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.

Effect of activation of protein kinase A and of protein kinase C on the kinetics of the renal basolateral PAH transporter.

The aim of the present study was to examine the effect of activation of the protein kinase A (PKA) and protein kinase C (PKC) pathways on 3H-p-aminohippurate (PAH) uptake of 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 3H-PAH reflects transport across the basolateral membrane. The phorbol ester phorbol 12-myristate 13-acetate (PMA) (10(-7) M), an activator of PKC, significantly increased tubular 3H-PAH uptake with steady state conditions (by 115%), whereas dibutyryl cyclic adenosine monophosphate (db-cAMP) (10(-4) M) and forskolin (10(-4) M) significantly inhibited it (by 42% and 52%, respectively). Kinetic data, which were based on 15 sec PAH uptake measurements, revealed that PMA, after a 10 min incubation period, significantly enhanced Km and Vmax of the PAH transporter (Km from 174 +/- 22 to 447 +/- 91 microM, Vmax from 2.76 +/- 0.24 to 16.67 +/- 1.85 pmol nL-1 min-1), whereas db-cAMP significantly decreased Vmax (from 2.76 +/- 0.24 to 1.82 +/- 0.19 pmol nL-1 min-1). The Km value was also numerically lowered by dibutyryl-cAMP (from 174 +/- 22 to 139 +/- 21 microM), but this change did not reach statistical significance. The data provide evidence that short time activation of the PKC pathway 1) enhances the effectiveness of PAH transport into proximal S2 segments across the basolateral cell membrane, 2) increases the maximum transport rate of the PAH transporter and 3) decreases its affinity for PAH. Activation of the cAMP/PKA pathway induces the opposite effects.