%0 Journal Article %1 Nehe_1992_273 %A Neher, E. %A Augustine, G. J. %D 1992 %J J. Physiol. %K 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. %P 273--301 %T Calcium gradients and buffers in bovine chromaffin cells. %V 450 %X 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.

@article{Nehe_1992_273, abstract = {1. Digital imaging and photometry were used in conjunction with the fluorescent {C}a$^{2+}$ indicator, Fura-2, to examine intracellular {C}a$^{2+}$ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of {C}a$^{2+}$ within the cell, with {C}a$^{2+}$ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated {C}a$^{2+}$ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced {C}a$^{2+}$ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic {C}a$^{2+}$ buffer with which Fura-2 competes for {C}a$^{2+}$. 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 {C}a$^{2+}$ buffer suggest that 98-99\% of the {C}a$^{2+}$ 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 {C}a$^{2+}$ ions, because it does not saturate with 1 microM-{C}a$^{2+}$ inside the cell. 6. The low capacity, affinity and mobility of the endogenous {C}a$^{2+}$ buffer makes it possible for relatively small amounts of exogenous {C}a$^{2+}$ buffers, such as Fura-2, to exert a significant influence on the characteristics of the {C}a$^{2+}$ 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 {C}a$^{2+}$ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Neher, E. and Augustine, G. J.}, biburl = {https://www.bibsonomy.org/bibtex/27b25ab911f1d2ba3d85b0c46d286ee16/hake}, description = {The whole bibliography file I use.}, interhash = {5bcef8176c40991091de71d34ec1ce84}, intrahash = {7b25ab911f1d2ba3d85b0c46d286ee16}, journal = {J. Physiol.}, keywords = {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.}, month = May, pages = {273--301}, pmid = {1331424}, timestamp = {2009-06-03T11:21:23.000+0200}, title = {Calcium gradients and buffers in bovine chromaffin cells.}, volume = 450, year = 1992 }