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

Danielle M Mcdonald, Chris M. Wood

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16 Citations (Scopus)

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 (K0.5=0. 35±0.01 mmol·l-1; Vmax=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.

Original languageEnglish
Pages (from-to)89-96
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1663
Issue number1-2
DOIs
StatePublished - May 27 2004

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Facilitated Diffusion
Oncorhynchus mykiss
Urea
Membranes
Carrier Proteins
Proteins

Keywords

  • Membrane vesicle
  • Nitrogen excretion
  • Phloretin
  • Teleost
  • Urea analogue
  • UT-A

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Cite this

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title = "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 (K0.5=0. 35±0.01 mmol·l-1; Vmax=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.",
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AU - Mcdonald, Danielle M

AU - Wood, Chris M.

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N2 - 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 (K0.5=0. 35±0.01 mmol·l-1; Vmax=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.

AB - 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 (K0.5=0. 35±0.01 mmol·l-1; Vmax=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.

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KW - Teleost

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