Arachidonic acid directly activates members of the mitogen-activated protein kinase superfamily in rabbit proximal tubule cells.
BACKGROUND
To explore the roles of eicosanoids in arachidonic acid-induced mitogen-activated protein kinase (MAPK) signal transduction, we have shown that exposure of proximal tubular cells to arachidonic acid induces phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two members of the MAPK superfamily. We observed that ketoconazole, an inhibitor of the cytochrome P450 pathway, blocked ERK but not JNK activation.


METHODS
Direct regulation of arachidonic acid on mitogen-activated protein kinase (MAPK) signaling pathways was evaluated more directly by utilizing specific enzyme inhibitors of the cytochrome P450 metabolic pathway and by comparing the relative efficacy of arachidonic acid versus its cytochrome P450 metabolites (exogenous and endogenous), eicosatetraynoic acid (ETYA), and other fatty acids on the phosphorylation of members of the MAPK superfamily (ERKs, JNK, and p38(MAPK)), by utilizing early passage rabbit proximal tubular epithelial cells.


RESULTS
Arachidonic acid activated p38(MAPK), a third member of the MAPK superfamily, in a time- and concentration-dependent manner. Studies designed to evaluate the ability of arachidonic acid and its cytochrome P450 metabolites (endogenously and exogenously) to stimulate ERKs, JNK, and p38(MAPK) found four conclusions. First, the metabolites of arachidonic acid generated endogenously by cytochrome P450 2C1 significantly augmented basal ERK activity, whereas the metabolites generated by the 2C2 isozyme significantly augmented basal p38(MAPK) activity. However, their effects were less profound than arachidonic acid itself. In contrast, there were no significant effects with transfection of either isozyme on basal JNK activity. Second, a variety of exogenous cytochrome P450 products were less potent than arachidonic acid on a molar basis in stimulating the activity of all three MAPKs. Third, ketoconazole and 17-octadecynoic acid, inhibitors of the cytochrome P450 pathway, as well as PPOH and DDMS, inhibitors of the epoxygenase and omega-hydroxylase pathways, respectively, failed to significantly reduce the effects of arachidonic acid to activate ERK and p38(MAPK) (JNK was not evaluated). Finally, arachidonic acid, its inactive analog ETYA, and other fatty acids with differing chain lengths and degrees of saturation stimulated the activity of all three MAPKs.


CONCLUSIONS
These observations substantiate a role for arachidonic acid and other fatty acids in signaling linked to the MAPK superfamily in rabbit proximal tubular epithelium without the necessity of conversion to cytochrome P450 metabolites.
Alexander
L D
LD
Department of Medicine, Division of Hypertension, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106-4982, USA.
Cui
X L
XL
Falck
J R
JR
Douglas
J G
JG
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D016718:Arachidonic Acid / Q000032:analysis / Q000493:pharmacokinetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005227:Fatty Acids / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D007654:Ketoconazole / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D048051:p38 Mitogen-Activated Protein Kinases
D000935:Antifungal Agents; D002250:Carbon Radioisotopes; D005227:Fatty Acids; D015117:5,8,11,14-Eicosatetraynoic Acid; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020929:Mitogen-Activated Protein Kinase Kinases; D007654:Ketoconazole