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 (I(SC)) 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 I(SC) whereas subsequent basolateral application elicited a transient increase in I(SC). This increase was not due to a stimulation of electrogenic Na⁺ absorption since the response was preserved in the presence of amiloride. 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 whole-cell currents or I(SC). 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.