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 confir med renal expression of proteinase-activated receptor (PAR-2) and demonstra ted its presence in native renal epithelial and in cultured M-1. mouse cort ical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), correspo nding 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 (I-SC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent t ransient increase in the whole-cell conductance. Removal of extracellular N a+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly u pon application of AP while a Ca2+-free pipette solution completely abolish ed the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no ef fect on I-SC whereas subsequent basolateral application elicited a transien t increase in I-SC. This increase was not due to a stimulation of electroge nic Na+ absorption since the response was preserved in the presence of amil oride. 5. The I-SC, 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 w hole-cell currents or I-SC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secret ion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolater al PAR-2.