Evidence for facilitated diffusion of urea across the gill basolateral membrane of the rainbow trout ( Oncorhynchus mykiss)

Abstract

Recent in vivo evidence suggests that the mechanism of branchial urea excretion in the ammoniotelic rainbow trout ( Oncorhynchus mykiss) is carrier-mediated. Further characterization of this proposed mechanism was achieved by using an in vitro isolated basolateral membrane vesicle (BLMV) preparation in which isolated gill membranes were used to determine a variety of physiological properties of the transporter. BLMV demonstrated two components of urea uptake, a linear component at concentrations up to 17.5 mmol·l −1 and a saturable component ( K 0.5=0.35±0.01 mmol·l −1; V max=0.14±0.02 μmol mg protein −1 h −1) with a Hill constant of 1.35±0.18 at low, physiologically relevant urea concentrations (<2 mmol·l −1). Saturable uptake of urea at 1 mmol·l −1 by BLMV was reduced by 88.5% when incubated with 0.25 mmol·l −1 phloretin, a potent blocker of UT-type facilitated diffusion urea transport mechanisms. BLMV also demonstrated differential handling of urea versus urea analogues at 1 mmol·l −1 concentrations and total analogue/total urea uptake ratios were 32% for acetamide and 84% for thiourea. Saturable urea uptake at 1 mmol·l −1 was significantly reduced by almost 100% in the presence of 5 mmol·l −1 thiourea but was not affected by 5 mmol·l −1 acetamide or 5 mmol·l −1 N-methylurea. Lastly, total urea uptake at 1 mmol·l −1 by BLMV was sensitive to temperatures above and below the temperature of acclimation with a Q 10>2 suggesting a protein carrier-mediated process. Combined, this evidence indicates that a facilitated diffusion urea transport mechanism is likely present in the basolateral membrane of the rainbow trout gill.