TY - JOUR
T1 - Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation
AU - Palmer, Melissa L.
AU - So, Yeong Lee
AU - Maniak, Peter J.
AU - Carlson, Dan
AU - Fahrenkrug, Scott C.
AU - O'Grady, Scott M.
PY - 2006/4
Y1 - 2006/4
N2 - Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl- secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (Isc: thrombin, 21 ± 2 μA; SLIGRL, 83 ± 22 μA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 μM) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 μM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE2 receptor antagonist AH-6809 (25 μM) significantly inhibited the effects of SLIGRL on Isc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca2+-activated KCNN4 K+ channel, and the KCNQ1 K+ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl- conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl- secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl- secretion requires activation of CFTR and at least two distinct K+ channels located in the basolateral membrane.
AB - Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl- secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (Isc: thrombin, 21 ± 2 μA; SLIGRL, 83 ± 22 μA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 μM) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 μM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE2 receptor antagonist AH-6809 (25 μM) significantly inhibited the effects of SLIGRL on Isc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca2+-activated KCNN4 K+ channel, and the KCNQ1 K+ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl- conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl- secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl- secretion requires activation of CFTR and at least two distinct K+ channels located in the basolateral membrane.
KW - Calciumactivated potassium channels
KW - Cystic fibrosis transmembrane conductance regulator
KW - KCNN4
KW - KCNQ1
KW - cAMP
UR - http://www.scopus.com/inward/record.url?scp=33646394610&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646394610&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00464.2005
DO - 10.1152/ajpcell.00464.2005
M3 - Article
C2 - 16531569
AN - SCOPUS:33646394610
SN - 0363-6143
VL - 290
SP - C1189-C1198
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 4
ER -