Two types of protease-activated receptors (PAR-1 and PAR-2) mediate calcium signaling in rat retinal ganglion cells RGC-5.

Protease-activated receptors (PARs), G-protein-coupled receptors, are widely expressed in various tissues, where they participate in physiological and pathological processes, such as hemostasis, proliferation, tissue repair, and inflammation. Recently, we found that PARs were upregulated in the rat retina following optic nerve crush injury. However, the role of PAR in retinal ganglion cells following optic nerve crush still remains unknown. Here, we studied PAR-mediated calcium signaling in retinal ganglion cells, RGC-5. Using reverse transcription-polymerase chain reaction, we demonstrate that RGC-5 cells mainly express PAR-1 and to a lower extent PAR-2, which was further confirmed by indirect immunofluorescence. Short-term stimulation of RGC-5 cells with thrombin (0.001-1 U/ml) and trypsin (1-100 nM) concentration-dependently induced a transient increase in intracellular calcium concentration (Ca(2+)). An increase in Ca(2+) was also induced by both TRag (PAR-1 activating peptide) and PAR-2 activating peptide (PAR-2 AP). The EC(50) values were 0.3 nM for thrombin, 12.0 nM for trypsin, 1.3 microM for TRag, and 1.6 microM for PAR-2 AP, respectively. Desensitization was studied using two successive pulses of agonists. The thrombin-induced calcium response was significantly reduced by PAR-1 desensitization caused by pre-challenging RGC-5 cells with thrombin or TRag, but not by PAR-2 desensitization. On the other hand, pretreatment with trypsin, TRag or PAR-2 AP desensitized the cells since the calcium response to a second exposure to trypsin was significantly reduced. Calcium source studies revealed that PAR-induced Ca(2+) rise mainly comes from intracellular stores in RGC-5 cells. Thus, we demonstrate that PAR-1 and PAR-2 are functionally expressed in retinal ganglion cells, mediating calcium mobilization mainly from intracellular stores.