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

Abstract

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 microg/ml) transiently increased intracellular Ca(2+) concentrations ([Ca(2+)]i), this effect being abolished by a Ca(2+) channel blocker, verapamil, and Ca(2+) removal. Prestimulation of PAR-2 with trypsin (0.1-1 U/ml) or an agonist peptide (SLIGRL-NH(2), 1 microM) for 60 min inhibited the LPS-induced [Ca(2+)]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 [Ca(2+)]i caused by a Ca(2+) 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 Ca(2+) 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 PKCalpha and -epsilon. Thus, PAR-2 stimulation reduced the epithelial cell response to LPS, probably through the inactivation of Ca(2+) channels via PKC-mediated phosphorylation.

Involvement of Protein Kinase C and RhoA in Protease-Activated Receptor 1–Mediated F-Actin Reorganization and Cell Growth in Rat Cardiomyocytes

“…It is possible that cytochalasin D inhibited hypertrophic cell growth through another mechanism such as reduction of glucose uptake resulting from inhibition of actin polymerization because actin organization has been demonstrated to regulate glucose uptake by modulating glucose transporter trafficking (27). In previous reports using airway epithelial cells, we showed that PAR regulates cytoskeletal structure as well as cell function such as neutrophil adhesion through PKC-and/or Rho-dependent signaling (28,29). Furthermore, it was demonstrated that PKC and RhoA act in concert as mediators of actin cytoskeleton organization (18,30).…”

Lipopolysaccharide hyperpolarizes guinea pig airway epithelium by increasing the activities of the epithelial Na+ channel and the Na+-K+ pump

“…Trypsin is an agonist of PARs, which stimulate an increase in the intracellular concentration of Ca 2ϩ and PKC activation. The activation of PAR receptors has been found to inhibit the Ca 2ϩ response of guinea pig tracheal epithelial cells in culture to LPS (45). Small concentrations of trypsin (1-10 ng/ml) below those that cleave ENaC directly were found to activate ENaC via an indirect, receptor-mediated mechanism (3).…”

A Novel TLR4-Mediated Signaling Pathway Leading to IL-6 Responses in Human Bladder Epithelial Cells

“…For a long time it was assumed that PRRs were exclusively found on immune cells of hematopoietic origin and, therefore, most of our current information regarding PRR-mediated signal transduction is largely based on these cells [1,47]. Although there is no conclusive data suggesting cell-specific TLR4 signaling, there have been suggestions that LPSs evoke intracellular signaling reactions in Kupffer cells [48] and tracheal epithelial cells [49] that are absent in polymorphonuclear leukocytes [50]. Here, we report the existence of a distinct TLR4-mediated signaling pathway leading to IL-6 secretion that is present in BECs but absent in other human cell types.…”