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Am J Physiol Lung Cell Mol Physiol 277: L457-L464, 1999;
1040-0605/99 $5.00
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Vol. 277, Issue 3, L457-L464, September 1999

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

Stephen E. Alpert1, Ronald W. Walenga1, Atashi Mandal1, Nicole Bourbon2, and Mark Kester2

1 Pediatric Pulmonary Division, Case Western Reserve University, Cleveland, Ohio 44106; and 2 Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033

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 µM 15-[3H]-HETE or 15-[14C]HETE for 1-2 h were stimulated with 1 nM to 1 µM 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 µM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]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-[14C]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.

15-hydroxyeicosatetraenoic acid; protein kinase C; human airway epithelial cells; signal transduction; monohydroxy-substituted diacylglycerols


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