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

Cellular Physiology and Biochemistry

关键词: Substrate affinities;    Proximal tubule;    Microfluorimetry;    Protein kinases;    Organic cation transport;    Trafficking;   

DOI  :  10.1159/000339063

学科分类：分子生物学，细胞生物学和基因

来源: S Karger AG

【 摘 要 】

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 Ca²⁺/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 Ca²⁺/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.

【 授权许可】

CC BY-NC-ND