Biology Reference
In-Depth Information
Involvement of Cytoskeleton Contraction in Thrombin-Induced Effects on IC
Thrombin induces actomyosin contraction in corneal endothelial cells, leading to
significant changes in the organization of the cortical actin cytoskeleton. Such
changes in the cytoskeleton have been implicated in loss of barrier integrity of
corneal endothelial monolayers [94]. Specifically, thrombin, which can be gener-
ated in the cornea [6], may lead to onset of centripetal forces by the peri-junctional
actomyosin ring (PAMR), a dense band of actin at the apical junctional complex.
These forces are opposed by intercellular tethering forces, which are essential to
establish interactions of the transmembrane proteins that make up the tight junc-
tions. Since the formation of intercellular gaps due to actomyosin contraction could
also influence GJIC, we investigated a possible role of contractility in the propaga-
tion of intercellular Ca
2+
waves by using blebbistatin, a selective myosin II ATPase
inhibitor [109, 62, 73].
Pretreatment with blebbistatin (5
M for 20 min) or its nitro derivative [64]
prevented the thrombin-induced inhibition of the Ca
2+
wave [79]. Neither photo-
inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect.
Blebbistatin also prevented thrombin-induced inhibition of lucifer yellow uptake,
ATP release and FRAP, indicating that it prevented the thrombin effect on PIC and
GJIC. In the absence of thrombin, blebbistatin had no significant effect on PIC
or GJIC. The drug had no influence on MLC phosphorylation or on [Ca
2+
]
i
tran-
sients in response to thrombin or ATP [79]. Our findings demonstrate that myosin
II-mediated actomyosin contractility plays a central role in thrombin-induced
inhibition of GJIC and of hemichannel-mediated PIC.
μ
10.4 Discussion
10.4.1 Contribution of Cx Hemichannels Towards IC in BCEC
Our experiments demonstrate that intercellular Ca
2+
wave propagation in BCEC
consists of both PIC and GJIC, and that PIC is the main contributor to IC.
Furthermore, our experiments show that ATP and ADP are the mediators of this
PIC, acting via activation of P2Y receptors, and that ATP is released via hemichan-
nels. Our results also provide evidence that the hemichannels involved in PIC in
corneal endothelial cells are of Cx43 isoform.
Recent experiments from other laboratories have provided strong evidence for
Panx hemichannels [17] as pathways for ATP release in many different cell types
(for review see [95]). The Panx subtype Panx1 is widely distributed among tissues
that exhibit IC via Ca
2+
waves [17]. The channel formed by Panx1 can be opened
by mechanical perturbation at the resting membrane potential [7]. The channel is
permeable to ATP and can be opened at physiological Ca
2+
levels [17]. Since high
concentrations of Cx mimetic peptides were shown to reduce the current through
Panx hemichannels, and since opening of Cx hemichannels has not been demon-
strated by electrophysiological methods under physiological conditions, it has been
Search WWH ::
Custom Search