significant interference from bicarbonate, chloride, phosphate, ascorbate, lactate, glutamate, acetate, or hydroxyl ions at concentrations expected in vivo. Intracellular and triple-barrel ISMs, consisting of a CO3(2-) sensitive, pH-sensitive, and reference barrel, were fabricated. Skeletal muscle cells (n = 17) were penetrated in vivo and showed values of 74 +/- 7 mV for membrane potential, 6.94 +/- 0.09 pHi, and 11 +/- 5 microM intracellular alpha CO3(2-), from which intracellular alpha HCO3- of 25 +/- 10 mM and CO2 tension of 120 +/- 55 Torr were calculated. All ion measurements reached a new steady state in 9 +/- 2 s after cell penetration. Thus measurements of intracellular alpha CO3(2-) and pH and associated levels of alpha HCO3(2-) and CO2 tension can be determined in biological tissues and cells with a spatial and temporal resolution previously unattainable.
Wietasch
K
K
Department of Neurology, University of Chicago, Illinois 60637.
Kraig
R P
RP
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002021:Buffers; D004594:Electrophysiology / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D008839:Microelectrodes; D009132:Muscles / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D001639:Bicarbonates; D002021:Buffers; D007202:Indicators and Reagents