RESUMO
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.
