Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123

Reducing intracellular xenobiotic concentration is an important defence strategy used by cells challenged with foreign chemicals. One mechanism used to achieve this goal is via the use of P-glycoproteins (P-gps), ATP-dependent transporters that mediate the removal of hydrophobic compounds from cells. The energetic costs of this mechanism are unknown, therefore, the activity and respiratory costs associated with the P-gp-mediated efflux of rhodamine 123 (R123) was measured in isolated rainbow trout hepatocytes. The accumulation of R123 was rapid and concentration-dependent. Initial accumulation rates were 1.79 ± 0.41, 7.29 ± 1.06 and 15.30 ± 1.74 ng R123/min/106 cells when exposed to 1, 5 and 10 μM R123, respectively. Efflux was measured in cells 'pre-loaded' with R123 at each concentration, resulting in initial efflux rates of 0.77 ± 0.12, 2.02 ± 0.35 and 3.51 ± 0.84 ng R123/min/106 cells, respectively. The baseline oxygen consumption rate of hepatocytes was 33.21 ± 1.09 ng O2/min/106 cells. Respiration rates were significantly higher in cells exposed to 5 and 10 μM R123 (39.08 ± 0.80 and 41.72 ± 0.61 ng O2/min/106 cells), representing increases over basal rates of 18.5 and 25.7%, respectively. Measurements of isolated mitochondrial respiration established that changes in hepatocyte oxygen consumption were not through the direct effects of R123 on mitochondria. The P-gp inhibitor, XR9576 significantly inhibited R123 efflux from cells with a concomitant return of respiration rates to baseline values. This study demonstrates that increased P-gp transport of xenobiotics can significantly raise cellular respiration rates and may result in higher energy costs for organisms living in P-gp-substrate contaminated environments.