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Control of ion transport in mammalian airways by protease activated receptors type 2 (PAR-2)

Karl Kunzelmann^*,1, Jane Sun, Daniel Markovich, Jens König, Bettina Mürle, Marcus Mall and Rainer Schreiber^*

^* Institut für Physiologie, Universität Regensburg, Regensburg, Germany;
School of Biomedical Sciences, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Brisbane, Australia; and
Mukoviszidose-Zentrum, Universitäts-Kinderklinik III, Heidelberg, Germany

¹Correspondence: E-mail: uqkkunze{at}mailbox.uq.edu.au

SPECIFIC AIMS

PROTEASE activated receptors type 2 (PAR-2) are expressed abundantly in the respiratory tract. However, virtually nothing is known about their role for the fluid transport in native airways. This prompted us to examine the role of PAR-2 for electrolyte transport in the respiratory tract and to unmask the underlying second messenger pathways and contributing ion conductances.

PRINCIPAL FINDINGS

1. PAR-2 are expressed in airway epithelial cells, where they are activated by mast cell tryptase and neutrophil proteinase 3 or trypsin
Although PAR-2 appear to be expressed predominantly on the apical side of the epithelium, only basolateral stimulation results in inhibition of amiloride sensitive Na⁺ conductance and stimulation of both luminal Cl^– channels and basolateral K⁺ channels (Fig. 1 ).

View larger version (15K): [in this window] [in a new window]   Figure 1. Activation of ion transport in mouse airways due to stimulation of PAR-2. Summary of the effects of luminal and basolateral application of activating (AP) and reverse (RP) peptides (both 100 µmol/L) on short circuit currents (Isc). *Significant difference compared with control (paired t test).

2. The presents data indicate that these changes occur through activation of phospholipase C and increase of intracellular Ca²⁺, which activates basolateral SK4 K⁺ channels and luminal Ca²⁺-dependent Cl^– channels

3. The secretory response in tracheas upon stimulation of PAR-2 was comparable for wild-type (WT) mice, CF mice homozygous for the severe CFTR mutation G551D (cftr^G551D/G551D), and bitransgenic cftr^G551D/G551D K18-GFP-CFTR^+/– (G551D/CFTR) animals

4. Evidence is presented that stimulation of PAR-2 in the airways mediates release of prostaglandin E2, which contributes to the secretory response by increasing intracellular cAMP in columnar epithelial cells, thus activating luminal CFTR Cl^–channels and basolateral KCNQ1 K⁺ channels

5. Ion transport was activated by trypsin, tryptase, and PAR-2-activating peptide and was independent of simultaneous application of tetrodotoxin, indicating stimulation of electrolyte secretion by direct binding to epithelial PAR-2 receptors

CONCLUSIONS AND SIGNIFICANCE

Taken together, the present data show multiple effects on airway electrolyte transport by stimulation of PAR-2 receptors (Fig. 2 ). The results provide further evidence for a role of PAR-2 in inflammatory airway disease. PAR-2 may participate in the hypersecretion and bronchorrhea observed in asthma and other inflammatory diseases such as bronchitis. Stimulation of PAR-2 in columnar epithelial cells induces electrolyte secretion and inhibition of electrogenic Na⁺ absorption. PAR-2 expressed on basal cells of the airway epithelium may be in charge of PGE₂ release, indirectly contributing to electrolyte secretion. Activation of electrolyte secretion and inhibition of absorption may have a protective rather than a proinflammatory function. The bronchodilation and thus protective role of PAR-2 may be accomplished by a switch of epithelial transport from absorption toward secretion, which will increase airway surface liquid. This will improve mucociliary clearance and thus may flush noxious stimuli and bacteria away from the affected area.

View larger version (15K): [in this window] [in a new window]   Figure 2. Continuous original recording of the transepithelial voltage (Vte) in mouse trachea in a perfused micro-Ussing chamber. Effects of amiloride (A, 10 µmol/L) before and after stimulation by basolateral application of trypsin (1 µmol/L).

View larger version (57K): [in this window] [in a new window]   Figure 3. Model for the effects of PAR-2 activation in airways. Autocrine release of activators of PAR-2 from airway cells (trypsin, proteinase 3) and inflammatory cells (tryptase, proteinase 3). Activation of PAR-2 in columnar epithelial cells leads to electrolyte secretion, whereas binding to PAR-2 in basal cells leads to release of PGE2, which binds to columnar cells and thus further enhances Cl– secretion. PGE2 binding to airway smooth muscle cells leads to relaxation, whereas activation of PAR-2 leads to contraction. Increase in electrolyte secretion improves mucociliary clearance and flushing of the airways by enlarging the airway surface liquid. The insert shows ion conductances activated in columnar epithelial cells during parallel PAR-2 and PGE2 stimulation. Second messengers Ca2+ and cAMP are increased, which activates luminal Ca2+ and cAMP-dependent Cl– channels and basolateral Ca2+ and cAMP-dependent K+ channels. Whereas NaCl secretion is induced, Na+ absorption by ENaC is inhibited.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2469fje; doi: 10.1096/fj.04-2469fje

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