Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.
1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.
Bertog
M
M
University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Letz
B
B
Kong
W
W
Steinhoff
M
M
Higgins
M A
MA
Bielfeld-Ackermann
A
A
Frömter
E
E
Bunnett
N W
NW
Korbmacher
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology
D002712:Chlorides; D017931:DNA Primers; D008024:Ligands; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014357:Trypsin