20-HYDROXYEICOSATETRAENOIC ACID IS FORMED IN RESPONSE TO EGF AND IS AMITOGEN IN RAT PROXIMAL TUBULE

20-Hydroxyeicosatetraenoic acid (20-HETE) is a major cytochrome P-450- arachidonic acid metabolite in the rat kidney, and its synthesis along the nephron is specifically localized to the proximal tubule, where r eceptor density for epidermal growth factor (EGF) is the highest. EGF stimulated endogenous 20-HETE formation in a concentration and time-de pendent manner, i.e., from 1.6 to 2.6 +/- 0.3 and 3.0 +/- 0.6 pmol 20- HETE . mg(-1). min(-1) at 10(-8) and 10(-7) M EGF, respectively. The e ffect of 20-HETE on proximal tubular cell proliferation was examined u sing primary cultures of rat proximal tubular cells and proximal tubul ar-derived cell lines, LLC-PK1 and opossum kidney OK. In both cell lin es, 20-HETE increased thymidine incorporation into DNA with maximal ef fect at 10(-9) M. Addition of 20-HETE to serum-deprived LLC-PK1 or OK cells for 48 h caused a concentration-dependent increase in cell numbe r with maximal effect at 10(-9) M. This effect was specific, as struct urally similar eicosanoids such as 20-COOH-arachidonic acid, 19(R)-HET E, and 19(S)-HETE did not increase cell number. In 4-day primary cultu res of proximal tubular cells, EGF (10(-9) M) and 20-HETE (10(-9) M) i ncreased bromodeoxyuridine (BrdU) incorporation by 40 and 28%, respect ively. Addition of both resulted in a twofold increase in BrdU incorpo ration. Although 20-HETE synthesis in cultured cells is greatly dimini shed with time,significant picomolar concentrations can be obtained in 4-day cultures. Addition of 17-octadecynoic acid (17-ODYA), an inhibi tor of 20-HETE synthesis, significantly inhibited EGF-stimulated BrdU incorporation. The demonstrations that EGF stimulates proximal tubular 20-HETE production and that the latter is a potent mitogen to these c ells suggests that 20-HETE may act as a mediator of the EGF effect on cellular growth in the proximal tubule.