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Protease-Activated Receptor-2–Mediated Inhibition for Ca²⁺ Response to Lipopolysaccharide in Guinea Pig Tracheal Epithelial Cells

Department of Pharmacology and Department of Internal Medicine 1, Kansai Medical University, Osaka, Japan

Address correspondence to: Chiyoko Inagaki, M.D., Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan. E-mail: inagaki{at}takii.kmu.ac.jp

The protease-activated receptor-2 (PAR-2) has been implicated in airway inflammation. Here, we examined the interaction between PAR-2 and lipopolysaccharide (LPS), a major proinflammatory factor, using cultured guinea pig tracheal epithelial cells. In fura2-loaded cells, LPS (1 µg/ml) transiently increased intracellular Ca²⁺ concentrations ([Ca²⁺]i), this effect being abolished by a Ca²⁺ channel blocker, verapamil, and Ca²⁺ removal. Prestimulation of PAR-2 with trypsin (0.1–1 U/ml) or an agonist peptide (SLIGRL-NH₂, 1 µM) for 60 min inhibited the LPS-induced [Ca²⁺]i increase. Such an inhibitory effect of trypsin was abolished by inhibitors of protein kinase C (PKC), chelerythrine and staurosporine. A PKC activator, phorbol 12,13-dibutylate, also reduced the LPS response. Trypsin also inhibited a transient increase in [Ca²⁺]i caused by a Ca²⁺ channel opener, Bay K 8644. When the trypsin-pretreated cells were incubated in normal buffer for 10–60 min before LPS exposure, the effect of trypsin on the Ca²⁺ response to LPS diminished in a time-dependent manner. Such a recovery was slowed by incubation with a protein phosphatase inhibitor, okadaic acid. Further, trypsin induced sustained activations of PKC and -. Thus, PAR-2 stimulation reduced the epithelial cell response to LPS, probably through the inactivation of Ca²⁺ channels via PKC-mediated phosphorylation.

Abbreviations: 1,4-Dihydro-2, 6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester, (±)-Bay K 8644 • fetal calf serum, FCS • intracellular Ca²⁺ concentration, [Ca²⁺]i • Joklik's modified Eagle's medium, JMEM • lipopolysaccharide, LPS • protease-activated receptor, PAR • phorbol 12, 13-dibutylate, PDBu • cAMP-dependent protein kinase, PKA • protein kinase C, PKC • cGMP-dependent protein kinase, PKG • soybean trypsin inhibitor, SBTI

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