title: Regulation of organic cation transport in isolated mouse proximal tubules involves complex changes in protein trafficking and substrate affinity.
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.
authors: Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universität Münster, Münster, Germany.
authors: Denise
authors: D
authors: Guckel
authors: Giuliano
authors: G
authors: Ciarimboli
authors: Hermann
authors: H
authors: Pavenstädt
authors: Eberhard
authors: E
authors: Schlatter
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000502:physiology; D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002927:Cimetidine / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D027701:Organic Cation Transport Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011726:Pyridinium Compounds / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D050257:Tubulin Modulators / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000804:Angiotensin II; D002147:Calmodulin; D002927:Cimetidine; D003078:Colchicine; D017868:Cyclic AMP-Dependent Protein Kinases; D005456:Fluorescent Dyes; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D027701:Organic Cation Transport Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D011726:Pyridinium Compounds; D050257:Tubulin Modulators; D000077191:Wortmannin; C052247:4-(4-diethylaminostyryl)-N-methylpyridinium