Na+-DependentL-Arginine Transport in the Pigmented Rabbit Conjunctiva

Abstract

The objective of this study was to characterize Na+-coupledL-arginine (L-Arg) transport in the pigmented rabbit conjunctiva. The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber for measurement of short-circuit current (Isc),3H-L-arginine (3H-L-Arg) flux, and22Na flux.L-Arg when added to the mucosal side led to 0.32–2.65 μA cm−2increases in the Isc at 37°C, but not at 4°C or in a Na+-free solution.L-Arg at 1 mMstimulated net Na+absorption by 0.12 μEq cm−2h−1. The evidence for carrier-mediated transport ofL-Arg includes: (1) temperature dependence and saturability over 0.01–10 mM, (2) Na+dependence and ouabain sensitivity, (3) 84±2% reduction in the apparent permeability coefficient (Papp) of3H-L-Arg in the presence of excess unlabeledL-Arg (1 mM), and (4) 16-fold difference inL-Arg transport (at 0.1 mM) between the mucosal-to-serosal and the serosal-to-mucosal direction. Moreover,L-Arg transport was inhibited by basic amino acids, large neutral amino acids, and nitric oxide synthase inhibitors, but not by acidic and small neutral amino acids. Kinetic analysis revealed the possible existence of both high and low affinity processes forL-Arg transport. A half maximal concentration (Km) and maximalL-Arg flux (Jmax) values of the low and high affinity processes were 5.90 and 0.07 mM, and 1,248 and 111 pmol cm−2min−1, respectively. Hill analysis ofL-Arg transport at 0.1 mMin the presence of varying Na+concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.93, suggesting a 1:1 coupling between Na+andL-Arg. In conclusion, Na+-coupled transport process(es) forL-Arg in accordance with a 1:1 stoichiometry appear to be present on the mucosal side of the pigmented rabbit conjunctiva. The pattern of inhibition by basic and large neutral amino acids and Na+dependency are suggestive of system B0,+-mediatedL-Arg transport.