Copper uptake kinetics across the gills of rainbow trout ( Oncorhynchus mykiss) measured using an improved isolated perfused head technique

Abstract

Measurements of branchial Cu uptake in vivo are complicated by rapid changes in the Cu stores of other body compartments such as the blood and liver. This study eliminates these problems by adopting an in vitro approach. The perfused head technique was improved and viability extended to 2 h or more, so that kinetic studies on Cu uptake could be performed. The improvements included the use of Leibovitz L-15 culture medium instead of saline perfusates, ventilation of the gills with single pass flowing water to allow control of metal speciation, and the use of surgical glue rather than sutures to greatly reduce surgery time. Viability was assessed by perfusion pressure of <50 cm H 2O, demonstration of net Na influx, <7.1 IU ml −1 of lactate dehydrogenase (LDH) activity in the perfusate, and a steady-state ethanol permeability of the gills to indicate the absence of changes in functional gill area. Gill morphology was also checked histologically. Concentrations of Cu >10 μmol l −1 caused only slight effects on some secondary lamellae, but this did not effect gill haemodynamics, gill permeability (ethanol uptake), or perfusate LDH activity when compared to similar measurements in non-viable preparations. Cumulative Cu uptake into the perfusate did not follow changes in perfusate flow and reached a steady state in about 1 h. Copper uptake kinetics based on the total Cu concentration in the water fitted a rectangular hyperbole (the Michaelis equation) with a regression coefficient ( r 2) of 0.98. The K m and V max values were 11.85±2.8 μmol l −1 and 52.2±4.9 nmol kg −1 h −1, respectively (mean±SE, n=5). Copper uptake rate plotted against free divalent Cu (Cu 2+) in the water did not fit the Michaelis equation, but showed second order reaction kinetics ( r 2=0.87) with a rate constant ( k) of 4.43. Copper uptake was abolished by the serosal application of 50 μmol l −1 vanadate. These observations suggest that Cu uptake by the gills is mediated through a vanadate sensitive P-type ATPase which has a similar K m (total Cu) to the mammalian Cu-ATPase. The second order reaction kinetics for Cu 2+ uptake from the water is consistent with the reduction of Cu 2+ to Cu + prior to membrane transport, as observed in mammalian cells.