ATP Release Via Connexin Hemichannels Controls Intercellular Propagation of Ca Waves in Corneal Endothelial Cells - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 175

10.3.3.2 Effect of Thrombin on PIC is Inhibited by Adenosine

Adenosine was previously shown to decrease the permeability of the corneal
endothelial barrier [87]. We therefore investigated the effect of adenosine on IC.
Pretreatment with adenosine prevented the inhibitory effect of thrombin on inter-
cellular Ca 2+ wave propagation, while it had no significant effect on Ca 2+ wave
propagation in control conditions (Fig. 10.8). NECA (a potent A2B agonist) and
forskolin, agents known to elevate cAMP in BCEC, also suppressed the effect of
thrombin. The A1 receptor agonist CPA (N6-cyclopentyladenosine) failed to inhibit
the effect of thrombin [27].
Similarly to the effects on Ca 2+ wave propagation, adenosine prevented the
thrombin-induced reduction in the fluorescence recovery during photobleaching
experiments, providing evidence for inhibition by adenosine of the effect of
thrombin on GJIC. Pretreatment with adenosine also prevented both thrombin
and TRAP-6 from blocking the uptake of lucifer yellow in a Ca 2+ -free medium.
Adenosine was ineffective in overcoming the inhibition of lucifer yellow by
43 Gap26 [27]. These experiments provide evidence that the main mechanism of
the effect of adenosine in overcoming the inhibition of IC by thrombin involves
the hemichannel-mediated paracrine pathway. In consistence with this conclusion,
the thrombin-induced inhibition of ATP release was also overcome by pretreatment
with adenosine.

10.3.3.3 Mechanism of Effect of Effects of Thrombin and Adenosine on
Intercellular Communication

Signaling Pathways Initiated by Thrombin and Adenosine

It has been demonstrated that the thrombin-induced decrease in barrier integrity
of BCEC is mediated via myosin light chain (MLC) phosphorylation [94]. MLC
phosphorylation is driven by MLC kinase (MLCK), while its dephosphorylation
is brought about by myosin light chain phosphatase (MLCP). MLCK can be acti-
vated by thrombin, since PAR-1 is coupled to G α q/11 , which results in Ca 2+ release,
and hence Ca 2+ -calmodulin binding to MLCK, which activates the kinase. PAR-1
receptors also activate PKC via G

12/13 ; both pathways
contribute to inactivation of MLCP. The transduction of the thrombin effect on MLC
phosphorylation can thus occur via two pathways, namely inhibition of the MLCP
or stimulation of the MLCK (Fig. 10.9). We performed experiments to investigate
whether thrombin exerts its effect on the Ca 2+ wave propagation through the same
transduction cascade that decreases the barrier integrity.
Pretreatment with MLCK inhibitor ML-7, with the Rho kinase inhibitor Y-27632
or with the PKC inhibitor chelerythrine caused a significant suppression of the effect
of thrombin or TRAP-6 on the propagation of the Ca 2+ wave. Upon pretreatment of
the cells with a combination of Y-27632 plus chelerythrine, or with the combination
of ML-7 plus Y-27632 plus chelerythrine, application of thrombin did not signifi-
cantly decrease the active area. The lack of a significant effect of thrombin in the

q/11 and Rho kinase via G α α