15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells

Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273-281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 mu M 15-[H-3]-HETE or 15-[C-14] HETE for 1-2 h were stimulated with 1 nM to 1 mu M platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 +/- 5% decrease after 1 mu M PAF) without a concomitant release of free intra- or extracellular 15-HETE. C-14-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major C-14 band, identified as sn-1,2-15-[C-14]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes alpha, beta(I), beta(II), delta, epsilon, and zeta. In vitro, cell-generated sn-1,2-15-[C-14] HETE-DG selectively activated immunoprecipitated PKC-alpha and inhibited diacylglycerol-induced activation of PKC-alpha, -delta, -beta(I), and -beta(II) Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.