Abstract
1. Digital imaging and photometry were used in conjunction with the
fluorescent Ca$^2+$ indicator, Fura-2, to examine intracellular
Ca$^2+$ signals produced by depolarization of single adrenal
chromaffin cells. 2. Depolarization with a patch pipette produced
radial gradients of Ca$^2+$ within the cell, with Ca$^2+$
concentration highest in the vicinity of the plasma membrane. These
gradients dissipated within a few hundred milliseconds when the voltage-gated
Ca$^2+$ channels were closed. 3. Dialysis of Fura-2 into the
chromaffin cell caused concentration-dependent changes in the depolarization-induced
Ca$^2+$ signal, decreasing its magnitude and slowing its recovery
time course. These changes were used to estimate the properties of
the endogenous cytoplasmic Ca$^2+$ buffer with which Fura-2 competes
for Ca$^2+$. 4. The spatially averaged Fura-2 signal was well
described by a model assuming fast competition between Fura-2 and
an endogenous buffer on a millisecond time scale. Retrieval of calcium
by pumps and slow buffers occurs on a seconds-long time scale. No
temporal changes indicative of buffers with intermediate kinetics
could be detected. 5. Two independent estimates of the capacity of
the fast endogenous Ca$^2+$ buffer suggest that 98-99\% of the
Ca$^2+$ entering the cell normally is taken up by this buffer.
This buffer appears to be immobile, because it does not wash out
of the cell during dialysis. It has a low affinity for Ca$^2+$
ions, because it does not saturate with 1 microM-Ca$^2+$ inside
the cell. 6. The low capacity, affinity and mobility of the endogenous
Ca$^2+$ buffer makes it possible for relatively small amounts
of exogenous Ca$^2+$ buffers, such as Fura-2, to exert a significant
influence on the characteristics of the Ca$^2+$ concentration
signal as measured by fluorescence ratios. On the other hand, even
at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over
the endogenous buffers. Under these conditions radiometric Ca$^2+$
concentration signals are largely attenuated, but absolute fluorescence
changes (at 390 nm) accurately reflect calcium fluxes.
- 1331424
- adrenal
- animals,
- biological,
- calcium
- calcium,
- cattle,
- cells,
- channels,
- cultured,
- dose-response
- drug,
- electrophysiology,
- exocytosis,
- fluorescence,
- fura-2,
- glands,
- gov't,
- humans,
- kinetics,
- mathematics,
- membrane
- microscopy,
- models,
- neurons,
- non-u.s.
- p.h.s.,
- potentials,
- relationship,
- research
- signal
- support,
- transduction,
- u.s.
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