Mg2+ transport in sheep rumen epithelium:: evidence for an electrodiffusive uptake mechanism

The potential difference (PD)-dependent component of transcellular Mg2+ uptake in sheep rumen epithelium was studied. Unidirectional Mg-28(2+) fluxes were measured at various transepithelial PD values, and the unidirectional mucosal-to-serosal Mg-28(2+) flux (J(ms)(Mg)) was correlated with the PD across the apical membrane (PDa) determined by mucosal impalement with microelectrodes. PDa was found to be -54 +/- 5 mV, and J(ms)(Mg) was 65.9 +/- 13.8 nmol.cm(-2).h(-1) under short-circuit conditions. Hyperpolarization of the ruminal epithelium (blood-side positive) depolarized PDa and, mast noticeably, decreased J(ms)(Mg). Further experiments were performed with cultured ruminal epithelial cells (REC). With the aid of the fluorescence probe mag-fura 2, we measured the intracellular free Mg2+ concentration (Mg2+](i)) of isolated REC under basal conditions at various extracellular Mg2+ concentrations ([Mg2+](e)) and after alterations of the transmembrane voltage. Basal [Mg2+](i) was 0.54 +/- 0.08 mM. REC suspended in media with [Mg2+](i) between 0.5 and 7.5 mM showed an increase in [Mg2+](i) that was dependent on [Mg2+](i) and that exhibited a saturable component (Michaelis-Menten constant = 1.2 mM; maximum [Mg2+](i) = 1.26 mM). Membrane depolarization with high extracellular K+ (40, 80, or 140 mM K+) and the K+ channel blocker quinidine (50 and 100 mu M) resulted in a decrease in [Mg2+](i). On the other hand, hyperpolarizatian created by K+ diffusion (intracellular K+ concentration > extracellular K+ concentration) in the presence of valinomycin induced a 15% increase in [Mg2+](i). None of the manipulations had any effect on intracellular Ca2+ concentration and intracellular pH. The results support the assumption that the membrane potential acts as a principal driving force for Mg2+ entry in REC and suggest that the pathway for this electrodiffusive Mg2+ uptake across the luminal membrane is a channel or a carrier.