Mechanisms of intracellular Mg2+ regulation affected by amiloride and ouabain in the guinea-pig taenia caeci

Abstract

1. The effects of amiloride and ouabain on the regulation of the intracellular, free Mg2+ concentration ([Mg2+]i) were investigated in the taenia isolated from the guinea-pig caecum, using nuclear magnetic resonance (NMR) techniques. 2. [Mg2+]i were mainly estimated from the separation of the alpha- and beta-ATP peaks observed in 31P NMR spectra. In normal (physiological) and nominally Ca(2+)-free solutions, [Mg2+]i was approximately 0.3-0.4 mM. Application of either amiloride or ouabain in Ca(2+)-free solutions significantly increased [Mg2+]i, with only a small change in ATP content. Washout of the drugs reversed the changes in [Mg2+]i. 3. Changes in pHi were estimated from: (1) the chemical shift of phosphoethanolamine, and (2) solving two relational equations of pHi and [Mg2+]i obtained from the beta- and gamma-ATP peaks. Both estimations revealed some intracellular alkalosis during application of these two drugs. After correction for pHi, a significant increase in [Mg2+]i was still obtained 150 min after application of either drug. 4. In the presence of amiloride, simultaneous removal of extracellular Mg2+ and Ca2+ significantly depleted intracellular Mg2+. This result suggests the presence of an amiloride-insensitive (or less sensitive) pathway which passively transports Mg2+ across the plasma membrane. 5. The intracellular Rb+ concentration was monitored as an index of Na(+)-K+ pump activity, using 87Rb NMR. In Ca(2+)-free solutions containing 5 mM Rb+, the intracellular Rb+ concentration was hardly changed by amiloride, but was depleted by additional applications of ouabain. Wash-out of ouabain restored the intracellular Rb+ in the presence of amiloride. 6. These results are consistent with the presence of Na(+)-Mg2+ exchange as an effective Mg(2+)-extruding mechanism in smooth muscle. Although many other factors may cause changes in [Mg2+]i, it seems likely that amiloride directly inhibits the Na(+)-Mg2+ exchanger, whilst ouabain does so indirectly through reduction of the Na+ gradient across the plasma membrane.