Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation.
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 (I(sc): thrombin, 21 +/- 2 microA; SLIGRL, 83 +/- 22 microA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca(2+)-chelating agent BAPTA-AM (50 microM) abolished the increase in I(sc) produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 microM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I(sc) was observed. In addition, basolateral treatment with the PGE(2) receptor antagonist AH-6809 (25 microM) significantly inhibited the effects of SLIGRL on I(sc). QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca(2+)-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.
Palmer
Melissa L
ML
Department of Physiology, 495 Animal Science/Veterinary Medicine Bldg., 1988 Fitch Ave., University of Minnesota, St. Paul, 55110, USA.
Lee
So Yeong
SY
Maniak
Peter J
PJ
Carlson
Dan
D
Fahrenkrug
Scott C
SC
O'Grady
Scott M
SM
D002460:Cell Line; D002614:Chelating Agents / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology*; D013917:Thrombin / Q000378:metabolism
C505032:CFTR protein, human; D002614:Chelating Agents; D002712:Chlorides; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D051657:KCNQ1 Potassium Channel; D009842:Oligopeptides; D011453:Prostaglandins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D044462:Receptors, Proteinase-Activated; C099570:seryl-leucyl-isoleucyl-glycyl-arginyl-leucine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D013917:Thrombin; D015232:Dinoprostone