Y Miyamoto

Na+ + Cl−-gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles

Uptake of taurine in human placental brush‐border membrane vesicles was greatly stimulated in the presence of an inwardly‐directed Na+ + Cl−‐gradient and uphill transport of taurine could be demonstrated under these conditions. Na+ as well as Cl− were obligatory for this uptake and both ion gradients could energize the uphill transport. This Na+ + Cl−‐gradient‐dependent taurine uptake was stimulated by an inside‐negative membrane potential, demonstrating the electrogenicity of the process. The uptake system was highly specific for β‐amino acids and the K m of the system for taurine was 6.5 ± 0.4 μM.

Fatty acid-induced alterations in transport systems of the small intestinal brush-border membrane

We have investigated the effects of fatty acids on the Na+-H+ exchanger and other carrier-mediated transport systems in intestinal brush-border membrane vesicles. The Na+-H+ exchanger (i.e. H+ gradient-dependent, dimethylamiloride-sensitive Na+ uptake) was strongly inhibited by fatty acids and the inhibition was concentration dependent. Unsaturated fatty acids showed more inhibition than saturated fatty acids. Among unsaturated fatty acids, ricinoleic acid was found to be the most potent inhibitor. Inhibition of the Na+-H+ exchanger by oleic acid was partially reversible, and the nature of the inhibition was found to be non-competitive with respect to Na+. The dimethylamiloride-sensitive Na+ uptake measured in the absence of an H+ gradient was also inhibited by oleic acid, suggesting that the inhibition of the Na+-H+ exchanger by fatty acids was not due to the accelerated dissipation of the H+ gradient. Treatment of the membrane vesicles with oleic acid also inhibited other carrier-mediated transport systems as well, such as the H+ gradient-driven transport of glycylsarcosine and the Na+ gradient-driven transport of D-glucose and L-alanine, whereas it did not affect the permeability of L-glucose, a non-carrier-mediated process. However, the inhibitory effects of oleic acid on the transport of D-glucose and L-alanine appeared to be related to the enhanced collapse of the Na+ gradient rather than a direct effect on the carrier systems because transport of these solutes when measured in the absence of a Na+ gradient ([Na+]i = [Na+]o) was not affected by oleic acid. These data demonstrate that fatty acids bring about significant alterations in the activities of various transport systems of the small intestinal brush-border membrane, either by directly interacting with the transport protein or by abolishing the energy source that is necessary for the transport process.

Properties of taurine transport in a human retinal pigment epithelial cell line

The characteristics of taurine transport were studied in a human retinal pigment epithelial cell line (HRPE). Uptake of taurine into monolayer cultures of the HRPE cells was markedly stimulated by the presence of NaCl in the uptake medium whereas the uptake was negligible in its absence. This NaCl-dependent uptake was an active process as the cells were able to accumulate taurine against a concentration gradient. The uptake rate of taurine was found to be many-fold greater than that of gamma-aminobutyric acid (GABA). Unlabeled taurine and GABA competed with radiolabeled taurine for the uptake process, the former being more effective than the latter. However, uptake of radiolabeled GABA was not affected by unlabeled taurine and GABA. Substrate specificity studies revealed strong interaction of beta-amino acids with the transport system responsible for taurine uptake. alpha-Amino acids failed to inhibit taurine uptake. A specific anion requirement was observed for optimal activity of the taurine transport system and Cl- was the most supportive among several anions tested. Kinetic analyses showed that multiple Na+ and one Cl- were involved in transfer of one taurine molecule. The transport process consisted of a single saturable system with a Michaelis-Menten constant of 2.0 +/- 0.1 microM. These results show that the HRPE cell line expresses a high-affinity taurine transport system. This is the first demonstration of the presence of the taurine transporter in the human retinal pigment epithelium and the HRPE cell line may provide a useful model system for future studies involving taurine transport in the retinal pigment epithelium.

Proton gradient-coupled uphill transport of glycylsarcosine in rabbit renal brush-border membrane vesicles

An inward-directed proton gradient energizes the transport of intact glycylsarcosine against a concentration gradient in rabbit renal brush-border membrane vesicles. Dissipation of the proton gradient abolishes the uphill transport. Generation of an inside-negative membrane potential nearly doubles the intravesicular concentration of the dipeptide at the peak of the overshoot without altering the equilibrium value. These data provide direct evidence for peptide-proton cotransport in the renal brush-border membrane.

Inactivation of taurine transporter by calcium in purified human placental brush border membrane vesicles

We investigated the effects of calcium on the activity of the taurine transporter in purified human placental brush border membrane vesicles. Treatment of the membrane vesicles with calcium markedly inhibited taurine uptake. The magnitude of inhibition was dependent on the calcium concentration and the treatment time. Free ionized Ca2+ was responsible for this effect because EGTA, a Ca2+ chelator, totally abolished the calcium-induced effect. Uptake of succinate, which occurs via a Na(+)-dependent process as does the uptake of taurine, was reduced only to a small extent by the calcium treatment. This result indicates that the effect of Ca2+ on taurine uptake was not due to an accelerated dissipation of the Na+ gradient as a result of an increased Na+ permeability of the membrane. Preloading the vesicles with phospholipase inhibitors such as neomycin and quinacrine significantly protected the taurine transporter from the Ca2+ effect, raising the possibility that Ca(2+)-activated phospholipases may mediate the Ca2+ effect. Kinetic analysis revealed that Ca2+ decreased the affinity of the transporter for taurine as well as the translocation rate of the taurine-loaded transporter complex.