Basolateral transport of glutarate in proximal S2 segments of rabbit kidney: kinetics of the uptake process and effect of activators of protein kinase A and C.
The kinetics of tubular glutarate uptake, the coupling of glutarate to p-aminohippurate (PAH) transport and the effect of activators of protein kinase A and C on glutarate uptake were studied using isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of [14C]glutarate reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s glutarate uptake measurements were performed. In a first set of experiments it could be shown that preloading proximal S2 segments with glutarate (10(-3 )M) stimulated [3H]PAH uptake indicating that glutarate may be a substrate of the PAH /dicarboxylate exchanger. The kinetic data revealed a Km value of 0. 62 mM and a Vmax value of 84.1 pmol nl-1min-1 for tubular [14C]glutarate uptake across the basolateral cell membrane. In contrast to basolateral PAH transport (previous studies from this laboratory), tubular 30 s [14C]glutarate uptake was not affected by either the phorbol ester phorbol 12-myristate 13-acetate (PMA, 10(-7 )M), an activator of protein kinase C, or by the membrane-permeant analogues of cAMP, dibutyryl cyclic AMP (db-cAMP, 10(-4 )M) and 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP, 10(-4 )M). The results indicate that the protein kinases A and C have no function in the regulation of the renal basolateral dicarboxylate transporter. This finding agrees well with the structural feature of the recently cloned rabbit renal dicarboxylate transporter which does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent kinase.
Röver
N
N
Department of Pharmacology and Toxicology, Rheinisch-Westfälische Technische Hochschule Aachen, Wendlingweg 2, D-52057 Aachen, Germany.
Krämer
C
C
Stärk
U
U
Gabriëls
G
G
Greven
J
J
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D003994:Bucladesine / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005977:Glutarates / Q000378:metabolism* / Q000494:pharmacology; D066298:In Vitro Techniques; D007684:Kidney Tubules / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D010130:p-Aminohippuric Acid / Q000378:metabolism
D005977:Glutarates; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C035736:glutaric acid; D013755:Tetradecanoylphorbol Acetate; D010130:p-Aminohippuric Acid