Component characteristics of the vectorial transport system for taurine in isolated bovine retinal pigment epithelium

1. A wide rang of substrate concentrations (5-1600 mu M) were used to screen for the presence of systems capable of transporting taurine into isolated and free-floating samples of bovine retinal pigment epithelium (RPE). Both high and low affinity systems displaying Michaelis-Menten saturation kinetics were identified. The high affinity system was characterized by a K-m of 23 mu M and a V-max of 86.7 pmol (5 min)(-1) (4 mm disc of tissue sample)(-1). Similarly, the low affinity system was characterized by a K-m of 507 mu M and a V-max of 344 pmol (5 min)(-1) (4 mm disc)(-1). 2. Ussing-type incubation chambers and double-label radiotracer techniques were used to assess the presence of specific taurine carriers on apical and basolateral surfaces of the RPE. High affinity carriers were shown to be present on both surfaces and the kinetic constants (K-m and V-max) for apical and basolateral systems were determined as 23.2 mu M and 34.8 pmol (5 min)(-1) (4 mm disc)(-1) and 29 mu M and 54.7 pmol (5 min)(-1) (4 mm disc)(-1), respectively. Both these high affinity systems were sodium dependent with a Hill coefficient of about 2.0 indicating that two sodium ions are required for the translocation of one molecule of taurine. The low affinity system was unevenly distributed over the two surfaces of the RPE, basolateral capacities being roughly twofold higher. The basolateral system was totally insensitive to sodium whereas the apical one with 50% sodium sensitivity suggested the presence of low affinity carrier heterogeneity. 3. A temperature-dependent mechanism for the release of pre-loaded taurine from bovine RPE was also demonstrated. 4. The effect of [K+](o) trans-RPE gradients on the vectorial transport of taurine across the isolated preparation was also investigated. The results demonstrated that the direction and magnitude of taurine transport could be controlled by physiological variations in the extracellular concentration of potassium. 5. The determined kinetic parameters of the carriers were used to construct a working model of the vectorial pathway for the translocation of taurine across bovine RPE. This estimated the level of free intracellular taurine to be in the micromolar range. However, direct measurements of total RPE taurine by high performance liquid chromatography showed levels of 19.5 +/- 3.6 mM indicating that the major taurine pool in bovine RPE exists in the bound form. The model also showed that the magnitude and direction of net transport was a function of the transepithelial taurine gradient.