Human and rat multidrug resistance-associated proteins (MRP/Mrp)

The multidrug resistance associated proteins (MRP(human)Mrp (rat) are ATP-dependent transporters responsible for the efflux of a wide range of substrates, including endogenous compounds e.g. bilirubin, drug metabolites e.g. paracetamol glucuronide and fluorescent dyes e.g. 5 (and 6)-carboxy-2’,7’-dichlorofluorescein (CDF). Mrp1-6 (abccl-6) are expressed in rat liver, with Mrp2 being expressed at the highest level. Isolation of hepatocytes by in situ collagenase perfusion causes bile canalicular disruption and internalisation of Mrp2. Cells cultured in a sandwich configuration of Matrigel or collagen (Type 1) showed bile canalicular reformation at different days in cell culture, depending on the extracellular matrix and time of overlay. We have developed a method for quantifying Mrp-mediated efflux in hepatocytes cultured in a sandwich configuration of collagen (Type 1). This method is unique in its ability to distinguish between sinusoidal efflux, canalicular efflux and diffusion in intact hepatocytes. Alternative in vitro models of Mrp2-mediated efflux include the vesicular (direct and indirect methods) and the ATPase assays. We have used these assays to identify atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin as substrates of human and rat MRP2/Mrp2. A close correlation was seen between the kinetic parameters of transport of the Mrp2 substrate; CDF determined in sandwich cultured rat hepatocytes using the method above (Km = 3.5 – 9.9 μM), vesicle preparations (Km = 37.9 μM) and membrane preparations (Km = 18.7 μM). We also present data to implicate the nuclear receptors, PXR, CAR and FXR in the regulation of abcc2 and abcc3 and PXR and CAR in abcc1 gene regulation. Abcc2 and abcc5 are also up-regulated in response to a toxic insult to the cell, probably via Nrf2 activation, suggesting a role in cell defence.EThOS - Electronic Theses Online ServiceGBUnited Kingdo