Effect of chemical hypoxia on intracellular ATP and cytosolic Mg2+ levels.

Intracellular magnesium is intimately associated with adenosine triphosphate (ATP) concentrations and energy utilization. We determined adenine nucleotide concentrations (ATP, adenosine diphosphate, and adenosine triphosphate) and the associated changes in intracellular free Mg2+ ([Mg2+]i) by high-performance liquid chromatography and fluorescent methods, respectively. Various mitochondrial inhibitors were used to deplete intracellular ATP and alter energy charge in epithelial cells. The opossum kidney (OK) cell line was used as a prototypic renal epithelial cell. These agents markedly deplete intracellular ATP levels with modest changes in [Mg2+]i and [Ca2+]i. Because these agents have disparate actions, it is likely that these changes were due to alterations in ATP rather than to selective drug effects. Cyanide resulted in a rapid (within 2 minutes) fall in ATP from 25.85 to 10.58 nmol/mg protein or about 3 mmol/L, whereas [Mg2+]i increased gradually (10 minutes), from 513 +/- 7 to 1096 +/- 105 mumol/L [Ca2+]i increased from 109 +/- 12 to 153 +/- 10 nmol/L within 20 seconds, then returned to basal concentrations. The changes in ATP, Mg2+, and Ca2+ were not altered by removing external Na+o, adding ruthenium red, or treating with vanadate. Antimycin diminished ATP levels in a manner similar to the effect of cyanide, but by contrast [Mg2+]i decreased to 436 +/- 13 mumol/L and [Ca2+]i transiently increased. These studies indicate that we are able to distinguish Mg2+ movements from those of Ca2+ by fluorescent techniques and suggest that intracellular regulation of [Mg2+]i is distinctive from that of [Ca2+]i. Oligomycin resulted in marked and rapid falls in [ATP]i with disproportionate increases in [Mg2+]i. The response of magnesium-depleted cells (basal [Mg2+]i, 231 +/- 10 mumol/L) after inhibitor-induced energy depletion was similar to that of control cells. These studies suggest that large changes in intracellular ATP levels do not markedly alter intracellular [Mg2+]i control and, in turn, that intracellular free Mg2+ is not a limiting factor in ATP metabolism after energy depletion with chemical hypoxia.