Determination of cytosolic Mg2+ activity and buffering in BC3H-1 cells with mag-fura-2.
The magnesium buffer coefficient (BMg) was calculated for BC3H-1 cells from the rise in cytosolic Mg2+ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg2+ activity (0.54 +/- 0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ATP: BMg was 12.9 indicating that a 1 mM increase in Mg2+ activity requires an addition of about 13 mM magnesium. The accuracy of this value depends on these assumptions: (a) all of the magnesium released from ATP stayed in the cells; (b) the rise in Mg2+ was not secondary to pH-induced changes in BMg; (c) mag-fura-2 measured Mg2+ and not Ca2+; and (d) the accuracy of the mag-fura-2 calibration. Total magnesium did not change in response to IAA/CN treatment, thus the change in Mg2+ activity reflected a redistribution of cell magnesium. pH changes induced by NH4Cl pulse and removal had little effect on Mg2+ activity and the changes were slower than and opposite to pH-induced changes in Ca2+ activity measured by fura-2. Ca2+ responses were temporally uncoupled from Mg2+ responses when the cells were treated with IAA only and in no cases did Ca2+ levels rise above 1 microM, showing that the mag-fura-2 is responding to Mg2+. Additional studies demonstrated that approximately 90% of the mag-fura-2 signal was cytosolic in origin. The remaining non-diffusible mag-fura-2 either was bound to cytosolic membranes or sequestered in organelles with the fluorescence characteristics of the Mg2(+)-complexed form, even when cytosolic free Mg2+ activity was approximately 0.5 mM. This bound mag-fura-2 would appear to increase the Kd and thus clearly limits the accuracy of our estimmate for BMg. Despite this limitation, we demonstrate that Mg2+ is tightly regulated in face of large changes in extracellular Mg2+, and that interplay observed between pH, Ca2+ and Mg2+ activities strongly supports the hypothesis that these factors interact through a shared buffer capacity of the cell.
Grubbs
R D
RD
Department of Pharmacology and Toxicology, School of Medicine, Wright State University, Dayton, Ohio 45435.
Walter
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002451:Cell Compartmentation; D002478:Cells, Cultured / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008274:Magnesium / Q000378:metabolism*; D009132:Muscles / Q000166:cytology*; D010693:Phloretin / Q000494:pharmacology; D012966:Sodium Cyanide / Q000494:pharmacology
D002021:Buffers; D005456:Fluorescent Dyes; D007461:Iodoacetates; D000643:Ammonium Chloride; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D004072:Digitonin; D012966:Sodium Cyanide; D010693:Phloretin; D016257:Fura-2; D019807:Iodoacetic Acid