AJP - Regulatory, Integrative and Comparative Physiology, Vol 261, Issue 3 760-R765, Copyright © 1991 by American Physiological Society
Carbonic acid buffer species measured in real time with an intracellular microelectrode array
K. Wietasch and R. P. Kraig
Department of Neurology, University of Chicago, Illinois 60637.
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.
