Regulation of Organic Cation Transport in Isolated Mouse Proximal Tubules Involves Complex Changes in Protein Trafficking and Substrate Affinity

This study characterizes the complex mechanisms of acute regulation of organic cation (OC) transport across the basolateral membrane of isolated mouse proximal tubules. The fluorescent substrate ASP(+), 4-(-4-(dimethylamino) styryl-N-methylpyridinium, was used to quantify OC transport using a microtiter plate based fluorescence reader method. Inhibition of phosphatidylinositol-3-kinase, of p56 tyrosine kinase, stimulation of PKC and inhibition of PKA reduced ASP(+)-uptake. ASP(+)-kinetic and Dixon plot analyses revealed effects on transporter trafficking as explanation for the inhibition of ASP(+)-uptake by these pathways. Angiotensin II (AII) via stimulation of Ca(2+)/calmodulin increased ASP(+)-uptake. This effect aroused from an altered substrate affinity. Bafilomycin, an inhibitor of the vacuolar H(+)-ATPase and thus endosomal and lysosomal function, reduced ASP(+)-uptake, but did not prevent the AII effect on ASP(+)-uptake. Bafilomycin seemed to diminish the recycling rate of OCTs and hence to reduce the amount of transporters in the membrane. AII via Ca(2+)/calmodulin increased the substrate affinity of the remaining OCTs. The involvement of the cytoskeleton in acute regulation of OCTs became obvious as colchicine induced inhibition of microtubule polymerisation reduced ASP(+)-uptake. Acute regulation of mouse OCTs mostly involves changes in trafficking from and to the plasma membrane and only in the case of AII/CaM changes in substrate affinity.