Intracellular Mg2+ concentrations following metabolic inhibition in opossum kidney cells

Intracellular magnesium is associated with intracellular ATP concentrations as Mg-ATP2- and is involved with many enzymes in energy utilization. Intracellular Mg2+ has also been postulated to be involved with various Ca2+ actions. We determined adenine nucleotide concentrations (ATP, ADP and AMP) by HPLC and the associated changes in intracellular free Mg2+ ([Mg2+]i) by fluorescent methods in an epithelial cell line (opossum kidney cells). CCCP (a mitochondrial uncoupler), iodoacetate and amobarbital resulted in marked and rapid falls in [ATP]i with disproportionate increases in [Mg2+]i. These studies indicate that we are able to distinguish Mg2+ movements from Ca2+ by fluorescent techniques and suggests that intracellular regulation of [Mg2+]i is distinctive from those of [Ca2+]i. As CCCP plus amobarbital are reversible, we removed these inhibitors and tested the effect of Mg(2+)-availability on ATP depletion and recovery. The response of magnesium-depleted cells (basal [Mg2+]i 231 +/- 10 microM) following inhibitor-induced energy depletion and ATP recovery were similar to control cells. Accordingly, intracellular [Mg2+]i does not appear to be a limiting factor in ATP regeneration following removal of the chemical hypoxic insult. Finally, exogenous application of Na2ATP2- altered intracellular energy levels in normal and energy depleted cells but was without effect on [Mg2+]i. These studies suggest that intracellular ATP levels do not directly alter intracellular [Mg2+]i control and, in turn, intracellular free Mg2+ is not a limiting factor in ATP regeneration following energy depletion with chemical hypoxia.