Regulation of organic cation transport in isolated mouse proximal tubules involves complex changes in protein trafficking and substrate affinity.

Denise Guckel, Giuliano Ciarimboli, Hermann Pavenstädt, Eberhard Schlatter

Index: Cell Physiol. Biochem. 30(1) , 269-81, (2012)

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Abstract

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.Copyright © 2012 S. Karger AG, Basel.