Mg(2+) transport in sheep rumen epithelium: evidence for an electrodiffusive uptake mechanism

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