%0 Journal Article %1 Zhou_1993_245 %A Zhou, Z. %A Neher, E. %D 1993 %J J. Physiol. %K 8271200 Adrenal Animals, Buffers, Calcium Calcium, Calcium-Binding Cattle, Cell Cells, Channel Channels, Chromaffin Cultured, Dyes, El, Electric Enterochromaffin Fluorescent Fura-2, Gating, Glands, Ion Ligands, Membrane, Molecular Nicotinic, Nystatin, Permeability, Proteins, Receptors, Stimulation, System, Weight, ectrophysiology, %P 245--273 %T Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. %V 469 %X 1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca$^2+$ binding proteins in the range of 7-20 kDa. 4. The average Ca$^2+$ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca$^2+$ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca$^2+$ affinity, since kappa S did not change in the range of Ca$^2+$ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)

@article{Zhou_1993_245, abstract = {1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble {C}a$^{2+}$ binding proteins in the range of 7-20 kDa. 4. The average {C}a$^{2+}$ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile {C}a$^{2+}$ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low {C}a$^{2+}$ affinity, since kappa S did not change in the range of {C}a$^{2+}$ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Zhou, Z. and Neher, E.}, biburl = {https://www.bibsonomy.org/bibtex/230cfa8362dd6833735ddcb1db64eea31/hake}, description = {The whole bibliography file I use.}, interhash = {ab73dca3a8e4f659b005173fe8d7146d}, intrahash = {30cfa8362dd6833735ddcb1db64eea31}, journal = {J. Physiol.}, key = 275, keywords = {8271200 Adrenal Animals, Buffers, Calcium Calcium, Calcium-Binding Cattle, Cell Cells, Channel Channels, Chromaffin Cultured, Dyes, El, Electric Enterochromaffin Fluorescent Fura-2, Gating, Glands, Ion Ligands, Membrane, Molecular Nicotinic, Nystatin, Permeability, Proteins, Receptors, Stimulation, System, Weight, ectrophysiology,}, month = Sep, pages = {245--273}, pmid = {8271200}, timestamp = {2009-06-03T11:21:39.000+0200}, title = {Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.}, volume = 469, year = 1993 }