A Kinetic Method for the Measurement of Zinc Influx In Vivo in the Rainbow Trout, and the Effects of Waterborne Calcium on Flux Rates

Three methods were evaluated to measure rate of influx of Zn into rainbow trout. The first two, disappearance of 65Zn from the water and whole-body counting, overestimated influx when compared with a third method which used a terminal plasma sample to calculate influx. The cause of the overestimate was a short-term adsorption phenomenon to both the experimental apparatus and the exterior of the fish. The third method measured only Zn which entered the fish. This method entailed ‘calibration’ of cannulated trout by constant infusion of small amounts of radiolabelled Zn. This was analogous to the entry of Zn into fish across the gill. After 24–36 h of infusion, plasma radioactivity reached a steadystate concentration which was a simple linear function of the rate of infusion. This relationship was then used to predict influx from a single terminal plasma sample from uncannulated trout exposed to radiolabelled Zn in the water. Trout acclimated to tapwater (Ca2+ = 2.0 mequivl−1) and exposed to Zn (1.5-45.9 μequivl−1; 0.05-1.5 mgl−1) showed saturable uptake which was apparently first order with no significant linear component. The apparent Jmax and Km were 314 nequiv kg−1 h−1 and 7.3 μequivl−1 (0.24 mgl−1), respectively. Acutely raising the waterborne [Ca2+] (4.7 and 9.7 mequivl−1) over the same range of [Zn] revealed a competitive type of interaction - little change in Jmax, with increased Km. When Ca2+ was acutely removed (0.05 and 1.02 mequivl−1) by the use of artificial soft water, significant linear influx occurred in addition to the saturable uptake noted at higher [Ca2+], suggesting the opening of a paracellular leak. Calculation of the inhibitor constant for Ca2+ yielded a value of 0.48 mequivl−1. This value is similar to the Km for Ca2+ when it was a transported substrate (0.28 ± 0.07 mequivl−1). The true Km for Zn transport in the absence of Ca2+ was 1.0 μequivl−1 (0.06 mgl−1). These data showed Zn influx to be saturable and strongly dependent upon waterborne [Ca2+], perhaps traversing the gill in a manner similar to Ca2+.