Short-term regulation of basolateral organic anion uptake in proximal tubular OK cells: EGF acts via MAPK, PLA(2), and COX1.
The organic anion transport system of the kidney is of major importance for the excretion of a variety of endogenous compounds, drugs, and potentially toxic substances. The basolateral uptake into proximal tubular cells is mediated by a tertiary active transport system. Epidermal growth factor (EGF) leads to an increase in the basolateral uptake rate of the model substrate para-aminohippuric acid (PAH) in opossum kidney (OK) cells. This stimulation is mediated by successive activation of the mitogen-activated protein kinases,mitogen-activated/extracellular signal-regulated kinase kinase (MEK) and extracellular regulated kinase isoforms 1 and 2 (ERK1/2). This study investigates the regulatory network of EGF action on PAH uptake downstream ERK1/2 in more detail. EGF stimulation of the basolateral uptake rate of [(14)C]PAH was abolished by the phospholipase A(2) inhibitor AACOCF3.[(14)C]PAH uptake was enhanced by arachidonic acid. Furthermore, EGF led to an increase in arachidonic acid release and to the generation of prostaglandins. AACOCF3 did not influence EGF-induced ERK1/2 activation, indicating that ERK1/2 is upstream of PLA(2). In addition, EGF stimulated the influx of extracellular Ca(2+). However, Ca(2+)-influx was not required for the stimulatory action of EGF on [(14)C]PAH uptake. Inhibitors of COX and lipoxygenases reduced [(14)C]PAH uptake dose-dependently, whereas inhibition of cytochrome P450 did not. In the presence of indomethacin, EGF had no stimulatory effect on [(14)C]PAH uptake. The inhibitory effect of indomethacin was not due to competitive action on PAH uptake. Furthermore, prostaglandin E(2) (PGE(2)) increased basolateral [(14)C]PAH uptake rate dose-dependently, and this increase was also observed in the presence of indomethacin. Selective inhibition of COX2 by indomethacin amid or indomethacin n-heptyl ester did not inhibit [(14)C]PAH uptake, whereas selective inhibition of COX1 dose-dependently inhibited [(14)C]PAH uptake. This and previous data lead to the conclusion that EGF successively activates MEK, ERK1/2, and PLA(2), leading to an increased release of arachidonic acid. Subsequently, arachidonic acid is metabolized to prostaglandins via COX1, which then mediate EGF-induced stimulation of basolateral organic anion uptake rate.
Physiologisches Institut der Universität Würzburg, Würzburg, Germany. christoph.sauvant@mail.uni-wuerzburg.de
Christoph
C
Sauvant
Hildegard
H
Holzinger
Michael
M
Gekle
D000818:Animals; D016718:Arachidonic Acid / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D051545:Cyclooxygenase 1; D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology / Q000502:physiology*; D005110:Extracellular Space / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism* / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009893:Opossums; D027361:Organic Anion Transporters / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism* / Q000502:physiology; D011453:Prostaglandins / Q000096:biosynthesis; D013997:Time Factors; D010130:p-Aminohippuric Acid / Q000378:metabolism
D007527:Isoenzymes; D027361:Organic Anion Transporters; D011453:Prostaglandins; D016718:Arachidonic Acid; D004815:Epidermal Growth Factor; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D015232:Dinoprostone; D002118:Calcium; D010130:p-Aminohippuric Acid