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Calcium gradients and buffers in bovine chromaffin cells.
Author(s) -
Neher E,
Augustine G J
Publication year - 1992
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1992.sp019127
Subject(s) - depolarization , biophysics , chemistry , calcium , intracellular , fura 2 , endogeny , chromaffin cell , pipette , analytical chemistry (journal) , biochemistry , chromatography , biology , adrenal medulla , endocrinology , catecholamine , organic chemistry , cytosol , enzyme
1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura‐2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage‐gated Ca2+ channels were closed. 3. Dialysis of Fura‐2 into the chromaffin cell caused concentration‐dependent changes in the depolarization‐induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura‐2 competes for Ca2+. 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 Ca2+ buffer suggest that 98‐99% of the Ca2+ 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 Ca2+ ions, because it does not saturate with 1 microM‐Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura‐2, to exert a significant influence on the characteristics of the Ca2+ 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 Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.