Rapid Calcium Signaling by Novel Lacrimal Prosecretory Mitogen ‘Lacritin’ Involves Galpha, Phospholipase C and Protein Kinase C

Abstract

Abstract: : Purpose:Addition of prosecretory mitogen lacritin provokes a rapid calcium transient in rabbit lacrimal, human ductal (salivary) and human corneal cell lines – possibly as an early signaling step in its promotion of secretion and mitogenesis. To efficiently elucidate how lacritin signals through calcium and to gain insight into its putative receptor, inhibitor studies were performed on lacritin–treated human ductal cells in the presence of calcium indicator dye fluo–4, with fluorescence changes monitored by confocal microscopy.Methods:Subconfluent cultures were loaded for 1 hr with 2 µM fluo–4 AM, washed repeatedly with HBSS and then treated at t=20 sec with 0.1 nM – 10 µM lacritin in calcium–containing or calcium–free media with or without inhibitors. Images were obtained at <2 sec intervals (485 nm excitation; 525 nm emission) for up to 330 sec.Results:Cells pretreated with pertussis toxin (100 ng/ml) were completely unresponsive to lacritin, but not to positive control carbachol, indicating involvement of Gαi or Gαo. Also unresponsive were U73122– and heparin–treated cells respectively indicating roles for G subunit binding of phopholipase C, and inositol 3–phosphate (IP3) binding of the endoplasmic reticulum IP3 receptor in lacritin–induced calcium signaling. Interestingly, no lacritin transient was observed in the presence of PKC inhibitor G06976 (carbachol transient unaffected), apparently in keeping with blot evidence of lacritin–dependent PKC phosphorylation. Lacritin stimulated [35S]GTPγS binding at levels comparable to carbachol, albeit low in these cells. No significant cAMP changes or apparent effects of L–type channel blocker nifedipine were noted.Conclusions: Lacritin–dependent calcium signaling is mediated by GTP binding of a pertussis toxin–sensitive Gα subunit, a PLC isomer (likely PLCγ), IP3 ligation of endoplasmic reticulum IP3 receptors, and PKC. Parallels studies suggest no involvement of ERK1/2, JNK, c–Raf, Cdc42, Rho or Rac.

Keywords: lacrimal gland • signal transduction • calcium