The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin alpha3beta3gamma2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC).
Protease Activated Receptor Type 2 (PAR2) is known to play an important role in inflammatory, visceral and cancer-evoked pain based on studies using PAR2 knockout (PAR2) mice. Here we have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state via extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF) / tropomyosin related kinase B (trkB) / atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel, highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (ED50 ∼ 12 pmoles), facial grimacing and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The pro-mechanical hypersensitivity effect of 2-at is completely absent in PAR2 mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126 and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming following 2-at injection. Systemic injection of the trkB antagonist ANA-12 likewise inhibited PAR2-mediated mechanical hypersensitivity, grimacing and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.
Myoendothelial junctions (MEJs) represent a specialized signaling domain between vascular smooth muscle cells (VSMC) and endothelial cells (EC). The functional consequences of phosphorylation state of the connexins (Cx) at the MEJ have not been explored.
