Thrombin, a serine protease, is known to break down the barrier integrity in corneal endothelial cells by inducing formation of interendothelial gaps. We hypothesize that intercellular gap-formation can also affect gap junctions and hence influence intercellular communication (IC). In this study we investigated the effect of thrombin on gap junctional (GJIC) and paracrine intercellular communication (PIC) in cultured bovine corneal endothelial cells (BCEC).

Cell-cell communication was investigated by intercellular Ca²⁺ wave propagation, elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca²⁺]_i in the mechanically stimulated cell and in the neighboring cells were visualized with a confocal microscope using the fluorescent dye Fluo-4. Normalized fluorescence (calculated as the ratio of the average fluorescence of a cell to the average value under resting conditions) and the area reached by the Ca²⁺ wave (active area) were used as a measure of [Ca²⁺]_i. GJIC was assessed by fluorescence recovery after photobleaching (FRAP). Activity of hemichannels was assayed by Lucifer Yellow (LY) uptake and also by ATP release using the Luciferin-Luciferase technique. RT-PCR and immunocytochemistry techniques were used to examine the expression of proteinase-activated receptors (PARs).

RT-PCR showed transcripts for PAR-1 and PAR-2, but not for PAR-4. Immunocytochemistry showed thrombin-sensitive PARs as well as trypsin-sensitive PAR-2. Both thrombin and the selective PAR-1 agonist TRAP-6 reduced the active area of the Ca²⁺ wave. These agents also reduced the fluorescence recovery in FRAP experiments. The effect of thrombin on the Ca²⁺ wave was inhibited by a peptide antagonist of PAR-1, but not by a PAR-4 antagonist. Activation of PAR-1 did not affect the Ca²⁺ wave propagation in cells pretreated with Gap26, which blocks Connexin43 hemichannels. However, pretreatment with Gap27, which inhibits Connexin43 gap junctions, decreased the effect of PAR-1 activation on the active area. Thrombin abolished the enhancement of the Ca²⁺ wave propagation by the ecto-nucleotidase inhibitor ARL-67156. The effect of the PAR-1 agonists on the Ca²⁺ wave was not detectable in cells pretreated with exogenous apyrases.

In order to investigate whether thrombin inhibits hemichannel-mediated PIC, we examined the effects of thrombin and TRAP-6 on LY uptake. Exposure of BCEC to both agonists under Ca²⁺-free medium led to complete inhibition of LY uptake, indicating that PAR-1 activation blocks LY uptake. Therefore, our results indicate that thrombin inhibits the connexin hemichannel-mediated PIC pathway. In order to determine if PAR-1 agonists inhibit ATP release upon mechanical stimulation, we measured extracellular ATP levels by Luciferin-Luciferase technique. Thrombin markedly reduced ATP release upon mechanical stimulation (median reduction of 69%; N = 55). The reduction was only 9% after pretreatment with PAR-1 antagonist. The PAR-4 antagonist YD-3 did not influence the effect of thrombin on ATP release. Similar to thrombin, TRAP-6 caused a marked inhibition of ATP release upon mechanical stimulation (median reduction of 50%; N =16).

In conclusion thrombin or TRAP-6 inhibit IC through activation of PAR-1 receptors. The effect is mainly through inhibition of the ATP-mediated PIC pathway but GJIC is also inhibited although to a minor extent.