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of contractility of the cytoskeleton in the effect of thrombin. Inhibition of the myosin
II ATPase activity by blebbistatin inhibited the effect of thrombin, suggesting that
contractility of the actin cytoskeleton, including that of PAMR, plays an important
regulatory role in the activity of the hemichannels (Fig. 10.9).
Our findings on thrombin-mediated inhibition of hemichannel-mediated IC help
us understand the role of IC in corneal endothelial cells under physiological and
pathological situations. Thus, our results suggest that thrombin, besides enhancing
the permeability of the corneal endothelial monolayer, decreases the IC in corneal
endothelial cells, which may relate to pathological ophthalmic conditions that result
in inflammation, e.g., uveitis and infection. Endotoxin (LPS), a component of the
cell wall of gram-negative bacteria, triggers a cascade for macrophagal/endothelial
cells to secrete pro-inflammatory cytokines, such as TNF
, IL-1, IL-6 and IL-8.
These proinflammatory cytokines can induce the expression of tissue factor on
monocytes [68] and endothelial cells [118] (for review see [63]) via Toll-like recep-
tors (TLRs) [33] leading to thrombin formation. LPS can also cause activation of
factor XII, which is involved in the formation of thrombin. Since it has been shown
that all extrinsic and intrinsic tenase factors for generation of thrombin are present
in the cornea, thrombin can be formed in the cornea in situations of infection and
inflammation, where it causes permeabilization of the endothelial monolayer [6].
Our experiments demonstrate that thrombin also inhibits intercellular propaga-
tion of Ca
2+
waves, and that the inhibition is mainly due to a reduction of PIC,
but that also GJIC is reduced. The role of the reduction of IC between corneal
endothelial cells in situations of inflammation is not yet clear. However, IC may
control the spread and release of inflammatory mediators, thereby coordinating the
corneal inflammatory responses. At least in the lung capillary bed, Cx43-dependent
intercellular Ca
2+
wave propagation conveys the spread of inflammation by control-
ling expression of inflammatory mediators (P-selectin) [72]. Inhibition of Cx43 by
the Cx43-derived Cx-mimetic peptides
43
Gap26 or
43
Gap27 blocked the increase
of lung micro-vascular permeability by thrombin. It therefore seems plausible that
Ca
2+
wave propagation in corneal endothelial cells via PIC and/or GJIC may also
coordinate the inflammatory response of cells in the cornea.
Our experiments revealed that increased MLC phosphorylation inhibits IC in
the corneal endothelium, thereby suggesting a role of contractility of the actin
cytoskeleton in the regulation of IC. The role of the actin cytoskeleton and teth-
ering forces for gap junctions is reminiscent of interactions of the cytoskeleton with
tight junctions and adherens junctions in the regulation of barrier integrity [94, 105,
93]. However, the block of hemichannels involved in PIC in response to increased
actomyosin contraction suggests that the disposition of cortical actin also plays an
important role in the functioning and regulation of endothelial hemichannels. In sit-
uations of inflammatory stress, which often result in MLC phosphorylation [117,
121, 8, 103], block of hemichannels could be thought of as being a defense mech-
anism. Thus, such a block could prevent potential “bystander effects” [2, 102] by
limiting the release of signaling molecules. While IC could contribute to the spread
of inflammatory mediators, thrombin might act as a negative feedback mechanism,
limiting the spreading of the response of the cells to inflammation by reducing
α
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